EA046439B1 - METHODS AND COMPOSITIONS FOR TREATING MALIGNANT TUMORS - Google Patents

Description

Technical field

The present invention provides methods and compounds for treating cancer by administering to a subject a therapeutically effective amount of an inhibitor of G protein-coupled receptor 174 (GPR174)-mediated signaling, or a combination of an inhibitor of GPR174-mediated signaling and an inhibitor of ATP-adenosine-A2aR or -A2bR-mediated signaling signals (such as an A2aR antagonist and/or an A2bR antagonist, or a combination of an inhibitor of GPR174-mediated signaling and an inhibitor of an enzyme pathway involved in the production of adenosine, e.g. CD38, CD39 and/or CD73 (such as a CD73 inhibitor and/or a CD38 inhibitor, and /or a CD39 inhibitor), or an inhibitor of GPR174-mediated signaling and a T regulatory cell (Treg) attenuating agent, thereby stimulating an immune response in a mammalian subject in need thereof, such as a subject suffering from a cancer.

State of the art

Mammalian G protein-coupled receptors (GPCRs) constitute a superfamily of diverse proteins with hundreds of members. GPCRs act as receptors for many different signals. Sensory GPCRs (spurs) are receptors for sensory signals of external origin, which are sensed as light of various colors, odors, pheromones or tastes. Most other GPCRs respond to endogenous signals such as peptides, lipids, neurotransmitters, or nucleotides. GPCRs falling into the latter group are involved in numerous physiological processes, including the regulation of neuronal excitability, metabolism, reproduction, development, cell division, hormonal homeostasis, and behavior, and are differentially expressed in many cell types in the body.

Ligands have been identified for several GPCRs. GPCRs for which an endogenous or cognate ligand has not yet been identified are known as orphan GPCRs. As of 2019, approximately 120 GPCRs were considered to be orphan GPCRs, including GPR174 (see Orphan and other 7TM receptors, IUPHAR/BPS Guide to Pharmacology, accessed on 6/8/2019; Alexander S. et al., Br. J Pharmacol 174 Suppl 1:S17-S129, 2017). Of all drugs currently sold on the market, 30-40% are modulators of specific GPCRs. Of the approximately 240 non-orphan GPCRs, more than half are targeted by drugs marketed or currently in clinical development (see Hauser A. S. et al., Nature Reviews Drug Discovery 16(12):829–842, 2017).

Drug development to target a specific GPCR typically requires knowledge of the endogenous ligand or surrogate ligand, e.g. a non-endogenous molecule that binds and functionally interacts with a specific GPCR, which can serve as a screening control and/or as a medicinal chemistry basis for the development of synthetic drug molecules that functionally interact with that specific GPCR. Given the potential use of orphan GPCRs as targets for new drugs, the identification of ligands or surrogate ligands for orphan GPCRs may accelerate the development of new drugs for these receptors. However, despite significant efforts to identify such ligands, a large number of orphan receptors remain (see, for example, Levoye et al., Drug Discov. Today 13(1-2):52-8, 2008). Conventional GPCR screening efforts typically rely on drug discovery screening using both biochemical and functional assays, such as those measuring Ca signaling in response to GPCR activation. However, these conventional methods rely on the presence of an endogenous ligand or a surrogate ligand. When used for orphan GPCRs, these assays can only identify GPCR agonists. Using conventional assay technology, neither inverse agonists nor antagonists can be identified until the agonist is available. Importantly, many FDA-approved drugs targeting GPCRs are functional inhibitors (eg, partial agonists, inverse agonists, partial inverse agonists, or antagonists). Thus, conventional methods miss detection of this common and critical class of GPCR ligands.

GPR174, also known as FKSG79 and GPCR17, is expressed in a relatively small number of tissues, including the spleen, thymus, bone marrow, and lymph node (Regard et al., Cell 135:561-71, 2008; Chu et al., J Med Genet 50 :479-85, 2013; Sugita et al., Biochem Biophys Res Commun 430:190-5, 2013). In lymphoid tissues, GPR174 is expressed to high levels in naive B and T cells, especially regulatory T cells (Treg) (Barnes et al., J Exp Med 212:1011-20, 2015).

As described herein, GPR174 activity has been associated with cancer. Given the role that GPR174 appears to play in cancer, it is desirable to identify inhibitors of this receptor. There is a need in the art for effective treatments for malignant tumors. The methods and compositions presented herein address these and other shortcomings in the field.

Summary of the invention

The present inventors have identified chemical compounds that function

- 1046439 naturally interact with GPR174 and inhibit one or more GPR174-mediated signaling pathways and further have a characterized GPR174 receptor signaling profile that includes the Gs signaling pathway. The inventors have shown that representative GPR174 inhibitory compounds are capable of reducing the proportion of highly suppressive regulatory T cells or T-reg (FoxP3+Helios+) in human peripheral blood mononuclear cells (PBMCs), as described in Examples 6, 8 and 9. The inventors of the present invention further determined that inhibition of GPR174 stimulates IL-2 production in a dose-dependent manner in PBMC, as described in Examples 6-11. We have also determined that inhibition of GPR174 reduces the expression of immune cell-associated programmed death ligand-1 (PD-L1), the expression of cytotoxic T-lymphocyte-associated antigen 4 (CTLA4), and the expression of T-cell immunoreceptor with Ig and ITIM domains (TIGIT) ), and amphiregulin (AREG) expression as described in Examples 13 and 14. Thus, GPR174 inhibitory compounds can be used as drugs for use in stimulating an immune response in a mammalian subject and can form the basis for additional drugs. Based on these discoveries, the present invention provides methods in vivo and in vitro for inhibiting the GPR174 receptor and thereby stimulating an immune response, particularly in a subject suffering from non-malignant neoplasia or malignant neoplasia (ie, cancer).

As further described herein, the present inventors have also shown that combined inhibition of GPR174 and inhibition of adenosine 2a receptor (A2aR) and/or inhibition of adenosine 2b receptor (A2bR) results in a synergistic induction of Th1 cytokine production (e.g., IFN-γ, IL-2, TNF and GM-CSF) in human PBMC as described in Examples 15, 18-23, 27 and 29.

As further described herein, the present inventors also showed that mouse colon carcinoma tumor growth and melanoma tumor growth were reduced in GPR174-deficient mice treated with an anti-GITR antibody (ie, a T-reg attenuating agent) by compared to wild-type mice as described in Examples 24 and 25.

As further described herein, the present inventors have determined that cancer cell-derived exosomes stimulate GPR174, and inhibition of GPR174 in the presence of cancer cell-derived exosomes increases Th1 cytokine levels, thereby stimulating and/or enhancing the immune system, as described. in example 26.

In general, as shown herein, GPR174 is an immune-restricted Gas-coupled GPCR, and PS exposed on liposomes and cell membranes stimulates GPR174, supporting a model of active GPR174-mediated immunosuppression in the tumor microenvironment. In conditions where both PS/lysoPS and adenosine are present, inhibition of both axes is necessary to effectively restore T cell function. As further demonstrated herein, GPR174 deficiency enhances antitumor immune responses in mice.

Thus, in one aspect, the present invention provides a method of treating cancer, comprising administering to a patient in need thereof a therapeutically effective amount of a GPR174 inhibitor selected from the group consisting of:

i. compound 10, having the following structure:

ii. compound 6, having the following structure o

iii. compound 11, having the following structure:

iv. compound 20 having the following structure:

- 2 046439

and an adenosine-A2A (A2A) receptor antagonist selected from the group consisting of:

i. A (ZM-241385);

ii. (SCH-58261); And

In a preferred aspect of the present invention, the GPR174 inhibitor inhibits PS- or LysoPS-dependent activation of GPR174 signaling in a cell expressing GPR174 by at least 25%.

In another preferred aspect of the present invention, a GPR174 inhibitor stimulates an immune response in a patient by inhibiting GPR174 G-alpha-s signaling.

Preferably, the stimulated immune response includes an NK cell-mediated immune response and an antitumor cytotoxic T cell-mediated immune response.

In yet another aspect, the present invention provides a method of increasing the level of Th1 cytokines in human peripheral blood mononuclear cells (PBMCs), comprising contacting the human PBMCs with a GPR174 inhibitor selected from the group consisting of:

i. compound 10, having the following structure:

ii. compound 6, having the following structure:

iii. compound 11, having the following structure:

iv. compound 20 having the following structure:

- 3 046439

and an adenosine-A2A (A2A) receptor antagonist selected from the group consisting of:

i. ZM-241385;

ii. SCH-58261; and iii. PBF-509.

In a preferred aspect of the present invention, contacting is carried out in vitro.

In another preferred aspect of the present invention, the PBMC contain T cells or NK cells.

Preferably, the level of at least one of the Th1 cytokines IFN-γ, IL-2, TNF or GM-CSF is increased by at least 20%.

In yet another aspect, the present invention relates to a pharmaceutical composition for the treatment of cancer, comprising a combination of a GPR174 signaling inhibitor selected from the group consisting of:

i. compound 10, having the following structure:

ii. compound 6, having the following structure:

iii. compound 11, having the following structure:

iv. compound 20 having the following structure:

and an adenosine-A2A (A2A) receptor antagonist selected from the group consisting of:

i. ZM-241385;

ii. SCH-58261; and iii. PBF-509;

and a pharmaceutically acceptable excipient.

In yet another aspect, the present invention provides a method of enhancing an antitumor immune response in a subject who is currently being treated with an A2aR antagonist selected from the group consisting of:

i. ZM-241385;

ii. SCH-58261; and iii. PBF-509, comprising administering an effective amount of a GPR174 inhibitor selected from the group consisting of: i. compound 10, having the following structure:

- 4 046439

ii. compound 6, having the following structure:

iii. compound 11, having the following structure:

iv. compound 20 having the following structure:

to stimulate an enhanced antitumor response in a subject.

Adenosine signaling through the A2A receptor and/or A2B receptor pathway in particular has been identified as a promising modality for immunotherapy of malignancies. Leone et al., Targeting adenosine for cancer immunotherapy, J. ImmunoTher. Cancer., 2018, 5:57. Allard et al, The ectonucleotidases CD39 and CD73: novel checkpoint inhibitor targets, Immunol. Rev., 2017, 275(1), 121-144 (see also Table 15 below). Adenosine is an immunosuppressive metabolite produced at high levels in the tumor microenvironment. Adenosine signaling has emerged as a key metabolic pathway regulating tumor immunity. In particular, adenosine signaling through the A2A receptor expressed on immunocytes strongly attenuates immune responses in inflamed tissues. Adenosine is produced under hypoxic conditions in inflamed and malignant tissues through the conversion of ATP mediated by the enzymes CD73 (NT5E, 5'-nucleotidase (5'NT) or ecto-5'-nucleotidase) and CD39 (ectonucleoside triphosphate diphosphohydrolase-1, NTPDase1) . Adenosine can also be produced from NAD+ in an axis centered on the NAD+ metabolizing CD38 producing adenosine diphosphate ribose (ADPR). Adenosine produced through the CD38-mediated pathway has been shown to correlate with the progression of human myeloma (Horenstein et al., Mol Med 22:694-704, 2016). Adenosine production and signaling, particularly through the A2A receptor, plays a role in resolving inflammation in response to tissue damage by dampening the immune response. This association of wound healing and immunosuppression, however, represents a mechanism for malignant tumor evasion from immunological surveillance. Accordingly, inhibition of the hypoxia-CD38CD39-CD73-A2AR pathway has been identified as a promising target for immunotherapy of malignant tumors.

Inhibitors of the ATP adenosine receptor A2A and/or A2B pathway.

Inhibitors of the adenosine A2A receptor ATP pathway and/or adenosine A2B receptor ATP pathway inhibitors that can be used in combination with GPR174 inhibitors in the compositions, methods and uses described herein include adenosine A2A receptor antagonists, A2B receptor antagonists and enzyme inhibitors agents that degrade ATP to adenosine, such as CD73 inhibitors and CD38 inhibitors, and CD39 inhibitors, and combinations of such agents.

Adenosine A2A receptor antagonists.

Adenosine A2A receptor antagonists include compounds ATL-444, AZD4635, caffeine, cyforadenant (CPI-444; V81444), CPI-445, EOS100850, MK-3814, istradefylline (KW-6002), MSX-3, PBF-509 (NIR178) , preladenant (SCH-420,814), SCH-58261, SCH-412,348, SCH-442,416, ST-1535, ST-4206, theophylline, tosadenant (SYN115), VER-6623, VER-6947, VER-7835, vipadenant (BIIB -014) and ZM-241,385, the structures of which are given below.

- 5 046439

V81444J

Additional A2A receptor antagonists are described in Preti, et al., History and Perspectives of A2A Adenosine Receptor Antagonists as Potential Therapeutic Agents, Med. Res. Rev., 2015, 35(4), 790-848, co-Preladenant (SCH-420,814)

- 6 046439 the contents of which are incorporated herein by reference, and Congreve et al., Targeting adenosine A2A receptor antagonism for the treatment of cancer, Exp. Opin. DrugDiscov., 2018, 73(11), 997-1003.

Other A2A receptor antagonists are described in the following international patent applications, the contents of which are incorporated herein by reference.

Publications

PCT No. WO 2019/007140; WO 2018/166493; WO 2018/161910; WO 2018/130184; WO 2018/059531; WO 2017/136375; WO2 017/112917; WO 2017/008205;WO

2017/011214; WO 2016/200717; WO 2016/126570; WO 2016/087429; WO 2016/081290;WO

2015/020565; WO 2014/105664; WO 2014/105666; WO 2014/101120; WO 2014/101113;WO

2013/156614, WO 2013/058681; WO 2012/129381; WO 2012/112962; WO 2012/061787;WO

2012/060844; WO 2012/038980; WO 2011/061527; WO 2011/060207; WO 2011/053507;WO

2010/040003; WO 2010/037122; WO 2009/055308; WO 2009/050198; WO 2008/055711;WO

2007/047293; WO 2007/038212; WO 2006/137527; WO 2006/129626; WO 2006/124770;WO

2006/083949; WO 2012/03898; WO 2011/06152; WO 2011/06020; WO 2011/05350;WO

2010/04000; WO 2010/03712; WO 2009/055308; WO 2009/050198; WO 2008/055711; WO 2007/047293; WO 2007/038212; WO 2006/137527; WO 2006/129626; WO 2006/124770; and WO 2006/083949.

Adenosine A2B receptor antagonists.

Adenosine A2B receptor antagonists include compounds MRS-1754, GS-6201, ISAM-140, PSB0788, PSB-1115 and PSB-603 whose structures are listed below:

Additional A2B receptor antagonists include ATL-801, CVT-6883, MRS-1706, OSIP339.391, PSB-1901, PBF-1129 and additional A2B receptor antagonists as described in Vigano S. et al., Frontiers in Immunology vol 10: 925, 2019; Volpini R. et al., Journal of Med Chem 45(15):3271-9, 2002; Volpini R. et al., Current Pharmaceutical Design 8(25):2285-98, 2002; Baraldi P.G. et al., Journal of Med Chem 47(6): 1434-47, 2004; Cacciari B. et al., Mini Reviews in Med Chem 5(12): 1053-60, 2005; Baraldi P.G. et al., Current Med Chem 13(28):3467-82, 2006; Beukers M.W. et al., Medical Research Reviews, 26(5):667-98, 2006; Elzein E. et al., Bioorganic & Medicinal Chemistry Letters 16(2):302-6, 2006; Carotti A. et al., Journal of Med Chem 49(1):282-99, 2006; Tabrizi M.A. et al., Bioorganic & Medicinal Chemistry 16(5):2419-30, 2008; Stefanachi A. et al., Bioorganic & Medicinal Chemistry 16(6):2852-69, 2008 and Jiang et al., Journal of Med Chem 62(8):4032-4055, 2019, as well as compound 38 described in Stefanachi A. et al., Bioorganic & Medicinal Chemistry 16(22):9780-9, 2008.

Other A2B receptor antagonists are described in the following international patent applications, the contents of which are incorporated herein by reference.

Publications

PCT No. WO 1995/011681; WO 1999/042093; WO 2000/049051; WO 2000/073307; WO 2009/157938; WO 2011/005871; WO 2012/112964; WO 2007/149277; WO 2019/123482; WO 2007/134958; WO 2005/051951; WO 2003/042214; WO 2007/039297; WO 2005/040155; WO 2005/042534; WO 2004/106337; WO 2001/016134; WO 2008/027585; WO 2003/053366; WO 2003/053361; WO 2005/070926; WO 2008/080461; WO 2016/150901; WO 2005/051951; WO 2003/042214; WO 2003/063800; and WO 2016/164838.

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CD73 inhibitors (NT5E, 5'-nucleotidase (5'-NT) or ecto-5'-nucleotidase) CD73 inhibitors may include anti-CD73 antibodies, nucleotides (eg, inhibitory RNA) that inhibit CD73 expression, as well as chemicals (eg , low molecular weight) inhibitors.

Antibodies against CD73, including oleclumab, are described in the following international patent applications, the contents of which are incorporated herein by reference.

Publications

PCT No. WO 2018/237157; WO 2018/237173; WO 2018/215535; WO

2018/187484; WO 2018/137598; WO 2018/013611; WO 2017/152085; WO 2017/118613; WO

2017/100670; WO 2017/064043; WO 2016/131950; WO 2016/081748; WO 2016/075176; WO 2016/075099; and WO 2016/055609.

Nucleotides that inhibit CD73 expression are described in the following international patent applications, the contents of which are incorporated herein by reference. PCT Publication No. WO 2018/065627.

CD73 inhibitors include benzyl-a,β-methyladenosine 5'-diphosphate sodium and α,β-methyladenosine 5'-diphosphate sodium (PSB 12379), BMS-986179, MEDI9447, CPI-006 and NZV930. CD73 inhibitors are also described in the following international patent applications, the contents of which are incorporated herein by reference.

Publications

PCT No WO 2018/208727; WO 2018/208980; WO

2018/187512; WO 2018/183635; WO 2018/110555; WO 2018/094148; WO 2018/067424; WO

2017/153952; WO 2017/120508; WO 2017/098421; WO 2015/164573; WO 2015/049447; and WO 2007/135195.

It is noted that while CD73 inhibitors are useful in the compositions and methods described herein for the treatment of cancer, the use of a CD73 inhibitor is preferably avoided during early tumor formation and in metastatic settings due to the fact that formed through CD73 adenosine may help prevent metastasis (may act as a barrier in the vascular endothelium), so it may be preferable to have CD73 remaining active in such conditions.

CD38 (cyclic ADP-ribose hydrolase) inhibitors.

CD38 is an ectoenzyme that catalyzes the synthesis and hydrolysis of cyclic ADPribose (cADPR) from NAD+ to ADP-ribose, in addition to the synthesis of NAADP from NADP+.

CD38 inhibitors may include anti-CD38 antibodies, nucleotides (eg, inhibitory RNA) that inhibit CD38 expression, as well as chemical (eg, small molecule) inhibitors.

Antibodies against CD38, including daratumumab, and other CD38 inhibitors, are described in the following publications and international patent applications, the contents of which are incorporated herein by reference; Xia S, et al, Drugs of Today. 52 (10): 551-560, 2016; Escande C et al., Diabetes 62(4): 1084-1093, 2013;

US Patent No. 9840496.

US Patent Publications No. 2018/85383; 2017/0260164.

Publications

PCT No. WO

2019/140410; WO 20190/74973; WO 2019/0904931; WO 2019/087151; WO 2019/074973; WO

2019/034753; WO 2019/034752; WO 2019/020643; WO 2018/224685; WO 2018/224683; WO

2018/224682; WO 2016/087975; and WO 2013/002879

CD39 Inhibitors (ectonucleoside triphosphate diphosphohydrolase-1, NTPDase1) CD39 inhibitors may include anti-CD39 antibodies, nucleotides (eg, inhibitory RNA) that inhibit CD39 expression, as well as chemical (eg, small molecule) inhibitors.

Antibodies against CD39, including TTX-030, are described in the following international patent applications, the contents of which are incorporated herein by reference.

Publications

PCT No. WO 2018/167267; WO 2018/065552; WO 2017/191300; WO

2017/157948; WO 2017/089334; WO 2017/064043; WO 2016/073845; WO 2012/085132; and WO 2009/095478.

Nucleotides that inhibit CD39 expression are described in the following international patent applications, the contents of which are incorporated herein by reference. PCT Publication No. WO 2018/065622.

CD39 inhibitors include sodium metatungstate (POM 1); trisodium salt 6-Ν,Ν-,·ιη)τηί-Ωβ,γ-dibromomethylene-ATP (ARL-67156); 1-amino-4-(1-naphthyl)aminoanthraquinone-2-sulfonic acid sodium salt (PSB 06126), 1-amino-4-(4-chlorophenyl)aminoanthraquinone-2-sulfonic acid sodium salt (PSB069) and IPH52. CD39 inhibitors are also described in the following international patents:

- 8 046439 applications, the contents of which are given in this description by reference: PCT Publication No. WO 2007/135195.

Other features and advantages of the present invention will be apparent from the following detailed description, drawings and claims.

Brief description of drawings

Fig. 1A shows the amino acid sequence of human GPR174 (NP_115942.1), indicated as SEQ ID NO: 1.

Fig. 1B shows a cDNA encoding human GPR174 (NM_032553.1) listed as SEQ ID NO: 2.

Fig. 1C shows the amino acid sequence of mouse GPR174 (NP_001028423.1), indicated as SEQ ID NO: 3.

Fig. 1D shows the amino acid sequence of rat GPR174 (NP_001100408.1), indicated as SEQ ID NO: 4.

Fig. 2 is a graph showing that the ranking order of reported activity of a variety of known muscarinic acetylcholine receptor M1 (CHRM1) agonists is maintained in a cellular redistribution assay (CRA).

Fig. 3 is a schematic diagram showing the algorithm used to determine the G protein signaling profile for an orphan GPCR.

Fig. 4A is a graph showing the effect of various compounds on adrenergic receptor beta 2 (ADBR2) signaling.

Fig. 4B is a graph showing the effects of ADBR2 overexpression on the CRE-luc reporter, as determined by transient transfection with increasing amounts (ng) of DNA encoding ADBR2.

Fig. 4C is a graph showing the effects of ADBR2 overexpression on the NFAT-luc reporter, as determined by transient transfection with increasing amounts (ng) of DNA encoding ADBR2.

Fig. 4D is a graph showing that ADBR2 inverse agonist compound ICI 118551 specifically inhibits CRE-luc reporter activity in cells transiently transfected with ADBR2.

Fig. 4E is a graph showing that compound ICI 118551 does not inhibit NFAT-luc reporter activity in cells transiently transfected with ADBR2.

Fig. 5A is a graph illustrating dose response curves of representative compounds, including compounds 1, 2, 3, 4 and 20, against GPR174 in a cell redistribution assay (CRA).

Fig. 5B is a graph illustrating the dose response curve of representative compounds, including compound 1, 2, 3, 4 and 20, against CHRM1 in the CRA assay. A dose-response curve for the CHRM1-interacting compound pirenzepine is also shown.

Fig. 6A-6B are graphs showing that compound 1 (group I) inhibited the Gs pathway in the presence of GPR174 (Fig. 6A) but not in the presence of ADBR2 (Fig. 6B).

Fig. 7A-7B are graphs showing that compound 2 (group I) inhibited the Gs pathway in the presence of GPR174 (Fig. 7A) but not in the presence of ADBR2 (Fig. 7B).

Fig. 8A-8B are graphs showing that compound 4 (group I) did not modulate the Gs pathway in the presence of GPR174 (Fig. 8A) and did not modulate the Gs pathway in the presence of ADBR2 (Fig. 8B).

Fig. 9A-9B are graphs showing that compound 6 (group I) inhibited the Gs pathway in the presence of GPR174 (Fig. 9A) but not in the presence of ADBR2 (Fig. 9B).

Fig. 10A-10B are graphs showing that compound 7 (group I) inhibited the Gs pathway in the presence of GPR174 (Fig. 10A) but not in the presence of ADBR2 (Fig. 10B).

Fig. 11A-11B are graphs showing that compound 10 (group I) inhibited the Gs pathway in the presence of GPR174 (FIG. 11A) but not in the presence of ADBR2 (FIG. 11B).

Fig. 12A-12B are graphs showing that compound 11 (group I) inhibited the Gs pathway in the presence of GPR174 (Fig. 12A) but not in the presence of ADBR2 (Fig. 12B).

Fig. 13A-13B are graphs showing that compound 19 (group II) did not modulate the Gs pathway in the presence of GPR174 (Fig. 13A) and did not modulate the Gs pathway in the presence of ADBR2 (Fig. 13B).

Fig. 14A-14B are graphs showing that compound 21 (group III) did not modulate the Gs pathway in the presence of GPR174 (Fig. 14A) and did not modulate the Gs pathway in the presence of ADBR2 (Fig. 14B).

Fig. 15A-15B are graphs showing that compound 22 (group IV) inhibited the Gs pathway in the presence of GPR174 (FIG. 15A) but not in the presence of ADBR2 (FIG. 15B).

Fig. 16A-16B are graphs showing that compound 23 (group IV) inhibited the Gs pathway in the presence of GPR174 (FIG. 16A) but not in the presence of ADBR2 (FIG. 16B).

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Fig. 17A-17B are graphs showing that compound 31 (group IV) inhibited the Gs pathway in the presence of GPR174 (FIG. 17A) but not in the presence of ADBR2 (FIG. 17B).

Fig. 18A-18B are graphs showing that compound 33 (group IV) inhibited the Gs pathway in the presence of GPR174 (FIG. 18A) but not in the presence of ADBR2 (FIG. 18B).

Fig. 19A-19B are graphs showing that compound 36 (group IV) inhibited the Gs pathway in the presence of GPR174 (FIG. 19A) but not in the presence of ADBR2 (FIG. 19B).

Fig. 20A-20B are graphs showing that compound 42 (group IV) inhibited the Gs pathway in the presence of GPR174 (FIG. 20A) but not in the presence of ADBR2 (FIG. 20B).

Fig. 21A-21B are graphs showing that compound 4 (group I) does not modulate the Gs pathway in the presence of GPR174, but competes with the GPR174 agonist LysoPS (Fig. 21A), and neither compound 4 nor LysoPS modulates Gs signaling in the presence ADBR (Fig. 21B).

Fig. 22 graphically illustrates the relative abundance of GPR174 transcript in human tissues as measured by qPCR as described in Example 5.

Fig. 23 graphically illustrates the relative abundance of GPR174 transcript in human lymphoid cells as measured by c.|PCR. as described in example 5.

Fig. 24A graphically illustrates the percentage of FoxP3'Helios ⁻ cells in the CD4+ cell population on day 3 after stimulation in human peripheral blood mononuclear cell (PBMC) cultures treated with vehicle, compound 10, or interleukin-2 (IL-2) (n=0.03 for compound 10 versus vehicle; p=0.003 for IL-2 versus vehicle) as described in Example 6.

Fig. 24B graphically illustrates the percentage of FoxP3'Helios' cells in the CD4+ cell population at day 7 post-stimulation in PBMC cultures treated with vehicle, compound 10, or IL-2 (p=0.01 for compound 10 vs. vehicle; p=0.01 for IL -2 against the carrier), as described in example 6.

Fig. 25 graphically illustrates the amount of IL-2 in culture supernatants (fold over vehicle levels) on day 2 post-stimulation in PBMC cultures treated with vehicle or Compound 10 (1, 3, or 10 μM) as described in Example 6.

Fig. 26 graphically illustrates the amount of IFN-γ in culture supernatants (fold over vehicle levels) on day 2 post-stimulation in PBMC cultures treated with vehicle or Compound 10 (1, 3, or 10 μM) as described in Example 6.

Fig. 27 graphically illustrates the amount of IL-2 (pg/ml) in culture supernatants 48 hours after stimulation in splenocyte cultures from WT or GPR174 KO mice treated with vehicle or Compound 10 (10 μM) as described in Example 7.

Fig. 28 graphically illustrates the percentage of FoxP3'Helios' cells in the CD4+ cell population at day 7 post-stimulation in single-donor PBMC cultures treated with vehicle or Compound 10 (3 μM, 10 μM, or 30 μM) (n=3, *n<0, 05; **n<0.01), as described in example 8.

Fig. 29 graphically illustrates the amount of IL-2 (pg/ml) in culture supernatants on day 3 post-stimulation in single-donor PBMC cultures treated with vehicle or GPR174 inhibitory compound 10 (3, 10, 30, or 60 μM) as described in Example 8 .

Fig. 30 graphically illustrates the proportion of the vehicle value of FoxP3'Helios' cells in the CD4+ cell population at day 7 post-stimulation in PBMC cultures from eight different donors treated with vehicle or Compound 10 (30 μM) as described in Example 9.

Fig. 31 graphically illustrates the amount of IL-2 (fold over vehicle IL-2 production) in culture supernatants on day 2 post-stimulation in PBMC cultures from eight different donors treated with vehicle or Compound 10 (30 μM) as described in Example 9 .

Fig. 32 graphically illustrates the amount of different cytokines (fold compared to vehicle cytokine production) in culture supernatants on day 2 post-stimulation for human PBMCs obtained from eight donors treated with vehicle or compound 10 (30 μM), where a statistically significant fold increase was observed for interleukin-6 (IL-6) (***p<0.001), interleukin-10 (IL-10) (*p<0.05), interferon gamma (IFN-γ) (*p<0.05) and tumor necrosis factor (TNF-a)(*p<0.05), compared with vehicle control, and where a statistically significant fold reduction was observed for interleukin 17 (EL-17A) (***p<0.001), compared with control media as described in example 9.

Fig. 33 graphically illustrates the fold induction by compound 10 (30 μM) in a panel of cytokines in co-cultured C57BL/6+DBA1 mouse splenocytes on day 6 after mixing, compared to cytokine levels measured in co-cultured C57BL/6+DBA1 splenocytes on day 6 in the absence of compound 10, as described in example 10.

In fig. 34A shows the CD4+ (46.2%), CD8+ (17.1%) and non-T cell (33.9%) populations present in a representative culture of PBMC from a single donor, four hours after stimulation with anti-CD3 antibody and against CD28 in the presence of compound 10 (10 μM), as described in example 11.

Fig. 34B graphically illustrates the proportion of cells with intracellular IL-2 in the CD4 ⁺ T cell population

- 10 046439 current treated with compound 10 (10 μM) shown in FIG. 34A, compared to the vehicle control as described in Example 11.

Fig. 34C graphically illustrates the proportion of cells with intracellular IL-2 in the CD8+ T cell population treated with Compound 10 (10 μM) shown in FIG. 34A, compared to the vehicle control as described in Example 11.

In fig. 35A shows the CD4+ (31.8%), CD8+ (27.5%) and non-T cell (33.1%) populations present in a representative culture of PBMC from a single donor, two days after stimulation with anti-CD3 antibody and against CD28, as described in example 11.

Fig. 35B graphically illustrates the proportion of cells with intracellular IL-2 staining in the CD4 ⁺ T cell population in PBMC cultures treated with Compound 10 (0, 0.1, 0.3, 1.0, or 3.0 μM), representative of which shown in Fig. 35 A, as described in example 11.

Fig. 35C graphically illustrates the proportion of cells with intracellular IL-2 staining in the CD8 ⁺ T cell population in PBMC cultures treated with Compound 10 (0, 0.1, 0.3, 1.0, or 3.0 μM), representative of which are shown in Fig. 35A, as described in example 11.

Fig. 35D graphically illustrates the proportion of cells with intracellular IL-2 staining in non-T cell populations in PBMC cultures treated with Compound 10 (0, 0.1, 0.3, 1.0, or 3.0 μM), representative of which are shown in Fig. 35A, as described in example 11.

Fig. 35E graphically illustrates the proportion of cells with intracellular IL-10 staining in the CD4 ⁺ T cell population in PBMC cultures treated with Compound 10 (0, 0.1, 0.3, 1.0, or 3.0 μM), representative of which shown in Fig. 35A, as described in example 11.

Fig. 35F graphically illustrates the proportion of cells with intracellular IL-10 staining in the CD8 ⁺ T cell population in PBMC cultures treated with Compound 10 (0, 0.1, 0.3, 1.0, or 3.0 μM), representative of which shown in Fig. 35A, as described in example 11.

Fig. 35G graphically illustrates the proportion of cells with intracellular IL-10 staining in non-T cell populations in PBMC cultures treated with Compound 10 (0, 0.1, 0.3, 1.0, or 3.0 μM), representative of which are shown in Fig. 35A, as described in example 11.

Fig. 35H graphically illustrates the proportion of cells with intracellular IFN-γ staining in the CD4 ⁺ T cell population in PBMC cultures treated with Compound 10 (0, 0.1, 0.3, 1.0, or 3.0 μM), representative of which are shown in Fig. 35A, as described in example 11.

Fig. 35I graphically illustrates the proportion of cells with intracellular IFN-γ staining in the CD8 ⁺ T cell population in PBMC cultures treated with Compound 10 (0, 0.1, 0.3, 1.0, or 3.0 μM), representative of which shown in Fig. 35A, as described in example 11.

Fig. 35J graphically illustrates the proportion of cells with intracellular IFN-γ staining in non-T cell populations in PBMC cultures treated with Compound 10 (0, 0.1, 0.3, 1.0, or 3.0 μM), representative of which shown in Fig. 35A, as described in example 11.

Fig. 35K graphically illustrates the proportion of cells with intracellular TNF-α staining in the CD4 ⁺ T cell population in PBMC cultures treated with Compound 10 (0, 0.1, 0.3, 1.0, 3.0, or 10 μM), representative for which are shown in Fig. 35 A, as described in example 11.

Fig. 35L graphically illustrates the proportion of cells with intracellular TNF-α staining in the CD8 ⁺ T cell population in PBMC cultures treated with Compound 10 (0, 0.1, 0.3, 1.0, 3.0, or 10 μM), representative of which are shown in Fig. 35A, as described in example 11.

Fig. 35M graphically illustrates the proportion of cells with intracellular TNF-α staining in non-T cell populations in PBMC cultures treated with Compound 10 (0, 0.1, 0.3, 1.0, 3.0, or 10 μM), representative for which are shown in Fig. 35A, as described in example 11.

In fig. 36A shows additional analysis of the non-T cell population from FIG. 35A, sorted into the CD56+CD16 ^- (2.27%), CD56+CD16+ (11.8%) and non-NK (78.5%) populations present in the non-T cell population in a representative PBMC culture after 2 days after stimulation with anti-CD3 antibody and anti-CD28 antibody, as described in example 11.

Fig. 36B graphically illustrates the proportion of cells with intracellular IFN-γ staining in He-T-CD56 ⁺ CD16 ⁺ cell populations in PBMC cultures treated with Compound 10 (0, 0.1, 0.3, 1.0, or 3.0 μM). representative ones of which are shown in FIG. 36A, as described in example 11.

Fig. 36C graphically illustrates the proportion of cells with intracellular IFN-γ staining in He-T-CD56 ⁺ CD16 ⁻ cell populations in PBMC cultures treated with Compound 10 (0, 0.1, 0.3, 1.0, or 3.0 μM). representative ones of which are shown in Fig. 36A, as described in example 11.

Fig. 36D graphically illustrates the proportion of cells with intracellular TNF-α staining in He-T-CD56 ⁺ CD16 ⁺ cell populations in PBMC cultures treated with Compound 10 (0, 0.1, 0.3, 1.0, 3.0, or 10 μM ), representative ones of which are shown in FIG. 36A, as described in example 11.

Fig. 36E graphically illustrates the proportion of cells with intracellular TNF-α staining in non-T-CD56 ⁺ CD16 ⁻ cell populations in PBMC cultures treated with Compound 10 (0, 0.1, 0.3, 1.0, 3.0, or 10 μM ), representative ones of which are shown in Fig. 36A, as described in example 11.

Fig. 37 graphically illustrates the number of cells stained with dextramer (antigen specific

- 11 046439 human T cells) as a percentage of total CD8+ cells in splenocyte cultures obtained from WT and GPR174 KO mice three weeks after immunization with 1000 HAU of influenza A/PR/8/34 virus, and cultured for 5 days in the presence of the antigenic peptide NP, where splenocytes obtained from naïve WT mice are included as a control as described in Example 12.

Fig. 38 graphically illustrates the number of dextramer-stained cells (antigen-specific T cells) as a percentage of total CD8+ cells in splenocyte cultures obtained from WT and GPR174 KO mice immunized and boosted three weeks later with 1000 HAU (hemagglutinating units) of influenza A virus /PR/8/34, where splenocytes were analyzed 11 days after boost, and where splenocytes obtained from naive WT mice are included as a control, as described in example 12.

Fig. 39A graphically illustrates the proportion of naïve regulatory T cells (Treg) with CTLA-4 positive staining (percentage of naïve Treg cells (CD45RA+FOXP3+) in PBMC cultures treated with GPR174 inhibitory compound 10 (0, 0.19, 0.38, 0.75, 1.5, 3.0 or 6.0 μM) or equivalent amounts of vehicle control (DMSO), one day after stimulation, as described in example 13;

Fig. 39B graphically illustrates the proportion of non-regulatory T cells (non-Treg) with CTLA-4 positive staining (percentage of naïve non-Treg cells (CD45RA+FOXP3-) in PBMC cultures treated with the GPR174 inhibitory compound 10 (0 , 0.19, 0.38, 0.75, 1.5, 3.0 or 6.0 μM) or equivalent amounts of control vehicle (DMSO), one day after stimulation, as described in example 13;

Fig. 40A graphically illustrates the proportion of memory T-regs with CTLA-4 positive staining (percentage of memory T-regs (CD45RA-FOXP3+) in PBMC cultures treated with GPR174 inhibitory compound 10 (0, 0.19, 0.38, 0.75 , 1.5, 3.0 or 6.0 μM) or equivalent amounts of control vehicle (DMSO), one day after stimulation, as described in example 13;

Fig. 40B graphically illustrates the proportion of non-Treg memory cells with CTLA-4 positive staining (percentage of non-Treg memory (CD45RA-FOXP3-)) in PBMC cultures treated with GPR174 inhibitory compound 10 (0, 0.19, 0 .38, 0.75, 1.5, 3.0 or 6.0 μM) or equivalent amounts of control vehicle (DMSO), one day after stimulation, as described in example 13;

Fig. 41 graphically illustrates the proportion of human CD4+ T cells with positive PD-L1 staining in PBMC cultures treated with GPR174 inhibitory compound 10 (0, 1.25 or 5 μM), one day after stimulation, showing that PD-L1 expression is reduced in CD4+ T cells treated with compound 10 in a dose-dependent manner compared to vehicle control-treated cells as described in Example 14;

Fig. 42 graphically illustrates the proportion of human CD8+ T cells with TGIT positive staining in PBMC cultures treated with GPR174 inhibitory compound 10 (0, 1.25 or 5 μM) one day after stimulation, showing that TGIT expression is reduced in CD8+ T cell treated compound 10, in a dose-dependent manner, compared to vehicle control-treated cells as described in Example 14; And

Fig. 43 graphically illustrates the proportion of human CD4+ T cells with positive AREG staining in PBMC cultures treated with GPR174 inhibitory compound 10 (0, 1.25 or 5 μM) one day after stimulation, showing that AREG expression is reduced in CD4+ T cells treated compound 10, in a dose-dependent manner, compared to vehicle control-treated cells as described in Example 14;

Fig. 44 graphically illustrates the amount of IFN-γ in culture supernatants 24 hours after stimulation in PBMC cultures from a single donor cultured in the presence of GPR174 inhibitory compound 10 (3 μM); A2aR inhibitory compound (ZM241385, 1 μM or 10 μM); combinations of compound 10 (3 μM) plus 1 μM ZM241385; combinations of compound 10 (3 μM) plus 10 μM ZM241385; or vehicle control (DMSO), showing that combined inhibition of GPR174 and A2aR results in a synergistic induction of IFN-γ production as described in Example 15.

Fig. 45A graphically illustrates the fold induction of Gs signaling activity in HEK293 cells transfected with wild-type GPR174 expression plasmid and cAMP biosensor expression plasmid pGlo22F in the presence of increasing concentrations of phosphatidylserine (PS) or lysophosphatidylserine (Lyso-PS) liposomes as described in Example 16.

Fig. 45B graphically illustrates the fold induction of Gs signaling activity in HEK293 cells transfected with the GPR174-v38 mutant expression plasmid and the cAMP biosensor expression plasmid pGlo22F, in the presence of increasing concentrations of phosphatidylserine (PS) or lysophosphatidylserine (Lyso-PS) liposomes as described in Example 16 .

Fig. 46 graphically illustrates the level of GPR174 Gs signaling activity in HEK293 cells transfected with the GPR174 expression plasmid and the cAMP biosensor expression plasmid pGlo22F (shown as the ratio of luminescence after adding phospholipid to the pre-read luminescence value) in the presence of PS liposomes, phosphatidyl

- 12 046439 choline (PC), phosphatidylethanolamine (PE) and phosphatidylinositol (PI), as described in example 16.

Fig. 47A graphically illustrates the inhibition of PS-induced GPR174 Gs signaling by GPR174 inhibitory compound 10 (group I) in a dose-dependent manner, with an apparent IC ₅₀ of approximately 20-40 nM, as described in Example 16.

Fig. 47B graphically illustrates the inhibition of PS-induced GPR174 Gs signaling by GPR174 inhibitory compound 6 (group I) in a dose-dependent manner, with an apparent IC ₅₀ of approximately 0.1 μM, as described in Example 16.

Fig. 47C graphically illustrates the inhibition of PS-induced GPR174 Gs signaling by GPR174 inhibitory compound 11 (group I) in a dose-dependent manner, with an apparent IC50 of approximately 0.1 μM, as described in Example 16.

Fig. 47D graphically illustrates the inhibition of PS-induced GPR174 Gs signaling by GPR174 inhibitory compound 20 (group II) in a dose-dependent manner, with an apparent IC ₅₀ of approximately 3.0 μM, as described in Example 16.

Fig. 47E graphically illustrates the inhibition of PS-induced GPR174 Gs signaling by GPR174 inhibitory compound 30 (group IV) in a dose-dependent manner, with an apparent IC ₅₀ of approximately 1.3 μM, as described in Example 16.

Fig. 47F graphically illustrates the inhibition of PS-induced GPR174 Gs signaling by GPR174 inhibitory compound 23 (group IV) in a dose-dependent manner, with an apparent IC ₅₀ of approximately 0.15 μM, as described in Example 16.

Fig. 48A graphically illustrates the ratio of luminescence after addition to pre-readout luminescence for GPR174 and GloSensor expressing HEK293 cells exposed to culture medium, or untreated (non-apoptotic) K562 cells, or apoptotic K562 cells (treated with H ₂ O ₂ for 20 hours) as described. in example 17.

Fig. 48B graphically illustrates the ratio of luminescence after addition to pre-read luminescence for GPR174 and GloSensor expressing HEK293 cells exposed to culture medium, or freshly isolated neutrophils, or apoptotic neutrophils (treated with anti-Fas antibodies) as described in Example 17.

Fig. 48C graphically illustrates the ratio of luminescence after addition to pre-read luminescence for GPR174 and GloSensor expressing HEK293 cells exposed to culture supernatant (sup.) or platelets (P1) as described in Example 17.

Fig. 49A graphically illustrates the amount of IFN-γ (pg/ml) in culture supernatants 24 hours after stimulation in PBMC cultures from a single donor (donor 1) cultured in the presence of GPR174 inhibitory compound 10 (2 μM); an A2aR inhibitory compound (SCH-58261, 0.2 µM, 0.6 µM or 2 µM); combinations of compound 10 (2 μM) plus 0.2 μM SCH-58261; combinations of compound 10 (2 μM) plus 0.6 μM SCH-58261 or combinations of compound 10 (2 μM) plus 2 μM SCH-58261; or vehicle control (DMSO) as described in Example 18.

Fig. 49B graphically illustrates the amount of IL-2 (pg/ml) in culture supernatants 24 hours after stimulation in PBMC cultures from a single donor (donor 1) cultured in the presence of GPR174 inhibitory compound 10 (2 μM); an A2aR inhibitory compound (SCH-58261, 0.2 µM, 0.6 µM or 2 µM); combinations of compound 10 (2 μM) plus 0.2 μM SCH-58261; combinations of compound 10 (2 μM) plus 0.6 μM SCH-58261 or combinations of compound 10 (2 μM) plus 2 μM SCH-58261; or vehicle control (DMSO) as described in Example 18.

Fig. 49C graphically illustrates the amount of TNF (pg/ml) in culture supernatants 24 hours after stimulation in PBMC cultures from a single donor (donor 1) cultured in the presence of GPR174 inhibitory compound 10 (2 μM); an A2aR inhibitory compound (SCH-58261, 0.2 µM, 0.6 µM or 2 µM); combinations of compound 10 (2 μM) plus 0.2 μM SCH-58261; combinations of compound 10 (2 μM) plus 0.6 μM SCH-58261 or combinations of compound 10 (2 μM) plus 2 μM SCH-58261; or vehicle control (DMSO) as described in Example 18.

Fig. 50A graphically illustrates the amount of IFN-γ (pg/ml) in culture supernatants 24 hours after stimulation in PBMC cultures from a single donor (donor 2) cultured in the presence of GPR174 inhibitory compound 10 (1 μM); A2aR inhibitory compound (ZM, 0.2 μM); A2aR inhibitory compound (PBF-509 0.1 μM), combination of compound 10 (1 μM) plus 0.2 μM ZM; combinations of compound 10 (1 μM) plus 0.1 μM PBF-509; or vehicle control (DMSO) as described in Example 18.

Fig. 50B graphically illustrates the amount of IL-2 (pg/ml) in culture supernatants 24 hours after stimulation in PBMC cultures from a single donor (Donor 2) cultured in the presence of GPR174 inhibitory compound 10 (1 μM); A2aR inhibitory compound (ZM, 0.2 μM); A2aR inhibitory compound (PBF-509 0.1 μM), combination of compound 10 (1 μM) plus 0.2 μM ZM; combinations of compound 10 (1 μM) plus 0.1 μM PBF-509; or vehicle control (DMSO) as described in Example 18.

Fig. 50C graphically illustrates the amount of TNF (pg/ml) in culture supernatants through

- 13046439 hours after stimulation in PBMC cultures from one donor (donor 2), cultured in the presence of GPR174 inhibitory compound 10 (1 μM); A2aR inhibitory compound (ZM, 0.2 μM); A2aR inhibitory compound (PBF-509 0.1 μM), combination of compound 10 (1 μM) plus 0.2 μM ZM; combinations of compound 10 (1 μM) plus 0.1 μM PBF-509; or vehicle control (DMSO) as described in Example 18.

Fig. 51A graphically illustrates the amount of IFN-γ (pg/ml) in culture supernatants 24 hours after stimulation in PBMC cultures from a single donor (donor 3) cultured in the presence of GPR174 inhibitory compound 10 (1 μM); A2aR inhibitory compound (ZM, 0.2 μM); A2aR inhibitory compound (PBF-509 0.2 μM), combination of compound 10 (1 μM) plus 0.2 μM ZM; combinations of compound 10 (1 μM) plus 0.2 μM PBF-509; or vehicle control (DMSO) as described in Example 18.

Fig. 51B graphically illustrates the amount of IL-2 (pg/ml) in culture supernatants 24 hours after stimulation in PBMC cultures from a single donor (donor 3) cultured in the presence of GPR174 inhibitory compound 10 (1 μM); A2aR inhibitory compound (ZM, 0.2 μM); A2aR inhibitory compound (PBF-509 0.2 μM), combination of compound 10 (1 μM) plus 0.2 μM ZM; combinations of compound 10 (1 μM) plus 0.2 μM PBF-509; or vehicle control (DMSO) as described in Example 18.

Fig. 51C graphically illustrates the amount of TNF (pg/ml) in culture supernatants 24 hours after stimulation in PBMC cultures from a single donor (donor 3) cultured in the presence of GPR174 inhibitory compound 10 (1 μM); A2aR inhibitory compound (ZM, 0.2 μM); A2aR inhibitory compound (PBF-509 0.2 μM), combination of compound 10 (1 μM) plus 0.2 μM ZM; combinations of compound 10 (1 μM) plus 0.2 μM PBF-509; or vehicle control (DMSO) as described in Example 18.

Fig. 52A graphically illustrates the fold change, relative to vehicle, in the amount of IFN-γ in culture supernatants 24 hours post-stimulation in PBMC cultures from 12 donors cultured in the presence of GPR174 inhibitory compound 10 (1-2 μM); A2aR inhibitory compound (ZM, 0.2-1 μM); combinations of compound 10 (1-2 µM) plus 0.2-1 µM ZM; or vehicle control (DMSO) as described in Example 19.

Fig. 52B graphically illustrates the fold change, relative to vehicle, in the amount of IL-2 in culture supernatants 24 hours post-stimulation in PBMC cultures from 12 donors cultured in the presence of GPR174 inhibitory compound 10 (1-2 μM); A2aR inhibitory compound (ZM, 0.2-1 μM); combinations of compound 10 (1-2 µM) plus 0.2-1 µM ZM; or vehicle control (DMSO) as described in Example 19.

Fig. 52C graphically illustrates the fold change, relative to vehicle, in the amount of TNF in culture supernatants 24 hours after stimulation in PBMC cultures from 12 donors cultured in the presence of GPR174 inhibitory compound 10 (1-2 μM); A2aR inhibitory compound (ZM, 0.2-1 μM); combinations of compound 10 (1-2 µM) plus 0.2-1 µM ZM; or vehicle control (DMSO) as described in Example 19.

Fig. 52D graphically illustrates the fold change, relative to vehicle, in the amount of GM-CSF in culture supernatants 24 hours post-stimulation in PBMC cultures from 12 donors cultured in the presence of GPR174 inhibitory compound 10 (1-2 μM); A2aR inhibitory compound (ZM, 0.2-1 μM); combinations of compound 10 (1-2 µM) plus 0.2-1 µM ZM; or vehicle control (DMSO) as described in Example 19.

Fig. 53A graphically illustrates the amount of IFN-γ (pg/ml) in culture supernatants 24 hours after stimulation of human PBMC cultures from a single donor (donor 3) cultured in the presence of GPR174 inhibitory compound 10 (1 μM); ZM (0.1 µM); PBF (0.1 μM), SCH (0.2 μM), MRS (1 μM), adenosine deaminase (ADA, 0.75 μg/ml), alone or in combination with compound 10, or vehicle control as described in the example 20.

Fig. 53B graphically illustrates the amount of IL-2 (pg/ml) in culture supernatants 24 hours after stimulation of human PBMC cultures from a single donor (donor 3) cultured in the presence of GPR174 inhibitory compound 10 (1 μM); ZM (0.1 µM); PBF (0.1 μM), SCH (0.2 μM), MRS (1 μM), adenosine deaminase (ADA, 0.75 μg/ml), alone or in combination with compound 10, or vehicle control as described in the example 20.

Fig. 53C graphically illustrates the amount of TNF (pg/ml) in culture supernatants 24 hours after stimulation of human PBMC cultures from a single donor (donor 3) cultured in the presence of GPR174 inhibitory compound 10 (1 μM); ZM (0.1 µM); PBF (0.1 μM), SCH (0.2 μM), MRS (1 μM), adenosine deaminase (ADA, 0.75 μg/ml), alone or in combination with compound 10, or vehicle control as described in the example 20.

Fig. 54A graphically illustrates the amount of IFN-γ (pg/ml) in culture supernatants 24 hours after stimulation of human PBMC cultures from a single donor (donor 6) cultured in the presence of GPR174 inhibitory compound 10 (1 μM); ZM (0.1 µM); PBF (0.1 µM), SCH (0.2 µM),

- 14 046439

MRS (1 μM), adenosine deaminase (ADA, 0.75 μg/ml), alone or in combination with compound 10, or vehicle control as described in Example 20.

Fig. 54B graphically illustrates the amount of IL-2 (pg/ml) in culture supernatants 24 hours after stimulation of human PBMC cultures from a single donor (donor 6) cultured in the presence of GPR174 inhibitory compound 10 (1 μM); ZM (0.1 µM); PBF (0.1 μM), SCH (0.2 μM), MRS (1 μM), adenosine deaminase (ADA, 0.75 μg/ml), alone or in combination with compound 10, or vehicle control as described in the example 20.

Fig. 54C graphically illustrates the amount of TNF (pg/ml) in culture supernatants 24 hours after stimulation of human PBMC cultures from a single donor (donor 6) cultured in the presence of GPR174 inhibitory compound 10 (1 μM); ZM (0.1 µM); PBF (0.1 μM), SCH (0.2 μM), MRS (1 μM), adenosine deaminase (ADA, 0.75 μg/ml), alone or in combination with compound 10, or vehicle control as described in the example 20.

Fig. 55A graphically illustrates the amount of IFN-γ (pg/ml) in supernatants 24 hours after stimulation of human PBMC (donor 7) in the presence of GPR174 inhibitory compounds 6, 10, 11, or 20, each in the presence or absence of ZM, as described in Example 21.

Fig. 55B graphically illustrates the amount of IL-2 (pg/ml) in supernatants 24 hours after stimulation of human PBMC (donor 7) in the presence of GPR174 inhibitory compounds 6, 10, 11, or 20, each in the presence or absence of ZM, as described in Example 21.

Fig. 55C graphically illustrates the amount of TNF (pg/ml) in supernatants 24 hours after stimulation of human PBMC (donor 7) in the presence of GPR174 inhibitory compounds 6, 10, 11 or 20, each in the presence or absence of ZM, as described in Example 21.

Fig. 55D graphically illustrates the amount of GM-CSF (pg/ml) in supernatants 24 hours after stimulation of human PBMC (donor 7) in the presence of GPR174 inhibitory compounds 6, 10, 11, or 20, each in the presence or absence of ZM, as described in Example 21.

Fig. 56A graphically illustrates the fold change, relative to vehicle, in the amount of IFN-γ in culture supernatants 24 hours post-stimulation in human PBMC cultures from 5 donors cultured in the presence of GPR174 inhibitory compound 10 (1 μM); A2aR inhibitory compound (ZM, 0.2 μM); A2bR inhibitory compound MRS (2 μM) or a combination of compound 10 (1 μM) plus 0.2 μM ZM plus 2 μM MRS, or vehicle control (DMSO) as described in Example 22.

Fig. 56B graphically illustrates the fold change, relative to vehicle, in the amount of IL-2 in culture supernatants 24 hours after stimulation in human PBMC cultures from 5 donors cultured in the presence of GPR174 inhibitory compound 10 (1 μM); A2aR inhibitory compound (ZM, 0.2 μM); A2bR inhibitory compound MRS (2 μM) or a combination of compound 10 (1 μM) plus 0.2 μM ZM plus 2 μM MRS, or vehicle control (DMSO) as described in Example 22.

Fig. 56C graphically illustrates the fold change, relative to vehicle, in the amount of TNF in culture supernatants 24 hours after stimulation in human PBMC cultures from 5 donors cultured in the presence of GPR174 inhibitory compound 10 (1 μM); A2aR inhibitory compound (ZM, 0.2 μM); A2bR inhibitory compound MRS (2 μM) or a combination of compound 10 (1 μM) plus 0.2 μM ZM plus 2 μM MRS, or vehicle control (DMSO) as described in Example 22.

Fig. 57 graphically illustrates the fold reduction in the number of AREG+ cells in the presence of vehicle; GPR174 inhibitory compound 10 (1 μM); ZM-241385 (0.2 µM); or a combination of compound 10 (1 μM) and ZM (0.2 μM) as described in Example 23.

Fig. 58 is a schematic diagram illustrating how both GPR174 and adenosine receptors respond to stress and cell death products as described in Example 23.

Fig. 59A graphically illustrates the growth of CT26 tumors in individual wild-type (WT) mice inoculated with CT26.c125 murine colon carcinoma cells on day 0 and treated with anti-GITR antibody on days 7 and 9 as described in Example 24.

Fig. 59B graphically illustrates the growth of CT26 tumors in individual GPR174-KO mice inoculated with CT26.c125 murine colon carcinoma cells on days 0 and treated with anti-GITR antibody on days 7 and 9; as described in example 24.

Fig. 60 graphically illustrates the percentage of survival of tumor-bearing WT and GPR174KO mice inoculated with CT26.c125 murine colon carcinoma cells on day 0 and treated with anti-GITR antibody on days 7 and 9, showing that GPR174-KO mice had a higher percentage of survival (t .e. were euthanized significantly later) than WT mice (p=0.03, log-rank test) as described in Example 24.

Fig. 61A graphically illustrates the growth of CT26 tumors in individual WT mice inoculated with CT26.c125 murine colon carcinoma cells on day 0 and treated with an

- 15 046439 with anti-GITR antibody on days 7, 9 and 14, as described in example 24.

Fig. 61B graphically illustrates tumor growth in individual GPR174-KO mice inoculated with CT26.c125 murine colon carcinoma cells on day 0 and treated with anti-GITR antibody on days 7, 9, and 14; as described in example 24.

Fig. 62 graphically illustrates the survival rate of tumor-bearing WT and GPR174KO mice inoculated with CT26.c125 murine colon carcinoma cells on day 0 and treated with anti-GITR antibody on days 7, 9, and 14, showing that GPR174-KO mice had a higher survival rate (i.e., were euthanized significantly later) than WT mice as described in Example 24.

Fig. 63A graphically illustrates the growth of B16F10-Kb melanoma tumors in individual WT mice inoculated with B16F10-Kb cells on day 0 and treated with anti-GITR antibody on days 4 and 14 as described in Example 25.

Fig. 63B graphically illustrates the growth of B16F10-Kb melanoma tumors in individual GPR174-KO mice inoculated with B16F10-Kb cells on day 0 and treated with anti-GITR antibody on days 4 and 14 as described in Example 25.

Fig. 63 C graphically illustrates the mean tumor volume for B16F10-Kb melanoma tumors in WT and GPR174-KO mice inoculated with B16F10-Kb cells on day 0 and treated with anti-GITR antibody on days 4 and 14, showing that the mean tumor sizes were significantly smaller in GPR174 KO mice on days 14 (where * denotes: p=0.01) and 16 (where *** denotes: p=0.00005), as described in example 25.

Fig. 64 graphically illustrates the survival rate of B16F10-Kb melanoma tumor-bearing WT and GPR174-KO mice inoculated with B16F10-Bb cells on day 0 and treated with anti-GITR antibody on days 4 and 14, showing that GPR174-KO mice had a higher survival rate (i.e., were euthanized significantly later) than WT mice (p=0.006, log-rank test) as described in Example 25.

Fig. 65 graphically illustrates the concentrations of IL-2 in cultures of stimulated human T cells in the presence or absence of PS liposomes (PSL) or GPR174 inhibitor (compound 10, 3 μM) as described in Example 26.

Fig. 66A graphically illustrates the concentration of IL-2 in cultures of stimulated human T cells in the presence or absence of PS liposomes, tumor exosomes, or a GPR174 inhibitory compound (compound 10, 0.5 μM) as described in Example 26.

Fig. 66B graphically illustrates the concentration of IFN-γ in cultures of stimulated human T cells in the presence or absence of PS liposomes, tumor exosomes, or a GPR174 inhibitory compound (compound 10, 0.5 μM) as described in Example 26.

Fig. 66C graphically illustrates the concentration of TNF in cultures of stimulated human T cells in the presence or absence of PS liposomes, tumor exosomes, or a GPR174 inhibitory compound (compound 10, 0.5 μM) as described in Example 26.

Fig. 67 graphically illustrates IL-2 levels (fold change relative to vehicle) in cultures of stimulated WT or GPR174-KO mouse T cells in the presence or absence of PS liposomes (PSL) or GPR174 inhibitor (compound 10, 1 μM) as described in example 26.

Fig. 68A graphically illustrates the effects of the adenosine A2a/A2b receptor antagonist ZM241385 (0.1 μM), the GPR174 inhibitory compound 10 (1 μM), or both compounds (ZN-241385 plus compound 10) on IL-2 levels in supernatants of cultured splenocytes isolated from n =3 WT mice after 2 days of stimulation with anti-CD3 and anti-CD28 as described in Example 27.

Fig. 68B graphically illustrates the effects of the adenosine A2a/A2b receptor antagonist ZM-241385 (0.1 μM), the GPR174 inhibitory compound 10 (1 μM), or both compounds (ZN-241385 plus compound 10) on IL-2 levels in supernatants of cultured splenocytes. isolated from n=3 GPR174-KO mice after 2 days of stimulation with anti-CD3 antibody and anti-CD28 antibody, as described in example 27.

Fig. 69 graphically illustrates the effects of adenosine A2a/A2b receptor antagonist ZM-241385 (0.2 µM), GPR174 inhibitory compound 10 (0.3 µM) or GPR174 inhibitory compound 6 (0.3 µM), or ZM-241385 plus compound 10 or ZM-241385 plus Compound 6 on IL-2 levels in supernatants of cultured splenocytes isolated from 3 WT mice after 2 days of stimulation with anti-CD3 and anti-CD28 in the presence of the adenosine receptor agonist NECA (0.1 μM) as described in Example 27 .

Fig. 70A graphically illustrates the effects of adenosine A2a/A2b receptor antagonist ZM241385 (0.2 μM), GPR174 inhibitory compound 10 (0.3 μM) or compound 6 (0.3 μM), or ZM-241385 alone or in combination with any GPR174 inhibitory compound compound on IL-2 levels in the supernatants of cultured splenocytes isolated from 3 WT mice after 2 days of stimulation with anti-CD3 and anti-CD28 in the presence of the adenosine receptor agonist NECA (0.1 μM), as described in Example 27.

- 16 046439

Fig. 70B graphically illustrates the effects of adenosine A2a/A2b receptor antagonist ZM-241385 (0.2 μM), GPR174 inhibitory compound 10 (0.3 μM) or compound 6 (0.3 μM), or ZM241385 alone or in combination with any GPR174 inhibitory agent. compound on IL-2 levels in the supernatants of cultured splenocytes isolated from 3 GPR174-KO mice after 2 days of stimulation with anti-CD3 and anti-CD28 in the presence of the adenosine receptor agonist NECA (0.1 μM), as described in Example 27.

Fig. 71 graphically illustrates the levels of IL-2 in supernatants of human PBMCs stimulated with anti-CD3 antibody and anti-CD28 antibody in the presence or absence of GPR174 inhibitory compound 10 (1 μM); A2aR/A2bR inhibitory compound ZM-241385 (0.1 μM), or CD73 inhibitory compound APCP (10 μM), collected 24 hours after stimulation as described in Example 28.

Fig. 72A graphically illustrates the concentration of IL-2 in purified human T cells stimulated with CD3/CD28 activator in the presence of PS liposome (PS, 1 μM), adenosine receptor agonist NECA (0.1 μM), or a combination of PS (1 μM) plus NECA (0.1 µM), compared to media alone as described in Example 29.

Fig. 72B graphically illustrates the concentration of IFN-γ (FIG. 72B) in purified human T cells stimulated with CD3/CD28 activator in the presence of PS liposome (PS, 1 μM), NECA adenosine receptor agonist (0.1 μM), or a combination of PS ( 1 µM) plus NECA (0.1 µM), compared to media alone as described in Example 29.

Fig. 72C graphically illustrates the concentration of IFN-γ (FIG. 72C) in purified human T cells stimulated with CD3/C28 activator in the presence of PS liposome (PS, 1 μM), NECA adenosine receptor agonist (0.05 μM), or a combination of PS ( 1 µM) plus NECA (0.05 µM), compared to media alone as described in Example 29.

Detailed description

As described in detail below, the present inventors have identified compounds that functionally interact with GPR174 and are capable of inhibiting one or more GPR174-mediated signaling pathways. The present inventors further characterized the signaling pathways activated by GPR174 and determined the signaling profile for this receptor, which includes the Gs signaling pathway. The present inventors have further determined that combined inhibition of GPR174 and inhibition of adenosine 2a receptor (A2aR) and/or adenosine 2b receptor (A2bR), or a combination of inhibition of GPR174 and an inhibitor of ATP hydrolysis to adenosine by CD38, CD39 and CD73 (such as a CD73 inhibitor and/or a CD38 inhibitor and/or a CD39 inhibitor) and/or a T-reg attenuating agent leads to a synergistic induction of the production of IFN-γ, IL-2, TNF and GM-CSF in human PBMC. The present inventors have further discovered that phosphatidylserine (PS) is an agonist of GPR174-mediated Gs signaling and have shown that PS signaling through GPR174 is inhibited by a variety of GPR174-inhibiting small molecules with diverse chemical structures. As further demonstrated herein, GPR174 deficiency enhances antitumor immune responses in mice.

Based on these findings, the present invention provides methods in vivo and in vitro for inhibiting GPR174-mediated signaling pathways, either alone or in combination with inhibition of ATP-adenosine-A2aR and/or A2bR mediated signaling (e.g., an A2aR antagonist and/or A2bR antagonist), or a combination of an inhibitor of GPR174-mediated signaling and an inhibitor of the hydrolysis of ATP to adenosine by CD38, CD39 and CD73 (for example, a CD73 inhibitor or a CD38 inhibitor or a CD39 inhibitor) and/or a T-reg attenuating agent, and thus stimulation immune response in a mammalian subject, in particular in subjects suffering from conditions such as cancer, as described herein.

Definitions

The term G protein-coupled receptor or GPCR or GPR refers to a transmembrane receptor capable of transmitting a signal from the environment outside the cell to the inside of the cell via the G protein pathway and/or the arrestin pathway. Hundreds of such receptors are known in the art; see, for example, Fredriksson et al., Mol. Pharmacol. 63:1256-1272, 2003, and Vassilatis, D.K., Proc Natl Acad Sci USA 100: 4903-4908 (2003), the contents of each of which are therefore incorporated by reference. These references characterize human and mouse GPCRs based on sequence homology and function. Human GPCRs can be divided into five classes: secretin, rhodopsin, glutamate, frizzled/Tas2 and adhesion molecules. Alternatively, receptors can be classified by their ligands, such as peptide hormones or small molecules (eg, biogenic amines). Other classification schemes include classification A-F, where class A represents rhodopsin- and adrenergic-related receptors, class B, calcitonin- and parathyroid hormone-related receptors, class C, metabotropic receptor-related receptors, and classes D-F represent receptors found in fungi and archaebacteria.

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The terms G protein-coupled receptor 174, GPR174, FKSG79, or GPCR17 refer to any naturally occurring forms of the GPR174 protein, such as SEQ ID NO: 1 shown in Figure 1, or naturally occurring variants thereof, such as variants having at least 90% identity (e.g., at least 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity) to SEQ ID NO: 1. Preferred forms of GPR174 have the ability to signal through at least one G-coupled pathway -receptor protein such as Gs.

The term G protein refers to a heterotrimeric protein complex that transduces a signal from an activated GPCR to effector molecule(s) within the cell, such as enzymes and ion channels. G proteins are composed of Ga, Ge and θγ subunits. The Ga subunit families include Gq, Gi, Gs and Ga12/13. G protein signaling pathways are named after the activated Ga subunit, i.e. Gas, Gai, Gaq and Ga12/13. The heterotrimeric G protein binds the activated GPCR protein, i.e. a GPCR protein bound to a ligand or surrogate ligand. When bound to a GPCR protein, the Ga subunit replaces guanosine diphosphate (GDP) with guanosine 5'-triphosphate (GTP) and dissociates from the Ge and Θγ subunits, which typically associate into a heterodimeric complex. Once dissociated, both the Ga-GTP-bound protein and the Gey complex can activate the signaling pathway. The Gq family includes Gaq, Ga11, Gal4 and Ga15/16. The Gi family includes Gai1-3, Gao, Gat, Gagust and Gaz. The Gs family includes Gas and Gaolf. G12/13 includes Ga12 and Ga13.

The term signal transduction pathway refers to the intracellular response to a signal. For the purposes of this description, the G protein signaling pathway refers to the intracellular response that occurs following binding of a ligand, surrogate ligand, or any other functional activator or functional inactivator to a G protein-coupled receptor, such as GPR174, and activates the G protein-coupled receptor. protein way. G protein signaling pathways include the Gq, Gi, Gs, G12/13, and βγ pathways. The arrestin signaling pathway refers to the intracellular response that occurs following binding of a ligand, surrogate ligand, or any other functional activator or functional inactivator to a G protein-coupled receptor, such as GPR174, and activation of an arrestin-mediated signaling pathway, such as arrestin 1, arrestin 2, arrestin 3 and arrestin 4.

The Gq signaling pathway or Gaq signaling pathway refers to an intracellular signaling pathway activated through activated Gq protein alpha. The Gaq protein is activated by replacing GTP with GDP after binding to an activated GPCR protein. Activation of the Gq signaling pathway typically activates phospholipase C β-isoforms (PLCe), which hydrolyze phosphatidylinositol to produce diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP ₃ ). IP ₃ is soluble and diffuses through the cytoplasm, and interacts with IP ₃ receptors on the endoplasmic reticulum, causing calcium release and an increase in intracellular calcium levels. DAG remains associated with the inner leaflet of the plasma membrane due to its hydrophobic nature, where it attracts protein kinase C (PKC), which becomes activated in conjunction with the binding of calcium ions. PKC then phosphorylates other proteins to control their function. Downstream effects, for example reporter gene expression regulated by a suitable transcription factor, can also be detected.

The Gi signaling pathway or Gai signaling pathway refers to an intracellular signaling pathway activated through activated Gi alpha protein. The Gai protein is activated after binding to an activated GPCR protein. Gai1-3, Gao and Gaz inhibit adenylyl cyclase (AC) activity. The Gi sensor protein, Gat, activates cyclic guanosine monophosphate (cGMP)-dependent phosphodiesterase, causing a decrease in intracellular cGMP levels, and Gagust activates phospholipase C (PLC). All members of the Gi family, with the exception of Gaz, are susceptible to pertussis toxin inhibition via adenosine diphosphate (ADP) ribosylation of their subunit! a (Siehler, Biotechnology 3:471-83, 2008).

The Gs signaling pathway or Gas signaling pathway refers to an intracellular signaling pathway activated through activated Gs alpha protein. The Gas protein is activated after binding to an activated GPCR protein. The Gs family of G proteins (Gas and Gaolf) activate AS, which forms the second messenger 3',5'-cyclic adenosine monophosphate (cAMP).

The G12/13 signaling pathway or Ga12/13 signaling pathway refers to an intracellular signaling pathway activated through activated G12/13 alpha protein. The Ga12/13 protein is activated after binding to the GPCR protein. The G12/13 family of G proteins (Ga12 and Ga13) activate Rho-guanine exchange factor (Rho-GEF) proteins. Rho-GEF proteins catalyze the exchange of GTP for GDP to activate RhoA. RhoA, in turn, activates Rho kinase, which further leads to activation of activating transcription factor 2 (ATF2) and leads to cellular responses (Liu et al., Methods Mol. Biol. 237:145-9, 2004).

The βγ signaling pathway refers to an intracellular pathway activated by the free (ie, not bound to Ga protein) βγ complex. βγ complexes can, for example, activate

- 18 046439 phospholipase A2, control the opening of G-protein coupled inward rectifying potassium channels (GIRK) upon binding to muscarinic acetylcholine receptors, activate L-type calcium channels, and initiate the phospholipase C pathway through activation of PLC.

The arrestin signaling pathway refers to an intracellular signaling pathway activated by arrestin 1, arrestin 2, arrestin 3, or arrestin 4. Arrestins are involved in signaling through ERK, JNK, p38, Akt, PI3-kinase, and RhoA (DeWire et al., Annu. Physiol. 69:483-510, 2007).

The term contacting is used interchangeably herein with the following: combining with, adding to, mixing with, introducing into, passing through, incubating with, flowing over, etc. For purposes of clarity, the phrase cell contacting includes administering a compound to a mammal (eg, orally, plasma, or intramuscularly) such that the compound contacts cells of the mammal in vivo.

The term GPR174-mediated signaling pathway refers to a signaling pathway whose activity is modulated, at least in part, by GPR174.

As used herein, the phrase modulate at least one GPR174-mediated signaling pathway refers to modulating (activating or inhibiting) at least a Gs signaling pathway that is functional in a cell expressing GPR174 by bringing the cell into contact with a compound that is functional interacts with GPR174.

A modulator is a compound that operably interacts with a GPCR and affects GPCR-mediated signaling activity, either alone or by altering the ability of another compound to influence GPCR-mediated signaling activity. Modulators include activators and inhibitors of the GPCR-mediated signaling pathway.

An activator is a compound that increases GPCR-mediated signaling in a signaling pathway. Activators can activate the receptor directly (eg, an agonist) or can increase receptor activation through another compound (eg, a positive allosteric modulator). Activators can bind a receptor and increase receptor activity, increase, open, activate, facilitate, enhance activation, sensitize, agonize, convert the receptor conformation to an active state, or up-regulate receptor-protein activity. Activators include agonists, partial agonists, and positive allosteric modulators.

The agonist, which may be a full agonist or partial agonist, binds the GPCR, activates the receptor and initiates a response, typically through G protein activation. The agonist increases receptor activity above normal baseline levels. A compound appears to be a receptor agonist if it increases the activity of the GPCR-mediated signaling pathway by at least 20% above the corresponding control (eg, background or baseline activity) at concentrations up to 50 μM. A full agonist is always an activator. A partial agonist has less intrinsic activity than a full agonist but increases receptor activity above baseline activity. A partial agonist can act as a functional inhibitor in the presence of a full agonist.

A positive allosteric modulator or PAM is a compound that binds a GPCR at a site other than the ligand binding site and increases GPCR-mediated signaling activity in response to agonists.

An inhibitor is a compound that reduces GPCR-mediated signaling in a signaling pathway. Inhibitors are compounds that functionally interact with GPCRs and partially or completely block activity, reduce, prevent, slow activation, inactivate, antagonize, desensitize, render the receptor conformation inactive, or block the ability of another compound (for example, an endogenous agonist ligand) to interact with receptor or otherwise down-regulate receptor activity. Inhibitors may reduce the original activity of the receptor (eg, an inverse agonist), or may block or reduce the activity of another compound (eg, a partial agonist or antagonist). Inhibitors include antagonists, inverse agonists, partial agonists, partial inverse agonists, and negative allosteric modulators. Inhibitors do not include compounds that act solely by reducing the expression of a receptor nucleic acid or protein.

An antagonist is an inhibitor that binds a GPCR, usually at the same site as the ligand or agonist. By itself, the antagonist does not activate or inhibit receptor signaling activity and does not change receptor activity from baseline levels. However, an antagonist is capable of inhibiting or blocking receptor activation in the presence of an agonist or ligand, or inhibiting or blocking receptor inhibition in the presence of an inverse agonist. A compound appears to be a receptor antagonist or allosteric modulator if, at concentrations up to 50 μM, it has activity in the CRA assay and it increases

- 19 046439 activity of the GPCR receptor-mediated signaling pathway with less than 20% increase, compared to the corresponding control (for example, background or baseline), or decreases the activity of the GPCR-mediated signaling pathway with less than 10% inhibition, compared to the corresponding control (for example, background or source). Additionally, a compound that inhibits the effect of an agonist or inverse agonist may be considered an antagonist.

An inverse agonist, which may be a full inverse agonist or a partial inverse agonist, is an inhibitor that binds the receptor and reduces the initial signaling of the GPCR-mediated signaling pathway. A compound appears to be an inverse agonist if, at concentrations up to 50 μM, it reduces baseline GPCR signaling pathway signaling by at least 10%. A partial inverse agonist has less intrinsic inhibitory activity than a full inverse agonist.

A negative allosteric modulator is a compound that binds a receptor at a site separate from the ligand binding site and reduces GPCR-mediated signaling activity in response to agonists.

A partial agonist may be a functional activator or a functional inhibitor, depending on the presence or absence of a full agonist in a given biological environment. Partial agonists bind to the receptor and increase receptor activity above the baseline level of activity, but are only partially as effective as a full agonist. For example, in the presence of a full agonist, a partial agonist can reduce receptor activation, thereby acting as a functional inhibitor. In the absence of a full agonist, a partial agonist can increase receptor activation, thereby acting as a functional activator.

A partial inverse agonist may be a functional activator or a functional inhibitor, depending on the presence or absence of a full inverse agonist in a given biological environment. Partial inverse agonists bind to the receptor and reduce receptor activity from baseline activity, but are only partially as effective as a full inverse agonist. For example, in the presence of a full inverse agonist, a partial inverse agonist can increase receptor activation, thereby acting as a functional activator. In the absence of a full inverse agonist, a partial inverse agonist can reduce receptor activation, thereby acting as a functional inhibitor.

As used herein, the term baseline activity or baseline signaling activity, or baseline activity refers to the level of GPCR-mediated signaling activity in the absence of a modulatory compound. In one embodiment, the baseline level of activity of the GPR174-mediated signaling pathway is determined in relation to a specific cell line, where the cell line is known to have functional GPR174 activity or defective GPR174 activity. In another embodiment, the baseline level of activity of the GPR174-mediated signaling pathway is determined in relation to a cell overexpressing GPR174 (eg, as described in Examples 2, 3 and 4 herein). In some embodiments, the baseline level of GPR174-mediated signaling pathway activity is determined relative to a cell known to interact with an endogenous ligand, or known to contain an endogenous GPR174 ligand, or relative to a cell not known to contain endogenous GPR174 -ligand, assuming that such an endogenous GPR174 ligand will be identified later.

A ligand is a compound that binds to a receptor and modulates the activity of the receptor.

An endogenous ligand is an endogenous entity (eg, molecule, peptide, or ion) that modulates a receptor in a cell, tissue, or source organism.

A surrogate ligand is a non-endogenous molecule that binds to a receptor and modulates its activity. The surrogate ligand may be natural or may be synthetic. A surrogate ligand can activate or inhibit receptor activity. In one embodiment, the surrogate ligand is a small molecule. In a further embodiment, the surrogate ligand is a small organic molecule.

A biased ligand is a compound that binds GPCRs and selectively modulates (more than 2-fold) the activity of one of the GPCR-mediated signaling pathways (e.g., Gq, Gi, Gs, G12/13, βγ, or arrestin signaling pathways) by compared to other GPCR-mediated signaling pathway(s).

As used herein, the term apparent binding affinity refers to the observed change in binding affinity of a GPR174 compound in the presence of an additional compound that binds GPR174 compared to the binding affinity observed for a GPR174 compound without the presence of an additional compound. In some embodiments, the compound and the additional compound compete for binding to the same site on GPR174. Although the binding affinities of each compound for GPR174 are not changed, the determination

- 20 046439 the binding affinities of one compound for GPR174 in the presence of an additional compound that binds GPR174 may result in a decrease in the measured binding affinity of the compound for GPR174. In some embodiments, the two compounds do not bind at the same site, and allosteric binding of one compound to GPR174 may result in decreased or increased binding affinity for GPR174 of the other compound. This may be due to conformational changes in GPR174 caused by the binding of one of the compounds to GPR174, which affects the binding site of the other compound.

A compound is specific for GPR174 if it is capable of modulating (e.g., inhibiting) the activity of GPR174 but does not have similar activity toward other proteins, such as other G protein-coupled receptors, e.g., compared to a reference panel of proteins (e.g. including GPCRs other than GPR174, and optionally also including ion channels and/or other types of transporters). In one example, the reference panel includes the following receptors: muscarinic Ml, CCRL2, CMKOR1, GPR3, GPR4, GPR12, GPR17, GPR18, GPR19, GPR20, GPR21, GPR22, GPR25, GPR26, GPR27, GPR31, GPR32, GPR34, GPR37, GPR37L1 , GPR39, GPR43, GPR45, GPR48, GPR50, GPR52, GPR61, GPR62, GPR63, GPR65, GPR68, GPR78, GPR80, GPR83, GPR85, GPR87, GPR88, GPR101, GPR132, GPR135, GPR139, GPR141, GPR148, GPR149 , GPR150, GPR151, GPR152, GPR153, GPR160, GPR161, GPR162, GPR173, GPR182, GPR183, LGR5, LGR6, MAS1, MRGD, MRGE, MRGF, MRGI4, OPN3, OPN4, OPN5, P2Y8, P2Y10, TAAR6 and TAAR8 .

In another example, the reference panel includes the following receptors: human adenosine A1; human adenosine A2A; rat adrenergic a1A; rat adrenergic a1B; human adrenergic a2A; human adrenergic β1; human adrenergic β2; rat L-type calcium channel dihydropyridine-sensitive; human dopamine D1; human dopamine D2; and human G protein coupled receptor, GPR103. In another example, the reference panel includes the following receptors: rat GABA A; rat NMDA glutamate; human histamine H1; human muscarinic _M2 ; human nicotinic acetylcholine α1; human opiate μ (OP3, MOP); human potassium channel [ _KATP ]; human potassium channel hERG; human sigma σ1; rat sigma σ ₂ ; rat sodium channel site 2; and the human transporter, norepinephrine.

In specific embodiments, a compound that is specific for GPR174 has 2, 5, 10, 25, 50, 100, 500, 1000, 5000, or 10,000 times greater activity in inhibiting GPR174 activity compared to other G-coupled protein receptors, for example, other GPCRs in one or more of the reference panels described above.

In specific embodiments, a compound that is specific for GPR174 has 2, 5, 10, 25, 50, 100, 500, 1000, 5000, or 10,000 times more activity in a Gs signaling assay that is performed in a cell expressing GPR174 , compared to Gs signaling activity in a cell expressing other GPCRs, for example, other GPCRs in each of the reference panels described above.

An agency-approved compound refers to a compound that was approved for clinical use for human or veterinary purposes before November 4, 2016, by the United States Food and Drug Administration (FDA) or a similar government regulatory agency (e.g., the European Medicines Agency). EMA), Health Canada, Ministry of Health and Welfare of Japan).

A compound not approved for use in GPR174-associated disease refers to a compound that was not approved for therapeutic use before November 4, 2016 by the FDA or similar government regulatory agency (e.g., EMA, Health Canada, and the Japanese Ministry of Health and Welfare) for an indication associated with the activity of GPR174 (eg, any of those described herein, such as a cancer or a disease or disorder of the nervous system).

A non-approved compound that targets GPR174 is defined as a compound that is not listed in a pharmacological data sheet accepted by the US FDA or similar government regulatory agency (e.g., EMA, Health Canada, and the Japanese Ministry of Health and Welfare) as targeting GPR174. until November 4, 2016. Indication of such targeting is often found in the labeling of an approved drug.

The term compound or grammatical equivalents, as used herein, means a molecule, either natural or synthetic, such as a protein; antibody, oligopeptide (eg, about 5 to about 25 amino acids long, eg, about 10 to 20 or 12 to 18 amino acids long, eg, 12, 15 or 18 amino acids long); nucleotides (eg, inhibitory RNA) that inhibit the expression of GPR174, a small molecule chemical compound, eg, a small organic, organometallic, or inorganic molecule; polysaccharide; oligonucleotides; lipid; and fatty acid. The compound may be included in a library of compounds such as combinatorial, synthetic, natural, heterocyclic, drug-like

- 21 046439 means, similar to the lead compounds, an organic, inorganic, non-randomized or randomized library that provides a sufficient range of diversity, or it may be a focused or directed collection of the above compounds. The compounds are optionally associated with a fusion partner, for example, targeting compounds, activity-reducing compounds, dimerizing compounds, stabilizing compounds, targeting compounds and other functional groups. Conventionally, new chemical compounds with useful properties are obtained by identifying a compound (termed a lead compound) having some desired property or activity, eg, inhibitory activity, producing variants of the lead compound, and evaluating the properties and activities of these variant compounds. Often, high throughput screening (HTS) methods are used for such analysis.

The terms small molecule, small organic molecule, and small inorganic molecule refer to molecules (organic, organometallic, or inorganic), organic molecules, and inorganic molecules, respectively, that are either natural or synthetic, and that have a molecular weight of greater than about 50 Da or less. than approximately 2500 Da. Small organic (for example) molecules may be less than about 2000 Da, between about 100 Da and about 1000 Da, or between about 100 and about 600 Da, or between about 200 and 500 Da.

The term compound previously determined to be functionally interacting with GPR174 is a compound that is determined, prior to contact with a cell expressing GPR174 or administration of the compound to a subject, to modulate the GPR174-mediated signaling pathway by functionally interacting with GPR174. This preliminary determination can be made either by:

(i) directly obtaining information that a compound functionally interacts with GPR174 by physically demonstrating that the compound functionally interacts with GPR174, by performing an assay measuring functional interaction with GPR174, or by performing an assay measuring GPR174-mediated signaling activity, or ( ii) by indirectly obtaining knowledge that a compound functionally interacts with GPR174 by receiving or obtaining such knowledge or information, in relation to the results of other investigators physically demonstrating such interaction using an assay measuring the functional interaction of the compound with GPR174 or measuring GPR174-mediated signaling activity , from another group or source (e.g., knowledge or information, in relation to the results of a study conducted by other researchers physically demonstrating such an interaction using any assay that measures the functional interaction of a compound with GPR174 or measures the activity of GPR174-mediated signaling derived from, e.g. publication in a scientific journal, or in a published patent application, or by reading the instructions for use of the drug, or obtained through private channels (for example, through personal communication), in each case, before contacting a cell expressing GPR174 with the compound or administering the compound subject. In one embodiment, the compound is preliminarily determined to operably interact with human GPR174 by directly or indirectly obtaining information that the compound operably interacts with human GPR174 shown as SEQ ID NO: 1, or a naturally occurring variant of GPR174 having at least 95% identity with SEQ ID NO: 1.

By a therapeutically effective amount is meant an amount that produces the desired effect for which it is administered, such as improving or delaying at least one symptom associated with the disease or condition being treated. The exact dosage may depend on the purpose of treatment and can be determined by one skilled in the art using known methods (see, for example, Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); and Pickar, Dosage Calculations (1999)).

By substantially pure or isolated is meant a compound (eg, a polypeptide or conjugate) separated from other chemical components. Typically, a compound is substantially pure when it is at least 30% by weight free from other components. In specific embodiments, the formulation is at least 50, 60, 75, 85, 90, 95, 96, 97, 98, or 99% by weight free from other components. A purified polypeptide can be obtained, for example, by expression of a recombinant polynucleotide encoding such a polypeptide, or by chemical synthesis of the polypeptide. Purity can be measured by any suitable method, for example, column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

In the context of a natural compound, the term isolated is a compound that has been altered or removed from its natural state (eg, through human intervention).

The term mammal includes all mammals, including, without limitation, humans, non-human primates, dogs, cats, horses, sheep, goats, cows, rabbits, pigs and rodents.

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It is within the scope of the invention, and as is well known in the art, to treat a disease, disorder or condition, treatment of a disease, disorder or condition (for example, the conditions described herein, such as inflammatory conditions), or therapy is a method of obtaining beneficial or desired outcomes, such as clinical outcomes, and can be carried out either prophylactically or during the course of clinical pathology. Providing benefit or desired results may include, but is not limited to, mitigation or alleviation of one or more symptoms or conditions; reduction in the severity of a disease, disorder or condition; reducing the likelihood of developing a disease, disorder or condition; stabilization (i.e., no worsening) of the disease, disorder, or condition; preventing the development or spread of a disease, disorder or condition; delaying or slowing the progression of a disease, disorder or condition; alleviation or mitigation of a disease, disorder or condition; and remission (either partial or complete), either detectable or undetectable. Alleviation of a disease, disorder or condition means that the severity and/or undesirable clinical manifestations of the disease, disorder or condition are reduced and/or its progression is slowed or prolonged, compared to the extent or progression without treatment.

As used herein, the terms subject or patient refer to any organism to which a compound or composition of the present invention can be administered, for example, for experimental, diagnostic, prophylactic and/or therapeutic purposes. A subject to be treated with a compound or composition described herein may be a subject diagnosed by a medical practitioner as having a disease, disorder, or condition described herein, or at risk of developing a disease, disorder, or condition described herein. in the present description. Diagnostics can be performed by any method or technique known in the art. One of ordinary skill in the art will appreciate that a subject may be diagnosed as having a disease, disorder or condition, using a standard test or examination, or may be identified, without examination, as being at high risk due to the presence of one or more risk factors. Typical subjects include animals (eg, mammals such as mice, rats, rabbits, non-human primates and humans).

As used herein, the term neoplasia refers to any new and abnormal cell growth, especially growth in which cell proliferation is uncontrolled and progressive. Neoplasias can be noncancerous (ie, benign) or malignant.

As used herein, the term tumors means neoplasia, including solid and liquid (ie blood) neoplasia, and benign and malignant neoplasia, including primary and/or metastatic neoplasia.

The term alkanoyl, as used herein, refers to a group having the structure -C(O)-R, in which R represents alkyl. Alkanoyl may be unsubstituted or substituted (eg, optionally substituted alkanoyl) as described for alkyl. The suffix oil can be used to identify other groups having the structure -C(O)-R. For example, in an alkenyl group, R represents alkenyl; in the alkynyl group, R represents alkynyl; in the cycloalkanoyl group, R represents cycloalkyl; in the cycloalkenoyl group, R represents cycloalkenyl; and in the cycloalkynoyl group, R represents cycloalkynyl (all groups are as defined herein). In addition, groups defined using the suffix oil can further be used to define groups having the structure -O-C(O)R' by adding the suffix oxy, for example, when R' is alkyl, the group is alkanoyloxy. For example, in an alkenoyloxy group, R' represents alkenyl; in the alkinoyloxy group, R' represents alkynyl; in the cycloalkanoyloxy group, R' represents cycloalkanyl; in the cycloalkenoyloxy group, R' represents cycloalkenyl; and in a cycloalkynoyloxy group, R' represents cycloalkynyl (all groups are as defined herein). Each of these groups may be unsubstituted or substituted (eg, optionally substituted), as described for each respective group.

The term alkenyl, as used herein, refers to a monovalent straight chain or branched chain substituent containing one or two carbon-carbon double bonds and containing only C and H in the absence of substitution. An alkenyl group may contain, unless otherwise indicated, 2, 3, 4, 5 or 6 carbon atoms, excluding carbon atoms from any substituents if present. Non-limiting examples of alkenyl groups include ethenyl, prop-1-enyl, prop-2enyl, 1-methylethenyl, but-1-enyl, but-2-enyl, but-3-enyl, 1-methylprop-1-enyl, 2-methylprop -1-enyl and 1methylprop-2-enyl. Alkenyl may be unsubstituted or substituted (eg, optionally substituted alkenyl) as described for alkyl.

The term alkenylene, as used herein, refers to a divalent straight chain or branched chain substituent containing one or two carbon-carbon double bonds and containing only C and H in the absence of substitution. An alkenylene group may contain, if not indicated

- 23 046439 otherwise specified, 2, 3, 4, 5 or 6 carbon atoms, excluding carbon atoms from any substituents, if present. Non-limiting examples of alkenylene groups include ethene-1,1-diyl; ethene-1,2diyl; prop-1-ene-1,1-diyl, prop-2-ene-1,1-diyl; prop-1-ene-1,2-diyl, prop-1-ene-1,3-diyl; prop-2-ene-1,1diyl; prop-2-ene-1,2-diyl; but-1-ene-1,1-diyl; but-1-ene-1,2-diyl; but-1-ene-1,3-diyl; but-1-ene-1,4-diyl; but-2-ene-1,1-diyl; but-2-ene-1,2-diyl; but-2-ene-1,3-diyl; but-2-ene-1,4-diyl; but-2-ene-2,3-diyl; but-3-ene1,1-diyl; but-3-ene-1,2-diyl; but-3-ene-1,3-diyl; but-3-ene-2,3-diyl; buta-1,2-diene-1,1-diyl; buta-1,2-diene1,3-diyl; buta-1,2-diene-1,4-diyl; buta-1,3-diene-1,1-diyl; buta-1,3-diene-1,2-diyl; buta-1,3-diene-1,3-diyl; buta-1,3-diene-1,4-diyl; buta-1,3-diene-2,3-diyl; buta-2,3-diene-1,1-diyl; and buta-2,3-diene-1,2-diyl. Alkenylene may be unsubstituted or substituted (eg, optionally substituted alkenylene) as described for alkylene.

The term alkoxy represents a chemical substituent of the formula -OR, where R represents an optionally substituted alkyl group (eg, an optionally substituted C ₁ -C ₆ alkyl group). A substituted alkoxy group may have 1, 2, 3, 4, 5 or 6 substituent groups as defined herein. Likewise, the term arylalkoxy represents a chemical substituent of the formula -OR, where R represents an optionally substituted arylalkyl group. The term cycloalkoxy represents a substituent of the formula -OR', where R' represents an optionally substituted cycloalkyl group, as described herein. Likewise, the term alkenoxy represents a chemical substituent of the formula -OR, where R represents an optionally substituted alkenyl group, as described herein.

The term alkyl, as used herein, refers to a saturated monovalent straight chain or branched chain substituent containing only C and H in the absence of substitution. An alkyl group may contain, unless otherwise indicated, 1, 2, 3, 4, 5 or 6 carbon atoms, excluding carbon atoms from any substituents if present. Non-limiting examples of the alkyl group include methyl, ethyl, isobutyl, t-butyl and the like. The alkyl group may be unsubstituted or substituted (eg, optionally substituted alkyl) with 1,2, 3, 4, 5 or 6 substituents independently selected from the group consisting of: halo (eg, F, Cl, Br or I), CN, NO2, CF ₃ , OCF ₃ , COOR', CONR'2, OR', SR', SOR, SO2R', NR' ₂ , NR'(CO)R', NR'C(O)OR', NR'C (O)NR' ₂ , NR'SO ₂ NR' ₂ , NR'SO ₂ R', oxo (=O) or oxymido (=NOR), wherein each R' independently represents H or an optionally substituted group selected from alkyl , alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl and aryl (all as defined herein); and R represents H or an optionally substituted group selected from alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl and aryl (all as defined herein). Alternatively, the substituted alkyl group may be a perfluoroalkyl group. In particular embodiments, when at least one of the substituents on the alkyl group is oxo, the oxo group is not bonded to the carbon atom bonded to the parent molecular group.

The term alkylene, as used herein, refers to a saturated divalent straight chain or branched chain substituent containing only C and H in the absence of substitution. An alkylene group may contain, unless otherwise indicated, 1, 2, 3, 4, 5 or 6 carbon atoms, excluding carbon atoms from any substituents if present. Non-limiting examples of the alkylene group include methylene, ethane-1,2-diyl, ethane-1,1-diyl, propan-1,3-diyl, propan-1,2-diyl, propan1,1-diyl, propan-2,2 -diyl, butane-1,4-diyl, butane-1,3-diyl, butane-1,2-diyl, butane-1,1-diyl and butane-2,2diyl, butane-2,3-diyl. The alkylene group may be unsubstituted or substituted (eg, optionally substituted alkylene) with 1, 2, 3, 4, 5, or 6 substituents independently selected from the group consisting of: halo (eg, F, Cl, Br, or I), CN , NO2, CF ₃ , OCF ₃ , COOR', CONR' ₂ , OR', SR', SOR', SO2R', NR'2, NR'(CO)R', NR'C(O)OR', NR 'C(O)NR'2, NR'SO2NR'2, NR'SO2R', oxo (=O) or oxymido (=NOR), where each R' independently represents H or an optionally substituted group selected from alkyl, alkenyl , alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl and aryl (all as defined herein); and R represents H or an optionally substituted group selected from alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl and aryl (all as defined herein). Alternatively, the substituted alkylene group may be a perfluoroalkylene group.

The term alkylsulfinyl refers to a group having the structure alkyl-SiO)-. wherein alkyl is as described herein. Alkylsulfinyl may be unsubstituted or substituted (eg, optionally substituted alkylsulfinyl) as described for alkyl.

The term alkylsulfonyl refers to a group having the structure alkyl^(O) ₂ -, in which alkyl is as described herein. Alkylsulfonyl may be unsubstituted or substituted (eg, optionally substituted alkylsulfonyl) as described for alkyl.

The term alkynyl, as used herein, refers to a monovalent straight chain or branched chain substituent comprising one or two carbon-carbon triple bonds and containing only C and H in the absence of substitution. An alkynyl group may, unless otherwise specified, contain

- 24 046439 other, 2, 3, 4, 5 or 6 carbon atoms, excluding carbon atoms from any substituents, if present. Non-limiting examples of alkynyl groups include ethynyl, prop-1-ynyl, prop-2-ynyl, butyn-1-nyl, but-2-ynyl, but-3-ynyl, 1-methylprop-2-ynyl and the like. Alkynyl may be unsubstituted or substituted (eg, optionally substituted alkynyl) as described for alkyl.

The term alkynylene, as used herein, refers to a divalent straight chain or branched chain substituent containing one or two carbon-carbon triple bonds and containing only C and H in the absence of substitution. An alkynylene group may contain, unless otherwise indicated, 2, 3, 4, 5 or 6 carbon atoms, excluding carbon atoms from any substituents if present. Non-limiting examples of alkenylene groups include ethyn-1,2-diyl; prop-1-yne1,3-diyl; prop-2-yn-1,1-diyl; but-1-yne-1,3-diyl; but-1-yne-1,4-diyl; but-2-yn-1,1-diyl; but-2-yn-1,4diyl; but-3-yn-1,1-diyl; but-3-yn-1,2-diyl; but-3-yn-2,2-diyl; and buta-1,3-diyne-1,4-diyl. Alkenylene may be unsubstituted or substituted (eg, optionally substituted alkenylene) as described for alkylene.

The term amido, for the purposes of the invention, refers to a group having the structure -N(R ^N1 )RN ² , in which RN ¹ represents -H, OH, -N(R ^N3 ) ₂ , -C(O)RN ⁴ , - SO2ORN ⁴ , -SO2RN ⁴ , -SoR ^N4 , alkyl, alkenyl, alkynyl, alkoxy, aryl, arylalkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl (eg heteroaryl) or heterocyclylalkyl (eg heteroarylalkyl); RN ² is C(O)RN ⁵ , SO2ORN ⁵ , SO2RN ⁵ or SORN ⁵ ; or RN ¹ and RN ⁵ combine to form a 5-, 6-, 7-, or 8-membered ring. RN ³ represents H, alkyl, aryl, arylalkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl (eg heteroaryl), or heterocyclylalkyl (eg heteroarylalkyl); each of RN ⁴ and RN ⁵ independently represents alkyl, alkenyl, alkynyl, alkoxy, aryl, arylalkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl (eg, heteroaryl) or heterocyclylalkyl (eg, heteroarylalkyl). In a preferred embodiment, RN ¹ is H. Amido may be unsubstituted when RN ¹ is H and the group on RN ² is unsubstituted (e.g., RN ³ is H, unsubstituted alkyl, unsubstituted aryl, unsubstituted arylalkyl, unsubstituted cycloalkyl , unsubstituted cycloalkylalkyl, unsubstituted heterocyclyl (eg, unsubstituted heteroaryl) or unsubstituted heterocyclylalkyl (eg, unsubstituted heteroarylalkyl); or each of RN ⁴ and RN ⁵ is unsubstituted alkyl, unsubstituted alkenyl, unsubstituted alkynyl, unsubstituted alkoxy, unsubstituted aryl, unsubstituted arylalkyl, unsubstituted cycloalkyl, unsubstituted cycloalkylalkyl, unsubstituted heterocyclyl (eg, unsubstituted heteroaryl), or unsubstituted heterocyclylalkyl (eg, unsubstituted heteroarylalkyl)). Alternatively, amido may be substituted when at least one of the groups listed under RN ³ , RN ⁴ or RN ⁵ is substituted, and/or when RN ¹ is not H.

The term amino, for the purposes of the invention, represents -N(R ^N1 )2, where each R ^N1 independently represents H, OH, NO2, N(RN ² )2, N-protecting group, alkyl, alkenyl, alkynyl, alkoxy, aryl, arylalkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl (eg, heteroaryl), heterocyclylalkyl (eg, heteroarylalkyl), or two R ^N1 are combined to form a heterocyclyl or N protecting group, and wherein each R ^N2 is independently H, alkyl, or aryl. An amino may be unsubstituted when each R ^N1 is H, or substituted when at least one R ^N1 is not H (eg, optionally substituted amino). In a preferred embodiment, amino is -NH2 or -NHRN ¹ where RN ¹ is independently OH, NO2, NH2, NR ^N2 2, SO ₂ OR ^N2 , SO ₂ ^RN2 , SOR ^N2 , alkyl or aryl, and each R ^N2 may be H, alkyl or aryl.

The terms aromatic group and aryl, as used herein, refer to a carbocyclic monovalent group (monocyclic or fused ring bicyclic) in which the carbocycle satisfies Hückel's rule (4n+2 electrons in one π system) and has aromatic stabilization characteristics compared to the hypothetical a molecule that is not aromatically stabilized (eg benzene versus cyclohexatriene). Aryl may contain 6-10 carbon atoms, excluding carbon atoms from any substituents if present. Non-limiting examples of monocyclic and fused bicyclic aromatic groups include phenyl and naphthyl, respectively. Aryl may be unsubstituted or substituted, as defined herein. The term arylene refers to an aryl group as described herein, except that arylene is a divalent substituent. Arylene may be unsubstituted or substituted, as defined herein.

The term arylalkyl, as used herein, is a chemical substituent (aryl)(alkylene)-, in which each of the aryl and alkylene groups is as described herein. The arylalkyl group may be unsubstituted or substituted (eg, optionally substituted C ₆ -C ₁₀ -aryl C 1 -C ₆ -alkyl). A non-limiting example of arylalkyl is phenylmethyl, commonly referred to as benzyl. Arylalkenyl (eg, C ₆ -C ₁₀ -aryl C ₂ -C ₆ alkenyl) and arylalkynyl (eg, C ₆ -C ₁₀ -aryl C ₂ -C ₆ -kynyl) are similarly defined as having the general structure (aryl)-( alkenylene)- and (aryl)(alkynylene)-, respectively. Arylheteroalkyl, arylheteroalkenyl, and arylheteroalkynyl are similarly defined as having the structure (aryl)

- 25 046439 (heteroalkylene)-, (aryl)-(heteroalkenylene)- and (aryl)-(heteroalkynylene)-, respectively. Likewise, other groups can be defined by combining the group defining term with alkyl. For example, heteroarylalkyl is a chemical substituent having the general structure (heteroaryl)-(alkylene)-, which may be unsubstituted or substituted (e.g., optionally substituted C.'|-C _y -heteroaryl C.'|-C' ₆ -alkyl ) according to the respective definitions of each part of the heteroarylalkyl group. Each of the groups may be unsubstituted or substituted (eg, optionally substituted). Substituents for the aryl or heteroaryl moiety are those described for aromatic groups. Substituents for the alkyl, heteroalkyl, alkenyl, heteroalkenyl, alkynyl or heteroalkynyl moieties are those described in the respective definitions of these groups.

The term aryloyl, as used herein, refers to a group having the structure (C ₆ -C ₁₀ -aryl)C(O)-. The aryloyl may be unsubstituted or substituted according to the definition of an aryl group (eg, optionally substituted aryloyl). A typical example of an aryloyl group is a benzoyl group. Likewise, the term heteroaryloyl, as used herein, refers to a group having the structure (C ₁ -C ₉ -heteroaryl)-C(O)-. Heteroaryloyl may be unsubstituted or substituted (eg, optionally substituted heteroaryloyl) as described for heteroaryl.

The term aryloxy, as used herein, refers to a carbocyclic aromatic system linked to another moiety via an oxygen atom, for example, (C ₆ -C ₁₀ -aryl)-O-. The aryloxy group may be unsubstituted or substituted (eg, optionally substituted aryl), as described for aromatic groups. A typical example of aryloxy is phenoxy (eg, optionally substituted phenoxy).

The term aryloyloxy, as used herein, refers to a group having the structure (C ₆ -C ₁₀ aryl)-C(O)-O-. Aryloxy may be unsubstituted or substituted according to the definition of aryl group (eg, optionally substituted aryloyloxy). A typical example of an aryloyloxy group is benzoate. Likewise, the term heteroaryloyloxy, as used herein, refers to a group having the structure (C ₁ -C ₉ -heteroaryl)-C(O)-O-. Heteroaryloyloxy may be unsubstituted or substituted (eg, optionally substituted heteroaryloyloxy) as described for heteroaryl.

The term arylsulfinyl refers to a group having the structure (C^C^-aryl)^^)-. The arylsulfinyl group may be unsubstituted or substituted, as described herein (eg, optionally substituted arylsulfinyl).

A non-limiting example of arylsulfinyl is phenylsulfinyl.

The term arylsulfonyl refers to a group having the structure (C^C^-aryl)^^^-. The arylsulfonyl group may be unsubstituted or substituted, as described herein (eg, optionally substituted arylsulfonyl). A non-limiting example of arylsulfonyl is phenylsulfonyl.

The term arylthio refers to a group having the structure (C ₆ -C ₁₀ -aryl)^-. The arylthio group may be unsubstituted or substituted, as described herein (eg, optionally substituted arylthio). A non-limiting example of an arylthio is phenylthio.

The term carbocyclic, as used herein, represents an optionally substituted C _3-12 monocyclic, bicyclic or tricyclic structure in which rings, which may be aromatic or non-aromatic, are formed by carbon atoms. Carbocyclic structures include cycloalkyl, cycloalkenyl, cycloalkynyl and aryl groups.

The term carbonyl, as used herein, refers to a divalent group consisting of C=O in which two valencies are present on the carbon atom. This term can be used to define other groups that have the general structure R-C(O)-. Thus, in an alkoxycarbonyl group, R represents alkoxy; in an aryloxycarbonyl group, R is aryloxy; in an aminocarbonyl group, R is amino; in the heteroaryloxycarbonyl group, R represents heteroaryloxy; in the heterocyclyloxycarbonyl group, R represents heterocyclyloxy; or in a hydroxycarbonyl group, R represents hydroxy. Each of the groups may be unsubstituted or substituted as defined herein. For example, an alkoxycarbonyl group may be unsubstituted or substituted, as defined for an alkoxy group.

The terms carboxamide and carboxylic acid amide, as used herein, refer to the group having the structure CONR'R, wherein each R and R are independently selected from H, optionally substituted C _1-6 alkyl, optionally substituted C ₃ -C ₁₀ -cycloalkyl , optionally substituted C ₁ -C ₉ - heterocyclyl, optionally substituted C ₆ -C ₁₀ -aryl, optionally substituted C ₁ -C ₉ - heteroaryl, or R' and R combine to form an optionally substituted heterocyclyl. The carboxamide may be unsubstituted when the R' group and the R group are unsubstituted or substituted when at least one of R' and R is a substituted group as defined herein

- 26 046439 Saniy. Accordingly, an optionally substituted carboxamide is a carboxamide that may be unsubstituted or substituted.

The terms carboxylic acid ester and ester, as used herein, refer to a group having the structure -CO2R', where R' is selected from optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl. An ester may be unsubstituted when the R' group is an unsubstituted group, or substituted when the R' group is a substituted group, as defined herein. Accordingly, an optionally substituted ester is an ester that may be unsubstituted or substituted.

By cyano we mean a group having the structure -CN.

The term cycloalkenyl, as used herein, refers to a non-aromatic carbocyclic group having one, two or three carbon-carbon double bonds and having from three to ten carbon atoms (eg, C ₃ -C ₁₀ -cycloalkylene), unless otherwise specified. Non-limiting examples of cycloalkenyl include cycloprop-1-enyl, cycloprop-2-enyl, cyclobut-1-enyl, cyclobut-1-enyl, cyclobut-2enyl, cyclopent-1-enyl, cyclopent-2-enyl, cyclopent-3-enyl, norbornen-1-yl, norbornen-2-yl, norbornen-5-yl and norbornen-7-yl. The cycloalkenyl group may be unsubstituted or substituted (eg, optionally substituted cycloalkenyl) as described for cycloalkyl.

The term cycloalkenylene, as used herein, refers to a divalent non-aromatic carbocyclic group having one, two, or three carbon-carbon double bonds and having from three to ten carbon atoms (for example, C ₃ -C1 ₀ -cycloalkenylene), unless otherwise noted . Non-limiting examples of cycloalkenylene include cycloprop-1-ene-1,2-diyl; cycloprop-2-ene-1,1-diyl; cycloprop-2-ene-1,2-diyl; cyclobut-1-ene-1,2-diyl; cyclobut-1-ene-1,3-diyl; cyclobut-1-ene-1,4-diyl; cyclobut2-ene-1,1-diyl; cyclobut-2-ene-1,4-diyl; cyclopent-1-ene-1,2-diyl; cyclopent-1-ene-1,3-diyl; cyclopent-1ene-1,4-diyl; cyclopent-1-ene-1,5-diyl; cyclopent-2-ene-1,1-diyl; cyclopent-2-ene-1,4-diyl; cyclopent-2ene-1,5-diyl; cyclopent-3-ene-1,1-diylcyclopent-1,3-diene-1,2-diyl; cyclopent-1,3-diene-1,3-diyl; cyclopent-1,3-diene-1,4-diyl; cyclopent-1,3-diene-1,5-diyl; cyclopent-1,3-diene-5,5-diyl; norbornadiene-1,2diyl; norbornadiene-1,3-diyl; norbornadiene-1,4-diyl; norbornadiene-1,7-diyl; norbornadiene-2,3-diyl; norbornadiene-2,5-diyl; norbornadiene-2,6-diyl; norbornadiene-2,7-diyl; and norbornadiene-7,7-diyl. Cycloalkenylene may be unsubstituted or substituted (eg, optionally substituted cycloalkenylene) as described for cycloalkyl.

The term cycloalkyl, as used herein, refers to a monovalent carbocyclic group having from three to ten carbon atoms (eg, C ₃ -C1 ₀ -cycloalkyl), unless otherwise specified. Cycloalkyl groups may be monocyclic or bicyclic. Bicyclic cycloalkyl groups may be of the bicyclo[rd.0]alkyl type, in which p and q are each independently 1, 2, 3, 4, 5, 6 or 7, provided that the sum of p and q is 2. 3, 4, 5, 6, 7 or 8. Alternatively, bicyclic cycloalkyl groups may include bridged cycloalkyl structures, for example bicyclo[rdt]alkyl, in which r is 1, 2, or 3, each of p and q is independently 1, 2, 3, 4, 5 or 6, provided that the sum of p, q and t is 3, 4, 5, 6, 7 or 8. The cycloalkyl group may be a spirocyclic group, for example spiro |р.с.|]alkyl. wherein p and q are each independently 2, 3, 4, 5, 6, or 7, provided that the sum of p and q is 4, 5, 6, 7, 8, or 9. Non-limiting examples of cycloalkyl include cyclopropyl, cyclobutyl , cyclopentyl, cyclohexyl, cycloheptyl, 1-bicyclo[2,2,1]heptyl, 2bicyclo|2,2,1]heptyl, 5-bicyclo|2,2,1]heptyl, 7-bicyclo|2,2,1 ]heptyl and decalinyl. The cycloalkyl group may be unsubstituted or substituted (e.g., optionally substituted cycloalkyl) with 1, 2, 3, 4, 5, or 6 substituents independently selected from the group consisting of: alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, aryl, arylalkyl, heteroaryl, halo (for example, F, Cl, W or I), CN, NO2, CF ₃ , OCF ₃ , COOR', CONR'2, OR', SR', SOR, SO2R, NR' ₂ , NR'(CO)R',NR'C(O)OR,NR'C(O)NR' ₂ , NR'SO ₂ NR' ₂ , NR'SO ₂ R, oxo (=O) or oxymido (=NOR), wherein each R independently represents H or an optionally substituted group selected from alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl and aryl (all as defined herein); and R represents H or an optionally substituted group selected from alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl and aryl (all as defined herein). Alternatively, the substituted cycloalkyl group may be a perfluorocycloalkyl group.

The term cycloalkylene, as used herein, refers to a divalent carbocyclic group having from three to ten carbon atoms (eg, C ₃ -C1 ₀ -cycloalkyl), unless otherwise specified. Non-limiting examples of cycloalkylene include cyclopropane-1,1-diyl; cyclopropane-1,2diyl; cyclobutane-1,1-diyl; cyclobutane-1,2-diyl; cyclobutane-1,3-diyl; bicyclo[2,2,1]hepta-1,2-diyl; bicyclo[2,2,1]hepta-1,3-diyl; bicyclo[2,2,1]hepta-1,4-diyl; bicyclo[2,2,1]hepta-1,7-diyl; bicyclo|2,2,1]hepta-2,2-diyl; bicyclo|2,2,1]hepta-2,3-diyl; bicyclo|2,2,1]hepta-2,7-diyl; decalin-1,2-diyl; decalin-1,3-diyl; decalin-1,4-diyl; decalin-1,5-diyl; decalin-1,6-diyl; decalin-2,2-diyl; decalin-2,3

- 27 046439 diyl; decalin-2,4-diyl; and decalin-2,5-diyl. The cycloalkylene group may be unsubstituted or substituted (eg, optionally substituted cycloalkylene) as described for cycloalkyl.

The term cycloalkynyl, as used herein, refers to a monovalent carbocyclic group having one or two non-contiguous carbon-carbon triple bonds and having from eight to ten carbon atoms (for example, ^-C^-cycloalkyl), unless otherwise specified. Non-limiting examples of cycloalkynyl include cyclooctynyl, cyclononinyl, cyclodecynyl and cyclodecadinyl. The cycloalkynyl group may be unsubstituted or substituted (eg, optionally substituted cycloalkynyl) as described for cycloalkyl.

The halo can be any halogen atom, especially F, Cl, Br or I, and more specifically it is fluorine or chlorine.

The term haloalkyl, as used herein, is an alkyl group, as defined herein, substituted with a halogen group (ie F, Cl, Br or I). A haloalkyl may be substituted with one, two, three, or, in the case of alkyl groups of two or more carbon atoms, four halogens. Haloalkyl groups include perfluoroalkyl groups. In some embodiments, a haloalkyl group may be further substituted with 1, 2, 3, or 4 substituent groups as described herein for alkyl groups.

The term heteroalkenyl, as used herein, refers to an alkenyl group in which the alkenyl chain is interrupted once by one, two or three heteroatoms; twice, each time, independently, with one, two or three heteroatoms; three times, each time, independently, with one, two or three heteroatoms; or four times, each time, independently, with one, two or three heteroatoms. Each heteroatom is independently O, N, or S. No heteroalkenyl group contains more than two contiguous oxygen atoms. The heteroalkenyl group may be unsubstituted or substituted (eg, optionally substituted heteroalkenyl). When the heteroalkenyl group is substituted and the substituent is bonded to the heteroatom, the substituent is selected accordingly. The substituent bonded to the heteroatom, as valence allows, is selected from the group consisting of: alkyl, alkanoyl, alkenyl, alkenoyl, alkynyl, alkinoyl, cycloalkyl, cycloalkanoyl, cycloalkenyl, cycloalkenoyl, cycloalkynyl, cycloalkynoyl, aryl, aryloyl, heteroaryl, heteroaryloyl, heterocyclyl , heterocycloyl, amino, aminocarbonyl, alkoxycarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl and heterocyclyloxycarbonyl. When the heteroalkenyl group is substituted and the substituent is bonded to carbon, the substituent is selected from those described for alkyl, provided that the substituent on the carbon atom bonded to the heteroatom is not a halo. In some embodiments, the heteroalkenyl group has a C at the end attached to other groups. In some embodiments, the heteroatom is O or N.

The term heteroalkenylene, as used herein, refers to an alkenylene group in which the alkenylene chain is interrupted once by one, two or three heteroatoms; twice, each time, independently, with one, two or three heteroatoms; three times, each time, independently, with one, two or three heteroatoms; or four times, each time, independently, with one, two or three heteroatoms. Each heteroatom is independently O, N or S. No heteroalkenylene group contains more than two contiguous oxygen atoms. The heteroalkenylene group may be unsubstituted or substituted (eg, optionally substituted heteroalkenylene). When the heteroalkenylene group is substituted and the substituent is bonded to the heteroatom, the substituent is selected accordingly. The substituent bonded to the heteroatom, as valence allows, is selected from the group consisting of: alkyl, alkanoyl, alkenyl, alkenoyl, alkynyl, alkinoyl, cycloalkyl, cycloalkanoyl, cycloalkenyl, cycloalkenoyl, cycloalkynyl, cycloalkynoyl, aryl, aryloyl, heteroaryl, heteroaryloyl, heterocyclyl , heterocycloyl, amino, aminocarbonyl, alkoxycarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl and heterocyclyloxycarbonyl. When the heteroalkenylene group is substituted and the substituent is bonded to carbon, the substituent is selected from those described for alkyl, provided that the substituent on the carbon atom bonded to the heteroatom is not a halo. In some embodiments, the heteroalkenylene group has a C at each end attached to other groups. In some embodiments, the heteroatom is O or N.

The term heteroalkyl, as used herein, refers to an alkyl group in which the alkyl chain is interrupted once by one, two or three heteroatoms; twice, each time, independently, with one, two or three heteroatoms; three times, each time, independently, with one, two or three heteroatoms; or four times, each time, independently, with one, two or three heteroatoms. Each heteroatom is independently O, N, or S. No heteroalkyl group contains more than two adjacent oxygen atoms. A heteroalkyl group may be unsubstituted or substituted (eg, optionally substituted heteroalkyl). When the heteroalkyl group is substituted and the substituent is bonded to the heteroatom, the substituent is selected accordingly. The substituent bonded to the heteroatom, as valence allows, is selected from the group consisting of: alkyl, alkanoyl, alkenyl, alkenoyl, alkynyl, alkinoyl, cycloalkyl, cycloalkanoyl, cycloalkenyl, cycloalkenoyl, cycloalkynyl, cycloalkynoyl, aryl, aryloyl, heteroaryl, heteroaryloyl, heterocyclyl ,

- 28 046439 heterocycloyl, amino, aminocarbonyl, alkoxycarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl and heterocyclyloxycarbonyl. When the heteroalkyl group is substituted and the substituent is bonded to carbon, the substituent is selected from those described for alkyl, provided that the substituent on the carbon atom bonded to the heteroatom is not a halo. In some embodiments, the heteroalkyl group has a C at the end attached to another group. In some embodiments, the heteroatom is O or N.

The term heteroalkylene, as used herein, refers to an alkylene group in which the alkylene chain is interrupted once by one, two or three heteroatoms; twice, each time, independently, with one, two or three heteroatoms; three times, each time, independently, with one, two or three heteroatoms; or four times, each time, independently, with one, two or three heteroatoms. Each heteroatom is independently O, N, or S. No heteroalkylene group contains more than two contiguous oxygen atoms. The heteroalkylene group may be unsubstituted or substituted (eg, optionally substituted heteroalkylene). When the heteroalkylene group is substituted and the substituent is bonded to the heteroatom, the substituent is selected accordingly. The substituent associated with the heteroatom, as valence allows, is selected from the group consisting of: alkyl, alkanoyl, alkenyl, alkenoyl, alkynyl, alkinoyl, cycloalkyl, cycloalkanoyl, cycloalkenyl, cycloalkenoyl, cycloalkynyl, cycloalkynoyl, aryl, aryloyl, heteroaryl, heteroaryloyl, heterocyclyl, heterocycloyl, amino, aminocarbonyl, alkoxycarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl and heterocyclyloxycarbonyl. When the heteroalkylene group is substituted and the substituent is bonded to carbon, the substituent is selected from those described for alkylene, provided that the substituent on the carbon atom bonded to the heteroatom is not a halo. In some embodiments, the heteroalkylene group has a C at each end attached to other groups. In some embodiments, the heteroatom is O or N.

The term heteroalkynyl, as used herein, refers to an alkynyl group in which the alkynyl chain is interrupted once by one, two or three heteroatoms; twice, each time, independently, with one, two or three heteroatoms; three times, each time, independently, with one, two or three heteroatoms; or four times, each time, independently, with one, two or three heteroatoms. Each heteroatom is independently O, N, or S. No heteroalkynyl group contains more than two contiguous oxygen atoms. The heteroalkynyl group may be unsubstituted or substituted (eg, optionally substituted heteroalkynyl) as described for heteroalkenyl.

The term heteroalkynylene, within the scope of the invention, refers to an alkynylene group in which the alkynylene chain is interrupted once by one, two or three heteroatoms; twice, each time, independently, with one, two or three heteroatoms; three times, each time, independently, with one, two or three heteroatoms; or four times, each time, independently, with one, two or three heteroatoms. Each heteroatom is independently O, N, or S. No heteroalkynylene group contains more than two contiguous oxygen atoms. The heteroalkynylene group may be unsubstituted or substituted (eg, optionally substituted heteroalkynylene). The heteroalkynylene group may be unsubstituted or substituted (eg, optionally substituted heteroalkynylene) as described for heteroalkenylene.

The terms heteroaromatic group and heteroaryl, as used herein, refer to a heterocyclic structure (monocyclic or fused bicyclic) satisfying Hückel's rule (4n+2 electrons in one r system) and thus having aromatic stabilization characteristics. Excluding heteroatoms from any substituents, if present, a heteroaryl group contains one, two, three, or four heteroatoms selected from the group consisting of O, S, and N. A heteroaryl group contains 1, 2, 3, 4, 5, 6, 7, 8, or 9 carbon atoms, excluding carbon atoms from any substituents if present. The inclusion of a heteroatom allows the inclusion of 5-membered rings, considered aromatic, as well as 6-membered rings. Thus, non-limiting examples of heteroaromatic groups include pyridyl, pyrimidyl, indolyl, benzimidazolyl, benzotriazolyl, isoquinolyl, quinolyl, benzothiazolyl, benzofuranyl, thienyl, furyl, pyrrolyl, thiazolyl, oxazolyl, isoxazolyl, benzoxazolyl, benzoisoxazolyl and imidazolyl. Since tautomers are theoretically possible, phthalimido is also considered aromatic. As a rule, heteroaryl ring systems contain 5-12 ring member atoms. For example, the heteroaryl group may be a 5-12 membered ring system. In some embodiments, the heteroaromatic group is a 6-membered aromatic ring system containing 1-2 nitrogen atoms. In some embodiments, the heteroaryl group is an optionally substituted pyridyl, indolyl, pyrimidyl, pyridazinyl, benzothiazolyl, benzimidazolyl, pyrazolyl, imidazolyl, isoxazolyl, thiazolyl, benzothiazolyl, or indolyl. In specific embodiments, the heteroaromatic group is pyridyl or pyrimidyl. The term heteroarylene refers to a heteroaryl group as described herein, except that the heteroarylene is a divalent substituent.

The term heteroarylalkylthio, within the scope of the invention, represents a chemical substituent

- 29 046439 formula -SR, where R represents a heteroarylalkyl group. In some embodiments, the heteroarylalkyl group may be further substituted with 1, 2, 3, or 4 groups of substituents, as described herein.

The term heteroarylsulfinyl refers to a group having the structure heteroaryl^(O)-, in which heteroaryl is as described herein. The heteroarylsulfinyl group may be unsubstituted or substituted, as described herein.

The term heteroarylsulfonyl refers to a group having the structure heteroaryl^(O) ₂ -, in which heteroaryl is as described herein. The heteroarylsulfonyl group may be unsubstituted or substituted, as described herein.

The term heteroarylthio refers to a group having the structure heteroaryl-S-, in which heteroaryl is as described herein. The heteroarylthio group may be unsubstituted or substituted as described herein.

The term heterocyclyl, within the scope of the invention, represents cyclic heteroalkyl or heteroalkenyl, i.e. for example, a 3-, 4-, 5-, 6-, or 7-membered ring, unless otherwise noted. Excluding heteroatoms from any substituents, if present, a heterocyclic group contains one, two, three, or four heteroatoms selected from the group consisting of O, S, and N. A heterocyclic group contains, unless otherwise noted, 1, 2, 3, 4, 5, 6, 7, 8 or 9 carbon atoms (e.g. C1-C9 heterocyclyl), excluding carbon atoms from any substituents if present. Sulfur may be included as divalent sulfur (-S-), tetravalent sulfur (-S(=O)-) or hexavalent sulfur (-S(=O) ₂ -). A 5-membered ring has zero to two double bonds, and 6- and 7-membered rings have zero to three double bonds. The term heterocyclyl also represents a heterocyclic compound having a bridged multicyclic structure in which one or more carbon atoms and/or heteroatoms bridge two non-adjacent monocyclic ring members, such as a quinuclidinyl group. The term heterocyclyl includes bicyclic, tricyclic and tetracyclic groups, in which any of the above heterocyclic rings is fused with one, two or three carbocyclic rings, for example, an aryl ring, a cyclohexane ring, a cyclohexene ring, a cyclopentane ring, a cyclopentene ring, or other monocyclic heterocyclic ring, such as indolyl, quinolyl, isoquinolyl, tetrahydroquinolyl, benzofuryl, benzothienyl and the like. Exemplary heterocycles include pyrrolyl, pyrrolinyl, pyrrolidinyl, pyrazolyl, pyrazolinyl, pyrazolidinyl, imidazolyl, imidazolinyl, imidazolidinyl, pyridyl, piperidinyl, homopiperidinyl, pyrazinyl, piperazinyl, pyrimidinyl, pyridazinyl, oxazolyl, oxazolidinyl, isoxazolyl, isoxazolidinyl, morpholinyl, thiomorpholinyl, thiazolyl, thiazolidinyl , isothiazolyl, isothiazolidinyl, indolyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzothiazolyl, benzoxazolyl, furyl, thienyl, thiazolidinyl, isothiazolyl, isoindazoyl, triazolyl, tetrazolyl, oxadiazolyl, purinyl, thiadiazolyl (e.g. 1,3,4-thiadiazole), hydrofuranyl, dihydrofuranyl, tetrahydrothienyl, dihydrothienyl, dihydroindolyl, tetrahydroquinolyl, tetrahydroisoquinolyl, pyranyl, dihydropyranyl, dithiazolyl, benzofuranyl, benzothienyl, etc. Other exemplary heterocyclyls include: 2,3,4,5-tetrahydro-2-oxo-oxazolyl; 2,3-dihydro-2-oxo-1H-imidazolyl; 2,3,4,5-tetrahydro-5-oxo-1H-pyrazolyl (for example, 2,3,4,5-tetrahydro-2-phenyl-5-oxo-1H-pyrazolyl); 2,3,4,5-tetrahydro-2,4-dioxo-1H-imidazolyl (e.g. 2,3,4,5-tetrahydro-2,4-dioxo-5-methyl-5-phenyl-1H-imidazolyl) ; 2,3-dihydro-2-thioxo-1,3,4-oxadiazolyl (for example, 2,3-dihydro-2-thioxo-5phenyl-1,3,4-oxadiazolyl); 4,5-dihydro-5-oxo-III-triazolyl (for example, 4,5-dihydro-3-methyl-4-amino 5oxo-III-triazolyl); 1,2,3,4-tetrahydro-2,4-dioxopyridinyl (for example, 1,2,3,4-tetrahydro-2,4-dioxo3,3-diethylpyridinyl); 2,6-dioxo-piperidinyl (for example, 2,6-dioxo-3-ethyl-3-phenylpiperidinyl); 1,6-dihydro-6-oxopyridiminyl; 1,6-dihydro-4-oxopyrimidinyl (for example, 2-(methylthio)-1,6-dihydro4-oxo-5-methylpyrimidin-1-yl); 1,2,3,4-tetrahydro-2,4-dioxopyrimidinyl (for example, 1,2,3,4tetrahydro-2,4-dioxo-3-ethylpyrimidinyl); 1,6-dihydro-6-oxo-pyridazinyl (for example, 1,6-dihydro-6oxo-3-ethylpyridazinyl); 1,6-dihydro-6-oxo-1,2,4-triazinyl (for example, 1,6-dihydro-5-isopropyl-6oxo-1,2,4-triazinyl); 2,3-dihydro-2-oxo-III-indolyl (for example, 3,3-dimethyl-2,3-dihydro-2-oxo-shindolyl and 2,3-dihydro-2-oxo-3,3'-spiropropane -SH-indol-1-yl); 1,3-dihydro-1-oxo-2H-iso-indolyl; 1,3-dihydro-1,3-dioxo-2H-iso-indolyl; N-benzopyrazolyl (for example, 1-(ethoxycarbonyl)-N-benzopyrazolyl); 2,3-dihydro-2-oxo-N-benzimidazolyl (for example, 3-ethyl-2,3-dihydro-2-oxo-N-benzimidazolyl); 2,3-dihydro-2-oxo-benzoxazolyl (eg 5-chloro-2,3-dihydro-2-oxo-benzoxazolyl); 2,3-dihydro-2-oxo-benzoxazolyl; 2-oxo-2H-benzopyranyl; 1,4-benzodioxanyl; 1,3-benzodioxanyl; 2,3-dihydro-3-oxo,4H-1,3-benzothiazinyl; 3,4-dihydro-4-oxo-3R-quinazolinyl (for example, 2methyl-3,4-dihydro-4-oxo-3H-quinazolinyl); 1,2,3,4-tetrahydro-2,4-dioxo-3H-quinazolyl (for example, 1ethyl-1,2,3,4-tetrahydro-2,4-dioxo-3R-quinazolyl); 1,2,3,6-tetrahydro-2,6-dioxo-7H-purinyl (for example, 1,2,3,6-tetrahydro-1,3-dimethyl-2,6-dioxo-7H-purinyl); 1,2,3,6-tetrahydro-2,6-dioxo-III-purinyl (for example, 1,2,3,6-tetrahydro-3,7-dimethyl-2,6-dioxo-III-purinyl); 2-oxobenz[c, d]indolyl; 1,1-dioxo-2Hnaph[1,8-c,d]isothiazolyl; and 1,8-naphthylenedicarboxamido. The heterocyclic group may be unsubstituted or substituted (eg, optionally substituted heterocyclyl). The term heterocyclylene refers to a heterocyclic group as described herein, except

- 30 046439 that heterocyclylene is a divalent substituent.

The term heterocyclyloxy, within the scope of the invention, refers to a group having the structure (C1-C ₉ heterocyclyl)-O-. Heterocyclyloxy may be unsubstituted or substituted (eg, optionally substituted heterocyclyloxy), according to the definition of heterocyclyl.

The term heterocycloyl, within the scope of the invention, refers to a group having the structure (C1-C9heterocyclyl)-C(O). Heterocycloyl may be unsubstituted or substituted (eg, optionally substituted heterocycloyl), according to the definition of heterocyclyl.

The term heterocycloyloxy, within the scope of the invention, refers to a group having the structure (C1-C9-heterocyclyl)-C(O)-O-. Heterocycloyloxy may be unsubstituted or substituted (eg, optionally substituted heterocycloyloxy), as defined by heterocyclyl.

The term heterocyclylsulfinyl refers to a group having the structure heterocyclyl^(O)-, in which the heterocyclyl is as described herein. The heterocyclylsulfinyl group may be unsubstituted or substituted, as described herein.

The term heterocyclylsulfonyl refers to a group having the structure heterocyclyl^(O) ₂ -, in which heterocyclyl is as described herein. The heterocyclylsulfonyl group may be unsubstituted or substituted, as described herein.

The term heterocyclylthio refers to a group having the structure heterocyclyl-S-, in which heterocyclyl is as described herein. The heterocyclylthio group may be unsubstituted or substituted as described herein.

The term hydroxy, as used herein, represents an -OH group.

The term hydroxyalkyl, as used herein, is an alkyl group, as defined herein, substituted with one to three hydroxy groups, with the proviso that no more than one hydroxy group may be attached to one carbon atom of the alkyl group, and examples thereof are hydroxymethyl, dihydroxypropyl, etc.

The term nitro, within the scope of the invention, refers to the group -NO ₂ .

The term n-membered ring, in which n is 5,6,7 or 8, as used herein, refers to a carbocyclic or heterocyclic structure, which may be aromatic or non-aromatic. When an n-membered ring is carbocyclic aromatic, it is subject to definition as an aromatic group. When the n-membered ring is carbocyclic non-aromatic, it is subject to definition as a cycloalkylene. When the n-membered ring is heterocyclic aromatic, it is to be defined as heteroarylene. When the n-membered ring is heterocyclic, non-aromatic, it is to be defined as heterocyclylene. An n-membered ring may be unsubstituted or substituted (eg, an optionally substituted n-membered ring), as defined herein unless otherwise indicated. In some embodiments, the n-membered ring may be substituted with 1, 2, 3, 4, or 5 substituents, where each substituent is independently selected from the group consisting of H, halo, hydroxy, optionally substituted amino, optionally substituted amido, thiol, cyano , optionally substituted C ₂ -C ₆ -alkyl, optionally substituted C ₂ -C ₆ -alkenyl, optionally substituted C ₂ -C ₆ -alkynyl, optionally substituted C 1 -C ₆ -alkoxy, optionally substituted C ₆ -C ₁₀ -aryloxy, optionally substituted C1- _C9 -heteroaryloxy, optionally substituted _C2 - _C6 -alkanoyl, optionally substituted _C7 - _C11 -aryloyl, optionally substituted C2-C10-heteroaryloyl, optionally substituted C2-C10-heterocycloyl, hydroxycarbonyl, optionally substituted complex ether, optionally substituted carboxamide, optionally substituted C1- _C6 -alkanoyloxy, optionally substituted _C7 - _C11 -aryloyloxy, optionally substituted _C2 - _C10 -heteroaryloyloxy, optionally substituted _C2 - _C10 -heterocycloyloxy, optionally substituted C1-C ₆ -thioalkyl, optionally substituted ^-^-alkylsulfinyl, optionally substituted C1-C ₆ -alkylsulfonyl, optionally substituted C ₆ -C ₁₀ -arylthio, optionally substituted C ₆ -C ₁₀ -arylsulfinyl, optionally substituted C ₆ -C ₁₀ -arylsulfonyl , optionally substituted C ₁ -C ₉ -heteroarylthio, optionally substituted C ₁ -C ₉ -heteroarylsulfinyl, optionally substituted ^-^-heteroarylsulfonyl, optionally substituted ^-^-heterocyclylsulfinyl, optionally substituted C ₁ -C ₉ -heterocyclylsulfonyl, optionally substituted sulfamoyl , optionally substituted C ₁ -C ₆ -heteroalkyl, optionally substituted C ₂ -C ₆ heteroalkenyl, optionally substituted C ₂ -C ₆ -heteroalkynyl, optionally substituted C ₃ -C ₁₀ cycloalkyl, optionally substituted C ₄ -C ₁₀ -cycloalkenyl, optional substituted C ₈ -C ₁₀ cycloalkynyl, optionally substituted C ₆ -C ₁₀ -aryl, optionally substituted C ₆ -C ₁₀ -aryl C1-C6 alkyl, optionally substituted C ₆ -C ₁₀ -aryl C ₂ -C ₆ -alkenyl, optionally substituted C ₆ -C ₁₀ -aryl C ₂ -C ₆ -alkynyl, optionally substituted C1-C ₉ -heteroaryl, optionally substituted C ₁ -C ₉ heteroaryl C1-C6-alkyl, optionally substituted C1-C9-heteroaryl C2-C6-alkenyl , optionally substituted C'i-C'9-heteroaryl C ₂ -C ₆ -alkynyl, optionally substituted ^-^-heterocyclyl, optionally substituted C ₂ -C ₉ -heterocyclyl C1-C ₆ -alkyl, optionally substituted C ₂ -C ₉ -heterocyclyl

- 31 046439

C2-C ₆ -alkenyl and optionally substituted C1-C ₉ -heterocyclyl C2-C ₆ -alkynyl.

An oxo group is a divalent substituent consisting of an oxygen atom, for example =O.

The term pharmaceutically acceptable salt, for the purposes of the invention, represents salts which, within the bounds of informed medical judgment, are suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic response, etc., and are consistent with an appropriate ratio benefit/risk. Pharmaceutically acceptable salts are well known in the art. For example, S.M. Berge et al. described pharmaceutically acceptable salts in detail in J. Pharm. Sci. 66:1-19, 1977. Salts can be prepared in situ during the final isolation and purification of the compounds of the present invention or separately by reaction of the free base group with a suitable organic acid. Representative acid-addratic salts include acetate salts, adipate, alginate, ascorbate, aspartate, benzulusphone, benzoate, bisulfate, borage, boutirath, campfire, campfer sulfate, citrate, cyclopentantanate, diglouconate, milk-and-dimensional sulfate, etansyphonate, fumarate, glucogepe tonat, glycerophosphate, hemysulfate, heptonate 3 -phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate, etc. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium and the like cations, as well as non-toxic ammonium, quaternary ammonium and amine cations, including, but not limited to, ammonium, tetramethylammonium, tetraethylammonium, methylammonium, dimethylammonium , trimethylammonium, triethylammonium, ethylammonium, etc.

The term protecting group, as used herein, is a group designed to protect a functional group (eg, hydroxy, amino or carbonyl) from participating in one or more unwanted reactions during chemical synthesis (eg, polynucleotide synthesis). The term O-protecting group, as used herein, is a group designed to protect an oxygen-containing (eg, phenolic, hydroxyl or carbonyl) group from participating in one or more undesired reactions during chemical synthesis. The term N-protecting group, as used herein, is a group designed to protect a nitrogen-containing (eg, amino or hydrazine) group from participating in one or more undesired reactions during chemical synthesis. Common O- and N-protecting groups are described in Greene, Protective Groups in Organic Synthesis, ^3rd Edition (John Wiley & Sons, New York, 1999), the contents of which are incorporated herein by reference. Exemplary O- and N-protecting groups include acyl, aryloyl, or carbamyl groups such as formyl, acetyl, propionyl, pivaloyl, t-butylacetyl, 2-chloroacetyl, 2-bromoacetyl, trifluoroacetyl, trichloroacetyl, phthalyl, o-nitrophenoxyacetyl, α- chlorobutyryl, benzoyl, 4-chlorobenzoyl, 4-bromobenzoyl, m-butyldimethylsilyl, tri-iso-propylsilyloxymethyl, 4,4'-dimethoxytrityl, isobutyryl, phenoxyacetyl, 4-isopropylphenoxyacetyl, dimethylformamidino and 4-nitrobenzoyl.

Exemplary O-protecting groups for protecting carbonyl-containing groups include, but are not limited to: acetals, acylas, 1,3-dithiane, 1,3-dioxane, 1,3-dioxolane and 1,3-dithiolane.

Other O-protecting groups include, but are not limited to: substituted alkyl, aryl and aryl-alkylene ethers (e.g., trityl; methylthiomethyl; methoxymethyl; benzyloxymethyl; siloxymethyl; 2,2,2,-trichloroethoxymethyl; tetrahydropyranyl; tetrahydrofuranyl; ethoxyethyl; 1- [2-(trimethylsilyl)ethoxy]ethyl; 2-trimethylsilylethyl; p-chlorophenyl, p-methoxyphenyl, p-nitrophenyl, benzyl, p-methoxybenzyl, and nitrobenzyl); silyl ethers (eg, trimethylsilyl; triethylsilyl; triisopropylsilyl; dimethylisopropylsilyl; t-butyldimethylsilyl; t-butyldiphenylsilyl; tribenzylsilyl; triphenylsilyl; and diphenylmethylsilyl); carbonates (e.g. methyl, methoxymethyl, 9fluorenylmethyl; ethyl; 2,2,2-trichloroethyl; 2-(trimethylsilyl)ethyl; vinyl, allyl, nitrophenyl; benzyl; methoxybenzyl; 3,4-dimethoxybenzyl; and nitrobenzyl).

Other N-protecting groups include, but are not limited to, chiral auxiliaries such as protected or unprotected D, L or D, L-amino acids such as alanine, leucine, phenylalanine and the like; containing sulfonyl groups such as benzenesulfonyl, p-toluenesulfonyl, etc.; carbamate-forming groups such as benzyloxycarbonyl, p-chlorobenzyloxycarbonyl, pmethoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, 3,4-dimethoxybenzyloxycarbonyl, 3,5-dimethoxybenzyloxycarbonyl, 2,4-dimethoxybenzyloxycarbonyl, 4-methoxybenyl, zyloxycarbonyl, 2- nitro-4,5-dimethoxybenzyloxycarbonyl, 3,4,5trimethoxybenzyloxycarbonyl, 1-(p-biphenylyl)-1-methylethoxycarbonyl, a,a-dimethyl-3,5-dimethoxybenzyloxycarbonyl, benzhydryloxycarbonyl, t-butyloxycarbonyl, diisopropylmethoxycarbonyl, isopropyloxycarbonyl, ethoxycarbonyl, methoxycarbonyl, allyloxycarbonyl, 2,2,2,-trichloroethoxycarbonyl, phenoxycarbonyl, 4-nitrophenoxycarbonyl, fluorenyl-9-methoxycarbonyl, cyclopentyloxycarbonyl, adamantyloxycarbonyl, cyclohexyloxycarbonyl, phenylthiocarbonyl, etc., aryl

- 32 046439 alkylene groups such as benzyl, triphenylmethyl, benzyloxymethyl, etc., and silyl groups such as trimethylsilyl and the like. Useful N-protecting groups are formyl, acetyl, benzoyl, pivaloyl, t-butylacetyl, alanyl, phenylsulfonyl, benzyl, t-butyloxycarbonyl (Boc) and benzyloxycarbonyl (Cbz).

The term sulfamoyl, as used herein, refers to a group having the structure -SO2-N(R ^N1 )2, where each RN ¹ is independently H, alkyl, alkenyl, alkynyl, aryl, arylalkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl (e.g. heteroaryl), heterocyclylalkyl (eg, heteroarylalkyl), or two RN ^1s combine to form a heterocyclyl. The sulfamoyl group may be unsubstituted when each RN ¹ is H, or substituted when at least one RN ¹ is not H (eg, optionally substituted sulfamoyl). In a preferred embodiment, sulfamoyl is -SO2NH2 or -SO2NHRN ¹ where RN ¹ is independently alkyl, aryl, arylalkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl (eg heteroaryl), heterocyclylalkyl (eg heteroarylalkyl).

The term thioalkyl, as used herein, is a chemical substituent of the formula SR, where R represents an alkyl group. In some embodiments, the alkyl group may be further substituted with 1, 2, 3, or 4 substituent groups as described herein.

The term thiol represents a -SH group.

Typical optional substituents on aromatic or heteroaromatic groups independently include halo (e.g., F, Cl, Br, or I), optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted cycloalkenyl, optionally substituted cycloalkynyl, CN, NO2 , CF ₃ , OCF ₃ , COOR, CONR'2, OR, SR', SOR', SO2R', NR'2, NR'(CO)R', NR'C(O)OR', NR'C(O )NR'2, NR'SO2NR'2 or NR'SO2R, wherein each R' independently represents H or an optionally substituted group selected from alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl and aryla (all as defined above); or the substituent may be an optionally substituted group selected from alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, aryl, heteroaryl, O-aryl, O-heteroaryl and arylalkyl.

Unless otherwise noted, typical optional substituents on non-aromatic groups include, independently, halo (eg, F, Cl, Br or I), CN, NO ₂ , CF ₃ , OCF ₃ , COOR', CONR' ₂ , OR', SR ', SOR', SO2R', NR'2, NR'(CO)R, NR'C(O)OR', NR'C(O)NR'2, NR'SO2NR'2 or NR'SO2R', where each R independently represents H or an optionally substituted group selected from alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkenyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl and aryl (all as defined above); or the substituent may be an optionally substituted group selected from alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, aryl, heteroaryl, O-aryl, O-heteroaryl and arylalkyl. A non-aromatic group may also include a substituent selected from =O and =NOR', where R' is H or an optionally substituted group selected from alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl and aryl (all as defined above) .

In general, a substituent group (eg, alkyl, alkenyl, alkynyl, or aryl (including all heteroforms as defined above) may itself be optionally substituted with additional substituents. The nature of these substituents is similar to the substituents listed in relation to substituents on the basic structures above. Thus Thus, where an embodiment of the substituent is an alkyl, the alkyl may be optionally substituted with the remaining substituents listed as substituents where it makes chemical sense and where it does not violate the size limit of the alkyl itself, for example, alkyl substituted with alkyl or alkenyl, simply; may exceed the upper limit of the number of carbon atoms for these embodiments, and is not included. However, alkyl substituted with aryl, amino, halo, etc. may be included. For example, where the group is substituted, the group may be substituted 1, 2. , 3, 4, 5 or 6 substituents. Optional substituents include, but are not limited to: C ₁ -C ₆ -alkyl or heteroalkyl, C ₂ -C ₆ -alkenyl or heteroalkenyl, C ₂ -C ₆ -alkynyl or heteroalkynyl, halogen; aryl, heteroaryl, azido(-Н ₃ ), nitro (-NO2), cyano (-CN), acyloxy(-OC(=O^'), acyl (-C(=O)R'), alkoxy (-OR '), amido (-NR'C(=O)R), carboxamide (e.g. -C(=O)NRR'), amino (-NRR'), carboxylic acid (-CO ₂ H), carboxylic ester ( -CO2R'), carbamoyl (-OC(=O)NR'R or -NRC(=O)OR'), hydroxy (-OH), isocyano (-NC), sulfonate (-S(=O)2OR), sulfonamide (-S(=O)2NRR' or -NRS(=O)2R'), or sulfonyl (-S(=O)2R), where each R or R' is independently selected from H, C1- _C6 alkyl or heteroalkyl, C ₂ -C ₆ -alkenyl or heteroalkenyl, C ₂ -C ₆ -alkynyl or heteroalkynyl, aryl or heteroaryl. The substituted group may have, for example, 1, 2, 3, 4, 5, 6, 7, 8. or 9 deputies.

In some embodiments, the present invention relates to groups that are amino acid residues. The amino acid residue may be a naturally occurring amino acid (e.g., Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,

- 33 046439

Thr, Trp, Tyr or Val), or the amino acid residue may be a non-natural amino acid. A non-naturally occurring amino acid is an amino acid that is not naturally produced or found in a mammal. Examples of unnatural amino acids include D amino acids; an amino acid having an acetylaminomethyl group attached to a cysteine sulfur atom; pegylated amino acid; omega amino acids of the formula NH2(CH2) _n COOH where n represents 2-6, neutral non-polar amino acids such as sarcosine, t-butylalanine, tbutylglycine, N-methylisoleucine and norleucine; phenylglycine; citrulline; methionine sulfoxide; cysteic acid; ornithine; and hydroxyproline.

Inhibitory antibodies against GPR174.

In specific embodiments, the GPR174 inhibitory compound is an antibody or antibody fragment that binds GPR174. In specific embodiments, the anti-GPR174 antibody or receptor binding fragment binds to an epitope bound by any of the compounds identified herein. Such antibodies include polyclonal, monoclonal and recombinant antibodies derived from any antibody-producing vertebrate and may be multispecific, chimeric, humanized, anti-idiotypic antibodies and antibody fragments. Antibody fragments include Fab, Fab', F(ab) ₂ , F(ab') ₂ , Fv fragments, scFv fragments and single chain antibodies.

Inhibitory antibodies against GPR174 can be obtained using any methods known in the art. Polyclonal antibodies, for example, can be obtained by immunizing an animal with the GPR174 polypeptide or an immunogenic portion thereof. GPR174 inhibitory monoclonal antibodies include not only intact monoclonal antibodies and full-length monoclonal antibodies, but also fragments thereof (such as Fab, Fab', F(ab')2, Fv), single chain antibodies (ScFv), variants thereof, fusion proteins including an antigen-binding moiety, humanized monoclonal antibodies, chimeric monoclonal antibodies, and any other modified configuration of an immunoglobulin molecule that contains an antigen-binding moiety (epitope recognition region) of the required specificity and has the ability to bind an epitope. This is not limiting as to the source of the antibody or the method in which it is produced (eg, through hybridoma, phage selection, recombinant expression, transgenic animals, etc.). The term includes full-length immunoglobulins, as well as fragments, etc., described above under the definition of antibody.

GPR174 inhibitory monoclonal antibodies can be obtained from naïve or immunized animals, or B cell lines derived from them, using well-known hybridoma technology, or by screening populations of cultured or immortalized cells from these animals. Monoclonal antibodies can be produced directly from selected hybridomas or can be produced recombinantly. Humanized monoclonal antibodies are produced by transferring non-human (eg, murine) complementarity determining regions (CDRs) from mouse immunoglobulin heavy and light variable chains into the human variable domain. Human monoclonal antibodies can also be produced through the use of transgenic mice engineered to produce specific human antibodies in response to antigenic stimulation. In addition, human monoclonal antibodies can be generated by screening cultured human B lymphocytes or screening libraries of human antibodies exposed in various contexts, including phages, yeast or vertebrate cells. Humanized or fully human antibodies specific for human GPR101, consisting of IgG2 or IgG4 isotypes, can be produced by one of several methods known to one of ordinary skill in the art, as described in Vaughan et al., Nature Biotechnical 16:535 539, 1998.

The formation of antibodies against GPR174 can be induced using GPR174 polypeptides (eg, full-length human GPR174 listed as SEQ ID NO: 1) or using peptides bearing the GPR174 antigenic epitope (eg, part of a GPR174 polypeptide). Immunogenic peptides can be as small as five amino acid residues. For example, a GPR174 polypeptide comprising the entire amino acid sequence of SEQ ID NO: 1 can be used to induce the formation of anti-GPR174 antibodies that can be used in the method of the present invention. Specific domains of GPR174 known to be involved in receptor-ligand interactions can be expressed in the form of recombinant polypeptides and used as antigens. In addition, peptides comprising a portion of at least 6 amino acids from the GPR174 polypeptide (SEQ ID NO: 1) can also be used to induce the formation of anti-GPR174 antibodies. GPR174 peptides and polypeptides used to induce antibody formation can be isolated in the form of natural, recombinant or synthetic polypeptides.

Antigens that can be used to induce the formation of antibodies against GPR174 also include fusion polypeptides, such as fusions of GPR174 or portions thereof with an immunoglobulin polypeptide or maltose binding protein. The polypeptide immunogen may be a full-length molecule or a portion thereof. If part of the polypeptide is hapten-like,

- 34 046439 such a portion may advantageously be combined or associated with a macromolecular carrier (such as limpet hemocyanin (KLH), bovine serum albumin (BSA), or tetanus toxoid) for immunization.

GPR174 antigens may also include a polypeptide or portion thereof presented within a lipid membrane or detergent. As a G protein-coupled receptor, GPR174 contains seven hydrophobic transmembrane domains that make the protein sparingly soluble in an aqueous environment. Various methods can be adopted to adapt to the hydrophobic nature of GPR174. For example, such methods include immunizing an animal or screening an antibody library using hydrophilic receptor subdomains in aqueous solutions. Alternatively, immunogens comprising the full-length protein or subdomains containing hydrophobic moieties can be solubilized in detergent, or can be presented to an animal or antibody library in the membrane of whole cells modified to overexpress the protein, or in membrane preparations or exosomes derived from such cells, or in liposomes.

In some embodiments, the GPR174 inhibitory compound is an anti-GPR174 monoclonal antibody. Anti-GPR174 monoclonal antibodies are highly specific, being directed against a single epitope of GPR174. As used herein, the designation monoclonal refers to the nature of the antibody as being derived from a substantially homogeneous population of antibodies, and does not imply that the antibody must be produced by any particular method. Monoclonal antibodies can be produced using any method that produces antibody molecules in culture of stable cell lines, such as the hybridoma method described in Kohler et al., Nature 256:495, 1975, or they can be produced by recombinant DNA methods (see, for example, US Patent No. 4816567 from Cabilly). Monoclonal antibodies can also be isolated from phage antibody libraries using methods described in Clackson et al., Nature 352:624 8, 1991, and Marks et al., J. Mol. Biol. 222:581 97, 1991. Such antibodies may belong to any class of immunoglobulins, including IgG, IgM, IgE, IgA, IgD, and any subclass thereof.

For example, monoclonal antibodies can be produced by injecting a suitable mammal (eg, BALB/c mouse) with a composition containing a GPR174 polypeptide or a portion thereof. After a predetermined period of time, splenocytes are isolated from the mouse and resuspended in culture medium. The splenocytes are then fused with the immortalized cell line to form a hybridoma. The formed hybridomas are grown in cell culture and screened for their ability to produce a monoclonal antibody against GPR174. (See also Current Protocols in Immunology, Vol. 1., John Wiley & Sons, pages 2.5.1 2.6.7, 1991.)

A number of ex vivo display methods have been developed for the detection of human or non-human monoclonal antibodies. In such methods, antibody polynucleotide libraries are prepared by cloning a repertoire of natural or synthetic immunoglobulin heavy and light chains into vectors containing elements for expression in the host, such as an enhancer and promoter for transcription, and a polyadenylation signal sequence, as well as a polynucleotide sequence encoding the domain , such as a phage surface protein or transmembrane domain, to mediate expression of the antibody on the surface of the host. The antibody library is introduced into host cells through methods such as transfection, transduction, or site-specific genomic targeting. Methods such as the above have been used to display antibody libraries on phage (see, e.g., Clackson et al., Nature 352:624-8, 1991; Marks et al., J Mol Biol 222:581-97, 1991) yeast (see, for example, Barnard et al., J Ind Microbiol Biotechnol 37:961-71, 2010), mammalian cells (see, for example, Bowers et al., PNAS 108:20455-60, 2011) and chicken ( see, for example, Yabuki et al. PLoS One 7:e36032, 2012). Monoclonal antibodies can be isolated and purified from hybridoma cultures through a variety of well-established methods. Such isolation methods include affinity chromatography using Protein A Sepharose, size exclusion chromatography, and ion exchange chromatography (see, for example, Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3; Baines et al., Purification of Immunoglobulin G (IgG), in Methods in Molecular Biology, The Humana Press, Inc., Vol. 10, pages 79-104, 1992).

Once produced, polyclonal, monoclonal or phage-derived antibodies are first tested for specific binding to GPR174. A variety of assays known to one skilled in the art can be used to detect antibodies that specifically bind to GPR174. Exemplary assays include Western blot or immunoprecipitation assays by standard methods (eg, as described in Ausubel et al.), immunoelectrophoresis, enzyme-linked immunosorbent assays, dot blot assays, inhibition or competition assays, and sandwich assays (as described in Harlow and Land, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1988). Once antibodies that specifically bind to GPR174 are identified, the antibodies are tested for their ability to function as a GPR174 inhibitory compound in one of several assays, such as those described herein, for example.

Chimeric/humanized antibodies.

GPR174 inhibitory monoclonal antibodies that can be used in the method according to ISO

- 35 046439 bretenium include chimeric antibodies in which part of the heavy and/or light chain is identical or homologous to the corresponding sequences in antibodies derived from a particular species or belonging to a particular class or subclass of antibodies, while the rest of the chain(s) ) is identical or homologous to corresponding sequences in antibodies derived from other species or belonging to another class or subclass of antibodies, as well as fragments of such antibodies (U.S. Patent No. 4,816,567, from Cabilly; and Morrison, S.L., et al., Proc. Nat. 'l Acad. Sci. USA #7:6851 6855, (1984)). One form of chimeric antibody that can be used in the present invention is a humanized monoclonal antibody against GPR174. Humanized forms of non-human (eg, murine) antibodies are chimeric antibodies that contain minimal sequence derived from a non-human immunoglobulin. Humanized monoclonal antibodies are produced by transferring non-human (eg, murine) complementarity determining regions (CDRs) from mouse immunoglobulin heavy and light variable chains into the human variable domain. Typically, substitutions are then made with human antibody residues in the framework regions of the non-human equivalents. In addition, humanized antibodies may contain residues not found in the recipient antibody or the donor antibody. These modifications are made to further refine the activity of the antibody. Typically, a humanized antibody contains substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to loops from a non-human immunoglobulin, and all or substantially all of the framework Fv regions are regions from the human immunoglobulin sequence. The humanized antibody optionally also contains at least a portion of an immunoglobulin constant region (Fc), typically from human immunoglobulin. For further details, see Jones, P.T., et al., Nature 321:522525, (1986); Reichmann, L., et al., Nature 332:323 329, (1988); and Presta, Curr. Op. Struct. Biol. 2:593596, (1992).

Humanized antibodies that can be used in the invention include human monoclonal antibodies containing at least the GPR174 binding CDR3 region. In addition, the Fc portions can be replaced to produce IgA or IgM, just like human IgG antibodies. Such humanized antibodies may be of particular clinical utility because they specifically recognize human GPR174 but do not provoke an immune response in the individual against the antibody itself. Therefore, they are better suited for in vivo administration to humans, especially when repeated or long-term administration is required. Methods for producing humanized monoclonal antibodies are also described, for example, in Jones, P.T., et al., Nature 321:522, (1986); Carter, P., et al., Proc. Nat'l. Acad. Sci. USA 89:4285, (1992); Sandhu, J. S., Crit. Rev. Biotech. 12:437, (1992); Singer, I. I., et al.,J. Immun. 750:2844, (1993); Sudhir (ed.), Antibody Engineering Protocols, Humana Press, Inc., (1995); Kelley, Engineering Therapeutic Antibodies, in Protein Engineering: Principles and Practice, Cleland et al. (eds.), John Wiley & Sons, Inc., pages 399-434, (1996); and in US Pat. No. 5,693,762, Queen's, 1997. In addition, there are commercial organizations that synthesize humanized antibodies from specific regions of murine antibodies, such as Protein Design Labs (Mountain View, CA).

Recombinant antibodies.

GPR174 inhibitory monoclonal antibodies can also be produced using recombinant methods. For example, human antibodies can be produced using human immunoglobulin expression libraries (available, for example, from Stratagene, Corp., La Jolla, Calif.) to produce human antibody fragments (VH, VL, Fv, factor D, Fab or F(ab') ₂ ). These fragments are then used to construct fully human antibodies using methods similar to those for producing chimeric antibodies.

Fragments of immunoglobulins.

GPR174 inhibitory compounds that can be used in the method of the present invention include not only intact immunoglobulin molecules, but also well known fragments, including Fab fragments, Fab', F(ab) ₂ , F(ab') ₂ and Fv fragments, fragments scFv, diabodies, linear antibodies, single-chain antibody molecules, and multispecific (eg, bispecific and trispecific) antibodies formed from antibody fragments. It is well known in the art that only a small region of the antibody molecule, the paratope, is involved in the binding of the antibody to its epitope (see, e.g., Clark, W.R., The Experimental Foundations of Modern Immunology, Wiley & Sons, Inc., NY, 1986) . The pFc' and Fc regions of the antibody are effectors of the classical complement pathway but are not involved in antigen binding. An antibody from which the pFc' region has been enzymatically excised, or which has been produced without the pFc' region, is designated an F(ab')2 fragment and retains both antigen-binding regions of the intact antibody. The isolated F(ab')2 fragment is designated a bivalent monoclonal fragment due to its two antigen binding sites. Likewise, an antibody from which the Fc region has been enzymatically excised, or which is produced without the Fc region, is designated a Fab fragment, and retains one of the antigen-binding regions of the intact antibody molecule. Antibody fragments can be obtained through proteolytic hydrolysis,

- 36 046439 such as cleavage of full-length antibodies with pepsin or papain by conventional methods. For example, antibody fragments can be produced by enzymatic digestion of antibodies using pepsin to produce a 5S fragment designated F(ab')2. This fragment can be further cleaved using a thiol reducing agent to produce 3.5S monovalent Fab' fragments. Optionally, the cleavage reaction can be carried out using a blocking group for sulfhydryl groups that are formed by cleavage of disulfide bonds. Alternatively, enzymatic cleavage using pepsin directly produces two monovalent Fab fragments and an Fc fragment. These methods are described, for example, in US Patent No. 4331647 from Goldenberg; Nisonoff, A., et al., Arch. Biochem. Biophys. 89:230, (1960); Porter, R.R., Biochem. J. 75:119, (1959); Edelman, et al., in Methods in Enzymology 7:422, Academic Press, (1967); and in Coligan at pages 2.8.1-2.8.10 and 2.10.-2.10.4.

Single chain antibody fragments.

Alternatively, single polypeptide chain binding molecules specific for GPR174 can be prepared in which the heavy and light chain Fv regions are connected. Fv fragments can be joined via a peptide linker to form a single chain antigen binding protein (scFv). These single-stranded antigen-binding proteins are produced by constructing a structural gene containing DNA sequences encoding the VH and VL domains linked by an oligonucleotide. The structural gene is inserted into an expression vector, which is then introduced into a host cell such as E. coli. Recombinant host cells synthesize a single polypeptide chain with a peptide linker bridging the two V domains. Methods for preparing scFv are described, for example, in Whitlow, et al., Methods: A Companion to Methods in Enzymology 2:97, (1991); Bird, et al., Science 242:423, (1988); US Patent No. 4,946,778, from Ladner; Pack, P., et al., Bio/Technology 77:1271, (1993).

As an illustrative example, a GPR174-specific scFv can be generated by exposing lymphocytes to a GPR174 polypeptide in vitro and selecting from antibody display libraries in phage or similar vectors (eg, by using an immobilized or tagged GPR174 protein or peptide). Genes encoding polypeptides having potential GPR174 polypeptide binding domains can be obtained by screening libraries of random peptides displayed on phage or bacteria such as E. coli. These random peptide display libraries can be used to screen for peptides that interact with GPR174. Methods for generating and screening such random peptide display libraries are well known in the art (US Patent No. 5,223,409, from Lardner; US Patent No. 4,946,778, from Ladner; US Patent No. 5,403,484, from Lardner; US Patent No. 5,571,698, from Lardner; and Kay et al., Phage Display of Peptides and Proteins Academic Press, Inc., 1996), and random peptide display libraries and screening kits for such libraries are commercially available, for example, from CLONTECH Laboratories, Inc. (Palo Alto, Calif.), Invitrogen Inc. (San Diego, Calif.), New England Biolabs, Inc. (Ipswich, Mass.), and Pharmacia LKB Biotechnology Inc. (Piscataway, N.J.).

B. Screening methods.

As described herein, we have identified a number of compounds capable of inhibiting GPR174-mediated signaling activity and identified G protein signaling pathways amenable to modulation by GPR174. Screening methods that can be used to identify such compounds and methods for measuring signaling pathway activation are described below.

1. CRA.

One way to identify compounds capable of modulating the receptor is to use a cellular redistribution assay (CRA). Exemplary CRA methods are found in US Patent No. 7309576 issued to O'Dowd et al., and O'Dowd et al., J. Biomol. Screen. 12:175-85, 2007. Briefly, this method involves inserting a nuclear localization sequence (NLS) into a transmembrane protein that does not contain an endogenous functional NLS. NLS insertion alters the cellular localization of GPCRs such that the majority of the NLS-modified receptor is present within the cell. An NLS-modified GPCR, when interacting with a functionally active compound, is largely localized to the cell surface. Thus, it becomes possible to identify receptor modulators without knowledge of the endogenous ligand or surrogate ligand.

The present inventors have identified a number of compounds that inhibit GPR174 using a CRA assay similar to that described in US Pat. No. 7,309,576 and O'Dowd et al., J. Biomol. Screen. 12:175-85, 2007. These compounds are described in detail below.

2. CRA confirmation.

To demonstrate that the compounds identified in the CRA assay are capable of modulating G protein signaling pathways, we tested compounds identified in the CRA assays measuring multiple non-orphan receptor signaling. As shown in Example 1, the inventors of the present invention have shown that all of the compounds

- 37 046439 identified by screening libraries of compounds against CHRM1, NPSR and ADRAla receptors using CRA, modulated downstream signaling pathways of these receptors. These results indicate that the best CRA screening candidates tend to be those essential for receptor activity.

3. Overview of G protein-coupled receptor-regulated signal transduction pathways.

While hundreds of GPCR proteins have been identified, only a small number of heterotrimeric G proteins are activated by GPCR proteins. Heterotrimeric G proteins include three subunits, alpha, beta and gamma subunits. The nomenclature of G proteins is determined by alpha subunits, for example, Gaq, Gas, Gai, Gaz, Gao, Ga12, Ga13, Ga15 and Ga16. G proteins are grouped into families based on homology, which also share some signaling characteristics. G protein signaling pathways include the Gq, Gs, Gi, and Ga12/13 signaling pathways. In addition to G protein signaling pathways, GPCRs can signal through the βγ subunit complexes, arrestin 1, arrestin 2, arrestin 3, and arrestin 4.

Numerous assays have been developed to measure GPCR stimulation and signaling pathways activated by GPCRs (Siehler S., Biotechnology 3:471-83, 2008 and references therein). There are several common assay technologies that measure GPCR activation. Binding of guanosine-5'-O-(3'-[%]thio)-triphosphate ([ ³⁵ S]GTPyS) to Ga subunits has been widely used to measure receptor activation (Milligan, Trends Pharmocol. Sci. 24:87-90, 2003 ).

Another common way to measure receptor activation is to measure ligand-induced internalization of the receptor. GPCRs are typically internalized within 1–2 hours of agonist stimulation through the process of GPCR phosphorylation and subsequent binding of β-arrestin, which acts as an adapter to bind the receptor to clathrin-coated pits and targets the receptor for internalization into endosomes (Kahout et al. , Mol. Pharmacol. 63:9-18, 2003). Several technologies have been developed that utilize some aspect of receptor internalization to exhibit agonist-induced receptor activation (Siehler, Biotechnology 3:471-83, 2008 and references therein). These general assays are limited to measuring agonist-induced receptor activation and do not consider which G proteins are coupled to the GPCR and thus the signaling pathways that the GPCR activates to elicit its biological response.

A. Gq signaling pathway.

G proteins Gaq, Ga11, Ga14, Ga15/16 (collectively, the Gq family) activate the Gq signaling pathway and are associated with the activation of the PLC enzyme, which, in turn, hydrolyzes the phospholipid PIP2, releasing two intracellular messengers: DAG and IP ₃ . IP ₃ is soluble and diffuses through the cytoplasm and interacts with IP3 receptors on the endoplasmic reticulum, causing calcium release and an increase in cytosolic calcium levels. This increase can be measured directly using fluorescent dyes that detect intracellular calcium. DAG remains associated with the inner leaflet of the plasma membrane due to its hydrophobic nature, where it attracts protein kinase C (PKC), which becomes activated in conjunction with the binding of calcium ions. This leads to cellular responses in the host through stimulation of calcium-sensing proteins such as calmodulin (Kawakami et al., J. Biochem. 132:677-82, 2002).

Increased IP3 accumulation is associated with activation of Gq-associated receptors. Assays that detect IP3 accumulation can be used to determine whether a candidate compound is, for example, a Gq-associated receptor agonist (ie, such a compound may increase IP3 levels). Gq-associated receptors can also be analyzed using a transcription reporter assay. For example, Gq-dependent PLC causes the activation of genes containing activator protein 1 (AP1) response elements, also known as AP-1 elements; thus, activated Gq-associated receptors may support increased expression of such genes. Inverse agonists of the Gq-associated receptor can lead to a decrease in such expression, and agonists can lead to an increase in such expression. AP1 and other Gq-regulated transcriptional activators such as serum response element (SRE) and nuclear factor of activated T cell (NFAT) response element may be operably linked to a nucleic acid encoding a conveniently analyzable reporter enzyme such as luciferase, β β-galactosidase, alkaline phosphatase or other detectable output. Assays for such detection are commercially available, for example from Promega and Stratagene.

b. Gs signaling pathway.

Activated Gs protein (GaS and Gaolf) stimulates the AC enzyme, which catalyzes the conversion of ATP to cAMP. GPCRs coupled to the Gs protein are associated with increases in cellular levels of cAMP, which can be measured directly. Increasing cAMP levels activates protein kinase A (PKA) and has other downstream effects. PKA, for example, phosphorylates and thereby activates transcription of genes regulated by cAMP response element binding (CREB) proteins (Neves et al., Science 296:1636-39, 2002). Gs activity can be analyzed in a reporter assay

- 38 046439 transcription using a CREB element operably linked to a nucleic acid encoding a conveniently analyzable reporter enzyme such as luciferase, β-galactosidase, or other detectable output. In addition, assays that detect intracellular levels of cAMP can be used to assess Gs signaling activity. A variety of methods known in the art for measuring cAMP can be used; in some embodiments, the preferred method is based on the use of anti-cAMP antibodies in an ELISA-based format.

c. Gi signaling pathways.

G proteins G1 (Gai1, Gai2, and Gai3), Gao, Gat1, Gat2, Gagust, Gaz (collectively, the Gi family) activate the Gi signaling pathway and are associated with inhibition of AS and suppression of cAMP formation. The Gi sensor protein, Gat, activates cGMP-dependent phosphodiesterase, causing a decrease in intracellular cGMP levels, and Gagust activates PLC. Thus, assays that detect intracellular levels of cAMP, as described above, can be used to assess the activity of the Gi signaling pathway. Some cells have relatively low levels of cAMP, so increasing cellular cAMP levels may help determine the baseline level of Gi signaling activity. One skilled in the art is able to increase cellular levels of cAMP by contacting cells with, for example, phorbol esters or forskolin. Alternatively, receptor activity

Gi can be analyzed using G protein fusions to redirect Gi signaling to either Gq or Gs and using the methods described herein. All members of the Gi family, with the exception of Gaz, are susceptible to inhibition by pertussis toxin via ADP-ribosylation of their corresponding a subunit (Siehler S., Biotechnology 3:471-83 (2008)).

d. Other G protein signaling pathways.

Ga12/13 proteins are involved in Rho family GTPase signaling (via RhoGEF) and control of cell cytoskeletal remodeling, thereby regulating cell migration. Rho-GEF proteins catalyze the exchange of GTP for GDP to activate RhoA. RhoA, in turn, activates Rho kinase, which further leads to activation of activating transcription factor 2 (ATF2) and leads to cellular responses (Liu et al., Methods Mol. Biol. 237:145-9, 2004).

e. The role of β- and γ-subunits.

In addition to the 16 Ga subunits, there are five different β and 12 γ subunits that have been described (Milligan et al., Br. J. Pharmacol. 147:S46-S55, 2006). The βγ subunits form a heterodimer and were initially thought to be nothing more than a binding partner for the Ga subunit to suppress spontaneous signaling and to provide a membrane anchor for the Ga subunit. Although the role of βγ subunits is still emerging, it is clear that βγ subunits play a more important role in GPCR signaling and can directly activate enzymes and ion channels (Dupre et al., Annu. Rev. Pharmacol. Toxicol. 49:31-56, 2009) .

f. Arrestin signaling pathways.

In addition to G protein signaling pathways, GPCRs can signal through arrestin 1, arrestin 2, arrestin 3, and arrestin 4. In addition to their role in the termination of G protein signaling and GPCR internalization, arrestins have been implicated in signaling through ERK, JNK, p38, Akt, PI3 kinase and RhoA (DeWire et al., Annu. Rev. Physiol. 69:483-510, 2007).

4. Testing the initial best candidate CRAs for signaling activity.

The present inventors confirmed that the initial best candidates identified using the CRA assay for GPR174 interacting compounds are capable of inhibiting the Gs signaling pathway through GPR174. Accordingly, the present inventors have clearly determined that the identified compounds functionally interact with GPR174. In particular (in relation to Table 1), compounds 1, 2, 518 and 22-56 were found to inhibit the initial activity of GPR174, indicating that, in this system, these compounds are inverse agonists of GPR174. These results are detailed in Example 4 below. Compounds 3, 19-21 and 57-58 were found to have activity in the CRA and were not found to be modulators in the GPR174 Gs signaling assay, thus these compounds are characterized as GPR174 antagonists or allosteric modulators. Compound 4, which has activity in CRA, was found not to be a modulator in the GPR174 Gs signaling assay and was found to compete with the GPR174 agonist LysoPS, thus Compound 4 is characterized as a GPR174 antagonist. These results are detailed in Example 4 below.

C. GPR174.

GPCRs have seven transmembrane alpha helices. GPCRs have an extracellular N-terminus, three extracellular loops, three intracellular loops, and an intracellular C-terminus. Extracellular loops and transmembrane domains are involved in ligand binding, while transmembrane domains and intracellular loops contribute to signal transduction. See, for example, Luttrell, Methods Mol. Biol.

- 39 046439

332:3–49, 2006. Most GPCR proteins have conserved cysteine residues that form disulfide bridges in extracellular loops. Many GPCR proteins are N-terminally glycosylated. Conserved cysteine residues are also found at the C-terminus and may serve as a site for palmitoylation. See, for example, Lutrell, supra. Structures have been resolved for some GPCR proteins, and consensus models of GPCR structures are available. See, for example, Lagerstrom and Schioth, Nat. Rev. 7:339-57, 2008.

GPR174, also known as FKSG79 and GPCR17, is an orphan G protein-coupled receptor (GPCR) (Davenport et al., IUPHAR/BPS, Class A orphans: GPR174, accessed 11/1/2016). The human GPR174 protein has 333 amino acids as indicated by SEQ ID NO: 1 (NP_115942.1) shown in FIG. 1A coded by SEQ ID NO: 2 (NM_032553.1) as shown in FIG. 1B. The GPR174 protein has seven transmembrane domains. The human GPR174 gene has a single coding exon (Takeda et al., FEBS Lett 520:97-101, 2002) and is mapped to chromosome Xq21.1 (Sugita et al., Biochem Biophys Res Commun 430:190-195, 2013). The amino acid sequence of the mouse GPR174 protein (NP_001028423.1) is indicated as SEQ ID NO: 3 (shown in Fig. 1C). The amino acid sequence of the rat GPR174 protein (NP_001100408.1) is indicated as SEQ ID NO: 4 (shown in Fig. 1D).

GPR174 is expressed in relatively few tissues. By quantitative real-time PCR (qnU,P) detection, GPR174 was found to be most abundantly expressed in the thymus, lymph nodes, spleen and bone marrow, as shown in FIG. 22 (see also Regard et al., Cell 135:561-71, 2008; Chu et al., J Med Genet 50:479-85, 2013; Sugita et al., Biochem Biophys Res Commun 430:190-195, 2013). In lymphoid tissues, GPR174 is expressed at high levels in naïve B and T cells, especially regulatory T cells, as shown in FIG. 23 (see also Barnes et al., J Exp Med 212:1011–20, 2015). A recent report identified lysophosphatidylserine (LysoPS) as a ligand for GPR174 (Inoue et al., Nat Methods 9:1021-9, 2012). LysoPS is a lysophospholipid mediator formed through enzymatic hydrolysis of the membrane phospholipid, phosphatidylserine (PS). LysoPS is secreted by cells of the immune system and can induce multiple cellular responses, including T cell suppression and mast cell degranulation (Makide et al., Prostaglandins Other Lipid Mediat 89:135-9, 2009). The EC ₅₀ of LysoPS for GPR174 varies from 80 nM to 520 nM, depending on the assay used (Inoue et al., Nat Methods 9:1021-9, 2012; Uwamizu et al., J Biochem 157(3):151-160, 2015; Ikubo et al., J Med Chem 58(10):4204-19, 2015). In addition, LysoPS is a ligand for two other GPCRs, namely GPR34 and P2Y10. The EC ₅₀ of LysoPS for P2Y10 is 28 nM, assuming that the EC ₅₀ for GPR174 is 520 nM. It should be noted that the International Union of Basic and Clinical Pharmacology (IUPHAR) continues to list GPR174 as an orphan GPCR (guidetopharmacology.org/GRAC).

GPR174 is associated with several autoimmune diseases. The GPR174 gene is located on chromosome X, where a cluster of single nucleotide polymorphisms (SNPs) near or within the gene is associated with susceptibility to Grave's disease in both Asian and Caucasian populations (Chu et al., J Med Genet 50:479-85, Szymanski et al. al., Tissue Antigens 83:41-4, 2014). The strength of association of the leading SNP, rs3827440, with Grave's disease is similar to the strength of association for a specific allele of HLADRB1, the strongest known genetic risk factor for the disease (Kula et al., Thyroid 16:447-53, 2006; Barlow et al., Clin Endocrinol (Oxf) 44:73-7,1996). The same SNP is also significantly associated with autoimmune Addison's disease, another autoimmune endocrinopathy resulting from damage to the adrenal glands (Napier et al., J Clin Endocrinol Metab 100:E18790, 2015).

Barnes et al. (J Exp Med 212:1011-20, 2015) showed that in the mouse, GPR174 is expressed in naïve B and T cells and is also highly and preferentially expressed in a subset of T cells known as regulatory T cells (Treg). . In male mice lacking GPR174 (Gpr174 ^-/Y ), T-reg cell numbers were significantly increased in specific tissues, consistent with the observation that proliferation and differentiation of CD4+ T cells into T-reg cells is suppressed as a consequence of GPR174 signaling (Barnes et al., 2015 above). In a model of experimental autoimmune encephalomyelitis, peak disease severity was significantly reduced in Gpr174 ^-/Y mice (Barnes et al., 2015 supra). Together, these results highlight the importance of GPR174 in the modulation of T-reg cells and suggest possible therapeutic utility for the use of GPR174 antagonists in the suppression of autoimmunity.

The association between GPR174 and specific malignancies has been published. Qin et al., (2011) discovered that GPR174 is expressed in metastatic melanoma, particularly in subcutaneous metastases, compared to either lymph node or brain metastases (Qin et al., Pigment Cell Melanoma Res 24:207-18 , 2011). Sugita et al., (2013, supra), observed that ectopic overexpression of GPR174 in CHO cells results in an elongated, spindle-shaped morphology and delayed proliferation, indicating that the receptor can influence phenotypic change. Also, moderate expression of messenger RNA GPR174 was noted in lymphoma (Unigene EST Profile database).

Taking into account the oncogenic contribution of intratumoral T-regs (Savage et al., 2013, supra), we investigated the potential of GPR174 inhibitors to modulate the number

- 40 046439 and/or T-reg activity. As described in Example 6 and shown in FIG. 24A, 24B, 25 and 26, the present inventors surprisingly determined that in human PBMC, inhibition of GPR174 enhances immune system activation while also reducing T-reg cell content. This is a surprising finding given the mouse data reported in Barnes et al. (2015, supra), showing that in male mice lacking GPR174, the numbers of T-reg cells were significantly increased in specific tissues.

As further described herein in Examples 3 and 4 shown in Table 1 and FIG. 5-21, the present inventors have determined that GPR174 is a Gs-coupled receptor that is expected, either constitutively or through ligand activation, to increase intracellular cAMP levels. cAMP is a key regulator of the immune system (Mosenden and Tasken, Cellular Signaling 23:1009–1016, 2011). cAMP is known to inhibit IL-2 production and T cell receptor (TCR)-mediated signaling, and subsequently inhibit T cell activation (see Vang et al., J Exp Med 193:497-507, 2001; Ruppelt et al. ., J Immunol 179:5159-5168, 2007). IL-2 is a major cytokine that determines T cell survival and differentiation into effector T cells, memory T cells, and regulatory T cells. GPR174 inhibitors, such as those described herein, are expected to reduce cAMP concentrations and thus stimulate T cell activation through stimulation of TCR signaling and IL-2 secretion, a mechanism that accounts for the downregulation of T-reg cells and activation of effector T cells Thus, overall stimulation of T cell-mediated immunity is a critical component in the immunotherapy of malignancies. It is expected that a large number of malignant tumors may be more sensitive to a more active immune system stimulated by an agent, such as a GPR174 inhibitor, which stimulates T-cell mediated immunity (see Jiang et al., Oncoimmunology 5(6):el 163462, 2016 ; Papaioannou et al., Ann Transl Med 4(14):261, 2016; Park et al., Immune checkpoint inhibitors for cancer treatment, Arch Pharm Res, Oct 21, 2016; 5606, 2016).

As further described herein in Examples 16 and 17, the present inventors have discovered that phosphatidylserine (PS) is an agonist of GPR174-mediated Gs signaling. Example 17 presents experimental results showing that apoptotic cells stimulate the GPR174 Gs signaling pathway in cells expressing GPR174. As further described in Example 16 and shown in FIG. 47A-47F, PS-mediated GPR174 signaling of Gs is inhibited by representative GPR174 inhibitory compounds 6, 10, 11, 20, 23 and 30, belonging to various chemical classes (ie, groups I, II and IV). These data indicate that compounds 6,10,11,20,23 and 30 act as GPR174 antagonists and inhibit PS-liposome-mediated cAMP signaling. Thus, PS-liposome signaling through GPR174 is inhibited by a variety of GPR174-inhibiting small molecules with diverse chemical structures. As described herein, extracellular PS is highly enriched in the tumor microenvironment and is found on the surface of tumor cells as well as endothelial cells of blood vessels infiltrating solid tumors. In addition, apoptotic neutrophils and activated platelets, both of which exhibit PS, are also recruited to solid tumors (see A.K. and Rao D.A., Blood 120:4667-4668, 2012; Schlesinger, M., Journal of Hematology and Oncology (11) 125, 2018; Treffers L.W. et al., Immunological Reviews vol 273: 312-328, 2016; and Gregory A. D. and Houghton A. M., Cancer Research Vol 71 (7): 2411-6, 2011). Thus, high concentrations of PS are considered to be a major source of tumor-mediated immunosuppression and may play a role in resistance to cancer immunotherapies such as checkpoint inhibitors.

Accordingly, one embodiment provides a method of treating a cancer comprising administering to a patient a therapeutically effective amount of a GPR174 inhibitor that inhibits GPR174 G-alpha-s signaling thereby stimulating an immune response in the patient. In another more specific embodiment, GPR174 expressed on immunocytes is brought into contact with phosphatidylserine (PS) or lysophosphatidylserine (lysoPS) in the microenvironment of tumors or associated lymphoid tissues, and wherein said GPR174 inhibitor inhibits PS- or lysoPS-mediated GPR174 signaling. In specific embodiments, the cancer contains living cells, dying cells, or extracellular vesicles having phosphatidylserine (PS) on their surface.

In some embodiments, the patient is a mammalian patient. In specific embodiments, the method further includes administering at least one additional agent selected from the group consisting of:

i. adenosine-A2A (A2A) receptor antagonist;

ii. adenosine-A2B (A2B) receptor antagonist;

iii. CD73 inhibitor;

iv. CD38 inhibitor;

v. CD39 inhibitor; or

- 41 046439 vi. a T-reg attenuating agent, wherein the GPR174 inhibitor and at least one additional agent are administered simultaneously, or sequentially in any order, provided that the effects of the first aforementioned inhibitor or antagonist remain present at the time of administration of the second inhibitor or antagonist.

Additionally, in one embodiment, the present invention provides a method of stimulating T cell-mediated immunity in a subject suffering from cancer, comprising administering a GPR174 inhibitor to said subject in an amount effective to stimulate T cell mediated immunity. In some embodiments, the subject suffers from a malignancy selected from the group consisting of: breast cancer, melanoma, colorectal cancer, urological malignancy, lung cancer, small cell and non-small cell lung cancer, recurrent or refractory malignancies, non-Hodgkin's lymphomas, and Hodgkin's lymphoma, lymphoma, follicular lymphoma, lymphocytic lymphoma, central nervous system lymphoma, T-cell lymphoma, AIDS-associated lymphoma, acute lymphoblastic leukemia, gastrointestinal malignancies, liver cancer, hepatocellular carcinoma, ovarian cancer, pancreatic cancer, biliary cancer duct, prostate cancer, renal carcinoma, bladder cancer, colorectal cancer, multiple myeloma, mesothelioma, cervical cancer, vaginal cancer, anal cancer, oropharyngeal cancer, myelogenous leukemia, chronic myeloid leukemia, stomach cancer, nasopharyngeal carcinoma, head carcinoma and neck, glioblastoma, gliosarcoma, squamous cell malignant tumor of the brain, malignant glioma, diffuse brain stem gliomas, esophageal cancer, thyroid cancer, astrocytoma, malignant tumor of the chest, endometrial cancer, skin cell carcinoma, leukemia, acinar cell carcinoma, adenocarcinoma, bronchioloalveolar carcinoma, cholangiocarcinoma, chordoma, giant cell carcinoma, intestinal carcinoma, major salivary gland carcinoma, malignant odontogenic neoplasia, malignant peripheral nerve sheath tumor, skin cancer, testicular cancer, germ cell tumor, neuroendocrine carcinoma, parathyroid carcinoma, pituitary carcinoma, placental choriocarcinoma, malignant scrotal tumor, tracheal carcinoma, transitional cell carcinoma, uterine body cancer, vulvar cancer, kidney cancer, rectal cancer, fallopian tube carcinoma, peritoneal carcinoma, epithelial malignant tumor, pleural mesothelioma, sarcomatoid carcinoma, synovial sarcoma, nephroblastoma, neuroblastoma, acute myeloid adult leukemia, myelodysplastic/myeloproliferative neoplasia, embryonal carcinoma, Kaposi's sarcoma, malignant bone tumor, uterine cancer, gastric cancer, endometrial carcinoma, malignant tumor of the small intestine, malignant tumor of the endocrine system, parathyroid cancer, adrenal cancer, soft tissue sarcoma, cancer urethra, penile cancer, ureteral cancer, renal pelvic carcinoma, central nervous system neoplasia, primary tumor angiogenesis, spinal axis tumor, epidermoid malignancy, environmentally induced tumors including asbestos-induced tumors, adenosarcoma, adenosquamous carcinoma, adrenocortical carcinoma, Astrocitic tumors, basal -cell carcinoma, chondrosarcoma, sarcoma of the Juing, cancer of the gallbladder, hypopharyngeal malignant tumor, intraocular melanoma, layomiosarcoma cancer, malignant tumor of the lips and oral cavities, malignant mesothelial tumors, malignant Timoma, and honey , Maul -epitheliomas, merckel cells from Merkel cells, mucoepidermoid carcinoma, myelodysplastic syndrome, cancer of the nasal cavity and paranasal sinuses, osteosarcoma, pulmonary blastoma, pineal and supratentorial primitive neuroectodermal tumors, plasma cell neoplasia, retinoblastoma, rhabdomyosarcoma, sarcoma, neuroectodermal tumors and Wilms tumor.

Animal experiments have shown that, in addition to their immune function, T cells and cytokines also contribute to CNS functions. T cell deficiency leads to cognitive impairment (Kipnis et al., PNAS 101:8180-8185, 2004), an effect mediated by IL-4 produced by T cells residing in the meninges (Derecki et al., J Exp Med 207 :1067-1080, 2010). IL-4 has also been implicated in stimulating axonal regrowth after spinal cord or optic nerve injury (Walsh et al., J Clin Invest 125:699-714, 2015). Lymphocyte-deficient mice are less social, and this effect is mediated by IFN-γ produced by T cells residing in the choroid plexus (Filano et al., Nature 535:425-429, 2016). Stimulation of T cells either through Treg depletion (Baruch et al., Nat Commun 6:7967, 2015) or by blocking the T cell checkpoint protein PD-1 (Baruch et al., Nat Med 22:135-137 , 2016), reduced amyloid-β plaques in the brains of mice and improved learning and memory performance. These data demonstrate the involvement of T cells and their corresponding cytokines in the normal functioning of the central nervous system, in neurodegenerative diseases, and in the repair of neural tissue after injury. Thus, GPR174 inhibitory compounds affecting T cell activity, differentiation and cytokine production, as shown in Examples 3 and 4, have therapeutic potential for the treatment of mental illness and neurodegeneration

- 42 046439 non-operative diseases such as schizophrenia, Parkinson's disease, autism, mild cognitive impairment, age-related cognitive decline, Alzheimer's disease, frontotemporal dementia, amyotrophic lateral sclerosis, as well as traumatic injury to the spinal cord and brain.

Natural killer cells (NK cells) play a critical role in the innate immune system, and unlike other cells in the immune system, NK cells can be cytotoxic without prior stimulation (Mandal et al., Hematol Oncol Stem Cell Ther. (2): 47 -55, 2015). For example, they can attack tumor cells even when the expression of MHC class I molecules is absent or down-regulated. Tumor cells can also be attacked by NK cells when NKG2D ligands on tumor cells are up-regulated (Mandal et al., Hematol Oncol Stem Cell Ther. (2): 47-55, 2015). However, NK cells can be suppressed by T-reg cells, and there is a correlation between increased frequency of T-reg cells and cancer progression (Pedroza-Pacheco, et al., Cell Mol Immunol. 10(3): 222-9, 2013 ). Accordingly, many therapies are considered to suppress T-reg cells to stimulate NK cells and improve patient prognosis. NK cells are also important for the immune response against viruses and other pathogens, as well as being involved in autoimmune processes (Zwimer, et al., Front Immunol. 8:25, 2017). NK cell function, differentiation, growth and/or proliferation can be activated by cytokines such as IL-2, IL-21, EL-12, IL-15, IL-18, IL-10, IFN-alpha, IFN-beta and TGF -β (Wu, et al., Front Immunol. 8: 930, 2017). Thus, NK cell function, differentiation, growth and/or proliferation may be affected by GPR174 inhibitory compounds, for example, through the effects of GPR174 inhibition on T cell differentiation and/or cytokine production. Thus, GPR174 inhibitory compounds may have additional therapeutic potential for diseases such as cancer.

In accordance with the above, in one aspect, the present invention relates to a method of treating, preventing and/or reducing the likelihood of developing a malignant tumor in a subject in need of such treatment. The method includes administering a compound that inhibits the GPR174-mediated signaling pathway to a subject or into cells obtained from the subject and reintroduced to the subject, wherein inhibition of the GPR174-mediated signaling pathway results in the treatment or prevention of, or reduction in the severity of, a disease, disorder or condition. Examples of indications that can be treated or prevented using the methods herein are discussed below.

D. GPR174-interacting compounds.

We have identified the following compounds as functionally interacting with the GPR174 receptor using a modified form of the CRA assay described above. The structures of identified representative compounds are shown in Table 1 below. 1.

- 43 046439

Table 1. Representative compounds

No. compounds/group of formulas Structure CRA ec ₅₀ (mkM) Human GPR174 CRE activity, EC ₅₀ ; Fold reduction in Gs signaling activity 1 (group I) f\<' ? \ ₂ -η \ / .—Ζ _ζ / 7 gР ζ-—· O ( o < 2,1,2,1, 1.5, 0.9 ΙΑ; reduction factor 0.4, 0.8; 2.9 2 (group I) ) about......and O \ Z Z— \/' ts-z < S 3.3, 3.0, 5.1,0.4 ΙΑ; reduction ratio 0.4, 0.3; 1.7 3 (group I) 0 n. JV -γ·· -> .- N I ¹ JN O 4,1.2,0, >10 Antagonist or allosteric modulator 4 (group I) 0 I ,,. °V ^N -''b 0.2, 2.0, Antagonist 5 (group I) G z z / \ o V. / Z— ABOUT ( >1.2 ΙΑ; 0.9, reduction factor 2.4 6 (group I) F, ,NO, ,, 1; ,, c: ' ^N '; rj ,, ,, , 0 1.4, >0.4 ΙΑ; 0.4, 0.3; reduction factor 3.8

- 44 046439

7 (group I) F. ,.,.NO ₂ .. 11 1 . γ Ί k...--·' ^N. 0 >0.6, >0.7 IA; 0.3, 0.5; reduction ratio 2.4 8 (group I) ^{F NO} C ''NN - ..J I. ,N. - .- - - ABOUT >0.7, >1.6 ΙΑ; 0.7, 0.4; reduction factor 3.3 9 (group I) ^F '·-.. ...·ΝΟ ₂ L. ,-- -. N.--. -...- -.....· _¥ ·... Oh >2 ΙΑ; 0.7, 0.5; reduction factor 2.1 10 (group I) F... ...-,.. ,.NO ₂ ... 11 .. -· ' ^N ' ^N _l ..---. ...- -- ,N ⁰ il I 0.6, 0.5 ΙΑ, 0.2, 0.5; reduction factor 5.1 11 (group I) F... .NO·; ,, 11 .: _r - Ν'Ν' ·- 1.....,.- -/-/., 0· Il 'Ί 1.5, >1.1 ΙΑ, 0.5,1.3, 0.6; reduction factor 3.1 12 (group I) F-. ,·... ,NO ₂ Ϊ TN^''/O^··· _N ''\ 0 0.3 ΙΑ, 0.4, 0.7, reduction factor 3.9

- 45 046439

13 (group I) F.. ,N0 ₂ . ii: . 'Ν'N' А N 0 >2 IA, 0.8, 1.2; reduction factor 2.2 14 (group I) FNCl N •'•'cA·····'·· _N o' ND ΙΑ, 1.1; reduction factor 3 15 (group I) '- r τί V, .·'/ ,. zz ..-\o\y rr. o, ' ω. . / '° / \ ^CH X A ND ΙΑ, 1.7, reduction factor 2.6 16 (group I) F.. .NO, , T I . r' 'N N l L. -,. . N. 0 ND ΙΑ, 0.8, reduction factor 2.1 17 (group I) F.., ,.-... „.NO, , ϊ :i : -FT '-V- γ·- I·. ,.1. -. .N .-...... -.. .-..,. _¥ . 0 ND ΙΑ, 3.0, reduction factor 2.0 18 (group I) Q ) ABOUT .—z Ο ·\/ z z— ’ \___/ 111 z < ND ΙΑ, 1.0; reduction ratio 1.9

- 46 046439

53 (Group I) \ / / \ o \ .= p: o.. , ^ζ ' . /° ND ΙΑ, 1.8; reduction ratio 3.4 19 (group II) X ² -O /A ..from 7 O ζ Ζ ) ^y/ ----// --·\----/7 V--Z \ / \ 7 T , - ь < bh // 1.0, 2.0, 1.6 Antagonist or allosteric modulator 20 (group II) 1 ·' s ·<.. ,-.. ,,Ν .... _t HN 0.5. 1.0, 5.0 Antagonist or allosteric modulator 21 (group III) O _o Ch / -L- w γ- 'Ν'y'· Η 1 Η V. JZ 2.7, 0.5, >0.5 Antagonist or allosteric modulator

- 47 046439

22 (group IV) /X Н γν ⁰ о Г 1'· ,...0 6.7, 5.3 IA, 1.0, 1.5; reduction factor 10.2 23 (group IV) ( >=o \/ / < / \ IZ )-- A ! O >10, 2.9 ΙΑ, 0.4, 0.9, 1.2, 1.0; reduction factor 14.1 24 (group IV) U -λ N h X- ..J .l, Ίι: .1 / ΥΙ but^Х··'·'' ND ΙΑ, 4.4; reduction ratio 2.8 25 (group IV) . _n ΓΎΎ ° ...X-.. ° ND ΙΑ, 4.6, reduction factor 4.2 26 (group IV) y - λ Η (·. V .Ν. -Ο _: J Τ ΊΓ ⁰ X-.... ο ⁰ V ΟΗ ND ΙΑ, 3.6, reduction factor 1.7 27 (group IV) /==λ Η at Α..^. Ν ND ΙΑ, 0.8, 0.5, reduction factor 6.2 28 (group IV) λ Η y V ,N . .. Μ Ί ϊ 1 ° χι ''Ό' X-···'· ND ΙΑ, 3.6, reduction factor 1.9 29 (group IV) ί =ο \/ ΧΖ ')--4 —( Η 1 π ί ND ΙΑ, 1.2, reduction factor 5.3

- 48 046439

30 (group IV) /—λ N h V ,Ν, -ri:) / XI •'Χγ .,.0 ND IA, 3.8, reduction factor 2.2 31 (group IV) /<·· A G ί °vV ( b—7 b—\ > ND ΙΑ, 1.2, reduction factor 8.9 32 (group IV) Οχ ' χ ' b 4—// /.—/ _ZT o=^f \ ND ΙΑ, 2.5, reduction factor 3.9

- 49 046439

33 (group IV) ==- N V.XN ΜΊ 7 '1 / I 0 'Y' ,..ABOUT No' ND ΙΑ, 4.4, reduction factor 9.2 34 (group IV) — \n '· iJ Ο'^ζ;: ·Ν W ND ΙΑ, 2.8, reduction factor 8.4 35 (group IV) r = - _n \\ ,κ ...-n F ND ΙΑ, 4.3, reduction factor 2.8 36 (group IV) /=Η δ ,1. 0 _Γ ·>· °'V •••....° ND ΙΑ, 2.6, reduction factor 8.3

- 50 046439

37 (group IV) N'. ^N. T Y ° J-·,. o N ND ΙΑ, 5.4, reduction factor 2.8 38 (group IV) /N Q λ. .N. ί : I y 1 g ΎNO, ND ΙΑ, 2.7, reduction factor 8.6 39 (group IV) —·. N 41.....1. J T ND ΙΑ, 3, reduction factor 7.8 40 (group IV) s... _;g ' , _g . O .-. 14 11 \\ / ............ω-κ/' ζτ О ^Х >< о^К ) ND ΙΑ, 3.3, reduction factor 8.8

- 51 046439

41 (group IV) L\. ,th......... -gi: ί / t t s .0 ND ΙΑ, 1.0; reduction ratio 13.7 42 (group IV) o _h ' J ζ Х—( ζτ О=Х ) ND ΙΑ, 6.6, reduction factor 4.3 43 (group IV) 1 Ιί J,, and cVv |· ’ — ζτ o o=G ) ND ΙΑ, 5.2, reduction factor 5.1 44 (group IV) GD. .Y. ...... ΜΊ Г j о Т ΊΓ WITH; 0 ND ΙΑ, 1.3, reduction factor 4.7

- 52 046439

45 (group IV) ......and '-,J·. 4 I J ς; O ! .l. ND ΙΑ, 1.0; reduction ratio 7.6 46 (group IV) m j : j / X X That F^F ND ΙΑ, 1.2, reduction factor 8.1 47 (group IV) / = = 1. N X X. X m 1 : j / eleven ° ,.H. O Article 1 WITH ND ΙΑ, 1.0, reduction factor 7.1

- 53 046439

48 (group IV) k k .N md.....:d O.1. 0 d Oh sr. ND ΙΑ, 1.2, reduction factor 5 49 (group IV) / - k n 4 A...N, Ϊ " ; O Ύ ...........ABOUT ND ΙΑ, 0.6; reduction factor 14.3 50 (group IV) =\n o ϊ Υ .0 ND ΙΑ, 1.2, reduction factor 10.1 51 (group IV) —' n \\ .J. .,uu.. _O ^VY ⁰ Ju., o ND ΙΑ, 1.6, reduction factor 1.7

- 54 046439

52 (group IV) N V. N. M'D 7 Ί E I Ϊ 1 Ϊ ABOUT ND IA, 0.5, reduction factor 3.1 54 (group IV) n u. ND ΙΑ, 2.0, reduction factor 7 55 (group IV) n at a.-···'-'··... »' 1U L. .Ν g ND ΙΑ, 0.6, reduction factor 7 56 (Group V) ex J.L. c7 g g~ -kg -Ν n n 0.5 ΙΑ, 0.5, reduction factor 3 57 (Group VI) ' n ,Ν ......... ,.C1 ~ _i Ν ... ....... ⁰ 5.6 Antagonist or allosteric modulator 58 (Group VI) ° _V :A. . N. ; ,Вг Г П ^N || | ' ⁰ no-^M 3.7 Antagonist or allosteric modulator 59 (Group Va) 1. S .0 ^{n n} ND ΝΜ

*For EC50 values, it should be noted that for all compounds included in the table. 1 shows at least a minimum level of activity greater than 3 times the average background of the assay when tested at 40 µM. When present, many EC50 values correspond to those obtained in individual experiments. IA stands for inverse agonist. NM stands for non-modulator. Compound 4 is labeled as an antagonist because it was found to compete with the GPR174 agonist LysoPS (see Example 4).

GPR174-interacting compounds described herein (e.g., a compound according to any of formulas I, II, III, IV, V or VI (or (I), (II), (III), (IV), (V ), (Va) or (VI)) or any of the illustrative compounds 1-59 of Table 1) can be prepared in accordance with methods known in the art using conventional starting materials. Illustrative syntheses are presented herein.

The compounds of formula (I) can be prepared according to methods known in the art. As shown in Scheme 1A, piperazine derivative A and electrophile B can undergo a cross-coupling reaction or a nucleophilic aromatic substitution reaction (eg, when one or both of X ³ and X ⁷ is N). Cross-coupling reactions may be C-C cross-coupling reactions (eg, Suzuki coupling, Hiyama coupling, Still coupling, Negishi coupling, Tamao-Kumada coupling, or Murahashi coupling), cross-coupling reactions

- 55 046439 C-N combinations (for example, Buchwald-Hartwig or Ullmann combinations), C-O cross-coupling reactions, etc. Typical cross-coupling reactions involve the reaction between an electrophile (eg, compound B) and a nucleophile (eg, compound A) in the presence of catalytic amounts of a metal salt, such as a palladium, copper, iron or nickel salt (eg, PdCl2, Pd(OAc)2 ,

CuBr, CuI, complex (CuOTf) ₂ -toluene, Fe(OTf) ₃ , FeCl ₃ , FeBr ₃ , NiCl2 or NiBr2). Optional ligands, e.g. phosphine (e.g. PPh ₃ , P(2-furyl) ₃ , P(1:-Bu) ₃ , dppf, dppb or BINAP), N-heterocyclic carbene (e.g. SIMes or SIPr) or dipyridine (e.g. 2,2'-bipyridyl or 1,10-phenanthroline) may be added to stimulate the response. Alternatively, an organometallic complex such as Pd(PPh ₃ ) ₄ or (dppf)PdCl ₂ can be used directly in the presence or absence of additional ligands. Additives such as tetrabutylammonium fluoride, LiCl, KOAc or AgOTf can be added to minimize dehalogenation or to facilitate cross-coupling reactions. One skilled in the art will be able to determine a suitable solvent for the reaction through conventional screening. Non-limiting examples of solvents used in cross-coupling reactions include water, ethanol, acetone, tetrahydrofuran, toluene, 1,4-dioxane, and mixtures thereof. For non-limiting examples of conditions and catalysts that can be used in chemical cross-coupling reactions, see Miyaura et al., CrossCoupling Reactions: A Practical Guide to Topics in Current Chemistry, Springer, 2002; Nicolaou et al., Angew. Chem. Int. Ed., 44:4442-4489, 2005; Maiti et al., Chem. Set, 2:57-68, 2011, the full contents of which are provided herein by reference. A suitable base may also be necessary for catalyst turnover, for example in a Suzuki cross-coupling or a Buchwald-Hartwig cross-coupling. Typical bases include K3PO ₄ , Na ₂ CO3 , CS2CO3 , trialkylamine (eg, Hunig's base or triethylamine), and pyridine or substituted pyridine (eg, lutidine or collidine). Nucleophilic aromatic substitution reaction conditions are well known in the art and typically involve the use of F or Cl as the leaving group (LG). A non-limiting example of nucleophilic aromatic substitution reaction conditions is presented in Petit and Vo-Thanh, ^15th International Electronic Conference on Synthetic Organic Chemistry, Section C004, 2011, the entire contents of which are incorporated herein by reference. The product of this transformation can be deprotected (PGN removed) to produce C, which can be further functionalized to include R ¹ that is not H, through reductive amination, amidation, cross-coupling, acylation, or sulfonation. Typical amidation conditions include the use of reagents such as EDC/DMAP, HATU/HOAt or HBTU/HOAt. One skilled in the art will know the conditions for the reductive amination state and suitable hydride sources (eg NaBH(OAc)3 or NaBH3CN). Exemplary synthetic methods for preparing compounds of formula (I) can be found in Sythana S. et al., Organic Process Research and Development, (2014), 18, 912-918 and Garino et al., J. Med. Chem. (2006), 49, 4275-4285, the contents of which are incorporated herein by reference. In addition, the compounds of formula (I) can be further modified to produce other compounds of formula (I) shown in Scheme 1B.

Scheme 1A

1) nucleophilic aromatic, χ ⁴ reductive _L substitution „d·- amination .. _; 7h, ^or T ^{1 RJR} r3 or X ^x x _r5r 4 cross D D. D, amidation ।/ r3

2) deprotection ^NH cross-I (removal of PG ^N ) ^R t?8 in9 combination ^to or ^{to R8 R9} acylation

C or compound of formula (I) sulfonation

LG = halide or pseudohalide (e.g. phosphate, OTf, ONf, OTs or OMs) PG ⁿ = /V-protecting group

- 56 046439

Scheme 1B no ₂ ¹ Вг

NO

HCI, MeOH

NO. I ' NH HCI 4 J

F

C.I.

Toluene K _? CO

100 degrees ,°s ¹ o

NO..

R no ₂ X .,,Ν ^S o

TEA, CH ₂ C1 ₂

F

C.I.

DMSO, KCO, 95 degrees compound of formula (I)

Compounds of formula (II) can be prepared into compounds of formula (I) according to methods known in the art. As shown in Scheme 2, cyclic anhydride A can be alkylated with B or C to produce compound D. The reaction with B can be carried out under cross-coupling conditions as described herein (for example, when [M] is Cu, Sn , Si or B). A non-limiting example of cross-coupling reaction conditions suitable for this transformation is presented in Xin et al., Synthesis, 1970-1978, 2007, the entire contents of which are incorporated herein by reference. When [M] of B is Li, Mg or Zn (eg diaryl-Zn or diheteroaryl-Zn), the reaction between A and B can occur without a catalyst. One skilled in the art will be able to select appropriate conditions based on the presence or absence of specific substituents (e.g., the presence of carbonyl groups that are not part of the group undergoing the reaction may preclude the use of, for example, Li, Mg and diaryl- or diheteroaryl-Zn reagents) . Alternatively, when D is prepared from cyclic anhydride A and compound C, appropriate conditions may be Friedel Crafts reaction conditions (eg, in the presence of catalytic or stoichiometric amounts of oxophilic Lewis acid (eg, AlCl ₃ or BF 3 )). Compound D, after esterification and condensation with hydrazine (eg, hydrazine hydrate), can form compound E. Esterification reaction conditions may include Steglich esterification (eg, EDC/DMAP) or treatment of compound D with isobutyl chloroformate and N-methylmorpholine to produce a mixed anhydride intermediate, which then react with a nucleophile (eg alcohol). Alternatively, the esterification reaction may involve the use of a weak base (eg K ₂ CO ₃ ) and an electrophile (eg Me ₂ SO ₄ ) in a polar aprotic solvent (eg acetone). A non-limiting example of reaction conditions for the formation of phthalazine from an o-acylbenzoic acid ester is presented in Li et al., Molecules, 11:574-582, 2006, the entire contents of which are incorporated herein by reference. Compound E can be subjected to a halogenation reaction (eg using PCl ₅ , POCl ₃ , PCl ₃ or a mixture of two or three reactants) and subsequent nucleophilic aromatic substitution or cross-coupling to introduce R ¹ , thereby obtaining a compound of formula (II). The conditions for cross-coupling reactions and nucleophilic aromatic substitution reactions are as described herein.

- 57 046439

Scheme 2 uncatalyzed organometallic addition or cross-coupling or Friedel-Crafts reaction

О x ¹⁰ ~х” _χ5 ί ^ν _χ 8

2) hydrazine

1) esterification _Χ 1Ο _Χ 11 x ⁸

BUT

N-N

X ⁴ ^ ⁵

Ch X ^e x ³ - x ⁷ or

XW x ³ - x ⁷ C _χ 10χ11

X ^th X ^s

R ¹ - ·;

IM-N

X ⁴ = X ⁵

X ^e

X ³ X ⁷

E

1) halogenation

2) cross-coupling or nucleophilic aromatic substitution of a compound of formula (II)

[M] = Li, Mg, Zn, Cu, Sn, Si or B (depending on the substitution pattern of the reactants) L _n = auxiliary ligands

The compounds of formula (III) can be prepared according to methods known in the art. General methods for preparing compounds of formula (III) are described in Endo et al., Journal of Organic Chemistry, 77(17): 7223-7231 2012; the entire contents of which are incorporated herein by reference. A non-limiting example of a method for synthesizing a compound of formula (III) is shown in Scheme 3, Method 1.

As shown in Scheme 3, a compound of formula (III) (eg, compound D) can be prepared from simple starting materials such as aryl halides, preferably bromides, A and B, through sequential Suzuki-Miyaura cross-coupling in one reaction vessel using diborylmethane C. Compound D can be further modified, for example, by removing optional protecting groups or modifying substituents on the aryl rings. Alternatively, as shown in Scheme 3, Method 2, Compound E can be used as a starting material as described in Wang et al., Organic & Biomolecular Chemistry, (2015), 13(17), 4925-4930;

the entire contents of which are incorporated herein by reference. Scheme 3

The compounds of formula (IV) can be prepared according to methods known in the art. For example, as shown in Scheme 4, compound A and compound B can be reacted with aryl aldehyde C and amine D under suitable conditions to obtain compound E (compound of formula (IV)). Non-limiting examples of reaction conditions that can be used in the synthesis of compound E are presented in Tu et al., Organic & Biomolecular Chemistry, 4(21): 3980-3985; 2006; Kosal et al., Angew. Chem. Int. Ed., 51, 12036-12040, 2012; and Heravi et al., Synthetic Communications, 40(15): 2191-2200; 2010; Chen et al., Journal of Heterocyclic Chemistry, (2007), 44(5), 1201-1205; Abdolmohammadi, Chinese Chemical Letters, 24(4), 318-320; 2013; Heravi et al., Synthetic Communications (2010), 40(15), 2191-2200; Quang et al., Bulletin of the Korean Chemical Society, (2012), 33(4), 1170-1176; Laengle et al., European J.

- 58 046439

Med. Chem, (2015), 95, 249-266, and Shirini et al., Dyes and Pigments (2013), 97, 19-25, the entire contents of which are incorporated herein by reference.

Scheme 4

A B C

In the reactions described above, it may be necessary to protect reactive functional groups (eg hydroxy, amino, thio or carboxy groups) to avoid their unwanted participation in the reactions. The inclusion of such groups and the methods necessary for their introduction and removal are known to one skilled in the art (eg, Greene, supra). The deprotection step may be the final step in the synthesis such that removal of the protecting groups results in compounds of formula (I), (II), (III), (IV), (V), (Va) or (VI) as described in the present description. The starting materials used in any of the above schemes can be purchased or prepared by methods described in the chemical literature, or by adaptation thereof, using methods known to one skilled in the art. The order in which the steps are carried out may vary depending on the groups introduced and the reagents used, but will be apparent to one skilled in the art.

Compounds of any of formulas (I), (II), (III), (IV), (V), (Va) or (VI), or any of the intermediates described in the schemes above, can be further derivatized using one or several standard synthesis methods known to one skilled in the art. Such methods may involve displacement, oxidation or reduction reactions. These methods can also be used to prepare or modify compounds of formulas (I), (II), (III), (IV), (V), (Va) or (VI), or any preceding intermediates by modification, introduction or removal suitable functional groups. Specific substitution methods include alkylation, arylation, heteroarylation, acylation, thioacylation, halogenation, sulfonylation, nitration, formylation, hydrolysis, and coupling methods. These methods can be used to introduce a functional group into the parent molecule (for example, nitration or sulfonylation of an aromatic ring) or to link two molecules together (for example, to couple an amine with a carboxylic acid to form an amide; or to form a carbon-carbon bond between two heterocycles) . For example, alcohol or phenol groups can be converted to ester groups by coupling the phenol with the alcohol in a solvent, such as tetrahydrofuran, in the presence of a phosphine (eg, triphenylphosphine) and a dehydrating agent (eg, diethyl, diisopropyl, or dimethyl azodicarboxylate). Alternatively, ester groups can be prepared by deprotonation of the alcohol using a suitable base (eg sodium hydride) followed by the addition of an alkylating agent (eg an alkyl halide or alkyl sulfonate).

In another example, a primary or secondary amine can be alkylated using a reductive alkylation method. For example, the amine can be treated with an aldehyde and a borohydride (eg, sodium triacetoxyborohydride or sodium cyanoborohydride) in a solvent (eg, a halogenated hydrocarbon, eg, dichloromethane, or an alcohol, eg, ethanol) and, where necessary, in the presence of an acid (eg, acetic acid).

In another example, -OH groups can be obtained from the corresponding ester, acid, acid chloride or aldehyde by reduction using a suitable reducing agent, for example, a complex metal hydride, for example lithium aluminum hydride, in a solvent (for example, tetrahydrofuran).

In another example, hydroxy groups (including phenolic OH groups) can be converted to leaving groups, e.g., halogen atoms or sulfonyloxy groups (e.g., alkylsulfonyloxy, e.g., trifluoromethylsulfonyloxy, or arylsulfonyl, e.g., p-toluenesulfonyloxy) using conditions known to one skilled in the art. areas. For example, an aliphatic alcohol can be reacted with thionyl chloride in a halogenated hydrocarbon (eg, dichloromethane) to produce the corresponding alkyl chloride. A base (eg triethylamine) can also be used in the reaction.

In another example, an ester can be converted to the corresponding carboxylic acid via acid- or base-catalyzed hydrolysis, depending on the nature of the ester group. Acid-catalyzed hydrolysis can be achieved by treatment with an organic or inorganic acid (eg trifluoroacetic acid in an aqueous solvent, or an inorganic acid such as hydrochloric acid in a solvent such as dioxane). Base-catalyzed hydrolysis can be achieved by treatment with an alkali metal hydroxide (eg lithium hydroxide in an aqueous alcohol, eg methanol).

In another example, aromatic halogen substituents in compounds can be subjected to

- 59 046439 halogen-metal exchange by treatment with a base (eg lithium base, eg nbutyl- or t-butyllithium) optionally, at low temperature (eg -78°C) in a solvent (eg tetrahydrofuran), and reaction in the mixture can then be terminated using an electrophile to introduce the desired substituent. Thus, for example, a formyl group can be introduced using dimethylformamide as an electrophile. Aromatic halogen substituents can also be subjected to palladium catalyzed reactions to introduce groups such as carboxylic acids, esters, cyano or amino substituents.

In another example, aromatic halogen substituents on compounds can participate in a number of metal-catalyzed reactions to introduce alternative functional groups, for example, amines, amides, ethers, thiols, aryl groups or heteroaryl groups.

Specific oxidation methods include dehydrogenation and aromatization, and the addition of oxygen to specific functional groups. For example, aldehyde groups can be produced by oxidation of the corresponding alcohol using conditions well known to one skilled in the art. For example, the alcohol can be treated with an oxidizing agent (eg, Dess-Martin reagent) in a solvent (eg, a halogenated hydrocarbon, eg, dichloromethane). Alternative oxidation conditions can be used, for example treatment with oxalyl chloride and an activating amount of dimethyl sulfoxide and then stopping the reaction by adding an amine (eg triethylamine). Such a reaction can be carried out in a suitable solvent (eg, a halogenated hydrocarbon, eg, dichloromethane) and under suitable conditions (eg, cooling below room temperature, eg -78°C, followed by heating to room temperature). In another example, sulfur atoms can be oxidized to the corresponding sulfoxide or sulfone using an oxidizing agent (eg, a peroxyacid, eg, 3-chloroperoxybenzoic acid) in an inert solvent (eg, a halogenated hydrocarbon, eg, dichloromethane) at approximately ambient temperature.

Specific reduction methods include removal of oxygen atoms from specific functional groups, saturation (or partial saturation) of unsaturated compounds, including aromatic rings. For example, primary alcohols can be prepared from the corresponding ester or aldehyde by a reaction using a metal hydride (eg lithium aluminum hydride or sodium borohydride in a solvent such as methanol). Alternatively, -OH groups can be obtained from the corresponding carboxylic acid by reduction using a metal hydride (eg lithium aluminum hydride in a solvent such as tetrahydrofuran). In another example, a nitro group can be reduced to an amine by catalytic hydrogenation in the presence of a metal catalyst (eg, palladium on a solid support, such as carbon) in a solvent (eg, an ether, such as tetrahydrofuran, or an alcohol, such as methanol), or by chemical reduction using a metal (eg tin or iron) in the presence of an acid (eg hydrochloric acid). In the following example, the amine can be prepared by reduction of a nitrile, for example, by catalytic hydrogenation in the presence of a metal catalyst (eg, palladium on a solid support, such as carbon) or Raney nickel in a solvent (eg, tetrahydrofuran) and other suitable conditions (eg, when cooling below room temperature, for example to -78°C, or heating, for example, for reflux).

GPR174-interacting reference compounds.

As described herein, in various embodiments, the present invention provides reference compounds clearly predefined as functionally interacting with GPR174 containing a structure in accordance with formula I, II, III, IV, V or VI (for example, such as compounds 1-59 in Table 1 or formula (I), (II), (III), (IV), (V), (Va) or (VI)), and optionally further containing a detectable group also designated in herein as reference compounds comprising a detectable group. GPR174-reactive reference compounds comprising a detectable moiety, such as, for example, a radioactive isotope, a fluorescent label, a bioluminescent label, a chemiluminescent label, a photoaffinity label, and the like, can be prepared according to methods known in the art using generally accepted starting materials.

For example, in one embodiment, the GPR174-reactive reference compound is labeled using, or synthesized under conditions suitable for incorporating a molecular label(s) including a radioactive isotope such as ¹²⁵ 1, ¹⁴ C, ³ H, ¹¹ C, ¹⁸ F , ^99m Tc or other suitable radiometric tag, using methods known in the art. See, for example, Seevers RH and Counsell, RE, Radioiodination Techniques for Small Organic Molecules, Chem Rev 82:575590, 1982; Voges R. et al., Preparation of Compounds Labeled with Tritium and Carbon-14, John Wiley & Sons, 2009; Ametamey, S.M., et al., Molecular Imaging with PET, Chem Rev 108:1501-1516, 2008; and Cheng Y. et al., J Med Chem, 55:2279-2286, 2012, each of which is incorporated herein by reference.

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In another embodiment, the GPR174-interacting reference compound is labeled using, or synthesized under conditions suitable for incorporating a photoaffinity tag, using methods known in the art. See, for example, Spletstoser J.T. et al., J. Med. Chem. 47:6459-6465, 2004, the contents of which are incorporated herein by reference.

In another embodiment, the GPR174-interacting reference compound is labeled using, or synthesized under conditions suitable for incorporating a fluorescent label, such as 6-carboxyfluorescein (FAM), Alexa Fluor® (Molecular Probes, Inc), fluorescein isothiocyanate (FITC), and the like, using methods known in the art. See, for example, Goncalves, M.S., Chem Rev 109:190-212, 2009, the contents of which are incorporated herein by reference.

In another embodiment, the GPR174-interacting reference compound is labeled using a bioluminescent or chemiluminescent label, such as isoluminol or acridinium ester, or an enzyme label, such as horseradish peroxidase (HRP) or alkaline phosphatase (AP), to further enhance readout. using enhanced chemiluminescence (ECL) substrates using methods known in the art. See, for example, Barton V. et al., J Med Chem 53:4555-4559, 2010, the contents of which are incorporated herein by reference.

E. GPR174 signaling pathway.

As described herein, the present inventors have discovered that GPR174 modulates the Gs signaling pathway. The assignment of GPR174 to the Gs signaling pathway followed the determination of the initial activity of GPR174 in a transcriptional reporter panel assay, followed by confirmation of this assignment in the presence and absence of small molecule surrogate ligands for GPR174, which were identified in a separate screening assay for their ability to functionally interact with associated membrane GPR174 in CRA. As described in detail herein, CRA is based on the observation that NLS replacement of a partial sequence in the intracellular portion of a GPCR effectively removes the modified and unbound receptor from the cell surface and that this effect is reversible through the presence of a receptor-specific ligand.

Briefly, to determine the baseline activity of GPR174 in the Gs signaling pathway, GPR174 was overexpressed in cultured mammalian tissue cells that also included an expression cassette with a luciferase reporter protein under the transcriptional control of one of five promoters. The promoters were cAMP response element (CRE), NFAT, AP1, serum response element (SRE), and serum response factor (SRF). The Gs signaling pathway induces transcription from the CRE promoter. The Gq signaling pathway induces transcription from the promoters of NFAT, AP1, SRE, CRE and SRF. The Gi signaling pathway can induce transcription from CRE in the presence of Gs chimeric G proteins or induce transcription of the NFAT AP1, SRE, CRE and SRF promoters using G15, G16 or Gq chimeric G proteins. The G12/13 signaling pathway induces transcription from the SRE and SRF promoters. One skilled in the art will appreciate that other reporters may be used in the assay, such as β-galactosidase or other detectable or analyzable proteins.

As shown in the examples below, GPR174 has been determined to modulate the Gs signaling pathway.

Activity of compounds interacting with GPR174.

In one embodiment, a GPR174 inhibitor that can be used in the methods of the present invention is a compound that inhibits a GPR174-mediated signaling pathway (e.g., Gs pathway) by at least 0.5-fold (e.g., at least 1.5-fold). 5 times, at least 2 times, at least 5 times, at least 10 times, at least 50 times, or at least 100 times) compared to the initial activity of the receptor itself at a compound concentration of less less than 40 µM (eg, less than 25.10, 5.1, 0.5, 0.25, 0.1 or less than 0.5 µM).

In some embodiments, the GPR174 modulator is an inhibitor of the GPR174-mediated signaling pathway and reduces the activity of at least one GPR174-mediated signaling pathway (e.g., the Gs pathway) by at least 10% (e.g., at least 0.5-fold , at least 2-fold, at least 5-fold, at least 10-fold, at least 50-fold, or at least 100-fold) compared to the initial activity of the receptor itself at a compound concentration of less than 40 µM (eg, less than 25.10, 5.1, 0.5, 0.25, 0.1 or less than 0.5 µM).

In some embodiments, the GPR174 modulator is an inhibitor of the GPR174-mediated signaling pathway and reduces the activity of at least one GPR174-mediated signaling pathway (e.g., the Gs pathway) in the presence of an agonist by at least 20% (e.g., at least 0 .5-fold, at least 2-fold, at least 5-fold, at least 10-fold, at least 50-fold, or at least 100-fold) at an inhibitor concentration of less than 40 μM (e.g., less than 25, 10, 5, 1, 0.5, 0.25, 0.1, or less than 0.5 μM) compared to receptor activity without agonist and without inhibitor.

- 61 046439

In some embodiments, the GPR174 modulator is an inhibitor of the GPR174-mediated signaling pathway and reduces the activity of at least one GPR174-mediated signaling pathway (e.g., the Gs pathway) in the presence of an activator by at least 20% (e.g., at least 0. 5-fold, at least 2-fold, at least 5-fold, at least 10-fold, at least 50-fold, or at least 100-fold) at an inhibitor concentration of less than 40 μM (e.g., less than 25.10, 5.1, 0.5, 0.25, 0.1, or less than 0.5 μM), compared with receptor activity with an activator and without an inhibitor.

An exemplary assay useful for determining GPR174 modulator (activator or inhibitor) activity relative to baseline GPR174 activity is presented in Examples 3 and 4 herein, which describe the use of reporter assays to measure the activity of a GPR174-mediated signaling pathway (e.g., pathway activity Gs) in the absence (to determine baseline activity) or in the presence of a GPR174 modulating compound.

F. Screening methods to identify additional compounds.

With compounds now identified that inhibit GPR174 and the signaling pathways modulated by this receptor as described herein, additional methods may be used to identify other GPR174 modulators.

In the case of orphan GPCRs where the natural ligand remains unknown, labeled surrogate ligands can be used as reference compounds as a means of measuring binding to natural receptors and/or engineered receptor constructs so that competition (i.e. competitive binding) or non-competitive binding can be determined binding to other receptor modulating agents. Surrogate ligands (eg, compounds 1-59 presented in Table 1) can bind to the natural binding site (orthosteric site) or can bind to the allosteric binding site(s) on the GPR174 receptor.

Examples of such screening methods to identify agents that modulate GPR174 signaling activity are described below.

As used herein, the term modulatory agent candidate, in the context of screening methods for identifying agents that modulate GPR174 activity, refers to any modulatory agent, including compounds as defined herein (i.e., including molecules, either natural or synthetic, for example, a protein; a peptide (eg, about 5 to about 25 amino acids long, eg about 10 to 20 amino acids long, or 12 to 18 amino acids long, eg 12, 15 or 18 amino acids long); or a fragment or mimetic thereof; an aptamer, a small molecular weight chemical compound, such as a small organic, organometallic or inorganic molecule; an oligonucleotide; a fatty acid compound may be included in a compound library such as a combinatorial, synthetic, natural, drug-like compound. compounds, an organic, inorganic, non-randomized or randomized library that provides a sufficient range of diversity, or it may be a focused or directed collection of the above compounds). Candidate modulatory agents also include natural extracts, nucleotides, nucleic acid molecules (including a library of nucleic acid molecules, such as a library of cDNA encoding the modulating agent), nucleotide analogues, nucleosides and nucleoside analogues, and gases (for example, such as hydrogen sulfide (H2S ), or nitric oxide (NO)).

As used herein, the term natural extract means any extract obtained from a natural source, such as a eukaryotic cell (e.g., a mammalian cell (including a mammalian cell line) or mammalian tissue), bacteria, animal, plant, fruit, tree, and the like.

In accordance with the above, in one aspect, the present invention relates to a method for identifying a modulatory agent capable of modulating a GPR174-mediated signaling pathway (eg, Gs pathway) comprising: (a) bringing a cell expressing GPR174 into contact with (i) at least at least one candidate modulatory agent and (ii) a reference compound comprising a detectable moiety wherein the reference compound (i.e., a surrogate ligand) is known to modulate the activity of the GPR174-mediated signaling pathway, and (b) determining whether a candidate compound modulates (i.e., increases or decreases) the binding affinity of the reference compound to GPR174. In some embodiments, a candidate modulatory compound that reduces the binding affinity (ie, inhibits binding) of the reference compound to GPR174 is identified as a modulatory agent capable of modulating the GPR174-mediated signaling pathway. In some embodiments, a candidate compound that competitively reduces the binding affinity (i.e., competitively inhibits the binding) of a reference compound to GPR174 is identified as a compound that binds to the same region of GPR174 as the reference compound and is capable of modulating mediated GPR174 signaling pathway. In some embodiments, a candidate modulatory compound that increases the binding affinity (i.e., enhances binding) of the reference

- 62 046439 compounds with GPR174 are identified as a modulating agent acting as an allosteric modulating agent capable of modulating the GPR174-mediated signaling pathway. In some embodiments, the candidate modulatory agent reduces the binding affinity of the reference compound to GPR174 by at least 2-fold. In some embodiments, the candidate modulatory agent increases the binding affinity of the reference compound to GPR174 by at least 2-fold. In some embodiments, the method further includes performing a signaling assay to measure the effect of the identified compound on a GPR174-mediated signaling pathway, optionally in the presence of a reference compound known to modulate at least one GPR174-mediated signaling pathway.

In accordance with the above, in one aspect, the present invention relates to a method for identifying a modulatory agent capable of modulating a GPR174-mediated signaling pathway (eg, Gs pathway) comprising: (a) bringing a cell expressing GPR174 into contact with (i) at least at least one candidate modulatory agent and (ii) a reference compound comprising a detectable moiety wherein the reference compound (i.e., a surrogate ligand) is known to modulate the activity of the GPR174-mediated signaling pathway, and (b) determining whether whether the candidate compound modulates (i.e., increases or decreases) the apparent binding affinity of the reference compound to GPR174. In some embodiments, a candidate modulatory compound that reduces the apparent binding affinity of the reference compound for GPR174 is identified as a modulatory agent capable of modulating the GPR174-mediated signaling pathway. In some embodiments, a candidate compound that competitively reduces the apparent binding affinity of a reference compound for GPR174 is identified as a compound that binds to the same region of GPR174 as the reference compound and is capable of modulating the GPR174-mediated signaling pathway. In some embodiments, a candidate modulatory compound that reduces the binding affinity of a reference compound for GPR174 is identified as a modulator that acts as a negative allosteric modulator that binds an allosteric site on GPR174. In some embodiments, a candidate modulatory compound that increases the apparent binding affinity of a reference compound for GPR174 is identified as a modulatory agent that acts as a positive allosteric modulatory agent capable of modulating the GPR174-mediated signaling pathway. In some embodiments, the candidate modulatory agent reduces the apparent binding affinity of the reference compound for GPR174 by at least 2-fold. In some embodiments, the candidate modulatory agent increases the apparent binding affinity of the reference compound for GPR174 by at least 2-fold. In some embodiments, the method further includes performing a signaling assay to measure the effect of the identified compound on a GPR174-mediated signaling pathway, optionally in the presence of a reference compound known to modulate at least one GPR174-mediated signaling pathway. In some embodiments, a candidate modulatory compound that reduces the apparent activity of GPR174-mediated signaling compared to a reference GPR174 compound is identified as a modulatory agent capable of modulating the GPR174-mediated signaling pathway. In some embodiments, the candidate modulatory agent reduces the apparent activity of GPR174-mediated signaling with the reference compound by at least 10%. In some embodiments, a candidate modulatory compound that increases the apparent activity of GPR174-mediated signaling compared to a reference GPR174 compound is identified as a modulatory agent capable of activating the GPR174-mediated signaling pathway. In some embodiments, the candidate modulatory agent increases the apparent activity of GPR174-mediated signaling with the reference compound by at least 20%.

In some embodiments, the present invention provides a method for identifying a modulatory agent capable of modulating a GPR174-mediated signaling pathway (e.g., the Gs pathway), comprising:

(a) bringing a cell expressing GPR174 into contact with:

(i) at least one candidate modulatory agent and (ii) a reference compound known to modulate the activity of the GPR174-mediated signaling pathway, and (b) comparing the activity of GPR174-mediated signaling in a cell brought into contact with the candidate modulatory compound and a reference compound, with GPR174-mediated signaling activity in a cell brought into contact with the reference compound alone; wherein the difference (ie, a difference of at least a factor of two) in signaling activity in a cell contacted with the reference compound combination indicates that the candidate modulatory compound is a compound that modulates the activity of the GPR174-mediated signaling pathway.

In accordance with this aspect of the present invention, the reference compound is known to be

- 63 046439 duplicating at least one GPR174-mediated signaling pathway, contains a structure in accordance with any of formulas I, II, III, IV, V or VI (or (I), (II), (III), (IV) , (V), (Va) or (VI)) described herein. In one embodiment, the reference compound known to modulate at least one GPR174-mediated signaling pathway is any of compounds 1-59 presented in Table. 1.

The term reference compound including a detectable moiety refers to a reference compound that is modified to include, or is covalently linked to, any detectable moiety suitable to provide readout of the GPR174-linked reference compound in any of the wide variety of different binding assay formats well known to one of ordinary skill in the art. in the field, including, but not limited to, the use of radioactive isotopes, fluorescence, fluorescence polarization (FP), fluorescence resonance energy transfer (FRET), bioluminescence, bioluminescence resonance energy transfer (BRET), chemiluminescence, photoaffinity labeling, time-resolved fluorometry ( TRF), etc. For example, in one embodiment, the reference compound is labeled with a molecular tag(s) including a radioactive isotope such as ¹²⁵ I, ¹⁴ C, ³ H, ¹¹ C, ¹⁸ F ^99m Tc, or other suitable radiometric label. In another embodiment, the reference compound is labeled with a fluorescent label such as 6-carboxyfluorescein (FAM), Alexa Fluor® (Molecular Probes, Inc), fluorescein isothiocyanate (FITC), and the like. In another embodiment, the reference compound is labeled with a bioluminescent or chemiluminescent label such as isoluminol or acridinium ester, or an enzyme label such as horseradish peroxidase (HRP) or alkaline phosphatase (AP) to further enhance readout using improved chemiluminescence substrates (ECL). In another embodiment, the reference compound is covalently linked to a high affinity binding molecule, such as biotin, to utilize various streptavidin-linked tags or enzymes as described above.

In accordance with one embodiment of this aspect of the present invention, the binding assay includes contacting a cell expressing GPR174, or a cell membrane derived therefrom, with at least one candidate modulator compound and a reference compound comprising a detectable moiety. Typically, cells expressing GPR174 are cultured using standard cell culture techniques to 80-90% confluency and then used to prepare whole cells, disrupted cell preparations, or cell membrane preparations, which are then frozen at -80°C. Alternatively, whole cells or cell preparations can be used fresh, without refrigeration. Cell or membrane preparations, thawed or used fresh, are incubated with an optimal concentration of a reference compound (surrogate ligand) (e.g., 0.01 to 10,000 nM) in the presence or absence of varying concentrations of a candidate competitive compound (i.e., candidate modulatory agent) , preferably in multi-well format. After incubation in a suitable binding buffer for a suitable time (i.e. 10 min to 2 h) at a suitable temperature (i.e. ~2037°C), the cells are transferred to a multiwell filtration plate and washed multiple times with cold buffer. for washing. A bound reference compound comprising a detectable group is measured using a suitable device to provide a binding affinity readout. Alternatively, the bound reference compound can be separated from the free compound by a variety of separation techniques, including centrifugation, dialysis, resin binding, and various types of chromatography (affinity, thin layer, ion exchange, HPLC, FPLC, etc.). When using homogeneous analysis technologies, the separation and washing stages can be eliminated. The amount of bound reference compound is assessed to detect dissociation of the labeled reference compound from GPR174 after incubation with the candidate modulator(s), compared to control wells incubated in the absence of the candidate modulator(s).

An excess of unlabeled reference compound (surrogate ligand) can be used to assess nonspecific binding (NSB) in negative control wells (without added competitive agent), and positive control wells (without added competitive agent) are used to determine the maximum signal from the labeled surrogate ligand. The percentage of activity is calculated as

100 x (competitive signal - NSB)/(positive control - NSB) for each competitive concentration used, and IC ₅₀ values (competitor concentration resulting in 50% activity) are obtained using a suitable curve fitting model such as 4 - or 5-parameter logistic fitting, Hill's equation, or other equations based on binding saturation data models. In one embodiment, the assays are configured such that the IC ₅₀ values reflect the dissociation constants for the inhibitory molecules (eg, IC ₅₀ =K ₁ (1+[reference compound]/^), where [reference compound] << KD). In cases where competitive inhibition is weak and the IC value is ₅₀

- 64 046439 cannot be obtained, values are recorded as > highest concentration tested. Compounds are considered active competitors when the % activity at the highest concentration tested is significantly less (P > 95%, n=3) than 100% activity. In other cases, such as positive allosteric modulation, binding may be characterized by an EC50 (half maximum effective concentration) greater than that of the reference compound alone due to increased affinity and/or accessibility of binding sites. In cases where EC50 values cannot be obtained for positive modulators, compounds are considered active positive modulators when the % activity at the highest concentration tested is significantly greater (P>95%, n=3) than 100%.

In one embodiment, the identified modulatory agents are small molecule compounds that bind competitively to the same GPR174 region as the reference compound and that show a corresponding dose response curve when used to compete with the reference compound (surrogate ligand), with the resulting IC ₅₀ <100 µM, or more preferably IC ₅₀ <10 µM.

As described herein, we have identified chemical compounds (i.e., surrogate ligands) that functionally interact with GPR174 and inhibit one or more GPR174-mediated signaling pathways and, in addition, have a characterized GPR174 receptor signaling profile that includes the Gs signaling pathway. With compounds now identified that modulate GPR174, these newly identified surrogate ligands for GPR174 can be used as reference compounds in the competitive binding assays described herein, using conventional methods known to one of ordinary skill in the art to identify additional compounds. that bind to a precise region of interest on GPR174, for use in modulating GPR174-mediated signaling. As is well known in the art, binding assays are capable of detecting competitive as well as non-competitive or non-competitive inhibitors. These binding assays can also detect allosteric modulators that can promote or inhibit binding with additive or synergistic effects.

In accordance with the above, in one embodiment, the present invention provides a method of using a small molecule chemical compound to inhibit a GPR174-mediated cell signaling pathway, comprising the steps of: (a) providing a small molecule chemical compound that operably interacts with GPR174 and inhibits the GPR174-mediated pathway signaling (for example, the Gs pathway) in cells expressing GPR174, where the specified connection is characterized by at least one of the following criteria:

said compound has a structure selected from the group consisting of formula I, II, III, IV, V or VI (or (I), (II), (III), (IV), (V), (Va) or ( VI)); or the specified compound changes the binding affinity of any of the reference compounds 1-59 (as indicated in Table 1) with GPR174; or the specified compound causes a difference in the modulatory activity of any of the reference compounds 1-59 (as indicated in Table 1), in the GPR174-mediated signaling pathway assay, when tested in the presence of the specified reference compound compared to the modulatory activity of the reference compound alone; and contacting a cell expressing GPR174 that includes the GPR174-mediated signaling pathway with said compound in accordance with step (a), thereby modulating the GPR174-mediated signaling pathway in said cell.

In some embodiments, the compound modifies (ie, increases or decreases) the binding affinity of any of reference compounds 1-59 to GPR174, in accordance with criteria (ii). In some embodiments, the compound reduces (ie, inhibits) the binding affinity of any of reference compounds 1-59 to GPR174. In some embodiments, the compound competitively inhibits (ie, reduces the binding affinity of) at least one of reference compounds 1-59 with GPR174. In some embodiments, the compound increases the binding affinity (enhances binding) of any of reference compounds 1-59 to GPR174.

In accordance with the above, in one embodiment, the present invention relates to a method of using a small molecule chemical compound for inhibiting a GPR174-mediated cell signaling pathway, wherein the method includes the steps of: (a) providing a small molecule chemical compound that operably interacts with GPR174 and inhibits a GPR174-mediated signaling pathway (e.g., Gs pathway) in cells expressing GPR174, wherein the compound is characterized by at least one of the following criteria:

said compound has a structure selected from the group consisting of formula I, II, III, IV, V or VI (or (I), (II), (III), (IV), (V), (Va) or ( VI)); or

- 65 046439 the specified compound changes the apparent binding affinity of any of the reference compounds 1-59 (as listed in Table 1) with GPR174; or the specified compound causes a difference in the modulatory activity of any of the reference compounds 1-59 (as indicated in Table 1), in the GPR174-mediated signaling pathway assay, when tested in the presence of the specified reference compound, compared with the modulatory activity of the reference compound alone; and contacting a cell expressing GPR174 that includes the GPR174-mediated signaling pathway with said compound in accordance with step (a), thereby modulating the GPR174-mediated signaling pathway in said cell.

In some embodiments, the compound modifies (ie, increases or decreases) the apparent binding affinity of any of reference compounds 1-59 to GPR174, in accordance with criteria (ii). In some embodiments, the compound reduces the apparent binding affinity of any of reference compounds 1-59 for GPR174. In some embodiments, the compound binds competitively to GPR174 (ie, has higher binding affinity) than at least one of reference compounds 1-59 to GPR174. In some embodiments, the compound binds non-competitively or noncompetitively to GPR174 (ie, binds to an allosteric site and reduces apparent binding affinity) relative to at least one of reference compounds 1-59 to GPR174. In some embodiments, the compound increases the binding affinity (ie, enhances binding and is a positive allosteric modulator) of any of reference compounds 1-59 to GPR174.

In another embodiment, the present invention provides a compound comprising a detectable moiety, wherein the compound contains a structure according to any one of formulas I, II, III, IV, V or VI (or (I), (II), (III) , (IV), (V), (Va) or (VI)) described herein, and modulates the GPR174-mediated signaling pathway (eg, Gs pathway). In one embodiment, the compound comprising the detectable group contains a structure in accordance with any of compounds 1-59 described in table. 1. In one embodiment, the detectable group is at least one of a radioactive isotope(s), a fluorescent label, a chemiluminescent label, a bioluminescent label, or a photoaffinity label. In one embodiment, the reference compound contains a radioactive isotope selected from the group consisting of ¹²⁵ 1, ¹⁴ C, ³ H, ¹¹ C, ¹⁸ F and ^99m Tc. Compounds having a structure according to any of formulas I, II, III, IV, V or VI (or (I), (II), (III), (IV), (V), (Va) or (VI) ), and further including a detectable group (e.g., compounds 1-59 in Table 1), can be used in methods for identifying one or more modulatory agents capable of modulating the GPR174-mediated signaling pathway, and can also be used as diagnostic probes for detection the presence or amount of GPR174 receptor in a test sample in vitro, or as probes for in vivo molecular imaging, such as positron emission tomography (PET).

In accordance with the above, in one aspect, the present invention provides a method for identifying a modulatory agent capable of modulating a GPR174-mediated signaling pathway (eg, the Gs pathway), comprising: (a) contacting a cell with a candidate modulatory agent that is administered into a cell, wherein said cell expresses GPR174 and includes a signaling pathway, and (b) determining whether the candidate modulator modulates at least one of the GPR174-mediated signaling pathways compared to a cell brought into contact with a control or, compared to reference standard. In one embodiment, step (b) includes performing an assay to detect at least one of the following: transcriptional reporter gene expression, GTPase activity, cAMP level, intracellular messenger level, calcium level, downstream kinase activity, transcriptional activation of downstream genes, changes in morphology cells, changes in the rate of cell growth, arachidonic acid release, or the rate of extracellular acidification. In one embodiment, the candidate modulatory agent is selected from the group consisting of a compound, a nucleic acid, a natural extract, and a gas. In one embodiment, said modulating agent candidate is a chemical compound. In one embodiment, the specified chemical compound contains a structure in accordance with any of formulas I, II, III, IV, V or VI (or (I), (II), (III), (IV), (V), ( Va) or (VI)) described herein. For example, methods in accordance with this aspect of the present invention can be carried out to evaluate the GPR174 modulatory activity of specific chemical compounds comprising a structure in accordance with any of formulas I, II, III, IV or V (or (I), (II), (III ), (IV), (V), (Va) or (VI)) described herein.

In one embodiment, said chemical compound contains a structure in accordance with any of formulas (I), (II), (III), (IV), (V), (Va) or (VI) described herein. For example, methods in accordance with this aspect of the present invention can be carried out to evaluate the GPR174 modulating activity of specific chemical compounds comprising the structure

- 66 046439 ru in accordance with any of formulas (I), (II), (III), (IV), (V), (Va) or (VI) described in the present description.

A candidate modulatory agent may modulate (ie, inhibit or activate) at least one of the GPR174-mediated pathways (eg, the Gs pathway), compared to a cell contacted with a control or compared to a reference standard. Gs signaling pathway activity in the presence of a candidate agent is present relative to the activity of a control, which may be a cell contacted with an agent known to be a GPR174 modulating agent, or a reference value obtained for a cell contacted with an agent known to be a GPR174 modulating agent. Alternatively, the control may be the activity of the Gs signaling pathway in the cell in the absence of the candidate agent. A candidate modulator used in such an assay may be free in solution, attached to a solid support, carried on the cell surface or localized within the cell, or associated with a portion of the cell.

In another embodiment, the present invention provides a method for identifying a modulatory agent capable of modulating the activity of a GPR174-mediated signaling pathway (eg, Gs pathway activity), comprising:

(a) bringing a cell expressing GPR174 into contact with:

(i) at least one candidate modulatory agent; and (ii) a reference chemical compound known to modulate the activity of the GPR174-mediated signaling pathway;

(b) determining the level of activity of the GPR174-mediated signaling pathway in the cell brought into contact according to step (a); and (c) comparing the level of activity of the GPR174-mediated signaling pathway in a cell brought into contact with only the reference chemical with the activity of the GPR174-mediated signaling pathway determined in step (b); wherein the difference in GPR174-mediated signaling activity between a cell containing a candidate modulatory agent in the presence of a reference compound and a cell brought into contact with only the reference compound indicates that the candidate modulatory agent is capable of modulating GPR174 activity. In one embodiment, the reference chemical compound contains a structure in accordance with any of formulas (I), (II), (III), (IV), (V), (Va) or (VI) described herein.

In one embodiment, step (b) comprises performing an assay to detect at least one of the following: transcriptional reporter gene expression, GTPase activity, cAMP level, intracellular messenger level, calcium level, downstream kinase activity, transcriptional activation of downstream genes, changes in morphology cells, changes in cell growth rate, arachidonic acid release, or extracellular acidification rate. In one embodiment, the candidate modulatory agent is selected from the group consisting of a compound, a nucleic acid, a natural extract, and a gas. In one embodiment, said compound is a chemical compound.

Assays for use in accordance with the screening methods of the present invention can measure the output of GPCR protein signaling by detecting, for example, GTPase activity, intracellular messenger levels, downstream kinase activity, or transcriptional activation of downstream genes. The choice of assay may depend on the signaling pathway activated by the GPCR protein and the needs of the user.

Typically, samples or assays involving GPR174 proteins are treated with a candidate modulatory agent, such as a candidate compound. These samples are then compared to control samples not treated with the candidate compound, and optionally treated with a control agent to test the effect on GPR174 activity, in the case of agonists. Control samples (untreated, for example, with activators, inhibitors, agonists, inverse agonists or antagonists) are assigned a relative protein activity value of 100%. The activity of an inhibitor, such as an antagonist or inverse agonist, is typically determined in the presence of an agonist or ligand, such as the GPR174 agonists described herein, or can be determined in overexpression studies. Inhibition of GPR174 is achieved when the activity value, compared to the control, is approximately 80%, preferably approximately 50%, and more preferably approximately 25-0%, depending on the conditions tested. Activation of GPR174 is achieved when the value of activity, compared to the control (untreated with activators or agonists) is at least 110%, more preferably 150%, more preferably 200-500% (i.e. two to five times higher , compared to control), and more preferably 1000-3000% higher. In some embodiments, the methods use a reference standard, which may be a numerical value (eg, a threshold value) determined by averaging a plurality of control samples (untreated, for example, with activators, inhibitors, agonists, inverse agonists, or antagonists).

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1. Transcription reporter assay.

Analyzable reporter proteins such as luciferase provide a useful tool for analyzing GPCR activity measured by transcriptional reporter assays. Cells (e.g., HEK293 cells, CHO cells, or COS 7 cells) are transiently co-transfected with both a GPR174 expression construct and a reporter construct that includes a cDNA for the reporter protein downstream of a transcription factor binding site, such as a cAMP response element (CRE), AP-1, serum response element (SRE), or NFAT or serum response factor (SRF) binding sites. Binding of the agonist to receptors coupled to the Gs subtype of G proteins leads to an increase in cAMP levels, thereby activating the CRE transcription factor and resulting in reporter gene expression. Binding of the agonist to receptors coupled to the Gs subtype of G proteins leads to the production of diacylglycerol, which activates protein kinase C, which leads to the activation of the transcription factors SRE, AP-1 NFAT, SRF and CRE, in turn resulting in reporter gene expression. Activation of Gi can be detected by a CRE reporter system, if necessary by first incubating the cells with forskolin or the like. to increase intracellular cAMP levels. Gi expression can also be detected by using chimeric Gq and Gs proteins. Activation of G12/13 can be detected through the reporter genes SRF and SRE. Reporter protein expression levels reflect the activation status of signaling events. See, for example, George et al., J. Biomol. Screen. 2:235-40 (1997) and Stratowa et al., Curr. Opin. Biotechnol. 6:574-81 (1995). Comparisons are made with cells transfected with a GPR174 expression construct or a control expression construct to determine the extent to which GPR174 activity is inhibited or activated.

To assess GPR174 activity, cells are transiently co-transfected with both a GPR174 expression construct and a reporter construct that includes cDNA for the reporter protein downstream of transcription factor binding sites, i.e., SFE, NFAT, SRE, SRF or CRE. Comparisons are made with cells transfected with a GPR174 expression construct or a control expression construct to determine the extent to which GPR174 activity is inhibited or activated.

2. Label-free biosensor measurement of GPCR activation.

Measuring GPCR activity results in changes in cell morphology that can be detected using biosensors. Such systems do not require labeling of either the GPCR protein or the test compound. Cell morphology can be detected using either resistance measurements or optical signals. See, for example, Fang et al., Comb. Chem. High Throughput Screen. 11:357-68, 2008 and Peters et al., Assay Drug Dev. Technol. 8:219-27, 2010. Systems for measuring resistance include, for example, the CellKey™ system (MDS Sciex, South San Francisco, CA). Optical signal measurements are provided by an Epic™ system (Corning, NY) or a BIND® analysis system (SRU Biosystems, Inc.). These systems require a ligand or surrogate ligand to detect activation of a specific GPCR protein. Cells expressing the GPCR protein of interest, ie, GPR174, are contacted with a ligand or surrogate ligand and monitored for changes in resistance or optical signal. Comparisons are made with cells that are not treated with the ligand or surrogate ligand.

3. Tagged ligand or surrogate ligand binding assay.

Cells are analyzed for their ability to specifically bind GPR174 ligand or a surrogate ligand, such as an inhibitor or activator. Cells that transiently or stably express GPR174 protein are grown to subconfluency, collected from flasks in PBS, and pelleted. Cell pellets are homogenized. The homogenate is centrifuged at 47,000 g for 15 minutes. The membrane pellet is resuspended in 1 ml of tissue. An aliquot of the membrane preparation is used to determine the protein concentration. To measure binding saturation, cell membranes are incubated with varying concentrations of labeled ligand or surrogate ligand. Nonspecific binding is determined by inclusion of an unlabeled ligand or a surrogate ligand. After binding incubation, the contents of the plates were collected onto GF/C filters pre-soaked in 0.3% nonfat dry milk. The filters are dried and counted in a 96-well microplate scintillation counter. Calculate Kd values.

4. GTPyS binding assay.

Because G protein-coupled receptors such as GPR174 signal through intracellular G proteins, whose activities include binding and hydrolysis of GTP, to produce bound GDP, measuring the binding of the non-hydrolyzable GTP analogue [ ³⁵ S]-GTPyS in the presence and in the absence of candidate inhibitors or activators provides a different analysis of GPR174 activity. See, for example, Kowal et al., Neuropharmacology 37:179-187 (1998). In one exemplary assay, cells stably transfected with a GPR174 expression vector are grown in 10 cm culture dishes to subconfluence, washed once with 5 ml of ice-cold Ca ²⁺ /Mg ²⁺ -free phosphate-buffered saline, and scraped into 5 ml of such same buffer. Cells are pelleted by

- 68 046439 centrifugation (500 g, 5 min), resuspended in TEE buffer (25 mM Tris, pH 7.5, 5 mM EDTA, 5 mM EGTA), and frozen in liquid nitrogen. After thawing, cells are homogenized using a Dounce homogenizer (1 ml TEE per cell plate) and centrifuged at 1000 g for 5 min to remove nuclei; and undestroyed cells.

The supernatant of the homogenate is centrifuged at 20,000 g for 20 min to isolate the membrane fraction and the membrane pellet is washed once with TEE and resuspended in binding buffer (20 mM HEPES, pH 7.5, 150 mM NaCl, 10 mM MgCl ₂ , 1 mM EDTA). Resuspended membranes can be frozen in liquid nitrogen and stored at -70°C until use. Aliquots of cell membranes obtained as described above and stored at -70°C are thawed, homogenized and diluted. The final homogenates are incubated with various concentrations of candidate compounds or guanosine 5'-triphosphate (GTP) for 30 min at 30°C and then placed on ice. To each sample, guanosine 5'-0-(3[ ³⁵ S thio)triphosphate (NEN, 1200 Ci/mmol; [ ³⁵ S]-GTPyS) was added to a final concentration of 100-200 pM. Samples are incubated at 30°C for an additional 30 minutes, add 1 ml of 10 mm HEPES, pH 7.4, 10 mm MgCl ₂ at 4°C, and the reaction is stopped by filtration.

Samples are filtered through Whatman GF/B filters and the filters are washed with 20 ml of ice-cold 10 mM HEPES, pH 7.4, 10 mM MgCl ₂ . Filters are counted by liquid scintillation spectroscopy. Nonspecific [ ³⁵ S]-GTPyS binding is measured in the presence of GTP and subtracted from the total. Comparisons are made with untransfected control cells to determine changes in GPR174 activity.

5. Measurement of intracellular calcium using FLIPR.

Changes in intracellular calcium levels are another known indicator of G-protein coupled receptor activity, and such assays can be used to screen for modulators of GPR174 activity. In one specific example of such an assay, mammalian cells stably transfected with a GPR174 expression vector are seeded at a density of 40,000 cells/well in 96-well plates specifically designed to distinguish between fluorescent signals emitted from different wells on the plate. Cells are incubated for 60 min at 37°C in Dulbecco's modified PBS containing pyruvate and 1 g/L glucose supplemented with 1% fetal bovine serum and a calcium indicator dye such as Fluo-3™ AM, Fluo-4™ AM , Calcium Green™-1 AM, or Oregon Green™ BAPTA-1 AM. The plates are washed once with modified Dulbecco's PBS without 1% fetal bovine serum and incubated for 10 min at 37°C to remove residual dye from the cell membrane. The calcium response is initiated by the addition of one or more candidate GPR174 inhibitors or activators, calcium ionophore A23187 (positive control), or ATP (positive control). Fluorescence is measured using the Molecular Device's FLIPR with an argon laser (excitation 144 at 488 nm). See, for example, Kuntzweiler et al., Drug Dev. Res. 44:14-20 (1998). Comparisons are made with untransfected control cells to determine changes in GPR174 activity.

6. Aequorin assays.

The protein aequorin provides another way to measure intracellular calcium following activation of the Gq signaling pathway. In the presence of the cofactor coelenterazine, aequorin can emit measurable luminescence that is proportional to the amount of intracellular (cytoplasmic) free calcium. See, for example, Cobbold et al. Aequorin measurements of cytoplasmic free calcium, In: McCormack J.G. and Cobbold P.H., eds., Cellular Calcium: A Practical Approach. Oxford: IRL Press (1991); Stables et al., Anal. Biochem. 252:115-26 (1997); and Haugland, Handbook of Fluorescent Probes and Research Chemicals, Sixth edition. Eugene Ore.: Molecular Probes (1996).

In a typical assay, mammalian cells are transiently co-transfected with both a GPR174 expression construct and a construct encoding the photoprotein apoaequorin. Cells are cultured for twenty-four hours at 37°C, for example, in MEM (Gibco/BRL, Gaithersburg, MD) supplemented with 10% fetal bovine serum, 2 mM glutamine, 10 U/ml penicillin and 10 μg/ml streptomycin, at this time, the medium is replaced with serum-free medium containing coelenterazine (Molecular Probes, Eugene, OR). Cells are incubated for two additional hours at 37°C. Cells are then detached from the plate, washed, and resuspended at 200,000 cells/ml in serum-free MEM.

GPR174 inhibitors or activators are prepared in serum-free MEM and dispensed into wells of an opaque 96-well assay plate. The plates are then loaded into an MLX Microtiter Plate Luminometer (Dynex Technologies, Inc., Chantilly, VA). The device is programmed to dispense cell suspensions into each well, one well at a time, and the luminescence is immediately read for 15 seconds. Dose response curves for GPR174 inhibitors or activators are constructed using the area under the curve for each light signal peak. The data is analyzed and EC50 values are obtained. Compound-induced changes in luminescence indicate changes in GPR174 activity.

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7. Release of arachidonic acid.

It was also observed that GPCR activation activates the release of arachidonic acid in cells, providing another useful assay for identifying inhibitors or activators of GPCR activity. See, for example, Kanterman et al., Mol. Pharmacol. 39:364-9 (1991). For example, CHO cells stably transfected with a GPR174 expression vector are seeded into 24-well plates at a density of 15,000 cells/well and grown in MEM supplemented with 10% fetal bovine serum, 2 mM glutamine, 10 U/ml penicillin and streptomycin for 48 hours at 37°C before use. Cells from each well were labeled by incubation with [ ³ H]-arachidonic acid (Amersham Corp., 210 Ci/mmol) for 2 hours at 37°C. The cells are then washed twice with 1 ml of buffer. Candidate compounds are added to 1 ml of the same buffer, either alone or with ATP, and the cells are incubated at 37°C for 30 min. Buffer alone and mock-transfected cells are used as controls. Samples (0.5 ml) from each well were counted by liquid scintillation spectroscopy.

8. Rate of extracellular acidification.

In another assay, the effects of candidate inhibitors or activators on GPCR activity are analyzed by monitoring extracellular pH changes induced by the test compounds. See, for example, Dunlop et al., J. Pharmacol. Toxicol. Methods 40:47-55 (1998). In one embodiment, cells transfected with a GPCR expression vector are seeded into 12 mm capsule dishes (Molecular Devices Corp.) at 400,000 cells/dish in MEM supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 10 units/ ml penicillin and 10 µg/ml streptomycin. Cells are incubated in this medium at 37°C in 5% CO2 for 24 hours. The rate of extracellular acidification is measured using a Cytosensor microphysiometer (Molecular Devices Corp.). Candidate agonists or other agents are diluted in a running buffer and perfused through a second fluid pathway. The pH of the running buffer in the sensor chamber is recorded for a cycle of 43-58 s, and the pump is restarted after 60 s to begin the next cycle. The rate of acidification of the working buffer during the recording time is calculated using the Cytosoft program. Changes in acidification rate are calculated by subtracting the initial value (the average of the four rate measurements immediately before addition of the candidate compound) from the highest rate measurement obtained after addition of the candidate compound.

9. cAMP assays.

In one type of assay, cyclic adenosine monophosphate (cAMP) levels are measured in cells transfected with a GPCR expression vector. GPCR-expressing cells are exposed to candidate compounds. Protocols for cAMP assays are described in the literature. See, for example, Sutherland et al., Circulation 37:279-306, 1968; Frandsen et al., Life Sci. 18:529-41, 1976; Dooley et al., J. Pharmacol. Exp. Ther. 283:735-41, 1997; and George et al., J. Biomol. Screen. 2:235-40, 1997. Additional protocols describing cAMP assays include: Hill et al., Br. J. Pharmacol. 161(6): 1266-1275, 2010; Smith et al., Appl. Biochem. Biotechnol. 41(3):189-218, 1993; and Berrera et al., Handb. Exp. Pharmacol. (186):285-98, 2008. An exemplary protocol for such an assay, using the FlashPlate® Adenylyl Cyclase Activation Assay from NEN® Life Science Products, is listed below.

Briefly, a GPCR coding sequence (eg, cDNA or intronless genomic DNA) is subcloned into an expression vector and transiently transfected into a host cell line, eg, Chinese hamster ovary (CHO) cells, using known methods. Transfected CHO cells are seeded into 96-well microplates from FlashPlate® microplates (coated with solid scintillator to which anti-cAMP antiserum is bound). As a control, wild-type (untransfected) CHO cells were seeded into some wells. Various amounts of cAMP standard solution are added to other wells in the plate for use in preparing the standard curve.

One or more test compounds (ie candidate inhibitors or activators) are added to the cells in each well, where water and/or compound-free media/diluent serve as a control or controls. After treatment, cAMP is allowed to accumulate in cells for exactly 15 min at room temperature. The assay is stopped by adding lysis buffer containing labeled cAMP and the plate is counted using a Packard Topcount® 96-well microplate scintillation counter. Unlabeled cAMP from lysed cells (or from standards) and fixed amounts of cAMP compete for binding to antibody bound to the plate. A standard curve is constructed and cAMP values for unknown points are obtained by interpolation. Changes in intracellular cAMP levels of cells in response to exposure to a test compound are indicative of GPCR inhibitory or activating activity. Other assays can be used to detect cAMP levels, such as tagged cAMP detection.

In addition, many other kits for measuring cAMP levels are commercially available, such as the cAMP Direct Immunoassay Detection Kit from Abcam (cat. no.

- 70 046439 ab138880), LANCE® cAMP Detection Kit from Perkin Elmer, Direct cAMP ELISA Kit from Enzo Life Sciences, Inc., and CatchPoint® Fluorescent cAMP Assay Kit from Molecular Devices.

In some embodiments, the level of cAMP is reduced by at least 20%, at least 50%, or at least 75% in a GPR174-expressing cell contacted with a small molecule inhibitor of GPR174 signaling, compared to a control cell, expressing GPR174, not brought into contact with a small molecule inhibitor.

10. GPR174 protein expression.

Recombinant GPR174 polypeptides, either full-length or fragments thereof, can be produced using standard methods known in the art. These proteins can then be used in the assays described herein. In particular, such recombinant GPR174 polypeptides, for example, can be used in in vitro assays to identify GPR174 inhibitors or activators. Recombinant production can occur in host cells or in cell-free translation systems, as is known in the art.

Typical fusion protein expression vectors include pGEX (Pharmacia Biotech Inc; Smith et al., Gene 67:31-40, 1988), pMAL (New England Biolabs, Ipswich, MA), pRIT5 (Pharmacia, Piscataway, N.J.), and pFUSE (Invivogen , San Diego, CA) in which glutathione B-transferase (GST), maltose binding protein E, protein A, or IgG Fc, respectively, is fused to a recombinant target protein.

If the GPR174 polypeptide is to be expressed for use in screening assays, it can be produced on the cell surface. In this case, the cells can be collected before use in the screening assay. If the polypeptide is secreted into the medium, the medium can be isolated to isolate and purify the polypeptide. For intracellular production, cells must first be lysed before the polypeptide is isolated.

Recombinant polypeptides (or alternatively, GPR174 polypeptides isolated from the body) can be targeted to the cell membrane. Membrane-bound GPR174 can be produced by expressing GPR174 in a suitable cell or cell line, for example, Pichia pastoris cells, oocytes or COS cells. Membranes containing the recombinant polypeptide can then be isolated from other cellular components by standard methods known in the art.

11. Kinase activity assays.

In another type of assay, the activity of kinases downstream of GPR174 in a signaling pathway, such as the Gs signaling pathway, can be measured. Kinases downstream of GPR174 include, but are not limited to, PKA, and the activity of one or more of the kinases downstream of GPR174 can be modulated in the presence of a GPR174 inhibitor. For example, protein kinase A (PKA) activity can be used to detect changes in the GPR174-mediated Gs signaling pathway, since cAMP, the level of which is modulated by Gs signaling, activates PKA. Protocols for PKA assays are described in the literature. See, for example, Karege et al., Brain Res. 903(1-2):86-93, 2001. A variety of kits are also commercially available for measuring PKA activity, including the cPKA Kinase Activity Assay Kit from Abcam (Cat. No. ab139435), the Colorimetric PKA Activity Assay Kit from Life Technologies Corporation and the PKA Kinase Activity Assay Kit from Enzo Life Sciences, Inc. These and similar assays can be used, for example, to compare the difference in PKA activity between cells transfected with a GPCR expression vector and exposed to the small molecule inhibitor of GPR174 signaling and cells transfected with a GPCR expression vector but not exposed to the small molecule inhibitor of GPR174 signaling.

In some embodiments, PKA activity is reduced by at least 20%, at least 50%, or at least 75% in a GPR174-expressing cell contacted with a small molecule inhibitor of GPR174 signaling, compared to a control cell, expressing GPR174, not brought into contact with a small molecule inhibitor.

12. Cytokine production assays.

Levels of cytokines such as IL-2 can be modulated through cAMP levels and Gs signaling. Assays that measure the levels of one or more cytokines can be used to detect changes in the GPR174-mediated Gs signaling pathway. The level of one or more cytokines can be measured using a sandwich ELISA immunoassay. For example, IL-2 and IFN-γ can be measured using ELISA kits from ThermoFisher Scientific. The level of one or more cytokines can also be measured using bead-based immunoassays, which use beads specific for different cytokines that are differentiated by size and fluorescence intensity using flow cytometry. Bead-based assay kits are also commercially available, for example BioLegend's LEGENDplex™ Human Th Cytokine Panel allows simultaneous measurement of IL-2, L-4, IL-5, IL-6, L-9, IL-10, IL -12, IL13, IL-17A, L-17F, IL-21, IL-22, GM-CSF, IFN-γ and TNF-α. Alternatively, these cytokines can also be detected in supernatants using the MSD platform (MesoScale).

Intracellular levels of cytokines can also be determined. For example, mononuclear cells

- 71 046439 peripheral blood cells (PBMC) can be treated with BD GolgiStop reagent (BD Biosciences) to block vesicular transport and cause accumulation of intracellular cytokine-filled secretory vesicles. These cells can then be stained using fluorescent antibodies for cell type-specific surface markers and antibodies against specific cytokines (eg, from BioLegend), such as IL-2, IL-10, GM-CSF, IFN-γ and TNF-α. Stained cells can be analyzed by flow cytometry to determine the level of one or more cytokines.

These and similar assays can be used, as another example, to compare differences in the levels of one or more cytokines between a population of PBMCs exposed to the small molecule inhibitor of GPR174 signaling and a population of PBMCs not exposed to the small molecule inhibitor of GPR174 signaling.

In some embodiments, the level of one or more cytokines is reduced by at least 20%, at least 50%, or at least 80% in a population of PBMCs exposed to the small molecule inhibitor of GPR174 signaling compared to a population of PBMCs not exposed to the small molecule inhibitor of GPR174 signaling. In some embodiments, the level of IL-17A is reduced by at least 20%, at least 50%, or at least 80% in a PBMC population exposed to the small molecule inhibitor of GPR174 signaling compared to a PBMC population not exposed exposure to the small molecule inhibitor of signaling GPR174.

In some embodiments, the level of one or more cytokines is increased by at least 20%, at least 50%, or at least 80% in a PBMC population exposed to the small molecule inhibitor of GPR174 signaling, either in the form of a single agent, or in combination with at least one of an A2aR antagonist, an A2bR antagonist, a CD38 inhibitor, a CD39 inhibitor, and/or a CD73 inhibitor, compared to a PBMC population not exposed to the small molecule inhibitor of GPR174 signaling. In some embodiments, the cytokine is selected from IL-2, IFN-γ, TNF, and GM-CSF.

In some embodiments, the level of one or more cytokines is increased by at least 20%, at least 50%, at least 100%, at least 500%, or at least 1000% in the PBMC population subjected to exposure to the small molecule inhibitor of GPR174 signaling, either as a single agent or in combination with at least one of an A2aR antagonist, an A2bR antagonist, a CD38 inhibitor, a CD39 inhibitor, and/or a CD73 inhibitor, compared with a PBMC population not exposed to the small molecule inhibitor GPR174 signaling. In some embodiments, the cytokine is selected from IL-2, IFN-γ, TNF, GM-CSF, IL-6, IL-12, IL-17A, and IL-10. In some embodiments, the level of one or more of IL-2, IFN-γ, TNF, or GM-CSF is increased by at least 20%, at least 50%, at least 100%, at least 500 % or at least 1000% in a PBMC population exposed to the small molecule inhibitor of GPR174 signaling, either as a single agent or in combination with at least one of an A2aR antagonist, an A2bR antagonist, a CD38 inhibitor, a CD39 inhibitor and/or an inhibitor CD73 compared to a PBMC population not exposed to the small molecule signaling inhibitor GPR174.

In some embodiments, the level of IL-2 is increased by at least 100%, at least 500%, or at least 1000% in a PBMC population exposed to the small molecule inhibitor of GPR174 signaling, either in the form of a single agent or in combinations with at least one of an A2aR antagonist, an A2bR antagonist, a CD38 inhibitor, a CD39 inhibitor, and/or a CD73 inhibitor, compared to a PBMC population not exposed to the small molecule inhibitor of GPR174 signaling. In some embodiments, the level of IFN-γ is increased by at least 50%, at least 100%, or at least 500% in a PBMC population exposed to the small molecule inhibitor of GPR174 signaling, either in the form of a single agent or in combinations with at least one of an A2aR antagonist, an A2bR antagonist, a CD38 inhibitor, a CD39 inhibitor, and/or a CD73 inhibitor, compared to a PBMC population not exposed to the small molecule inhibitor of GPR174 signaling. In some embodiments, the level of TNF-α is increased by at least 50%, at least 100%, or at least 500% in a population of PBMC exposed to the small molecule inhibitor of GPR174 signaling, either in the form of a single agent or in combinations with at least one of an A2aR antagonist, an A2bR antagonist, a CD38 inhibitor, a CD39 inhibitor, and/or a CD73 inhibitor, compared to a PBMC population not exposed to the small molecule inhibitor of GPR174 signaling.

In some embodiments, the level of one or more cytokines is not modulated and is not increased or decreased by more than 20% in a population of PBMCs exposed to the small molecule signaling inhibitor GPR174 compared to the population

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PBMC not exposed to the small molecule inhibitor of GPR174 signaling. In some embodiments, the cytokine or cytokines are selected from the group consisting of: IL-4, L5, IL-9, IL-13, IL-17F, IL-21, and IL-22.

In addition, these and similar assays can be used, for example, to compare the difference in the level of one or more cytokines between cells expressing GPR174 and exposed to a small molecule inhibitor of GPR174 signaling, either in the form of a single agent or in combination with at least one antagonist A2aR, an A2bR antagonist, a CD38 inhibitor, a CD39 inhibitor, and/or a CD73 inhibitor, and cells expressing GPR174 but not exposed to the small molecule inhibitor of GPR174 signaling.

In some embodiments, production of one or more cytokines is reduced by at least 20%, at least 50%, or at least 80% in a GPR174-expressing cell contacted with a small molecule inhibitor compared to a control cell, expressing GPR174, not brought into contact with a small molecule inhibitor. In some embodiments, IL-17A production is reduced by at least 20%, at least 50%, or at least 80% in a GPR174-expressing cell contacted with a small molecule inhibitor compared to a control cell expressing GPR174 not brought into contact with a small molecule inhibitor

In some embodiments, production of one or more cytokines is increased by at least 20%, at least 50%, at least 100%, at least 500%, or at least 1000% in a cell expressing GPR174 , brought into contact with the small molecule inhibitor of GPR174 signaling, either in the form of a single agent or in combination with at least one of an A2aR antagonist, an A2bR antagonist, a CD38 inhibitor, a CD39 inhibitor and/or a CD73 inhibitor, compared with a control cell expressing GPR174 not brought into contact with a small molecule inhibitor of GPR174. In some embodiments, production of one or more of IL-2, IFN-γ, TNF, or GM-CSF is increased by at least 20%, at least 50%, at least 100%, at least 500 % or at least 1000% in a cell expressing GPR174 brought into contact with a small molecule inhibitor of GPR174, either in the form of a single agent or in combination with at least one of an A2aR antagonist, an A2bR antagonist, a CD38 inhibitor, a CD39 inhibitor and/ or a CD73 inhibitor, compared with a control cell expressing GPR174 not exposed to a small molecule GPR174 inhibitor.

In some embodiments, IL-2 production is increased by at least 100%, at least 500%, or at least 1000% in a GPR174-expressing cell contacted with a small molecule inhibitor of GPR174 signaling, or in the form of a single agent, or in combination with at least one of an A2aR antagonist, an A2bR antagonist, a CD38 inhibitor, a CD39 inhibitor, and/or a CD73 inhibitor, compared to a control cell expressing GPR174 not contacted with a small molecule GPR174 inhibitor. In some embodiments, IFN-γ production is increased by at least 50%, at least 100%, or at least 500% in a cell expressing GPR174 contacted with a small molecule inhibitor of GPR174 signaling, or in the form of a single agent, or in combination with at least one of an A2aR antagonist, an A2bR antagonist, a CD38 inhibitor, a CD39 inhibitor, and/or a CD73 inhibitor, compared to a control cell expressing GPR174 not contacted with a small molecule GPR174 inhibitor. In some embodiments, TNF-α production is increased by at least 50%, at least 100%, or at least 500% in a GPR174-expressing cell contacted with a small molecule inhibitor of GPR174 signaling, or in the form of a single agent, or in combination with at least one of an A2aR antagonist, an A2bR antagonist, a CD38 inhibitor, a CD39 inhibitor, and/or a CD73 inhibitor, compared to a control cell expressing GPR174 not contacted with a small molecule GPR174 inhibitor.

In some embodiments, the production of one or more of IL-4, L-5, L-9, IL-13, IL-17F, IL-21, and IL-22 is not modulated and is not increased or decreased by more than 20 % in a GPR174-expressing cell contacted with a small molecule inhibitor of GPR174 signaling compared to a control GPR174-expressing cell not contacted with a small molecule inhibitor.

13. Receptor internalization assays.

GPCR-agonist interactions in the context of a living cell can activate G proteins and their corresponding downstream signaling cascades, as well as the recruitment of β-arrestins, which can recruit additional signaling and induce receptor internalization (see, e.g., Paterson et al. Pharmacol Rev 69(3): 256-297, 2017). There are many assays that use arrestin engagement and receptor internalization to assess agonist-receptor interaction or antagonist inhibition of agonist-receptor interaction. There are many

- 73 046439 available versions of both assays, including some that are commercially available. For example, Discoverx provides both β-arrestin recruitment and receptor internalization assays, and ThermoFisher Scientific and Molecular Devices provide the Tango GPCR assay and TransFluor technology, respectively, to quantify β-arrestin recruitment.

In some embodiments, receptor internalization is reduced by at least 10%, at least 20%, at least 50%, or at least 80% in a cell expressing GPR174 brought into contact with a small molecule inhibitor of GPR174 signaling , compared to a control cell expressing GPR174 not exposed to the small molecule inhibitor. In some embodiments, receptor internalization is reduced by 1-99% in a GPR174-expressing cell contacted with a small molecule inhibitor of GPR174 signaling compared to a control GPR174-expressing cell not contacted with a small molecule inhibitor. In some embodiments, receptor internalization is reduced by 1-20%, 20-40%, 40-60%, 60-80%, or 80-99% in a GPR174-expressing cell contacted with a small molecule inhibitor of GPR174 signaling , compared to a control cell expressing GPR174 not exposed to the small molecule inhibitor.

In some embodiments, β-arrestin recruitment is reduced by at least 10%, at least 20%, at least 50%, or at least 80% in a GPR174-expressing cell contacted with a small molecule transmission inhibitor GPR174 signals compared to a control cell expressing GPR174 not exposed to the small molecule inhibitor. In some embodiments, β-arrestin recruitment is reduced by 1-99% in a GPR174-expressing cell contacted with a small molecule inhibitor of GPR174 signaling compared to a control GPR174-expressing cell not contacted with a small molecule inhibitor. In some embodiments, β-arrestin recruitment is reduced by 1-20%, 20-40%, 40-60%, 60-80%, or 80-99% in a GPR174-expressing cell contacted with a small molecule transmission inhibitor GPR174 signals compared to a control cell expressing GPR174 not exposed to the small molecule inhibitor.

G. Connections.

The compounds used in the present invention include small molecules. Such compounds can be identified using screening assays (eg, the assays described herein). Specific types of compounds, as well as methods for preparing such compounds, are described below.

1. Small molecules.

Additional inhibitors include large or small inorganic, organometallic or organic molecules. In specific embodiments, the inhibitor is a small organic molecule or a derivative or analog thereof. Such small molecules preferably have a molecular weight below 2000 Daltons, for example between 200 and 1000 Daltons or between 400 and 700 Daltons. It is preferred that these small molecules are organic molecules. Examples of small molecules that inhibit GPR174 activity are described above.

In specific embodiments, the inhibitor includes a protecting group. The term protecting group refers to chemical groups that block at least some reactive groups and prevent such groups from participating in chemical reactions until the protecting group is removed (or cleaved). Examples of blocking/protecting groups are described, for example, in Greene and Wuts, Protective Groups in Organic Synthesis, 3rd Ed., John Wiley & Sons, New York, NY, 1999.

Any inhibitors may have one or more chiral centers, and each center may exist in an R or S configuration. Inhibitors herein include all diastereomeric, enantiomeric and epimeric forms, as well as mixtures thereof. Stereoisomers can be prepared, if desired, by methods known in the art, for example, such as the separation of stereoisomers by means of chiral chromatography columns. Inhibitors further include Noxides, crystalline forms (also known as polymorphs) and pharmaceutically acceptable salts, as well as active metabolites of any inhibitor or activator. All tautomers are included within the scope of the inhibitors or activators provided herein. In addition, the inhibitors described herein may exist in unsolvated as well as solvated forms with pharmaceutically acceptable solvents such as water, ethanol, and the like. Solvated forms of the inhibitors presented herein are also included within the scope of the present invention. Inhibitors may include isotopically labeled compounds. Isotopes that can be used include hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine and chlorine (e.g. ^2H , ^3H , ^13C , ^14C , ^15N , ^18O , ^17O , ^31P , ^32P , ³⁵ S, ¹⁸ F and ³⁶ Cl). Isotopically labeled compounds can be prepared by synthesizing the compound using a readily available isotope labeling reagent including

- 74 046439 one hundred labeled with a reagent label different from isotopes.

In a specific embodiment, the small molecule inhibitor binds to GPR174. The small molecule can bind to the extracellular or transmembrane region of GPR174 and interfere with binding or reduce ligand binding to GPR174, thereby acting as an inhibitor. In a further embodiment, the small molecule inhibitor binds to the extracellular region of GPR174 and acts as an antagonist to inhibit the activity of GPR174, ie, signaling in the associated G protein-associated signaling pathway. In another embodiment, the small molecule is a small organic molecule that inhibits the activity of GPR174, i.e., signaling in the associated G protein-associated signaling pathway. Other mechanisms of action are also included, such as positive or negative allosteric modulation.

2. Polypeptides and polynucleotides.

In specific embodiments, the methods of the present invention are practiced using peptide or polypeptide GPR174 modulators. Peptides and polypeptides can be easily synthesized or produced recombinantly using conventional methods known and available in the art. For example, polynucleotides can be used as a tool for expressing a polypeptide in a suitable cell. Methods well known to one skilled in the art can be used to construct expression vectors containing sequences encoding the polynucleotide or polypeptide of interest and suitable transcriptional and translational control elements. These methods include in vitro recombinant DNA methods, synthetic methods and in vivo genetic recombination. Such methods are described, for example, in Sambrook, J. et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y., and Ausubel, F.M. et al. (1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York. N.Y.

H. Therapeutic modalities.

Based on our identification of GPR174 receptor inhibitors, coupled with their discovery of the GPR174 signaling pathway, methods for inhibiting GPR174 and its associated G protein-regulated signaling pathways are described herein; wherein the GPR174 inhibitory compound is used in the form of a single agent or in combination with at least one of an A2aR antagonist, an A2bR antagonist, a CD38 inhibitor, a CD39 inhibitor and/or a CD73 inhibitor. The methods can be used to increase or decrease signaling in a cell in vitro, ex vivo, or in vivo (eg, in a mammalian cell, such as a human cell). In specific embodiments, the compound is administered to a subject having a disease associated with GPR174 activity to reduce or alleviate symptoms of the disease or underlying disease pathology. In other embodiments, the compound is administered to a subject at risk of developing a disease associated with GPR174 activity to reduce the risk of developing the disease. In ex vivo applications, cells are brought into contact with a compound outside the subject's body. These cells are then transplanted back into the subject. Medical conditions that are associated with GPR174 activity, or in which GPR174 may play a role in the disorder and/or its treatment, include cancer.

As further described herein, the present inventors have shown that combined inhibition of GPR174 and inhibition of A2aR and/or A2bR, or a combination of a GPR174 inhibitor and an inhibitor of an enzyme pathway involved in the production of adenosine such as CD38, CD39 and CD73 (e.g., a CD73 inhibitor and/or a CD38 inhibitor, and/or a CD39 inhibitor) leads to a synergistic induction of the production of IFN-γ, IL-2, TNF and GM-CSF in human PBMC, thus, in particular embodiments, the present invention relates to methods of treating cancer by administering to a subject a therapeutically effective amount of a GPR174 inhibitor and an inhibitor of ATP-adenosine-A2aR and/or A2bR mediated signaling (such as an A2aR antagonist and/or an A2bR antagonist and/or a CD73 inhibitor and/or a CD38 inhibitor and/or an CD39) thereby stimulating an immune response in said subject.

In some embodiments, the cancer is selected from the group consisting of breast cancer, melanoma, colon cancer, urological malignancy, lung cancer, small cell and non-small cell lung cancer, recurrent or refractory malignancies, non-Hodgkin's lymphoma and Hodgkin's lymphoma, lymphoma, follicular lymphoma, lymphocytic lymphoma, central nervous system lymphoma, T-cell lymphoma, AIDS-associated lymphoma, acute lymphoblastic leukemia, gastrointestinal malignancies, liver cancer, hepatocellular carcinoma, ovarian cancer, pancreatic cancer, bile duct cancer, prostate cancer , kidney carcinoma, bladder cancer, colorectal cancer, multiple myeloma, mesothelioma, cervical cancer, vaginal cancer, anal cancer, oropharyngeal cancer, myelogenous leukemia, stomach cancer, nasopharyngeal carcinoma, head and neck carcinoma, glioblastoma, gliosarcoma, squamous cell malignancy brain tumors, malignant gliomas, diffuse brainstem gliomas, esophageal cancer, thyroid cancer,

- 75 046439 astrocytoma, malignant tumor of the chest, endometrial cancer, skin cell carcinoma, leukemia, acinar cell carcinoma, adenocarcinoma, bronchioloalveolar carcinoma, cholangiocarcinoma, chordoma, giant cell carcinoma, intestinal carcinoma, carcinoma of the major salivary gland, malignant odontogenic neoplasia , malignant tumor of the peripheral membrane nerve, skin cancer, testicular cancer, germ cell tumor, neuroendocrine carcinoma, parathyroid carcinoma, pituitary carcinoma, placental choriocarcinoma, scrotal cancer, tracheal carcinoma, transitional cell carcinoma, uterine body cancer, vulvar cancer, kidney cancer, rectal cancer, carcinoma fallopian tubes, peritoneal carcinoma, epithelial malignancy, pleural mesothelioma, sarcomatoid carcinoma, synovial sarcoma, nephroblastoma, neuroblastoma, adult acute myeloid leukemia, myelodysplastic/myeloproliferative neoplasia, embryonal carcinoma, Kaposi's sarcoma, malignant bone tumor, body cancer ki, stomach cancer, endometrial carcinoma, small intestinal malignancy, endocrine system malignancy, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, ureteral cancer, renal pelvic carcinoma, central nervous system neoplasia, primary tumor angiogenesis, axis tumor spine, epidermoid malignancy, environmentally induced tumors, including asbestos-induced tumors, adenosarcomas, adenosquamous carcinoma, adrenocortical carcinoma, astrocytic tumors, basal cell carcinoma, chondrosarcoma, Ewing's sarcoma, gallbladder cancer, hypopharyngeal malignancy, intraocular melanoma, laryngeal cancer, leiomyosarcoma, malignant tumor of the lips and oral cavity, malignant mesothelial tumors, malignant thymoma, medulloblastoma, medulloepithelioma, Merkel cell carcinoma, mucoepidermoid carcinoma, myelodysplastic syndrome, cancer of the nasal cavity and paranasal sinuses, osteosarcoma, pulmonary blastoma, pineal and supratentorial tive neuroectodermal tumors, plasma cell neoplasia, retinoblastoma, rhabdomyosarcoma, sarcoma, neuroectodermal tumors and Wilms tumor.

In some embodiments, the subject suffering from a cancer has one or more tumors infiltrated by regulatory T cells, such as breast cancer, lung cancer (such as small cell lung cancer or non-small cell lung cancer), colorectal cancer, cervical cancer , kidney, ovarian, melanoma, pancreatic cancer, hepatocellular cancer, stomach cancer, glioblastoma, glioma, bladder cancer, myeloma (such as multiple myeloma), prostate, thyroid, testicular and esophageal cancer.

In some embodiments, the subject suffering from cancer is resistant to checkpoint inhibitors, such as anti-PD-1 (eg, Keytruda® and Opdivo®) and anti-CTLA-4 (Ervoy®), and/or such cell therapies such as CAR-T cell and adoptive T-cell therapy. Checkpoint inhibitors are effective in only a minority of patients, and high levels of adenosine-forming molecules have been observed in non-responding patients. Additionally, overcoming natural immunosuppression in solid tumors poses a major challenge for cell therapy. Since PS and adenosine are both products of stress and cell death in solid tumors, it is expected that patients resistant to checkpoint inhibitor therapy and/or cell therapy may greatly benefit from combined inhibition of the GPR174 and adenosine pathways.

In some embodiments, the method further includes administering one or more additional drugs. In specific embodiments, the cancer cell is further brought into contact with a chemotherapeutic agent.

2. Administration and dosing.

The compounds (in their composition forms) are administered to patients by conventional methods known in the art, for example, by injection, oral, intravenous, subcutaneous, intraperitoneal, intramuscular, infusion, infiltration, irrigation, intra-articular, inhalation, subcutaneous, topical, rectal, vaginal, dermal, nasal, transdermal or ocular administration, etc. For administration by injection and/or infiltration or infusion, the compositions or formulations of the present invention can be suspended or dissolved, as is known in the art, in a vehicle suitable for injection and/or infiltration or infusion. Such vehicles include isotonic saline, buffered or unbuffered, and the like. Depending on the intended use, they may also contain other ingredients, including other active ingredients such as isotonic agents, sodium chloride, pH modifiers, dyes, preservatives, antibodies, enzymes, antibiotics, antifungals, antivirals, other anti-infectives and/ or diagnostic agents such as radiation impervious dyes, radiolabeled agents, and the like, as is known in the art. However, the compositions of the present invention may contain a simple solution or suspension of the compound, or a pharmaceutically acceptable salt of the compound, in distilled water or saline.

- 76 046439

Alternatively, therapeutic compounds can be delivered by other routes, such as intranasal administration, administration by inhalation, or in the form of liposomes, nanocapsules, vesicles, and the like. Compositions for intranasal administration usually take the form of drops or sprays containing liquid forms (solutions, suspensions, emulsions, liposomes, etc.) of the active compounds. Administration by inhalation typically involves the formation of vapors, mists, dry powders or aerosols and again may include solutions, suspensions, emulsions and the like containing the active drugs.

The modes and frequency of administration of the therapeutic compositions described herein, as well as the dose, can vary from individual to individual, and can be easily determined using standard methods. Preferably, between 1 and 100 doses may be administered over a 52-week period. A suitable dose is an amount of compound which, when administered as described above, is capable of killing or inhibiting the growth of cancerous tumors or cancer cells.

Typically, a suitable dose and treatment regimen provides the active compound(s) in an amount sufficient to provide therapeutic and/or prophylactic benefit. In specific embodiments, the dose is 0.1-500 mg, such as about 0.1-50, 0.140, 0.1-20, 0.1-10, 0.2-20, 0.3-15, 0. 4-10, 0.5-1, 0.5-100, 0.5-50, 0.5-30, 0.5-20, 0.5-10, 0.5-5, 1-50, 1-30, 1-20, 1-10, 1-5, 5-50, 5-20, 5-10, 10-100, 20-200, 30-150, 40-100, 50-100, 50- 300, 50-250, 100-300 and 100-250 mg of each active ingredient(s). This response can be monitored by determining improved clinical outcome (eg, reduced inflammation, inhibition of cancer cell growth, more frequent remissions, complete or partial, longer disease-free survival, reduced morbidity, or improvement in the results of one or more standard tests or assays known in the field to assess the status of a disease, condition or disorder) in treated patients compared to untreated patients.

A therapeutic amount of a compound as described herein means an amount effective to produce a desired therapeutic response, for example, an amount effective to reduce inflammation or to delay the growth of a cancer, or to cause a cancer to shrink or not metastasize. If what is administered is not a compound (or compounds) but an enantiomer, prodrug, salt or metabolite of the compound (or compounds), then the term therapeutically effective amount means the amount of such material that produces in the patient the same blood concentration of the compound in question. which is obtained by administering a therapeutically effective amount of the compound itself.

Patients who can be treated using a compound described herein, and pharmaceutically acceptable salts, prodrugs, enantiomers and metabolites of such compounds, in accordance with the methods of the present invention include, for example, patients diagnosed as having any of the diseases or disorders, described herein.

In such methods, pharmaceutical compositions are typically administered to a patient. For the purposes of the invention, patient or subject refers to any warm-blooded animal, preferably a human. The patient may or may not be affected by an inflammatory condition or an autoimmune disorder, or a malignancy. Accordingly, the above pharmaceutical compositions can be used to prevent the development of an inflammatory condition or an autoimmune disorder or a cancer, or to treat a patient affected by an inflammatory condition or an autoimmune disorder or a cancer. The inflammatory condition or autoimmune disorder, or malignancy, can be diagnosed using criteria generally accepted in the art. In the case of a malignant tumor, the pharmaceutical compositions can be administered either before or after surgical removal of primary tumors and/or treatment, such as radiotherapy or the administration of conventional chemotherapeutic drugs, or bone marrow transplantation (autologous, allogeneic or syngeneic).

The compounds or compositions provided herein may be used alone or in combination with one or more additional drugs suitable for the treatment of a particular indication. For example, a compound or composition of the present invention can be administered to a subject who has a cancer or is at risk of developing a cancer, in conjunction with conventional anticancer therapy regimens such as surgery, radiation, chemotherapy and/or bone marrow transplantation (autologous, syngeneic, allogeneic or unrelated) or in combination with conventional therapeutic regimens for the treatment of an inflammatory condition or autoimmune disorder, or malignancy. Compound administration kits may be prepared containing a composition or composition of the subject compound, or an enantiomer, prodrug, metabolite, or pharmaceutically acceptable salt of any of them, together with conventional means for administering the therapeutic ingredient.

- 77 046439

3. Combination therapy.

GPR174 inhibitors (including antagonists, inverse agonists, partial agonists, and negative allosteric modulators) can be used in combination with an inhibitor of an enzyme pathway involved in adenosine production and/or an inhibitor of ATP-adenosine-A2aR and/or A2bR-mediated signaling (such as an A2aR antagonist and/or an A2bR antagonist and/or a CD73 inhibitor and/or a CD38 inhibitor and/or a CD39 inhibitor), and optionally with one or more additional drugs (e.g., therapeutic compounds, compositions, treatments, therapies and/or medical procedures). In such combination therapies, the drugs of the present invention can be formulated with or administered simultaneously with, before, or after one or more other desired therapies. For a particular combination of therapies to be used in a combination regimen, the compatibility of the desired therapeutic agents and/or procedures and the desired therapeutic effect to be achieved are taken into account. It should also be understood that the therapies used may achieve a desired effect for the same condition, disease or disorder, or they may achieve different effects (eg, control of any adverse effects).

In general, for use in treatment, the compounds described herein may be used alone, in the form of mixtures of two or more compounds, or in combination with other agents, compounds and/or pharmaceuticals. Examples of other agents that can be combined with the compounds described herein include agents known to be used to treat inflammatory conditions, autoimmune disorders, or malignancies. Another example of a potential agent for combination with the compounds described herein may include agents for the treatment of different but associated or related symptoms or indications. Depending on the route of administration, the agents can be formulated into suitable compositions to ensure easy delivery. Each component of the combination therapy can be formulated in a variety of ways known in the art. For example, the first and second agents of the combination therapy may be formulated together or separately. The compound of the present disclosure and the additional agent can suitably be administered to a patient at one time or over a series of treatments.

As described herein, in some embodiments, combination therapy can provide synergism and has been proven to be synergistic, i.e., the effect achieved when the active ingredients are used together exceeds the sum of the effects resulting from using the compounds separately. A synergistic effect can be obtained when the active ingredients: (1) are co-formulated and administered or delivered simultaneously in a combined, unit dosage formulation; (2) delivered alternately or in parallel in the form of separate formulations; or (3) administered through some other mode. When delivered in sequential therapy, a synergistic effect can be obtained when the compounds, agents and/or treatments are administered or delivered sequentially, for example, through different injections in separate syringes. Typically, during alternation therapy, an effective dose of each active ingredient is administered sequentially, i.e., serially, while in combination therapy, an effective dose of two or more active ingredients is administered together. Suitable dosages for any of the above co-administered agents are those currently used and may be reduced due to the combined effect (synergism) of the compound herein and other co-administered agents or treatments.

Each combination therapy compound as described herein can be formulated in a variety of ways known in the art. For example, the first and second agents of the combination therapy may be formulated together or separately. Individually or separately formulated products can be packaged together in kit form. Non-limiting examples include, but are not limited to, kits containing, for example, two pills, a pill and a powder, a suppository and a liquid in a vial, two topical creams, etc. The kit may include optional components to facilitate the administration of a single dose to patients, such as powder reconstitution vials, injection syringes, custom IV delivery systems, inhalers, etc. In addition, the unit dose kit may contain instructions for preparing and administering the compositions. The kit can be formulated in the form of a single single dose for a single patient, multiple doses for a specific patient (at a constant dose or where the activity of individual compounds can be changed as therapy progresses); or the kit may contain multiple doses suitable for administration to multiple patients (bulk packaging). The kit components can be assembled into boxes, blister packs, bottles, test tubes, etc. Two or more compounds may be mixed together in a tablet, capsule, or other carrier, or may be separated. In one example, the first compound is contained within the tablet and the second compound is external such that a significant portion of the second compound is released before the first compound is released.

4. Compositions.

For administration to a subject, for example, for the treatment of diseases such as cancer,

- 78 046439 a GPR174 inhibitor is included or formulated, optionally, in combination with at least one of an inhibitor of ATP-adenosine-A2aR- and/or A2bR-mediated signaling (such as an A2aR antagonist, and/or an A2bR antagonist, and/or CD73 inhibitor, and/or CD38 inhibitor, and/or CD39 inhibitor), where the composition includes a pharmaceutical carrier for packaging, storage, transportation and administration. In addition, racemic mixtures, enantiomers, prodrugs of either a racemic mixture or a stereoisomer, a metabolite of either a racemic mixture or a stereoisomer, or a salt of any of them may be included in a formulation or composition, including a pharmaceutical carrier. The compositions contain one or more pharmaceutically acceptable carrier(s) and may also contain other therapeutically active ingredients, as well as adjuvants and other ingredients that may be found in pharmaceutical compositions.

Thus, the compounds of the present invention can be formulated with a pharmaceutically acceptable carrier for administration to a subject. While any suitable carrier known to one skilled in the art may be used in the pharmaceutical compositions of the present invention, the type of carrier may vary depending on the route of administration. The pharmaceutical composition is typically formulated such that the compound in question is present in a therapeutically effective amount, that is, the amount of the compound necessary to achieve the desired effect in treating a subject.

For the preparation of pharmaceutical compositions, pharmaceutically acceptable carriers can be either solid or liquid. Solid form preparations include powders, tablets, pills, capsules, sachets, suppositories and dispersible granules. A solid carrier may be one or more substance(s) that may also act as diluents, flavoring agents, binders, preservatives, tablet disintegrants, or encapsulating material.

In powders, the carrier is a finely divided solid that is mixed with the finely divided active ingredient. In tablets, the active ingredient is mixed with a carrier having the necessary binding properties in suitable proportions and compressed into the desired shape and size.

Suitable carriers for the solid compositions of the present invention include, for example, magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, low melting wax, cocoa butter and the like. Alternatively, the compositions can be formulated using an encapsulating material as a carrier, yielding a capsule in which the active ingredient, in the presence or absence of other carriers, is surrounded by a carrier that is thereby associated with it. Likewise, sachets and lozenges are included. Tablets, powders, capsules, pills, sachets and lozenges can be used as solid dosage forms suitable for oral administration.

In specific embodiments, a GPR174 inhibitor, either in the form of a single agent or in combination with at least one of an A2aR antagonist, an A2bR antagonist, a CD38 inhibitor, a CD39 inhibitor, a CD73 inhibitor, and/or a T-reg attenuating agent, can be dispersed in one or several pharmaceutically acceptable polymers or waxes and, for example, obtained in solid dosage forms. The GPR174 inhibitor or activator can be dispersed in one or more pharmaceutically acceptable polymers through various methods. For example, a solvent-based method, a fusion-fusion method, a hybrid fusion-dissolution method, or other dispersion methods can be used to obtain one or more pharmaceutically active drug substances in a solid dispersion. Both melt and solvent based methods define methods for dissolving one or both of the active ingredient and the polymer.

In one aspect, the solvent-based method uses a solvent, such as water, inorganic solvents, and organic solvents, to dissolve and thoroughly disperse or dissolve the drug and one or more pharmaceutically acceptable polymers. The solvent is later removed by evaporation or other means while the solid drug/polymer dispersion is collected into a solid dosage form. The use of organic solvents may result in the generation of hazardous and toxic waste in the environment. If possible, use water for water-soluble drugs to obtain a dispersion. Other suitable solvents may be, for example, alcohols and acetone for the use of water-insoluble polymers.

The resulting dispersion can be mixed with additional polymers, controlled release agents, binders, lubricant and/or excipients to form solid dosage forms. For example, the resulting dispersion can be mixed with a mixture of polymers, controlled release agents, binders, lubricants and/or excipients by granulation before being compressed into tablets or other solid dosage forms.

In another aspect, the fusion-fusion method comprises melting a GPR174 inhibitor and one or

- 79 046439 several pharmaceutically acceptable polymers together at temperatures equal to or higher than the melting point of either one or more pharmaceutically acceptable polymers and/or an inhibitor or activator. In the fusion-fusion process, the inhibitor or activator and one or more pharmaceutically acceptable polymers can first be crushed and melted in a suitable mixer. The molten mixture is then quickly cooled to form a thickened mass. Alternatively, one or more pharmaceutically acceptable polymers can be melted to a molten state before being mixed homogeneously with the inhibitor or activator. The molten mixture of the inhibitor or activator and one or more pharmaceutically acceptable polymers can be concentrated by reducing the temperature and then prepared in pharmaceutical dosage forms, such as solid dosage form, for example, powder and tablets. For example, the cooled mixture can then be ground to obtain a powder form. Alternatively, the cooled mixture may be milled and mixed with additional excipients, lubricants, or binders and compressed into tablets.

In another aspect, a hybrid fusion-dissolution method can be used. For example, if there is thermal instability and immiscibility between the inhibitor or activator and one or more pharmaceutically acceptable polymers, the inhibitor or activator can first be dissolved in a small amount of solvent and added to the molten pharmaceutically acceptable polymer. The solvent is then evaporated to obtain the product, which is then milled into a solid dosage form, such as a powder form, or compressed into tablets.

Preparations in liquid form include solutions, suspensions and emulsions, for example, solutions or suspensions in water or water/propylene glycol. Aqueous suspensions suitable for oral use can be prepared by dispersing the finely divided compound in water with a viscous material such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose and other well known suspending agents. For parenteral injection, liquid preparations can be dissolved in an aqueous solution of polyethylene glycol. In specific embodiments, the pharmaceutical compositions are formulated into a stable emulsion formulation (eg, a water-in-oil emulsion or an oil-in-water emulsion) or an aqueous formulation, which preferably contains one or more surfactants. Suitable surfactants well known to one skilled in the art can be used in such emulsions. In one embodiment, the composition comprising the subject compound is in the form of a micellar dispersion comprising at least one suitable surfactant. Surfactants that can be used in such micellar dispersions include phospholipids. Examples of phospholipids include: diacylphosphatidylglycerols such as dimyristoylphosphatidylglycerol (DPMG), dipalmitoylphosphatidylglycerol (DPPG) and distearoylphosphatidylglycerol (DSPG); diacylphosphatidylcholines such as: dimyristoylphosphatidylcholine (DPMC), dipalmitoylphosphatidylcholine (DPPC) and distearoylphosphatidylcholine (DSPC); diacylphosphatidic acids such as: dimyristoylphosphatidic acid (DPMA), dipalmitoylphosphatidic acid (DPPA) and distearoylphosphatidic acid (DSPA); and diacylphosphatidylethanolamines such as: dimyristoylphosphatidylethanolamine (DPME), dipalmitoylphosphatidylethanolamine (DPPE) and distearoylphosphatidylethanolamine (DSPE). Other examples include, but are not limited to, ethanolamine derivatives (such as phosphatidylethanolamine, as mentioned above, or cephalin), serine (such as phosphatidylserine), and 3'-O-lysylglycerol (such as 3'-O-lysylphosphatidylglycerol).

Also included in the compositions for use according to the present invention are solid form preparations intended to be converted, immediately prior to use, into liquid form preparations for oral administration. Such liquid forms include solutions, suspensions and emulsions. These preparations may contain, in addition to the active compound, coloring agents, flavoring agents, stabilizers, buffers, artificial and natural sweeteners, dispersing agents, thickeners, solubilizing agents, and the like.

The compositions of the present invention may also be in controlled release or sustained release formulations, as is known in the art, for example, in matrices of biodegradable or non-biodegradable injectable polymer microspheres or microcapsules, liposomes, emulsions, and the like, including compositions for use as subcutaneous depots.

A pharmaceutical preparation comprising a GPR174 inhibitor, either in the form of a single agent or in combination with at least one of an A2aR antagonist, an A2bR antagonist, a CD38 inhibitor, a CD39 inhibitor, a CD73 inhibitor and/or a T-reg attenuating agent, may be contained in a unit dosage form. form. In this form, the drug is divided into unit doses containing appropriate amounts of the active component. The unit dosage form may be a packaged preparation, where the package contains discrete quantities of the drug, such as packaged tablets, capsules and powders in vials or ampoules. Also, the unit dosage form may be a capsule, tablet, sachet or lozenge itself, or may be

- 80 046439 provide the appropriate quantity of any of them in packaged form.

In some embodiments, a pharmaceutical preparation comprising a GPR174 inhibitor, either in the form of a single agent or in combination with at least one of an A2aR antagonist, an A2bR antagonist, a CD38 inhibitor, a CD39 inhibitor, a CD73 inhibitor, and/or a T-reg attenuator, can be formulated for topical, subcutaneous, intradermal, subdermal, subcutaneous or transdermal administration. Topical administration refers to the use of a composition applied to the surface of the skin (eg, the site of a wound, injury, or defect) to exert local action. Accordingly, such topical compositions include pharmaceutical or cosmetic forms in which the composition is applied externally by direct contact with the surface of the skin to be treated, for example, the face, neck, arms, legs and/or torso. Common pharmaceutical or cosmetic forms for this purpose include ointments, liniments, creams, shampoos, lotions, pastes, gels, sprays, aerosols, etc., and may further be applied directly or in patches or impregnated dressings, e.g. depending on the wound/defect and area of skin to be treated. The term ointment includes compositions (including creams) having oily, water-soluble and emulsion-type bases, for example, petroleum jelly, lanolin, polyethylene glycols, as well as mixtures thereof. In some embodiments, a pharmaceutical preparation comprising a GPR174 inhibitor for use in promoting wound healing is formulated in the form of a wound dressing including any of a variety of materials used to cover and protect the wound. Examples include occlusive dressings, adhesive dressings, antiseptic dressings and protective dressings.

In pharmaceutical preparations, the cream is an oil-in-oil or water-in-oil semi-solid emulsion suitable for topical administration. In accordance with the present description, the creams and foams used may also be suitable for use with the drugs described in the present description.

The following examples are intended to be illustrative and not limiting of the invention.

Examples

Example 1.

CRA and CRA Confirmation.

The inventors of the present invention used a CRA similar to that described in US Patent No. 7309576 and O'Dowd et al., J. Biomol. Screen. 12:175-85, 2007, to identify compounds capable of interacting with GPR174 using a library of approximately 370,000 chemical compounds. Briefly described, the CRA assay, described in US Patent No. 7309576, involves the artificial insertion of an NLS into the dopamine D1 receptor (DRD1), which allows the receptor to migrate from the cell membrane to the cell interior, while the binding of antagonists to the receptor retains the receptor on the cell surface , while removal of the ligand allows the receptor to continue internalization from the cell surface. Both antagonists and agonists that selectively bound the receptor retained the receptor at the plasma membrane. GPR174 constructs were prepared for use in a CRA-based compound screening assay. From this screening, the present inventors first identified compounds of formula I-VI as described herein and included them in Table 1. 1.

It should be further noted that the compounds identified by the present inventors as having activity in the CRA assay for GPR174 (i.e., compounds of formula I-VI) specifically interact with GPR174, compared to a reference panel of 68 other GPCRs, against which also screened these compounds. For example, compounds identified as having activity in the CRA assay for GPR174 are found to have no activity when tested in the CRA assay (e.g., in the CRA assay as described in Example 1, when tested at a concentration of 2 μM) for other GPCRs in a reference panel including: muscarinic M1, CCRL2, CMKOR1, GPR3, GPR4, GPR12, GPR15, GPR17, GPR18, GPR19, GPR20, GPR21, GPR22, GPR25, GPR26, GPR27, GPR31, GPR37, GPR37L1, GPR39, GPR43, GPR45 , GPR48, GPR50, GPR52, GPR61, GPR62, GPR63, GPR65, GPR68, GPR78, GPR80, GPR83, GPR85, GPR87, GPR88, GPR101, GPR132, GPR135, GPR139, GPR141, GPR146, GPR148, GPR149, 50, GPR151, GPR152 , GPR153, GPR160, GPR161, GPR162, GPR173, GPR182, GPR183, LGR5, LGR6, MAS1, MRGD, MRGE, MRGF, MRGI4, OPN3, OPN4, OPN5, P2Y8, P2Y10, TAAR6 and TAAR8.

The present inventors have also sought to confirm that the compounds identified using CRA are modulatory compounds. For this purpose, the above-described CRA was used to screen a library for compounds that interact with three control non-orphan receptors, and the results of this screen were compared with the results of screening the same library for compounds that were identified using a fluorimetric imaging plate reader (FLIPR) assay, a commonly used method commonly used to identify compounds that interact with non-orphan GPCRs through Ca signaling. Three non-orphan GPCRs—muscarinic acetylcholine receptor M1 (CHRM1), adrenergic receptor alpha 1a (ADRA1a), and neuropeptide S receptor (NPSR)—were used in a screen against targeted GPCR libraries of 10,000 compounds. The output of these systems

- 81 046439 traces are summarized in table. 2. Thirty top interactors were identified using CRA, and eighty-one top interactors were identified using FLIPR for the CHRM1 receptor; 9 top compounds in CRA and 29 top compounds in FLIPR were identified for the NPS receptor; and 110 best compounds in CRA and 110 best compounds in FLIPR for ADRA1a. All compounds that were identified in the CRA screen were also the top compounds in the FLIPR for the corresponding receptor, indicating that each of the top compounds in the CRA affects receptor signaling.

Table 2. Comparison of CRA and FLIPR using three different receptors, each screened against a GPCR-focused library of 10,000 compounds

GPCR The best connections in FLIPR Best connections in CRA Best connections in CRA, effective in FLIPR CHRM1 81 thirty thirty NPSR 29 9 9 ADRAla BY BY BY

In addition, CRA can also provide relative activities of compounds against the target receptor. This is demonstrated using the CHRM1 receptor and a variety of CHRM1-targeting compounds. As can be seen in FIG. 2, the reported activities of the compounds have the same ranking order as the ranking order of the concentration at which the fluorescent signal is observed in the CRA. As shown in FIG. 5A, for representative compound 1 (group I), compound 2 (group I), compound 3 (group I), compound 4 (group I) and compound 20 (group II) identified as the best in the CRA analysis as compounds that interact with GPR174, a dose response curve against GPR174 is shown, while the same group of compounds showed no activity against CHRM1 (Fig. 5B).

Example 2.

Assays to identify the GPCR signaling pathway.

A variety of assay technologies have been developed to identify the signaling pathway(s) that an activated GPCR uses to elicit its biological response (Siehler, Biotechnology 3:471-83, 2008 and references therein). The Gas family of G proteins stimulates AS activity, while members Gai1-3, Gao, and Gaz of the Gai family of G proteins inhibit AS activity. AS form the second messenger cAMP using ATP as a substrate. A number of cAMP assay kits are available that measure cAMP levels (Siehler, Biotechnology 3:471-83, 2008 and references therein). To detect activation of Gas-coupled GPCRs, increases in cAMP levels are typically measured 15-60 minutes after agonist stimulation (Williams, Nat. Rev. Drug. Discov. 3:125-35, 2004). To measure inhibition of AS by the Gai family of G proteins, cells are typically treated with forskolin, a nonspecific activator of AS, to increase intracellular cAMP levels. The activity of the Gai family of G proteins is determined by measuring the decrease in cAMP levels in forskolin-stimulated cells (Wang et al., Assay Drug. Dev. Technol. 9:522-31, 2011).

The Gaq family of G proteins (Gaq, Ga11, Ga14 and Ga15/16) activate the β isoforms of phospholipase C (PLCP) to produce the second messengers DAG and IP ₃ . IP ₃ is soluble and diffuses through the cytoplasm and interacts with IP ₃ receptors on the endoplasmic reticulum, causing calcium release and an increase in intracellular calcium levels. DAG recruits PKC, which becomes activated in conjunction with calcium ion binding. Cellular assays are available to identify the second messengers IP3 and calcium. Assays to measure inositol phosphate accumulation typically detect inositol monophosphate (IP1), since IP ₃ and IP2 are rapidly degraded by cellular phosphatases (Liu et al., Anal. Biochem. 318:91-99, 2003; Trinquet et al., Anal. Biochem. 358:126-135, 2006). Calcium detection is a well-developed method for monitoring the activation of Gaq-coupled GPCRs. Calcium responses are temporary, and calcium levels usually return to baseline within a few minutes. Calcium assays are based on the response to Ca ²⁺ binding to fluorescent dyes that are loaded into cells (Merit et al., J. Biomol. Screen. 10:780-7, 2005; Xin et al., J. Biomol. Screen. 12 :705-14, 2007).

The G12/13 family of G proteins (Ga 12 and Ga 13) activate Rho-GEF proteins. Rho-GEF proteins catalyze the exchange of GTP for GDP to activate RhoA. RhoA, in turn, activates Rho kinase, which further leads to the activation of ATF2. Assays for directly measuring RhoA activation by Ga12/13 are limited by their lack of quantitativeness, low throughput, and the need for specialized imaging devices (Ren et al., Methods Enzymol. 325:264-72, 2000).

In addition to the signaling pathway activated by GPCRs through activation or inhibition of adenylyl cyclases by Gas and Gai, stimulation of PLC-β by Gaq, and activation of RhoA kinase by Ga12/13, the mitogen-activated protein kinase (MAPK) signaling cascade is activated by G proteins (Siehler , Biotechnology 3:471-83, 2008 and references therein). Activation of the MARK signaling cascade can occur through Gaq via

- 82 046439 Ca ²⁺ -mediated activation of proline-rich tyrosine kinase 2 (Pyk2) or activation of Raf-1 by PKC (Marinissen et al., Trends Pharmacol. Sci. 22:368-76, 2001; Pierce et al., Oncogene 20: 1532-9, 2001). In addition to activation by Gaq of the MaRK signaling cascade, βγ subunits released from Gi-coupled GPCRs activate either receptor tyrosine kinases (RTKs) or non-receptor tyrosine kinases (Src), resulting in activation of the Ras and MARK cascades (Marinissen et al., Trends Pharmacol. Sci 22:368-76, 2001; Pierce et al., Oncogene 20:1532-9, 2001).

As described above, there are numerous assays available to identify the signaling pathway(s) associated with activated GPCRs. However, these conventional assays rely on an agonist to stimulate the receptor. For orphan GPCRs, receptors whose agonists are unknown, these assays are not suitable. Of the approximately 370 human GPCRs that respond to endogenous signals such as peptides, lipids, neurotransmitters, or nucleotides (Vassilatis et al., Proc. Natl. Acad. Sci. USA 100:4903-8, 2003), it is estimated that >120 GPCRs are orphan (Howard et al., Trends Pharmacol. Sci. 22:132-40, 2001; www.iuphar-db.org). Traditional assays require an agonist to activate GPCRs to measure increases in levels of second messenger molecules or to identify components of activated signaling. Although the development of reporter assays and the use of receptor overexpression allows the identification of possible signaling pathways for orphan GPCRs (Bresnick et al., Assay Drug Dev. Technol. 1:239-49, 2003), as described below, it has been observed that overexpression GPCRs can lead to spurious activation of reporter genes. Thus, to obtain an accurate definition of the GPR174 signaling profile, we first identified signaling pathways in reporter assays that are influenced by overexpressed GPR174 and then confirmed these results using compounds identified in the CRA assay as interacting with GPR174.

A reporter assay uses a reporter gene that contains a DNA sequence whose product is synthesized in response to activation of the signal transduction cascade of interest. The DNA sequence contains a transcription factor-specific promoter to control transcription of the reporter gene. A reporter gene ideally has low baseline expression, is specific for a signaling pathway, has a large increase in reporter gene transcription upon activation of the signaling pathway of interest, and can be reproducibly measured (Dinger et al., Molecular Biology in Medicinal Chemistry 73-94, 2004; Schenborn et al., Mol. Biotechnol. 13:29-44. Several reporter genes are commercially available. Firefly luciferase has been identified as a good reporter gene for signaling pathway studies due to its low endogenous activity, high sensitivity, wide dynamic range, and ease of detection (Cheng, Curr. Chem. Genomics 4:84-91, 2010; Dinger et al., Molecular Biology in Medicinal Chemistry 73-94, 2004). Another advantage of firefly luciferase is that it can be measured in the presence of Renilla luciferase. The Renilla luciferase substrate, coelenterazine, is different from the firefly luciferase substrate, D-luciferin. Due to substrate differences, Renilla is used as an internal control for luciferase-based assay systems for mammalian gene expression (Shifera et al. Anal. Biochem. 396:167-72, 2010). In these assays, Renilla luciferase is expressed under the control of a constitutive promoter, and firefly luciferase is expressed under the control of an inducible promoter. This system, developed at Promega (Madison, WI, USA), allows the normalization of measurement results in the firefly luciferase assay, thereby reducing variability that may be caused by experimental differences such as transfection efficiency or toxicity due to transfected gene products and for treating cells with compounds.

To identify a GPCR signaling pathway, synthetic promoters containing only one type of transcription factor binding site are introduced into a reporter gene such that the reporter gene can only be activated by specific signaling components from the activated signaling pathway (Hill et al., Curr Opin. Pharmacol. 1:526-32, 2001; Cheng. Chem. 4:84-91. Reporter constructs containing promoter elements (CRE, SRE, NFAT, AP1, and SRF) associated with firefly luciferase have been developed that can be used to identify and distinguish each of the signaling pathways activated by GPCRs (Stratagene, CA and Promega, WI).

GPCRs coupled to the Gs family of G proteins activate AC to acquire AMP. cAMP activates protein kinase A (PKA) and leads to phosphorylation of CREB. Phosphorylated CREB binds to the cAMP response element (CRE) and activates CRE-dependent promoters. To identify activation by GPCRs of the Gs signaling pathway, CRE-dependent promoters drive expression of the firefly luciferase (Luc) reporter gene. In one example, we used the pCRE-Luc (Stratagene, CA) and pGL4.29 (Promega, WI) CRE-Luc reporter plasmids, which are sensitive to cAMP levels and result in transcriptional activation and increased luciferase synthesis when cAMP levels increase (Table 3). The algorithm is also shown schematically in FIG. 3.

- 83 046439

Table 3. Reporter constructs used to study orphan GPCR signaling pathways

Gs G12/13 Gq Gi Gi Gi Gi No chimeric G proteins Chimeras Gs Chimeras Gq Gal 5/16 CRE-Luc + + + + + API-Luc + + + NFAT-Luc + + + SRE-Luc + + + + SRF-Luc + + + +

GPCRs coupled to the Gq family of G proteins (Gaq, Ga11, Ga14, and Ga16) activate PLCe, which hydrolyzes phosphatidylinositol to produce DAG and IP ₃ . DAG and IP ₃ control the release of calcium from the endoplasmic reticulum. In turn, DAG and calcium control the activity of several PKC isoforms. PKC isoforms activate the FOS and JUN proteins, which in combination bind and activate the activator protein response element 1 (AP1). Calcium also binds and activates calcineurin, which dephosphorylates the transcription factor nuclear factor of activated T cells (NFAT). Dephosphorylated NFAT binds and activates the NFAT response element. To measure PLCa activation by Gq-coupled GPCRs, we used AP1-dependent and NFAT promoters to drive Luc reporter gene expression. Specifically, we used the AP1-Luc reporter construct pAP1-Luc (Stratagene, CA) and the NFAT-Luc reporter constructs pNFAT-Luc and pGL4.30 (Stratagene, CA and Promega, WI). Activation of AP1-Luc and NFAT-Luc reporter constructs results in transcriptional activation of firefly luciferase (Stratagene, CA and Promega, WI). In addition to stimulating AP1-Luc and NFAT-Luc reporter constructs through activation of the Gs family of G proteins, signaling components from Gq-activated GPCRs can activate MaRK, RhoA, and Gs signaling pathways. Reporter constructs for the MaRK, RhoA, and Gs signaling pathways are also activated by Gq-coupled GPCRs (Siehler S., Biotechnology 3:471-83, 2008 and references therein; Mao et al., J. Biol. Chem. 273: 118-27, 1998; Hill et al., Curr. Pharmacol. 1:526-532, 2001; Promega, Cell notes 16:22-25, 2006). Reporter constructs for these signaling pathways, pCRE-Luc, pGL4.29, pSRE-Luc, pGL4.33, and pGL4.34 (Stratagene, CA and Promega, WI) are used in reporter assays to identify Gq signaling by GPCRs (Table .3).

GPCRs coupled to the Gi family of G proteins (Gail-3, Gao and Gaz) inhibit AC activity, which suppresses cAMP formation (Siehler S., Biotechnology 3:471-83, 2008). Because of the difficulty of measuring inhibition of AC activity, we used chimeric G proteins that retarget Gi-coupled GPCRs to activate either PLC or AC (Coward et al., Anal. Biochem. 270:242-8, 1999). Chimeric G proteins were originally developed after the observation that the carboxy terminus of the G protein is involved in receptor recognition while the rest of the G protein is involved in effector activation. Because of the ease of measuring the activation of signaling molecules in the Gq and Gs pathways, and the difficulty of measuring the activation of Gi-coupled GPCRs, chimeric G for Gq proteins have been developed in which the last five amino acids from Gaq are replaced with the last five amino acids from either Gai or Gao for formation of chimeric G proteins with Gqi5 or Gqo5 (Coward et al., Anal. Biochem. 270:242-8, 1999; Molecular Devices, CA). Chimeric G proteins with Gqi5 and Gqo5 are capable of retargeting Gi-coupled GPCRs to activate phospholipase C. Using chimeric G proteins with Gq, GPCRs that normally inhibit AS can now be measured using reporter constructs that measure Gaq activation. Similarly, chimeric G proteins for Gs are prepared in which the last thirteen carboxy-terminal amino acids of Gas are replaced with the last thirteen carboxy-terminal amino acids of any of Gat, Gai, Gao or Gaz to form chimeric G proteins Gas-t, Gas-i, Gas -o and Gas-z. The chimeric G proteins Gas-t, Gas-i, Gas-o, and Gas-z are capable of retargeting Gi-coupled GPCRs to activate AS. Using chimeric G proteins with Gs, GPCRs that normally inhibit AS can now be measured using reporter constructs that measure Gas activation. Just as chimeric G proteins are useful tools for coupling Gi-activated GPCRs to other signaling pathways that are more suitable for measurement, the diverse G proteins Ga 15 and Ga 16 have been reported to couple a wide variety of GPCRs to PLCe (Offermanns et al. , J. Biol. Chem. 270:15175-80, 1995). Ga15/16, in addition to chimeric G proteins with Gq, are useful tools for coupling Gi GPCRs to the Gq signaling pathway. Activation of Gi-coupled GPCRs does not activate CRE, SRE, NFAT, AP1, or SRF reporter constructs. Although it has been published that Gi-coupled GPCRs activate the MARK signaling cascade through

- 84 046439 units of βγ, the present inventors do not observe activation of the MARK-dependent reporter construct SRE-Luc or any of the reporter constructs after activation of Gi by GPCR (Table 3). When Gi-coupled GPCRs are activated using a co-expressed Gs-chimeric G protein, Cre-Luc is stimulated through AC activation via the Gs-chimeric G protein. Similarly, when Gi-coupled GPCRs are activated using co-expressed Gq chimeric G proteins or diverse G proteins with Ga16, the reporter constructs CRE, SRE, NFAT, AP1 and SRF are stimulated through PLCe activation via G protein chimeras with Gq or Ga16 ( table 3).

The G12/13 family of G proteins (Ga12 and Ga13) activate the Rho-GEF protein, which catalyzes the exchange of GTP for GDP to activate RhoA-GTPase. RhoA, in turn, activates Rho kinase, which further leads to the activation of ATF2. ATF2 binds the serum factor response element (SRE) and activates SRF- and SRE-dependent promoters (Siehler, Biotechnology 3:471-83, 2008 and references therein). Reporter constructs for these signaling pathways, pSRE-Luc, pGL4.33, and pGL4.34 (Stratagene, CA and Promega, WI) are used in reporter assays to identify Ga12/13 GPCR signaling (Table 3).

Orphan GPCRs do not have an agonist and rely on the original activity of the orphan GPCR to activate the signaling pathway. GPCRs in their initial state are in equilibrium between active and inactive states (Bond et al., Trends Pharmacol. Sci. 27:92-96, 2006). The degree of initial activation depends on the receptor, with some GPCRs being constitutively active in the absence of the agonist and other GPCRs having very low levels of initial activity (Hebert, Biochem. Cell. Biol. 76:1-11, 1998). Due to the original GPCR activity, GPCRs that are overexpressed in transient transfections can activate their expected signaling pathways in the absence of an activating ligand (Bresnick et al., Assay Drug Dev. Technol. 1:239-249, 2003). Reporter assays can be used as a tool to study the signaling pathways of orphan GPCRs by enhancing the original activity through reporter gene induction. However, as discussed below, reporter assays based solely on transient transfection-based GPCR overexpression may not be reliable for accurately determining the signaling profile of a GPCR of interest, such as a class A GPCR (eg, GPR174).

High-throughput screens of anti-GPCR compound libraries have identified compounds that interact with GPCRs and modulate GPCR activity. Traditional high-throughput screens typically involve assays that displace a radiolabeled ligand or antagonize the functional activator response to a GPCR (Xiao, Comb. Chem. High Throughput Screen. 11:195-215, 2008). It is now known that GPCR interacting compounds modulate the initial activity of the receptor in various ways (Bond et al., Trends Pharmacol. Sci. 27:92-96, 2006). Agonists convert the receptor conformation to an active state, inverse agonists convert the receptor conformation to an inactive conformation, and antagonists do not affect the initial equilibrium of the receptor. By overexpressing GPCRs in a cell-based system, the GPCR signaling pathway can initially be predicted using reporter constructs as described above. Compounds identified in high-throughput assays as interacting with GPCRs can be tested to identify the natural compound by modulating the predicted parent signal by the overexpressed GPCR, and thus confirm the GPCR signaling profile originally predicted by overexpressing the GPCR in the reporter assay. If the signal is enhanced, the compound is an agonist, if the signal is inhibited, the compound is an inverse agonist, and if the compound has no effect on the signal, it is an antagonist or allosteric modulator. In our reporter assay system, we used, among others, the β-2-adrenergic (ADBR2) GPCR and several compounds that interact with this receptor to illustrate the expected effects on signaling (Fig. 4A).

The use of compounds that interact with a specific GPCR is essential to accurately determine the receptor signaling profile. As shown below, overexpression of GPCRs can lead to spurious activation of reporter genes. Thus, only reporter assays susceptible to influence of both the overexpressed receptor and its corresponding compounds provide accurate profiling of GPCR signaling.

The results of experiments using ADBR2 highlight the need to validate the results of signaling pathway assays through transient transfection using junction-mediated modulation.

The β-2-adrenergic (ADBR2) GPCR, class A, is known to stimulate adenylate cyclase activity through activation of the Gs signaling pathway (Wenzel-Seifert, K. et al., Biochem Pharmacol, 64: 9-20 (2002)). A compound (ICI118551) known to act as an inverse agonist on ADBR2 has been published (Hothersall J.D. et al., Br J Pharmacol., 164(2):317-31 (2011)). An experiment was performed to compare the results of (1) ADBR2 overexpression in a reporter assay using different

- 85 046439 reporter constructs and (2) overexpression of ADBR2 in the presence/absence of compound ICI 118551, known to interact with ADBR2 and act as an inverse agonist, to determine whether transfection studies alone can accurately identify the ADBR2 signaling pathway.

Reporter analyses.

Reporter assays were performed by transient transfection with increasing amounts of the ADBR2 construct pCMV6-XL4-ADBR2 (SC107904, Origene, MD), pNFAT-Luc, pGL4.30, pAP1Luc, pCRE-Luc, pGL4.29 (100 ng), pSRE-Luc, pGL4.33 or pGL4.34 and TK-renilla as an internal control (0.3 ng) (Promega) with Lipofectamine 2000 (2 μg) (Invitrogen, CA), according to the manufacturer's instructions. The DNA/Lipofectamine 2000 mixture (100 μl) was incubated at room temperature for 30 min and then added to 24-well culture plates (Corning, NY) containing 70-90% confluent, cultured cells (HEK293 or CHO cell lines). ATCC, VA)), in 500 µl of media. The cells were further incubated for 4-6 hours at 37°C and then 500-1000 μl of additional media was added to each well of transfected cells. The next day, the media was removed and the cells were lysed. Luciferase and renilla activities were measured using a dual luciferase assay system (Promega, WI), according to the manufacturer's instructions. Lysis buffer (100 μl) was added to the cells and incubated at room temperature for 20-30 min to allow cell lysis. Luciferase and renilla activities were measured in the lysate (10 μl) using LARII and Stop&Glo reagent (50 μl each) using SpectraMax L (Molecular Devices, CA). Data were analyzed using either Excel (Microsoft, WA) or Prism 4.03 (GraphPad, CA).

Compound analyses.

Compound assays were performed by transiently transfecting the ADBR2 construct pCMV6-XL4-ADBR2 (SC107904, Origene, MD), pNFAT-Luc, pGL4.30, or pGL4.29 (3000 ng), and TK-renilla as an internal control (10 ng; Promega ) with Lipofectamine 2000 (60 mcg) (Invitrogen, CA), according to the manufacturer's instructions. The DNA/Lipofectamine 2000 mixture (3000 μl) was incubated at room temperature for 30 min and then added to a 10 cm culture dish (Corning, NY) containing 70-90% confluent, cultured cells (HEK293 or CHO cell lines (ATCC) , VA)), in 15 ml of medium. The cells were further incubated for 4-6 hours at 37°C and then the media was removed and the cells detached using 2 ml trypsin (Life Technologies, CA). Media (26 ml) was added to detached cells, and 120 μl of transfected cells was transferred to each well in a 96-well plate. Increasing concentrations of ICI 118551 or pirenzepine in DMSO (1.2 μL) were added to the cells after seeding into a 96-well plate. The cells were further incubated at 37°C for various periods of time. The media was removed and the cells were lysed. Luciferase and renilla activities were measured using a dual luciferase assay system (Promega, WI), according to the manufacturer's instructions. Lysis buffer (25 μl) was added to the cells and incubated at room temperature for 20–30 min to allow cell lysis. Luciferase and renilla activities were measured in the lysate (10 μl) using LARII and Stop&Glo reagent (50 μl each) using SpectraMax L (Molecular Devices, CA). Data were analyzed using either Excel (Microsoft, WA) or Prism 4.03 (GraphPad, CA).

Overexpression of ADBR2 in the reporter assay resulted in strong stimulation of the CRE-Luc reporter from Stratagene (pGL4.29), as expected (Fig. 4B). However, NFAT-Luc reporters were also stimulated, although to a lesser extent (Fig. 4C). When the compound ICI 118551, known to act as an inverse agonist on ADBR2, was added to cells transfected with ADBR2 and CRE-Luc, ICI118551 was determined to specifically inhibit ADBR2 activity using the CRE-Luc reporter, compared to a nonspecific control compound (pirenzepine) , as shown in Fig. 4D. In contrast, when compound ICI 118551 was added to cells transfected with ADBR2 and NFAT-luc, it was determined that compound ICI 118551 did not modulate the activity of NFAT-Luc reporters using ADBR2 overexpression (Fig. 4E), indicating that activation of NFAT-luc reporters Luc via transiently transfected ADBR2 was nonspecific.

This experiment shows that overexpression of GPCRs can lead to spurious activation of reporter genes. Thus, modulation by a GPCR compound is necessary to confirm the accuracy of the signaling pathway originally predicted based on the activation of reporter constructs in an assay based solely on GPCR overexpression.

Example 3.

Identification of GPR174 signaling pathways.

To determine which GPR174 signaling pathways may be activated, GPR174 was tested using the reporter constructs pAPI-Luc, pNFAT-Luc, pGL4.30, pCRE-Luc, pGL4.29, pSRE-Luc, pGL4.33, and pGL4.34 (Stratagene, CA and Promega, WI). The algorithm used is shown in Fig. 3.

Reporter assays were performed by transient transfection with increasing amounts of

- 86 046439 properties of the GPR174 construct pCMV6-XL4-GPR174 (SC104514, Origene, MD), pNFAT-Luc, pGL4.30, pAPl-Luc, pCRE-Luc, pGL4.29 (100 ng), pSRE-Luc, pGL4.33 or pGL4.34 and TK-renilla as an internal control (0.3 ng, (Promega) with Lipofectamine 2000 (2 μg, Invitrogen CA), according to the manufacturer's instructions. The DNA/Lipofectamine 2000 mixture (100 μl) was incubated at room temperature temperature for 30 min and then added to 24-well culture plates (Corning, NY) containing 70-90% confluent, cultured cells, typically HEK293 or CHO cell lines (ATCC, VA), in 500 μl of media. The cells were further incubated for 4-6 hours at 37°C and then 5,000 1,000 μl of additional media was added to each well of the transfected cells. The next day, the media was removed and the cells were lysed using a dual luciferase assay system. Promega, WI) according to the manufacturer's instructions Typically, lysis buffer (100 μl) was added to the cells and incubated at room temperature for 20-30 min to allow cell lysis. Luciferase and renilla activities were measured in the lysate (10 μl) using LARII and Stop&Glo reagent (50 μl each) using SpectraMax L (Molecular Devices, CA). Data were analyzed using either Excel (Microsoft, WA) or Prism 4.03 (GraphPad, CA).

Overexpression of GPR174 strongly activated two Cre-luc reporter constructs (Table 4), suggesting that GPR174 activates the Gs signaling pathway to activate adenylate cyclase. The data also show that GPR174 does not activate the Gq family or G12/13 G proteins. As described herein, GPR174 was determined not to activate the Gi signaling pathway.

Table 4. Fold of stimulation of reporter constructs using GPR174 titration

GPR174 (ng DNA) S-Cre S-NFAT S-Sre AR-1 R-Cge P-NFAT P-Sre Srf 0 ng 1 1 1 1 1 1 1 1 5 ng 40 2.1 0.9 2.0 52 2.2 1.0 0.9 25 ng 52 1.8 0.8 2.6 145 8.0 1.4 1.1 100 ng 93 2.2 1.0 2.6 252 4.2 1.5 1.2 250 ng 102 2.4 1.1 3.3 298 3.2 1.6 1.2

Note: S denotes reporter constructs from Stratagene. P stands for reporter constructs from Promega.

To observe whether GPR174 could be coupled to the Gi signaling pathway in addition to the Gs signaling pathway, GPR174 was tested using chimeric G proteins with Gq and Gs as follows.

Gi reporter assays using chimeric Gs proteins were performed by transient transfection with increasing amounts of the GPR174 construct pCMV6-XL4-GPR174 (SC104514, Origene, MD), varying amounts of Gas-t, Gas-i, Gas-o, or Gas-z, pCRE-Luc or pGL4.29 (100 ng) and TK-renilla as an internal control (0.3 ng) with Lipofectamine 2000 (2 μg) (Invitrogen, CA), according to the manufacturer's specifications.

The DNA/Lipofectamine 2000 mixture (100 μl) was incubated at room temperature for 30 min and then added to 24-well culture plates (Corning, NY) containing 70-90% confluent, cultured cells, typically HEK293 or CHO (ATCC, VA), in 500 µl of media. The cells were further incubated for 4-6 hours at 37°C and then 500-1000 μl of additional media was added to each well of transfected cells. The next day, the media was removed and the cells were lysed. Luciferase and renilla activities were measured using a dual luciferase assay system (Promega, WI), according to the manufacturer's instructions. Typically, lysis buffer (100 μl) was added to the cells and incubated at room temperature for 20–30 min to allow cell lysis. Luciferase and renilla activities were measured in the lysate (100 μl) using LARII and Stop&Glo reagent (50 μl each) using SpectraMax L (Molecular Devices, CA). Data were analyzed using either Excel (Microsoft, WA) or Prism 4.03 (GraphPad, CA). Gi reporter assays using chimeric Gq proteins were performed by transiently transfecting increasing amounts of the GPR174 construct pCMV6-XL4-GPR174, varying amounts of either RD-PGQI5 (Gqi5) or RD-PGQO5 (Gqo5) (Molecular Devices, CA), pNFAT-Luc, pGL4.30, pAP1-Luc, and TK-renilla as internal control (0.3 ng, Promega) with Lipofectamine 2000 (2 μg) (Invitrogen, CA), according to the manufacturer's instructions. The DNA/Lipofectamine 2000 mixture (100 μl) was incubated at room temperature for 30 min and then added to 24-well culture plates (Corning, NY) containing 70-90% confluent, cultured cells, typically HEK293 or CHO (ATCC, VA), in 500 µl of media. The cells were further incubated for 4-6 hours at 37°C and then 500-1000 μl of additional media was added to each well of transfected cells. The next day, the media was removed and the cells were lysed. Luciferase and renilla activities were measured using a dual luciferase assay system (Promega, WI), according to the manufacturer's instructions. Typically, lysis buffer (100 μl) was added to the cells and incubated at room temperature for 20-30 min to allow cell lysis. In the lysate (10 μl), the activity of luciferase and renilla was measured using

- 87 046439 using LARII and Stop&Glo reagent (50 µl using SpectraMax L (Molecular Devices, CA). Data were analyzed using either Excel (Microsoft, WA) or Prism 4.03 (GraphPad, CA).

As shown in table. 5, titration of GPR174 using the chimeric Gq proteins Gaq-i5 and Gaqo5 did not significantly activate AP1, NFAT, or SRE reporters. Similarly, titration of GPR174 with the chimeric Gs G, Gas-t, Gas-i, Gas-o, or Gas-z proteins did not further activate the Cre reporter compared with the absence of the Gs G chimeric protein (Table 6). These results indicate that GPR174 is not coupled to the Gi signaling pathway.

Table 5. Fold stimulation of reporter constructs using titration of GPR174 and either Gqi5 or Gqo5

GPR174 (ng DNA) AP1Gqi5 AP1- Gqo5 APIcontrol NFATGqi5 NFATGqo5 NFATcontrol SreGqi5 SreGqo5 SREcontrol 0 ng 1 1 1 1 1 1 1 1 1 5 ng 2.1 2.2 1.9 1.7 0.8 2.6 1.3 1.6 1.1 25 ng 3.7 4.6 2.2 2.1 1.1 2.6 1.5 2.0 1.3 100 ng 3.1 4.4 1.7 2.0 1.3 2.4 2.1 2.7 1.5 250 ng 2.6 4.3 2.1 1.6 1.1 2.3 2.6 2.7 1.6

Table 6. Fold of stimulation of the Cre reporter construct using titration of GPR174 and either Gs-t, Gs-o, Gs-1, Gs-z, or no Gs chimera

GPR174 (ng DNA) Cre/Gs-t Cre/Gs-o Cre/Gs-i Cre/Gs-z Cre/absence of Gs chimera 0 ng 1 1 1 1 1 5 ng 8.8 5.6 101 2.2 134 25 ng 13.7 19.3 184 3.7 198 100 ng 16.9 22.4 263 4.9 270 250 ng 19.7 20.9 259 3.7 210

Overall, the results described in the reporter assays described above indicate that GPR174 stimulates the Gs signaling pathway and is not coupled to the Gi, Gq, or G12/13 signaling pathways.

Example 4.

Functional interaction of identified compounds.

The reporter assays described above in Example 3 indicate that GPR174 stimulates the Gs signaling pathway. Representative compounds from each of the chemical groups I-VI (shown in Table 1) that were identified as interacting with GPR174 in the CRA assay were tested in the reporter assay and to determine the specificity of GPR174 signaling and the nature of the GPR174 interacting compound.

Reporter analyses.

Compounds were tested in a reporter system using increasing concentrations of GPR174 interacting compounds. GPR174 construct pCMV6-XL4-GPR174 or ADBR2 reporter of interest, pCRE-Luc or GL4.29 (3000 ng), and TK-renilla as internal control (10 ng) with Lipofectamine 2000 (60 μg) (Invitrogen, CA) , in accordance with the manufacturer's instructions.

The DNA/Lipofectamine 2000 mixture (3000 μl) was incubated at room temperature for 30 min and then added to a 10 cm culture dish (Corning, NY) containing 70-90% confluent, cultured cells, typically HEK293 or CHO cell lines (ATCC, VA), in 15 ml of media. The cells were further incubated for 4-6 hours at 37°C and then the media was removed and the cells detached using 2 ml trypsin (Life Technologies, CA). Media (26 ml) was added to detached cells, and 120 μl of transfected cells was transferred to each well in a 96-well plate. Increasing concentrations of each compound in DMSO (1.2 μL) were added to the cells after seeding into a 96-well plate. The cells were further incubated at 37°C for various periods of time. The media was removed and the cells were lysed. Luciferase and renilla activities were measured using a dual luciferase assay system (Promega, WI), according to the manufacturer's instructions. Typically, lysis buffer (25 μl) was added to the cells and incubated at room temperature for 20–30 min to allow cell lysis. Luciferase and renilla activities were measured in the lysate (10 μl) using LARII and Stop&Glo reagent (50 μl each) using SpectraMax L (Molecular Devices, CA). Data were analyzed using either Excel (Microsoft, WA) or Prism 4.03 (GraphPad, CA).

Each of compounds 1-59 interacting with GPR174, as shown in table. 1, titrated against GPR174 and the control receptor, ADBR2. ADBR2 is a GPCR similar to GPR174 that activates the Gs signaling pathway.

Results of a signaling assay titrating human GPR174 using pCRE-Luc and increasing concentrations of compounds shown in Table. 1, are presented in table. 1 (values

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EC50 and effect multiplicity). As shown in table. 1, in group I, compounds 1, 2, 5-18 and 53 are inverse agonists, and compounds 3-4 are not found to be modulators in signaling assays and are thus characterized as either antagonists or allosteric modulators of GPR174 . As further described in this example, Compound 4 was determined to indeed compete with the GPR174 agonist LysoPS and was thus confirmed to be a GPR174 antagonist. In group II, both compounds 19 and 20 were found to be non-modulators in the signaling assay and were thus characterized as either antagonists or allosteric modulators of GPR174. In group III, compound 21 was confirmed to be a non-modulator in the signaling assay and was thus characterized as a GPR174 antagonist or allosteric modulator. In group IV, compounds 22-55 are inverse agonists. In group V, compound 56 was confirmed to be an inverse agonist. In group VI, compounds 57 and 58 were found to be antagonists or allosteric modulators of GPR174.

Signaling assay results for representative GPR174 interacting compounds are presented in FIG. 6-20.

As shown in FIG. 6A, compound 1 (group I) inhibited the Gs pathway in the presence of GPR174, but did not inhibit the Gs pathway in the presence of ADBR2 (Fig. 6B), indicating that compound 1 is a specific inverse agonist of GPR174.

As shown in FIG. 7A, compound 2 (group I) inhibited the Gs pathway in the presence of GPR174, but did not inhibit the Gs pathway in the presence of ADBR2 (Fig. 7B), indicating that compound 2 is a specific inverse agonist of GPR174.

As shown in FIG. 5A and in table. 1, compound 4 shows a dose response curve against GPR174 in the CRA assay. As shown in FIG. 8A, compound 4 (group I) did not modulate the Gs pathway in the presence of GPR174 and did not modulate the Gs pathway in the presence of ADBR2 (Fig. 8B), thus compound 4 is characterized as a GPR174 antagonist.

As shown in FIG. 9A, compound 6 (group I) inhibited the Gs pathway in the presence of GPR174, but did not inhibit the Gs pathway in the presence of ADBR2 (Fig. 9B), indicating that compound 6 is a specific inverse agonist of GPR174.

As shown in FIG. 10A, compound 7 (group I) inhibited the Gs pathway in the presence of GPR174, but did not inhibit the Gs pathway in the presence of ADBR2 (Fig. 10B), indicating that compound 7 is a specific inverse agonist of GPR174.

As shown in FIG. 11A, compound 10 (group I) inhibited the Gs pathway in the presence of GPR174, but did not inhibit the Gs pathway in the presence of ADBR2 (Fig. 11B), indicating that compound 10 is a specific inverse agonist of GPR174.

As shown in FIG. 12A, compound 11 (group I) inhibited the Gs pathway in the presence of GPR174 but did not inhibit the Gs pathway in the presence of ADBR2 (Fig. 12B), indicating that compound 11 is a specific inverse agonist of GPR174.

As shown in table. 1, compound 19 has activity in the CRA assay. As shown in FIG. 13A, compound 19 (group II) did not modulate the Gs pathway in the presence of GPR174 and did not modulate the Gs pathway in the presence of ADBR2 (Fig. 13B), thus compound 19 is characterized as an antagonist or allosteric modulator of GPR174.

As shown in table. 1, compound 21 has activity in the CRA assay. As shown in FIG. 14A, compound 21 (group III) did not modulate the Gs pathway in the presence of GPR174 and did not modulate the Gs pathway in the presence of ADBR2 (Fig. 14B), thus compound 21 is characterized as an antagonist or allosteric modulator of GPR174.

As shown in FIG. 15A, compound 22 (group IV) inhibited the Gs pathway in the presence of GPR174, but did not inhibit the Gs pathway in the presence of ADBR2 (Fig. 15B), indicating that compound 22 is a specific inverse agonist of GPR174.

As shown in FIG. 16A, compound 23 (group IV) inhibited the Gs pathway in the presence of GPR174, but did not inhibit the Gs pathway in the presence of ADBR2 (Fig. 16B), indicating that compound 23 is a specific inverse agonist of GPR174.

As shown in FIG. 17A, compound 31 (group IV) inhibited the Gs pathway in the presence of GPR174, but did not inhibit the Gs pathway in the presence of ADBR2 (Fig. 17B), indicating that compound 31 is a specific inverse agonist of GPR174.

As shown in FIG. 18A, compound 33 (group IV) inhibited the Gs pathway in the presence of GPR174, but did not inhibit the Gs pathway in the presence of ADBR2 (Fig. 18B), indicating that compound 33 is a specific inverse agonist of GPR174.

As shown in FIG. 19A, compound 36 (group IV) inhibited the Gs pathway in the presence of GPR174, but did not inhibit the Gs pathway in the presence of ADBR2 (Fig. 19B), indicating that compound 36 is a specific inverse agonist of GPR174.

As shown in FIG. 20A, compound 42 (group IV) inhibited the Gs pathway in the presence of GPR174, but did not inhibit the Gs pathway in the presence of ADBR2 (Fig. 20B), indicating that compound 42 is a specific inverse agonist of GPR174.

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Compound 4 was confirmed to be a GPR174 antagonist.

To confirm that GPR174-interacting compound 4 is an antagonist of GPR174-mediated Gs signaling, the GloSensor assay was performed using increasing concentrations of compound 4 in the presence or absence of a fixed concentration (1 μM) of the GPR174 agonist LysoPS as follows.

The GloSensor assay was performed by transiently transfecting the GPR174 construct, pCMV6XL4-GPR174, or ADBR2, pGlo22 (300 ng) (Promega) with Lipofectamine 2000 (60 μg) (Invitrogen, CA), according to the manufacturer's instructions. The DNA/Lipofectamine 2000 mixture (3000 μl) was incubated at room temperature for 30 min and then added to a 10 cm culture dish (Corning, NY) containing 70-90% confluent, cultured cells, typically HEK293 or CHO cell lines (ATCC, VA), in 15 ml of media. The cells were further incubated for 4-6 hours at 37°C and then the media was removed and the cells detached using 2 ml trypsin (Life Technologies, CA). Media (26 ml) was added to detached cells, and 100 μl of transfected cells was transferred to each well in a 96-well plate. Cells were further incubated at 37°C overnight. The next day, the media was removed and 100 μl of equilibration media (Promega) was added to each well according to the manufacturer's instructions. Cells were then incubated at room temperature for 2 hours, and cell plates were preread using SpectraMax L (Molecular Devices, CA). After prereading, increasing concentrations of each compound in DMSO (1.0 μl) were added to the cells. Cell plates containing compound were read immediately after addition of compound and at various time points up to 30 minutes after addition of compound using SpectraMax L (Molecular Devices, CA). Data were analyzed using either Excel (Microsoft, WA) or Prism 4.03 (GraphPad, CA).

As shown in FIG. 21A, compound 4 does not modulate the Gs pathway in the presence of GPR174, but competes with the GPR174 agonist LysoPS. As shown in FIG. 21B, neither compound 4 nor LysoPS modulated Gs signaling in the presence of ADBR, thus confirming that compound 4 is a specific antagonist for GPR174.

Based on these results, compounds 1, 2, 5-18 and 22-55 are characterized as GPR174 inverse agonists. Compounds 3, 19–21 and 57–58 are characterized as antagonists or allosteric modulators of GPR174. Compound 4 is characterized as a GPR174 antagonist. The modulation of GPR174 signaling observed in CRE-Luc reporter assays indicates that GPR174 signals through Gs. As described in this example, the present inventors have clearly determined that compounds that operably interact with GPR174 modulate Gs signaling through GPR174.

Example 5.

Expression profile of gpr174 in human tissues by RT-PCR.

The expression profile of GPR174 was analyzed in human tissues as follows.

Human normal cDNA array was purchased from OriGene (cat. #HMRT103).

Quantitative PCR (q.|PCR) was performed for each cDNA sample in four replicates using GAPDH (normalization control) and GPR174 specific primers. Fig. 22 graphically illustrates the relative abundance of GPR174 transcript in human tissues as measured by c.|PCR. As shown in FIG. 22, GPR174 is highly expressed in a variety of tissues, including the thymus, lymph nodes, spleen, and bone marrow.

To determine the expression profile of GPR174 in human lymphoid cells, human neutrophils, dendritic cells, B cells, CD4+ T cells and CD8+ T cells were purchased from Astarte Biologies. RNA was isolated using the PureLink RNA Micro Scale kit (Life Technologies). cDNA was prepared from total RNA using the Superscript III First-Strand Synthesis Kit (Life Technologies) using random primers. Quantitative PCR (q.|PCR) was performed for each cDNA sample in four replicates using GAPDH (normalization control) and GPR174 specific primers. Fig. 23 graphically illustrates the relative abundance of GPR174 transcript in human lymphoid cells as measured by c.|PCR. As shown in FIG. 23, in human lymphoid tissues, GPR174 is expressed at high levels in naive B and T cells, and especially in regulatory T cells (see Barnes et al., 2015, above).

Example 6.

This example shows that representative GPR174 inhibitory compound 10 reduces the proportion of highly suppressive T-regs (FoxP3+Helios ⁺ ) in cultured human PBMCs.

The following experiments were performed to analyze whether representative GPR174 inhibitory compound 10 (Group I) was able to modulate regulatory T cell (Treg) behavior.

An experiment was performed with human peripheral blood mononuclear cells (PBMCs) to test T-reg formation and survival using the methods described in Gavin et al., Proc Natl Acad Sci 103:6659-664, 2006, the contents of which are given herein as

- 90 046439 links.

Briefly, human PBMCs obtained from a volunteer donor were adjusted to a density of 2 x 10 ⁶ cells/ml, stimulated with 100 ng/ml anti-CD3 antibody and 100 ng/ml anti-CD28 antibody, and treated with 100 μM GPR174 inverse agonist compound 10 (shown in Table 1) or 100 units/ml IL-2. A fraction of cells was selected for staining on day 3 after stimulation, while the remaining cells were diluted 1:4 with fresh medium and used for staining on day 7 after stimulation. To determine the proportion of natural T-reg (nT-reg) cells (FoxP3'Helios'). PBMCs were washed, stained with fluorescently labeled anti-CD4 antibodies, fixed, permeabilized, and stained with a mixture of fluorescent anti-FoxP3 and anti-Helios antibodies. Labeled cells were analyzed by flow cytometry against populations stained for these three markers. Each experiment was performed in three replicates and repeated five times.

The effect of compound 10 on IL-2 and IFN-γ production in human PBMC was determined as follows. PBMCs from a single donor were adjusted to a density of 2 x 10 ⁶ cells/ml, stimulated with 100 ng/ml anti-CD3 and 100 ng/ml anti-CD28, and treated with 1 μM, 3 μM, or 10 μM compound 10. Supernatant from these cells were collected on day 2 post-stimulation, and IL-2 and IFN-γ levels were determined by ELISA assay using Ready-SET-Go ELISA kits from Affimetrix Ebioscience, according to the manufacturer's instructions.

Fig. 24A graphically illustrates the percentage of FoxP3+Helios- cells in the CD4+ cell population at day 3 post-stimulation in PBMC cultures treated with vehicle, compound 10, or IL-2 (p=0.03 for compound 10 vs. vehicle; p=0.003 for IL-2 against the carrier). Fig. 24B graphically illustrates the percentage of FoxP3'Helios' cells in the D4+ cell population at day 7 post-stimulation in PBMC cultures treated with vehicle, compound 10, or IL-2 (p=0.01 for compound 10 vs. vehicle; p=0.01 for IL -2 against the wearer). As shown in FIG. 24A, GPR174 inverse agonist compound 10 significantly increased the proportion of conventional (FoxP3+Helios-) T cells; while on day 7, compound 10 significantly reduced the content of double positive (FoxP3'Helios') nT-reg cells (Fig. 24B).

Fig. 25 graphically illustrates the amount of IL-2 in culture supernatants (fold over vehicle levels) on day 2 post-stimulation in PBMC cultures treated with vehicle or Compound 10 (1, 3 or 10 μM). Fig. 26 graphically illustrates the amount of IFN-γ in culture supernatants (fold over vehicle levels) on day 2 post-stimulation in PBMC cultures treated with vehicle or Compound 10 (1, 3, or 10 μM). As shown in FIG. 25, compound 10 increased IL-2 levels in a dose-dependent manner and at concentrations as low as 1 μM. Similar effects, showing increased levels of IL-2, were observed in an experiment using PBMC cultures after treatment with compound 6 (data not shown). Likewise, as shown in FIG. 26, compound 10 increased the amount of interferon gamma (IFN-γ) in a dose-dependent manner.

As described in this example, cell-based assays were performed using human PBMCs to test T-reg formation and survival, consistent with the observations made in Gavin et al. (PNAS 103:6659-6665, 2006). To mimic T cell activation by antigen presenting cells, PBMCs were stimulated with anti-CD3 antibody and anti-CD28 antibody, and test substances (GPR174-targeting compound 10; IL-2; or vehicle control) were added immediately thereafter. Cells were collected after 3 and 7 days, fixed, and stained with fluorescent antibodies against the marker of helper T cells CD4 and the T-reg transcription factors, forkhead box factor P3 (FoxP3) and Helios factor.

Following T cell activation, FoxP3 is transiently up-regulated in conventional (i.e., effector T cells) (FoxP3 ^- Helios ^- ) T cells, forming a FoxP3 ⁺ Helios - population on day 3, which is reduced or absent by day 7. In contrast, the preexisting natural T-reg (nT-reg) population, defined as FoxP3+Helios ⁺ , persists during this time frame and still represents 1-5% of CD4 ⁺ T cells at day 7. Because nT-reg cells do not proliferate as quickly as conventional T cells after T cell activation, the relative abundance of nT-reg may decrease within this time frame, depending on the strength of T cell stimulation.

As shown in FIG. 24A, GPR174 inverse agonist compound 10 significantly increased the proportion of conventional (FoxP3'Helios ^- ) T cells; while on day 7, compound 10 significantly reduced the content of double positive (FoxP3+Helios ⁺ ) nT-reg cells (Fig. 24B). These results indicate that in human PBMC, inhibition of GPR174 enhances immune system activation while also reducing nT-reg cell content. This is a surprising finding given the mouse data reported in Barnes et al. (2015, above), which show that in male mice lacking GPR174, the numbers of T-reg cells were significantly increased in specific tissues. As shown in this example, in human cells, inhibition of GPR174 has the opposite effect on T-reg compared to inhibition of GPR174 in mouse cells. It was noted that the differences between the subgroups of effector and regulatory T cells in humans from groups in mice are also described for another

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GPCR, GPR15 (see Nguyen et al., Nat Immunol 16:207-213, 2015).

To further evaluate the GPR174 inhibitory properties in human cells, we examined the levels of selected cytokines produced in response to treatment of PBMCs using increasing concentrations of Compound 10. As shown in FIG. 25, compound 10 increased IL-2 levels in a dose-dependent manner and at concentrations as low as 1 μM. Likewise, as shown in FIG. 26, compound 10 increased interferon gamma (IFN-γ) in a dose-dependent manner.

These results indicate that inhibition of GPR174 enhances the activity of effector T cells, a cell population important for the treatment of inflammatory conditions and malignancies (Ramsay et al., British Journal of Hematology 162:313-325, 2013). In addition, the results show that inhibition of GPR174 reduces T-reg content. Reducing the number of immunosuppressive Tregs should also be beneficial for the immunotherapy of malignant tumors, since it is well known that the tumor microenvironment is selectively enriched for T-regs, which in turn contribute to the failure of immunosurveillance to detect these tumors (see, for example, Takeuchi and Nishikawa, Roles of regulatory T cells in cancer immunity, International Immunol 28:401–409, 2016). Reducing the number of T-reg cells should improve the outcome of treatment of malignant tumors. Tumors use multiple pathways to evade immunosurveillance. One of these strategies is to create an immunosuppressive microenvironment by stimulating T-reg development through TGF-β induction or other means. T-regs are found in a variety of malignant tumors and can specifically multiply in response to tumor antigens. In mouse models, reduction or elimination of T-regs increases the organism's resistance to malignant tumors (Klages et al., Cancer Res 70:7788-7799, 2010; Li et al., 2010; Teng et al., Cancer Res 70: 78007809, 2010; Bos et al., J Exp Med, 210:2435-2466, 2013). High frequency of T-reg cells in solid organ tumors most often correlates with poor patient outcomes. A meta-analysis of multiple studies found that T-reg infiltration in tumors correlates with poor prognosis for overall survival in breast and lung cancer, as well as in other malignancies such as colorectal, cervical, kidney, ovarian, melanoma, pancreatic cancer, hepatocellular cancer and gastric cancer (Shang et al., Sci Rep 5:15179-15187, 2015).

Clinical efficacy following immune checkpoint blockade is associated with somatic mutation burden in tumor cells (Rizvi et al., Science 348:124-128, 2015; Snyder et al., N. Engl. J. Med. 371:2189-2199, 2014 ; Van Allen et al., Science 350: 207-211, 2015; Le et al., N. J. Med. 372:25092520, 2015). That is, the clinical benefit of such therapy is limited to those patients whose malignant tumor cells carry neoantigens resulting from mutations and are recognized as non-self by the immune system (Matsushita et al., Nature 482:400-404, 2012; Gubin et al., Nature 515:577-581, 2014). T-regs involved in self-tolerance advantageously control the activation of T-cell responses to cancer antigens derived from self-constituents (common antigens) but are less suppressive of T-cells recognizing foreign antigens (Maeda et al. , Science 346:1536-1540, 2014). Thus, it is predicted that combining methods of reducing suppressive activity and/or T-reg numbers with methods of blocking immune checkpoint molecules may expand the therapeutic spectrum of cancer immunotherapy to patients with cancers having lower numbers of neoantigens.

Example 7.

This example describes an analysis of the effect of GPR174 inhibitory compound 10 on IL-2 production in mouse splenocytes obtained from wild-type or GPR174 knockout mice.

GPR174 knockout (KO) mice (also designated FKSG79) were generated as described in Gragerov A. et al., PNAS vol 104(36): 14406-14411, 2007. Splenocyte cultures were obtained from four GPR174 KO and four wild-type (WT) mice. Single cell suspensions from the spleens of individual mice were pelleted by centrifugation, resuspended in RBC lysis buffer to remove red blood cells, pelleted by centrifugation again, and resuspended in T cell medium (RPMI-1640, 10% FBS, 6 mM L-glutamine, 12.5 mM HEPES , 50 µM 2-mercaptoethanol, pen/strep). Thus, prepared splenocytes were plated in 96-well plates at a density of 3 x 10 ⁶ cells/ml, 250 μl/well and stimulated with 100 ng/ml anti-CD3 antibody and 100 ng/ml anti-CD28 antibody. Cells were treated with either vehicle (0.1% DMSO) or GPR174 inhibitory compound 10 (10 μM). After 48 h, cell supernatants were analyzed for cytokine levels using the LEGENDplex Mouse Th Cytokine Detection Kit (BioLegend). Four GPR174 KO and four WT spleens were analyzed, each in three replicates.

Fig. 27 graphically illustrates the amount of IL-2 (pg/ml) in culture supernatants 48 hours after stimulation in splenocyte cultures from WT or GPR174 KO mice treated with vehicle or Compound 10 (10 μM). As shown in FIG. 27, IL-2 levels are increased by treatment with GPR174 inhibitory compound 10 in WT but not GPR174 KO splenocytes, where IL-2 is high

- 92 046439 kim without processing. The p value for the difference between KO and WT in vehicle-treated splenocytes was p=0.02. There is no significant difference between KO and WT in the Compound 10-treated group, or between Compound 10-treated cells and vehicle-treated KO cells.

These results indicate that the effect of compound 10 on IL-2 production (described in Example 6 and shown in FIG. 25) is mediated by inhibition of GPR174.

Example 8.

This example shows that representative GPR174 inhibitory compound 10 reduces the proportion of immunosuppressive T-regs (FoxP3'Helios') and stimulates IL-2 production in a dose-dependent manner in cultured single-donor human peripheral blood mononuclear cells (PBMCs).

An experiment was carried out with human peripheral blood mononuclear cells using methods similar to those described above in example 6, with the difference that the effect of GPR174 inhibitory compound 10 was analyzed over a concentration range (3 μM - 60 μM).

Briefly, human PBMCs obtained from a volunteer donor were adjusted to a density of 2 x 10 ⁶ cells/ml, stimulated with 100 ng/ml anti-CD3 antibody and 100 ng/ml anti-CD28 antibody, and treated with 3, 10, or 30 µM GPR174 inverse agonist compound 10 (shown in Table 1), or vehicle control. A fraction of cells was selected for staining on day 3 after stimulation, while the remaining cells were diluted 1:4 with fresh medium and used for staining on day 7 after stimulation. To determine the proportion of natural T-reg (nT-reg) cells (FoxP3'Helios'), PBMCs were washed, stained with fluorescently labeled anti-CD4 antibodies, fixed, permeabilized, and stained with a mixture of fluorescent anti-FoxP3 and anti-Helios antibodies. Labeled cells were analyzed by flow cytometry against populations stained for these three markers. Each experiment was carried out in three replicates and repeated three times.

The effect of compound 10 on IL-2 production in human PBMC was determined as follows. PBMCs from a single donor were adjusted to a density of 2 x 10 ⁶ cells/ml, stimulated with 100 ng/ml anti-CD3 antibody and 100 ng/ml anti-CD28 antibody, and treated with 3 μM, 10 μM, or 30 μM compound 10. Supernatant from these cells were collected on day 3 post-stimulation, and IL-2 levels were determined by ELISA assay using the Ready-SETGo™ ELISA kits from Affimetrix Ebioscience and the LEGENDplex™ bead-based immunoassay kits from BioLegend, according to the manufacturer's instructions.

Fig. 28 graphically illustrates the percentage of FoxP3+Helios ⁺ cells in the CD4 ⁺ cell population on day 7 after stimulation in PBMC cultures treated with vehicle or compound 10 (3, 10 or 30 μM) (n=3, *p<0.05; ** p<0.01). As shown in FIG. 28, on day 7 after stimulation, the inverse GPR174 agonist compound 10 significantly reduced the content of double-positive (FoxP3+Helios ⁺ ) nT-reg cells in a dose-dependent manner, the result is consistent with the results described in example 6.

Fig. 29 graphically illustrates the amount of IL-2 (pg/ml) in culture supernatants on day 3 post-stimulation in PBMC cultures from an individual volunteer where PBMC were treated with vehicle or GPR174 inhibitory compound 10 (3, 10, 30 or 60 μM). As shown in FIG. 29, compound 10 increased IL-2 levels in culture supernatants in a dose-dependent manner, which is consistent with the results described in example 6. IL-2 is a major cytokine determining T cell survival and differentiation for effector T cells, T cells memory and regulatory T cells.

Example 9.

This example describes an analysis of the effect of representative GPR174 inhibitory compound 10 on T-reg, IL-2 and other cytokines in PBMC obtained from multiple donors.

To confirm and further extend the results described in Examples 6 and 8, the effect of representative GPR174 inhibitory compound 10 on T-reg, IL-2 and other cytokines was analyzed in cultured PBMCs obtained from multiple volunteer donors.

Briefly, human PBMC obtained from eight volunteer donors were adjusted to a density of 2 x 10 ⁶ cells/ml, stimulated with 100 ng/ml anti-CD3 antibody and 100 ng/ml anti-CD28 antibody, and treated with 30 μM inverse agonist GPR174 compound 10 (shown in Table 1). A fraction of cells was selected for staining on day 2 after stimulation, while the remaining cells were diluted 1:4 with fresh medium and used for staining on day 7 after stimulation. To determine the proportion of natural T-reg (nT-reg) cells (FoxP3'Helios'). PBMCs were washed, stained with fluorescently labeled anti-CD4 antibodies, fixed, permeabilized, and stained with a mixture of fluorescent anti-FoxP3 and anti-Helios antibodies. Labeled cells were analyzed by flow cytometry against populations stained for these three markers. Each experiment was performed in three replicates and repeated five times.

The effect of compound 10 on IL-2 production in human PBMC from each donor was determined as follows. PBMCs from each donor were brought to a density of 2x10 ⁶ cells/ml, stimulated with

- 93 046439 using 100 ng/ml anti-CD3 antibody and 100 ng/ml anti-CD28 antibody, and treated with 30 μM compound 10. The supernatant from these cells was collected on day 2 after stimulation, and IL-2 levels were determined by ELISA assay with using LEGENDplex™ bead-based immunoassays from BioLegend (San Diego, CA), according to the manufacturer's instructions.

Effect of compound 10 on the production of a panel of additional cytokines (interleukin 5 (IL-5), interleukin 13 (IL-13), interleukin 6 (IL-6), interleukin 9 (IL-9), interleukin 10 (IL-10), interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), interleukin 17A (IL-17A), interleukin 17F (IL-17F), interleukin 4 (IL-4) and interleukin 22 (IL-22)) in pooled human PBMCs obtained from eight donors were determined as follows. PBMCs from eight donors were pooled, then adjusted to a density of 2 x 10 ⁶ cells/ml, stimulated with 100 ng/ml anti-CD3 antibody and 100 ng/ml anti-CD28 antibody, and treated with 30 μM compound 10. The supernatant from these cells was collected on day 2 after stimulation, and the levels of IL-5, IL-13, IL-6, IL-9, IL10, IFN-γ, TNF-α, IL-17A, IL-17F, IL-4 and IL-22 were determined by ELISA using LEGENDplex™ bead-based immunoassays from BioLegend (San Diego, CA), according to the manufacturer's instructions.

Fig. 30 graphically illustrates the proportion of the vehicle value of FoxP3'Helios' cells in the CD4+ cell population at day 7 post-stimulation in PBMC cultures from eight different donors treated with vehicle or Compound 10 (30 μM). As shown in FIG. 30, on day 7 after stimulation, the inverse GPR174 agonist compound 10 significantly reduced the content of double-positive (FoxP3+Helios ⁺ ) nT-reg cells in PBMC cultures from all eight donors, the result is consistent with the results described in examples 6 and 8.

Fig. 31 graphically illustrates the amount of IL-2 (fold over vehicle) in culture supernatants on day 2 post-stimulation in PBMC cultures from eight different donors treated with vehicle or Compound 10 (30 μM). As shown in FIG. 31, compound 10 increased IL-2 levels in each of the PBMC cultures from eight donors by an average of approximately 5-fold, consistent with the results described in Examples 6 and 8.

Fig. 32 graphically illustrates the amounts of various cytokines (fold over vehicle) in culture supernatants on day 2 post-stimulation for human PBMCs obtained from eight donors treated with vehicle or Compound 10 (30 μM). As shown in FIG. 32, a statistically significant fold increase was observed for IL-6 (***p<0.001), IL-10 (*p<0.05), IFN-γ (*p<0.05) and TNF-α (*p <0.05) compared to vehicle control. As further shown in FIG. 32, a statistically significant fold reduction was observed for IL-17A (***p<0.001) compared to vehicle control.

It was noted that IFN-γ induction was also observed in PBMCs of non-human primates, dogs and rats, but not mice.

The results obtained in this example confirm and further extend the results described in examples 6 and 8 and show that a representative GPR174 inhibitor, compound 10, significantly reduced the T-reg population in each of the human PBMC cultures from eight different donors. Additionally, in pooled human PBMCs obtained from eight donors, inhibition of GPR174 was shown to increase, with statistical significance, the levels of the cytokines IL-2 and IFN-γ, as well as EL-6, IL-10 and TNF-α. The increase in this subset of cytokines (IL-2, IFN-γ, IL-6, IL-10, and TNF-α) indicates that GPR174 inhibition activates the T111 subset of T cells, which primarily mediate responses against intracellular infectious agents.

Example 10.

This example describes an analysis of the effect of representative GPR174 inhibitory compound 10 on cytokines produced in mixed cultures of splenocytes obtained from different strains of mice.

To stimulate T cells in mice, splenocytes obtained from two different strains of mice, C57BL/6 and DBA1, were mixed in culture.

An initial experiment was performed to determine the time frame of cytokine production for co-cultured splenocytes in which cytokine production (IFN-γ, IL-5, TNF-α, IL-2, IL-6, IL-4, IL-10, IL-9, IL-17A, IL-17F, IL-21, IL-22 and IL-13) were measured in the co-culture supernatant on day 2, day 4, and day 6 after mixing in the absence of compound 10. The results are shown below in table. 7.

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Table 7. Co-culture of splenocytes C57BL/6+DBA1

cytokine Day 2 (pg/ml) Day 4 (pg/ml) Day 6 (pg/ml) IFN-γ 871 36030 8648 IL-5 18 18 20 TNF-a 15 96 46 IL-2 263 1079 504 IL-6 14 369 106 IL-4 eleven thirty 13 IL-10 22 93 50 IL-9 25 78 78 IL-17A 18 161 49 IL-17F 16 eleven 7 IL-21 26 26 26 IL-22 24 1098 288 IL-13 18 33 32

As shown in the table above. 7, peak levels for each cytokine measured in the C57BL/6+DBA1 splenocyte coculture were observed on day 4, and they persisted on day 6 after mixing.

Effect of compound 10 on the production of cytokines (IFN-γ, L-5, TNF-α, IL-2, L-6, IL-4, IL-10, L-9, IL17A, IL-17F, IL-21, IL -22 and IL-13) in co-cultures of mouse splenocytes were determined as follows. Splenocytes obtained from two different strains of mice, C57BL/6 and DBA1, were mixed in culture in the presence or absence of 30 μM compound 10. Cytokine production IFN-γ, IL-5, TNF-α, IL-2, IL-6, L-4, IL-10, IL-9, IL-17A, IL-17F, IL-21, IL-22 and EL-13 were measured in the co-culture supernatant on day 4 and day 6 after mixing. Cytokine levels were determined using LEGENDplex™ bead-based immunoassays from BioLegend (San Diego, CA), according to the manufacturer's instructions.

Fig. 33 graphically illustrates the fold induction by compound 10 (30 μM) in a panel of cytokines in co-cultured mouse C57BL/6+DBA1 splenocytes on day 6 post-mixing, compared to cytokine levels measured in co-cultured C57BL/6+DBA1 splenocytes on day 6 in the absence of compound 10. These results show that compound 10 causes a strong change in the amounts of cytokines produced during allogeneic splenocyte stimulation, indicating that inhibition of GPR174 significantly modulates the immune response. It is noted that the apparent difference between the groups of cytokines induced in human PBMC (as described in Example 9) compared to mouse splenocytes is not necessarily informative, since it is difficult to compare the responses of relatively naive immune systems of animals bred in a virtually sterile and protected vivarium , with the immune system responses of human volunteers exposed to multiple antigens.

Example 11.

This example describes a study that was conducted to determine which cell types in human PBMC cultures are responsible for cytokine production induced by a representative GPR174 inhibitory compound 10.

In the first experiment, human PBMCs from two different donors were incubated overnight in the presence or absence of compound 10 (10 μM). PBMC cultures were stimulated using anti-CD3 antibody and anti-CD28 antibody, and vesicular transport was simultaneously blocked using GolgiStop™. Cells were fixed after four hours and stained with antibodies for cell type markers (FITC anti-CD3 and PerCP-Cy5.5 anti-CD4) and IL2 (PE anti-IL-2). Cells were analyzed by flow cytometry. The proportion of cells with intracellular IL-2 (PE-labeled) was then determined in the CD4 and CD8 T cell populations as well as in non-T cells.

In the second experiment, human PBMCs from two different donors were stimulated with anti-CD3 antibody and anti-CD28 antibody and incubated in the absence or presence of increasing concentrations of compound 10 (0, 0.1, 0.3, 1.0 or 3.0 μM ). Vesicular transport was blocked after two days using GolgiStop™. Cells were fixed after four hours and stained with antibodies for cell type markers (FITC anti-CD3, PerCP-Cy5.5 anti-CD4, PE anti-CD56, and Pacific Blue anti-CD16) and any of the IL-1 cytokines. 2, IL-10 and IFN-γ (all anti-cytokine antibodies were APC-labeled). Cells were analyzed by flow cytometry. The proportion of cells with intracellular IL-2, IFN-γ, IL-10, and TNF-α was then determined in different cell populations.

The results of the first experiment for one of the two donors are shown in FIG. 34A-34C. Similar results were also observed for the second donor (data not shown).

In fig. 34A shows CD4+ (46.2%), CD8+ (17.1%) and non-T cell populations (33.9%) present

- 95 046439 growing in a representative culture of PBMC four hours after stimulation with anti-CD3 antibody and anti-CD28 antibody in the presence of compound 10 (10 μM).

Fig. 34B graphically illustrates the proportion of cells with intracellular IL-2 staining in the CD4+ T cell population shown in FIG. 34A treated with compound 10 (10 μM) compared to vehicle control; And

Fig. 34C graphically illustrates the proportion of cells with intracellular IL-2 staining in the CD8+ T cell population shown in FIG. 34A treated with compound 10 (10 μM) compared to vehicle control.

The results of the second experiment for one of the two donors are shown in FIG. 35A-35M. Similar results were also observed for the second donor (data not shown).

In fig. 35A shows the CD4+ (31.8%), CD8+ (27.5%) and non-T cell (33.1%) populations present in a representative PBMC culture 2 days after stimulation with anti-CD3 and anti-CD28 in presence of compound 10.

Fig. 35B graphically illustrates the proportion of cells with intracellular IL-2 staining in the CD4 ⁺ T cell population in PBMC cultures treated with Compound 10 (0, 0.1, 0.3, 1.0, or 3.0 μM), representative of which are shown in fig. 35A.

Fig. 35C graphically illustrates the proportion of cells with intracellular IL-2 staining in the CD8 ⁺ T cell population in PBMC cultures treated with Compound 10 (0, 0.1, 0.3, 1.0, or 3.0 μM), representative of which are shown in fig. 35A.

Fig. 35D graphically illustrates the proportion of cells with intracellular IL-2 staining in non-T cell populations in PBMC cultures treated with Compound 10 (0, 0.1, 0.3, 1.0, or 3.0 μM), representative of which are shown in fig. 35A.

Fig. 35E graphically illustrates the proportion of cells with intracellular IL-10 staining in the CD4 ⁺ T cell population in PBMC cultures treated with Compound 10 (0, 0.1, 0.3, 1.0, or 3.0 μM), representative of which are shown in fig. 35A.

Fig. 35F graphically illustrates the proportion of cells with intracellular IL-10 staining in CD8 ⁺ T cell populations in PBMC cultures treated with Compound 10 (0, 0.1, 0.3, 1.0, or 3.0 μM), representative of which are shown in fig. 35A; And

Fig. 35G graphically illustrates the proportion of cells with intracellular IL-10 staining in non-T cell populations in PBMC cultures treated with Compound 10 (0, 0.1, 0.3, 1.0, or 3.0 μM), representative of which are shown in fig. 35A.

Fig. 35H graphically illustrates the proportion of cells with intracellular IFN-γ staining in the CD4 ⁺ T cell population in PBMC cultures treated with Compound 10 (0, 0.1, 0.3, 1.0, or 3.0 μM), representative of which are shown in fig. 35A.

Fig. 35I graphically illustrates the proportion of cells with intracellular IFN-γ staining in the CD8 ⁺ T cell population in PBMC cultures treated with Compound 10 (0, 0.1, 0.3, 1.0, or 3.0 μM), representative of which are shown in fig. 35A.

Fig. 35J graphically illustrates the proportion of cells with intracellular IFN-γ staining in non-T cell populations in PBMC cultures treated with Compound 10 (0, 0.1, 0.3, 1.0, or 3.0 μM), representative of which are shown in fig. 35A.

Fig. 35K graphically illustrates the proportion of cells with intracellular TNF-α staining in the CD4 ⁺ T cell population in PBMC cultures treated with Compound 10 (0, 0.1, 0.3, 1.0, 3.0, or 10 μM), representative of which are shown in Fig. 35A.

Fig. 35L graphically illustrates the proportion of cells with intracellular TNF-α staining in the CD8 ⁺ T cell population in PBMC cultures treated with Compound 10 (0, 0.1, 0.3, 1.0, 3.0, or 10 μM), representative of which are shown in Fig. 35A.

Fig. 35M graphically illustrates the proportion of cells with intracellular TNF-α staining in non-T cell populations in PBMC cultures treated with Compound 10 (0, 0.1, 0.3, 1.0, 3.0, or 10 μM), representative of which are shown in Fig. 35A.

Results from further characterization of IFNy and TNF-α expression in non-T cells (from FIGS. 35A, 35J and 35M) are shown in FIG. 36A-36E. These results were obtained for a single donor. Similar results were also observed for the second donor (data not shown).

In FIG. 36A shows the non-T cell population from FIG. 35A, sorted into the CD56+CD16 ^- (2.27%), C.'D56'C.'D16' (11.8%) and non-NK (78.5%) populations present in the non-T- population cells in a representative PBMC culture two days after stimulation with anti-CD3 antibody and anti-CD28 antibody.

Fig. 36B graphically illustrates the proportion of cells with intracellular IFN-γ staining in He-T-CD56 ⁺ CD16 ⁺ cell populations in PBMC cultures treated with Compound 10 (0, 0.1, 0.3, 1.0, or 3.0 μM). representative ones of which are shown in FIG. 36A, and FIG. 36C graphically illustrates the proportion of cells with intracellular IFN-γ staining in He-T-CD56 ⁺ CD16 ⁻ cell populations in PBMC cultures treated with Compound 10 (0, 0.1, 0.3, 1.0, or

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3.0 μM), representative of which are shown in FIG. 36A.

Fig. 36D graphically illustrates the proportion of cells with intracellular TNF-α staining in He-T-CD56 ⁺ CD16 ⁺ cell populations in PBMC cultures treated with Compound 10 (0, 0.1, 0.3, 1.0, 3.0, or 10 μM ), representative ones of which are shown in FIG. 36A, and FIG. 36E graphically illustrates the proportion of cells with intracellular TNF-α staining in He-T-CD56 ⁺ CD16 ⁻ cell populations in PBMC cultures treated with Compound 10 (0, 0.1, 0.3, 1.0, 3.0, or 10 μM ), representative ones of which are shown in FIG. 36A.

The results shown in FIG. 35A-M and figs. 36A-E identify cell types affected by GPR174 inhibition using compound 10: IL-2 and IL-10 production were induced specifically in both CD4 and CD8 positive T cells, while IFN-γ production and TNF-α was upregulated in CD4 and CD8 positive T cells as well as in NK cells. These data establish a mechanism of immune stimulation through GPR174 inhibition.

Example 12.

This example describes a study that was conducted to evaluate the in vivo consequences of the absence of GPR174 activity by comparing the effect of influenza virus immunization on the level of influenza virus antigen-specific CD8 ⁺ T cells in wild-type and GPR174 knockout (KO) mice.

Animal exposure to the mouse-adapted influenza virus HIN1 A/PR/8/34 is a widely used model for assessing the immune response to acute infection (see Allen IC, Mouse Models of Innate Immunity: Methods and Protocols, Methods in Molecular Biology, vol 1031 : pp 177-188 (2013); Castiglioni P. et al., J Immunol 180:4956-64, 2008; Zak O., Sande MA (eds), Handbook of Animal Models of Infection, Academic Press: pp981-986, 1999). Accordingly, as described in this example, a study was conducted to evaluate the in vivo consequences of the absence of GPR174 activity by comparing the effect of influenza virus immunization on the level of influenza virus antigen-specific CD8 ⁺ T cells in wild-type and GPR174 knockout (KO) mice.

In the first experiment, wild-type (WT) mice and GPR174 KO mice (generated as described in Example 7) were immunized by intraperitoneal (ip) injection with 1000 HAU (hemagglutinating units) of influenza A/PR/8/34 virus. Three weeks later, splenocytes were obtained from two naive, four immunized WT, and four immunized GPR174 KO mice and exposed to 2 μM of the influenza virus nucleoprotein (NP) known antigenic peptide ASNENMETM (SEQ ID NO: 5) to induce preferential proliferation of CD8 ⁺ T -cells recognizing this antigen in complex with MHC class I presenting molecules. After five days, the number of CD8 ⁺ cells exhibiting the T-cell receptor (TCR) recognizing the MHC-peptide complex of the influenza virus was assessed by staining splenocytes using antibodies against CD8 and dextramers MHC-ASNENMETM (Immudex, Denmark). Fluorescently labeled MHC dextramers are reagents that carry multiple MHC-peptide complexes and thus have the ability to interact simultaneously with multiple TCRs on a single T cell, allowing stable interaction between the reagent and the T cell, and which as a result can be used to detection and quantification of antigen-specific T cells.

In the second experiment, four WT and four GPR174 KO mice were immunized by IP injection with 1000 HAU of influenza A/PR/8/34 virus. Three weeks after immunization, these mice were boosted with a second injection of 1000 HAU of influenza A/PR/8/34 virus, and their splenocytes were analyzed 11 days later, along with splenocytes from two naïve animals, by dextramer staining without prior incubation in vitro.

The results of the first experiment are shown in Fig. 37.

Fig. 37 graphically illustrates the number of dextramer-stained cells (antigen-specific T cells) as a percentage of total CD8 ⁺ cells in splenocyte cultures obtained from WT and GPR174 KO mice three weeks after immunization with influenza A/PR/8/34 virus, and cultured for five days in the presence of the antigenic peptide NP. Splenocytes obtained from control naïve WT mice are also shown in Fig. 37. As shown in FIG. 37, splenocyte cultures from GPR174 KO mice have a higher percentage of dextramer-stained CD8 ⁺ cells (antigen-specific T cells) compared with the percentage of dextramer-stained CD8 ⁺ cells in splenocyte cultures obtained from naïve and WT mice.

The results of the second experiment are shown in Fig. 38. Fig. 38 graphically illustrates the number of dextramer-stained cells (antigen-specific T cells) as a percentage of total CD8 ⁺ cells in splenocytes obtained from WT and GPR174 KO mice immunized and boosted three weeks later with 1000 HAU of influenza A/PR/8/34 virus , where splenocytes were analyzed 11 days after the boost. Splenocytes obtained from control naïve (N) WT mice are also shown in Fig. 38. As shown in FIG. 38, splenocytes from GPR174 KO mice have a higher percentage of dextramer-stained CD8 ⁺ cells (antigen-specific T

- 97,046,439 cells) compared with the number of dextramer-stained CD8+ cells in splenocytes obtained from WT and naïve mice.

Overall, the results in this example show that in both experimental conditions, i.e. in the presence or absence of prior in vitro incubation of antigen-specific T cells, there is a trend toward increased numbers of influenza antigen-specific CD8+ T cells in splenocytes from GPR174 KO mice compared with WT mice, indicating that GPR174 limits the immune response, and inhibition of GPR174 is immunostimulatory.

Example 13.

This example describes a study conducted to evaluate the effect of representative GPR174 inhibitory compound 10 on the expression of cytotoxic T lymphocyte associated antigen 4 (CTLA4) on naive human T cells and human memory T cells.

BACKGROUND/BACKGROUND: CTLA4 is expressed by T cells activated through the T cell receptor and CD28. CTLA4 is homologous to the T cell co-stimulatory protein, CD28, and both molecules bind to CD80 and CD86, also called B7-1 and B7-2, respectively, on antigen presenting cells. CTLA-4 binds CD80 and CD86 with greater affinity and avidity than CD28, thus allowing it to outcompete CD28 for its ligands. CTLA4 is also found in regulatory T cells and contributes to their inhibitory function. Blocking or inhibiting CTLA4 serves as a means of reversing the immune system's tolerance to a tumor, and thus provides an immunotherapy option for patients with cancer (see, e.g., Baumeister S.H. et al., Ann Rev Immunol 34:539-73, 2016) . This method is called immune checkpoint blockade therapy.

PBMCs from a single donor were cultured overnight with additional irradiated antigen presenting cells (APCs) in the presence of varying concentrations of GPR174 inhibitory compound 10 (0.019-6 μM) or equivalent concentrations of vehicle (DMSO). Anti-CD3 antibody (OKT3) was then added to the cultures for an additional 24 hours. Cells were analyzed by flow cytometry for the expression of FOXP3, CTLA-4, CD4, CD3, CD45RA, and CD25.

Fig. 39A graphically illustrates the proportion of naïve regulatory T cells (Treg) with CTLA-4 positive staining (percentage of naïve Treg cells (CD45RA+FOXP3+) in PBMC cultures treated with Compound 10 (0, 0.19, 0. 38, 0.75, 1.5, 3.0 or 6.0 μM), one day after stimulation, showing that CTLA4 expression is reduced in naïve T-reg cells treated with GPR174 inhibitory compound 10 in a dose-dependent manner. compared to vehicle control-treated cells.

Fig. 39B graphically illustrates the proportion of non-regulatory T cells (non-Treg) with CTLA-4 positive staining (percentage of naïve non-Treg cells (CD45RA+FOXP3-) in PBMC cultures treated with compound 10 (0, 0 ,19, 0.38, 0.75, 1.5, 3.0 or 6.0 μM) one day after stimulation, showing that CTLA4 expression is reduced in non-Treg cells treated with the GPR174 inhibitory compound 10 dependent in a dose-dependent manner compared to vehicle control-treated cells.

Fig. 40A graphically illustrates the proportion of memory T-regs with CTLA-4 positive staining (percentage of memory T-regs (CD45RA-FOXP3+) in PBMC cultures treated with GPR174 inhibitory compound 10 (0, 0.19, 0.38, 0.75 , 1.5, 3.0, or 6.0 μM) one day after stimulation, showing that CTLA4 expression is reduced in memory T-reg cells treated with compound 10 in a dose-dependent manner compared to vehicle control-treated cells.

Fig. 40B graphically illustrates the proportion of non-Treg memory cells with CTLA-4 positive staining (percentage of non-Treg memory (CD45RA-FOXP3-)) in PBMC cultures treated with GPR174 inhibitory compound 10 (0, 0.19, 0 .38, 0.75, 1.5, 3.0 or 6.0 μM), one day after stimulation, showing that CTLA4 expression is reduced in non-Treg memory cells treated with compound 10 in a dose-dependent manner , compared to control vehicle-treated cells.

Overall, the results in this example indicate that inhibition of GPR174 not only activates the immune response in T cells stimulated through recruitment of the TCR and its co-stimulatory protein CD28, but also prevents the induction of the immune checkpoint CTLA-4, which normally limits T cell activation. cells.

Example 14.

This example describes a study conducted to evaluate the effect of representative GPR174 inhibitory compound 10 on the expression of PD-L1, TIGIT, and other T cell-expressed immune checkpoint molecules on human T cells.

Programmed death ligand 1 (PD-L1), also known as CD274 or B7-H1, is a transmembrane protein that plays a role in immune system suppression. Binding of PD-L1 to PD-1 or B7.1 transmits an inhibitory signal that reduces T cell proliferation and may also induce apoptosis. Upregulation of PD-L1 appears to allow malignant tumors to evade the host immune system. Multiple PD-L1 inhibitors are in development

- 98 046439 botka as immuno-oncology therapeutics and have shown good results (see, for example, Baumeister S.H. et al., Ann Rev Immunol 34:539-73, 2016; Teng F. et al., Cancer Lett. 414 :166-173, 2017). Clinically available examples include durvalumab, atezolizumab, and avelumab.

T cell immunoreceptor with Ig and ITIM domains (TIGIT) is expressed by activated cytotoxic T cells and regulatory T cells and has also been shown to be up-regulated on T cells in a variety of cancer models. The ligands CD155 and CD112 are found on dendritic cells and macrophages, and are also expressed at high levels in several types of malignant tumors. TIGIT expression is highly correlated with the expression of other coinhibitory molecules, including PD-1. Studies have identified TIGIT as a unique and potentially complementary target to other inhibitory immune checkpoints. First, in addition to directly inhibiting cytotoxic T cell activity, TIGIT may fuel an immunosuppressive microenvironment through its effects on other immunocytes. For example, by binding to CD155 on the surface of dendritic cells, TIGIT increases the secretion of the immunosuppressive cytokine interleukin-10, and the recruitment of TIGIT on regulatory T cells enhances their immunosuppressive functions (see, for example, Blake S.J. et al., Clin Cancer Res 22(21): 5183-5188, 2016; Grogan J. et al. J Immunother Cancer 4(suppl l):P209, 2016; Kurtulis S. et al., J Clin Invest 125(11):4053-4062, 2015; al., J Immunol 188(8):3869–3875, 2012).

Recent studies suggest that T-reg cells may participate in tissue repair in a manner separate from their immunosuppressive capacity. Using lung tumor transplantation models in mice, it was found that amphiregulin (AREG), a member of the epidermal growth factor family, was markedly up-regulated in intratumoral T-reg cells. In addition, T cell-restricted amphiregulin deficiency resulted in markedly delayed lung tumor progression. This observed difference in tumor progression was not associated with detectable changes in the ability of T cells to mount an immune response or in the number of T cells and effector T cells. These observations suggest a new nonimmune mode of action for intratumoral T cells and effector T cells to stimulate tumor growth through the production of factors normally involved in tissue repair and maintenance (see, for example, Green J.A. et al., J Exp Med Oct 16 2017, doi:10.1084/jem.20170356. Epub ahead of print).

PBMCs from a single donor mixed with additional irradiated antigen presenting cells (APCs) and anti-CD3 (UCHT1) antibodies were incubated in the presence of varying concentrations of GPR174 inhibitory compound 10 (1.25-5 μM) or equivalent concentrations of vehicle (DMSO). Cells were analyzed by flow cytometry for expression of CD4, CD8, PD-L1, TIGIT, and AREG.

Fig. 41 graphically illustrates the proportion of human CD4+ T cells with positive PD-L1 staining in PBMC cultures treated with GPR174 inhibitory compound 10 (0, 1.25 or 5 μM), one day after stimulation, showing that PD-L1 expression is reduced in CD4+ T cells treated with compound 10 in a dose-dependent manner compared to vehicle control-treated cells.

Fig. 42 graphically illustrates the proportion of human CD8+ T cells with TGIT positive staining in PBMC cultures treated with GPR174 inhibitory compound 10 (0, 1.25 or 5 μM), one day after stimulation, showing that TGIT expression is reduced in CD8+ T cells. treated with compound 10 in a dose-dependent manner compared to vehicle control-treated cells.

Fig. 43 graphically illustrates the proportion of human CD4+ T cells with AREG positive staining in PBMC cultures treated with GPR174 inhibitory compound 10 (0, 1.25 or 5 μM), one day after stimulation, showing that AREG expression is reduced in CD4+ T -cells treated with compound 10 in a dose-dependent manner compared to vehicle control-treated cells.

As described above in Example 13, GPR174 inhibitory compound 10 was shown to prevent induction of the CTLA-4 immune checkpoint, which normally limits T cell activation. As shown in this example, the GPR174 inhibitory compound 10 reduced the expression of two additional immune checkpoint molecules, PD-L1 and TIGIT. These results indicate that the GPR174 inhibitory compound is capable of down-regulating molecules responsible for tumor immune tolerance, which should further enhance T cell mobilization against malignant cells. In addition, the T cell-produced tumor-promoting growth factor amphiregulin (AREG) is also down-regulated following treatment with a GPR174 inhibitory compound, which is expected to also limit tumor growth.

Example 15.

This example shows that the combination of a GPR174 inhibitor and an adenosine 2a receptor (A2aR) inhibitor results in a synergistic induction of IFN-γ production in human PBMC.

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Human PBMCs, obtained from a voluntary human donor, were distributed at a density of 1 x 10 ⁶ cells/well in a 96-well flat-bottom plate and stimulated in triplicate with 1 μg/ml anti-CD3 antibody (UCHT1) and 0.1 μg/ml antibodies against CD28 (CD28.2) in the presence of:

GPR174 inhibitory compound 10 (3 μM);

A2aR inhibitory compound (ZM241385, 1 μM or 10 μM);

combinations of compound 10 (3 μM) plus 1 μM ZM241385;

combinations of compound 10 (3 μM) plus 10 μM ZM241385; or control media (DMSO).

Supernatants from these cells were collected 24 h after stimulation, and IFN-γ levels were determined by ELISA assay. Statistical analysis was performed using one-way ANOVA with Tukey's post hoc multiple comparison correction.

Fig. 44 graphically illustrates the amount of IFN-γ in culture supernatants 24 hours after stimulation in PBMC cultures from a single donor cultured in the presence of GPR174 inhibitory compound 10 (3 μM); A2aR inhibitory compound (ZM241385, 1 μM or 10 μM); combinations of compound 10 (3 μM) plus 1 μM ZM241385; combinations of compound 10 (3 μM) plus 10 μM ZM241385; or control media (DMSO).

As shown in FIG. 44, a 2.3-fold increase was observed for GPR174i (3 μM compound 10); a 1.2-fold increase was observed for A2Ai (ZM241385, 1 μM, and 10 μM); a 9.3-fold increase was observed for the combination of GPR174i (3 μM compound 10) plus A2Ai (ZM241385, 1 μM); and a 17.1-fold increase was observed for the combination of GPR174i (3 μM compound 10) plus A2Ai (ZM241385, 10 μM). As shown in table. 8 below, statistical analysis (one-way ANOVA with Tukey's post hoc multiple comparison correction) shows that only the combination of inhibitors resulted in a significant increase in IFN-γ production compared to each inhibitor alone or vehicle control.

Table 8. Statistical analysis of the results shown in FIG. 44

Tukey's multiple comparison test Generalization of significance Corrected p value Carrier vs GPR174i ns 0.7772 Anti-A2Ai vehicle (1 µM) ns 0.9999 Anti-A2Ai vehicle (10 µM) ns >0.9999 Anti-GPR174i+A2Ai vehicle (1 µM) **** <0.0001 Anti-GPR174i+A2Ai vehicle (10 µM) **** <0.0001 GPR174i vs A2Ai (1 µM) ns 0.8851 GPR174i vs A2Ai (10 µM) ns 0.8745 GPR174i vs GPR174i +A2Ai (1 µM) *** 0.0002 GPR174i vs GPR174i+A2Ai (10 µM) **** <0.0001 Α2ΑΪ (1 µM) vs A2Ai (10 µM) ns >0.9999 Α2ΑΪ (1 µM) vs. GPR174i+A2Ai (1 µM) **** <0.0001 Α2ΑΪ (1 µM) vs. GPR174i+A2Ai (10 µM) **** <0.0001 Α2ΑΪ (10 µM) vs. GPR174i+A2Ai (1 µM) **** <0.0001 Α2ΑΪ (10 µM) vs. GPR174i+A2Ai (10 µM) **** <0.0001 GPR174i+A2Ai (1 µM) vs GPR174i+A2Ai (10 µM) **** <0.0001

ns = not significant.

The results shown in FIG. 44 and in table. 8 show that combined inhibition of GPR174 and A2aR stimulates synergistic induction of IFN-γ production. Because IFN-γ is a central mediator of antitumor immune responses, this result indicates that combined GPR174 inhibition and A2aR inhibition significantly enhances a type of immunocyte function known as helper T cell 1, or Th1, which is important for effective immunotherapy of malignancies. Importantly, both GPR174 and A2aR activate the GaS/cyclic AMP signaling pathway, which is well characterized as a suppressive checkpoint for Th1 immune responses. In addition, A2aR antagonists are currently in clinical development as checkpoint inhibitors for cancer immunotherapy, as are inhibitors of CD38, CD39, and CD73, the enzymes that convert ATP to the A2aR ligand adenosine (see Table 15 below). Cellular stress and cell death in the tumor microenvironment lead to increased concentrations of both adenosine and the GPR174 ligand lysophosphatidylserine (lysoPS), a metabolite of the cell membrane lipid phosphatidylserine (PS). Thus, immunocytes in and around tumor tissue

- 100 046439 can be suppressed through the combined activity of adenosine, PS and lysophosphatidylserine, and effective release of Th1 responses from this suppression appears to require combined inhibition of both GPR174 and the CD38/CD39/CD73/A2aR/A2bR axis. Thus, the results presented in this example demonstrate that to reduce cyclic AMP signaling to levels allowing robust Th1 immunity, both GPR174 and adenosine signaling must be inhibited.

Example 16.

This example describes the discovery that phosphatidylserine (PS) is an agonist for GPR174-mediated Gs signaling.

As described herein, a recent report identified lysophosphatidylserine (LysoPS) as a ligand for GPR174 (Inoue et al., Nat Methods 9:1021-9, 2012). LysoPS is a lysophospholipid mediator formed through enzymatic hydrolysis of the membrane phospholipid, phosphatidylserine (PS). LysoPS is secreted by cells of the immune system and can induce multiple cellular responses, including T cell suppression and mast cell degranulation (Makide et al., Prostaglandins Other LipidMediat 89:135-9, 2009).

As described in this example, the present inventors have determined that phosphatidylserine (PS) is also a GPR174 agonist. PS is a phospholipid and is a component of the cell membrane. PS is actively held towards the cytosolic (inner) side of the cell membrane by the enzyme flippase. However, when a cell undergoes apoptosis, PS is no longer restricted to the cytosolic side by flippase. Instead, scramblase catalyzes the rapid exchange of PS between the two sides of the cell membrane. When PS flips to the extracellular (outer) surface of a cell, it acts as a signal for macrophages to engulf these cells (Verhoven B et al., J of Exp Med. 182(5): 1597-601, 1995.

Several transmembrane receptors—mostly expressed by phagocytes such as monocytes, macrophages, and dendritic cells—bind PS or PS/protein complexes and mediate efferocytosis. These include the TAM family of receptor tyrosine kinases (Tyro3, Ax1, MerTK), which recognize PS in complex with the serum proteins GAS6 or PROS1; proteins TEM (T-cell immunoglobulin and mucin domain-containing molecule), CD300, and BAI1, which directly interact with PS; and MFG-E8, which bridges PS with integrins (see Arandjelovic S. and Ravichandran K., Nature Immunology 16:907-917, 2015; Lemke G. and Burstyn-Cohen T., Ann NY Acad Sci 1209:23- 29, 2010; Morizono K. and Chen I.S.Y., Journal of Virology, Vol 88 (8):4275-4290, 2014; and Park, S. and Kin, I., Experimental & Molecular Medicine 49 e331, 2017). The signals from these receptors, transmitted to cells that actively recognize PS and mediate efferocytosis, are largely immunosuppressive.

In addition to apoptotic cells, extracellular PS is highly enriched in the tumor microenvironment and is found on the surface of tumor cells as well as endothelial cells of blood vessels infiltrating solid tumors. In addition, apoptotic neutrophils and activated platelets, where both exhibit, are also recruited to solid tumors (see A.K. and Rao D.A., Blood 120:4667-4668, 2012; Schlesinger, M., Journal of Hematology and Oncology (11) 125, 2018; Treffers L.W. et al., Immunological Reviews vol 273: 312-328, 2016; and Gregory A.D. and Houghton A.M., Cancer Research Vol 71 (7): 2411-6, 2011). PS is also found in extracellular vesicles (EVs) derived from tumors and apoptotic cells, including microvesicles, exosomes and exomeres (see, for example, Zhang et al., Cell Reports 27:940-954, 2019 and Raposo G and Stoorvogel W. , Jour Cell Biol 200(4):373, 2013). Strikingly, PS binding proteins are highly concentrated in solid tumors in mouse models, as detected by whole body imaging (Li R. et al., Mol Cancer Ther 17(1): 169-82, 2018; Desai T.J. et al. al., Oncotarget 7:30678–30690, 2016; and Birge R.B. et al., Cell Death & Differentiation 23: 962–978, 2016). Thus, high concentrations of PS are considered to be a major source of tumor-mediated immunosuppression and may play a role in resistance to cancer immunotherapies such as checkpoint inhibitors.

Since lysoPS and PS are very similar in structure but differ in water solubility, we examined in this example whether liposomes formed from PS could agonize GPR174 to the same extent as lysoPS. The ability of PS-exposing membranes to agonize GPR174 may indicate that the enzymes required to hydrolyze PS to lysoPS may not be necessary for high levels of PS in tumors to act on GPR174-expressing cells.

Comparison of PS and LysoPS in Gs signaling in HEK293 cells transfected with wild-type GPR174 or mutant GPR174.

Single-layer liposomes of phosphatidylserine (PS) and other phospholipids were prepared by hydration in PBS and extrusion at 50-54°C through a 0.1 μm filter using a miniextruder (Avanti). Lysophosphatidylserine (Lyso-PS) is soluble.

HEK293 cells were grown in 6-well plates and transfected using lipo

- 101 046439 Fectamine 2000 (Thermo Fisher Scientific), using 300 ng/well cAMP biosensor-expressing plasmid pGlo22F (Promega) and 2 ng plasmid expressing wild-type GPR174, or 2 ng plasmid expressing a GPR174 mutant with lower initial activity, indicated like GPR174-v38.

5-6 hours after transfection, cells were trypsinized and plated at a density of 5000 cells/well in 96-well plates and incubated overnight. The next day, the culture medium was replaced using 100 μl/well of X-vivo15 serum-free medium (Lonza) with 2% GloSensor assay reagent (Promega). The plates were incubated for 1 hour at room temperature and measured in a luminometer to obtain the value before compound addition. Liposomes of phosphatidylserine (PS) in the concentration range from 10 to 10,000 nM or lysophosphatidylserine (Lyso-PS) in the concentration range from 10 to 10,000 nM were added to the wells and incubated for 15 min at room temperature. The plates were measured in a luminometer to obtain the value after compound addition. The ratio of measurements after the addition of the compound and before the addition of the compound (ie, the degree of induction of the Glossensor signal by the agonist) was determined.

Fig. 45A graphically illustrates the fold induction of Gs signaling activity in HEK293 cells transfected with wild-type GPR174 expression plasmid and cAMP biosensor expression plasmid pGlo22F in the presence of increasing concentrations of phosphatidylserine (PS) or lysophosphatidylserine (Lyso-PS) liposomes. As shown in FIG. 45A, both PS and Lyso-PS stimulate GPR174 signaling in a concentration-dependent manner (PS with EC ₅₀ =83 nM). It should be noted that the apparent activity of PS is 4-5 times higher than the apparent activity of LysoPS (i.e. the same signal is achieved at a lower concentration of PS), making it unlikely that the liposome-induced signal can be explained by the breakdown of PS to Lyso-PS during a signal transmission experiment. Moreover, in this regard, it is noted that direct measurement by LC-MS of the amounts of PS and Lyso-PS in the preparations used by the present inventors showed the presence of only small amounts of Lyso-PS (3%) in the PS preparation used by the present inventors, which is not increased after incubation of PS with cells and medium in a signaling experiment (data not shown).

Fig. 45B graphically illustrates using an enlarged signal window compared to FIG. 45A, Fold induction of Gs signaling activity in HEK293 cells transfected with the GPR174-v38 mutant expression plasmid and the cAMP biosensor expression plasmid pGlo22F in the presence of increasing concentrations of phosphatidylserine (PS) or lysophosphatidylserine (Lyso-PS) liposomes. As shown in FIG. 45B, both PS and Lyso-PS stimulate GPR174 signaling in a concentration-dependent manner. The signaling window for GPR174v38 is superior to that for the wild-type GPR174 receptor (GPR174-WT), but the lipid dose response is shifted slightly to the right, showing lower affinity for both PS and Lyso-PS. For this GPR174 mutant, similar to the results observed for GPR174-WT, PS liposomes serve as a more active agonist than Lyso-PS.

Analysis of additional phospholipids for ability to function as GPR174 agonists.

Additional phospholipids: phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI) and phosphatidylserine (PS): prepared in the form of unilamellar liposomes by hydration in PBS and extrusion at 50-54°C through a 0.1 μm filter using a mini-extruder ( Avanti) and analyzed in comparison to PS for their ability to function as GPR174 agonists. All lipids were used at a final concentration of 4 μM. The signaling experiment was performed as described above for FIG. 45, but instead of transient transfection, a pool of HEK293 cells stably transfected with GPR174 WT and the cAMP GloSensor biosensor was used.

Fig. 46 graphically illustrates the level of GPR174 Gs signaling activity in HEK293 cells transfected with the GPR174 expression plasmid and the cAMP biosensor expression plasmid pGlo22F (shown as the ratio of phospholipid-added luminescence to pre-read luminescence value) in the presence of phosphatidylserine (PS), phosphatidylcholine (PC) liposomes ), phosphatidylethanolamine (PE) and phosphatidylinositol (PI).

As shown in FIG. 46, GPR174 signaling was stimulated in the presence of PS, while phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylinositol (PI) liposomes were completely inactive. Thus, it has been shown that Gs signaling through GPR174 is specific to LysoPS and PS.

Analysis of PS-mediated GPR174 signaling in the presence of representative GPR174 inhibitory compounds.

The signaling experiment was performed as described for FIG. 45, but instead of transient transfection, a pool of HEK293 cells was used stably transfected with either GPR174 WT (for compound 10, as shown in FIG. 47A) or GPR174-v38 for the remaining compounds tested, as shown in FIG. 47B-47F)) and the cAMP GloSensor biosensor. Serial dilutions of each of the GPR174 inhibitory compounds were prepared: 10 (group I), 6 (group I), 11 (group I), 20 (group II), 23 (group

- 102 046439

IV) and 30 (group IV) in DMSO. After the plates were pre-read in the luminometer, 1 μl of compound dilution was added per well (with control wells containing 1 μl of DMSO), followed by the addition of PS liposomes to a final concentration of 1 μM. The plates were read in a luminometer after 15 min of incubation. The ratio of luminescence after addition of PS and pre-readout luminescence was used as an indicator of receptor-mediated signaling.

Fig. 47A graphically illustrates the inhibition of PS-induced GPR174 Gs signaling by GPR174 inhibitory compound 10 (group I) in a dose-dependent manner, with an apparent IC ₅₀ of approximately 20-40 nM. These data indicate that compound 10 is a relatively potent antagonist of PS-mediated GPR174 signaling. At concentrations of 1-2 μM, compound 10 achieved almost complete inhibition of PS signaling through the GPR174 receptor.

Fig. 47B graphically illustrates the inhibition of PS-induced GPR174 Gs signaling by GPR174 inhibitory compound 6 (group I) in a dose-dependent manner, with an apparent IC ₅₀ of approximately 0.1 μM.

Fig. 47C graphically illustrates the inhibition of PS-induced GPR174 Gs signaling by GPR174 inhibitory compound 11 (group I) in a dose-dependent manner, with an apparent IC ₅₀ of approximately 0.1 μM.

Fig. 47D graphically illustrates the inhibition of PS-induced GPR174 Gs signaling by GPR174 inhibitory compound 20 (group II) in a dose-dependent manner, with an apparent IC ₅₀ of approximately 3.0 μM.

Fig. 47E graphically illustrates the inhibition of PS-induced GPR174 Gs signaling by GPR174 inhibitory compound 30 (group IV) in a dose-dependent manner, with an apparent IC50 of approximately 1.3 μM.

Fig. 47F graphically illustrates the inhibition of PS-induced GPR174 Gs signaling by GPR174 inhibitory compound 23 (group IV) in a dose-dependent manner, with an apparent IC ₅₀ of approximately 0.15 μM.

It was also determined (data not shown) that inhibition of PS-induced GPR174 Gs signaling by GPR174 inhibitory compound 59 (group Va) occurred in a dose-dependent manner, with an apparent IC ₅₀ of approximately 10 μM.

As shown in FIG. 47A-47F, PS-mediated GPR174 signaling of Gs is inhibited by representative GPR174 inhibitory compounds 6, 10, 11, 20, 23 and 30, belonging to various chemical classes (ie, groups I, II and IV). These data indicate that compounds 6, 10, 11, 20, 23, and 30 act as GPR174 antagonists and inhibit PSliposome-mediated cAMP signaling. Thus, PS-liposome signaling through GPR174 is inhibited by a variety of GPR174-inhibiting small molecules with diverse chemical structures.

These data indicate that PS is a potent and specific GPR174 agonist and that compound 10 is a relatively potent GPR174 inhibitor. The ability of PS to directly stimulate GPR174 has many implications for immunotherapy of malignancies. PS is exposed to solid tumors, apoptotic cells including tumor cells and neutrophils, extracellular vesicles from tumor cells and other cells (exosomes), living tumor cells, activated T cells and B cells, activated platelets, monocytes and macrophages, and vascular endothelium . Because PS is thought to generally act on myeloid lineage immunocytes via efferocytosis receptors (described above), GPR174 is predominantly expressed on lymphocytes, including T cells, B cells and NK cells, providing the possibility that PS may directly modulate adaptive immune responses. GPR174 signals through the GscAMP pathway, which suppresses Th1 and NK cell function (Zidek K., Eur Cytokine Netw 1O(3):319-28, 1999; and Serezani S. N. et al., American Journal of Respiratory Cell and Molecular Biology Vol 39(2): 127-132, 2008); thus, inhibition of GPR174 should enhance antitumor responses of T cells and NK cells.

The present inventors have also shown in this example that using 1-2 μM compound 10 achieves almost complete inhibition of PS signaling by GPR174, thus these inhibitor concentrations are suitable for functional studies to study the modulation of T cell responses by GPR174 and PS .

Example 17.

GPR174 signaling in HEK293 cells following exposure to apoptotic K562 cells.

This example describes experimental results showing that apoptotic cells stimulate the GPR174 Gs signaling pathway in cells expressing GPR174.

As described in Example 16, the present inventors discovered that PS is a natural agonist of the GPR174 Gs signaling pathway. As provided in Example 16, PS can be exposed to a variety of cell types in the tumor microenvironment; however, in example 16 the authors

- 103 046439 of the present invention used only PS-containing liposomes to stimulate GPR174. In this example, we examined whether PS exposed on cells could also agonize GPR174 in our signaling assay. To do this, the present inventors used 1) an apoptotic cell line, 2) apoptotic primary neutrophils and 3) activated platelets.

Apoptosis was induced in K562 cells by overnight treatment with 0.8 mM H ₂ O ₂ . The presence of a high percentage of apoptotic cells after H ₂ O ₂ treatment was confirmed by flow cytometry after Annexin V staining (data not shown).

HEK293 cells stably transfected with wild-type GPR174 and GloSensor expression plasmids were seeded into 96-well plates at a density of 5000 cells/well and assayed using 50 μl/well of a 10 x 10 ⁶ /ml suspension of untreated K562 cells, or K562 cells treated with overnight using 0.8 mM H ₂ O ₂ or cell medium. The change in luminescence was measured after 15 minutes. HEK293 cells stably transfected with the GloSensor expression plasmid alone did not respond to apoptotic cells (data not shown).

Fig. 48A graphically illustrates the ratio of luminescence after addition to pre-readout luminescence for GPR174 and GloSensor expressing HEK293 cells exposed to culture media, or untreated (non-apoptotic) K562 cells, or apoptotic K562 cells (treated with H ₂ O ₂ for 20 hours). As shown in FIG. 48A, GPR174 signaling in HEK293 cells exposed to apoptotic K562 cells was significantly higher than in HEK293 cells exposed to untreated K562 cells (p<10 ^-4 ) or HEK293 cells exposed to media alone.

GPR174 signaling in HEK293 cells following exposure to apoptotic neutrophils.

Apoptosis was induced in donor blood neutrophils by overnight incubation with agonistic anti-Fas antibodies (Fisher Scientific). The high percentage of apoptotic cells after treatment with anti-Fas antibodies was confirmed by flow cytometry after Annexin V staining (data not shown).

HEK293 cells stably transfected with wild-type GPR174 and GloSensor expression plasmids were seeded into 96-well plates at a density of 5000 cells/well and assayed using 50 μl/well of 10 x 10 ⁶ cells/ml suspension of freshly isolated neutrophils from a donor, or incubated neutrophils overnight with anti-Fas antibodies or cell media.

Fig. 48B graphically illustrates the ratio of luminescence after addition to pre-readout luminescence for GPR174 and GloSensor expressing HEK293 cells exposed to culture medium or freshly isolated neutrophils or apoptotic neutrophils (treated with anti-Fas antibody). As shown in FIG. 48B, GPR174 signaling in HEK293 cells exposed to apoptotic neutrophils was significantly higher than in HEK293 cells exposed to freshly isolated neutrophils (p=0.0006) or HEK293 cells exposed to media alone.

GPR174 signaling in HEK293 cells following exposure to activated platelets.

Donor blood platelets were isolated by differential centrifugation. A high percentage of platelets isolated by this method were activated, as confirmed by flow cytometry after Annexin V staining (data not shown).

HEK293 cells stably transfected with wild-type GPR174 and GloSensor expression plasmids were seeded into 96-well plates at a density of 5000 cells/well and assayed using 50 μl/well of 5 x 108 cells/ml platelets (denoted as P1 in Fig. 48C) or platelet supernatants (indicated as sup. in Fig. 48C).

Fig. 48C graphically illustrates the ratio of luminescence after addition to pre-read luminescence for GPR174 and GloSensor expressing HEK293 cells exposed to culture supernatant (sup.) or platelets (P1). As shown in FIG. 48C, GPR174 signaling in HEK293 cells exposed to activated platelets was significantly higher than in HEK293 cells exposed to culture supernatants (p=0.03) or HEK293 cells.

Consistent with the ability of PS liposomes to act as a potent GPR174 agonist, the data in this example show that PS exposed at the cell surface—either through apoptosis, as in the case of K562 cells and neutrophils, or through platelet activation—serves to as a GPR174 agonist. These effects were observed at high cell concentrations, suggesting that cell-cell contact was important for this type of signal transduction. The discovery that PS is an active natural ligand for GPR174, and that PS stimulates an immunosuppressive GPCR, namely GPR174, on T lymphocytes represents a significant advance in our understanding of how PS can influence tumor immunity. Since GPR174 is a Gs-coupled receptor and cAMP is known to be an immunosuppressive signal, the present inventors believe that the ability

- 104 046439 GPR174 expressed on lymphocytes to respond to apoptosis-induced cell surface PS contributes to the ability of apoptotic cells to suppress the adaptive immune system. It is likely that some types of tumors and viral infections exploit this regulatory property to evade immunosurveillance by providing a PS-rich surface environment. Thus, GPR174 inhibitory compounds are believed to have therapeutic utility for inhibiting PS- and Lyso-PS-mediated GPR174 signaling and thereby avoiding the immunosuppression that is typically induced in the presence of apoptotic cells and/or tumors.

Example 18.

This example describes experimental results further demonstrating that a GPR174 inhibitory compound in combination with various adenosine 2a receptor (A2aR) inhibitors synergistically induce Thl cytokines in human PBMC.

As described in Example 15, it was determined that the combination of a representative GPR174 inhibitor (compound 10) and the adenosine 2a receptor (A2aR) inhibitor ZM-241385 resulted in a synergistic induction of IFN-γ production in human PBMC.

To extend our initial findings, additional experiments were performed to confirm that synergistic cytokine induction was observed using a representative GPR174 inhibitor (compound 10), in combination with various A2aR inhibitors.

A2aR inhibitor SCH-58261 (SCH).

Human PBMCs obtained from a volunteer human donor (donor 1) were spread in X-vivo15 media (Lonza) supplemented with Glutamax, penicillin and streptomycin at a density of 1x 10 ⁶ cells/well in a 96-well flat-bottom plate and stimulated with 1 µg/ml anti-CD3 antibody (UCHT1) and 0.1 µg/ml anti-CD28 antibody (CD28.2) in the presence of:

GPR174 inhibitory compound 10 (2 μM);

A2aR inhibitory compound (SCH-58261, 0.2, 0.6 or 2 μM);

GPR174 inhibitory compound 10 (2 μM) plus A2aR inhibitory compound (SCH58261, 0.2, 0.6 or 2 μM); or control media (DMSO).

Supernatants from these cells were collected 24 h after stimulation, and the levels of IFN-γ, IL-2, and TNF were determined by ELISA assays.

Fig. 49A graphically illustrates the amount of IFN-γ (pg/ml) in culture supernatants 24 hours after stimulation in PBMC cultures from a single donor (donor 1) cultured in the presence of GPR174 inhibitory compound 10 (2 μM); an A2aR inhibitory compound (SCH-58261, 0.2 µM, 0.6 µM or 2 µM); combinations of compound 10 (2 μM) plus 0.2 μM SCH-58261; combinations of compound 10 (2 μM) plus 0.6 μM SCH-58261 or combinations of compound 10 (2 μM) plus 2 μM SCH-58261; or control media (DMSO).

As shown in FIG. 49A, and summarized in table. 9, lower concentrations of A2aR inhibitor (0.2 μM and 0.6 μM SCH-58261) increased IFN-γ levels by less than 2-fold, and GPR174 inhibitory compound 10 (2 μM) increased IFN-γ levels by 4.9 once. In contrast, the combination of inhibitors increased IFN-γ levels 11- to 12-fold, showing synergistic activity between A2aRi and GPR174i, as these values were greater than the sum of the increases obtained for each inhibitor alone.

Fig. 49B graphically illustrates the amount of IL-2 (pg/ml) in culture supernatants 24 hours after stimulation in PBMC cultures from a single donor (donor 1) cultured in the presence of GPR174 inhibitory compound 10 (2 μM); an A2aR inhibitory compound (SCH-58261, 0.2 µM, 0.6 µM or 2 µM); combinations of compound 10 (2 μM) plus 0.2 μM SCH-58261; combinations of compound 10 (2 μM) plus 0.6 μM SCH-58261 or combinations of compound 10 (2 μM) plus 2 μM SCH-58261; or control media (DMSO).

As shown in FIG. 49B, and summarized in table. 9, all concentrations of the A2aR inhibitor (0.2 μM, 0.6 μM and 2 μM SCH-58261), as well as the GPR174 inhibitor (2 μM compound 10), had no effect on IL-2 accumulation. In contrast, the combination of inhibitors significantly increased IL-2 levels by 1.4- to 1.5-fold, demonstrating synergistic activity between A2aRi and GPR174i, as these values were greater than the sum of the increases obtained for each inhibitor alone.

Fig. 49C graphically illustrates the amount of TNF (pg/ml) in culture supernatants 24 hours after stimulation in PBMC cultures from a single donor (donor 1) cultured in the presence of GPR174 inhibitory compound 10 (2 μM); an A2aR inhibitory compound (SCH-58261, 0.2 µM, 0.6 µM or 2 µM); combinations of compound 10 (2 μM) plus 0.2 μM SCH-58261; combinations of compound 10 (2 μM) plus 0.6 μM SCH-58261 or combinations of compound 10 (2 μM) plus 2 μM SCH-58261; or control media (DMSO).

As shown in FIG. 49C and summarized in table. 9, lower concentrations of A2aR inhibitor (0.2 μM and 0.6 μM SCH-58261) increased TNF levels by less than 3-fold, and GPR174 inhibitor (2 μM

- 105 046439 compound 10) increased TNF levels by 4.2 times. In contrast, the combination of A2aRi and GPR174i increased TNF levels by 14- to 16-fold, indicating synergistic activity between the two inhibitors, as these values were greater than the sum of the increases obtained for each inhibitor alone.

Table 9. Summary of results from FIG. 49A-C

IFN-γ IL-2 Carrier 2 µM cond. 10 Carrier Media ¹ 1.0 4.9* 1.0 0.2 µM SCH ¹ 1.8 11.8* 1.0 0.6 µM SCH ¹ 1.9* 11.3* 0.9 2 µM SCH ¹ 5.9* 11.0* 0.8 2 µM conn. 10 0.9 1.5* 1.4* 1.4* TNF Carrier 1.0 2.2* 2.8* 4.1* 2 µM conn. 10 4.2* 14.2* 15.9* 14.7*

Fold change values for the GPR174 inhibitory compound 10 and the A2aR inhibitory compound SCH-58261 at the indicated concentrations, compared to vehicle control.

*, p<0.05, t-test, two-tailed distribution, two-sample t-test with equal variances.

A2aR inhibitor ZM-241385 (ZM) and A2aR inhibitor PBF-509 (PBF).

Human PBMCs obtained from two individual volunteer human donors (donor 2 and donor 3) were distributed at a density of 1 x 10 ⁶ cells/well in a 96-well flat-bottom plate and stimulated with 1 μg/ml anti-CD3 antibody (UCHT1) and 0. 1 μg/ml antibody against CD28 (CD28.2) in the presence of:

GPR174 inhibitory compound 10 (1 μM);

A2aR inhibitory compound (ZM-241385, 0.2 μM);

A2aR inhibitory compound (PBF-509, 0.1 μM or 0.2 μM); GPR174 inhibitory compound 10 (1 μM) plus A2aR inhibitory compound (ZM241385, 0.2 μM); or GPR174 inhibitory compound 10 (1 μM) plus A2aR inhibitory compound (PBF-509, 0.1 μM or 0.2 μM); or control media (DMSO).

Supernatants from these cells were collected 24 h after stimulation, and the levels of IFN-γ, IL-2, and TNF were determined by ELISA assays.

Fig. 50A graphically illustrates the amount of IFN-γ (pg/ml) in culture supernatants 24 hours after stimulation in PBMC cultures from a single donor (donor 2) cultured in the presence of GPR174 inhibitory compound 10 (1 μM); A2aR inhibitory compound (ZM, 0.2 μM); A2aR inhibitory compound (PBF-509 0.1 μM), combination of compound 10 (1 μM) plus 0.2 μM ZM; combinations of compound 10 (1 μM) plus 0.1 μM PBF-509; or control media (DMSO).

As shown in FIG. 50A, and summarized in table. 10, a high degree of IFN-γ induction was observed using ZM or PBF-509 in the presence of compound 10 (8.8- and 3.5-fold, respectively). In contrast, ZM or PBF-509 alone resulted in 1.8-fold and 1.4-fold induction of IFN-γ, respectively. Compound 10 alone induced IFN-γ by 1.6-fold. Thus, the values obtained using a combination of an A2aR inhibitory compound (eg, ZM or PBF-509) and a GPR174 inhibitory compound (eg, compound 10) showed synergistic activity because they were greater than the sum of the increases obtained for each inhibitor separately.

Fig. 50B graphically illustrates the amount of IL-2 (pg/ml) in culture supernatants 24 hours after stimulation in PBMC cultures from a single donor (Donor 2) cultured in the presence of GPR174 inhibitory compound 10 (1 μM); A2aR inhibitory compound (ZM, 0.2 μM); A2aR inhibitory compound (PBF-509 0.1 μM), combination of compound 10 (1 μM) plus 0.2 μM ZM; combinations of compound 10 (1 μM) plus 0.1 μM PBF-509; or control media (DMSO).

As shown in FIG. 50V and summarized in table. 10, a high degree of IL-2 induction was observed using ZM or PBF-509 in the presence of compound 10 (2.6-fold and 2.0-fold, respectively). In contrast, ZM or PBF-509 alone resulted in IL-2 induction by 1.6- and 1.3-fold, respectively. Compound 10 alone induced IL-2 by 1.1-fold. Thus, the values obtained using a combination of an A2aR inhibitory compound (eg, ZM or PBF-509) and a GPR174 inhibitory compound (eg, compound 10) showed synergistic activity because they were greater than the sum of the increases obtained for each inhibitor separately.

Fig. 50C graphically illustrates the amount of TNF (pg/ml) in culture supernatants 24 hours after stimulation in PBMC cultures from a single donor (donor 2) cultured in the presence of GPR174 inhibitory compound 10 (1 μM); A2aR inhibitory compound (ZM, 0.2 μM); A2aR inhibitory compound (PBF-509 0.1 μM), combination of compound 10 (1 μM) plus 0.2 μM ZM; combinations of compound 10 (1 μM) plus 0.1 μM PBF-509; or control media (DMSO).

- 106 046439

As shown in FIG. 50C and summarized in table. 10, a high degree of TNF induction was observed using ZM or PBF-509 in the presence of compound 10 (4.0-fold and 2.3-fold, respectively). In contrast, ZM or PBF-509 alone resulted in TNF induction of 1.7- and 1.3-fold, respectively. Compound 10 alone induced TNF by 1.5-fold. Thus, the values obtained using a combination of an A2aR inhibitory compound (eg, ZM or PBF-509) and a GPR174 inhibitory compound (eg, compound 10) showed synergistic activity because they were greater than the sum of the increases obtained for each inhibitor separately.

Fig. 51A graphically illustrates the amount of IFN-γ (pg/ml) in culture supernatants 24 hours after stimulation in PBMC cultures from a single donor (donor 3) cultured in the presence of GPR174 inhibitory compound 10 (1 μM); A2aR inhibitory compound (ZM, 0.2 μM); A2aR inhibitory compound (PBF-509 0.2 μM), combination of compound 10 (1 μM) plus 0.2 μM ZM; combinations of compound 10 (1 μM) plus 0.2 μM PBF-509; or control media (DMSO).

As shown in FIG. 51A and summarized in table. 10, significant induction of IFN-γ was observed using ZM or PBF-509 in the presence of compound 10 (2.5- and 1.8-fold, respectively). In contrast, ZM or PBF-509 alone resulted in IFN-γ induction by 1.3- and 1.2-fold, respectively. Compound 10 alone induced IFN-γ by 1.2-fold. Thus, the values obtained using a combination of an A2aR inhibitory compound (eg, ZM or PBF-509) and a GPR174 inhibitory compound (eg, compound 10) showed synergistic activity because they were greater than the sum of the increases obtained for each inhibitor separately.

Fig. 51B graphically illustrates the amount of IL-2 (pg/ml) in culture supernatants 24 hours after stimulation in PBMC cultures from a single donor (donor 3) cultured in the presence of GPR174 inhibitory compound 10 (1 μM); A2aR inhibitory compound (ZM, 0.2 μM); A2aR inhibitory compound (PBF-509 0.2 μM), combination of compound 10 (1 μM) plus 0.2 μM ZM; combinations of compound 10 (1 μM) plus 0.2 μM PBF-509; or control media (DMSO).

As shown in FIG. 51B and summarized in table. 10, a high degree of IL-2 induction was observed using ZM or PBF-509 in the presence of compound 10 (2.3 and 1.6 times, respectively). In contrast, ZM or PBF-509 alone resulted in IL-2 induction of 1.3- and 1.2-fold, respectively. Compound 10 alone induced IL-2 by 1.1-fold. Thus, the values obtained using a combination of an A2aR inhibitory compound (eg, ZM or PBF) and a GPR174 inhibitory compound (eg, compound 10) showed synergistic activity because they were greater than the sum of the increases obtained for each inhibitor separately.

Fig. 51C graphically illustrates the amount of TNF (pg/ml) in culture supernatants 24 hours after stimulation in PBMC cultures from a single donor (donor 3) cultured in the presence of GPR174 inhibitory compound 10 (1 μM); A2aR inhibitory compound (ZM, 0.2 μM); A2aR inhibitory compound (PBF-509 0.2 μM), combination of compound 10 (1 μM) plus 0.2 μM ZM; combinations of compound 10 (1 μM) plus 0.2 μM PBF-509; or control media (DMSO).

As shown in FIG. 51C and summarized in table. 10, a high degree of TNF induction was observed using ZM or PBF-509 in the presence of compound 10 (4.6- and 2.9-fold, respectively). In contrast, ZM or PBF-509 alone resulted in TNF induction by 2.1- and 1.8-fold, respectively. Compound 10 alone induced TNF by 1.5-fold. Thus, the values obtained using a combination of an A2aR inhibitory compound (eg, ZM or PBF-509) and a GPR174 inhibitory compound (eg, compound 10) showed synergistic activity because they were greater than the sum of the increases obtained for each inhibitor separately.

Table 10. Summary of results shown in FIG. 50А-С and 51А-С

Donor Cytokine Carrier ¹ ZM ¹ PBF ¹ 2 IFN-γ carrier 1.0 1.8* 1.4* 2 IFN-γ Conn. 10 1.6* 8.8* 3.5* 2 IL-2 carrier 1.0 1.6* 1.3* 2 IL-2 Conn. 10 1.1 2.6* 2.0* 2 TNF carrier 1.0 1.7* 1.3* 2 TNF Conn. 10 1.5* 4.0* 2.3* 3 IFN-γ carrier 1.0 1.2 0.9 3 IFN-γ Conn. 10 1.2 2.5* 1.8* 3 IL-2 carrier 1.0 1.3* 1.2 3 IL-2 Conn. 10 1.1 2.3* 1.6* 3 TNF carrier 1.0 2.1* 1.8* 3 TNF Conn. 10 1.5* 4.6* 2.9*

Fold change values for GPR174 inhibitory compound 10 and A2aR inhibitory compounds ZM-241385 and PBF-509 compared to vehicle control.

*, p<0.05, t-test, two-tailed distribution, two-sample t-test with equal variances.

- 107 046439

As described in Example 15, it was determined that the combination of a representative GPR174 inhibitor (compound 10) and the adenosine 2a receptor (A2aR) inhibitor ZM-241385 resulted in a synergistic induction of IFN-γ production in human PBMC. Using the same PBMC stimulation system as described in Example 15, as shown in this example, the present inventors have now observed synergistic induction of IFN-γ using three A2aR inhibitors: ZM241385, SCH-58261 and PBF-509 in combination with GPR174 inhibitory compound 10, as shown in FIG. 49A, 50A and 51A. In all cases, A2aR inhibitors showed greater induction of IFN-γ in the presence of the GPR174 inhibitory compound 10, and similarly, compound 10 was most effective when combined with an A2aR inhibitor. As further shown in FIG. 49B, 50B and 51B, the present inventors also observed synergistic induction of IL-2 using three A2aR inhibitors: ZM-241385, SCH-58261 and PBF-509 in combination with compound 10. As shown in FIG. 49C, 50C and 51C, synergistic induction of TNF was also observed using three A2aR inhibitors: ZM-241385, SCH-58261 and PBF-509 in combination with compound 10.

These findings reinforce our original finding that inhibition of A2aR and GPR174 results in a cooperative enhancement of T cell activation and cytokine production. Because the upregulated cytokines IFN-γ, IL-2, and TNF are characteristic of Th1 cells, these findings led to the conclusion that combined inhibition of the GPCR A2aR and GPR174 may enhance the antitumor immune response.

It should be noted that the A2aR inhibitors tested belong to four different chemical classes and that none of them affected GPR174 signaling when tested in signaling assays (data not shown). Likewise, GPR174 inhibitors do not affect A2aR signaling when assessed in signaling assays (data not shown). These data indicate that in all cases the observed effects are on target.

In the initial detections, we used doses of 1 and 10 μM ZM241385, which should inhibit both A2aR and A2bR. In contrast, the experiments present in this example show activity similar to two additional A2aR inhibitory compounds having greater specificity for A2aR than for A2bR (see Table 12 in Example 20).

In conclusion, the synergistic cytokine induction observed in the presence of A2aR-selective doses of PBF-509 (0.1 μM) and SCH-58261 (0.2 μM) in combination with the GPR174 inhibitory compound 10 indicates that inhibition of A2aR alone in the presence of the inhibitory compound 10 GPR174 compounds (eg, compound 10) can enhance Th1 responses and thus enhance antitumor responses.

Example 19.

This example describes an extended evaluation of synergistic Th1 cytokine induction in which a total of 44 individual assays of the A2aR inhibitor ZM-241385 and the GPR174 inhibitory compound 10 were assayed separately and together in PBMC isolated from 12 healthy donors.

State of the art.

As described above in Examples 15 and 18, synergistic induction of IFN-γ was observed in PBMCs isolated from several different donors using three different A2aR inhibitors: ZM241385, SCH-58261 and PBF-509, when each was combined with GPR174i (compound 10) , compared with cytokine induction in the presence of each inhibitor separately.

This example describes an extensive evaluation of synergistic IFN-γ induction in which a total of 44 individual assays of ZM-241385 and Compound 10 were assayed separately and together in PBMC isolated from 12 healthy donors.

Ways.

Human PBMCs obtained from twelve individual human donors were distributed at a density of 1x106 cells/well in a 96-well flat-bottom plate and stimulated in triplicate with 1 μg/ml anti-CD3 antibody (UCHT1) and 0.1 μg/ml anti-CD3 antibody (UCHT1). CD28 (CD28.2) in the presence of:

GPR174 inhibitory compound 10 (1.2 μM);

A2aR inhibitory compound (ZM-241385, 0.2, 1 μM);

GPR174 inhibitory compound 10 (1.2 μM) plus A2aR inhibitory compound (ZM241385, 0.2 μM, 1 μM); or control media (DMSO).

A total of 44 experiments were tested using PBMCs from 12 healthy donors (9 donors for GM-CSF), with each donor tested in 1-8 separate experiments, depending on the donor (1-4 experiments each, for GM-CSF). For donors tested multiple times, the average of replicate experiments was used for the data shown so that each donor is represented by a single value. Cytokines were detected in supernatants using Legendplex (Biolegend) or MSD (MesoScale) platforms. The median (line), mean (point), and range (error bars) of the summary data are shown in Fig. 52A-52E. Percentage of donors for whom synergistic ac is indicated

- 108 046439 activity of compound 10 and ZM together (defined as an increase in cytokine levels greater than the sum of the increases observed for each compound separately) are shown in each graph. One-way ANOVA with Tukey's post hoc multiple comparison correction was performed to identify significant differences (FC, mean fold change; ****, p<0.0001; ***, p<0.001; **, p<0.01; *, p<0.05, ns, not significant).

Results.

Fig. 52A graphically illustrates the fold change, relative to vehicle, in the amount of IFN-γ in culture supernatants 24 hours post-stimulation in PBMC cultures from 12 donors cultured in the presence of GPR174 inhibitory compound 10 (1-2 μM); A2aR inhibitory compound (ZM, 0.2-1 μM); combinations of compound 10 (1-2 µM) plus 0.2-1 µM ZM; or control media (DMSO).

As shown in FIG. 52A and summarized in table. 11, a high degree of IFN-γ induction was observed using ZM in the presence of compound 10 (8.8-fold average, 25-fold maximum). In contrast, ZM alone resulted in a mean IFN-γ induction of 2.1-fold. On its own, compound 10 was slightly more potent than ZM, with an average IFN-γ induction of 2.8-fold (p=0.06), as shown in Table 1. 11. Among individual donors, 75% (9/12) showed synergistic IFN-γ induction because the values obtained with both inhibitors were greater than the sum of the increases obtained with each inhibitor alone.

Fig. 52B graphically illustrates the fold change, relative to vehicle, in the amount of IL-2 in culture supernatants 24 hours post-stimulation in PBMC cultures from 12 donors cultured in the presence of GPR174 inhibitory compound 10 (1-2 μM); A2aR inhibitory compound (ZM, 0.2-1 μM); combinations of compound 10 (1-2 µM) plus 0.2-1 µM ZM; or control media (DMSO).

As shown in FIG. 52B and summarized in table. 11, a high degree of IL-2 induction was observed using ZM in the presence of compound 10 (2.7-fold mean, 4.3-fold maximum). In contrast, ZM alone induced IL-2 by a factor of 1.4 on average. Compound 10 alone was slightly more potent than ZM, producing an average of 1.7-fold IL-2 induction. Among individual donors, 92% (11/12) showed synergistic IL-2 induction because the values obtained using both inhibitors (i.e., A2aRi and GPR174i) were greater than the sum of the increases obtained using each inhibitor alone.

Fig. 52C graphically illustrates the fold change, compared to vehicle, in the amount of TNF in culture supernatants 24 hours after stimulation in PBMC cultures from 12 donors cultured in the presence of GPR174 inhibitory compound 10 (1-2 μM); A2aR inhibitory compound (ZM, 0.2-1 μM); combinations of compound 10 (1-2 µM) plus 0.2-1 µM ZM; or control media (DMSO).

As shown in FIG. 52C and summarized in table. 11, on average, a high degree of TNF induction was observed using ZM in the presence of compound 10 (8.0 times average, 25 times maximum). In contrast, ZM alone induced TNF by a factor of 2.1 on average. Compound 10 alone was slightly more potent than ZM, inducing TNF 2.7-fold on average. Among individual donors, 67% (8/12) showed synergistic TNF induction because the values obtained using both inhibitors (i.e., A2aRi and GPR174i) were greater than the sum of the increases obtained using each inhibitor alone.

Fig. 52D graphically illustrates the fold change, relative to vehicle, in the amount of GM-CSF in culture supernatants 24 hours post-stimulation in PBMC cultures from 12 donors cultured in the presence of GPR174 inhibitory compound 10 (1-2 μM); A2aR inhibitory compound (ZM, 0.2-1 μM); combinations of compound 10 (1-2 µM) plus 0.2-1 µM ZM; or control media (DMSO).

As shown in FIG. 52D and summarized in table. 11, on average, a high degree of granulocyte-macrophage colony-stimulating factor (GM-CSF) induction was observed using ZM in the presence of compound 10 (2.5-fold average, 5.5-fold maximum). In contrast, ZM alone induced GM-CSF by 1.3-fold. Compound 10 alone was slightly more potent than ZM, inducing GM-CSF 1.4-fold on average. Among individual donors, 89% (8/9) showed synergistic induction of GM-CSF because the values obtained using both inhibitors (i.e., A2aRi and GPR174i) were greater than the sum of the increases obtained using each inhibitor alone.

- 109 046439

Table 11. Summary of the results shown in FIG. 52A-52D

IFN-γ IL-2 TNF GM-CSF FC ¹ p2 F.C. R F.C. R F.C. R Carrier vs ZM 2.1 0.0412 1.4 <0.0001 2.1 0.0559 1.3 0.0116 Carrier vs. connection 10 2.8 0.0048 1.7 0.0004 2.7 0.0074 1.4 0.0076 Carrier vs. connection 10+ZM 8.8 0.0322 2.7 <0.0001 8.0 0.0365 2.5 0.0502 ZM vs. conn. 10 1.3 0.0625 1.2 0.2186 1.3 0.1131 1.1 0.6792 ZM vs. conn. 10+ZM 4.2 0.0376 1.9 0.0003 3.8 0.0451 1.9 0.0858 Conn. 10 vs. conn. 10+ZM 3.1 0.0549 1.6 <0.0001 3.0 0.0610 1.8 0.1049

1Fold change for the specified comparison of compounds.

² p value for Tukey's multiple comparison test.

For their extended assessment of synergistic cytokine induction in PBMC, we performed a total of 44 individual tests of the A2aR inhibitor ZM-241385 and the GPR174 inhibitory compound 10, separately and together. The present inventors used PBMCs from 12 healthy donors, where individual tests for the same donor ranged from 1 to 8 replicate experiments performed using individual vials of cryopreserved PBMCs. To present data and analyze significance, we first averaged replicate experiments for the same donor so that 12 data points for 12 donors were compared for the effects of a compound on cytokine production. In accordance with the initial experiments of the present inventors (described in Examples 15 and 17), ZM-241385 and compound 10 showed moderate induction of IFN-γ (on average 2.1 and 2.8 times, respectively), while how their combination caused an average IFN-γ induction of 8.8-fold, ranging up to 25-fold compared to vehicle control (see FIG. 52A). For 75% of donors (9/12), the amount of IFN-γ induced by the combination inhibitors exceeded the sum of the amounts of IFN-γ induced by each inhibitor alone, indicating that the GPR174 inhibitor and the A2aR inhibitor acted synergistically in the same pathway regulating production IFN-γ in human PBMCs. Similar synergistic effects were observed for the induction of IL-2 (FIG. 52B, 92% of donors), TNF (FIG. 52C, 67% of donors) and GM-CSF (FIG. 52D, 89% of donors), where synergistic effects were observed in the presence of the combination ZM-241385 and compounds 10 for most donors for each cytokine.

Consistent with inhibitors stimulating Th1 responses, it should be noted that additional cytokines characteristic of Th2 and TM7 cells, namely IL-4, IL-6, IL-10, L-13 and IL-17, were also measured in some of the experiments described in this example, all of which were largely unchanged or slightly reduced by treatment with the compound alone or in combination (data not shown).

It should also be noted that agonists for AdoR (eg, Ado or NECA) and GPR174 (PS or LysoPS) were not included in the experiments described in this example. In previous experiments, the present inventors found that addition of these agonists did not alter cytokine production, whereas AdoR and GPR174 inhibitors increased cytokine production to the same extent, regardless of agonist supplementation (data not shown), suggesting that The natural levels of adenosine and PS/LysoPS present in the PBMC cultures used by the present inventors were saturating. This finding also raises the possibility that the immunosuppressive activity of endogenous Ado and PS/LysoPS, where both are products of stress and cell death, is highly enriched in the tumor microenvironment (Vaupel, P. and Multhoff, G. Front Immunol (8): 1887 , 2017), can be effectively overcome by combined inhibition of their Gs-coupled GPCRs (i.e., A2aR and GPR174), while inhibition of their individual GPCRs results in suboptimal immune activation. It should be noted that, in most experiments, the increases in cytokines observed using compound 10 alone were greater than the increases obtained using ZM-241385 alone.

Since the present inventors observed that 0.2 μM ZM-241385 inhibited both A2aR and A2bR (see Table 12 in Example 20), they could not exclude the possibility that both receptors were inhibited for the data shown in this example. However, these findings strongly established that combined inhibition of GPR174 using A2aR, A2bR, or both (i.e., GPR174i plus A2aRi, or GPR174i plus A2bRi, or GPR174i plus A2aRi plus A2bRi) potently and synergistically induces TM cytokine responses in total human PBMCs from multiple human donors. Further evaluation of the contribution of A2aR and A2bR to synergy with GPR174 is presented below in Example 20.

Example 20.

This example describes further evaluation of the contribution of A2aRi and A2bRi to synergy with GPR174i.

Since most of the inventors' experiments use doses of ZM241385 predicted to inhibit both A2aR and A2bR, the present inventors then adjusted

- 110 046439 sought to determine whether the observed increase in cytokine levels is due to A2aR blockade alone, or whether A2bR inhibition also plays a role. Both receptors signal through the Gs-cAMP pathway and are known to suppress immune responses, and both receptors are considered important targets for immunotherapy, with receptor-specific inhibitors and inhibitors targeting both A2aR and A2bR (see e.g. Vigano S. et al., Front Immunol vol 10: 925, 2019). Determining whether inhibition of both or either receptor achieves maximum cytokine induction when combined with a GPR174 inhibitor should make an important contribution to the development of methods to optimally target both pathways.

To examine the relative contribution of A2aR and A2bR inhibition to synergy with compound 10, we evaluated A2aR-specific doses of PBF-509 (0.1 μM) and SCH58261 (0.2 μM), in the presence and absence of the A2bR inhibitor MRS-1754 (1 µM). In addition, the present inventors evaluated the effects of adenosine deaminase (ADA; 0.75 μg/ml), an enzyme that catabolizes adenosine to inosine and which should promote effects similar to dual inhibition of A2aR and A2bR.

Ways.

In an initial experiment, several A2aR and A2bR inhibitors were assayed for specificity for each receptor, and IC ₅₀ values were determined as follows: HEK293 cells were transiently cotransfected with A2aR or A2bR expression constructs (Origene, MD), Cgeluciferase, and TK-renilla as an internal control. (Promega) using Lipofectamine 2000 (Invitrogen, CA), according to the manufacturer's instructions. Transfected cells were incubated for 4-6 hours at 37°C and then treated with trypsin and transferred to a 96-well plate. Increasing concentrations of inhibitors (ZM-241385, SCH-58261, PBF-509 and MRS-1754) were added to the cells and then incubated overnight at 37°C. After overnight incubation, media was removed and cells were lysed. Luciferase and renilla activities were measured using a dual luciferase assay system (Promega, WI), according to the manufacturer's instructions. Lysis buffer (25 μl) was added to the cells and incubated at room temperature for 20-30 min to allow cell lysis. Luciferase and renilla activities were measured in the lysate (10 μl) using LARII and Stop&Glo reagent (50 μl each) using SpectraMax L (Molecular Devices, CA). The results are shown in table. 12 below.

Table 12. Specificity of various A2aR and A2bR inhibitors

A2A (1С ₅₀ nM) A2B (1C ₅₀ nM) ZM-241385 4.8 72 SCH-58261 40 >2000 PBF-509 5.6 680 MRS-1754 -1000 32

MRS-1754 was noted to have no effect on GPR174 signaling when tested in signaling assays (data not shown). Likewise, the GPR174 inhibitor had no effect on A2bR signaling when assessed in signaling assays (data not shown).

Human PBMCs (1 x 10 ⁶ cells/well in a 96-well flat-bottom plate) obtained from donor 3 and donor 6 were stimulated with 1 μg/ml anti-CD3 antibody (UCHT1) and 0.1 μg/ml anti-CD28 antibody ( CD28,2) in the presence of:

GPR174 inhibitory compound 10 (1 μM);

A2aR inhibitory compound ZM-241385 (0.1 μM);

A2aR inhibitory compound PBF-509 (0.1 μM);

A2aR inhibitory compound SCH-58261 (0.2 μM);

A2bR inhibitory compound MRS-1754 (1 μM);

adenosine deaminase (ADA) (0.75 μg/ml);

and combinations of these reagents, as indicated in table. 13 below.

Supernatants from these cells were collected 24 h after stimulation, and the levels of IFN-γ, IL-2, and TNF were determined using the MSD platform (MesoScale Discovery).

Fig. 53A graphically illustrates the amount of IFN-γ (pg/ml) in culture supernatants 24 hours after stimulation of human PBMC cultures from a single donor (donor 3) cultured in the presence of GPR174 inhibitory compound 10 (1 μM); ZM, 0.1 µM); PBF-509 (0.1 μM), SCH (0.2 μM), MRS (1 μM), adenosine deaminase (ADA, 0.75 μg/ml), alone or in combination with compound 10, or vehicle control.

As shown in FIG. 53A and summarized in table. 13, the highest degree of IFN-γ induction was observed when compound 10 was combined with an A2aR inhibitor (ZM, PBF-509 or SCH) or adenosine deaminase (ADA), resulting in a 3.3-4.5-fold increase in IFN-γ, compared to control media. Alone, compound 10 increased IFN-γ levels by 2.8-fold, while the A2aR and A2bR inhibitors and ADA alone had relatively modest effects (1.0- to 2.1-fold increase). Inclusion of an A2bR inhibitor (MRS) did not further increase IFN-γ levels in either cobalt.

- 111 046439 nations. Together, these findings indicate that the synergistic activity between GPR174 inhibition and A2aR/A2bR inhibition for IFN-γ production in PBMC from donor 3 is based on A2aR inhibition without involving A2bR.

Fig. 53B graphically illustrates the amount of IL-2 (pg/ml) in culture supernatants 24 hours after stimulation of human PBMC cultures from a single donor (donor 3) cultured in the presence of GPR174 inhibitory compound 10 (1 μM); ZM, 0.1 µM); PBF-509 (0.1 μM), SCH (0.2 μM), MRS (1 μM), adenosine deaminase (ADA, 0.75 μg/ml), alone or in combination with compound 10, or vehicle control.

As shown in FIG. 53B and summarized in table. 13, the highest degree of IL-2 induction was observed when compound 10 was combined with an A2aR inhibitor (ZM, PBF-509 or SCH) or adenosine deaminase (ADA), resulting in a 4.7-5.9-fold increase compared to control medium. Alone, compound 10 increased IL-2 levels by 2.9-fold, while A2aR inhibitors alone and ADA alone had relatively modest effects (1.6- to 2.3-fold increase). Incorporation of A2bR inhibitor (MRS) with A2aR inhibitors or ADA did not further increase IL-2 levels; however, in cultures lacking A2aR inhibitors, MRS alone increased IL-2 levels by 1.7-fold, and, with compound 10, by 4.3-fold. Together, these findings indicate that the synergistic activity between GPR174 inhibition and A2aR/A2bR inhibition for IL-2 production in PBMCs from donor 3 is based in large part on A2aR inhibition, but that A2bR inhibition may also cooperate with GPR174 inhibition to increase IL-2 levels. 2.

Fig. 53C graphically illustrates the amount of TNF (pg/ml) in culture supernatants 24 hours after stimulation of human PBMC cultures from a single donor (donor 3) cultured in the presence of GPR174 inhibitory compound 10 (1 μM); ZM, 0.1 µM); PBF-509 (0.1 μM), SCH (0.2 μM), MRS (1 μM), adenosine deaminase (ADA, 0.75 μg/ml), alone or in combination with compound 10, or vehicle control.

As shown in FIG. 53C and summarized in table. 13, the highest degree of TNF induction was observed when compound 10 was combined with an A2aR inhibitor (ZM, PBF-509 or SCH) or adenosine deaminase (ADA), resulting in a 6.0-8.6-fold increase compared to vehicle control . Alone, compound 10 increased TNF levels by 4.1-fold, while A2aR inhibitors alone and ADA alone had relatively modest effects (1.9- to 3.9-fold increase). Incorporation of A2bR inhibitor (MRS) with A2aR inhibitors or ADA did not further increase IL-2 levels; however, MRS alone increased IL-2 levels by 1.8-fold, and with PBF-509, MRS also slightly increased TNF levels. Together, these findings indicate that the synergistic activity between GPR174 inhibition and A2aR/A2bR inhibition for TNF production in PBMCs from donor 3 is based predominantly on A2aR inhibition.

Fig. 54A graphically illustrates the amount of IFN-γ (pg/ml) in culture supernatants 24 hours after stimulation of human PBMC cultures from a single donor (donor 6) cultured in the presence of GPR174 inhibitory compound 10 (1 μM); ZM, 0.1 µM); PBF-509 (0.1 μM), SCH (0.2 μM), MRS (1 μM), adenosine deaminase (ADA, 0.75 μg/ml), alone or in combination with compound 10, or vehicle control.

As shown in FIG. 54A and summarized in table. 13, the highest degree of IFN-γ induction was observed when compound 10 was combined with the A2bR inhibitor MRS and an A2aR inhibitor or adenosine deaminase (ADA), resulting in a 1.5- to 2.0-fold increase over the vehicle control. Alone, compound 10 did not significantly increase IFN-γ levels. Likewise, A2aR and A2bR inhibitors and ADA alone had no significant effect. The modest effect of all compounds on IFN-γ levels in this experiment is likely due to the fact that donor 6 responded strongly to anti-CD3, anti-CD28 stimulation, resulting in very high IFN-γ production (8000-16000 pg/ml) , which could not be further increased to a large extent by GPR174 and A2aR/A2bR inhibitors. However, involvement of A2bR inhibition was observed when combined with A2aR and GPR174 inhibitors. Similarly, the only significant increase in IFN-γ levels in the absence of an A2bR inhibitor was observed for the GPR174 inhibitor in combination with ADA (1.4-fold), which should reduce adenosine activity on both A2aR and A2bR. The further increase in IFN-γ levels observed when MRS was added to compound 10 and ADA (2.0-fold over vehicle control) may be due to the possibility that ADA was not used at saturating concentration for this donor, allowing some activity adenosine on A2bR to directly or indirectly suppress IFN-γ expression.

Fig. 54B graphically illustrates the amount of IL-2 (pg/ml) in culture supernatants 24 hours after stimulation of human PBMC cultures from a single donor (donor 6) cultured in the presence of GPR174 inhibitory compound 10 (1 μM); ZM, 0.1 µM); PBF-509 (0.1 μM), SCH (0.2 μM), MRS (1 μM), adenosine deaminase (ADA, 0.75 μg/ml), alone or in combination with compound 10, or vehicle control.

As shown in FIG. 54B and summarized in table. 13, the highest degree of IL-2 induction was observed when

- 112 046439 wherein compound 10 was combined with an A2bR inhibitor (MRS) or adenosine deaminase (ADA), resulting in a 2.3-2.7 fold increase over vehicle control. Alone, compound 10 increased IL-2 levels by 1.7-fold, while A2aR inhibitors alone and ADA alone had no or relatively modest effects (1.0- to 1.3-fold increase). Combining compound 10 with A2aR inhibitors increased IL-2 to intermediate levels (1.72.2-fold). In cultures lacking A2aR inhibitors, MRS alone increased IL-2 levels by 1.2-fold, and, with compound 10, by 1.4-fold. Together, these findings indicate that the synergistic activity between GPR174 inhibition and A2aR/A2bR inhibition for IL-2 production in PBMCs from donor 6 is based on the combined effects of both A2aR and A2bR inhibition.

Fig. 54C graphically illustrates the amount of TNF (pg/ml) in culture supernatants 24 hours after stimulation of human PBMC cultures from a single donor (donor 6) cultured in the presence of GPR174 inhibitory compound 10 (1 μM); ZM, 0.1 µM); PBF-509 (0.1 μM), SCH (0.2 μM), MRS (1 μM), adenosine deaminase (ADA, 0.75 μg/ml), alone or in combination with compound 10, or vehicle control.

As shown in FIG. 54C and summarized in table. 13, the highest degree of TNF induction was observed when compound 10 was combined with an A2bR inhibitor (MRS) or adenosine deaminase (ADA), resulting in a 2.1-2.5-fold increase over vehicle control. Alone, compound 10 increased TNF levels by 1.4-fold, while A2aR inhibitors alone and ADA alone had no or relatively modest effects (1.1-1.4-fold increase). Combining compound 10 with A2aR inhibitors increased TNF to intermediate levels (1.3- to 1.8-fold increase). In cultures lacking A2aR inhibitors, MRS alone increased TNF levels by 1.4-fold, and, with compound 10, by 2.1-fold. Together, these findings indicate that the synergistic activity between GPR174 inhibition and A2aR/A2bR inhibition for TNF production in PBMCs from donor 6 is based on the combined effects of both A2aR and A2bR inhibition.

Table 13. Summary of results shown in FIG. 53А-С and 54А-С

Donor 3 donor 6

Nose. ¹ MRS ¹ MRS/Hoc. ² Nose. ¹ MRS ¹ MRS/Hoc. ² Carrier 1.0 1.0 1.0 1.0 1.1 1.1 Conn. 10 2.8* 2.2* 0.8* 1.2 1.3* 1.1 ZM 1.4* 1.4* 1.0 1.3 1.2 0.9 Conn. 10+ZM 3.4* 3.6* 1.0 1.2 1.7* 1.5* PBF 1.3* 1.5* 1.1 1.3 1.0 0.8 Conn. 10+PBF 3.4* 3.3* 1.0 1.2 1.5* 1.3* IFN-γ SCH 2.1* 1.9* 0.9 0.8 1.3* 1.6* Conn. 10+SCH 4.5* 3.9* 0.9 1.1 1.5* 1.4* ADA 1.7* 1.1 0.7* 1.2 1.5* 1.3 Conn. 10+ADA 3.3* 2.7* 0.8 1.4* 2.0* 1.5* Carrier 1.0 1.7* 1.7* 1.0 1.2* 1.2* Conn. 10 2.9* 4.3* 1.5* 1.7* 2.3* 1.4* ZM 2.1* 1.9* 0.9 1.2* 1.3* 1.0 Conn. 10+ZM 5.9* 4.3* 0.7* 2.2* 2.7* 1.3* PBF 1.6* 1.7* 1.1 1.2 1.1* 1.0 IL-2 Conn. 10+PBF 4.7* 4.3* 0.9 1.9* 2.6* 1.3* SCH 2.3* 2.3* 1.0 1.0 1.1 1.2* Conn. 10+SCH 5.3* 4.6* 0.9 1.7* 2.5* 1.4* ADA 2.2* 1.9* 0.9 1.3* 1.3* 1.0 Conn. 10+ADA 5.2* 4.5* 0.9 2.4* 2.5* 1.0 Carrier 1.0 1.8* 1.8* 1.0 1.4* 1.4* TNF Conn. 10 4.1* 4.6* 1.1 1.4* 2.1* 1.4* ZM 2.4* 2.5* 1.1 1.2* 1.5* 1.2 Conn. 10+ZM 7.4* 6.9* 0.9 1.6* 2.4* 1.5*

The ratio of cytokine concentration values for the specified combination of compounds, compared with the control vehicle.

Ratio of cytokine concentration values for each compound combination in the presence versus absence of MRS-1754.

*, p<0.05, t-test, two-tailed distribution, two-sample t-test with equal variances.

- 113 046439

The findings presented in this example indicate that inhibition of both A2aR and A2bR can cooperate with GPR174 inhibition to produce maximal cytokine induction, and that there may be conditions under which inhibition of all 3 GPCRs results in the highest cytokine induction. For donor 3, A2aR inhibition alone provided maximal cytokine induction when combined with compound 10, as inclusion of an A2bR inhibitor (MRS) or removal of adenosine using ADA did not further increase cytokine levels (except for a small increase in IL-2 in the absence of A2aR inhibitor). For donor 6, addition of A2bR was necessary to achieve the highest levels of IFN-γ, IL-2, and TNF.

Three other targets for cancer immunotherapy in the adenosine pathway are CD38, CD39 and CD73, ectonucleotidases involved in adenosine production. CD39 and CD73 convert ATP to adenosine through phosphate removal. Adenosine can also be generated from NAD+ through an axis focused on NAD+ metabolizing CD38 to form adenosine diphosphate ribose (ADPR).

Our finding that in the presence of a GPR174 inhibitor, adenosine depletion using ADA results in maximal cytokine induction similar to that achieved using combined A2aR/A2bR inhibition indicates that combining a GPR174 inhibitor with CD38, CD39, or CD73 inhibitors should also enhance antitumor immune responses.

Example 21.

This example shows that four compounds in two chemical classes of GPR174 inhibitors induced almost identical Th1 cytokine profiles in total PBMCs stimulated with anti-CD3 mitogenic antibody and anti-CD28 antibody, and that similar degrees of synergy with the A2aR inhibitor ZM-241385 were obtained for all four GPR174 inhibitors.

Since the data presented to date in cytokine release assays from PBMCs have used a single GPR174 inhibitor (compound 10), it was important to show that the activity observed by the present inventors was due to the effect of the inhibitor on GPR174 and not to off-target activity unique to specific connection. To exclude the possibility of compound-specific off-target activity, we tested three additional GPR174 inhibitors: two from the same chemical series as compound 10 (compounds 6 and 11, group I), and a third compound from a different chemical class (compound 20, group I). group II). Based on the IC50 values obtained using these compounds in a signaling assay (see Example 16, FIG. 47), we tested these compounds at two concentrations: one predicted to effectively inhibit GPR174, and one that may exhibit partial inhibition in total PBMC stimulation assay.

Human PBMCs (1 x 10 ⁶ cells/well in a 96-well flat-bottom plate) obtained from donor 7 were stimulated with 1 μg/ml anti-CD3 antibody (UCHT1) and 0.1 μg/ml anti-CD28 antibody (CD28.2 ) in the presence:

GPR174 inhibitory compound 10 (1 and 0.3 μM);

GPR174 inhibitory compound 6 (1 and 0.3 μM);

GPR174 inhibitory compound 11 (1 and 0.3 μM);

GPR174 inhibitory compound 20 (10 and 3 μM);

each in the presence and absence of the A2aR inhibitory compound ZM-241385 (0.1 μM).

Supernatants from these cells were collected 24 h after stimulation, and the levels of IFN-γ, IL-2, TNF, and GM-CSF were determined using the MSD platform (MesoScale Discovery).

Results.

Fig. 55A graphically illustrates the amount of IFN-γ (pg/ml) in supernatants 24 hours after stimulation of human PBMC (donor 7) in the presence of GPR174 inhibitory compounds 6, 10, 11 or 20, each in the presence or absence of ZM.

As shown in FIG. 55A and summarized in table. 14, all four GPR174 inhibitory compounds 6, 10, 11 and 20 synergized with ZM to increase IFN-γ to levels greater than the sum of the increases obtained using each inhibitor alone. Synergy was observed between ZM and both high and low concentrations of each GPR174 inhibitor.

Fig. 55B graphically illustrates the amount of IL-2 (pg/ml) in supernatants 24 hours after stimulation of human PBMC (donor 7) in the presence of GPR174 inhibitory compounds 6, 10, 11, or 20, each in the presence or absence of ZM.

As shown in FIG. 55B and summarized in table. 14, all four GPR174 inhibitory compounds 6, 10, 11 and 20 synergized with ZM to increase IL-2 to levels greater than the sum of the increases obtained using each inhibitor alone. Synergy was observed between ZM and both high and low concentrations of each GPR174 inhibitor, with the exception of a low dose of compound 20, the least active of the four compounds (see example 16, Fig. 47).

Fig. 55C graphically illustrates the amount of TNF (pg/ml) in supernatants 24 hours after storage

- 114 046439 emulate human PBMC (donor 7) in the presence of GPR174 inhibitory compounds 6,10, 11 or 20, each in the presence or absence of ZM.

As shown in FIG. 55C and summarized in table. 14, all four GPR174 inhibitory compounds 6, 10, 11 and 20 synergized with ZM to increase TNF to levels greater than the sum of the increases obtained using each inhibitor alone. Synergy was observed between ZM and both high and low concentrations of each GPR174 inhibitor, with the exception of the low dose of compound 20, the least active of the 4 compounds (see example 16, Fig. 47).

Fig. 55D graphically illustrates the amount of GM-CSF (pg/ml) in supernatants 24 hours after stimulation of human PBMC (donor 7) in the presence of GPR174 inhibitory compounds 6, 10, 11 or 20, each in the presence or absence of ZM.

As shown in FIG. 55D and summarized in table. 14, all four GPR174 inhibitory compounds 6, 10, 11 and 20 synergized with ZM to increase the amount of GM-CSF to levels greater than the sum of the increases obtained using each inhibitor alone. Synergy was observed between ZM and both high and low concentrations of each GPR174 inhibitor, with the exception of a low dose of compound 20, the least active of the four compounds (see example 16, Fig. 47).

Table 14. Summary of results shown in FIG. 55A-D

Connection 10 Connection 6 Connection 11 Connection 20 Hoc. 1 µM ¹ 0.3 µM 1 µM 0.3 µM 1 µM 0.3 µM 10 µM 3 µM IFN-γ Hoc. 1.0 2.2* 2.1* 1.9* 2.0* 1.8* 1.9* 1.5* 1.2 ZM 1.4* 3.7* 3.7* 3.8* 3.1* 3.4* 2.9* 2.1* 1.8* IL-2 Hoc. 1.0 1.6* 1.7* 1.5* 1.5* 1.3 1.4* 1.4* 1.3 ZM 1.0 2.2* 2.0* 2.1* 1.9* 1.9* 1.8* 2.1* 1.6* TNF Hoc. 1.0 2.2* 2.2* 2.1* 2.1* 1.7* 1.7* 1.5* 1.4* ZM 1.3* 3.8* 3.6* 4.0* 3.4* 3.0* 2.6* 2.4* 1.9* GM-CSF Hoc. 1.0 3.1* 3.6* 2.9* 3.9* 2.4* 2.9* 2.3* 2.7* ZM 1.3 5.9* 6.2* 6.0* 6.2* 4.9* 4.4* 4.4* 4.6*

¹ Ratio of cytokine concentration values for the indicated combination of compounds, compared with the control vehicle (Nos, Nos).

*, p<0.05, t-test, two-tailed distribution, two-sample t-test with equal variances.

In this example, we showed that four compounds spanning two chemical classes of GPR174 inhibitors induced nearly identical TM cytokine profiles in total PBMCs stimulated with an anti-CD3 mitogenic antibody and an anti-CD28 antibody, and that similar degrees of synergy with the inhibitor A2aR ZM-241385 were obtained for all four GPR174 inhibitors. This finding further confirms that the observed effects are due to each of the four compounds acting on GPR174 and not on other molecular targets.

In general, our findings indicate that combined inhibition of GPR174 and the adenosine/A2aR/A2bR pathway results in a synergistic enhancement of immune responses. Synergy was observed when GPR174 and A2aR/A2bR inhibitors were combined, resulting in, in some cases, a remarkable 25-fold increase in IFN-γ and TNF levels. Because the enhanced responses were of TM polarity, and because both adenosine and GPR174 PS/lysoPS agonists are highly enriched in the tumor microenvironment (see, e.g., Vigano S. et al., Front Immunol vol 10: 925, 2019; Li et al., Molecular Cancer Therapeutics vol 17 (1): 169-82, 2017; and Desai T. J. et al., Oncotarget 7:30678-30690, 2016), the authors' findings suggest that combined inhibition of both pathways in patients with malignant tumors may be important to effectively counteract the immunosuppressive nature of the tumor microenvironment and stimulate immune responses to kill tumors.

Example 22.

This example describes an extended assessment of synergistic TM cytokine induction in which an A2aR inhibitor (ZM-241385), an A2bR inhibitor (MRS1754), and a GPR174 inhibitor (compound 10) were assayed individually and in combination in PBMC isolated from 5 healthy donors.

Ways.

The methods were carried out as described in Example 20, with the modification that human PBMCs from 5 healthy donors were stimulated in the presence of an A2aR inhibitor (ZM-24138: 0.2 μM), an A2bR inhibitor (MRS1754: 2 μM) and GPR174 inhibitory compound 10 ( 1 µM) were analyzed separately and in combination. Supernatants from these cells were collected 24 h after stimulation, and the levels of IFN-γ, IL-2, and TNF were determined using the MSD platform (MesoScale Discovery).

Results.

Results showing the effects of combined inhibition of GPR174, A2aR and A2bR in

- 115046439 normalized data for 5 human donors are shown in FIG. 56A-S. Note: GPR174i=CoeguHeHue 10.

Fig. 56A graphically illustrates the fold change, relative to vehicle, in the amount of IFN-γ in culture supernatants 24 hours post-stimulation in human PBMC cultures from 5 donors cultured in the presence of GPR174 inhibitory compound 10 (1 μM); A2aR inhibitory compound (ZM, 0.2 μM); A2bR inhibitory compound MRS (2 μM) or a combination of compound 10 (1 μM) plus 0.2 μM ZM plus 2 μM MRS, or vehicle control (DMSO).

Fig. 56B graphically illustrates the fold change, relative to vehicle, in the amount of IL-2 in culture supernatants 24 hours after stimulation in human PBMC cultures from 5 donors cultured in the presence of GPR174 inhibitory compound 10 (1 μM); A2aR inhibitory compound (ZM, 0.2 μM); A2bR inhibitory compound MRS (2 μM) or a combination of compound 10 (1 μM) plus 0.2 μM ZM plus 2 μM MRS, or vehicle control (DMSO).

Fig. 56C graphically illustrates the fold change, relative to vehicle, in the amount of TNF in culture supernatants 24 hours after stimulation in human PBMC cultures from 5 donors cultured in the presence of GPR174 inhibitory compound 10 (1 μM); A2aR inhibitory compound (ZM, 0.2 μM); A2bR inhibitory compound MRS (2 μM) or a combination of compound 10 (1-2 μM) plus 0.2 μM ZM plus 2 μM MRS, or vehicle control (DMSO).

Discussion.

The results shown in this example further confirm and extend the results shown in example 20 and show that inhibition of both A2aR and A2bR can cooperate with GPR174 inhibition to cause maximal cytokine induction, and that there may be conditions under which inhibition of all 3 GPCR results in the highest cytokine induction.

Example 23.

This example describes a study that was conducted to evaluate the effect of a representative GPR174 inhibitory compound 10, alone or in combination with an A2aR inhibitor (ZM-241385), on amphiregulin (AREG) expression.

Ways.

Methods were performed as described in Example 14, with the modification that cells were incubated in the presence of GPR174 inhibitory compound 10 at 1 μM (GPR174i), A2aR inhibitor ZM-241385 at 0.2 μM, or a combination of compound 10 (1 μM) plus ZM ( 0.2 µM).

Results.

Fig. 57 graphically illustrates the fold reduction in the number of AREG+ cells in the presence of vehicle; GPR174 inhibitory compound 10 (1 μM); ZM-241385 (0.2 µM); or a combination of compound 10 (1 μM) plus ZM (0.2 μM).

Discussion.

The results shown in this example are consistent with the results described in example 14 and show that the combination of a GPR174 inhibitor and an A2aR inhibitor further reduces AREG expression in activated CD4+ T cells compared to either agent alone.

Generalization.

As described and shown herein, GPR174 is an orphan GPCR that is expressed almost exclusively in immunocytes (T, B and NK cells), and numerous GPR174 inhibitors have been identified, falling into six different chemical classes as described herein. As further described herein, the present inventors have discovered that phosphatidylserine (PS) is a GPR174 agonist. As further described herein, inhibition of GPR174 stimulates the immune system, in particular the Th1 response, and suppresses tumor activators. As further described herein, combined inhibition of GPR174 and the adenosine pathway synergistically enhances ^1-^^^ production. These findings represent a new therapeutic option in cancer immunotherapy for all solid tumors. This therapeutic method includes the use of a GPR174 inhibitor in the form of a single agent or in combination with one or more A2aR inhibitors, A2bR inhibitors, CD38 inhibitors, CD39 inhibitors and/or CD73 inhibitors for the treatment of a solid tumor in a subject in need thereof. This method is also amenable to combination with checkpoint inhibitors and cell therapies.

Fig. 58 is a schematic diagram illustrating how both GPR174 and adenosine receptors respond to products of stress and cell death. As illustrated in FIG. 58, both GPR174 and adenosine receptors respond to products of stress and cell death. As shown in FIG. 58, in the GPR174-mediated pathway, apoptosis and stress lead to the production of phosphatidylserine (PS), which is a GPR174 agonist and induces Gs/cAMP signaling and leads to immunosuppression. As further shown in FIG. 58, in the adenosine pathway, exposure to cell destruction/activation/stress results in the production of extracellular ATP, which is converted to adenosine, which is an agonist for the A2A and A2B receptors, which also causes Gs/cAMP signaling and immunosuppression. As further shown in FIG. 58, the adenosine pathway can be inhibited by

- 116 046439 by means of CD39 inhibitors (for example, POM1, IPH52) or through CD73 inhibitors (for example, MEDI9447, BMS-986179), or through an A2aR inhibitor (for example, CPI-444, PBF-509, MK-3814 or AZD4635) or an inhibitor A2bR. As shown herein, inhibition of both the GPR174-mediated pathway and the adenosine pathway results in effective cAMP reduction and immunostimulation.

In general, the results described herein demonstrate (1) anticancer immune pathways controlled by GPR174; (2) identification of phosphatidylserine (PS) as a strong natural ligand for GPR174; (3) a collection of novel small molecule inhibitors of GPR174; and (4) a very large and synergistic increase in the production of tumor-fighting cytokines by T cells following combined inhibition of both GPR174 and the adenosine pathway, another key metabolic pathway regulating tumor immunity. Additionally, in this regard, as shown herein, GPR174 inhibitors enhance adenosine pathway inhibitor-induced production of tumor-fighting cytokines by up to 25-fold. This leads to a new cancer immunotherapy targeting GPR174 inhibition, which can be combined with adenosine pathway inhibitors to significantly improve tumor killing effects. It is expected that GPR174-targeted immunotherapy may be applicable to all solid tumors (eg, breast, lung, pancreas, colon, brain, etc.).

Like GPR174, adenosine receptors A2A/A2B are GPCRs. Central components of the adenosine pathway, the A2A/A2B receptors, have been targeted for immunotherapy of malignancies by several companies, as shown in Table 1. 15 below. GPR174 and the A2A/A2B receptors share certain characteristics. First, each increases the amount of intracellular cyclic adenosine monophosphate (cAMP), which is well known to suppress the type of immune response needed to kill tumor cells. Second, these receptors are activated by molecules (i.e., PS and adenosine, respectively) that are highly enriched in the tumor microenvironment. Strikingly, in experiments using total human peripheral blood mononuclear cells (PBMCs), where PS and adenosine are naturally abundant, GPR174 inhibitors synergize with A2A/A2B inhibitors to greatly enhance T cell responses.

Detections include the following:

inhibition of A2A/A2B alone resulted in, on average, a 2-fold increase in both interferon-gamma (IFN-γ) and tumor necrosis factor (TNF) and smaller increases in granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-2 (IL-2);

inhibition of GPR174 alone resulted in an average increase of 2.8-fold for IFN-γ, 2.7-fold for TNF, 1.7-fold for IL-2, and 1.4-fold for GM-CSF;

Combining a GPR174 inhibitor with A2A and/or A2B inhibition increased mean IFN-γ and TNF levels by 8- to 9-fold, and IL-2 and GM-CSF levels by nearly 3-fold, with a maximum increase reaching 25-fold for IFN-γ and TNF and more than 4 times for IL-2 and GM-CSF.

The adenosine pathway has been the focus of efforts for immunotherapeutic drug development in the pharmaceutical industry (see Table 15). The results described herein now indicate that the relatively modest acute increase in T cell cytokine production observed with inhibition of the A2A or A2B receptors, alone or both, can be markedly enhanced when combined with inhibition of the GPR174 pathway.

In addition, the discovery that PS independently stimulates an immunosuppressive GPCR—namely GPR174—on T lymphocytes represents a significant advance in our understanding of how PS may influence immunity against tumors.

These findings are particularly important for patients resistant to checkpoint inhibitors such as anti-PD-1 (eg, Keytruda® and Opdivo®) and anti-CTLA-4 (Ervoy®), and cell therapies such as CAR- T-cell and adoptive T-cell therapy. Checkpoint inhibitors are effective in only a minority of patients, and high levels of adenosine-forming molecules have been observed in non-responding patients. Additionally, overcoming natural immunosuppression in solid tumors poses a major challenge for cell therapy. Since PS and adenosine are both products of stress and cell death in solid tumors, it is expected that patients resistant to checkpoint inhibitor therapy or cell therapy may benefit greatly from combined inhibition of the GPR174 and adenosine pathways. Simply put, the present inventors have discovered that there are two pathways for cAMP to inhibit tumor immunity and that both GPR174 and the adenosine pathway need to be inhibited to provide more effective tumor killing activity, as illustrated in FIG. 58.

- 117 046439

Table 15. Adenosine pathway inhibitors in cancer immunotherapy studies ¹

Drug/Drug candidate Target Stage Design Indication Sponsor AZD4635 A2A Ph 2 Monotherapy, combination with Imfinzi® (anti-PD-L1 antibody) Solid malignant neoplasms in late stages AstraZeneca CPI-445 A2A Phlb/2 Monotherapy, combination with Tecentriq® (anti-PD-L1 antibody) Non-small cell lung cancer (NSCLC) Hoffinan-La Roche NIR178 A2A Ph lb Combination with Keytruda® and LAG525 (vs LAG3) Triple negative for breast cancer Novartis NIR178 A2A Ph 2 Monotherapy, combination with Solid malignant Novartis NIR178 CPI-444 EOS100850 PBF-1129 CPI-444 Daratumumab TTX-030 Oleklumab A2A A2A A2A A2B A2A/A2B CD38 CD39 CD73 Ph 1/lb Ph 1/lb Ph 1/lb Phi Ph 1/lb Phi Ph 1/lb Phi Keytruda® Monotherapy, combination with Keytruda® Monotherapy, combination with Tecentriq® Monotherapy Monotherapy Monotherapy, combination with Keytruda® or Tecentriq® Monotherapy Monotherapy, combination with Keytruda® and chemotherapy agents Monotherapy, combination with Imfinzi® (anti-PD-L1 antibody) advanced neoplasms Non-small cell lung cancer (NSCLC) Renal cell carcinoma; NSCLC Solid tumors Non-small cell lung carcinoma (NSCLC) Solid tumors Kidney carcinoma, bladder cancer Solid tumor, lymphoma Solid malignant neoplasms in late stages Palobiofarma Corvus Pharmaceuticals iTeos Therapeutics Palobiofarma Corvus Pharmaceuticals M.D. Anderson Tizona Therapeutics/AbbVie AstraZeneca Oleklumab CD73 Phi Monotherapy Solid malignant neoplasms in late stages AstraZeneca Oleklumab CD73 Phi Combination with Muscular Dana-Farber

- 118 046439

imfinzi® (anti-PD-L1 antibody) invasive bladder cancer Cancer Institute Oleklumab CD73 Ph 2 Combination with Imfinzi® (anti-PD-L1 antibody) Recurrent ovarian cancer Nordic Society of Gynecologic Oncology Oleklumab CD73 Ph 2 Combination with Imfinzi® (anti-PD-L1 antibody) PD-1/PD-L1 inhibition-resistant NSCLC AstraZeneca Oleklumab CD73 Ph lb/2 Combination with Imfinzi® (anti-PD-L1 antibody) and chemotherapy Triple negative for breast cancer AstraZeneca Oleklumab CD73 Ph lb/2 Combination with chemotherapy, combination with chemotherapy and Imfinzi® Metastasis of the pancreas AstraZeneca Oleklumab CD73 Ph lb/2 Combination with AZD4635 (Α2ΑΪ), combination with osimertinib (EGFR ^A T790M inhibitor) NSCLC in advanced stages AstraZeneca BMS-986179 CD73 Ph l/2a Monotherapy, combination with Opdivo® (anti-PD-1 antibody) Solid malignant neoplasms in late stages Bristol-Myers Squibb CPI-006 CD73 Ph 1/lb Monotherapy, combination with CPI-444 (Α2ΑΪ), combination with Keytruda® Solid malignant neoplasms in late stages Corvus Pharmaceuticals NZV930 CD73 Ph 1/lb Monotherapy, combination with Keytruda®, combination with NIR178 (Α2ΑΪ) Solid malignant neoplasms in late stages Novartis

1Vigano et al., Frontiers in Immunology, vol 10, Article 925, June 2019.

Example 24.

This example describes the effects of genetic deficiency of GPR174 on the growth of murine colon carcinoma CT26.c125 in mice treated with a T regulatory cell (Treg) depleting anti-GITR antibody.

Mouse models of tumor growth provide an experimental platform for monitoring and manipulating antitumor immunity. To assess whether a particular gene product is important in controlling the nature and intensity of antitumor immune responses, a common method is to inoculate normal or mutant mice lacking the gene of interest with a tumor cell line. The tumor grows to a volume necessitating euthanasia of the mouse unless the immune response reduces the rate of tumor growth or results in complete clearance of the tumors in the mice. In preliminary studies, the present inventors found that CT26.c125 colon carcinoma grew at an identical rate in WT mice and mice lacking an intact GPR174 gene (GPR174-KO mice). In vitro studies by the present inventors using mouse T cells and mouse splenocytes revealed that GPR174 deficiency or inhibition of GPR174 increased the production of IL-2 more significantly than other cytokines (see Example 7). Since IL-2 is a growth factor for both immunosuppressive T cells and tumor-killing Th1 and CD8 T cells, we reasoned that this increase in IL-2 in tumor-bearing mice may stimulate both tumor-promoting T cells. reg and tumor-killing Th1 and CD8 T cells, resulting in Treg-mediated neutralization of antitumor responses.

Regarding the balance of IL-2 consumption between Treg and Th1/CD8 T cells, there is a fundamental difference between humans and mice that should be taken into account during

- 119 046439 effective use and interpretation of mouse models of human disease. In naïve laboratory mice, including mice freshly inoculated with tumor cells, the vast majority of T cells expressing the high-affinity EL-2 receptor, CD25, are T-reg, with levels of CD25 expression markedly higher than for CD25 on other CD4 T cells. In contrast, the distribution of CD25 between T cells and other CD4 T cells in humans is reversed, where the majority of CD4 T cells expressing CD25 are effector T cells that express CD25 at levels highly overlapping with T cells. -reg (see Churlaud G. et al., Frontiers in Immunology vol 6, article 171, 2015; Akimova T. et al., PLoS ONE, Vol 6, Issue 8, 2011). Effector T cells respond to IL-2 in a proinflammatory manner, including the production of IFN-γ, an essential cytokine for antitumor immunity. Thus, in mice, increased levels of IL-2 due to GPR174 deficiency provide a major benefit to T-regs, while in humans, increased levels of IL-2 following administration of a GPR174 inhibitor have a greater potential to favor tumor-killing effector cells. , including Th1 cells, CD8 T cells and NK cells.

To allow the putative increased levels of IL-2 in GPR174-KO mice to act preferentially on tumor-killing immunocytes, we chose to treat mice with an antibody (DTA-1) against GITR (the gene product of glucocorticoid-induced TNFR-related protein), which is known to deplete T-reg cells and stimulate anti-tumor responses in other T cells (see Coe D. et al., Cancer Immunol Immunother 59(9): 1367-77, 2010). Complete depletion of T-reg cells using high doses of anti-GITR antibody is known to activate potent antitumor responses in WT mice, under conditions in which the present inventors may not have observed differences between WT and GPR174-KO mice. In contrast, partial or temporary depletion of T-reg cells stimulates partial antitumor immunity, which can be enhanced using other types of immune-stimulating therapy (see Zapposodi R. et al., Nat Med 25(5):759-766, 2019) . Because partial depletion of Treg allows for more efficient uptake of IL-2 by tumor-killing T cells, this method can be considered to form a humanizing mouse tumor model. Thus, we compared tumor cell line growth in WT or GPR174-KO mice that were treated with a low dose of the anti-GITR antibody DTA-1 for only 2-3 selected days after tumor inoculation.

Ways.

Colon carcinoma CT26.c125 originates from the BALB/c mouse strain. GPR174-KO mice were generated as described in Example 7. Since GPR174 is located on the X chromosome, we were able to use male WT and GPR174-KO mice with BALB/tolerant immune systems by crossing 129S GPR174-KO females with BALB/ males. c. Littermate F1 male WT or GPR174-KO mice (n=15) [BALB/cx129S] were inoculated subcutaneously with 500,000 CT26.c125 cells on day 0. In the first experiment, on days 7 and 9 (Fig. 59A and B), or on In the second experiment, on days 7, 9 and 14 (FIG. 61A and B), all mice received intraperitoneal injections of 200 μg of anti-GITR antibody (clone DTA-1; Bio-X-Cell). Tumor volume was calculated using the equation (length x width ² )/2), and mice with tumors measuring >1500 mm ³ were euthanized.

Results.

Fig. 59A and 59B graphically illustrate the growth of CT26 tumors in individual WT (FIG. 59A) and GPR174-KO (FIG. 59B) mice inoculated with CT26.c125 murine colon carcinoma cells on days 0 and treated with anti-GITR antibody on days 7 and 9 .

As shown in FIG. 59B, tumor growth was slowed on average in GPR174-KO mice compared to WT mice (Figure 59A).

Fig. 60 graphically illustrates the percentage of survival of tumor-bearing WT and GPR174KO mice inoculated with CT26.c125 murine colon carcinoma cells on days 0 and treated with anti-GITR antibody on days 7 and 9. As shown in FIG. 60, GPR174-KO mice had a higher survival rate (ie, were euthanized significantly later) than WT mice (p=0.03, log-rank test).

Fig. 61A and 61B graphically illustrate tumor growth in individual WT (FIG. 61A) and GPR174-KO (FIG. 61B) mice inoculated with CT26.c125 murine colon carcinoma cells on day 0 and treated with anti-GITR antibody on days 7, 9, and 14.

As shown in FIG. 61B, tumor growth was slowed on average in GPR174-KO mice compared to WT mice (Figure 61A).

Fig. 62 graphically illustrates the percentage of survival of tumor-bearing WT and GPR174KO mice inoculated with CT26.c125 murine colon carcinoma cells on day 0 and treated with anti-GITR antibody on days 7, 9, and 14.

As shown in FIG. 62, GPR174-KO mice had a higher survival rate (i.e., were euthanized significantly later) than WT mice.

- 120 046439

Discussion.

In these experiments, our goal was to determine whether GPR174-deficient mice induced more aggressive antitumor immune responses when T-reg cells were partially depleted using the anti-GITR antibody DTA-1. As shown in FIG. 59A-62, CT26.c125 murine colon carcinoma tumor growth was slowed in GPR174-KO mice compared with WT littermates, and survival was significantly improved.

Example 25.

This example describes the effects of genetic deficiency of GPR174 on the growth of B16F10 melanoma in mice treated with a T regulatory cell (Treg) depleting anti-GITR antibody.

In the B16F10 melanoma tumor model, it was previously observed that a single high dose of anti-GITR antibody administered on day 4 resulted in slower tumor growth but not complete tumor rejection in all mice (Zapposodi R. et al., Nature Medicine 25:759 -766, 2019). Taking into account the results described in Example 24, the following study was conducted to determine whether transient T-reg depletion using an anti-GITR antibody could slow tumor growth and improve survival of GPR174-KO mice, compared with WT mice, after inoculation with a more aggressive tumors: melanoma B16F10.

B16F10 melanoma originates from the C57BL/6 mouse strain. GPR174-KO mice were generated as described in Example 7. Since GPR174 is located on the X chromosome, we were able to use male WT and GPR174-KO mice with a C57Bb/6-tolerant immune system by crossing 129S GPR174-KO females with males C57BL/6. WT or GPR174-KO male littermates (n=12) [C57BL/6x129S]F1 were inoculated intradermally with 75,000 B16F10-Kb cells (B16F10 cells transfected with H-2Kb to increase antigen presentation to CD8 T cells) on day 0 (see Ohta A. et al., PNAS 103(35):13132-13137, 2006). On days 4 and 14, all mice received intraperitoneal injections of 500 μg of anti-GITR antibody (clone DTA-1; Bio-X-Cell). Tumor volume was calculated using the equation (length x width ² )/2), and mice with tumors measuring >1500 mm ³ were euthanized.

Fig. 63A and 63B graphically illustrate the growth of B16F10-Kb melanoma tumors in individual WT (FIG. 63A) and GPR174-KO (FIG. 63B) mice inoculated with B16F10-Kb cells on day 0 and treated with anti-GITR antibody on days 4 and 14.

As shown in FIG. 63B, GPR174 deficiency slowed tumor growth compared to tumor growth in WT mice (Figure 63A). Average tumor sizes were significantly smaller in KO mice on days 14 (p=0.01) and 16 (p=0.00005).

Fig. 63C graphically illustrates the average tumor volume for B16F10-Kb melanoma tumors in WT and GPR174-KO mice inoculated with B16F10-Kb cells on day 0 and treated with anti-GITR antibody on days 4 and 14. As shown in FIG. 63C, mean tumor sizes were significantly smaller in GPR174 KO mice on days 14 (* denotes: p=0.01) and 16 (*** denotes: p=0.00005).

Fig. 64 graphically illustrates the percentage of survival of B16F10-Kb melanoma tumor-bearing WT and GPR174-KO mice inoculated with B16F10-Bb cells on days 0 and treated with anti-GITR antibody on days 4 and 14. As shown in FIG. 64, GPR174-KO mice had a higher survival rate (i.e., were euthanized significantly later) than WT mice (p=0.006, log-rank test).

Discussion.

As shown in this example, in the B16F10 melanoma model, GPR174 deficiency resulted in significantly reduced tumor growth and prolonged survival following anti-GITR antibody treatment. Since T-reg attenuating therapies (i.e., agents that deplete, weaken, or otherwise damage T-reg cells) are currently in development as immunotherapies against cancer, our data suggests that a GPR174 inhibitor may be more effective in the presence of such co-therapy. Drugs that attenuate T-reg or both attenuate T-reg and enhance tumor killing activity of other T cells and/or NK cells include antibodies or other molecules that recruit one or more of the following: GITR, CTLA-4, CD25 , LAG3, TIGIT, NRP1, TGF-β, CCR2, CCR4, CCR8, TNFR2 and/or EZH2, such as described, for example, in Tanaka A and Sakaguchi S, European Journal of Immunology vol 49(8): 1140-1146 , 2019; Yano H. et al., Immunology vol 157(3): 232-247, 2019; Ohue Y and Nishikawa H., Cancer Science vol 110(7):2080-2089, 2019; Han S. et al., Front Oncol vol 9, Article 279, 2019, the contents of each of which are therefore incorporated by reference.

Accordingly, in one embodiment of the present invention, the GPR174 inhibitory agent is used in combination with one or more T-reg attenuating agent(s) (i.e., agents that deplete, attenuate, or otherwise impair tumor suppressor function of T-reg cells) . In some embodiments, the T-reg attenuating agent

- 121 046439 binds to one or more of GITR, CTLA-4, CD25, LAG3, TIGIT, NRP1, TGF-β, CCR2, CCR4, CCR8, TNFR2 and/or EZH2.

Exemplary T-reg attenuating agents for use in combination with GPR174 inhibitors in the compositions, methods and applications described herein are presented below.

GITR binding agents that can be used in combination with GPR174 inhibitors in the compositions, methods and applications described herein include anti-GITR antibodies and small molecules that bind GITR, such as: TRX518, DTA-1 and MK-4166 .

CTLA-4 (cytotoxic T lymphocyte associated protein 4) binders that can be used in combination with GPR174 inhibitors in the compositions, methods and applications described herein include anti-CTLA-4 antibodies and small molecules that bind CTLA-4 , such as ipilimumab (Bristol-Myers Squibb); tremelimumab (Medlmmune); AGEN1884 (Agenus); BCD-145 (Biocad); REGN4659 (Regeneron Pharmaceuticals); ADU-1604 (Aduro Biotech); and CS1002 (CStone Pharmaceuticals).

CD25 binding agents that can be used in combination with GPR174 inhibitors in the compositions, methods and uses described herein include anti-CD25 antibodies and small molecules that bind CD25, such as daclizumab; basiliximab and CD25-targeted NIR-PIT.

TIGIT (T cell immunoreceptor with Ig and ITEM domain) binding agents that can be used in combination with GPR174 inhibitors in the compositions, methods and applications described herein include anti-TIGIT antibodies and small molecules that bind TIGIT, such as , MK-7684 (Merck Sharp &Dohme); AB154 (Arcus Biosciences); MTIG7192A (Genentech); BMS-986207 (Bristol-Myers Squibb); and ASP8374 (Astellas Pharma).

NRP1 (neuropilin 1) binding agents that can be used in combination with GPR174 inhibitors in the compositions, methods and uses described herein include anti-NRP1 antibodies and small molecules that bind NRP1, such as ASP1948 (Astellas Pharma).

TGF-β binding agents that can be used in combination with GPR174 inhibitors in the compositions, methods and uses described herein include anti-TGF-β antibodies and small molecules that bind TGF-β, such as, for example, Galunisertib (Eli Lilly ); LY320082 (Eli Lilly); PF-06952229 (Pfizer) and Fresolimumab (Cambridge Antibody Technology).

LAG-3 binding agents that can be used in combination with GPR174 inhibitors in the compositions, methods and uses described herein include anti-LAG-3 antibodies and small molecules that bind LAG-3 such as, Sym022 (Symphogen ); relatlimab (Bristol-Myers Squibb); REGN3767 (Regeneron Pharmaceuticals); TSR-033 (Tesaro); IMP321 (Immutep); INCAGN02385 (Agenus); LAG525 (Novartis); and MK4280 (Merck Sharp & Dohme).

CCR2 binding agents that can be used in combination with GPR174 inhibitors in the compositions, methods and applications described herein include anti-CCR2 antibodies and small molecules that bind CCR2, such as, for example, BMS-813160 (Bristol-Myers Squibb), MLN1202 (Millennium Pharmaceuticals), C775, STI-B0201, STI-B0211, STI-B0221, STI-B0232, carlumab (CNTO 888; Centocor, Inc.), or STI-B0234.

CCR4 binding agents that can be used in combination with GPR174 inhibitors in the compositions, methods and uses described herein include anti-CCR4 antibodies and small molecules that bind CCR4, such as mogamulizumab (Kyowa Kirin), No.[(3- {[3-{[(5-chloro-2-thienyl)sulfonyl]amino}-4-(methyloxy)-1H-indazol-1-yl]methyl}phenyl)methyl]-2hydroxy-2-methylpropanamide, which are described in WIPO International Publication WO 2010/097395 and US Patent Publication No. 20100216860.

CCR8 binding agents that can be used in combination with GPR174 inhibitors in the compositions, methods and applications described herein include anti-CCR8 antibodies and small molecules that bind CCR8, such as those in US 10087259.

TNFR2 binding agents that can be used in combination with GPR174 inhibitors in the compositions, methods and applications described herein include anti-TNFR2 antibodies and small molecules that bind TNFR2, such as those in Vanamee E and Faustman D, Trends in Molecular Medicine vol 23, Issue 11:1037-1046, 2017, the contents of which are incorporated herein by reference.

EZH2 (enhancer of zeste homolog 2) binding agents that can be used in combination with GPR174 inhibitors in the compositions, methods and applications described herein include anti-EZH2 antibodies and small molecules that bind EZH2 for example, such as UNC1999, 3-deazaneplanocin A (DZNcp), E11, EPZ-5676, EPZ-6438, GSK343, EPZ005687, EPZ011989 and GSK126.

- 122 046439

In addition, taking into account the discovery of the present inventors using combined inhibition of A2aR/A2bR and GPR174, as described in Examples 15 and 18-23, the optimal method for patients with malignant tumors may be inhibition of both the adenosine pathway and GPR174 in the presence one or more T-reg weakening agents. Such a method may ensure that A2aR/A2bR/GPR174 inhibitor-induced IL-2 acts more on tumor-reactive effector T cells than on T cells.

Example 26.

This example describes the effects of PS liposomes and the GPR174 inhibitor on human and mouse T cell responses when isolated rather than in the presence of total PBMCs or splenocytes.

In the examples presented above, GPR174 inhibition was performed in cultures of mouse PBMC or splenocytes where cells were seeded at high density (1 million per well of a 96-well flat-bottom plate). Under these conditions, PS was considered to be present at a high level because the GPR174 inhibitors were active in the absence of additional PS liposomes, and because supplementation with 1 μM PS in the form of liposomes did not greatly alter T cell responses or responses to the GPR174 inhibitor. In contrast, in cultures from low numbers of purified T cells, GPR174 may not be exposed to PS to the same extent. Thus, as described in this example, we examined whether PS liposomes suppress purified human and murine T cell responses and whether a GPR174 inhibitor could reverse this suppression. In addition, we assessed the specificity of these effects by comparing responses in WT and GPR174-KO mouse T cells.

In addition to PS liposomes, we tested tumor exosomes for their ability to stimulate GPR174. Tumor exosomes (also referred to as cancer cell-derived exosomes) are small vesicles that are released in high quantities from tumor cells and that are enriched in exposed PS (see Kelleher R. J. et al., Cancer Immunology Research Vol 3(11 ):1269-78, 2015; Birge R.B. et al., Cell Death & Differentiation Vol 23:962-978, 2016). Tumor exosomes are believed to play a role in cancer-associated immunosuppression, so we hypothesized that they may suppress antitumor immunity, in part, through their activity on GPR174.

Purified human T cells (Astarte Biologics, Inc) were activated in the presence of K562 cells (artificial antigen presenting cells) presenting anti-CD3 and CD80.Fc antibody via Fc receptor binding in 96-well U-bottom plates containing Xvivo media -15, in the following conditions:

50,000 T cells per well

10,000 K562 cells per well anti-CD3 antibody (clone UCHT1; 0.5 μg/ml)

CD80.Fc fusion protein (R&D Systems; 2 μg/ml)

PS liposomes (1 µM) exosomes from the breast cancer cell line MDA-MB-231 (12 µg/ml) (EXOP-105A-1 from System Biosciences, LLC) GPR174 inhibitor (compound 10, 0.5 or 3 µM ) cells were cultured for 24 hours and cytokine concentrations were assessed in the supernatants (Legendplex, Biolegend).

Preparation of purified mouse T cells:

Mouse T cells (Stemcell Raw Pan T Cell Isolation Kit) from WT or GPR174-KO mice were cultured in X-vivo-15 media in 96-well flat-bottom plates precoated with 50 μg/ml goat anti-hamster IgG ( Jackson Immunochemicals) under the following conditions:

100,000 T cells per well anti-CD3 antibody (clone 2C11; 0.01 μg/ml) anti-CD28 antibody (clone 37.51; 0.1 μg/ml)

PS-liposomes (1 μM) GPR174 inhibitor (compound 10, 1 μM) cells were cultured for 24 h and cytokine concentrations in the supernatants were assessed (LegendPlex, Biolegend).

Fig. 65 graphically illustrates the concentrations of IL-2 in cultures of stimulated human T cells in the presence or absence of PS liposomes (PSL) or GPR174 inhibitor (compound 10, 3 μM).

As shown in FIG. 65, PS liposomes suppress IL-2 production (0.55-fold, p=4x10 ^-4 ), while GPR174 inhibitory compound 10 reverses this suppression (2.4-fold, p=3x10 ^-4 ). In contrast, in the absence of PS liposomes, compound 10 only increased IL-2 production by 1.3-fold (p=0.01).

Fig. 66A, B and C graphically illustrate the concentration of IL-2 (FIG. 66A), IFN-γ (FIG. 66B), and TNF (FIG. 66C) in cultures of stimulated human T cells in the presence or absence of PS liposomes.

- 123 046439 tumor exosomes, or GPR174 inhibitory compound (compound 10, 0.5 µM). As shown in FIG. 66A, B and C, human breast cancer tumor tumor-derived exosomes were more effective than PS liposomes in eliciting cytokine-inducing responses via GPR174 inhibition. The increase in IL-2 (FIG. 66A), IFN-γ (FIG. 66B), and TNF (FIG. 66C) in the presence of media, PS liposomes, or exosomes is summarized in Table 6 below. 16. Although exosomes did not suppress cytokine production like PS liposomes, exosomes may also have immunostimulatory proteins that counteract their PS-mediated suppression. Thus, the suppressive effects of exosome-associated PS and the hypothesized stimulatory effects of other exosome-associated traits together lead to minor net changes in cytokine production, and the addition of the GPR174 inhibitor neutralizes the effects of PS, resulting in a net increase in cytokine production greater than that observed without exosomes. .

Table 16. Summary of IL-2, IFN-γ and TNF production in the presence of media, PS liposomes or exosomes as shown in FIG. 66A-C

Compound 10 against the carrier, in the presence of: IL-2 IFN-γ TNF F.C. p value F.C. p value F.C. p value Wednesdays 1.2 3,ZE-OZ 1.1 ns 1.4 1,ZE-05 PS liposomes 1.4 1.6E-05 1.3 7,ZE-OZ 1.7 9.0E-07 Exosomasis 1.7 4.2E-04 1.4 1.5E-04 1.7 ZDE-06

FC, fold change; p value, T-test, 2-tailed, equal variances; ns, not significant.

Fig. 67 graphically illustrates the fold change in IL-2 levels, compared to vehicle, in cultures of stimulated WT or GPR174-KO mouse T cells in the presence or absence of PS liposomes (PSL) or GPR174 inhibitor (compound 10, 1 μM).

As shown in FIG. 67, PS liposomes suppress IL-2 production only from GPR174 WT T cells (WT: 0.19-fold, p=8x10 ⁴ ; KO: 1.8-fold, p=0.04). In addition, compound 10 reversed PS-liposome-mediated suppression in WT T cells (3.5-fold, p=5x10 ^-4 ), while having no effect on GPR174-KO T cells (0.9-fold, p=0.6). As noted above, PS liposomes slightly increased IL-2 levels in GPR174-KO T cells. This may be due to the action of PS on other lyso-PS receptors that are not Gas-coupled, such as P2Y10 and GPR34, and which may enhance rather than suppress cytokine production.

The results described in this example, using purified human and mouse T cells, established that PS liposomes suppress T cell responses specifically through GPR174, that the GPR174 inhibitory compound 10 reverses this suppression, and that compound 10 has little effect in the absence of PS or GPR174. Additionally, the results in this example show that tumor cell-derived exosomes stimulate GPR174, and inhibition of GPR174 in the presence of exosomes increases TM cytokine levels to a greater extent than in the presence of PS liposomes. Thus, reversing tumor cell-derived exosome-mediated immunosuppression using a GPR174 inhibitor may play a role in its antitumor efficacy. In addition, since tumor exosomes (i.e., cancer cell-derived exosomes) are known to migrate throughout the patient's body and stimulate tumor metastasis (see Wu M et al., Molecular Cancer, 18:53, 2019), the inhibitor GPR174 may enhance antitumor immune responses in tumor-draining lymph nodes, as well as reduce the formation and growth of metastases.

Example 27.

This example describes the effects on mouse T cell responses of GPR174 deficiency and GPR174 inhibition when combined with small molecule adenosine A2a/A2K receptor modulators using mouse total splenocyte cultures.

Both GPR174 and adenosine A2a/A2E receptors signal through the Gas/cAMP pathway, which suppresses T cell activation. Using human PBMC, we observed that inhibition of both axes resulted in a synergistic enhancement of Th1 cytokine production as described in Examples 15 and 18-22 herein. As described in this example, we extended these observations using splenocytes from WT mice and GPR174-deficient mice. GPR174 and adenosine receptor signaling preferentially suppresses IL-2 production from mouse splenocytes, so herein, we have only reported IL-2 levels for these experiments.

To obtain single cell suspensions from mouse splenocytes, spleens were cut into 12 mm ³ pieces using a razor blade and incubated at 37°C with 10 μg/ml DNase and 1 mg/ml collagenase D with shaking for 90 min. The spleen fragments and cells were then pressed through a nylon mesh and the resulting cell suspension was washed. In 96-well flat-bottom plates, splenocytes were cultured in quadruplicate using the following conditions for 2 days, after which the supernatants were tested for cytokine levels using the MSD platform.

- 124 046439

1,000,000 splenocytes per well anti-CD3 (clone 2C11; 0.1 µg/ml) anti-CD28 (clone 37.51; 0.1 µg/ml) GPR174 inhibitor (compound 10, 1 or 0.3 µM, or DMSO vehicle control) GPR174 inhibitor (Compound 6, 0.3 µM or DMSO vehicle control) Adenosine receptor agonist (NECA, 0.1 µM) Adenosine A2a/A2b receptor antagonist (ZM-241385, 0.1 or 0.2 µM, or DMSO vehicle control )

Fig. 68A and 68B graphically illustrate the effects of the adenosine A2a/A2b receptor antagonist ZM-241385 (0.1 μM), the GPR174 inhibitory compound 10 (1 μM), or both compounds (ZN241385 plus compound 10) on IL-2 levels in supernatants of cultured splenocytes. isolated from n=3 WT mice (Fig. 68A) or n=3 littermate GPR174-KO mice (Fig. 68B) after 2 days of stimulation with anti-CD3 antibody and anti-CD28 antibody.

As shown in FIG. 68A, compound 10 increased IL-2 levels by more than 4-fold (p<10 ^-5 ) in WT splenocyte cultures only, while vehicle control levels in GPR174KO cultures (FIG. 68B) averaged more than 6-fold. higher than levels for WT cultures (p<10 ^-9 ), indicating that this activity of compound 10 is specific for GPR174, and that GPR174 deficiency phenocopies the activity of compound 10. In contrast, ZM-241385 had a modest effect, increasing levels IL-2 increased on average 1.3-fold in both WT and GPR174-KO mice (p<0.05). The present inventors hypothesized that the poor response to A2b/A2b receptor antagonism was due to low levels of endogenous adenosine under culture conditions. Thus, as shown below, the present inventors repeated this experiment using WT splenocytes in the presence of the adenosine receptor agonist NECA.

Fig. 69 graphically illustrates the effects of adenosine A2a/A2b receptor antagonist ZM-241385 (0.2 µM), GPR174 inhibitory compound 10 (0.3 µM) or GPR174 inhibitory compound 6 (0.3 µM), or ZM-241385 plus compound 10, or ZM-241385 plus compound 6 on IL-2 levels in supernatants of cultured splenocytes isolated from 3 WT mice after 2 days of stimulation with anti-CD3 and anti-CD28 in the presence of the adenosine receptor agonist NECA (0.1 μM).

As shown in FIG. 69, both GPR174 inhibitory compounds 10 and 6 increased IL-2 levels 2-3-fold (p<0.05). When NECA was included in culture, ZM-241385 increased IL-2 levels by an average of 2-fold, in the absence or presence of GPR174 inhibitory compound 10 or 6 (p<0.01).

Fig. 70A and 70B graphically illustrate the effects of the adenosine A2a/A2b receptor antagonist ZM-241385 (0.2 μM), the GPR174 inhibitory compound 10 (0.3 μM) or compound 6 (0.3 μM), or ZM-241385 alone or in combination with any GPR174 inhibitory compound on IL-2 levels in supernatants of cultured splenocytes isolated from 3 WT mice (Fig. 70A) and 3 GPR174-KO mice (Fig. 70B) after 2 days of stimulation with anti-CD3 and anti-CD28 in the presence of agonist NECA adenosine receptor (0.1 µM).

As shown in FIG. 70A, in WT splenocytes using ZM-241383, IL-2 levels increased 2.3-2.6-fold (p<0.01), and GPR174 inhibitory compounds 10, and 6 increased IL-2 levels 1.7-fold. 2.0 times (p<0.02). In contrast, ZM in combination with GPR174 inhibitory compound 10 increased IL-2 levels by 5.7-6.7-fold (p<0.001), and

ZM in combination with GPR174 inhibitory compound 6 increased IL-2 levels by 4.9-5.9-fold, showing a synergistic enhancement of IL-2 production when both A2aR/A2bR and GPR174 were inhibited in the presence of NECA.

According to this synergy model, as shown in FIG. 70B, ZM-241385 was more effective using GPR174-KO splenocytes, increasing IL-2 levels by 3.5-4.1-fold (p<0.001). As expected, GPR174 inhibitory compounds were not active in GPR174-KO splenocytes, except for moderate off-target activity using compound 6 in 2 of 3 splenocyte cultures (KO1 and KO2, p<0.05).

In cultured mouse splenocytes, both GPR174 inhibition and GPR174 deficiency markedly increased IL-2 production, whereas GPR174 inhibitors were inactive in GPR174-KO splenocytes. When adenosine receptors exhibited agonism with NECA, inhibition of the two pathways was synergistic to enhance IL-2 production, that is, the levels achieved with both inhibitors were greater than the sum of the increases observed with each inhibitor alone. In addition, GPR174 deficiency phenocopied the effects of GPR174 inhibitors, both alone and in the presence of an A2aR/A2bR inhibitor, providing further support for the ability of the two pathways to act synergistically to regulate IL-2 production. Because GPR174 inhibitors reverse the effects of endogenous PS signaling in these splenocyte cultures, small differences in the amplitude of the effects of GPR174 inhibitors may arise from inter-experimental variability in the effects of PS on GPR174, potentially stemming from small differences in the compositions and activation states of the cells that difficult to control.

- 125 046439

Example 28.

This example describes the combined effects of a GPR174 inhibitor and two adenosine axis inhibitors on IL-2 induction in cultured human PBMCs.

Extracellular adenosine is formed primarily by enzymes that remove phosphate from extracellular ATP, which is released from activated or dying cells. Ectonucleotidases CD39 and CD73 cleave the first 2nd and 3rd phosphates from ATP, respectively, leaving adenosine for activation of adenosine receptors. In this example, we sought to determine whether inhibition of CD73 could have the same effect on PBMC cultures as inhibition of A2aR/A2bR, and whether inhibition of both CD73 and A2α/A2b could synergize with GPR174 inhibition in enhancing the T cell response. cells.

Human PBMCs obtained from a voluntary human donor (donor 8) were distributed in X-vivo15 media (Lonza) supplemented with Glutamax, penicillin, and streptomycin at a density of 1 × 10 ⁶ cells/well in a 96-well flat-bottom plate and cultured in four replicates in for 3 hours in the presence of:

GPR174 inhibitory compound 10 (1 μM);

A2aR/A2bR inhibitory compound ZM-241385 (0.1 μM);

CD73 inhibitory compound: sodium salt of adenosine-5'-(a,b-methylene) diphosphate (APCP, Tocris, 10 mKM);

combinations of the above means;

control media (DMSO).

Subsequently, 1 μg/ml anti-CD3 antibody (UCHT1) and 0.1 μg/ml anti-CD28 antibody (CD28.2) were added to activate T cells. Supernatants from these cells were collected 24 h after stimulation, and IL-2 levels were determined by ELISA.

Fig. 71 graphically illustrates the levels of IL-2 in supernatants of PBMCs stimulated with anti-CD3 antibody and anti-CD28 antibody in the presence of the indicated compounds.

As shown in FIG. 71, inhibition of GPR174 with compound 10 increased IL-2 levels by 1.4-fold, and inhibition of the adenosine axis using ZM-241385 or APCP increased IL-2 levels by 1.4- and 1.5-fold, respectively (p<0. 05 for each). Combination of GPR174 inhibitory compound 10 with either ZM-241385 or APCP increased IL-2 by 2.5-fold (p<0.001).

The results shown in this example demonstrate that both the A2aR/A2bR inhibitor (ZM241385) and the CD73 inhibitor (APCP) increased IL-2 levels to a similar extent, either alone or in the presence of GPR174 inhibitory compound 10. In addition, the extent The induction of IL-2 using combined inhibition of GPR174 and the adenosine axis was greater than the sum of the increases obtained using each inhibitor alone, indicating that inhibition of the adenosine axis using either inhibitor synergized with a GPR174 inhibitor to increase IL-2 production.

Example 29.

This example describes the suppression of cytokine production from purified human T cells by PS liposomes, a NECA adenosine receptor agonist, and a combination of both.

If, in the tumor microenvironment, immunosuppression due to high exposure to both adenosine and PS cannot be overcome unless both adenosine receptors A2aR/A2bR and GPR174 are inhibited, then it follows that exposure to the adenosine analogue NECA and PS liposomes together should suppress T cells in vitro to a degree similar to the suppression achieved with either inhibitor alone. In this example, we tested this hypothesis using full receptor agonist concentrations of NECA and PS for their ability to suppress IL-2 production from purified human T cells.

Purified human T cells (raw total T cells, Astarte Biologies, Inc.; CD3+ T cells in Figures 72A and 72B, and CD8+ T cells in Figure 72C) were stimulated in four replicates at 100,000 cells per well in 96- well flat-bottom plates and X-vivo 15 media using the following reagents:

human CD3/CD28 T cell activator ImmunoCult™ (StemCell) (1:50 dilution)

PS liposomes (1 µM) or adenosine receptor agonist control media (NECA, 0.1 µM or 0.05 µM) or control media a combination of both reagents.

After 24 hours of culture (44 hours for the results shown in Fig. 72C), cytokines were analyzed in the culture media (MesoScale Discovery).

Results.

Fig. 72A and 72B graphically illustrate the suppression of IL-2 (FIG. 72A) and IFN-γ (FIG. 72B) production from purified T cells by PS liposomes and NECA, alone or together. For CD8 T cell cultures (FIG. 72C), the indicated concentrations of ZM-241385 and Compound 10 were included.

As shown in FIG. 72A, 72B and 72C, and summarized below in table. 17, I will suppress both PS and NECA

- 126 046439 yt cytokine to almost the same extent as the suppression achieved using both together.

Table 17. Summary of IL-2 and IFN-γ production under the various conditions shown in FIG. 72A and 72B

IL-2 IFN-γ F.C. p value F.C. p value PS vs environments 0.51 3.0E-04 0.41 7.8E-04 NECA vs Wednesdays 0.42 1.0E-04 0.31 4.6E-04 PS +NECA vs Wednesdays 0.34 5,ZE-05 0.27 2.6E-04

FC, fold change; p, p value for T-test, 2-tailed, equal variances.

These findings further support the model that, in the presence of high degrees of GPR174 and A2aR/A2bR agonism, inhibition of both pathways is necessary to relieve T cells from cAMP-mediated suppression. This hypothesis is supported by the finding that the suppression achieved using NECA or PS liposomes alone was almost as strong as the suppression observed using a combination of both agonists. This suggests that both GPR174 and A2aR/A2bR feed the same cAMP compartment; or that their individual cAMP compartments may entirely involve cAMP-mediated suppression of cytokine production; and, to effectively counteract this suppression in the presence of PS/lysoPS and adenosine, both GPR174 and A2aR/A2bR must be inhibited.

In general, as shown herein, GPR174 is an immune-restricted Gas-coupled GPCR, and PS exposed on liposomes and cell membranes stimulates GPR174, supporting a model of active GPR174-mediated immunosuppression in the tumor microenvironment. In conditions where both PS/lysoPS and adenosine are present, inhibition of both axes is necessary for effective restoration of T cell function (e.g., reversal of PS+NECA-mediated suppression of IFN-γ production from human CD8 T cells appears to require synergistic activity of ZM and compound 10; see Fig. 72C). As further demonstrated herein, GPR174 deficiency enhances antitumor immune responses in mice.

As described herein, extracellular phosphatidylserine (PS) is a potent modulator of immune responses. Different phospholipid scramblases respond to different cellular processes to expose PS either through apoptosis or activation of a variety of cell types, including platelets, lymphocytes, endothelial cells and tumor cells. While it is well known that PS exposed during apoptosis suppresses inflammatory responses in phagocytes during efferocytosis, whether any form of exposed PS can act directly on T lymphocytes has not been widely investigated. As described herein, we show that PS suppresses T cells through the GPR174 Gas GPCR coupled previously described as a receptor for lysophosphatidylserine (lysoPS), a soluble catabolite of PS. PS liposomes were found to be 5x more active than lysoPS in stimulating GPR174-dependent cAMP formation, and PS exposed to apoptotic cells, platelets, and activated T cells all induced GPR174 signaling in a reporter cell line. Consistent with the well-described immunosuppressive nature of cAMP signaling, PS liposomes suppressed IL-2 production in human T cells and IL-2 production in mice from WT but not GPR174KO T cells. By effectively screening a novel library of GPCR modulating chemical compounds, we identified multiple GPR174 inhibitors spanning multiple chemical classes, and the GPR174 inhibitor reversed PS liposome-mediated suppression of human T cells and WT mouse T cells while having no effect on GPR174-KO mouse T cells. In a mouse syngeneic tumor model, GPR174 deficiency significantly increased tumor growth control in the presence of suboptimal anti-GITR antibody co-therapy. In many respects, GPR174 is similar to the A2A/A2B adenosine receptors in that both suppress T cells through cAMP signaling in response to stress and cell death products present at high levels in the tumor microenvironment, and the present authors inventions found that both pathways act synergistically to moderate T cell responses. In mouse splenocyte cultures containing endogenous levels of adenosine and PS, the A2A inhibitor enhanced T cell responses more effectively in GPR174 knockout cells than in WT cells. In similar cultures of human PBMC, inhibitors of GPR174 and A2A/A2B, or inhibitors of GPR174 and adenosine deaminase, synergistically increased IL-2 production. Our findings indicate that inhibition of both GPR174 and the adenosine pathway may be important for effectively overcoming cAMP-mediated immunosuppression in the tumor microenvironment.

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Claims (9)

CLAIM 1. A method of treating a malignant tumor, comprising administering to a patient in need thereof a therapeutically effective amount of a GPR174 inhibitor selected from the group consisting of: i. compound 10, having the following structure: ii. compound 6, having the following structure: iii. compound 11, having the following structure: iv. compound 20 having the following structure: and an adenosine-A2A receptor antagonist (A2A) selected from the group consisting of N (ZM-241385); (SCH-58261); and (PBF-509). 2. The method of claim 1, wherein the GPR174 inhibitor inhibits PS- or LysoPS-dependent activation of GPR174 signaling in a cell expressing GPR174 by at least 25%. 3. The method of claim 1, wherein the GPR174 inhibitor stimulates an immune response in the patient by inhibiting GPR174 signaling of G-alpha-s. 4. The method of claim 3, wherein the stimulated immune response includes an NK cell-mediated immune response and an antitumor cytotoxic T cell-mediated immune response. 5. A method for increasing the level of Th1 cytokines in human peripheral blood mononuclear cells (PBMCs), including bringing human PBMCs into contact with a GPR174 inhibitor, has been selected - 128 046439 nom from the group consisting of: i. compound 10, having the following structure: ii. compound 6, having the following structure: o iii. compound 11, having the following structure: iv. compound 20 having the following structure: and an adenosine-A2A (A2A) receptor antagonist selected from the group consisting of: i. ZM-241385; ii. SCH-58261; and iii. PBF-509. 6. The method according to claim 5, where the contacting is carried out in vitro. 7. The method of claim 5, wherein the PBMC contain T cells or NK cells. 8. The method according to any one of claims 5 to 7, wherein the level of at least one of the Th1 cytokines IFN-γ, IL-2, TNF or GM-CSF is increased by at least 20%. 9. A pharmaceutical composition for the treatment of a malignant tumor, containing a combination of a GPR174 signaling inhibitor selected from the group consisting of: i. compound 10, having the following structure: ii. compound 6, having the following structure: iii. compound 11, having the following structure: iv. compound 20 having the following structure: - 129 -