WO2024107118A1

WO2024107118A1 - Nor1 ligands and methods of their use

Info

Publication number: WO2024107118A1
Application number: PCT/SG2023/050766
Authority: WO
Inventors: Ho Sup Yoon; Jun Yeob YOO
Original assignee: Nanyang Technological University
Priority date: 2022-11-16
Filing date: 2023-11-15
Publication date: 2024-05-23

Abstract

Various embodiments relate generally to ligands that bind to the neuron-derived orphan receptor-1 protein-ligand binding domain (Nor1-LBD) and modulate the activity of Nor1, and methods of using Nor1-modulating ligands. In particular, through structural characterisation of the ligand binding domain of Nor1, ligands of the Nor1-LBD have been identified along with potential therapeutic uses in treating or preventing associated conditions or diseases thereof.

Description

NOR1 LIGANDS AND METHODS OF THEIR USE

CROSS-REFERENCE TO RELATED APPLICATION

[001] This application claims the benefit of priority of Singapore Patent Application No. 10202260107W filed 16 November 2022, the content of which being hereby incorporated by reference in its entirety for all purposes.

TECHNICAL FIELD

[002] Various embodiments relate generally to ligands that bind to the neuron-derived orphan receptor-1 protein-ligand binding domain (Nor1 -LBD) and modulate the activity of Nori , and methods of using Nori -modulating ligands.

BACKGROUND

[003] Nuclear receptors (NRs) are a family of transcriptional factors that play a crucial role in regulating cellular functions in response to environmental cues^(1,2). They comprise modular domains with distinct functions, including the ligand-binding (LBD) and DNA-binding (DBD) domains, which determine their ligand specificity and target gene expression⁽³’⁴⁾. While classical NRs are known to function as receptors for hormones, the discovery of orphan NRs through sequence homology screening has revealed a group of proteins with similar structural features but unknown native ligands^(5-7).

[004] The NR4A subfamily is one such group of orphan nuclear receptors, consisting of Nur77 (NR4A1 ), Nurrl (NR4A2), and Nori (NR4A3). Understanding the role of Nuclear Receptor (NR) during pathophysiological conditions, its regulatory mechanism and identifying ligands for orphan NRs is an emerging concept for potential therapeutics developments. Nori (Nuclear orphan receptorl ) is an orphan NR involved in pulmonary vascular remodeling, extracellular matrix regulation, neutrophil regulation, development of acute myeloid leukemia, and metabolic pathways. While NorTs associations with these biological pathways are reported earlier, currently the structure of Nori is not available and it still remains as an orphan nuclear receptor.

[005] Therefore, there is still need in the art to determine the structure of Nori ’s ligand binding domain, in order to identify ligands of Nori and their subsequent biological role and potential therapeutic use in treating or preventing associated conditions or diseases thereof.

SUMMARY OF INVENTION

[006] In a first aspect, there is provided a method of modulating insulin secretion from a p-cell, comprising administering an effective amount of a Nori ligand to the p-cell, wherein the Nori ligand directly binds to the ligand binding domain (LBD) of Nori . [007] In various embodiments, the Nori ligand, is an agonistic Nori ligand that induces: increased secretion of insulin from the p-cell relative to a p-cell not administered with the Nori ligand; and/or increased growth of the p-cell relative to a p-cell not administered with the Nori ligand.

[008] In various embodiments, the method is in vitro, and the Nori ligand contacts with the p-cell under suitable conditions to modulate the p-cell insulin secretion, preferably the p-cell is derived from a p-cell line or a biological sample obtained from a subject.

[009] In various embodiments, the method is in vivo, the p-cell is in a subject, preferably a mammal, more preferably a human, and the method further comprises administering to the subject the Nori ligand.

[010] In another aspect, there is provided a method of modulating glucose levels, preferably blood glucose levels, in a subject, comprising administering to the subject an effective amount of a Nori ligand, wherein the Nori ligand directly binds to the ligand binding domain (LBD) of Nori .

[011] In various embodiments, the method is for treating or preventing a glucose metabolism disorder or condition in the subject.

[012] In various embodiments, the glucose metabolism disorder is selected from the group consisting of: diabetes mellitus, glycosuria, hyperglycemia, hypoglycemia and hyperinsulinism.

[013] In various embodiments, the glucose metabolism disorder is diabetes mellitus, preferably Type 2 mellitus.

[014] In various embodiments, the Nori ligand is a small molecule, preferably the small molecule directly binds to the ligand-binding domain of Nori (Nori -LBD).

[015] In various embodiments, the Nori -LBD comprises or consists of an amino acid sequence set forth in SEQ ID NO:4 or variants thereof.

[016] In various embodiments, the Nori ligand directly binds to one or more amino acid residues in helix 10/1 1 of Nori -LBD, corresponding to positions 580-606 of SEQ ID NO: 1 , preferably the Nori ligand directly binds to one or more amino acid residues at positions corresponding to positions 591 - 603 of SEQ ID NO: 1 , more preferably the Nori ligand directly binds to one or more amino acid residues at positions corresponding to positions 594-600 of SEQ ID NO: 1 .

[017] In various embodiments, the Nori ligand directly binds with: (i) the amino acid residue C (Cys) at the position corresponding to position 594 of SEQ ID NO: 1 ; and/or

(ii) the amino acid residue T (Thr) at the position corresponding to position 595 of SEQ ID NO: 1 ; and/or

(iii) the amino acid residue L (Leu) at the position corresponding to position 596 of SEQ ID NO: 1 ; and/or

(iv) the amino acid residue G (Gly) at the position corresponding to position 597 of SEQ ID NO: 1 ; and/or

(v) the amino acid residue R (Arg) at the position corresponding to position 600 of SEQ ID NO: 1 .

[018] In various embodiments, the Nori ligand directly and covalently binds with the amino acid residue C (Cys) at the position corresponding to position 594 of SEQ ID NO: 1 .

[019] In various embodiments, the Nori ligand is an agonistic Nori ligand, preferably an agonistic Nori ligand that covalently binds to Nori , preferably the Nori ligand is PGA1 .

[020] In various embodiments, the Nori ligand is an antagonistic Nori ligand, preferably the Nori ligand is PGE1 .

[021] In various embodiments, the Nori ligand is a cyclopentenone prostaglandin or a derivative, or an analog thereof.

[022] In various embodiments, the cyclopentenone prostaglandin is selected from the group consisting of prostaglandin A1 (PGA1 ), prostaglandin A2 (PGA2), 15-deoxy-A12,14-prostaglandin J2 (15-d-A12,14-PGJ2), A12-Prostaglandin J2 (A12-PGJ2), prostaglandin J2 (PGJ2), prostaglandin E1 (PGE1 ), prostaglandin E2 (PGE2), derivatives and analogs thereof.

[023] In various embodiments, the cyclopentenone prostaglandin is prostaglandin A1 (PGA1 ) or prostaglandin A2 (PGA2) or a derivative, or an analog thereof.

[024] In various embodiments, the cyclopentenone prostaglandin is prostaglandin A1 (PGA1 ) or a derivative, or an analog thereof.

[025] In various embodiments, the Nori ligand is prostaglandin E1 (PGE1 ) or a derivative, or an analog thereof.

[026] In another aspect, there is provided a method of modulating Nori activity in a cell, comprising administering an effective amount of a prostaglandin or derivative, or analog thereof to the cell. [027] In another aspect, there is provided a computer-assisted method for screening, identifying or designing a candidate molecule or compound that modulates Nori activity, the method comprising the steps of: a) providing the structure of Nori -LBD having the amino acid sequence set forth in SEQ ID NO:4 or variants thereof, wherein the structure of Nor1 -LBD includes the following helical structures and their corresponding amino acid residues of SEQ ID NO: 4 or variants thereof: Helix 1 (H1 : residues 397-407), Helix 3 (H3: residues 430-454), Helix 4,5 (H4/5: residues 463-485), Helix 6 (H6: residues 504-510), Helix 7 (H7: residues 512-527), Helix 8 (H8: residues 531 -542), Helix 9 (H9: residues 552-572), Helix 10,1 1 (H10/1 1 : residues 580-606), and Helix 12 (H12: residues 614-622); b) providing the structure of the candidate molecule or compound; c) fitting the structure of the candidate molecule or compound to the Nori -LBD, wherein fitting comprises determining interactions between one or more atoms of the candidate molecule or compound and one or more atoms of Nori - LBD, to predict whether the candidate molecule or compound binds to or within Nor1 -LBD; and d) selecting the candidate molecule or compound if it is predicted to bind to or within Nori -LBD.

[028] In various embodiments, the candidate molecule or compound is selected from a virtual chemical library or is a prostaglandin or a derivative, or an analog thereof.

[029] In various embodiments, the Nor1 -LBD has the amino acid sequence set forth in SEQ ID NO:4, and the structure of the Nor1 -LBD is derived from, or characterized, by one or more of the distance constraints, dihedral constraints and structural statistics described in Table 4.

BRIEF DESCRIPTION OF THE DRAWINGS

[030] Various embodiments will be better understood with reference to the detailed description when considered in conjunction with the non-limiting examples and the accompanying drawings.

[031] FIG. 1 shows: (A) representative lowest energy calculated solution structure of Nor1 -LBD. Helical regions M397-D407 (a1), D430-K454 (a3), K463-R485 (a4/5), L504-F510 (a6), E512-L527 (a7), I531 -N542 (a8), P552-K572 (a9), E580-L606 (10/1 1 ), S614-D622 (a12) are shown in helical representations; (B) electrostatic potential surface of Nor1 -LBD is displayed. Positively charged residues are coloured white and negatively charged residues are coloured black; and (C) residues identified from chemical shift perturbation and peak intensity change were mapped onto the Nor1 - LBD NMR structure. A region of residues of C594, T595, L596, G597 and R600 (side chains shown) on helix-10/1 1 showed significant perturbation, suggesting the region of ligand-binding.

[032] FIG. 2 shows bar graphs of PGA1 activating the transcription function of Nori in C2C12 cell line: (A) Mouse myoblast C2C12 cells were transfected with plasmids (pSF-SV40-GAL4(DBD)- NOR1 (LBD), p9xUAS-Luc, and pRL-TK) and treated with a range of prostaglandins (concentration at 10 pM) to show Nori nuclear receptor transactivation specifically induced by PGA1/2; (B) Further analysis of the most effective activator, PGA1 , showed a dose-dependent activation effect on both hybrid Gal4-DBD + Nor1 -LBD system; and (C) full-length Nori system (pGL3 fN0R1 , p4xNL3-Luc, pRL-TK). (One-way ANOVA (vs. ligand non-treated control), n=3, error bar represents SEM, *<0.05, **<0.01 , ***<0.001 , ****<0.0001 ).

[033] FIG. 3 shows that PGA1 covalently binds to Nor1 -LBD. The purified Nor1 -LBD protein was preincubated with PGA1 to detect ligand-bound complex on the RESOURCE Q anion exchange chromatography column (Cytiva, MA). The ion exchange elution profile of the incubated sample detected an elution peak that contains covalently bonded PGA1 -Nor1 -LBD. The collected sample was concentrated and analyzed with mass spectrometry, confirming that the molecular weight of the new species was the sum of Nori -LBD and PGA1 .

[034] FIG. 4 shows PGA1/A2 treatment enhancing cell growth: (A) Each set of plots were fitted to an exponential doubling growth curve to calculate doubling time; and (B) Calculated growth rate (H- 1 ) from the exponential equation fitting show that PGA1 and PGA2 treatment can significantly increase cell growth compared to PGE1 , which shows an inhibitory effect (Statistical analysis done using one-way ANOVA, treated vs. (-) control, * < 0.05, **< 0.01).

[035] FIG. 5 shows PGA1 enhances insulin production in EndoC-pH1 cells: (A) GSIS assay shows that, depending on the type of ligand incubated with, cells exhibit different levels of insulin secretion upon glucose stimulation (11 .0 mM, non-white bars) compared to prestimulation (2.2 mM, white bar). The significant increase of insulin production from PGA1 and PGA2 treatments are noticeably comparable to PGE1 , a structurally similar molecule known to reduce insulin production by the EP3 receptor signalling pathway; and (B) GSIS assay shows that the ligand-stimulated cells exhibit different levels of insulin secretion upon glucose stimulation (11 .0 mM, non-white bars) compared to pre-stimulation (2.2 mM, white bar). The significant increase in insulin production from PGA1 treatment is noticeable. PGE1 , a structurally similar molecule, reduces insulin production. (One-way ANOVA (vs. ligand non-treated control), n=3, error bar represents SEM, *<0.05, **<0.01 , ***<0.001 , ****<0.0001 ).

[036] FIG. 6 shows PGA1 enhances insulin secretion in INS-1 E cells through Nori signaling. NR4A family members were knocked down by siRNAs. Only Nori knock-down cells abolished PGA1 - mediated insulin secretion enhancement, indicating that PGA1 effectively functions through Nori signaling.

[037] FIG. 7 shows PGA1 treatment has higher insulin expression and secretion than existing diabetes medications. EndoC-pH1 cells were pretreated with each compound overnight before performing a glucose-stimulated insulin secretion assay: (A) PGA1 , IBMX, Ex-4 (exendin-4), and nateglinide (Nate) all showed enhanced production of overall insulin; and (B) PGA1 excelled in increasing the cellular secretion of insulin compared to all other drugs. (Statistical analysis done using one-way ANOVA, vs. (-) control, * < 0.05, *“*< 0.0001 ).

[038] FIG. 8 shows isolated mouse pancreatic islets exhibit increased glucose-stimulated insulin secretion upon PGA1 pretreatment. Pancreatic islets isolated from C57BL/6 mice were pretreated with PGA1 , nateglinide, exendin-4, and PGE1 to show its insulin secretion modifying effects. While PGE1 treatment effectively inhibited insulin secretion from the islets, all other compounds exhibited increased insulin secretion, with PGA1 showing the most significant increase in insulin secretion. (One-way ANOVA (vs. ligand non-treated control), n=3, error bar represents SEM, *<0.05, **<0.01 , ***<0.001 , ****<0.0001 ).

DETAILED DESCRIPTION

[039] The following detailed description refers to, by way of illustration, specific details and embodiments in which the invention may be practiced. These embodiments are described in sufficient detail to enable those skilled in the art to practice the invention. Other embodiments may be utilized and structural and logical changes may be made without departing from the scope of the invention. Embodiments described below in context of the Nori -ligand binding domain and binding ligand are analogously valid for the respective methods, and vice versa. The various embodiments are not necessarily mutually exclusive, as some embodiments can be combined with one or more other embodiments to form new embodiments.

[040] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. The term "comprises" means "includes." In case of conflict, the present specification, including explanations of terms, will prevail. “About”, as used herein in connection with numerical values refers to the referenced numerical value ±10% or ±5%.

[041] The invention is based on the structural characterisation of the Nori ligand-binding domain (LBD) by nuclear magnetic resonance (NMR) spectroscopy, and the subsequent identification of ligands that interact with the Nori -LBD structure for elucidating the biological role of Nori and ligands in modulating cellular processes with potential therapeutic uses.

[042] The Nori is a protein and may be of any species of origin, and is preferably any one of the following in Table 1 below. Table 1

[043] In various embodiments, the Nori has the amino acid sequence set forth in SEQ ID NO:1 , 2 or 3, or variants thereof. In various embodiments, the Nori is of human origin and has the amino acid sequence set forth in SEQ ID NO:1 or variants thereof.

[044] Generally, the term “variant” covers such Nori proteins that have at least 80%, or at least 90% sequence identity with the reference amino acid sequence over their entire length, preferably at least 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99 % sequence identity. The identity of amino acid sequences is generally determined by means of a sequence comparison. This sequence comparison is based on the BLAST algorithm that is established in the existing art and commonly used (cf. e.g. Altschul et al. (1990) “Basic local alignment search tool”, J. Mol. Biol. 215:403-410, and Altschul et al. (1997): “Gapped BLAST and PSI-BLAST : a new generation of protein database search programs”; Nucleic Acids Res., 25, p. 3389-3402) and is effected in principle by mutually associating similar successions of amino acids in the amino acid sequences, respectively. A tabular association of the relevant positions is referred to as an "alignment." Sequence comparisons (alignments), in particular multiple sequence comparisons, are commonly prepared using computer programs which are available and known to those skilled in the art.

[045] As used herein, the term “amino acid” refers to natural and/or unnatural or synthetic amino acids, including both the D and L optical isomers, amino acid analogs (for example norleucine is an analog of leucine) and derivatives known in the art. The term “natural amino acid”, as used herein, relates to the 20 naturally occurring L-amino acids, namely Gly (G), Ala (A), Vai (V), Leu (L), lie (I), Phe (F), Cys (C), Met (M), Pro (P), Thr (T), Ser (S), Glu (E), Gin (Q), Asp (D), Asn (N), His (H), Lys (K), Arg (R), Tyr (Y), and Trp (W). Generally, in the context of the present application, the amino acid sequences are shown in the N- to C-terminal orientation.

[046] The ligand binding domain of Nori , i.e. Nor1 -LBD, in the context of the invention, refers to amino acid residues at positions corresponding to positions 379-626 of SEQ ID NO:1 and may be preferably any one of the following in Table 2. In this regard, the position numbering referenced herein is in accordance with SEQ ID NO:1 .

Table 2

[047] In various embodiments, the Nor1 -LBD has the amino acid sequence set forth in SEQ ID NO:4, 5 or 6, or variants thereof. In various embodiments, the Nor1 -LBD is of human origin and has the amino acid sequence set forth in SEQ ID NO:4 or variants thereof.

[048] In various embodiments, the structure of the Nori -LBD includes a triple layer of nine helices as follows: a Helix 1 (H1 : residues 397-407), a Helix 3 (H3: residues 430-454), a Helix 4,5 (H4/5: residues 463-485), a Helix 6 (H6: residues 504-510), a Helix 7 (H7: residues 512-527), a Helix 8 (H8: residues 531 -542), a Helix 9 (H9: residues 552-572), a Helix 10,1 1 (H10/1 1 : residues 580-606), and a Helix 12 (H12: residues 614-622). The position numbering is in accordance with SEQ ID NO:1 . In various embodiments, the variants include invariable amino acid residues corresponding to the nine helical structures, in particular, the amino acid residues corresponding to a Helix 10,1 1 are invariable.

[049] In various embodiments, the Nor1 -LBD has the amino acid sequence set forth in SEQ ID NO:4 and comprises the helical structures detailed in Table 3 below.

Table 3

[050] The 2-dimensional and 3-dimensional structure of the Nor1 -LBD has been characterised by NMR spectroscopy and using one or more computational methods. In various embodiments, the 3- dimensional conformation of the Nor1 -LBD is derived from a solution structure of the Nor1 -LBD determined by nuclear magnetic resonance (NMR) spectroscopy. The structural conformation may be defined by a set of NMR structure coordinates having a root mean square deviation (RMSD) of not more than about 1 .5 A (Angstroms), preferably not more than 1 .2 A, more preferably about 1 .05 A, in the backbone atoms; and a root mean square deviation (RMSD) of not more than about 2 A, preferably not more than 1 .8 A, more preferably about 1 .65 A, for all heavy atoms. As used herein, the term “root mean square deviation (RMSD)“ reflects and refers to a deviation in the distance between neighbouring carbon-backbone atoms. Related proteins with only local changes in conformation will be characterized by relatively low RMSD values whereas more changes will result in an increase of the RMSD value. A RMSD less than 2.0 A is considered accurate in the field.

[051] The NMR distance and dihedral constraints, and structural statistics of the human Nori -LBD is described in Table 4 below. The distance constraints refers to the limitations on the possible distances between pairs of atoms in the molecule, as determined by their NMR interactions. These constraints are derived from the NMR experimentally observable phenomenon called Nuclear Overhauser Effect (NOE). The NOE is a result of dipole-dipole interactions between nearby nuclei (typically protons) and leads to changes in the intensity of NMR signals. When two protons are close to each other (usually within about 5 A), their NMR signals can affect each other through space rather than through bond interactions. The distance constraints from NOE may be used in conjunction with other types of information (like torsion angle constraints, hydrogen bonds, etc.) to calculate the three- dimensional structure. The distances are not measured directly but are estimated based on the intensity of the NOE.

[052] Accordingly, in various embodiments, the Nor1 -LBD has the amino acid sequence set forth in SEQ ID NO:4 and has a structure derived from or characterised by the distance constraints, dihedral constraints and structural statistics described in Table 4.

Table 4

[053] In various embodiments, the Nor1 -LBD has the amino acid sequence set forth in SEQ ID NO:4 and has a structure derived from or characterised by one or more or all of the distance constraints, dihedral constraints and structural statistics described in Table 4.

[054] In various embodiments, the Nori -LBD comprises the amino acid sequences set forth in SEQ ID NO:7-15, or variants thereof, corresponding to the identified helical structures.

[055] In various embodiments, the Nori -LBD comprises the amino acid sequence set forth in SEQ ID NO:14, or variants thereof corresponding to the helix 10/1 1 structure. In particular, the inventors mapped onto the Nor1 -LBD structure amino acid residues identified from chemical shift perturbation and peak intensity change, whereby certain residues within the helix 10/1 1 showed significant perturbation, indicating the site of ligand binding. Accordingly, in various embodiments, the Nori -LBD comprises a ligand binding site within the helix 10/1 1 , corresponding to amino acid residues at positions corresponding to positions 580-606 of SEQ ID NO:1 . Further, amino acid residues within the helix 10/1 1 at the positions corresponding to positions C594, T595, L596, G597 and R600 (highlighted in bold in SEQ ID NO:14 in Table 3) of SEQ ID NO:1 showed significant perturbation. Accordingly, in various embodiments, the ligand binding domain comprises a ligand binding site within the helix 10/1 1 , corresponding to amino acid residues at positions corresponding to positions 594- 600 of SEQ ID NO:1 , more particularly a ligand binding site within the helix 10/1 1 , corresponding to amino acid residues at positions corresponding to positions 594-597 and 600 of SEQ ID NO:1 .

[056] In various embodiments, the Nor1 -LBD comprises amino acid residue C at the position corresponding to position 594 of SEQ ID NO:1 , and/or amino acid residue T at the position corresponding to position 595 of SEQ ID NO:1 , and/or amino acid residue L at the position corresponding to position 596 of SEQ ID NO:1 , and/or amino acid residue G at the position corresponding to position 597 of SEQ ID NO:1 , and/or amino acid residue R at the position corresponding to position 600 of SEQ ID NO:1 .

[057] Based on the foregoing structural characteristics of the Nor1 -LBD, the present invention relates to ligands that bind to Nori , more particularly the Nor1 -LBD, and their role and attributable effects in regulating associated cellular processes for therapeutic utility. The Nori ligand refers to a molecule that binds to the Nor1 -LBD, and initiates or interferes with the signal transduction process in a cell.

[058] As used herein, the term “binds” refers to the interaction between a molecule (the ligand) and a specific site on a larger molecule or protein (the receptor). The terms “interact” and “couple” may be used interchangeably with “bind’ in the context of the invention. In various embodiments, the nature of the binding interaction may be “direct binding”, meaning that the ligand is interacting with the receptor (i.e. Nori ) without any intermediaries or additional molecules to facilitate the binding, and is a one-on-one interaction. This is in contrast to indirect binding, where other molecules or co-factors might be needed for the ligand to effectively bind to the receptor. The nature and strength of this binding depend on various forces, such as electrostatic forces, hydrogen bonding, van der Waals forces, and hydrophobic interactions, that represent non-covalent interactions, however, it will be appreciated that the binding may include covalent interactions between the ligand and receptor that form covalent bonds. Accordingly, in various embodiments, the ligand may covalently and/or non- covalently bind to Nori or more preferably to Nor1 -LBD. In various embodiments, the ligand may covalently bind to at least one amino acid residue in the Nor1 -LBD. The region or pocket of one or more amino acid residues in the Nori -LBD where the ligand directly binds may be termed the “ligand binding site”. The “ligand binding site” may comprise a subset or region of amino acid residues that make direct or water-mediated interactions with the ligand.

[059] In various embodiments, the Nori ligand may be an agonistic Nori ligand or an antagonistic Nori ligand. The term “agonist” or “agonistic” as used herein, refers to a molecule or compound that binds to and activates Nori to induce its activation and function. The term “antagonist” or “antagonistic” as used herein, refers to a molecule that prevents, inhibits or reduces Nor 1 activation and function. [060] In various embodiments, the Nori ligand may be a small molecule such as organic compounds, inorganic compounds, small peptide molecules, peptide fragments, and the like. As used herein, the term “small molecule” may refer to molecules or compounds that may be natural or synthetic. A small molecule is typically characterized in that it contains several carbon-carbon bonds, and has a molecular weight of less than 5000 Daltons (5 kD), preferably less than 3 kD, still more preferably less than 2 kD, and most preferably less than 1 kD. In various embodiments, the small molecule has a molecular mass equal to or less than 700 Daltons.

[061] In various embodiments, the Nori ligand directly binds to the ligand-binding domain of Nori (Nor1 -LBD) corresponding to amino acid residues at the positions corresponding to positions 379- 626 of SEQ ID NO: 1 . In various embodiments, the Nori ligand binds to the ligand-binding domain of Nori (Nori -LBD) having the amino acid sequence set forth in SEQ ID NO:4 or variants thereof. The binding of the Nori ligand to the Nor1 -LBD may include covalent and/or noncovalent binding to the Nor1 -LBD.

[062] In various embodiments, the Nori ligand directly binds to one or more helix structures/regions of the Nor1 -LDB including H1 (residues 397-407), H3 (residues 430-454), H4,5 (residues 463-485), H6 (residues 504-510), H7 (residues 512-527), H8 (residues 531 -542), H9 (residues 552-572), 1-110,1 1 (residues 580-606), and H12 (residues 614-622), wherein position numbering is in accordance with SEQ ID NO:1 . As will be appreciated by those skilled in the art, the Nori ligand may bind to, or couple with, or interact with, one or more amino acids in a single helix region or multiple helix regions.

[063] In various embodiments, the Nori ligand directly binds to the helix 10/1 1 region of the ligand binding domain of Nori corresponding to amino acid residues at positions corresponding to positions 580-606 of SEQ ID NO: 1 . In this regard, the Nori ligand may directly bind to one or more amino acids within the helix 10/1 1 region. In various embodiments, the Nori ligand may directly bind to one or more amino acid residues at positions corresponding to positions 591 -603 of SEQ ID NO: 1 . In various embodiments, the Nori ligand may directly bind to one or more amino acid residues at positions corresponding to positions 594-600 of SEQ ID NO: 1 .

[064] The one or more amino acids may include but are not limited to (i) the amino acid residue C (Cys) at the position corresponding to position 594 of SEQ ID NO: 1 ; and/or (ii) the amino acid residue T (Thr) at the position corresponding to position 595 of SEQ ID NO: 1 ; and/or (iii) the amino acid residue L (Leu) at the position corresponding to position 596 of SEQ ID NO: 1 ; and/or (iv) the amino acid residue G (Gly) at the position corresponding to position 597 of SEQ ID NO: 1 ; and/or (v) the amino acid residue R (Arg) at the position corresponding to position 600 of SEQ ID NO: 1 . In various embodiments, the Nori ligand covalently binds to the helix 10/1 1 in the ligand binding domain of Nori , more specifically, to the amino acid residue C (Cys) at the position corresponding to position 594 of SEQ ID NO: 1 . [065] In various embodiments, the Nori ligand may be a compound or molecule identified in the screening methods disclosed herein.

[066] In various embodiments, the Nori ligand may be a prostanoid. In various embodiments, the prostanoid may be a prostaglandin, a prostacyclin, a thromboxane or a cyclopentenone prostaglandin

[067] In various embodiments, the Nori ligand may be a prostaglandin or a derivative, or an analog thereof.

[068] In various embodiments, the Nori ligand may be a prostanoid selected from prostaglandin A1 (PGA1 ), prostaglandin A2 (PGA2), 15-deoxy-A12,14-prostaglandin J2 (15-d-A12,14-PGJ2), A12- Prostaglandin J2 (A12-PGJ2), prostaglandin J2 (PGJ2), prostaglandin E1 (PGE1 ), prostaglandin E2 (PGE2), and a derivative or an analog thereof. In various embodiments, the prostaglandin may be a cyclopentenone prostaglandin or a derivative, or an analog thereof.

[069] Based upon the structural characterisation of the Nori -LBD, the inventors screened ligands and identified cyclopentenone prostaglandins (cyPGs), such as PGA1 and PGA2, as capable of binding to the Nor1 -LBD.

[070] The term “cyclopentenone prostaglandin” as used herein refers to a member of the subset of prostaglandins and prostanoids sharing a common mono-unsaturated cyclopentenone structure, as illustrated below. The cyclopentenone ring is a five-membered ring with a ketone (carbonyl group) that differentiates these compounds from other prostaglandins, which typically have a cyclopentane ring.

PGA1 :

PGA2:

[071] Accordingly, in various embodiments, the Nori ligand may be a cyclopentenone prostaglandin or a derivative, or an analog thereof. The cyclopentenone prostaglandin may be selected from the group consisting of prostaglandin A1 (PGA1 ), prostaglandin A2 (PGA2), 15-deoxy- A12,14-prostaglandin J2 (15-d-A12,14-PGJ2), A12-Prostaglandin J2 (A12-PGJ2), prostaglandin J2 (PGJ2), derivatives and analogs thereof. In various embodiments, the cyclopentenone prostaglandin may be selected from the group consisting of prostaglandin A1 (PGA1 ), and prostaglandin A2 (PGA2).

[072] The term “derivative” as used herein refers to a chemical substance related structurally to a parent compound, i.e., a cyclopentenone prostaglandin and may include synthetic and semi-synthetic derivatives. A derivative in the context of the present application comprises the same basic carbon skeleton and functionality as the parent compound, but can also bear one or more substituents or substitutions of the parent compound. The term “analog” as used herein refers to compound that acts similarly (i.e. mimic the action) to the reference compound in the body, binding to the same receptors and eliciting similar biological responses, but it might not be structurally related to the reference compound (i.e. prostaglandins). For example, an analog of prostaglandins, such as PGA1 , may be either capable of modulating Nori or serve as “prodrugs" converted in vivo into a biologically active compound capable of modulating Norf .

[073] In various embodiments, the cyclopentenone prostaglandin is prostaglandin A1 (PGA1 ) or prostaglandin A2 (PGA2) or a derivative, or an analog thereof. In various embodiments, the cyclopentenone prostaglandin is prostaglandin A1 (PGA1 ) or a derivative, or an analog thereof. PGA1 and PGA2 may be termed as agonistic Nori ligands that activate Nori activity.

[074] In various embodiments, the Nori ligand may be prostaglandin E1 (PGE1 ), prostaglandin E2 (PGE2) or a derivative, or an analog thereof, wherein PGE1 and PGE2 are illustrated below:

[075]

[076]

[077] In various embodiments, the Nori ligand may be PGE1 or a derivative, or an analog thereof.

PGE1 may be termed as an antagonistic Nori ligand that inhibits Nori activity.

[078] Furthermore, the present inventors discovered that prostaglandin A1 (PGA1 ) forms a covalent interaction with the Nori -LBD, specifically at Cys594 (position numbering corresponds to SEQ ID NO:1 ), acting as a potential naive ligand. Accordingly, in various embodiments, the Nor 1 ligand may be prostaglandin A1 (PGA1 ) or a derivative, or an analog thereof that covalently binds to Nor1 -LBD, more preferably covalently binds to at least the amino acid residue Cys at the position corresponding to position 594 in SEQ ID NO:1 . In particular, the PGA1 acts as a covalent agonist of Nori , by the covalent interaction with the Cys amino acid residue within the helix 10/1 1 of the Nori - LBD corresponding to positions 580-606 of SEQ ID NO: 1 .

[079] Accordingly, there is provided a method of modulating Nori activity in a cell, comprising administering an effective amount of a prostaglandin or derivative, or an analog thereof to the cell. In various embodiments, the prostaglandin may be PGA1 or a derivative or an analog thereof, that directly binds to the Nori -LBD, more preferably directly binds to one or more amino acid residues in helix 10/1 1 of the Nor1 -LBD, corresponding to positions 580-606 of SEQ ID NO: 1 . In various embodiments, the PGA1 or derivative, or an analog thereof covalently binds to at least the amino acid residue Cys at the position corresponding to position 594 in SEQ ID NO:1 .

[080] As used herein, the terms "modulate," "modulating," and like refer to an increase or decrease of the activity, amount or level of the referenced feature (i.e. Nori activity, insulin secretion, and/or glucose levels) following administration of the prostaglandin or derivative, or an analog thereof, or other suitable Nori ligands, relative to reference (control) activity, amount or level.

[081] In various embodiments, the modulation of Nori may be independent of the DNA binding domain (DBD) or any molecules or compounds directed against, binding to, interacting with, the Nori - DBD. The DBD is the region of Nori that recognizes and binds to specific DNA sequences, and contains two characteristic zinc-finger motifs that facilitate DNA binding (i.e. corresponding to amino acid residues at positions 292-312 and 328-352 of SEQ ID NO:1 ). It will be appreciated that the binding of Nori ligands to the Nor1 -LBD can induce conformational changes in the Nori protein, leading to altered DNA binding affinity or protein-protein interactions. This, in turn, may influence gene expression and cellular processes regulated by Nori .

[082] As used herein, the term "effective amount" and "therapeutically effective amount" of a Nori ligand of the invention refers to a nontoxic but sufficient amount of said Nori ligand to provide the desired effect.

[083] All embodiments disclosed above in relation to the Nori -LBD and Nori ligands similarly apply to the method of modulating Nori in a cell and vice versa.

[084] As used herein, the cell refers to any cell expressing Nori that contains, within its genome, a Nori gene that is expressed to produce the resulting Nori protein comprising at least the LBD. In various embodiments, the cell may be in vitro, ex vivo or in vivo, i.e. the cell may be within a subject.

[085] In various embodiments, the cell may be in vitro and the Nori ligand is administered such that the ligand contacts with the cell, and Nori protein, under suitable conditions, and the cell may be a cell from (or derived from, in case of cell cultures) a multicellular eukaryote such as a human cell line or another mammalian cell line. The mammalian cell lines can include, but are not limited to a human, simian, murine, mice, rat, monkey, rabbit, rodent, hamster, goat, bovine, sheep or pig cell lines. In various embodiments, the cell is from a cell line including, but not limited to Endoc-0H1 , INS- 1 , INS-1 E, MIN6, RIN, PTC, HIT-T15. In various embodiments, the cell may be in vitro and is a cell from a biological sample that has been obtained from, removed or isolated from the subject. The biological sample may be a sample of tissue or cells from the subject, for example, tissue or cells may be obtained and isolated from the pancreas of the subject, more preferably tissue or cells may be obtained and isolated from the islets of Langerhans within the pancreas.

[086] The “contacting” step in the in vitro methods generally refers to any suitable means for delivering, exposing or bringing the Nori and the ligand together in a controlled experimental setting under suitable conditions. As will be appreciated by the skilled person, the "contacting" step can vary depending on the nature of the cells used and Nori ligand involved, whereby the contacting may involve physically mixing the cells with the Nori ligand in a test tube, petri dish or other container, or it could involve applying the Nori ligand to the cells through techniques such as pipetting, incubation, or surface immobilization, or it could involve introducing the Nori ligand into the cells, such as through transfection. The purpose of the "contacting" step is to allow the Nori ligand to interact with the Nori - LBD and exert its modulatory effect. In various embodiments, contacting occurs in a solution in which the cells and Nori ligand are mixed in a common solution and are allowed to freely associate, or the contacting can occur at or otherwise within a suitable cell environment.

[087] In various embodiments, the cell may be in vivo, i.e. the cell may be within a subject, wherein the method further comprises administering to the subject an effective amount of the Nori ligand.

[088] As used herein, the term “subject” is used interchangeably with "individual" and "patient" herein and refers to a warm-blooded animal, preferably a mammal, more preferably a human. Said subject may be awaiting or receiving medical care or is or will become the subject of a medical procedure or is being monitored for the development of any condition or disease associated with aberrant Nori activity. In various embodiments, the subject has a glucose metabolism disorder or condition, selected from the group consisting of: diabetes mellitus, glycosuria, hyperglycemia, hypoglycemia and hyperinsulinism. In various embodiments, the subject has Type 2 mellitus. Tissues, cells and their progeny of a biological entity obtained in vivo or cultured in vitro are also encompassed.

[089] In various embodiments, the cell may be a pancreatic p-cell, more preferably a human pancreatic p-cell. In this regard, the inventors surprisingly discovered that the interaction and binding of PGA1 , as a representative Nori ligand to the Nor1 -LBD, in p-cells, resulted in enhancing Nori 's transcriptional function and stimulating p-cell proliferation and insulin secretion. Accordingly, this indicates a role for Nori in regulating glucose metabolism which offers potential therapeutic opportunities for treating and/or preventing glucose metabolism disorders or conditions. Thus, the modulation of Nori in p-cells through ligand binding, may be used for drug development and treatment strategies related to glucose regulation in a subject.

[090] Accordingly, there is provided a method of modulating insulin secretion from a p-cell, comprising administering an effective amount of a Nori ligand to the p-cell, wherein the Nori ligand directly binds to the ligand binding domain (LBD) of Nori . The invention also covers the use of said Nor 1 ligand in the manufacture of a medicament for modulating insulin secretion from a p-cell.

[091] In various embodiments, the administration of the Nori ligand induces: (i) increased secretion of insulin from the p-cell relative to a p-cell not administered with the Nori ligand; and/or (ii) increased growth of the p-cell relative to a p-cell not administered with the Nori ligand. It will be appreciated that those skilled in the art would readily understand suitable assays and tests known in the art for determining and measuring the secretion level of insulin and growth or proliferation rate of the cell prior to, during, and after the Nori ligand has been administered and bound to the Nori -LBD.

[092] As used herein, the phrase “secretion of insulin from the p-cell” refers to the cellular secretion of insulin from the p-cell that produces and releases insulin. This is in contrast to and distinct from “total insulin secretion” that refers to the sum amount of insulin secreted by all the p-cells. As readily known by those skilled in the art, "total insulin secretion" looks at the broader systemic picture of how much insulin is released into the body as a whole, while "cellular secretion of insulin" relates to biological and molecular mechanisms within individual p-cells that lead to insulin production and release from each p-cell.

[093] As used herein, the phrase “growth of the p-cell” refers to the proliferation, cellular replication and division of the cell, and may also refer to the viability of the cell. The growth of the cell does not refer to an increase in the actual size of the cells. As appreciated by those skilled in the art, the cell growth can be assayed in vitro by plotting cell populations over time. A cell population with a steeper growth curve can said to be growing faster than a cell population with a curve not as steep. Growth curves can be compared for various treatments between the same cell types, or growth curves can be compared for different cell types with the same conditions. Accordingly, there is also provided a method of modulating the growth of a p-cell, comprising administering an effective amount of a Nori ligand to the p-cell, wherein the Nori ligand directly binds to the ligand binding domain (LBD) of Nori . The invention also covers the use of said Nor 1 ligand in the manufacture of a medicament for modulating the growth of a p-cell in a subject.

[094] In various embodiments, the method is in vitro, and the Nori ligand is contacted with the p- cell under suitable conditions to modulate the p-cell insulin secretion, preferably the p-cell is derived from a p-cell line or a biological sample obtained from a subject. [095] In various embodiments, the method is in vivo, the p-cell is in a subject, preferably a mammal, more preferably a human, and the method further comprises administering to the subject the Nori ligand. Accordingly, by modulating insulin secretion from p-cells and/or p-cell growth, the use of Nori ligands, preferably PGA1 or derivatives, or analogs thereof, may improve insulin-deficient symptoms in subjects.

[096] In various embodiments, the Nori ligand directly binds to one or more amino acid residues in helix 10/1 1 of the Nor1 -LBD, corresponding to positions 580-606 of SEQ ID NO: 1 . In various embodiments, the Nori ligand directly binds to one or more amino acid residues at positions corresponding to positions 591 -603 of SEQ ID NO: 1 . In various embodiments, the Nori ligand directly binds to one or more amino acid residues at positions corresponding to positions 594-600 of SEQ ID NO: 1 .

[097] In various embodiments, the Nori ligand directly binds with: (i) the amino acid residue C (Cys) at the position corresponding to position 594 of SEQ ID NO: 1 ; and/or (ii) the amino acid residue T (Thr) at the position corresponding to position 595 of SEQ ID NO: 1 ; and/or (iii) the amino acid residue L (Leu) at the position corresponding to position 596 of SEQ ID NO: 1 ; and/or (iv) the amino acid residue G (Gly) at the position corresponding to position 597 of SEQ ID NO: 1 ; and/or (v) the amino acid residue R (Arg) at the position corresponding to position 600 of SEQ ID NO: 1 . In various embodiments, the Nori ligand covalently binds to at least the amino acid residue Cys at the position corresponding to position 594 in SEQ ID NO:1 .

[098] In various embodiments, the Nori ligand may be a cyclopentenone prostaglandin or a derivative, or an analog thereof. In various embodiments, the cyclopentenone prostaglandin is prostaglandin A1 (PGA1 ) or prostaglandin A2 (PGA2), whereby PGA1/A2 are agonistic Nori ligands that activate Nori and result in the increased secretion of insulin and/or increased cell growth. In various embodiments, the cyclopentenone prostaglandin is prostaglandin A1 (PGA1 ). In various embodiments, the Nori ligand is prostaglandin E1 (PGE1 ), whereby PGE1 is an antagonistic Nori ligand that inhibits Nori and results in the decreased secretion of insulin and/or reduced cell growth.

[099] In various embodiments, the p-cell may be a human p-cell.

[0100] In various embodiments, the method may be carried out in vitro, ex vivo, or in vivo.

[0101] All embodiments disclosed above in relation to the Nori -LBD and Nori ligands similarly apply to the method of modulating insulin secretion from a p-cell and vice versa. [0102] There is also provided a method of modulating glucose levels, preferably blood glucose levels, in a subject, comprising administering to the subject an effective amount of a Nori ligand, wherein the Nori ligand directly binds to the ligand binding domain (LBD) of Nori . The invention also covers the use of said Nor 1 ligand in the manufacture of a medicament for the treatment or prevention of a disease, disorder, or condition associated with modulating glucose levels.

[0103] In various embodiments, the subject may have a glucose metabolism disorder or condition, or the subject may be at risk of developing a glucose metabolism disorder or condition. Accordingly, the method of modulating glucose levels, through modulating insulin secretion levels, in the subject may also be for treating or preventing a glucose metabolism disorder or condition in the subject. The glucose metabolism disorder or condition may be selected from the group consisting of diabetes mellitus, glycosuria, hyperglycemia, hypoglycemia and hyperinsulinism.

[0104] As used herein, the terms "treating" and "treatment" refer to reduction in severity and/or frequency of symptoms, elimination of symptoms and/or underlying cause, prevention of the occurrence of symptoms and/or their underlying cause, and improvement or remediation of damage. As used herein, the term “preventing” refers to the prophylactic or preventative measures that prevent and/or slow the development of a targeted pathologic condition or disorder. Thus, those in need of treatment include those already with the disorder; those prone to have the disorder; and those in whom the disorder is to be prevented and those in whom reoccurrence of the disorder needs to be prevented. In various embodiments, the terms “treating”, “ameliorating”, “delaying” or “preventing”, as used herein refer to achieving one or more of the following in the subject: (a) reducing the severity of a given condition; (b) limiting or preventing the development of a condition; (c) removing a given condition; (d) limiting or preventing the recurrence of a given condition; (e) alleviation of the condition and/or its symptoms; and (f) delay the onset of a condition. Any one or more of these effects may be achieved in a subject who previously had or currently has or is suspected to develop a glucose metabolism disease or condition. In particular, the therapeutic terms “treating, ameliorating or preventing”, may refer to reducing the likelihood of a particular condition or disease state from occurring in a subject not presently experiencing or afflicted with the condition or disease state. The terms do not necessarily indicate complete or absolute prevention or treatment.

[0105] Type 2 diabetes mellitus (T2D) is a metabolic disorder that shows elevated blood glucose level due to insulin resistance and deficiency in secretion. As of 2014, over 422 million people were diagnosed with T2D and 1 .6 million patients died through direct causality, making this as the seventh leading cause of death (IDF 2017). Pathophysiology of diabetes is governed by the master regulator insulin, a hormone that is produced in pancreas. Over the years, understandings on the insulin secretion pathway(s) and its signalling mechanism have led to various anti-diabetic medications such as sulfonylureas, metformin, sitagliptin, incretin mimetics, etc. and non-medicinal treatments including bariatric surgery and insulin pumps. Prolonged usage of these drugs often becomes ineffective resulting in side-effects including weight-gain, risk of heart failure, liver-, kidney-malfunction, lactic acidosis, etc., and it is also evident that some of their earlier versions have been contraindicated or withdrawn from the market.

[0106] Accordingly, in various embodiments, the glucose metabolism disorder or condition may be diabetes mellitus, and more preferably Type 2 diabetes mellitus (T2D), and the method disclosed herein may be for treating or preventing diabetes mellitus, and more preferably Type 2 diabetes mellitus (T2D). In various embodiments, the subject may have been determined to display a poor insulin expression profile, which is commonly shown in pre-diabetic subjects, and also for those subjects with non-autoimmune p-cell degeneracy. Accordingly, by modulating, preferably increasing, insulin secretion and/or p-cell growth, the use of Nori ligands, preferably PGA1 or derivatives or analogs thereof, may improve insulin deficient symptoms in subjects with T2D.

[0107] A Nori ligand of the invention may be administered in the form of a salt, ester, amide, prodrug, active metabolite, analog, or the like, provided that the salt, ester, amide, prodrug, active metabolite or analog is pharmaceutically acceptable and pharmacologically active in the present context. Salts, esters, amides, prodrugs, active metabolites, analogs, and other derivatives of the Nori ligands may be prepared using standard procedures known to those skilled in the art of synthetic organic chemistry and described, for example, by J. March, Advanced Organic Chemistry: Reactions, Mechanisms and Structure, 4th Ed. (New York: Wiley-lnterscience, 1992). "Pharmacologically active" (or simply "active") as in a "pharmacologically active" derivative or analog, refers to a derivative or analog having the same type of pharmacological activity as the parent compound and approximately equivalent in degree.

[0108] Prior to being used in the treatment or prevention in vivo, pharmaceutical formulations composed of the Nori ligand in association with a pharmaceutically acceptable carrier may need to be formulated. See Remington: The Science and Practice of Pharmacy, 19th Ed. (Easton, Pa.: Mack Publishing Co., 1995), which discloses typical carriers and conventional methods of preparing pharmaceutical formulations.

[0109] Depending on the intended mode of administration, the pharmaceutical formulation may be a solid, semi-solid or liquid, such as, for example, a tablet, a capsule, caplets, a liquid, a suspension, an emulsion, a suppository, granules, pellets, beads, a powder, or the like, preferably in unit dosage form suitable for single administration of a precise dosage. Suitable pharmaceutical compositions and dosage forms may be prepared using conventional methods known to those in the field of pharmaceutical formulation and described in the pertinent texts and literature, e.g., in Remington: The Science and Practice of Pharmacy, cited above. [0110] The Nori ligand of the present invention may be administered orally, parenterally, rectally, vaginally, buccally, sublingually, nasally, by inhalation, topically, transdermally, or via an implanted reservoir in dosage forms containing conventional non-toxic pharmaceutically acceptable carriers and excipients. The term "parenteral" as used herein is intended to include subcutaneous, intravenous, and intramuscular injection. The amount of the Nori ligand administered will, of course, be dependent on the particular active agent, the condition or disorder being treated, the severity of the condition or disorder, the subject's weight, the mode of administration and other pertinent factors known to the prescribing physician.

[0111] Accordingly, there is also provided the use of a Nori ligand, preferably PGA1 , as a medicament or pharmaceutical composition to be administered to a subject. It will also be appreciated that there is also provided the use of a Nori ligand in the manufacture of a medicament or pharmaceutical composition, for use in the methods disclosed herein, and particularly for the treatment or prevention of a glucose metabolism disorder or condition in the subject.

[0112] As will be appreciated, the structural conformation of the Nor1 -LBD, as identified and disclosed herein, may be used for the discovery and identification of Nori ligands (agonists and antagonists for Nori ) for subsequent examination and investigation of interactions with the Norf -LBD, whereby such Nori ligands may be used in the methods disclosed herein.

[0113] in silica screening methods may be used to identify ligands that may be useful in the methods disclosed herein. Accordingly, the elucidated structure of Nor1 -LBD may be useful in methods for screening and identifying potential Nori ligands that are capable of enhancing or inhibiting Nori activity. In this context, the “screening" refers to the testing of synthetic chemical and natural databases (libraries) to discover compounds that bind to the Nori -LBD, and may serve as new therapeutic drug leads.

[0114] Accordingly, there is also provided a computer-assisted method for screening, identifying or designing a candidate molecule or compound that modulates Nori activity, the method comprising the steps of: a) providing the structure of Nori -LBD having the amino acid sequence set forth in SEQ ID NO:4 or variants thereof, wherein the structure of Nor1 -LBD includes the following helical structures and their corresponding amino acid residues of SEQ ID NO: 4 or variants thereof: Helix 1 (H1 : residues 397-407), Helix 3 (H3: residues 430-454), Helix 4,5 (H4/5: residues 463-485), Helix 6 (H6: residues 504-510), Helix 7 (H7: residues 512-527), Helix 8 (H8: residues 531 -542), Helix 9 (H9: residues 552-572), Helix 10,1 1 (H10/1 1 : residues 580-606), and Helix 12 (H12: residues 614-622); b) providing the structure of the candidate molecule or compound; c) fitting the structure of the candidate molecule or compound to the Nori -LBD, wherein fitting comprises determining interactions between one or more atoms of the candidate molecule or compound and one or more atoms of Nori - LBD, to predict whether the candidate molecule or compound binds to or within Nor1 -LBD; and d) selecting the candidate molecule or compound if it is predicted to bind to or within Nor1 -LBD.

[0115] In various embodiments, the candidate molecule or compound predicted to bind to or within Nori -LBD, may be used in any of the methods disclosed herein, i.e. in vitro, ex vivo or in vivo methods of use disclosed herein.

[0116] Structure-based compound design and identification methods are powerful techniques that can involve searching virtual chemical libraries (i.e. computer databases) containing a wide variety of potential modulators and chemical functional groups and fitting them into the active site or putative binding site of the protein structure. The term "fitting" as used herein can mean determining, by automatic or semi-automatic means, interactions between at least one atom of the candidate molecule/compound and at least one atom of the Nor1 -LBD and calculating the extent to which such an interaction is stable. Interactions can include attraction, repulsion, brought about by charge, steric considerations, and the like. The computerized design and identification of Nori ligands is useful as the virtual chemical libraries contain more molecules/compounds than the real chemical libraries, often by an order of magnitude. For reviews of structure-based drug design and identification (see Kuntz et al. (1994), Acc. Chem. Res. 27:1 17; Guida (1994) Current Opinion in Struc. Biol. 4: 777; Colman (1994) Current Opinion in Struc. Biol. 4: 868).

[0117] One method of rational design searches for Nori ligands by docking the computer representations of molecules/compounds from a virtual chemical library. Publicly available virtual chemical libraries include, with limitation, the following:

(a) ACD from Molecular Designs Limited;

(b) NCI from National Cancer Institute;

(c) CCDC from Cambridge Crystallographic Data Center;

(d) CAST from Chemical Abstract Service;

(e) Derwent from Derwent Information Limited;

(f) Maybridge from Maybridge Chemical Company LTD;

(g) Aldrich from Aldrich Chemical Company; and/or

(h) Directory of Natural Products from Chapman & Hall.

[0118] One such virtual chemical library (e.g. ACD) contains compounds that are synthetically derived or are natural products. Methods available to those skilled in the art can convert a data set represented in two dimensions to one represented in three dimensions. These methods are enabled by such computer programs as CONCORD from Tripos Associates or DE-Converter from Molecular Simulations Limited. Multiple methods of structure-based modulator design are known to those in the art (Kuntz et al., (1982), J. Mol. Biol. 162: 269; Kuntz et aZ., (1994), Acc. Chern. Res. 27:1 17; Meng et al., (1992), J. Compt. Chem. 13:505; Bohm, (1994), J. Comp. Aided Molec. Design 8: 623). [0119] A computer program widely utilized by those skilled in the art of rational modulator design is DOCK from the University of California in San Francisco. The general methods utilized by this computer program and programs like it are described below. More detailed information regarding some of these techniques can be found in the Accelerys User Guide, 1995. A typical computer program used for this purpose can perform a process comprising the following steps or functions:

(a) removing the existing compound from the protein;

(b) docking the structure of another compound into the active-site or putative binding site using the computer program (such as DOCK) or by interactively moving the compound into the active-site or putative binding site;

(c) characterizing the space between the compound and the atoms of the active-site or putative binding site;

(d) searching libraries for (i) compounds can fit into the empty space between the compound and the active-site or putative binding site, or (ii) molecular fragments which can be assembled to generate a candidate compound for further evaluation.

[0120] Part (c) refers to characterizing the geometry and the complementary interactions formed between the atoms of the active site or putative binding site and the compounds. A favourable geometric fit is attained when a significant surface area is shared between the compound and activesite atoms without forming unfavourable steric interactions. One skilled in the art would note that the method can be performed by skipping part (d) and screening a database of many compounds. Other methods of structure-based modulator design are reported in the literature and can be used, e.g.:

(1 ) CAVEAT: Bartlett et al., (1989), in Chemical and Biological Problems in Molecular Recognition, Roberts, S. M.; Ley, S. V.; Campbell, M. M. eds.; Royal Society of Chemistry: Cambridge, pp. 182- 196.

(2) FLOG: Miller et al., (1994), J. Comp. Aided Molec. Design 8:153.

(3) PRO Modulator: Clark et al., (1995), J. Comp. Aided Molec. Design 9:13.

(4) MCSS: Miranker and Karplus, (1991 ), Proteins: Structure, Function, and Genetics 1 1 :29.

(5) AUTODOCK: Goodsell and Olson, (1990), Proteins: Structure, Function, and Genetics 8:195.

(6) GRID: Goodford, (1985), J. Med. Chem. 28:849.

[0121] Another way of identifying compounds as potential Nori ligands is to modify an existing Nori ligand. For example, the computer representation of modulators can be modified within the computer representation of a prostanoid or a derivative, or an analog thereof, PGA1 or PGA2 in particular. Detailed instructions for this technique can be found, for example, in the Accelerys User Manual, 1995 in LUDI. The computer representation of a Nori ligand may be typically modified by the deletion of a chemical group or groups or by the addition of a chemical group or groups. In various embodiments, the candidate molecule/compound is a prostanoid or a derivative, or an analog thereof, preferably a cyclopentenone prostaglandin or derivative, or analog thereof. [0122] Upon each modification to the compound, the atoms of the modified compound and active site or putative binding site can be shifted in conformation and the distance between the Nori ligand and the active-site atoms may be scored along with any complementary interactions formed between the two molecules. Scoring can be complete when a favourable geometric fit and favourable complementary interactions are attained. Compounds that have favourable scores are potential Nori ligand.

[0123] In various embodiments, the step of fitting comprises predicting if the candidate compound binds to or within one or more amino acids in Helix 10,1 1 in the ligand binding domain of Nori corresponding to positions 580-606 of SEQ ID NO: 1 . Without wishing to be bound to any theory, it is believed that the potential Nori ligands may couple covalently to Nori , preferably to the amino acid residue Cys594 of Nori . In various embodiments, the candidate compound is predicted to bind to amino acid residue C (Cys) at the position corresponding to position 594 of SEQ ID NO: 1 . Alternatively, the potential Nori ligand may interact with Nori -LBD noncovalently.

[0124] In various embodiments, the Nor1 -LBD has the amino acid sequence set forth in SEQ ID NO:4, and the structure of the Nor1 -LBD is derived from, or characterized, by one or more of the distance constraints, dihedral constraints and structural statistics described in Table 4. In this regard, candidate molecules/compounds that satisfy the distance and dihedral constraints, without steric clashes may be ranked according to their predicted binding affinity.

[0125] As will be appreciated by those skilled in the art, the distance constraints can be used to develop a model of the binding domain and binding pockets therein. By knowing the exact distances between key residues in the binding domain, one can predict which candidate compound might fit within the pocket and make favorable contacts with the binding domain. The dihedral angles define the overall fold of the binding domain. Knowledge of the preferred dihedral angles can be used to predict the active conformation of the binding domain that a ligand would encounter. This information is important to ensure that potential ligands are screened against a conformationally relevant structure.

[0126] In conjunction with, or separate from, the computer-based method of virtual screening and/or rational design of Nori -LBD ligands, “wet” assay screening methods may also be employed. As large virtual chemical libraries of compounds can be searched in a matter of hours or even less, the computer-based methods can narrow the compounds tested as potential Nori -LBD ligands and verify their binding to Nor1 -LBD (e.g. ion exchange chromatography and mass spectrometry) and test their function in biochemical or cellular in vitro assays (e.g. luciferase reporter assay and/or glucose- stimulated insulin secreted (GSIS) assay, cell viability assays), and suitability for use in methods disclosed herein.

[0127] Accordingly, there is also provided a method of screening and identifying a ligand that modulates insulin secretion from a 0-cell, the method comprising the steps of: a) administrating to such assay system (i.e. GSIS assay) an effective amount of a candidate molecule or compound, sufficient to induce or reduce the secretion of insulin in said assay system; and b) comparing the measured level of insulin secretion with a reference Nori ligand and/or negative control (i.e., 0-cell not expressing Nori ), wherein if insulin secretion is increased, the candidate molecule or compound is determined as an agonist of Nori , or if insulin secretion is decreased, the candidate molecule or compound is determined as an antagonist of Nori .

[0128] In various embodiments, a molecule or compound identified or designed by the screening methods disclosed herein may be used in in vitro, ex vivo or in vivo methods of use disclosed herein as a Nori ligand.

[0129] In various embodiments, the candidate molecule or compound is selected from a virtual chemical library or is a cyclopentenone prostaglandin or a derivative, or an analog thereof.

EXAMPLES

Materials and Methods

[0130] Biochemicals: Ni²⁺-NTA agarose resins were purchased from Qiagen (Hilden, Germany). Isopropyl-thiogalactoside (IPTG) and Dithiothreitol (DTT) were purchased from Gold Biotechnology, Inc. (MO- USA). Chemicals for SDS-PAGE were obtained from BioRad (CA- USA). Isotopic chemicals used for NMR experiments such as D20, ¹⁵NH4CI and ¹³C D-glucose were purchased from Cambridge Isotope Laboratories (MA- USA). All other chemicals were at analytical grade and were obtained from Merck (Darmstadt, Germany).

[0131] Cloning of Nor1-LBD₃79-626: Nor1 -LBDs79-626 expressing vector, pSV40Nor1 LBD was kindly provided by Prof. Kim Kwang-Soo from Harvard Medical School (MA- USA). Nor1 -LBDs79-626 region was amplified using primer sequence. The PCR product incorporating Xbal and Hindlll restriction sites were digested and ligated to pE-SUMOstar vector (Life Sensors, PA-USA) with Kanamycin as the antibiotic selection marker.

[0132] Over-expression of Nor1-LBD₃79-626 protein: Recombinant Nor1 -LBDs79-626 was grown using Escherichia coli BL21 (DE3) cell in Lysogeny broth (LB) media with Kanamycin (30 pg/mL) incubated at 37 °C. The protein was over expressed with 1 mM of isopropyl-0-D-thio-galactoside (IPTG) and was incubated further overnight at 18 °C. Cells were then harvested from the grown culture by centrifugation at 8000 x g for 10 minutes and the pellet was frozen in -80°C for future use. The uniformly ¹⁵N- and ¹⁵N/¹³C-labelled Nori -LBD379-626 was expressed in Escherichia coli BL21 (DE3) using M9 minimal media containing either ¹⁵NH4CI (1 g/L) or both ¹⁵NH4CI (1 g/L) and [U-¹³C]- Glucose (2.5 g/L). Growth and over-expression of the recombinant protein protocol are same as described above. Nor1 -LBDs79-626 recombinant cells were inoculated to 50 mL M9 minimal media with Kanamycin as selection antibiotic and grown overnight at 37 °C. The cells were harvested at 3500 x g, suspended to M9 minimal media with 85 % D2O and further grown at 37 °C for 8 h. Five mL of the culture was transferred to 50 mL of M9 minimal media containing ¹⁵NH4CI (1 g/L), [U-¹³C]-glucose (2.5 g/L) in D2O and further grown overnight. The culture was then up-scaled to one L and grown at 37 °C until an optical density ODeoo of 0.6 was reached. 50 mg/L of ¹³C alpha-ketobutyrate and 85 mg/L of ¹³C alpha-ketoisovalerate were along with 1 mM IPTG for the over-expression of the recombinant protein.

[0133] Purification of No 1-LBD₃₇9-626: Nor1 -LBD379-626 recombinant cells were suspended in buffer (50 mM Tris-HCI, pH 8.0, 150 mM NaCI) and was lysed on ice by sonicating them at 25 % power for 30 min. Lysate was centrifuged at 40,000 x g for 30 minutes at 4 °C. Supernatant was added to the equilibrated Ni²⁺-NTA resin and was incubated for one hour at 4 °C. Recombinant protein was eluted using imidazole gradient (20 - 500 mM) in lysis buffer. Fractions containing His-SUMO tagged Nor1 - LBD379-626 protein were identified on SDS-PAGE and concentrated using Amicon® Ultra-15 Centrifugal Filter (Darmstadt, Germany). Imidazole was removed on PD10 desalting column (GE Healthcare, CT) as per the manufacturer’s protocol. To cleave Nor1 -LBD379-626 recombinant protein from the SUMO tag, one unit of SUMO protease was added per 20 g of protein sample and was incubated overnight at 18 °C. Free His-SUMO tag suspended in the solution were allowed to bind on to fresh Ni²⁺-NTA resin and pure protein was collected in flow-through. Samples were further purified using size exclusion column (Superdex 200 10/300 GL). Respective samples for NMR studies were also prepared in the same protocol.

[0134] Cloning, expression, and purification of Cys-Mutants of Nor1-LBD: Point mutation on Nor1 -LBD₃79-626 (Cyss94 to Ser) was performed using Q5® Site-Directed Mutagenesis Kit (NEB, MA- USA). Point mutation of Cys397, 420, 496, soe, 536, 559, 594 to Alanine were performed by Bio Basic (Markham, ON-Canada). All cloned constructs of Nor1 -LBD379-626 were transformed, over-expressed and the respective protein were purified as per the above-mentioned protocol.

[0135] Luciferase reporter assay: Luciferase reporter assay was performed using the Dual-Glo® Luciferase kit ordered from Promega (Madison, Wl). Cells (1 x 10⁴ ) were seeded into a 96-well transparent plate and incubated for 24 hours at 37 °C, 5 % CO2. 24-hour post-seeding, the cells were transfected with three plasmids (expression vector: pSF-SV40-GAL4 DBD-DHR38 LBD; firefly vector and renilla vector) and treated with drugs 6h post transfection and lysed using 75 pl of 1X Luciferase Assay Reagent (LAR) provided in the Dual-Glo® Luciferase kit Promega (Madison, Wl), together with 1X PBS buffer (150 pl/well), 24 h post transfection, and transferred to a Greiner CELLSTAR® 96-well white flat bottom polystyrene plate. The plate was then incubated for 10 minutes at room temperature on the shaker. The measurement was then taken using Tecan Infinite® 200 Pro or Tecan Safire2 microplate readers (Mannedorf, Switzerland). The signal integration period was set to 10-seconds. This step determines the luminescence measured in relative light units (RLU) for the firefly luciferase. Then, 75 pl of Dual- Gio® Stop & Gio® reagent was added into each well and incubated for 10 minutes before determining the RLU for the Renilla luciferase (with the same parameters as before). The averaged reading for the wells corresponding to non-transfected cells was first subtracted from each firefly luminescence reading and the triplicate readings were then averaged. Normalization of the luminescence reading for each well was carried out by dividing the firefly luciferase RLU with the corresponding Renilla luciferase RLU. Each drug-treated well was then normalized against the averaged DMSO-treated well reading.

[0136] NMR data collection and processing: Isotopically labeled Nor1 -LBD protein used in solution NMR spectroscopy were prepared in buffer containing 20 mM NaPCk, pH 7.4, 150 mM NaCI, 0.01 % NaNs, and 10 % D2O. All experiments were performed at 298 K. ²H/¹³C/¹⁵N-labeled protein sample were used to acquire HNCACB, HN(CO)CACB, HNCA, HN(CO)CA, HNCO, HN(CA)CO, TOCSY-HCCH, CCH-TOCSY, and ¹³C-NOESY spectra on Bruker Avance III 700 MHz equipped with 5 mm z-gradient TXI cryoprobe and Bruker Avance III HD 800 MHz with a 5 mm QCI H/P/C/N CryoProbe. All the 2D and 3D experiments made use of pulsed -field gradients for coherence selection and artifact suppression and utilized gradient sensitivity enhancement schemes. Quadrature detection in the indirectly detected dimensions was achieved using either the States/TPPI (timeproportional phase incrementation) or the echo/anti-echo method. Baseline corrections were applied where-ever necessary. All NMR data were processed using Bruker Avance Spectrometer in-built software Topspin v2.1 (Bruker, MA). Peak-picking and data analysis of the Fourier transformed spectra were performed using SPARKY-NMRFAM <⁸⁾.

[0137] Relaxation experiments for Nor1-LBD₃79-626: T , 77 and Heteronuciear NOE (HNOE) spectra for the Nor1 -LBD₃79-626 protein were recorded with spectral widths of 9615.85 Hz sampled over 2048 complex points in the ate f H) dimension and 2405.12 Hz over 128 complex points in the (¹⁵N) dimension with 16 scans for each increment in the indirect dimension. Relaxation delay for Ti and 77 measurements was 3.0 s. For 77 measurements, spectra were collected with relaxation delays of 50, 100, 300, 500, 800, 1000, 1200, 1600, 2000, 2500 ms. Experimental data for Ta were acquired with delays of 7.2, 14.4, 21 .6, 28.8, 36, 43.2, 50.4 and 57.6 ms. The delay between 180 pulses in the Carr-Purceii-Meiboom-Giii (CPMG) pulse train for Ta measurements was fixed at 0.9 ms. A total of 2048 complex data points with 128 complex increments were collected for the relaxation experiments. The relaxation rate constants were determined by fitting the cross-peak intensities to a mono exponential function.

[0138] Solution structure calculation for Nor1-LBD₃79-626: Chemical shift resonance data were used to calculate backbone dihedral angular constraints (phi, psi) using TALOS-N⁽¹⁰⁾. The PONDEROSA-CS software, which provides the AUDANA (Automated Database-Assisted NOE Assignment) algorithm, was used to do the initial automated assignment ⁽⁹⁾. Once the initial structure was generated, the NOE assignment was manually finished from identified cross-peaks in the ¹⁵N- and ¹³C-NOESY spectra. Subsequent structure calculations were done using the XPLOR-NIH⁽¹¹⁾. Iterations were repeated until a satisfactory structure was generated. Within the process, distance restraints were generated with each iteration. Cross-peaks on NOESY spectra were converted to distance restraint using the using sum over r⁶ calculation. Hydrogen bond restraints were obtained based on the helical formations from the structure iterations. Once all restraints were finalized, the last calculation of 100 conformers was performed along with an explicit water refinement by XPLOR- NIH. Finally, 20 structures were selected by their total energy values for display and structural analysis using Molmol ⁽¹²⁾ and Pymol ⁽¹³⁾. Structural validation was done using Protein Structure Validation Suite ⁽¹⁴⁾ and MolProbity ⁽¹⁵⁾.

[0139] Detection of ligand bound Nor1-LBD: Purified Nor1 -LBD proteins were incubated with prostaglandin A1 or E1 in 1 :3 molar ratio and incubated overnight. Afterward, samples were run through RESOURCE Q anion exchange chromatography column (Cytiva, MA) to separate the ligand bound and unbound Nor1 -LBD. Peaks by chromatography were gathered and analyzed by LC- MS/MS molecular weight determination. For NMR-based analysis, the ¹⁵N labeled No1 -LBD was titrated with different concentrations of PGA1 . The chemical shift perturbations were analyzed before and after the addition of apicidin. Weighted average of the ¹ H and ¹⁵N chemical shift changes, 5¹H and 5¹⁵N, respectively, were calculated with the function of the form, 6 = [(6¹H)²+ (6¹⁵N/6.51 )²]^1/2.

[0140] Cell viability assay: For determining the effective and nontoxic amount of drug to INS-1 E cells, the cell viability assay was performed. Cells were seeded by 25000 per well to 96-well transparent plate (Nunc, NY) and incubated overnight until 80% cell confluency. Varying concentrations of ligands were added and incubated for 24 hours or 48 hours. All media were removed and supplemented with 100 pl of fresh media and 10 pl of WST-1 Cell Proliferation Reagent (Roche, Basel), which were then further incubated for 30 minutes. After visually confirming the change of media color from pink to orange, the absorbance at 450 nm in reference to 600 nm.

[0141] Glucose-stimulated insulin secretion assay: For assessing the amount of insulin secreted from INS-1 E cells when exposed to high glucose levels, the GSIS assay was performed. 750000 cells/well were seeded to a 6-well culture plate and incubated overnight to achieve 80% confluency. Varying concentrations of ligands were added and incubated for 24 or 48 hours. Media in each well were replaced with 2.2 mM glucose-supplemented KRB buffer and incubated for 30 minutes. Media was removed afterward, replaced with 1 1 .0 mM glucose-supplemented KRB buffer, and incubated for 1 hour to trigger insulin secretion. Media was collected, centrifuged to remove suspended cells, and kept as an insulin secretion sample. The plated cells were subjected to acid-ethanol insulin extraction to assess insulin content within cells. 1 mL of 1 .5% HCI in 70% ethanol was added to each well and was incubated at 4°C overnight. The content of the well was moved to a 1 .6 mL Eppendorf tube and centrifuged at 14000 x g for 30 minutes. The supernatant was recollected and neutralized with a 1 :1 volume ratio of 1 M Tris pH 8.0 buffer. All samples were kept at 4°C until concentration measurement. Collected samples from INS-1 E cells were measured for their insulin content using an Insulin Rat ELISA Kit (Thermo Fisher Scientific, MA) or Insulin AlphaLISA Detection Kit (Perkin Elmer, MA). The concentration of the secreted insulin was divided by respective cell extracted insulin to obtain a normalized insulin secretion measurement. For GSIS assay in pancreatic islet cells, primary beta-cell islets were procured from healthy C57BL/6 mice. Briefly, HBSS dissolved 1 mg/ml collagenase type V (Sigma, MO) solution was perfused into the pancreas of a euthanized mouse after clamping the common bile duct. The removed whole pancreas was further subjected to collagenase digestion for 7 minutes in 37°C conditions until sieved and further isolated using histopaque 1077 (Sigma, MO) gradient. Islets were assessed by their shape and clarity by microscopic evaluation and further incubated in prepared media overnight for recovery. GSIS assay was performed as described above. All animal studies were approved by the CHA University Institutional Animal Care and Use Committee (IACUC2201 10).

[0142] Gene expression knockdown: siRNA transfection was performed to efficiently lower the expression of specific genes in INS-1 E cells. 750000 cells/well were seeded to a 6-well culture plate and incubated overnight to achieve 80% confluency. DsiRNA(rn.Ri.Nr4a3.13.1 ) was transfected in varying concentrations using Lipofectamine 3000 (Life Technologies, CA) and was incubated for 24 hours.

[0143] mRNA quantification by quantitative PCR: For measuring knockdown efficiency, real-time quantitative PCR was performed. Total mRNA was extracted by acid guanidinium thiocyanate- phenol-chloroform extraction using TRIzol reagent (Invitrogen, CA). The concentration of mRNA was measured using NanoDrop 1000 spectrophotometer. 5pg of total mRNA was reverse transcripted into cDNA using oligo-dT and reverse transcriptase. 10 pM of each primer and 20 ng of cDNA with 1x SYBR Green PCR Master Mix (Life Technologies, CA) were added to each well in the Framestar 96, semi-skirted PCR plate (4titude, Surrey). The plate went through 30 cycles of PCR amplification using the CFX96 Touch Real-Time PCR Detection System. The melt curve was analyzed by calculating AACq to quantify the knockdown efficiency.

Results

Example 1: Structural description of the Nor1-LBD

[0144] Initially, it was attempted to determine the Nor1 -LBD structure by employing X-ray crystallography. Surprisingly, unlike Nurrl and Nur77, the Nor1 -LBD exhibited poor crystallization properties, while it behaved well in solution and was suitable for structure studies by NMR. Thus, by NMR spectroscopy, for the first time the Nor1 -LBD structure has been determined.

[0145] Protein structures were calculated based on a total of 2,150 nontrivial NMR-derived distance constraints for Nor1 -LBD with the program PONDEROSA-CS⁽⁹⁾. FIG. 1A-C shows the ensemble of 20 low-energy structures. Excluding 19 residues in the N-terminal flexible region, the root-meansquare deviation (RMSD) for the Nori -LBD about the mean position is 1 .02 A for the backbone and 1 .63 A for all heavy atoms. The overall structure of the Nori -LBD shows a triple layer of nine helices: H1 (residues 397-D407), H3 (residues 430-454), H4,5 (residues 463-485), H6 (residues 504-510), H7 (residues 512-527), H8 (residues 531 -542), H9 (residues 552-572), H10,1 1 (residues 580-606), and H12 (residues 614-622), which are conserved not only in the NR4A family but also in other well- studied NRs.

[0146] NMR titration study performed on the calculated structure of Nori -LBD showed a specific site around Cys594 in which PGA1 binds to, and thus confirms molecular interaction. More specifically, a region of residues of C594, T595, L596, G597 and R600 (side chains shown) on helix-10/1 1 showed significant perturbation, suggesting the region of ligand-binding (FIG. 1C).

Example 2: Screening of Nori ligands

[0147] The availability and determination of the Nor1 -LBD structure enabled the screening of potential naive ligands of Nori .

[0148] To this aim, a luciferase reporter assay was employed, whereby the reporter assay results showed that PGA1 among the tested prostaglandins notably activates Nori ’s transcriptional function (FIG. 2A). The demonstrated dose-dependent response of PGA1 -mediated Nori activation was further shown to not be affected by the presence or lack of the DBD region (FIG. 2B-C).

Example 3: Ligand Binding to Nori -LBD

[0149] To understand the molecular basis of ligand binding, the purified Nor1 -LBD was incubated with PGA1 and analyzed the mixture on anion-exchange chromatography. The ion exchange elution profile of the incubated sample detected an elution peak that contains PGA1 -Nori -LBD complex. The collected sample was concentrated and analyzed with mass spectrometry (MS). The MS data indicated that the molecular weight of the new species was the sum of Nor1 -LBD and PGA1 , suggesting that PGA1 is covalently bound to Nori -LBD (FIG. 3). In particular, PGA1 forms a covalent interaction with the Nori -LBD, specifically at Cys594, acting as a potential naive ligand.

Example 4: Cyclopentenone prostaglandins biological role as a ligand of Nori

[0150] Human beta cells (EndoC-0H1 ) showed accelerated growth with PGA1/A2 treatment, which agrees with increased mRNA expression of cell cycle genes from PGA1/A2 pretreated EndoC-0H1 cells. In particular, PGA1/A2 treatment showed enhanced cell growth over time in a dose dependent manner, whereby cells treated with PGA1 /A2/E1 were observed for 4 days and assessed by WST-1 assay to quantify viability (FIG.4A-B). Subsequently, it was demonstrated that PGA1 and PGA2 treatment can significantly increase cell growth compared to PGE1 , which indicates an inhibitory effect (FIG.4B).

[0151] Human beta cells (EndoC-0H1) were used to evaluate if the Nor-1 ligands treatments influence insulin expression enhancement and improve cell survival. The incretin signaling pathway as previously described ⁶' was employed, whereby the insulin-secreting EndoC-0H1 cells were shown to be responsive to PGA1 /A2 treatment by dose-dependently increasing glucose-stimulated insulin secretion (GSIS) (FIG. 5A and 5B). [0152] It was then examined if PGA1 -mediated insulin secretion is specific to Nori signaling. To address this, siRNAs were designed against the NR4A members (NR4A1 , NR4A2, NR4A3) and siRNA-mediated knockdown studies were performed targeting each of the NR4A members (FIG. 6). The results showed that only NR4A3-knock-downed cells abolished PGA1 -mediated insulin secretion enhancement, suggesting that PGA1 selectively functions through Nori signaling, whereas NR4A1 and A2-knock-downed cells remain unaffected.

[0153] The effect of PGA1 -mediated enhancement of insulin production was compared with currently available secretagogues. While PGA1 , IBMX, Ex-4 (exendin-4), and nateglinide (Nate) all showed enhanced production of overall insulin (FIG. 7A), PGA1 excelled in increasing the cellular secretion of insulin compared to all other drugs (FIG. 7B).

[0154] Similar results were also obtained in primary beta cells isolated from C57BL/6 mice (FIG. 8), corroborating that the PGA1 and related PGs may provide ways to treat diabetes as alternative insulin secretagogues by activating Nori function.

[0155] Taken together, these results corroborate that the cyPG may provide alternative ways to treat diabetes as alternative insulin secretagogues by activating Nori function over currently available diabetes medications. In particular, these results suggest the use of PGA1/A2 and their derivatives as treatment for T2D, especially for those patients who display poor insulin expression profile, which is commonly shown in prediabetic patients, and also for those with non-autoimmune beta cell degeneracy. Based on these results on increased insulin secretion and beta cell protective effect, PGA1 /A2 and their derivative molecules can be expected to improve insulin-deficient symptoms in T2D patients.

[0156] The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein. Other embodiments are within the following claims.

[0157] One skilled in the art would readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. Further, it will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. The methods and uses described herein are presently representative of preferred embodiments are exemplary and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the invention are defined by the scope of the claims. The listing or discussion of a previously published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

[0158] The invention illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, it should be understood that although the present invention has been specifically disclosed by exemplary embodiments and optional features, modification and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

[0159] The content of all documents and patent documents cited herein is incorporated by reference in their entirety.

References:

1. Stienstra, R., Lichtenauer-Kaligis, E. & Muller, M. Stress- (and Diet-) Related Regulation of Hepatic Nuclear Receptors and Its Relevance for ABC-Transporter Functions. Drug Metabolism Reviews 36, 391 -406 (2004).

2. Taubenheim, J., Kortmann, C. & Fraune, S. Function and Evolution of Nuclear Receptors in Environmental-Dependent Postembryonic Development. Front Cell Dev Biol 9, 653792 (2021 ).

3. Mangelsdorf, D. J. et al. The nuclear receptor superfamily: the second decade. Cell 83, 835- 839 (1995).

4. Novac, N. & Heinzel, T. Nuclear receptors: overview and classification. Current Drug Targets- Inflammation & Allergy 3, 335-346 (2004).

5. Kliewer, S. A., Lehmann, J. M. & Willson, T. M. Orphan nuclear receptors: shifting endocrinology into reverse. Science 284, 757-760 (1999).

6. Gallastegui, N., Mackinnon, J. A., Fletterick, R. J. & Estebanez-Perpina, E. Advances in our structural understanding of orphan nuclear receptors. Trends in biochemical sciences 40, 25-35 (2015).

7. Giguere, V. Orphan nuclear receptors: from gene to function. Endocr Rev20, 689-725 (1999).

8. Lee, W., Tonelli, M. & Markley, J. L. NMRFAM-SPARKY: enhanced software for biomolecular NMR spectroscopy. Bioinformatics 31 , 1325-1327 (2015).

9. Lee, W., Petit, C. M., Cornilescu, G., Stark, J. L. & Markley, J. L. The AUDANA algorithm for automated protein 3D structure determination from NMR NOE data. Journal of biomolecular NMR 65 , 51 -57 (2016).

10. Shen, Y., Delaglio, F., Cornilescu, G. & Bax, A. TALOS+: a hybrid method for predicting protein backbone torsion angles from NMR chemical shifts. J Biomol NMR 44, 213-223 (2009).

11 . Schwieters, C. D., Kuszewski, J. J., Tjandra, N. & Clore, G. M. The Xplor-NIH NMR molecular structure determination package. Journal of magnetic resonance 160, 65-73 (2003).

12. Koradi, R., Billeter, M. & Wuthrich, K. MOLMOL: a program for display and analysis of macromolecular structures. Journal of molecular graphics 14, 51 -55 (1996).

13. DeLano, W. L. in Abstracts of Papers of the American Chemical Society. (AMER CHEMICAL SOC 1155 16TH ST, NW, WASHINGTON, DC 20036 USA).

14. Bhattacharya, A., Tejero, R. & Montelione, G. T. Evaluating protein structures determined by structural genomics consortia. Proteins: Structure, Function, and Bioinformatics 66, 778-795 (2007).

15. Williams, C. J. et al. MolProbity: More and better reference data for improved all-atom structure validation. Protein Science 27, 293-315 (2018).

16. Ordelheide, A. M. et al. Nor-1 , a novel incretin-responsive regulator of insulin genes and insulin secretion. Mol Metab 2, 243-255 (2013).

Claims

CLAIMS What is claimed is:

1 . A method of modulating insulin secretion from a p-cell, comprising administering an effective amount of a Nori ligand to the p-cell, wherein the Nori ligand directly binds to the ligand binding domain (LBD) of Nori .

2. The method of claim 1 , wherein the Nori ligand, is an agonistic Nori ligand that induces:

(i) increased secretion of insulin from the p-cell relative to a p-cell not administered the Nori ligand; and/or

(ii) increased growth of the p-cell relative to a p-cell not administered with the Nori ligand.

3. The method of claim 1 , wherein the method is in vitro, and the Nori ligand contacts with the P-cell under suitable conditions to modulate the p-cell insulin secretion, preferably the p-cell is derived from a p-cell line or a biological sample obtained from a subject.

4. The method of claim 1 , wherein the method is in vivo, the p-cell is in a subject, preferably a mammal, more preferably a human, and the method further comprises administering to the subject the Nori ligand.

5. A method of modulating glucose levels, preferably blood glucose levels, in a subject, comprising administering to the subject an effective amount of a Nori ligand, wherein the Nori ligand directly binds to the ligand binding domain (LBD) of Nori .

6. The method of claim 5, wherein the method is for treating or preventing a glucose metabolism disorder or condition in the subject.

7. The method of claim 6, wherein the glucose metabolism disorder is selected from the group consisting of: diabetes mellitus, glycosuria, hyperglycemia, hypoglycemia and hyperinsulinism.

8. The method of claim 7, wherein the glucose metabolism disorder is diabetes mellitus, preferably Type 2 mellitus.

9. The method of any one of claims 1 -8, wherein the Nori ligand is a small molecule, preferably the small molecule directly binds to the ligand binding domain of Nori (Nor1 -LBD).

10. The method of any one of claims 1 -9, wherein the Nor1 -LBD comprises or consists of an amino acid sequence set forth in SEQ ID NO:4 or variants thereof.

1 1 . The method of any one of claims 1 -10, wherein the Nori ligand directly binds to one or more amino acid residues in helix 10/1 1 of Nor1 -LBD, corresponding to positions 580-606 of SEQ ID NO: 1 , preferably the Nori ligand directly binds to one or more amino acid residues at positions corresponding to positions 591 -603 of SEQ ID NO: 1 , more preferably the Nori ligand directly binds to one or more amino acid residues at positions corresponding to positions 594-600 of SEQ ID NO: 1 .

12. The method of claim 1 1 , wherein the Nori ligand directly binds with:

(i) the amino acid residue C (Cys) at the position corresponding to position 594 of SEQ ID NO: 1 ; and/or

(Hi) the amino acid residue L (Leu) at the position corresponding to position 596 of SEQ ID NO: 1 ; and/or

13. The method of claim 12, wherein the Nori ligand directly and covalently binds with the amino acid residue C (Cys) at the position corresponding to position 594 of SEQ ID NO: 1 .

14. The method of any one of claims 1 -13, wherein the Nori ligand is an agonistic Nori ligand, preferably an agonistic Nori ligand that covalently binds to Nori , preferably the Nori ligand is PGA1 .

15. The method of any one of claims 1 -13, wherein the Nori ligand is an antagonistic Nori ligand, preferably the Nori ligand is PGE1 .

16. The method of any one of claims 1 -15, wherein the Nori ligand is a cyclopentenone prostaglandin or a derivative, or an analog thereof.

17. The method of claim 16, wherein the cyclopentenone prostaglandin is selected from the group consisting of prostaglandin A1 (PGA1 ), prostaglandin A2 (PGA2), 15-deoxy-A12,14-prostaglandin J2 (15-d-A12,14-PGJ2), A12-Prostaglandin J2 (A12-PGJ2), prostaglandin J2 (PGJ2), derivatives, and analogs thereof.

18. The method of claim 17, wherein the cyclopentenone prostaglandin is prostaglandin A1 (PGA1 ) or prostaglandin A2 (PGA2) or a derivative, or an analog thereof.

19. The method of claim 18, wherein the cyclopentenone prostaglandin is prostaglandin A1

(PGA1 ) or a derivative, or an analog thereof.

20. The method of claim 15, wherein the Nori ligand is prostaglandin E1 (PGE1 ) or a derivative, or an analog thereof.

21 . A method of modulating Nori activity in a cell, comprising administering an effective amount of a prostaglandin or derivative, or analog thereof to the cell.

22. A computer-assisted method for screening, identifying or designing a candidate molecule or compound that modulates Nori activity, the method comprising the steps of: a) providing the structure of Nori -LBD having the amino acid sequence set forth in SEQ ID NO:4 or variants thereof, wherein the structure of Nori -LBD includes the following helical structures and their corresponding amino acid residues of SEQ ID NO: 4 or variants thereof: Helix 1 (H1 : residues 397- 407), Helix 3 (H3: residues 430-454), Helix 4,5 (H4/5: residues 463-485), Helix 6 (H6: residues 504- 510), Helix 7 (H7: residues 512-527), Helix 8 (H8: residues 531 -542), Helix 9 (H9: residues 552-572), Helix 10,1 1 (H10/1 1 : residues 580-606), and Helix 12 (H12: residues 614-622); b) providing a structure of the candidate molecule or compound; c) fitting the structure of the candidate molecule or compound to the Nor1 -LBD, wherein fitting comprises determining interactions between one or more atoms of the candidate molecule or compound and one or more atoms of Nor1 -LBD, to predict whether the candidate molecule or compound binds to or within Nori -LBD; and d) selecting the candidate molecule or compound if it is predicted to bind to or within Nori -LBD.

23. The method of claim 22, wherein the candidate molecule or compound is selected from a virtual chemical library or is a cyclopentenone prostaglandin or a derivative, or an analog thereof.

24. The method of any one of claims 1 -23, wherein the Nori -LBD has the amino acid sequence set forth in SEQ ID NO:4, and the structure of the Nor1 -LBD is derived from, or characterized, by one or more of distance constraints, dihedral constraints and structural statistics described in Table

4.