WO2002063305A2 - Hemorphines servant de ligands endogenes pour recepteur de sous-type 3 de bombesine (brs-3) couple a la proteine g humaine - Google Patents
Hemorphines servant de ligands endogenes pour recepteur de sous-type 3 de bombesine (brs-3) couple a la proteine g humaine Download PDFInfo
- Publication number
- WO2002063305A2 WO2002063305A2 PCT/EP2002/000978 EP0200978W WO02063305A2 WO 2002063305 A2 WO2002063305 A2 WO 2002063305A2 EP 0200978 W EP0200978 W EP 0200978W WO 02063305 A2 WO02063305 A2 WO 02063305A2
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- brs
- receptor
- hemoφhin
- polypeptide
- ligands
- Prior art date
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
- G01N33/57492—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds localized on the membrane of tumor or cancer cells
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
- G01N2333/70571—Assays involving receptors, cell surface antigens or cell surface determinants for neuromediators, e.g. serotonin receptor, dopamine receptor
Definitions
- the present invention relates to the identification of a specific endogenous ligand for the human G-protein coupled receptor bombesin receptor subtype 3 (hereinafter referred to as BRS-3 receptor or BRS-3, and in particular the invention relates to the use of this receptor in drug discovery, preferably with respect to certain dysfunctions or diseases, and furthermore to the drugs that play a role in preventing, ameliorating or correcting said dysfunctions or diseases.
- BRS-3 receptor human G-protein coupled receptor bombesin receptor subtype 3
- BRS-3 receptor human G-protein coupled receptor bombesin receptor subtype 3
- the invention also relates to inhibiting or activating the action of such polynucleotides and polypeptides, to a vector containing said polynucleotides, a host cell containing such vector and transgenic animals where the BRS-3 gene is either overexpressed, misexpressed, underexpressed or suppressed (knock-out animals).
- the invention further relates to a method for screening compounds capable to act as an agonist or an antagonist of said G-protein coupled receptor BRS-3 or which modulate the activity of said G- protein coupled receptor BRS-3, and to an endogenous ligand of BRS-3 and the use of this ligand in the treating, preventing, ameliorating or correcting said dysfunctions or diseases related to the activities of BRS-3 in relation to its specific endogenous ligand.
- Hemorphins are known endogenous peptidic substances which are particularly described in the literature as being products of peptic hydrolysis of bovine hemoglobin. Thus, hemorphin peptides, issued from hemoglobin, have emerged as endogenous bioactive peptides derived from in vivo tissular degradation of hemoglobin. It is known that hemorphins may show specific binding to opioid receptors, but heretofore no other natural receptor has yet been clearly identified to which hemorphins show specific binding affinity and which therefore could also play a major role in several dysfunctions or diseases. Such endogenous substances which qualify as a potential receptor ligand, but for which the specific receptor is yet unknown, are called "orphan ligands".
- hemorphins Although various biological properties of hemorphins are described in the scientific literature showing potential implications with regard to several dysfunctions or diseases, heretofore hemorphins have not yet been identified to be potential ligands to a specific orphan receptor and thus playing also a role in particular dysfunctions or diseases associated with such an orphan receptor. With regard to hemo ⁇ hins a number of publications exist, some of which are referenced as representative literature below, and which are incorporated by reference herein.
- VV-hemo ⁇ hin-7 and LW-hemo ⁇ hin-7 released during in vitro peptic hemoglobin hydrolysis are confirmed by Garreau et al. (Neuropeptides (Edinburgh) (1995), 28(4), 243-50) to be mo ⁇ hinomimetic peptides. Binding experiments strongly confirm that VV-hemo ⁇ hin-7 and LW-hemo ⁇ hin-7 are opioid peptides since they inhibited 3 H-naloxone binding to rat brain membranes.
- VV-hemo ⁇ hin-7 and LW-hemo ⁇ hin-7 exhibit a lesser potency in binding test and in addition in a GPI test (guinea pig in vitro assay) as compared to hemo ⁇ hin-7, and selectivity and affinity of these purified peptides and synthetic hemo ⁇ hin-7 for opioid receptors is discussed by the authors.
- hemo ⁇ hins are described in a review as opioid peptides derived from hemoglobin (Zhao et al., Biopolymers (1997), 43(2), 75-98). Investigation of hemoglobin peptic hydrolyzate has revealed the presence of biologically active peptides with affinity for opioid receptors. Two peptides, VV-hemo ⁇ hin-7 and LVV-hemo ⁇ hin-7, were resolved by a combination of size exclusion and reversed phase HPLC, a new spectroscopic method based on the second order derivation spectra analysis of aromatic amino acids. This method allowed for qualitative and quantitative evaluation of hemo ⁇ hins generated by peptic hemoglobin hydrolysis.
- Dagouassat et al. (FEBS Lett. (1996), 382(1,2), 37-42) also described the generation of VV-hemo ⁇ hin-7 from globin by peritoneal macrophages.
- Bovine globin has been incubated with mice peritoneal macrophages in order to study its hydrolysis by lysosomal enzymes, among which chiefly cathepsin D.
- Analysis of resulting peptides by reversed-phase HPLC showed the release of the bioactive peptide W-hemo ⁇ hin-7.
- a carboxyl proteinase inhibitor such as pepstatin A was added, no hemo ⁇ hin was generated.
- Hemo ⁇ hins are also known to inhibit angiotensin IV binding and interact with aminopeptidase N (Garreau et al., Peptides (N. Y.) (1998), 19(8), 1339-1348). Thus, it was shown that 125 l-angiotensin IV binding to rabbit collecting duct cell membranes was inhibited by hemo ⁇ hins, a class of endogenous peptides obtained by hydrolysis of the ⁇ -chain of hemoglobin.
- hemo ⁇ hins i.e. about 0.3-2.6 nmol/g of tissue.
- the most represented hemo ⁇ hin in lung, heart and brain was VV-hemo ⁇ hin-5, while the content of other members of the hemo ⁇ hin family depended significantly on the tissue analyzed: lung extract was also rich in LVV-hemo ⁇ hin-5, heart contained similar amts. of LVV-hemo ⁇ hin-7 and LVV-hemo ⁇ hin-5 and brain of LW-hemo ⁇ hin-6.
- the hemo ⁇ hin family in spleen was represented mainly by .C-terminally shortened VV-hemo ⁇ hins, i.e. VV-hemo ⁇ hin-4 and VV- hemo ⁇ hin-3. The levels of hemo ⁇ hins in all cases were sufficient to activate the opioid receptors of the respective tissues.
- Endogenous hemo ⁇ hin-related hemoglobin fragments from bovine brain were isolated by Karelin et al. (Biochem. Biophys. Res. Commun. (1994), 202(1), 410-15). Thus, six short-chain peptides were isolated from an acidic extract of bovine brain in the course of total peptide screening. Their primary structures determined by Edman degradation were LVVYP, LWYPWT, LVVYPWTQ, LWYPWTQRF, WYPWTQ and VVYPWTQRF, which respectively corresponded to the fragments 31-35, 31-37, 31-38, 31-40, 32-38 and 32-40 of bovine hemoglobin ⁇ -chain. All these peptides contained sequences of opioid peptides-hemo ⁇ hins.
- the two opioid peptides LW-hemorphin-7 and VV-hemo ⁇ hin 7 were also isolated and characterized from a bovine hemoglobin peptic hydrolysate and investigated for their opioid characteristics (Piot et al., Biochem. Biophys. Res. Commun. (1992), 189(1), 101-10).
- the opioid potency of these peptides, exhibited by use of electrically stimulated muscle of isolated guinea pig ileum, were significant and comparable with some others previously described by the same authors.
- the location of the two opioid peptides, W-hemo ⁇ hin-7 and LW-hemo ⁇ hin- 7, revealed the existence of a strategic zone both in the bovine and human ⁇ -chains of hemoglobin.
- the fragments 31-40 (LW-hemo ⁇ hin-7) and 32-40 (W-hemo ⁇ hin 7) of the ⁇ -chain of bovine hemoglobin were found to be identical to hemo ⁇ hin fragments of human hemoglobin in positions 32-41 and 33-41 of the ⁇ -chain, respectively.
- a nonapeptide and a decapeptide of hemo ⁇ hin type were also isolated from porcine hypothalamus and structurally elucidated (Chang et al., Biochim. Biophys. Acta (1980), 625(2), 266-73).
- a tetrapeptide Gly-Lys-Val-Asn
- the nonapeptide, the decapeptide, and the hexadecapeptide were found to most probably represent artifact fragments of ⁇ - and ⁇ -chains of porcine hemoglobin.
- All of these peptides contained a Val-Val-Tyr-Pro-T ⁇ - Thr-Gln sequence which corresponds to the sequence at position 33-39 of the ⁇ -chain of human hemoglobin. They also have a strong inhibitory activity to angiotensin converting enzyme. An attempt to obtain similar peptides from bovine hemoglobin by enzymatic digestion is also described by the authors.
- GPCR G protein-coupled receptor
- proteins participating in signal transduction pathways that involve G-proteins and/or second messengers; e.g., cAMP (Lefkowitz, Nature, 1991 , 351:353-354).
- these proteins are referred to as proteins participating in pathways with G-proteins.
- Some examples of these proteins include the GPC receptors, such as those for adrenergic agents and dopamine (Kobilka, B.K., et al., Proc. Natl. Acad.
- G-proteins themselves, effector proteins, e.g., phospholipase C, adenyl cyclase, and phosphodiesterase, and actuator proteins, e.g., protein kinase A and protein kinase C (Simon, M.I., et al., Science, 1991, 252:802-8).
- effector proteins e.g., phospholipase C, adenyl cyclase, and phosphodiesterase
- actuator proteins e.g., protein kinase A and protein kinase C (Simon, M.I., et al., Science, 1991, 252:802-8).
- the receptor upon hormone binding to a GPCR the receptor interacts with the heterotrimeric G-protein and induces the dissociation of GDP from the guanine nucleotide-binding site.
- GTP fills the site immediately. Binding of GTP to the ⁇ subunit of the G-protein causes the dissociation of the G-protein from the receptor and the dissociation of the G-protein into ⁇ and ⁇ subunits. The GTP-carrying form then binds to activated adenylate cyclase.
- GTPase activity of the ⁇ subunit is, in essence, an internal clock that controls an on/off switch.
- the GDP bound form of the ⁇ subunit has high affinity for ⁇ and subsequent reassociation of ⁇ GDP with ⁇ returns the system to the basal state.
- the G- protein serves a dual role, as an intermediate that relays the signal from receptor to effector (in this example adenylate cyclase), and as a clock that controls the duration of the signal.
- the membrane bound superfamily of G-protein coupled receptors has been characterized as having seven putative transmembrane domains. The domains are believed to represent transmembrane ⁇ -helices connected by extracellular or cytoplasmic loops.
- G-protein coupled receptors include a wide range of biologically active receptors, such as hormone, viral, growth factor and neuroreceptors.
- the G-protein coupled receptor family includes dopamine receptors which bind to neuroleptic drugs used for treating CNS disorders.
- members of this family include, but are not limited to calcitonin, adrenergic, neuropeptide Y, somastotatin, neurotensin, neurokinin, capsaicin, VIP, CGRP, CRF, CCK, bradykinin, galanin, motilin, nociceptin, endothelin, cAMP, adenosine, muscarinic, acetylcholine, serotonin, histamine, thrombin, kinin, follicle stimulating hormone, opsin, endothelial differentiation gene-1 , rhodopsin, odorant, and cytomegalovirus receptors.
- G-protein coupled receptors share a common structural motif. All these receptors have seven sequences of between 22 to 24 hydrophobic amino acids that form seven alpha helices, each of which spans the membrane (each span is identified by number, i.e. transmembrane-1 (TM1), transmembrane-2 (TM2), etc.).
- the transmembrane helices are joined by strands of amino acids between TM2 and TM3, TM4 and TM5, and TM6 and TM7 on the exterior, or "extracellular" side, of the cell membrane (these are referred to as "extracellular" regions 1 , 2 and 3 (EC1 , EC2 and EC3), respectively).
- transmembrane helices are also joined by strands of amino acids between TM1 and TM2, TM3 and TM4, and TM5 and TM6 on the interior, or "intracellular" side, of the cell membrane (these are referred to as “intracellular” regions 1 , 2 and 3 (IC1 , IC2 and IC3), respectively).
- the "carboxy" (“C") terminus of the receptor lies in the intracellular space within the cell, and the "amino" (“N”) terminus of the receptor lies in the extracellular space outside of the cell.
- Most G-protein coupled receptors have single conserved cysteine residues in each of the first two extracellular loops which form disulfide bonds that are believed to stabilize functional protein structures.
- the 7 transmembrane regions are designated as TM1, TM2, TM3, TM4, TM5, TM6 and TM7.
- the cytoplasmic loop which connects TM5 and TM6 may be a major component of the G- protein binding domain.
- G-protein coupled receptor kinases constitute a family of six mammalian serine/threonine protein kinases that phosphorylate agonist- bound, or activated, G-protein coupled receptors (GPCRs) as their primary substrates.
- GPCRs G-protein coupled receptors
- GPCRs like the calcitonin-receptor like receptor, might interact with small single pass membrane proteins called receptor activity modifying proteins (RAMPs).
- RAMPs receptor activity modifying proteins
- the ligand binding sites of G-protein coupled receptors are believed to comprise hydrophilic sockets formed by several G-protein coupled receptor transmembrane domains, said sockets being surrounded by hydrophobic residues of the G-protein coupled receptors.
- the hydrophilic side of each G-protein coupled receptor transmembrane helix is postulated to face inward and form a polar ligand-binding site.
- TM3 has been implicated in several G-protein coupled receptors as having a ligand-binding site, such as the TM3 aspartate residue.
- TM5 serines, a TM6 asparagine and TM6 or TM7 phenylalanines or tyrosines are also implicated in ligand binding.
- G-protein coupled receptors can be intracellularly coupled by heterotrimeric G-proteins to various intracellular enzymes, ion channels and transporters (see, Johnson et al., Endoc. Rev., 1989, 10:317-331). Different G-protein ⁇ -subunits preferentially stimulate particular effectors to modulate various biological functions in a cell. Phosphorylation of cytoplasmic residues of G- protein coupled receptors has been identified as an important mechanism for the regulation of G- protein coupling of some G-protein coupled receptors.
- G-protein coupled receptor signal transduction involves the agonist-dependent interaction of GPCRs with heterotrimeric G-proteins at the plasma membrane and subsequent generation, by membrane- localized effectors, of soluble second messengers or ion currents. Termination of GPCR signals follows G-protein coupled receptor kinase (GRK)- and ⁇ -arrestin-mediated receptor uncoupling and internalization. But this classical paradigm is inadeqate to account for GPCR-mediated, RAS- dependent activation of mitogen-activated protein (MAP) kinases (Daaka et al., J. Biol. Chem. 1998, 273(2):685-688).
- MAP mitogen-activated protein
- GRKs and ⁇ -arrestins which uncouple GPCRs and target them for internalization, function as essetial elements in the GPCR-mediated MAP kinas signaling cascade.
- G-protein coupled receptors are found in numerous sites within a mammalian host.
- Receptors - primarily the GPCR class - have led to more than half of the currently known drugs (Drews, Nature Biotechnology, 1996, 14: 1516). This indicates that these receptors have an established, proven history as therapeutic targets.
- the GPCR BRS-3 described in the context of this invention below clearly satisfies a need in the art for identification and characterization of further receptors that can play a role in diagnosing, preventing, ameliorating or correcting a broad range of dysfunctions, disorders, or diseases.
- GPCRs are "promiscuous" with respect to G proteins, i.e., that a GPCR can interact with more than one G protein (see, Kenakin, T., 43 Life Sciences 1095 (1988)). Although other G- proteins exist, currently, Gq, Gs, Gi, Gz and Go are G-proteins that have been identified.
- signal transduction Endogenous ligand-activated GPCR coupling with the G-protein begins a signaling cascade process (referred to as "signal transduction"). Under normal conditions, signal transduction ultimately results in cellular activation or cellular inhibition. It is thought that the third intracellular (IC3) loop as well as the carboxy terminus of the receptor interact with the G protein.
- GPCRs exist in the cell membrane in equilibrium between two different conformations: an "inactive" state and an “active” state.
- a receptor in an inactive state is unable to link to the intracellular signaling transduction pathway to produce a biological response.
- Changing the receptor conformation to the active state allows linkage to the transduction pathway (via the G-protein) and produces a biological response.
- a receptor may be stabilized in an active state by an endogenous ligand or a compound such as a drug.
- the invention relates to the identification of the G-protein coupled receptor which specifically binds the endogenous ligands LW-hemo ⁇ hin-7 and/or VV-hemo ⁇ hin-7, in particular with substantial affinity.
- the receptor which is found according to the present invention for the first time to specifically bind the endogenous ligands LW-hemo ⁇ hin-7 and/or W- hemo ⁇ hin-7 was formerly described and designated as o ⁇ han receptor BRS-3 (bombesin receptor subtype 3).
- the o ⁇ han receptor BRS-3 was previously identified by Fathi et al. (J. Biol. Chem. (1993),
- BN-like gastrin- releasing peptide receptor
- NMB receptor neuromedin-B receptor
- BRS-3 bombesin-like peptide receptor subtype 3
- the invention relates also to the use of this BRS-3 receptor and the ligands LVV-H-7 and/or VV-H-7 in drug discovery with respect to certain dysfunctions or diseases related to any interaction of said ligands with BRS-3, and furthermore to the drugs that play a role in preventing, ameliorating or correcting said dysfunctions or diseases. Therefore, in the context the invention reference is made also to BRS-3 polynucleotides, BRS-3 polypeptides encoded by them, and to the use of such polynucleotides and polypeptides, and to their production.
- the invention relates to the treatment and/or prophylaxis of a dysfunction or disorder associated with or being implicated by pathophysiological conditions related to the activities of BRS-3, in particular hBRS-3, and its possible interrelation with the hemo ⁇ hin ligands VV-H-7 and LVV-H-7.
- pathophysiological conditions may evoke dysfunctions or disorders related to cell growth, cell proliferation, tumor development and cancer, e.g.
- SCLC small cell lung carcinoma
- neoplasm neoplasm
- immunology and inflammation etc.
- genitourinary system or any other dysfunction or disease related to the activities of BRS-3, in particular of hBRS-3, in connection with its interrelation with the hemo ⁇ hin ligands VV-H-7 and LVV-H-7.
- the invention may relate to methods for using such BRS-3 polypeptides and polynucleotides in the context of certain dysfunctions or disorders related to any interaction of the ligands VV-H-7 and/or LVV-H-7 with BRS-3 polypeptides, preferably in the fields of drug discovery (lead screening and/or lead structure design and optimization), diagnosis and treatment and /or prophylaxis of specific BRS-3/hemo ⁇ hin-related dysfunctions or disorders.
- Such specific BRS-3/hemo ⁇ hin-related uses include dysfunctions or disorders related to pathophysiological conditions subjected to the activities of BRS-3, in particular of hBRS-3, in connection with its interrelation with the hemo ⁇ hin ligands VV-H-7 and LVV-H-7; these dysfunctions or disorders may include, for example but without limitation, those evoked by pathophysiological conditions related to cell growth, cell proliferation, tumor development and cancer, e.g. such as small cell lung carcinoma (“SCLC”), neoplasm, immunology and inflammation etc., and to the genitourinary system.
- SCLC small cell lung carcinoma
- the invention relates to methods to identify agonists and antagonists using the materials provided by the invention, and treating conditions associated with BRS-3/hemo ⁇ hin imbalance with the identified compounds. Yet another aspect of the invention relates to diagnostic assays for detecting diseases associated with inappropriate BRS-3/hemo ⁇ hin activity or levels. A further aspect of the invention relates to animal-based systems which act as models for disorders arising from aberrant expression or activity of BRS-3/hemo ⁇ hin.
- Preferred agonists or antagonists identified according to the present invention are those which are suited for treating, preventing, ameliorating or correcting the said dysfunctions or diseases as mentioned before to be related to the activities of BRS-3 and its interrelation with the hemorphin ligands VV-H-7 and/or LVV-H-7.
- the invention relates to the use of (isolated) BRS-3/hemo ⁇ hin-ligand- complexes, e.g. BRS-3/W-H-7- or BRS-3/LW-H-7-complexes in the identification and optimization of lead structures which are peptides other than hemo ⁇ hin peptides or e.g. synthetic non-peptide organic molecules.
- the invention relates to the use of BRS-3 activators, inhibitors or modulators for the preparation of a pharmaceutical composition for the treatment and/or prophylaxis of BRS-3/hemo ⁇ hin-ligand-related dysfunctions or disorders as indicated above, preferably of dysfunctions or disorders, for example but without limitation, those evoked by pathophysiological conditions related to cell growth, cell proliferation, tumor development and cancer, e.g. such as small cell lung carcinoma ("SCLC"), neoplasm, immunology and inflammation etc., and to the genitourinary system.
- SCLC small cell lung carcinoma
- those BRS-3 activators, inhibitors or modulators are peptides other than hemo ⁇ hin peptides or e.g. synthetic non-peptide organic molecules.
- the invention relates to the use of BRS-3-polynucleotides, BRS-3- polypeptides and/or hemo ⁇ hin peptides VV-H-7 and/or LVV-H-7 in the diagnosis of BRS- 3/hemo ⁇ hin-ligand related dysfunctions or disorders.
- Values represent the maximal fluorescence change stimulated by the indicated peptides and are the means ⁇ SEM from at least eight independent experiments.
- Hemo ⁇ hins'' are a family of bioactive peptides generated by proteolytic cleavage of the ⁇ - chain of hemoglobin.
- the proteolytic fragments VV-H-7 and LVV-H-7 are hemorphins of particular interest in the context of the invention.
- BRS-3 (bombesin receptor subtype 3) refers to a polypeptide, in particular to a human polypeptide, from the bombesin receptor family, in particular comprising the amino acid sequence set forth in SEQ ID NO: 2, or a variant thereof, or a polypeptide essentially similar thereto, including polypeptides showing at least 80 % identity or any higher degree of identity as indicated below in the description of the invention.
- the term “hBRS-3” shall mean human BRS-3.
- Receptor Activity or "Biological Activity of the Receptor” refers to the metabolic or physiologic function of said BRS-3 including similar activities or improved activities or these activities with decreased undesirable side effects. Also included are antigenic and immunogenic activities of said BRS-3, in particular hBRS-3.
- Module shall mean materials (e.g. ligands, partial agonists, antagonists, inverse agonists, candidate compounds) that in any way “modulate” the natural or original. activity of a receptor e.g. a material that measurably influences the receptor's activity by e.g. evoking a total, partial or graded change or modification, preferably by evoking a partial or graded change or modification, of the natural or original activity of the receptor.
- Antists shall mean materials (e.g., ligands, candidate compounds) that activate the intracellular response when they bind to the receptor, or enhance GTP binding to membranes.
- Partial agonist shall mean materials (e.g., ligands, candidate compounds) that activate the intracellular response when they bind to the receptor to a lesser degree/extent than do agonists, or enhance GTP binding to membranes to a lesser degree/extent than do agonists.
- Antagonist shall mean materials (e.g., ligands, candidate compounds) that competitively bind to the receptor at the same site as the agonists but which do not activate the intracellular response initiated by the active form of the receptor, and can thereby inhibit the intracellular response in the presence of an agonist or partial agonist.
- Subject shall mean a molecule (for example, and not limitation, a chemical compound), preferably a candidate compound.
- Candidate compound shall mean a molecule (for example, and not limitation, a chemical compound) that is amenable to a screening technique.
- the phrase “candidate compound” does not include compounds which were publicly known to be compounds selected from the group consisting of modulator, agonist or antagonist, partial agonist or inverse agonist to a receptor, as previously determined by an indirect identification process ("indirectly identified compound”); more preferably, not including an indirectly identified compound which has previously been determined to have therapeutic efficacy in at least one mammal; and, most preferably, not including an indirectly identified compound which has previously been determined to have therapeutic utility in humans.
- Compound efficacy shall mean a measurement of the ability of a compound to inhibit or stimulate receptor functionality, as opposed to receptor binding affinity. Exemplary means of detecting compound efficacy are disclosed in the Example section of this patent document.
- Endogenous shall mean a material that a mammal naturally produces.
- Endogenous in reference to, for example and without limitation, the term “receptor”, shall mean that which is naturally produced by a mammal (for example and without limitation, a human) or a virus.
- non-endogenous in this context shall mean that which is not naturally produced by a mammal (for example and without limitation, a human) or a virus. Both terms can be utilized to describe both “in vivo” and “in vitro” systems.
- the endogenous or non-endogenous receptor may be in reference to an in vitro screening system.
- “Inverse agonists” shall mean materials (e.g., ligand, candidate compound) which bind to the endogenous form of the receptor and which inhibit the baseline intracellular response initiated by the active form of the receptor below the normal base level of activity which is observed in the absence of agonists or partial agonists, or decrease GTP binding to membranes.
- the baseline intracellular response is inhibited in the presence of the inverse agonist by at least 30 %, more preferably by at least 50 %, and most preferably by at least 75 %, as compared with the baseline response in the absence of the inverse agonist.
- Known receptor e.g. non-o ⁇ han receptor
- Known receptor shall mean an endogenous receptor for which the endogenous ligand specific for that receptor has been identified.
- Ligand shall mean an endogenous, naturally occurring molecule specific for an endogenous, naturally occurring receptor.
- Oleth receptor shall mean an endogenous receptor for which the endogenous ligand specific for that receptor has not been identified or is not known.
- BRS-3 gene refers to a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 1 (BRS-3), or allelic variants thereof and/or their complements.
- Antibodies as used herein includes polyclonal and monoclonal antibodies, chimeric, single chain, and humanized antibodies, as well as Fab fragments, including the products of a Fab or other immunoglobulin expression library.
- Isolated means altered “by the hand of man” from the natural state and/or separated from the natural environment. Thus, if an "isolated” composition or substance that occurs in nature has been “isolated”, it has been changed or removed from its original environment, or both. For example, a polynucleotide or a polypeptide naturally present in a living animal is not “isolated”, but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is "isolated", as the term is employed herein.
- Polynucleotide generally refers to any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA.
- Polynucleotides include, ithout limitation single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is a mixture of single-and double- stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions.
- polynucleotide may also include triple-stranded regions comprising RNA or DNA or both RNA and DNA.
- the term polynucleotide also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons.
- Modified bases include, for example, tritylated bases and unusual bases such as inosine.
- polynucleotide embraces chemically, enzymatically or metabolically modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells.
- Polynucleotide also embraces relatively short polynucleotides, often referred to as oligonucleotides.
- Polypeptide refers to any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres.
- Polypeptide refers to short chains, commonly referred to as peptides, oligopeptides or oligomers, and to longer chains, generally referred to as proteins, and/or to combinations thereof. Polypeptides may contain amino acids other than the 20 gene-encoded amino acids.
- Polypeptides include amino acid sequences modified either by natural processes, such as posttranslational processing, or by chemical modification techniques which are well known in the art.
- Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. Polypeptides may be branched as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched and branched cyclic polypeptides may result from posttranslation natural processes or may be made by synthetic methods.
- Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol; cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination.
- Variant is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide respectively, but retains essential properties.
- a typical variant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide. Changes in the nucleotide sequence of the variant may or may not alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide. Nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below.
- a typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical.
- a variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, and deletions in any combination.
- a substituted or inserted amino acid residue may or may not be one encoded by the genetic code.
- a variant of a polynucleotide or polypeptide may be a naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally. Non-naturally occurring variants of polynucleotides and polypeptides may be made by mutagenesis techniques or by direct synthesis.
- Identity is a measure of the identity of nucleotide sequences or amino acid sequences. In general, the sequences are aligned so that the highest order match is obtained. “Identity” per se has an art-recognized meaning and can be calculated using published techniques.
- identity is well known to skilled artisans (Carillo, H., and Lipton, D., SIAM J. Applied Math. (1988) 48:1073). Methods commonly employed to determine identity or similarity between two sequences include, but are not limited to, those disclosed in Guide to Huge Computers, Martin J. Bishop, ed., Academic Press, San Diego, 1994, and Carillo, H., and Lipton, D., SIAM J. Applied Math. (1988) 48:1073. Methods to determine identity and similarity are codified in computer programs.
- Preferred computer program methods to determine identity and similarity between two sequences include, but are not limited to, GCG program package (Devereux, J., et al., Nucleic Acids Research (1984) 12(1):387), BLASTP, BLASTN, FASTA (Atschul, S.F. et al., J. Molec. Biol. (1990) 215:403).
- GCG program package Digit, J., et al., Nucleic Acids Research (1984) 12(1):387
- BLASTP BLASTP
- BLASTN BLASTN
- FASTA Altschul, S.F. et al., J. Molec. Biol. (1990) 215:403
- the word “homology” may substitute for the word "identity”.
- a polynucleotide having a nucleotide sequence having at least, for example, 95% "identity" to a reference nucleotide sequence of SEQ ID NO: 1 is intended that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence of SEQ ID NO: 1.
- a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence, or in a number of nucleotides of up to 5% of the total nucleotides in the reference sequence there may be a combination of deletion, insertion and substitution.
- These mutations of the reference sequence may occur at the 5 or 3 terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.
- a polypeptide having an amino acid sequence having at least, for example, 95% "identity" to a reference amino acid sequence of SEQ ID NO: 2 is intended that the amino acid sequence of the polypeptide is identical to the reference sequence except that the polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the reference amino acid of SEQ ID NO: 2.
- up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 5% of the total amino acid residues in the reference sequence may be inserted into the reference sequence.
- These alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.
- the bombesin family of G-protein-coupled receptors includes the gastrin-releasing peptide receptor (GRP-R), the neuromedin receptor (NMB-R) and the o ⁇ han bombesin receptor subtype 3 (BRS-3).
- GRP-R gastrin-releasing peptide receptor
- NMB-R neuromedin receptor
- BRS-3 o ⁇ han bombesin receptor subtype 3
- BRS-3 shows a high affinity binding for the hemo ⁇ hin peptides LW-hemo ⁇ hin-7 (LVV-H-7) and/or W- hemo ⁇ hin-7 (VV-H-7).
- This affinity was only found with regard to the BRS-3 polypeptides dealt with in the present invention, but not for the other two receptors, GRP-R and NMB-R, which show BN-like ligand affinity.
- W-hemo ⁇ hin-7 (VV-H-7) and LW-hemo ⁇ hin-7 (LVV-H-7) are bioactive peptides and members of the hemo ⁇ hin peptide family that may be generated by proteolysis of the ⁇ -chain of hemoglobin, as already described supra.
- substantially affinity is understood as to describe a ligand binding showing log EC50 values of at least those found for the LVV-H-7 or VV- H-7 itself with regard to the BRS-3; more details about the affinity binding of LVV-H-7 and of VV- H-7 to BRS-3 is given in the experimental part and the Figures. Furthermore some characteristics of LVV-H-7 and of VV-H-7 known in the state of the art prior to this invention are described above in the Background section supra.
- VV-H-7 W-hemo ⁇ hin-7
- LW-hemo ⁇ hin-7 LW-hemo ⁇ hin-7
- LVV-H-7 LW-hemo ⁇ hin-7
- LVV-H-7 and VV-H-7 induced a dose-dependent increase in intracellular Ca 2+ concentration, e.g. EC 50 values were found of 19 ⁇ 6 ⁇ M for VV-H-7 and 38 ⁇ 18 ⁇ M for LVV-H-7. Again the other hemo ⁇ hins as indicated above had no effect.
- hemo ⁇ hins VV-H-7 and/or LVV-H-7 as endogenous ligands for the o ⁇ han receptor BRS-3, in particular for hBRS-3, and the physiological characterization on NCI-N417 cell line gives important insights into the possible interrelation between the receptor BRS-3, in particular hBRS-3, and the hemo ⁇ hin ligands VV-H-7 and LVV-H- 7 may further help defining the roles of these ligands in pathophysiological conditions related to the activities of BRS-3, in particular of hBRS-3, e.g. in pathophysiological conditions related to cell growth, cell proliferation, tumor development and cancer, e.g.
- SCLC small cell lung carcinoma
- neoplasm neoplasm
- immunology and inflammation etc.
- genitourinary system genitourinary system
- drugs for treating, preventing, ameliorating or correcting any other dysfunction or disease related to the activities of BRS-3, in particular of hBRS-3 in connection with its interrelation with the hemo ⁇ hin ligands VV-H-7 and LVV-H-7.
- the BRS-3 polypeptides have been as a G-protein coupled receptor responsive to LVV-H-7 and/or to VV-H-7.
- the finding of the present invention e.g. the identification of the responsiveness of the bombesin receptor subtype 3 polypeptides to LW-H-7 and/or to W-H-7 will greatly facilitate not only the understanding of the physiological role of LVV-H-7 and/or VV-H-7 but moreover also of other potential peptide ligands as well as of non-peptide ligands, e.g. of non- peptide organic molecules, with sufficiently similar binding to the BRS-3, as well as of the related physiological mechanisms and of the related diseases.
- the BRS-3 polypetides in particular the hBRS-3, identified in the context of the present invention as being responsive to VV-H-7 and/or LVV-H-7 belong to the bombesin family of G- protein-coupled receptors and they share about 50 % amino acid sequence identity to the gastrin- releasing peptide receptor (GRP-R) and the neuromedin receptor (NMB-R), the two other known members of this receptor family; for further details see literature Fathi et al. (J. Biol. Chem. (1993), 268(8), 5979 - 5984) which herein is incorporated by reference.
- GRP-R gastrin- releasing peptide receptor
- NMB-R neuromedin receptor
- hBRS-3 mRNA Studies of hBRS-3 mRNA revealed an expression pattern limited to secondary spermatocytes and tumor cell lines derived from human lung and testis (Fathi et al., 1993, see supra), a few brain regions, breast and epidermal tissue (Gorbulev et al., FEBS Lett. 1994, 340(3): 260-4; Organization and Chromosomal Localization of the Gene for the Human Bombesin Receptor Subtype Expressed in Pregnat Uterus) and placenta (Whitley et al., J. Clin. Endocrinol. Metab. 1996, 81(11): 3944-50; Expression of Gastrin-releasing Peptide (GRP) and GRP Receptors in the Pregnant Human Uterus at Term).
- GRP Gastrin-releasing Peptide
- BRS-3 polypeptides in particular hBRS-3 polypeptides
- the hemorphin ligands VV-H-7 and LVV-H-7 preferably play a role in dysfunctions and disorders being implicated by pathophysiological conditions related to the activities of BRS-3, in particular hBRS-3, and its possible interrelation with the hemo ⁇ hin ligands VV-H-7 and LVV-H-7.
- pathophysiological conditions may evoke dysfunctions or disorders related to cell growth, cell proliferation, tumor development and cancer, e.g.
- SCLC small cell lung carcinoma
- neoplasm neoplasm
- immunology and inflammation etc.
- VV-H-7 and LVV-H-7 any other dysfunction or disease related to the activities of BRS-3, in particular of hBRS-3, in connection with its interrelation with the hemo ⁇ hin ligands.
- BRS-3 polypeptides referred to in the context of the present invention are particularly described by Fathi et al. (J. Biol. Chem. (1993), 268(8), 5979 - 5984) and can be prepared in any suitable manner.
- BRS-3 receptor polypeptide sequences are also published in two international patent applications ("PCT applications") under publication no. WO 00/05244 (SKB) and under publication no. WO 92/16623 (Berlex Laboratories).
- PCT applications under publication no. WO 00/05244
- WO 92/16623 Bolex Laboratories.
- the BRS-3 receptor is a polypeptide of 399 amino acids.
- the BRS-3 polypeptides referred to in the context of the present invention may furthermore include isolated naturally occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. Methods for preparing such polypeptides are well known in the art.
- BRS-3 polynucleotides include isolated polynucleotides which encode the BRS-3 polypeptides, including fragments, and polynucleotides closely related thereto. More specifically, the BRS-3 polynucleotide includes a polynucleotide comprising the nucleotide sequence contained in SEQ ID NO: 1 or a sequence encoding a BRS-3 polypeptide of SEQ ID NO: 2. The polynucleotides having the particular sequence of SEQ ID NO: 1 or a sequence that essentially corresponds to the coding sequence contained in the 1413 nucleotides long DNA cloned by Fathi et al. (J. Biol. Chem. (1993), 268(8),
- BRS-3 receptor polynucleotide sequences are also published in two international patent applications under publication no. WO 00/05244 (SKB) and under publication no. WO 92/16623 (Berlex Laboratories).
- BRS-3 polynucleotides further include a polynucleotide comprising a nucleotide sequence that has at least 80% identity over its entire length to a nucleotide sequence encoding the BRS-3 polypeptide of SEQ ID NO: 2, a polynucleotide comprising a nucleotide sequence that is at least 80% identical to that of SEQ ID NO: 1 over its entire length and a polynucleotide.
- polynucleotides with at least 90% identity are particularly preferred within the context of the invention, and those with at least 95% are especially preferred.
- those with at least 97% are highly preferred and those with at least 98-99% are most highly preferred, with at least 99% being the most preferred.
- BRS-3 polynucleotides are a nucleotide sequences which have sufficient identity to a nucleotide sequence contained in SEQ ID NO: 1 to hybridize under conditions useable for amplification or for use as a probe or marker.
- the invention also may involve polynucleotides which are complementary to such BRS-3 polynucleotides.
- G-protein coupled receptors structurally related to BRS-3 proteins which may be of interest in the context of the present invention may be identified by the skilled artisan e.g. by BLAST searches (using BLAST, Altschul S.F. et al. [1997], Nucleic Acids Res. 25:3389-3402) in the public databases.
- polypeptides and polynucleotides sufficiently similar to the BRS-3 polypeptides and BRS-3 polynucleotides considered in the present invention are expected to have, inter alia, similar biological functions/properties to their homologous polypeptides and polynucleotides, and their utility is obvious to anyone skilled in the art.
- Polynucleotides used in the context of the invention can be obtained from natural sources such as genomic DNA.
- degenerated PCR primers can be designed that encode consen ed regions within a particular GPCR gene subfamily. PCR amplification reactions on genomic DNA or cDNA using the degenerate primers will result in the amplification of several members (both known and novel) of the gene family under consideration (the degenerated primers must be located within the same exon, when a genomic template is used). (Libert et al., Science, 1989, 244: 569-572). Polynucleotides used in the context of the invention can also be synthesized using well-known and commercially available techniques (e.g. F.M. Ausubel et al., 2000, Current Protocols in Molecular Biology).
- the nucleotide sequence encoding the BRS-3 polypeptide of SEQ ID NO:2 may be identical to the polypeptide encoding sequence contained in SEQ ID NO:1, or it may be a different nucleotide sequence, which as a result of the redundancy (degeneracy) of the genetic code might also show alterations compared to the polypeptide encoding sequence contained in SEQ ID NO:1 , but also encodes the polypeptide of SEQ ID NO:2.
- the polynucleotide may include the coding sequence for the mature polypeptide or a fragment thereof, by itself; the coding sequence for the mature polypeptide or fragment in reading frame with other coding sequences, such as those encoding a leader or secretory sequence, a pre-, or pro- or prepro-protein sequence, or other fusion peptide portions.
- a marker sequence which facilitates purification of the fused polypeptide can be encoded.
- the marker sequence is a hexa- histidine peptide, as provided in the pQE vector (Qiagen, Inc.) and described in Gentz et al., Proc. Natl. Acad. Sci USA (1989) 86:821-824, or is an HA tag.
- the polynucleotide may also contain non- coding 5' and 3' sequences, such as transcribed, non-translated sequences, splicing and polyadenylation signals, ribosome binding sites and sequences that stabilize mRNA.
- polynucleotides encoding BRS-3 variants comprising the amino acid sequence of the BRS-3 polypeptide of SEQ ID NO: 2 in which several, 5-10, 1-5, 1-3, 1-2 or 1 amino acid residues are substituted, deleted or added, in any combination.
- polynucleotides used in the context of the invention can be engineered using methods generally known in the art in order to alter BRS-3-encoding sequences for a variety of pu ⁇ oses including, but not limited to, modification of the cloning, processing, and/or expression of the gene product.
- DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides may be used to engineer the nucleotide sequences.
- oligonucleotide-mediated site-directed mutagenesis may be used to introduce mutations that create amino acid substitutions, create new restriction sites, alter modification (e.g. glycosylation or phosphorylation) patterns, change codon preference, produce splice variants, and so forth.
- the present invention further may involve polynucleotides that hybridize to the herein above-described sequences.
- the present invention especially may involve polynucleotides which hybridize under stringent conditions to the herein above-described polynucleotides.
- stringent conditions means hybridization will occur only if there is at least 80%, and preferably at least 90%, and more preferably at least 95%, yet even more preferably 97, in particular at least 99% identity between the sequences.
- Polynucleotides used in the context of the invention which are identical or sufficiently identical to a nucleotide sequence contained in SEQ ID NO: 1 , or a fragment thereof, may be used as hybridization probes for cDNA and genomic DNA, to isolate full-length cDNAs and genomic clones encoding BRS-3 receptor and to isolate cDNA and genomic clones of other genes (including genes encoding homologs and orthologs from species other than human) that have a high sequence similarity to the BRS-3 gene.
- these nucleotide sequences are 80% identical, preferably 90% identical, more preferably 95% identical to that of the referent.
- the probes generally will comprise at least 15 nucleotides.
- such probes will have at least 30 nucleotides and. may have at least 50 nucleotides. Particularly preferred probes will range between 30 and 50 nucleotides.
- One embodiment, to obtain a polynucleotide encoding the BRS-3 polypeptide, including homologs and orthologs from species other than human comprises the steps of screening an appropriate library under stringent hybridization conditions with a labeled probe having the SEQ ID NO: 1 , or a fragment thereof, and isolating full-length cDNA and genomic clones containing said polynucleotide sequence.
- Such hybridization techniques are well known to those of skill in the art.
- Stringent hybridization conditions are as defined above or alternatively conditions under overnight incubation at 42 °C in a solution comprising: 50% formamide, 5xSSC (150mM NaCI, 15mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5x Denhardt's solution, 10 % dextran sulfate (w/v), and 20 microgram/ml denatured, sheared salmon sperm DNA, followed by washing the filters in O.lxSSC at about 65 °C.
- polynucleotides and polypeptides described in the context of the present invention may be used as research reagents and materials for discovery of treatments and diagnostics to animal and human pathophysiological conditions, e.g. dysfunctions or diseases, such as in particular described supra with regard to the interrelation between BRS-3 receptor polypeptides and the hemo ⁇ hin ligands VV-H-7 and/or LVV-H-7.
- vectors which comprise a BRS-3 polynucleotide or BRS-3 polynucleotides
- host cells which are genetically engineered with said vectors and to the production of BRS-3 polypeptides by recombinant techniques.
- Cell-free translation systems can also be used to produce such proteins using RNAs derived from the DNA constructs described in the context of the present invention.
- host cells can be genetically engineered to inco ⁇ orate expression systems or portions thereof for said polynucleotides.
- Introduction of polynucleotides into host cells can be effected by methods described in many standard laboratory manuals, such as Davis et al., BASIC METHODS IN MOLECULAR BIOLOGY (1986) and Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Ha ⁇ or, N.Y. (1989) such as calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction or infection.
- bacterial cells such as streptococci, staphylococci, E. coli, Streptomyces and Bacillus subtilis cells
- fungal cells such as yeast cells and Aspergillus cells
- insect cells such as Drosophila S2 and Spodoptera Sf9 cells
- animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293, HL60, U937, Jurkat, mouse VMRO, MM39 human tracheal gland cells, rat mesangial cells, endothelia cells, Xenopus oocytes and Bowes melanoma cells
- plant cells include bacterial cells, such as streptococci, staphylococci, E. coli, Streptomyces and Bacillus subtilis cells; fungal cells, such as yeast cells and Aspergillus cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, HeLa, C127,
- Such systems include, among others, chromosomal, episomal and virus-derived systems, e.g., vectors derived from bacterial plasmids, from bacteriophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids.
- viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses
- vectors derived from combinations thereof such as those derived from plasmid and bacteriophage genetic elements, such as cosmid
- the expression systems may contain control regions that regulate as well as engender expression.
- any system or vector suitable to maintain, propagate or express polynucleotides to produce a polypeptide in a host may be used.
- the appropriate nucleotide sequence may be inserted into an expression system by any of a variety of well-known and routine techniques, such as, for example, those set forth in Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL (supra).
- secretion signals may be incorporated into the desired polypeptide. These signals may be endogenous to the polypeptide or they may be heterologous signals.
- the BRS-3 polypeptide is to be expressed for use in screening assays, generally, it is preferred that the polypeptide be exposed at the surface of the cell. In this event, the cells may be harvested prior to use in the screening assay. In case the affinity or functional activity of the BRS- 3 polypeptide is modified by receptor activity modifying proteins (RAMP), coexpression of the relevant RAMP most likely at the surface of the cell is preferred and often required. Also in this event harvesting of cells expressing the BRS-3 polypeptide and the relevant RAMP prior to use in screening assays is required. If the BRS-3 polypeptide is secreted into the medium, the medium can be recovered in order to recover and purify the polypeptide; if produced intracellularly, the cells must first be lysed before the polypeptide is recovered.
- RAMP receptor activity modifying proteins
- BRS-3 polypeptides can be recovered and purified from recombinant cell cultures by well- known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography is employed for purification. Well-known techniques for refolding proteins may be employed to regenerate active conformation when the polypeptide is denatured during isolation and or purification.
- This invention also relates to the use of BRS-3 polynucleotides for use as diagnostic reagents, in particular in the context of the dysfunctions, disorders or diseases as indicated supra in connection with the present invention.
- Detection of a mutated form of the BRS-3 gene associated with a dysfunction will provide a diagnostic tool that can add to or define a diagnosis of any of said disorders or diseases or susceptibility to any of said disorders or diseases which results from under-expression, over-expression or altered expression of BRS-3.
- co- expression of relevant receptor activity modifying proteins can be required to obtain diagnostic assays of desired quality.
- Individuals carrying mutations in the BRS-3 gene associated with a dysfunction, disorder or disease as indicated supra in the context of the invention may be detected at the DNA level by a variety of techniques.
- Nucleic acids for diagnosis may be obtained from a subject's cells, such as from blood, urine, saliva, tissue biopsy or autopsy material.
- the genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR or other amplification techniques prior to analysis.
- RNA or cDNA may also be used in similar fashion. Deletions and insertions can be detected by a change in size of the amplified product in comparison to the normal genotype. Point mutations can be identified by hybridizing amplified DNA to labeled BRS-3 nucleotide sequences. Perfectly matched sequences can be distinguished from mismatched duplexes by RNase digestion or by differences in melting temperatures.
- DNA sequence differences may also be detected by alterations in electrophoretic mobility of DNA fragments in gels, with or without denaturing agents, or by direct DNA sequencing. See, e.g., Myers et al., Science (1985) 230:1242. Sequence changes at specific locations may also be revealed by nuclease protection assays, such as RNase and S1 protection or the chemical cleavage method. See Cotton et al., Proc. Natl. Acad. Sci. USA (1985) 85: 4397-4401.
- an array of oligonucleotide probes comprising the BRS-3 nucleotide sequence or fragments thereof can be constructed to conduct efficient screening of e.g., genetic mutations.
- the diagnostic assays offer a process for diagnosing or determining a susceptibility to in particular BRS-3-receptor/hemo ⁇ hin-ligand related dysfunctions, disorders or diseases as stated supra.
- BRS-3-receptor/hemo ⁇ hin-ligand related dysfunctions, disorders or diseases are those related to pathophysiological conditions subjected to the activities of BRS-3, in particular of hBRS-3, in connection with its interrelation with the hemo ⁇ hin ligands VV-H-7 and LVV-H-7; these dysfunctions, disorders or dieseases may include, for example but without limitation, those evoked by pathophysiological conditions related to cell growth, cell proliferation, tumor development and cancer, e.g.
- the diagnostic assays offer in particular a process for diagnosing or determining a susceptibility to any of said dysfunctions, disorders and diseases.
- said dysfunctions, disorders or diseases can be diagnosed by methods comprising determining from a sample derived from a subject an abnormally decreased or increased level of the BRS-3 polypeptide or BRS-3 mRNA. Decreased or increased expression can be measured at the RNA level using any of the methods well known in the art for the quantitation of polynucleotides, such as, for example, PCR, RT-PCR, Taq-Man PCR analysis, RNase protection, Northern blotting and other hybridization methods. Assay techniques that can be used to determine levels of a protein, such as an BRS-3 receptor, in a sample derived from a host are well known to those of skill in the art. Such assay methods include radioimmunoassays, competitive-binding assays, Western Blot analysis and ELISA assays.
- the present invention relates to a diagonostic kit for a disease or susceptibility to a BRS-3/hemo ⁇ hin-related dysfunctions or disease, particularly dysfunctions or diseases related to pathophysiological conditions subjected to the activities of BRS-3, in particular of hBRS-3, in connection with its interrelation with the hemo ⁇ hin ligands VV-H-7 and LVV-H-7; these dysfunctions or disorders may include, for example but without limitation, those evoked by pathophysiological conditions related to cell growth, cell proliferation, tumor development and cancer, e.g. such as small cell lung carcinoma ("SCLC"), neoplasm, immunology and inflammation etc., and to the genitourinary system, which kit comprises:
- SCLC small cell lung carcinoma
- BRS-3 polynucleotide preferably the nucleotide sequence of SEQ ID NO: 1 , or a fragment thereof;
- a BRS-3 polypeptide preferably the polypeptide of SEQ ID NO: 2 or a variant thereof with amino acid replacement in position 26 of methionine by valine, or a fragment thereof; or (d) an antibody to a BRS-3 polypeptide, preferably to the polypeptide of SEQ ID NO: 2 or to a variant thereof with amino acid replacement in position 26 of methionine by valine, or to a fragment thereof; (e) a RAMP polypeptide required for the relevant biological or antigenic properties of a BRS-3 polypeptide
- kits for diagnosing or determining an BRS-3/hemo ⁇ hin-ligand related disease or a susceptibility to said an BRS- 3/hemorphin-ligand related dysfunctions or diseases.
- the BRS-3 nucleotide sequences are also valuable for chromosome identification.
- the sequence is specifically targeted to and can hybridize with a particular location on an individual human chromosome.
- the mapping of relevant sequences to chromosomes according to the present invention is an important first step in correlating those sequences with gene associated disease. Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. Such data are found, for example, in V. McKusick, Mendelian Inheritance in Man (available on line through Johns Hopkins University Welch Medical Library). The relationship between genes and diseases that have been mapped to the same chromosomal region are then identified through linkage analysis (coinheritance of physically adjacent genes).
- the differences in the cDNA or genomic sequence between affected and unaffected individuals can also be determined. If a mutation is observed in some or all of the affected individuals but not in any normal individuals, then the mutation is likely to be the causative agent of the disease.
- the BRS-3 polypeptides or their fragments or analogs thereof, or cells expressing them if required together with relevant RAMPs, may also be used as immunogens to produce antibodies immunospecific for the BRS-3 polypeptides.
- immunospecific means that the antibodies have substantially greater affinity for said BRS-3 polypeptides than their affinity for other related polypeptides in the prior art.
- Antibodies generated against the BRS-3 polypeptides may be obtained by administering the polypeptides or epitope-bearing fragments, analogs or cells to an animal, preferably a nonhuman, using routine protocols.
- an animal preferably a nonhuman
- any technique which provides antibodies produced by continuous cell line cultures, may be used. Examples include the hybridoma technique (Kohler, G.
- the above-described antibodies may be employed to isolate or to identify clones expressing the polypeptide or to purify the polypeptides by affinity chromatography.
- the antibodies may also be used to identify the presence of the BRS-3 receptor in membrane preparations, tissue cultures or isolated organs, which are e.g. foreseen for use in drug screening or drug design or drug profiling.
- Antibodies against BRS-3 polypeptides as such or against BRS-3 polypeptide-RAMP complexes may also be employed to treat the BRS-3/hemo ⁇ hin-ligand related dysfunctions or disorders as indicated supra.
- Another aspect of the invention relates to a animal-based systems which act as models for disorders arising from aberrant expression or activity of BRS-3 receptor.
- Animal based model systems may also be used to further characterize the activity of the BRS-3 gene.
- Such assays may be utilized as part of screening strategies designed to identify compounds which are capable to treat BRS-3/hemo ⁇ hin-ligand based disorders such as the BRS-3/hemo ⁇ hin-ligand related dysfunctions or disorders as indicated supra.
- the animal-based models may be used to identify pharmaceutical compounds, therapies and interventions which may be effective in treating disorders aberrant expression or activity of BRS-3 in connection with its interrelation with the hemo ⁇ hin-ligands VV-H-7 and/or LVV-H-7.
- animal models may be used to determine the LD50 and the ED50 in animal subjects. These data may be used to determine the in vivo efficacy of potential BRS-3/hemo ⁇ hin-ligand based disorder treatments.
- Animal-based model systems of BRS-3/hemo ⁇ hin-ligand based disorders may include both non-recombinant animals as well as recombinantly engineered transgenic animals.
- Animal models for BRS-3/hemorphin-ligand based disorders may include, for example, genetic models.
- Animal models exhibiting BRS-3/hemo ⁇ hin-ligand based disorder-like symptoms may be engineered by utilizing, for example, BRS-3 sequences such as those described, above, in conjunction with techniques for producing transgenic animals that are well known to persons skilled in the art.
- BRS-3 sequences may be introduced into, and overexpressed and/or misexpressed in, the genome of the animal of interest, or, if endogenous BRS-3 sequences are present, they may either be overexpressed, misexpressed, or, alternatively, may be disrupted in order to underexpress or inactivate BRS-3 gene expression.
- the coding portion of the BRS-3 gene sequence the coding portion of the
- BRS-3 gene sequence may be ligated to a regulatory sequence which is capable of driving high level gene expression or expression in a cell type in which the gene is not normally expressed in the animal type of interest.
- regulatory regions will be well known to those skilled in the art, and may be utilized in the absence of undue experimentation.
- an endogenous BRS-3 gene sequence such a sequence may be isolated and engineered such that when reintroduced into the genome of the animal of interest, the endogenous BRS-3 gene alleles will be inactivated, or "knocked-out".
- the engineered BRS-3 gene sequence is introduced via gene targeting such that the endogenous BRS-3 sequence is disrupted upon integration of the engineered BRS-3 gene sequence into the animal's genome. Gene targeting is discussed, below, in this section.
- Animals of any species including, but not limited to, mice, rats, rabbits, guinea pigs, pigs, micro-pigs, goats, and non-human primates, e.g., baboons, squirrels, monkeys, and chimpanzees may be used to generate animal models of BRS-3/hemo ⁇ hin-ligand related disorders.
- Any technique known in the art may be used to introduce a BRS-3 transgene into animals to produce the founder lines of transgenic animals.
- Such techniques include, but are not limited to pronuclear microinjection (Hoppe, P.C. and Wagner, T.E., 1989, U.S. Pat. No. 4,873,191); retrovirus mediated gene transfer into germ lines (van der Putten et al., Proc. Natl. Acad. Sci., USA 82:6148-6152, 1985); gene targeting in embryonic stem cells (Thompson et al., Cell 56:313- 321 , 1989,); electroporation of embryos (Lo, Mol. Cell. Biol.
- the present invention provides for transgenic animals that carry the BRS-3 transgene in all their cells, as well as animals which carry the transgene in some, but not all their cells, i.e., mosaic animals.
- the transgene may be integrated as a single transgene or in concatamers, e.g., head-to-head tandems or head-to-tail tandems.
- the transgene may also be selectively introduced into and activated in a particular cell type by following, for example, the teaching of Lasko et al. (Lasko, M. et al., Proc. Natl. Acad. Sci. USA 89:6232-6236, 1992).
- vectors containing some nucleotide sequences homologous to the endogenous BRS-3 gene of interest are designed for the purpose of integrating, via homologous recombination with chromosomal sequences, into and disrupting the function of, the nucleotide sequence of the endogenous BRS-3 gene.
- the transgene may also be selectively introduced into a particular cell type, thus inactivating the endogenous gene of interest in only that cell type, by following, for example, the teaching of Gu et al. (Gu, H. et al., Science 265:103-106, 1994).
- the regulatory sequences required for such a cell-type specific inactivation will depend upon the particular cell type of interest, and will be apparent to those of skill in the art.
- the expression of the recombinant BRS-3 gene and protein may be assayed utilizing standard techniques. Initial screening may be accomplished by Southern blot analysis or PCR techniques to analyze animal tissues to assay whether integration of the transgene has taken place. The level of mRNA expression of the BRS- 3 transgene in the tissues of the transgenic animals may also be assessed using techniques which include but are not limited to Northern blot analysis of tissue samples obtained from the animal, jn situ hybridization analysis, and RT-PCR. Samples of target gene-expressing tissue, may also be evaluated immunocytochemically using antibodies specific for the target gene transagene product of interest.
- BRS-3 transgenic animals that express BRS-3 gene mRNA or BRS-3 transgene peptide (detected immunocytochemically, using antibodies directed against target gene product epitopes) at easily detectable levels may then be further evaluated to identify those animals which display characteristic BRS-3/hemo ⁇ hin-ligand based disorder symptoms.
- BRS-3 transgenic founder animals are produced ⁇ le , those animals which express BRS-3 proteins in cells or tissues of interest, and which, preferably, exhibit symptoms of BRS- 3/hemorphin-ligand based disorders), they may be bred, inbred, outbred, or crossbred to produce colonies of the particular animal.
- breeding strategies include but are not limited to: outbreeding of founder animals with more than one integration site in order to establish separate lines; inbreeding of separate lines in order to produce compound BRS-3 transgenics that express the BRS-3 transgene of interest at higher levels because of the effects of additive expression of each BRS-3 transgene; crossing of heterozygous transgenic animals to produce animals homozygous for a given integration site in order to both augment expression and eliminate the possible need for screening of animals by DNA analysis; crossing of separate homozygous lines to produce compound heterozygous or homozygous lines; breeding animals to different inbred genetic backgrounds so as to examine effects of modifying alleles on expression of the BRS-3 transgene and the development of BRS-3/hemo ⁇ hin-ligand-like symptoms, one such approach is to cross the BRS-3 transgenic founder animals with a wild type strain to produce an F1 generation that exhibits BRS-3/hemo ⁇ hin-ligand related disorder-like symptoms, such as those described above. The F1 generation may then be inbre
- Another aspect of the invention relates to a method for inducing an immunological response in a mammal which comprises inoculating the mammal with the BRS-3 polypeptide, or a fragment thereof, if required together with a RAMP polypeptide, adequate to produce antibody and/or T cell immune response to protect said animal from said BRS-3/hemo ⁇ hin-ligand related dysfunctions or disorders as indicated supra.
- Yet another aspect of the invention relates to a method of inducing immunological response in a mammal which comprises delivering the BRS-3 polypeptide via a vector directing expression of the BRS-3 polynucleotide in vivo in order to induce such an immunological response to produce antibody to protect said animal from diseases.
- the invention relates to a method for inducing an immunological response in a mammal which comprises inoculating the mammal with the BRS-3 polypeptide, or a fragment thereof, if required together with a RAMP polypeptide, adequate to produce antibody and or T cell immune response to protect said animal from the BRS-3/hemo ⁇ hin-ligand related dysfunctions or disorders as indicated supra.
- a further aspect of the invention relates to an immunological/vaccine formulation (composition) which, when introduced into a mammalian host, induces an immunological response in that mammal to an BRS-3 polypeptide wherein the composition comprises an BRS-3 polypeptide or BRS-3 gene.
- the vaccine formulation may further comprise a suitable carrier.
- the BRS-3 polypeptide may be broken down in the stomach, it is preferably administered parenterally (including subcutaneous, intramuscular, intravenous, intradermal etc. injection).
- parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents or thickening agents.
- the formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampoules and vials and may be stored in a freeze-dried condition requiring only the addition of the sterile liquid carrier immediately prior to use.
- the vaccine formulation may also include adjuvant systems for enhancing the immunogenicity of the formulation, such as oil-in water systems and other systems known in the art. The dosage will depend on the specific activity of the vaccine and can be readily determined by routine experimentation.
- the BRS-3 polypeptide in the context of the present invention may be employed in a screening process for compounds which bind the receptor and which activate (agonists) or inhibit activation of (antagonists), or modulate the activity of (modulators) the receptor polypeptide of the present invention.
- polypeptides of the invention may also be used to assess the binding of small molecule substrates and ligands in, for example, cells, cell-free preparations, chemical libraries; and natural product mixtures. These substrates and ligands may be natural substrates and ligands or may be structural or functional mimetics.
- BRS-3 polypeptides are responsible for biological functions, including pathologies.
- agonists are employed for therapeutic and prophylactic purposes for such conditions as the BRS-3/hemo ⁇ hin-ligand related dysfunctions, disorders or diseases as indicated supra.
- Antagonists may be employed for a variety of therapeutic and prophylactic purposes for such conditions as the BRS-3/hemo ⁇ hin-ligand related dysfunctions, disorders or diseases as indicated supra.
- the present invention may be employed in a screening process for compounds which bind the receptor and which activate (agonists) or inhibit activation of (antagonists), or modulate the activity of (modulators) the BRS-3 receptor protein. These screening assays are particularly suitable for screening compounds which are effective with regard to the BRS- 3/hemo ⁇ hin-ligand related dysfunctions or disorders as indicated supra.
- such screening procedures involve producing appropriate cells, which express the receptor polypeptide used in the present invention on the surface thereof and, if essential co- expression of RAMP's at the surface thereof.
- Such cells include cells from mammals, yeast, Drosophila or E. coli.
- Cells expressing the receptor (or cell membrane containing the expressed receptor) are then contacted with a test compound to observe binding, or stimulation or inhibition of a functional response.
- One screening technique includes the use of cells which express the receptor used in this invention (for example, transfected CHO cells) in a system which measures extracellular pH or intracellular calcium changes caused by receptor activation.
- compounds may be contacted with cells expressing the receptor polypeptide of the present invention.
- a second messenger response e.g., signal transduction, pH changes, or changes in calcium level, is then measured to determine whether the potential compound activates or inhibits the receptor.
- Another method involves screening for receptor inhibitors by determining modulation of a receptor-mediated signal, such as cAMP accumulation and/or adenylate cyclase activity.
- a receptor-mediated signal such as cAMP accumulation and/or adenylate cyclase activity.
- Such a method involves transfecting an eukaryotic cell with the receptor used in this invention to express the receptor on the cell surface. The cell is then exposed to an agonist to the receptor in the presence of a potential antagonist.
- the potential antagonist binds the receptor, and thus inhibits receptor binding, the agonist- mediated signal will be modulated.
- Another method for detecting agonists or antagonists for the receptor of the present invention is the yeast-based technology as described in U.S. Patent 5,482,835, inco ⁇ orated by reference herein.
- the assays may simply test binding of a candidate compound wherein adherence to the cells bearing the receptor is detected by means of a label directly or indirectly associated with the candidate compound or in an assay involving competition with a labeled competitor. Further, these assays may test whether the candidate compound results in a signal generated by activation of the receptor, using detection systems appropriate to the cells bearing the receptor at their surfaces. Inhibitors of activation are generally assayed in the presence of a known agonist and the effect on activation by the agonist by the presence of the candidate compound is observed.
- candidate compounds may be screened which show ligand binding to the BRS-3 receptors of the present invention.
- ligand binding is understood as to describe compounds with affinity to the BRS-3 receptors showing log EC50 values at least in the range of those found for LVV-H-7 or VV-H-7.
- the invention concerns a method of determining whether a substance is a potential ligand of BRS-3 receptor comprising
- the assays may simply comprise the steps of mixing a candidate compound with a solution containing a BRS-3 polypeptide to form a mixture, measuring the BRS-3 activity in the mixture, and comparing the BRS-3 activity of the mixture to a standard.
- the BRS-3 cDNA, protein and antibodies to the protein may also be used to configure assays for detecting the effect of added compounds on the production of BRS-3 mRNA and protein in cells.
- an ELISA may be constructed for measuring secreted or cell associated levels of BRS-3 protein using monoclonal and polyclonal antibodies by standard methods known in the art, and this can be used to discover agents which may inhibit or enhance the production of BRS-3 (also called antagonist or agonist, respectively) from suitably manipulated cells or tissues. Standard methods for conducting screening assays are well known in the art.
- BRS-3 antagonists include antibodies or, in some cases, oligonucleotides or proteins which are closely related to the ligand of the BRS-3, e.g., a fragment of the ligand, or small molecules which bind to the receptor but do not elicit a response, so that the activity of the receptor is prevented.
- the present invention relates to a screening kit for identifying agonists, antagonists, ligands, receptors, substrates, enzymes, etc. for BRS-3 polypeptides; or compounds which decrease or enhance the production of BRS-3 polypeptides, which comprises:
- BRS-3 polypeptide preferably that of SEQ ID NO: 2 or a variant thereof with amino acid replacement in position 26 of methionine by valine, or
- a cell membrane expressing a BRS-3 polypeptide preferably that of SEQ ID NO: 2 or a variant thereof with amino acid replacement in position 26 of methionine by valine; or
- component (a), (b), (c) or (d) may comprise a substantial component, and that the kit is preferably related to the dysfunctions, disorders or diseases as indicated supra in the context of the present invention.
- the present invention enables the person skilled in the art to identify compounds, e.g. candidate compounds, by means of screening methods involving the findings of the present invention, said compounds may reveal as prospective drug candidates in particular with respect to dysfunctions, disorders or diseases evoked by pathophysiological conditions related to the activities of BRS-3, in particular of hBRS-3, e.g. in pathophysiological conditions related to cell growth, cell proliferation, tumor development and cancer, e.g.
- the invention also relates to a candidate compound that modulates the interaction of LW-hemo ⁇ hin-7 or W-hemo ⁇ hin-7 with a BRS-3 polypeptide receptor, to an agonistic candidate compound with comparable binding as the ligands LW-hemo ⁇ hin-7 or W-hemo ⁇ hin-7 and that activates a BRS-3 polypeptide receptor, and to an antagonistic candidate compound that inhibits the interaction of LW-hemo ⁇ hin-7 or W- hemo ⁇ hin-7 with a BRS-3 polypeptide receptor.
- SCLC small cell lung carcinoma
- candidate compounds may easily be analyzed in their structure and chemical properties by today's well-established analytical means such as e.g. mass spectroscopy, nuclear magnetic resonance, infrared spectra, melting points, optical rotation if chiral compounds are involved, and elemental analysis.
- analytical means such as e.g. mass spectroscopy, nuclear magnetic resonance, infrared spectra, melting points, optical rotation if chiral compounds are involved, and elemental analysis.
- the invention also pertains to a process for preparing a candidate compound with a defined chemical structure capable of activating, modulating or inhibiting the interaction of histamine or of an analog thereof with a BRS-3 polypeptide receptor, said process is comprising the manufacture of a compound or of a pharmaceutically acceptable salt thereof by means of chemical synthesis, provided that the activity of the compound to activate, to modulate or to inhibit the interaction of LW-hemo ⁇ hin-7 or W-hemo ⁇ hin-7 with an BRS-3 polypeptide receptor is identifiable by a screening method according to the present invention (see supra).
- the invention in another aspect relates to a protein-ligand-complex comprising a BRS-3 polypeptide of at least 80% identity to the polypeptide of SEQ ID NO: 2 and a BRS-3-binding compound, preferably a compound with BRS-3-binding affinity of at least that of or being comparable to that of VV-H-7 or LVV-H-7.
- Such protein-ligand-complexes are particularly useful in drug design methods, lead structure finding, lead structure optimization and modulation methods. The methods are well known in the state of the art.
- combinatorial synthesis and multidimensional NMR-spectroscopy and its contribution to the understanding of protein-ligand-interactions see literature concerning e.g. combinatorial
- the invention also pertains to the use of a protein-ligand-complex comprising a BRS-3 polypeptide of at least 80% identity to the polypeptide of SEQ ID NO: 2 and a BRS-3-binding compound for the design and modulation or optimization of lead structures with BRS-3-binding or BRS-3-activity related to the hemo ⁇ hin ligands VV-H-7 andor LVV-H-7.
- This invention provides methods of treating abnormal conditions as stated above in relation to the hemo ⁇ hin ligands and furthermore related to both an excess of and insufficient amounts of BRS-3 activity.
- BRS-3 activity of BRS-3 is in excess
- One approach comprises administering to a subject an inhibitor compound (antagonist) as herein above described along with a pharmaceutically acceptable carrier in an amount effective to inhibit activation by blocking binding of hemo ⁇ hin ligands to the BRS-3 receptor, or by inhibiting interaction with a RAMP polypeptide or a second signal, and thereby alleviating the abnormal condition.
- soluble forms of BRS-3 polypeptides still capable of binding the hemo ⁇ hin ligand in competition with endogenous BRS-3 may be administered. Typical embodiments of such competitors comprise fragments of the BRS-3 polypeptide.
- expression of the gene encoding endogenous BRS-3 receptor can be inhibited using expression-blocking techniques.
- Known such techniques involve the use of antisense sequences, either internally generated or separately administered. See, for example, O'Connor, J Neurochem (1991) 56:560 in Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Florida USA (1988).
- oligonucleotides which form triple helices with the gene, can be supplied.
- oligomers can be administered per se or the relevant oligomers can be expressed in vivo.
- a therapeutically effective amount of a compound which activates BRS-3 receptor i.e., an agonist as described above
- a pharmaceutically acceptable carrier i.e., a pharmaceutically acceptable carrier
- gene therapy may be employed to effect the endogenous production of BRS-3 receptor by the relevant cells in the subject.
- a polynucleotide of the invention may be engineered for expression in a replication defective retroviral vector, as discussed above.
- the retroviral expression construct may then be isolated and introduced into a packaging cell transduced with a retroviral plasmid vector containing RNA encoding a polypeptide of the present invention such that the packaging cell now produces infectious viral particles containing the gene of interest.
- These producer cells may be administered to a subject for engineering cells in vivo and expression of the polypeptide in vivo.
- gene therapy see Chapter 20, Gene Therapy and other Molecular Genetic-based Therapeutic
- Peptides such as the soluble form of BRS-3 polypeptides, and agonists and antagonist peptides or small molecules, may be formulated in combination with a suitable pharmaceutical carrier.
- suitable pharmaceutical carrier comprise a therapeutically effective amount of the polypeptide or compound, and a pharmaceutically acceptable carrier or excipient. Formulation should suit the mode of administration, and is well within the skill of the art.
- the invention further relates to pharmaceutical packs and kits comprising one or more containers filled with one or more of the ingredients of the aforementioned compositions of the invention.
- Polypeptides and other compounds of the present invention may be employed alone or in conjunction with other compounds, such as therapeutic compounds.
- systemic administration of the pharmaceutical compositions include injection, typically by intravenous injection.
- Other injection routes such as subcutaneous, intramuscular, or intraperitoneal, can be used.
- Alternative means for systemic administration include transmucosal and transdermal administration using penetrants such as bile salts or fusidic acids or other detergents.
- penetrants such as bile salts or fusidic acids or other detergents.
- oral administration may also be possible.
- the dosage range required depends on the choice of peptide, the route of administration, the nature of the formulation, the nature of the subject's condition, and the judgment of the attending practitioner. Suitable dosages, however, are in the range of 0.1-100 ⁇ g/kg of subject. Wide variations in the needed dosage, however, are to be expected in view of the variety of compounds available and the differing efficiencies of various routes of administration. For example, oral administration would be expected to require higher dosages than administration by intravenous injection. Variations in these dosage levels can be adjusted using standard empirical routines for optimization, as is well understood in the art.
- Polypeptides used in treatment can also be generated endogenously in the subject, in treatment modalities often referred to as "gene therapy" as described above.
- cells from a subject may be engineered with a polynucleotide, such as a DNA or RNA, to encode a polypeptide ex vivo, and for example, by the use of a retroviral plasmid vector. The cells are then introduced into the subject.
- a polynucleotide such as a DNA or RNA
- NMB neuromedin B
- GRP gastrin releasing peptide
- VV-H-7 W-hemo ⁇ hin-7 and (LVV-H- 7), LW-hemo ⁇ hin-7
- GPCR G-protein coupled receptor
- SCLC small cell lung carcinoma human
- hBRS-3 bombesin receptor subtype 3 receptor, (PCR) polymerase chain reaction
- bombesin receptor subtypes mRNA in NCI N417, CHO-G ⁇ (16)-BRS-3, BHY, and HT-29 human tumor cell lines by RT-PCR is shown in Fig. 2.
- Transfected CHO-G ⁇ (16) cells (Molecular Devices Co ⁇ ., Sunnyvale, Canada) were grown in nutrient mixture F12 (HAM), 200 ⁇ g/mL hygromycin, 400 ⁇ g/ml G418.
- NCI-N417 cells (ATCC Number: CRL-5809) were grown in RPMI-1640, HT-29 (ATTC, HTB-38) in McCoy's 5a medium with 1.5 mM L-glutamine and BHY cells (DSMZ, ACC 404) in Dulbecco's MEM (4.5 g/l glucose), with 1 % L-glutamine and 1 % penicilin/streptomycin. All cells were cultivated with 1 % penicillin/streptomycin, 5 % FCS at 37 °C in a 5% CO2 atmosphere.
- RNA extraction, cDNA first strand synthesis, polymerase chain reaction (PCR), reverse transcription/polymerase chain reaction (RT-PCR), and DNA fluorescence sequencing were performed as described by Pardigol et al. (Proc. Natl. Acad. Sci.USA. 95 (1998): 6308-13) or by Magert et al. (J. Biol. Chem. 274 (1999): 444-50; and Regul. Pept. 73 (1998): 165-76).
- human hBRS-3 cDNA (Ace. No. L08893) was subcloned into the expression vector pcDNA 3.1 and transfected into a CHO-G ⁇ (16) expressing cell line (Molecular Devices Corp., Sunnyvale, Canada) using Effectene (Qiagen, Hilden, Germany). Cells were selected by G418 and hygromycin, single cell clones were propagated and tested for stable expression by Northern blotting using a digoxigenin DNA labeling and detection kit following the manufacturer's instructions (Roche, Mannheim, Germany).
- hBRS-3 activation was determined using a WST-1 proliferation assay kit under the protocol as described by the manufacturer (Roche Molecular Biochemicals, Mannheim, Germany). The survival assay was performed as described earlier by Ryan et al. (J. Pharmacol. Exp. Ther. (1998), 287:366-80).
- the corresponding fractions were applied to a preparative RP-C18 chromatography column (PrepPak Cartridge 300 A, 15-30 ⁇ m, Baker, Phillipsburg, NJ), with a gradient from 20 - 70 % B in 45 min, solvent A: 30% MeOH, 10 mM HCI; solvent B: MeOH 100 %, 10 mM HCI) fractionated and tested in the bioassay.
- the bioactive material was further purified using the same column with different eluents (solvent A: water, 0.1 % trifluoroacetic acid; solvent B: 80 % acetonitrile, 0.1 % trifluoroacetic acid; gradient from 20 - 50 % B in 45 min).
- the final purification step was performed with an analytical RP C18 column (4,6 x 250 mm, Aqua RP C18, Phenomenex, Aillesburg, Germany, solvent A: 0.1 % trifluoroacetic acid; solvent B: 80 % acetonitrile, 0.085 % trifluoroacetic acid, isocratic at 32,5 % B, flow rate: 0,7 ml/min).
- the purified fraction was freeze-dried and analyzed.
- CZE capillary zone electrophoresis
- VV-H-7 W-hemo ⁇ hin-7
- LW-hemo ⁇ hin-7 LW-hemo ⁇ hin-7
- V-H-7 V-hemo ⁇ hin-7
- neuromedin B NMB
- neuromedin C GFP
- plates clear, flat-bottom, black well 96-well plates (Costar);
- Media growth medium: Nut-Mix F-12 (HAM) with Glutamax (Gibco) supplemented with 10% fetal calf serum (Gibco);
- Incubator 5% C0 2 , 37°C (Nuaire).
- the method was worked as follows: Cells were seeded 24 hours or 48 hours prior to the experiment into black wall microplates. The cell density was 0.4x10 "4 cells/well for 48 hour incubation and 1.5x10 ⁇ cells/well for 24 hour incubation. All steps were done under sterile conditions.
- FLUO-4 Molecular Probes
- FLUO-4 Molecular Probes
- Dve/pluronic acid mixture Immediately before use, equal volumes of the dye stock and 20% pluronic acid were mixed. The dye and pluronic acid had a final concentration of 1mM and 10%, respectively.
- Probenicid 250mM stock solution: 710mg probenicid (Sigma) solubilized in 5ml 1 N NaOH and mixed with 5ml Hank ' s BSS without phenol red (Gibco) supplemented with 20mM HEPES.
- Loading-Buffer 10.5ml Hank ' s BSS without phenol red (Gibco) supplemented with 20mM HEPES, 105 ⁇ l probenicid, 210 ⁇ l 1M HEPES.
- Wash-Buffer Hank ' s BSS without phenol red (Gibco) supplemented with 20mM HEPES (Gibco) and 2.5mM probenicid.
- the method was worked as follows: The 2mM stock of dye was mixed with an equal volume of 20% (w/v) pluronic acid immediately before adding to the loading-Buffer. The growth-medium was aspirated out of the well without distu ⁇ ing the confluent cell layer. 100 ⁇ l loading medium was dispensed into each well using a Multidrop (Labsystems). Cell were incubated in a 5% C0 2 , 37°C incubator for 30 minutes. In order to calculate the background fluorescence, some wells were not dye loaded. The background fluorescence in these wells results from autofluorescence of the cells. After dye loading, cell were washed three times with Wash-Buffer (automated Denley cell washer) to reduce the basal fluorescence to 20.000-25.000 counts above background. 100 ⁇ l buffer was added and cells were incubated at 37°C till start of the experiment.
- Wash-Buffer automated Denley cell washer
- NCI- N417 cells were loaded with medium containing 2 ⁇ M Fluo-4 (30 min, 37 °C), washed three times (centrifugation at 1200 ⁇ m, in HBSS/Hepes), seeded in 96-well plates (10 7 cells/ml) and centrifuged at 800 ⁇ m for 2 min.
- the cells were placed in the FLIPR (fluometric imaging plate reader system; Molecular Devices Co ⁇ ., Sunnyvale, Canada) and changes in cellular fluorescence were recorded after the addition of 50 ⁇ l tissue extract fractions or test ligands diluted in wash buffer.
- FLIPR fluometric imaging plate reader system
- Fig. 3 The effects of the peptides VVH-7, LVVH-7, GRP, NMB, VH-7 on intracellular Ca 2+ level changes in CHO-G ⁇ (16)-hBRS-3h cells (A) and in NCI N 417 cells (B) are shown in Fig. 3.
- the values given in Fig. 3 represent the maximal fluorescence change stimulated by the indicated peptides and are the means ⁇ SEM from at least eight independent experiments.
- CHO-G ⁇ (16)-hBRS-3 cells were generated and receptor activation was analyzed in the FLIPR assay. Based on hBRS-3 expression in placenta a peptide library was made to screen for endogenous ligands. Testing 240 fractions of this library on CHO- Galpha(16)-hBRS-3 cells, fractions 22 and 23 from pH-pool 4 generated an increase in intracellular Ca 2+ concentrations (Fig. 1 A). In contrast, these fractions were not active with control cells (CHO-G ⁇ (16)-expressing other GPCRs than BRS-3).
- W-hemo ⁇ hin-7 and LW-hemo ⁇ hin-7 were isolated from human placenta in four isolation steps. Over 80 % of the biologically inactive peptides were separated by a preparative C18 chromatography column; biological activity was detected in fractions 10 and 11 (Fig. 1 B). Changing the mobile phase from MeOH/HCI to acetonitrile/TFA subsequently the biological activity was detected in fractions 23 and 24 (Fig. 1 C). Finally, further purification steps using a semipreparative RP C4 chromatography column and an analytical C18 chromatography column resulted in two bioactive fractions (Fig. 1 D). Fraction 1 was pure as shown by capillary zone electrophoresis (CZE).
- CZE capillary zone electrophoresis
- bombesin peptides can bind with varying affinity to hGRP-R, hNMB-R and hBRS-3, it was examined by RT-PCR which receptors are expressed on the human cell lines studied, NCI-N417,
- the NCI-N417 cells and CHO-G ⁇ (16)-hBRS-3 were positive for hBRS-3, and negative for hGRP-R and hNMB-R.
- hGRP-R or hNMB-R could only be detected in
- GRP and NMB are present in low concentrations (pM) in plasma and tissue extracts (Haraguchi et al., (1988), Gastroenterol. Jpn. 23:247-50; and Namba et al., (1985) Neuroscience 15:1217-26), whereas LVV-H-7 was found in high concentrations (1-10 ⁇ M, approximately 100.000 fold higher) in bronchoalveolar lavage fluid of patients with non-small cell lung cancer (Duethman et al.,
- the isolated hemo ⁇ hin peptides W-hemo ⁇ hin-7 and LW-hemo ⁇ hin-7 qualify as endogenous ligands for the o ⁇ han receptor hBRS-3.
- the affinities of as deduced from the EC50 values are in the range of the previously known bombesin peptide agonists GRP and NMB.
- the biological effects of the isolated hemo ⁇ hin peptides W- hemo ⁇ hin-7 and LW-hemo ⁇ hin-7 are in the micromolar range, pathophysiological conditions showing comparable concentrations of hemo ⁇ hins have been described in e.g. alveloar lavage and indicate a potential functional role for these peptides and their receptor, hBRS-3.
- Values represent the mean values from at least eight independent experiments. The concentrations which induce a Ca 2+ response in the FLIPR assay are indicated. The highest concentration used in the bioassay (300 ⁇ M) induced no specific Ca 2+ signal.
- the findings of the present invention provide the possibility to distinguish between the effects of different hemo ⁇ hin peptides and to analyse the physiological effects of VV-H-7 and and/or LVV-H-7 in reespect to the BRS-3 receptor in detail, in particular in the context of drug discovery.
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Cell Biology (AREA)
- Molecular Biology (AREA)
- Urology & Nephrology (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- General Health & Medical Sciences (AREA)
- Analytical Chemistry (AREA)
- Oncology (AREA)
- Microbiology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Pathology (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- General Physics & Mathematics (AREA)
- Hospice & Palliative Care (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
La présente invention concerne l'identification d'un ligand endogène spécifique pour le récepteur de sous-type 3 de bombesine couplé à la protéine G humaine (ci-après appelé récepteur de BRS-3 ('BRS-3'). L'invention porte notamment sur l'utilisation dudit récepteur dans la découverte de médicaments, de préférence dirigés contre certains dysfonctionnements ou certaines maladies, et de plus, de médicaments qui jouent un rôle dans la prévention, la suppression ou la correction desdits dysfonctionnements ou maladies. Dans ledit contexte, sont également décrits des polynucléotides de BRS-3, des polypeptides de BRS-3 codés par ces derniers, et l'utilisation de tels polynucléotides et polypeptides et leur production. L'invention concerne également l'inhibition ou l'activation de l'action de tels polynucléotides et polypeptides, un vecteur contenant lesdits polynucléotides, une cellule hôte contenant un tel vecteur et de tels animaux transgéniques dans lesquels le gène de BRS-3 est soit surexprimé, mal exprimé, sous-exprimé ou supprimé (animaux « knock-out »). L'invention concerne encore une méthode de criblage de composés pouvant agir en tant qu'agonistes ou antagonistes dudit récepteur de BRS-3 couplé à la protéine G ou modulant l'activité dudit récepteur de BSR-3 couplé à la protéine G, et un ligand endogène de BRS-3 ainsi que l'utilisation dudit ligand dans le traitement, la prévention, la suppression ou la correction desdits dysfonctionnements ou desdites maladies associés aux activités de BRS-3 en relation avec son ligand endogène spécifique.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU2002250872A AU2002250872A1 (en) | 2001-02-02 | 2002-01-31 | Hemorphins as endogenous ligands for human g-protein coupled bombesin receptor subtype 3 (brs-3) |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP01102383 | 2001-02-02 | ||
EP01102383.5 | 2001-02-02 | ||
US26718601P | 2001-02-08 | 2001-02-08 | |
US60/267,186 | 2001-02-08 |
Publications (2)
Publication Number | Publication Date |
---|---|
WO2002063305A2 true WO2002063305A2 (fr) | 2002-08-15 |
WO2002063305A3 WO2002063305A3 (fr) | 2003-10-16 |
Family
ID=26076456
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/EP2002/000978 WO2002063305A2 (fr) | 2001-02-02 | 2002-01-31 | Hemorphines servant de ligands endogenes pour recepteur de sous-type 3 de bombesine (brs-3) couple a la proteine g humaine |
Country Status (2)
Country | Link |
---|---|
AU (1) | AU2002250872A1 (fr) |
WO (1) | WO2002063305A2 (fr) |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6143521A (en) * | 1998-07-22 | 2000-11-07 | Smithkline Beecham Corporation | Human bombesin receptor subtype-3sb |
-
2002
- 2002-01-31 AU AU2002250872A patent/AU2002250872A1/en not_active Abandoned
- 2002-01-31 WO PCT/EP2002/000978 patent/WO2002063305A2/fr not_active Application Discontinuation
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6143521A (en) * | 1998-07-22 | 2000-11-07 | Smithkline Beecham Corporation | Human bombesin receptor subtype-3sb |
Non-Patent Citations (6)
Title |
---|
MANTEY SAMUEL A ET AL: "Discovery of a high affinity radioligand for the human orphan receptor, bombesin receptor subtype 3, which demonstrates that it has a unique pharmacology compared with other mammalian bombesin receptors." JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 272, no. 41, 1997, pages 26062-26071, XP002186411 ISSN: 0021-9258 * |
MANTEY SAMUEL A ET AL: "Rational design of a peptide agonist that interacts selectively with the orphan receptor, bombesin receptor subtype 3." JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 276, no. 12, 23 March 2001 (2001-03-23), pages 9219-9229, XP002186408 ISSN: 0021-9258 * |
OHKI-HAMAZAKI HIROKO ET AL: "Mice lacking bombesin receptor subtype-3 develop metabolic defects and obesity." NATURE (LONDON), vol. 390, no. 6656, 13 November 1997 (1997-11-13), pages 165-169, XP002186409 ISSN: 0028-0836 * |
RYAN RICHARD R ET AL: "Pharmacology and intracellular signaling mechanisms of the native human orphan receptor BRS-3 in lung cancer cells." JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS, vol. 287, no. 1, October 1998 (1998-10), pages 366-380, XP002186410 ISSN: 0022-3565 * |
SANDERSON K ET AL: "Modulation of peripheral inflammation by locally administered hemorphin-7." INFLAMMATION RESEARCH, vol. 47, no. 2, February 1998 (1998-02), pages 49-55, XP002186495 ISSN: 1023-3830 * |
YATSKIN O N ET AL: "LVV- and VV-hemorphins: comparative levels in rat tissues" FEBS LETTERS, ELSEVIER SCIENCE PUBLISHERS, AMSTERDAM, NL, vol. 428, no. 3, 29 May 1998 (1998-05-29), pages 286-290, XP004257956 ISSN: 0014-5793 cited in the application * |
Also Published As
Publication number | Publication date |
---|---|
AU2002250872A1 (en) | 2002-08-19 |
WO2002063305A3 (fr) | 2003-10-16 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US5874243A (en) | OLRCC15 receptor | |
US6020157A (en) | Polynucleotides encoding HFGAN72X receptor | |
JP2002512781A (ja) | Gタンパク質共役型7tm受容体(axor−1) | |
US5851798A (en) | Nucleic acid encoding human GPR14 receptor | |
EP0875565A2 (fr) | Nouveau récepteur couplé à la protéine G (HFGAN72Y) | |
EP0892051A2 (fr) | Clone heoad54 de CADN codant pour un récepteur 7-transmembranaire humaine | |
EP0899332A2 (fr) | HFIAO41, un récepteur couplé à la protéine G | |
US6010877A (en) | cDNA clone HE8CS41 that encodes a novel 7-transmembrane receptor | |
US6221627B1 (en) | cDNA clone HDPB130 that encodes a novel human 7-transmembrane receptor | |
EP0885960A2 (fr) | Récepteur couplé à la protéine g (H7TBA62) | |
US5871967A (en) | Cloning of a novel G-Protein coupled 7TM receptor | |
EP0913471A2 (fr) | Clone HNEAA81 de CADN codant pour un récepteur 7-transmembranaire humain | |
US6174994B1 (en) | 7TM receptor (H2CAA71) | |
JP2002505872A (ja) | 7回膜貫通gタンパク質共役受容体ecr673 | |
US5874252A (en) | Splicing variant of the Epstein-Barr virus-induced G-protein coupled receptor | |
EP0877083A1 (fr) | Récepteur humain (HCEPT09), connecté à la protéine G | |
US5976834A (en) | cDNA clone HNFJD15 that encodes a novel human 7-transmembrane receptor | |
US6008050A (en) | Human neurotensin receptor type 2 and splice variants thereof | |
EP0878479A2 (fr) | Récepteur couplé à la protéine G (HOFNH30) | |
EP0878542A2 (fr) | Clone HMTMF81 de cADN codant pour un nouveau récepteur 7-transmembranaire humain | |
US5994098A (en) | Human 7-TM receptor similar to murine frizzled-6 gene | |
US6277960B1 (en) | cDNA clone HNFDY20 that encodes a novel human 7-transmembrane receptor | |
US6277977B1 (en) | cDNA clone HAPOI67 that encodes a human 7-transmembrane receptor | |
EP0884387A2 (fr) | Récepteur 7-transmembrane humain HLWAR77 | |
EP0855443A2 (fr) | Clone de cADN HE8CH90 qui code pour un récepteur 7-transmembranaire |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AK | Designated states |
Kind code of ref document: A2 Designated state(s): AU CA CN DE IL JP US |
|
AL | Designated countries for regional patents |
Kind code of ref document: A2 Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR |
|
121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
DFPE | Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101) | ||
REG | Reference to national code |
Ref country code: DE Ref legal event code: 8642 |
|
122 | Ep: pct application non-entry in european phase | ||
NENP | Non-entry into the national phase in: |
Ref country code: JP |
|
WWW | Wipo information: withdrawn in national office |
Country of ref document: JP |