AU748425B2 - Mammalian melanocortin receptor agonists and antagonists for modulating feeding behavior in animals - Google Patents

Description

T

S&F Ref:. 454793D1

AUSTRALIA

PATENTS ACT 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT

ORIGINAL

Name and Address of Applicant: Actual Inventor(s): Address for Service: Invention Title: Oregon Health Sciences University 3181 S.W. Sam Jackson Park Road Portland Oregon 97201-3098 United States of America Roger D. Cone, Wei Fan, Bruce A. Boston, Robert A.

Kesterton, Dongsi Lu, Wenbiao Chen Spruson Ferguson St Martins Tower 31 Market Street Sydney NSW 2000 Mammalian Melanocortin Receptor Agonists and Antagonists for Modulating Feeding Behavior in Animals The following statement is a full description of this invention, including the best method of performing it known to me/us:- P PAir 'X DocmCits received on: w 5845c 1 MAMMALIAN MELANOCORTIN RECEPTOR AGONISTS AND ANTAGONISTS FOR MODULATING FEEDING BEHAVIOR IN ANIMALS BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to the cloning, expression and functional characterization of mammalian melanocortin receptor genes. The invention provides nucleic acid encoding mammalian melanocortin receptors, recombinant expression constructs comprising said nucleic acid, and mammalian cells into which said recombinant expression constructs have been introduced, and that express functional mammalian melanocortin receptors. The invention also provides a panel of such transformed mammalian cells expressing melanocortin receptors for screening compounds for receptor agonist and antagonist activity. The invention provides methods for using such panels of melanocortin receptor-expressing mammalian cells to specifically detect and identify agonists and antagonists for each melanocortin receptor, as well as patterns of agonist and antagonist activity of said compounds for the class of melanocortin receptors. Such screening methods provide a means for identifying compounds with patterns of melanocortin agonist and antagonist activity which is associated with the capacity to influence or modify physiological function and behavior, particularly metabolism and feeding behavior.

2. Background of the Invention The proopiomelanocortin (POMC) gene product is processed to produce a large number of biologically active peptides. Two of these peptides, a-melanocyte stimulating hormone (aMSH), and adrenocorticotropic hormone (ACTH) have well understood roles in control of melanocyte and adrenocortical function, respectively. Both of these hormones 25 are also found in a variety of forms with unknown functions, for example, y-melanocyte stimulating hormone (yMSH), which has little or no ability to stimulate pigmentation (Ling et al., 1979, Life Sci. 25: 1773-1780; Slominski et al., 1992, Life Sci. 50. 1103-1108). A melanocortin receptor gene specific for each of the aMSH, ACTH and yMSH hormones has been discovered by some of the present inventors (see US. Patent Nos. 5.280,112, 5,532,347 30 and 5,837,521, incorporated by reference herein). In addition, two other melanocortin *r /receptor genes have been discovered by some of the present inventors (see Lu et al, 1994, Nature 371: 799-802; Mountjoy et al, 1994, Molec. Endocrinol. 8: 1298-1308) and others (see Gantz et al., 1993, J. Biol. Chem. 268: 15174-15179 and Labbe et al., 1994, Biochem.

31: 4543-4549).

Along with the well-recognized activities of cMSH in melanocytes and ACTH in adrenal and pituitary glands, the melanocortin peptides also have a diverse array of biological activities in other tissues, including the brain and-immune system, and bind to specific receptors in these tissues with a distinct pharmacology (see, Hanneman et al., in Peptide Hormone as Prohormones, G. Martinez, ed. (Ellis Horwood Ltd.: Chichester, UK) pp. 53-82; DeWied Jolles, 1982, Physiol. Rev. 2: 976-1059 for reviews). A complete understanding of these peptides and their diverse biological activities requires the.isolation and characterization of their corresponding receptors. Some biochemical studies have been reported in the prior art.

Shimuze, 1985, Yale J. Biol. Med. 58: 561-570 discusses the physiology of melanocyte stimulating hormone.

Tatro Reichlin, 1987, Endocrinology 121: 1900-1907 disclose that MSH receptors are widely distributed in rodent tissues.

Sola et al., 1989, J. Biol. Chem. 26: 14277-14280 disclose the molecular weight characterization of mouse and human MSH receptors linked to radioactively and photoaffinity labeled MSH analogues.

Siegrist et al., 1991, J. Receptor Res. 1: 323-331 disclose the quantification of receptors on mouse melanoma tissue by receptor autoradiography.

Cone Mountjoy, U.S. Patent No. 5,532,347 disclose the isolation of human and mouse c-MSH receptor genes and uses thereof (incorporated herein by reference).

25 Cone Mountjoy, U.S. Patent No. 5,280,112 disclose the isolation of human and bovine ACTH receptor genes and uses thereof (incorporated herein by reference).

Mountjoy et al., 1992, Science 257: 1248-1251 disclose the isolation of cDNAs encoding mammalian ACTH and MSH receptor proteins.

POMC neurons are present in only two regions of the brain, the arcuate nucleus of the hypothalamus, and the nucleus of the solitary tract of the brain stem. Neurons from both sites project to a number of hypothalamic nuclei known to be important in feeding behavior, including the paraventricular nucleus, lateral hypothalamic area, and ventromedial hypothalamic nucleus. While previous reports have claimed both stimulatory and inhibitory effects of a-MSH on feeding behavior (see Shimizu et al., 1989, Life Sci. 45: 543-552; Tsujii et al., 1989, Brian Res. Bull. 23: 165-169), knowledge of specific melanocortin receptors, their location within the central nervous system and the necessary pharmacological tools were not sufficiently developed at that time to allow the resolution of this issue. The present inventors have shown herein that a novel antagonist of the MC-3 and MC-4 melanocortin receptors can substantially increase food consumption in animals engaged in normal or fast-induced feeding behavior. This is consistent with expression of both MC-3 and MC-4 receptor mRNAs at these sites in in situ hybridization studies (Roselli-Rehfuss et al., 1993, Proc. Nail.

Acad. Sci. USA 9: 8856-8860; Mountjoy et al., 1994, Molec. Endocrinol. 8: 1298- 1308). Moreover, the regulation of arcuate nucleus POMC gene expression is consistent with an inhibitory role for POMC in feeding behavior. POMC mRNA levels arc decreased following a fast (Bergendahl et al., 1992, Neuroendocrinol. 56: 913-920; 15 Brady et al., 1990, Neuroendocrinol. 2: 441-447), and a significant diurnal variation in POMC mRNA levels in the arcuate nucleus is seen in rat, with the nadir occurring around the onset of nighttime feeding at 1800 hrs (Steiner et al., 1994, FASEB J. 8: 479- 488).

Thus, the experimental evidence indicates that POMC neurons play an important 20 role in tonic inhibition of feeding behavior, wherein obesity results from a chronic disruption of this inhibitory tone by antagonism of central melanocortin receptors in at least one animal model (agouti).

These results reveal for the first time a need in the art for a means for characterizing mammalian melanocortin receptor agonists and antagonists in vitro for 25 the development of compounds that affect feeding behavior in animals.

SUMMARY OF THE INVENTION The present invention provides a biological screening system for identifying and characterizing compounds that are agonists or antagonists of mammalian meianocortin receptors. The biological screening system of the invention comprises a panel of transformed mammalian cells comprising a recombinant expression construct encoding 4 a mammalian melanocortin receptor, and expressing said receptor thereby. The invention provides such a panel of transformed mammalian cells wherein the panel comprises cells expressing each type of mammalian melanocortin receptor. Thus, the invention also provides nucleic acid encoding mammalian melanocortin receptors, recombinant expression constructs comprising said nucleic acid, and mammalian cells into which said recombinant expression constructs have been introduced, and that express functional mammalian melanocortin receptors. Methods for using such panels of melanocortin receptor-expressing mammalian cells to specifically detect and identify agonists and antagonists for each melanocortin receptor, as well as patterns of agonist and antagonist activity of said compounds for the class of melanocortin receptors, are also provided. Such screening methods provide a means for identifying compounds with patterns of melanocortin agonist and antagonist activity which is associated with the capacity to influence or modify metabolism and behaviour in animal, particularly feeding behaviour.

Herein disclosed is a biological screening panel for determining the melanocortin receptor agonist/antagonist profile of a test compound. The panel comprises a first mammalian cell comprising a recombinant expression construct encoding a mammalian melanocortin receptor that is the a-MSH (MC-1) receptor. The panel also comprises a second mammalian cell comprising a recombinant expression construct encoding a mammalian melanocortin receptor that is the ACTH (MC-2) receptor. The panel also comprises a third mammalian cell comprising a recombinant expression construct encoding a mammalian melanocortin receptor that is the MC-3 receptor. The panel also comprises a fourth mammalian cell comprising a recombinant expression construct encoding a mammalian melanocortin receptor that is the MC-4 receptor. The panel also comprises a fifth mammalian cell comprising a recombinant expression construct encoding a mammalian melanocortin receptor that is the MC-5 receptor. As provided by the invention, each mammalian cell expresses the melanocortin receptor encoded by the recombinant expression construct comprising said cell.

The melanocortin receptors encoded by the recombinant expression constructs comprising the transformed mammalian cells comprising the panel are mouse MC-1 30 receptor (SEQ ID Nos.:3 and human MC-1 receptor (SEQ ID Nos.:5 and human MC- 2 (ACTH) receptor (SEQ ID Nos.:7 and bovine MC-2 receptor (SEQ ID Nos.:9 and rat MC-3 receptor (SEQ ID Nos.:ll and 12), human MC-4 receptor (SEQ ID Nos.:15 and 16) and mouse MC-5 receptor (SEQ ID Nos.:17 and 18).

Also herein disclosed is a method for using the melanocortin receptor panel to identify 35 and characterize test compounds as melanocortin receptor agonists. In this embodiment, the method provided by the invention identifies a melanocortin receptor agonist, and comprises the steps of contacting each of the cells of the panel with a test compound to be characterized as an agonist of a mammalian melanocortin receptor and detecting binding of the test compound to each of the mammalian melanocortin receptors by assaying for a 40 metabolite produced in the cells that bind the compound. In a preferred embodiment, the detected metabolite is cAMP.

Each of the cells of the panel of mammalian cells expressing mammalian melanocortin receptors further comprises a recombinant expression construct encoding a cyclic AMP responsive element (CRE) transcription factor binding site that is operatively linked to a nucleic acid sequence encoding a protein capable of producing a detectable metabolite. In preferred embodiments, said protein is P-galactosidase, most preferably encoded by a nucleic acid comprising the recombinant expression construct identified as pCRE/p-galactosidase (as disclosed in Chen et al., 1994, Analyt. Biochem. 226: 349-354).

As provided by the invention, expression of the protein that produces the detectable metabolite is dependent on binding of the test compound to the melanocortin receptor expressed by each cell in the panel and the intracellular product of the cAMP as a result. In this embodiment, cAMP production results in expression of a protein capable of producing a detectable metabolite, the protein most preferably being P-galactosidase. In preferred embodiments, the detectable metabolite absorbs light to produce a colored product. Thus, this embodiment of the invention provides a panel of melanocortin receptor-expressing cells whereby melanocortin hormone binding results in the production of a colored product in proportion to the extent of cAMP production in the cell as a result of hormone receptor binding.

Also herein disclosed is a method for characterizing a compound as an antagonist of a mammalian melanocortin receptor. In this embodiment, the method comprises the steps of contacting each of the cells of the panel with an agonist of the mammalian melanocortin receptor in an amount sufficient to produce a detectable amount of a metabolite produced in the cells that bind the agonist, in the presence or absence of a test compound to be characterized as an antagonist of a mammalian melanocortin receptor, and detecting the amount of the metabolite produced in each cell in the panel in the presence of the test compound with the amount of the metabolite produced in each cell in the panel in the absence of the test compound. As provided by the assay, inhibition of the production of the detectable metabolite is used as an indication that the tested compound is a melanocortin receptor antagonist, which is further characterized quantitatively by the extent of said inhibition.

Each of the cells of the panel of mammalian cells expressing mammalian melanocortin receptors further comprises a recombinant expression construct encoding a cyclic AMP responsive element (CRE) transcription factor binding site that is operatively 30 linked to a nucleic acid sequence encoding a protein capable of producing a detectable metabolite. In preferred embodiments, said protein is P-galactosidase, most preferably encoded by a nucleic acid comprising the recombinant expression construct identified as pCRE/p-galactosidase. As provided by the invention, expression of the protein that produces the detectable metabolite is dependent on binding of the test compound to the S. 35 melanocortin receptor expressed by each cell in the panel. In preferred embodiments, the detectable metabolite absorbs light to produce a colored product. Thus, this embodiment of the invention provides a panel of melanocortin receptor-expressing cells whereby melanocortin hormone binding results in the production of a coloured product in proportion to the extent of cAMP production in the cell as a result of hormone receptor binding.

40 Also disclosed are melanocortin receptor agonists identified by the methods and using the screening panel of the invention. The agonist may be an agonist of the MC-3 mammalian melanocortin receptor, or an agonist of the MC-4 mammalian melanocortin receptor.

6 Also disclosed are melanocortin receptor antagonists identified by the methods and using the screening panel of the invention. The antagonist may be an antagonist of the MC- 3 mammalian melanocortin receptor, or an antagonist of the MC-4 mammalian melanocortin receptor.

The invention provides methods for characterizing mammalian melanocortin receptor agonists for the capacity to modify or influence metabolism and feeding behavior in an animal. In a first aspect, the invention provides a method for characterizing melanocortin receptor MC-3 or MC-4 agonists as inhibitors of feeding behavior in an animal, the method comprising the steps of providing food to an animal that has been deprived of food for at least 12 hours, with or without administering to the animal an MC-3 or MC-4 receptor agonist of the invention, and comparing the amount of food eaten by the animal after administration of the MC-3 or MC-4 receptor agonist with the amount of food eaten by the animal without administration of the MC-3 or MC-4 receptor agonist.

Therefore, according to a first embodiment of the invention, there is provided a method for characterising a compound as a mammalian melanocortin MC-3 or MC-4 receptor agonist that inhibits feeding behaviour in an animal, the method comprising: providing food to an animal that has been deprived of food for at least 12 hours with or without administering to the animal a candidate mammalian melanocortin MC-3 or MC-4 receptor agonist; and comparing the amount of food eaten by the animal with and without administration of the mammalian melanocortin MC-3 or MC-4 receptor agonist.

In another aspect, the invention provides a method for characterizing a melanocortin MC-3 or MC-4 receptor antagonist as a stimulator of feeding behavior in an animal. In this embodiment, the method comprises the steps of providing food to an animal not deprived of food for at least 12 hours, with or without administering to the animal an MC-3 or MC-4 receptor antagonist, immediately prior to the onset of darkness or nighttime, and comparing the amount of food eaten by the animal after administration of the MC-3 or MC-4 receptor antagonist with the amount of food eaten by the animal without administration of the MC-3 or MC-4 receptor antagonist.

30 Therefore, according to a second embodiment of the invention, there is provided a method for characterising a compound as a mammalian melanocortin MC-3 or MC-4 receptor antagonist that stimulates feeding behaviour in an animal, the method comprising: providing food to an animal that has been deprived of food for at least 12 hours with or without administering to the animal a candidate mammalian melanocortin 35 MC-3 or MC-4 receptor antagonist; and comparing the amount of food eaten by the animal with and without administration of the mammalian melanocortin MC-3 or MC-4 receptor antagonist.

The invention also provides methods for using melanocortin receptor agonists and antagonists according to the invention for modifying feeding behavior in an animal.

In a first aspect, the invention provides a method for stimulating feeding in an animal, the method comprising administering to the animal an MC-3 or MC-4 receptor antagonist.

In a preferred embodiment, the antagonists are administered systemically. In additional embodiments, the antagonists are administered intracerebroventricularly.

LA. I 7 In another aspect, the invention provides a method for inhibiting feeding in an animal, the method comprising administering to the animal an MC-3 or MC-4 receptor agonist. In a preferred embodiment, the agonists are administered systemically. In additional embodiments, the agonists are administered intracerebroventricularly.

Also disclosed herein are mammalian melanocortin receptor agonists having the general formula: A-B-C-D-E-F-G-amide wherein A is an aliphatic amino acid residue, including for example Leu, Ile, Nle and Met, as well as analogues and substituted derivatives thereof; B is an acidic amino acid residue, including for example Asp and Glu; C is a basic amino acid residue, such as His; D is an aromatic amino acid residue having a D- conformation, including D-Phe, D-Tyr and substituted derivatives thereof; E is a basic amino acid residue, for example Arg, Lys, homoArg, homoLys, and analogues or substituted derivatives thereof; F is Trp or substituted derivatives thereof; and G is Lys, homoLys or a substituted derivative thereof. In the peptide embodiments of the melanocortin receptor agonists of the invention, the peptide is cyclized by the formation of an amide bond between the side chain carboxyl group of the Asp or Glu residue at position B in the peptide, and the side chain amino group of the Lys or homoLys residue at position G. In preferred embodiments, the melanocortin receptor agonists of the invention are agonists of the MC-3 or MC-4 receptor.

According to a third embodiment of the invention, there is provided a mammalian melanocortin MC-3 or MC-4 receptor agonist having the general formula: A-B-C-D-E-F-G-amide wherein A is Leu, Ile, Met, or substituted analogues thereof; B is Asp, Glu, or substituted analogues thereof; C is His or substituted analogues thereof; D is D-Phe, D-Tyr or substituted analogues thereof; E is Arg, Lys, homoArg, homoLys, or substituted analogues thereof; F is Trp or substituted analogues thereof; G is Lys, homoLys or substituted analogues thereof; 30 and wherein the peptide is cyclised by the formation of an amide bond between the side chain carboxyl group of the Asp or Glu residue at position B in the peptide, and the side chain amino group of the Lys or homoLys residue at position G. Preferably the mammalian melanocortin MC-3 or MC-4 receptor agonist has the formula Ac-A4-cyclo(AspS-His6-D- Phe -Arg 8 -Trp 9 -Lys')aMSH-(4-10)-amide, wherein A is Leu, Ile, Met, or substituted 35 analogues thereof.

Also disclosed herein are mammalian melanocortin receptor antagonists having the general formula: A-B-C-D-E-F-G-amide wherein A is an aliphatic amino acid residue, including for example Leu, Ile, Nle and Met, 40 as well as analogues and substituted derivatives thereof; B is an acidic amino acid residue, including for example Asp and Glu; C is a basic amino acid residue, such as His; D is an aromatic amino acid residue having a D- conformation, including D-Nal and substituted *..derivatives thereof; E is a basic amino acid residue, for example Arg, Lys, homoArg, homoLys, and analogues or substituted derivatives thereof; F is Trp or substituted 8 derivatives thereof; and G is Lys, homoLys or a substituted derivative thereof. In the peptide embodiments of the melanocortin receptor antagonists of the invention, the peptide is cyclized by the formation of an amide bond between the side chain carboxyl group of the Asp or Glu residue at position B in the peptide, and the side chain amino group of the Lys or homoLys residue at position G. In preferred embodiments, the melanocortin receptor antagonists of the invention are antagonists of the MC-3 or MC-4 receptor.

According to a fourth embodiment of the invention, there is provided a mammalian melanocortin MC-3 or MC-4 receptor antagonist having the general formula: A-B-C-D-E-F-G-amide wherein A is Leu, Ile, Met, or substituted analogues thereof; B is Asp, Glu, or substituted analogues thereof; C is His or substituted analogues thereof; D is D-Nal or substituted analogues thereof; E is Arg, Lys, homoArg, homoLys, or substituted analogues thereof; F is Trp or substituted analogues thereof; G is Lys, homoLys or substituted analogues thereof; and wherein the peptide is cyclised by the formation of an amide bond between the side chain carboxyl group of the Asp or Glu residue at position B in the peptide, and the side chain amino group of the Lys or homoLys residue at position G. Preferably, the mammalian melanocortin MC-3 or MC-4 receptor antagonist has the formula Ac-A 4 -cyclo(Asp 5 -His 6

-D-

Nal(2) 7 -Arg'-Trp 9 -Lys' o )aMSH-(4-10)-amide, wherein A is Leu, Ile, Met, or substituted analogues thereof.

According to a fifth embodiment of the invention, there is provided the use of a mammalian melanocortin MC-3 or MC-4 receptor agonist having the general formula: A-B-C-D-E-F-G-amide wherein A is Leu, Ile, Nle, Met, or substituted analogues thereof; B is Asp, Glu, or substituted analogues thereof; C is His or substituted analogues thereof; D is D-Phe, D-Tyr or substituted analogues thereof; S 30 E is Arg, Lys, homoArg, homoLys, or substituted analogues thereof; F is Trp or substituted analogues thereof; G is Lys, homoLys or substituted analogues thereof; and wherein the peptide is cyclised by the formation of an amide bond between the side chain carboxyl group of the Asp or Glu residue at position B in the peptide, and the side 35 chain amino group of the Lys or homoLys residue at position G; for the manufacture of a medicament for inhibiting feeding behaviour in an animal.

According to a sixth embodiment of the invention, there is provided a medicament for inhibiting feeding behaviour in an animal comprising a mammalian melanocortin MC-3 or MC-4 receptor agonist having the general formula: 40 A-B-C-D-E-F-G-amide 0 Oa^^r 8a wherein A is Leu, Ile, Nle, Met, or substituted analogues thereof; B is Asp, Glu, or substituted analogues thereof; C is His or substituted analogues thereof; D is D-Phe, D-Tyr or substituted analogues thereof; E is Arg, Lys, homoArg, homoLys, or substituted analogues thereof; F is Trp or substituted analogues thereof; G is Lys, homoLys or substituted analogues thereof; and wherein the peptide is cyclised by the formation of an amide bond between the side chain carboxyl group of the Asp or Glu residue at position B in the peptide, and the side to chain amino group of the Lys or homoLys residue at position G, and a pharmaceutically or veterinary acceptable carrier.

According to a seventh embodiment of the invention, there is provided a method of inhibiting feeding behaviour in an animal, the method comprising administering to said animal an effective amount of a mammalian melanocortin MC-3 or MC-4 receptor agonist having the general formula: A-B-C-D-E-F-G-amide wherein A is Leu, Ile, Nle, Met, or substituted analogues thereof; B is Asp, Glu, or substituted analogues thereof; C is His or substituted analogues thereof; D is D-Phe, D-Tyr or substituted analogues thereof; E is Arg, Lys, homoArg, homoLys, or substituted analogues thereof; F is Trp or substituted analogues thereof; G is Lys, homoLys or substituted analogues thereof; and wherein the peptide is cyclised by the formation of an amide bond between the side chain carboxyl group of the Asp or Glu residue at position B in the peptide, and the side chain amino group of the Lys or homoLys residue at position G; or of a medicament comprising said receptor agonist.

According to an eighth embodiment of the invention, there is provided a mammalian melanocortin MC-3 or MC-4 receptor agonist having the general formula: A-B-C-D-E-F-G-amide wherein A is Leu, Ile, Nle, Met, or substituted analogues thereof; B is Asp, Glu, or substituted analogues thereof; C is His or substituted analogues thereof; D is D-Phe, D-Tyr or substituted analogues thereof; 35 E is Arg, Lys, homoArg, homoLys, or substituted analogues thereof; F is Trp or substituted analogues thereof; G is Lys, homoLys or substituted analogues thereof; and wherein the peptide is cyclised by the formation of an amide bond between the side chain carboxyl group of the Asp or Glu residue at position B in the peptide, and the side o. 40 chain amino group of the Lys or homoLys residue at position G; or a medicament comprising said receptor agonist, when used for inhibiting feeding behaviour in an animal.

According to a ninth embodiment of the invention, there is provided the use of a 0 4 mammalian melanocortin MC-3 or MC-4 receptor antagonist having the general formula: 8b A-B-C-D-E-F-G-amide wherein A is Leu, Ile, Nle, Met, or substituted analogues thereof; B is Asp, Glu, or substituted analogues thereof; C is His or substituted analogues thereof; D is D-Nal or substituted analogues thereof; E is Arg, Lys, homoArg, homoLys, or substituted analogues thereof; F is Trp or substituted analogues thereof; G is Lys, homoLys or substituted analogues thereof; and wherein the peptide is cyclised by the formation of an amide bond between the side chain carboxyl group of the Asp or Glu residue at position B in the peptide, and the side chain amino group of the Lys or homoLys residue at position G; for the manufacture of a medicament for stimulating feeding behaviour in an animal.

According to a tenth embodiment of the invention, there is provided a medicament for stimulating feeding behaviour in an animal comprising a mammalian melanocortin MC-3 or MC-4 receptor antagonist having the general formula: A-B-C-D-E-F-G-amide wherein A is Leu, Ile, Nle, Met, or substituted analogues thereof; B is Asp, Glu, or substituted analogues thereof; C is His or substituted analogues thereof; D is D-Nal or substituted analogues thereof; E is Arg, Lys, homoArg, homoLys, or substituted analogues thereof; F is Trp or substituted analogues thereof; G is Lys, homoLys or substituted analogues thereof; and wherein the peptide is cyclised by the formation of an amide bond between the side chain carboxyl group of the Asp or Glu residue at position B in the peptide, and the side chain amino group of the Lys or homoLys residue at position G, and a pharmaceutically or veterinary acceptable carrier.

According to an eleventh embodiment of the invention, there is provided a method of stimulating feeding behaviour in an animal, the method comprising administering to said 30 animal an effective amount of a mammalian melanocortin MC-3 or MC-4 receptor antagonist having the general formula: A-B-C-D-E-F-G-amide wherein A is Leu, Ile, Nle, Met, or substituted analogues thereof; B is Asp, Glu, or substituted analogues thereof; C is His or substituted analogues thereof; *i D is D-Nal or substituted analogues thereof; E is Arg, Lys, homoArg, homoLys, or substituted analogues thereof; F is Trp or substituted analogues thereof; G is Lys, homoLys or substituted analogues thereof; 40 and wherein the peptide is cyclised by the formation of an amide bond between the side chain carboxyl group of the Asp or Glu residue at position B in the peptide, and the side chain amino group of the Lys or homoLys residue at position G; Se or of a medicament comprising said receptor antagonist.

8c According to a twelfth embodiment of the invention, there is provided a mammalian melanocortin MC-3 or MC-4 receptor antagonist having the general formula: A-B-C-D-E-F-G-amide wherein A is Leu, Ile, Nle, Met, or substituted analogues thereof; B is Asp, Glu, or substituted analogues thereof; C is His or substituted analogues thereof; D is D-Nal or substituted analogues thereof; E is Arg, Lys, homoArg, homoLys, or substituted analogues thereof; F is Trp or substituted analogues thereof; G is Lys, homoLys or substituted analogues thereof; and wherein the peptide is cyclised by the formation of an amide bond between the side chain carboxyl group of the Asp or Glu residue at position B in the peptide, and the side chain amino group of the Lys or homoLys residue at position G; or a medicament comprising said receptor antagonist, when used for stimulating feeding behaviour in an animal.

According to a thirteenth embodiment of the invention, there is provided a peptidomimetic or organomimetic compound having mammalian melanocortin MC-3 or MC-4 receptor agonist activity, and which mimics the three-dimensional arrangement of the chemical constituents and sidechains of a peptide having the general formula: A-B-C-D-E-F-G-amide wherein A is Leu, Ile, Nle, Met, or substituted analogues thereof; B is Asp, Glu, or substituted analogues thereof; C is His or substituted analogues thereof; D is D-Phe, D-Tyr or substituted analogues thereof; E is Arg, Lys, homoArg, homoLys, or substituted analogues thereof; F is Trp or substituted analogues thereof; G is Lys, homoLys or substituted analogues thereof; and wherein the peptide is cyclised by the formation of an amide bond between the side chain carboxyl group of the Asp or Glu residue at position B in the peptide, and the side 30 chain amino group of the Lys or homoLys residue at position G.

According to a fourteenth embodiment of the invention, there is provided a method of designing a peptidomimetic or organomimetic compound having mammalian melanocortin MC-3 or MC-4 receptor agonist activity, said method comprising ascertaining the i pharmacophore of a peptide having the general formula: A-B-C-D-E-F-G-amide wherein A is Leu, Ile, Nle, Met, or substituted analogues thereof; B is Asp, Glu, or substituted analogues thereof; C is His or substituted analogues thereof; S. D is D-Phe, D-Tyr or substituted analogues thereof; 40 E is Arg, Lys, homoArg, homoLys, or substituted analogues thereof; F is Trp or substituted analogues thereof; G is Lys, homoLys or substituted analogues thereof; and wherein the peptide is cyclised by the formation of an amide bond between the side chain carboxyl group of the Asp or Glu residue at position B in the peptide, and the side 8d chain amino group of the Lys or homoLys residue at position G, and fitting a combination of suitable groups to said pharmacophore using computer aided drug design.

Peptidomimetic or organomimetic compounds having mammalian melanocortin MC-3 or MC-4 receptor agonist activity designed by such methods are also provided.

According to a fifteenth embodiment of the invention, there is provided a peptidomimetic or organomimetic compound having mammalian melanocortin MC-3 or MC-4 receptor antagonist activity, and which mimics the three-dimensional arrangement of the chemical constituents and sidechains of a peptide having the general formula: A-B-C-D-E-F-G-amide wherein A is Leu, Ile, Nle, Met, or substituted analogues thereof; B is Asp, Glu, or substituted analogues thereof; C is His or substituted analogues thereof; D is D-Nal or substituted analogues thereof; E is Arg, Lys, homoArg, homoLys, or substituted analogues thereof; F is Trp or substituted analogues thereof; G is Lys, homoLys or substituted analogues thereof; and wherein the peptide is cyclised by the formation of an amide bond between the side chain carboxyl group of the Asp or Glu residue at position B in the peptide, and the side chain amino group of the Lys or homoLys residue at position G.

According to a sixteenth embodiment of the invention, there is provided a method of designing a peptidomimetic or organomimetic compound having mammalian melanocortin MC-3 or MC-4 receptor antagonist activity, said method comprising ascertaining the pharmacophore of a peptide having the general formula: A-B-C-D-E-F-G-amide wherein A is Leu, Ile, Nle, Met, or substituted analogues thereof; S: B is Asp, Glu, or substituted analogues thereof; C is His or substituted analogues thereof; D is D-Nal or substituted analogues thereof; E is Arg, Lys, homoArg, homoLys, or substituted analogues thereof; F is Trp or substituted analogues thereof; SG is Lys, homoLys or substituted analogues thereof; .and wherein the peptide is cyclised by the formation of an amide bond between the I::i side chain carboxyl group of the Asp or Glu residue at position B in the peptide, and the side chain amino group of the Lys or homoLys residue at position G, and fitting a combination 35 of suitable groups to said pharmacophore using computer aided drug design.

Peptidomimetic or organomimetic compounds having mammalian melanocortin MC-3 or MC-4 receptor antagonist activity designed by such methods are also provided.

8e According to a seventeenth embodiment of the invention, there is provided the use of a peptidomimetic or organomimetic compound according to the invention having mammalian melanocortin MC-3 or MC-4 receptor agonist activity, for the manufacture of a medicament for inhibiting feeding behaviour in an animal.

According to an eighteenth embodiment of the invention, there is provided a medicament for inhibiting feeding behaviour in an animal, comprising a peptidomimetic or organomimetic compound according to the invention having mammalian melanocortin MC- 3 or MC-4 receptor agonist activity, and a pharmaceutically or veterinary acceptable carrier.

According to a nineteenth embodiment of the invention, there is provided a method of o1 inhibiting feeding behaviour in an animal, the method comprising administering to said animal an effective amount of a peptidomimetic or organomimetic compound according to the invention having mammalian melanocortin MC-3 or MC-4 receptor agonist activity, or a medicament comprising said compound.

According to a twentieth embodiment of the invention, there is provided a peptidomimetic or organomimetic compound according to the invention having mammalian melanocortin MC-3 or MC-4 receptor agonist activity, or a medicament comprising said compound, when used for inhibiting feeding behaviour in an animal.

According to a twenty-first embodiment of the invention, there is provided the use of a peptidomimetic or organomimetic compound according to the invention having mammalian melanocortin MC-3 or MC-4 receptor antagonist activity for the manufacture of a medicament for stimulating feeding behaviour in an animal.

According to a twenty-second embodiment of the invention, there is provided a medicament for stimulating feeding behaviour in an animal, comprising a peptidomimetic or organomimetic compound according to the invention having mammalian melanocortin MC- 25 3 or MC-4 receptor antagonist activity, or a medicament comprising said compound, and a pharmaceutically or veterinary acceptable carrier.

According to a twenty-third embodiment of the invention, there is provided a method "of stimulating feeding behaviour in an animal, the method comprising administering to said animal an effective amount of a mammalian melanocortin MC-3 or MC-4 receptor 30 antagonist according to the invention having mammalian melanocortin MC-3 or MC-4 receptor antagonist activity, or a medicament comprising said compound.

According to a twenty-fourth embodiment of the invention, there is provided a peptidomimetic or organomimetic compound according to according to the invention having mammalian melanocortin MC-3 or MC-4 receptor antagonist activity, or a medicament comprising said compound, when used for stimulating feeding behaviour in an animal.

••go It is an advantage of the present invention that it provides an in vitro screening method for characterizing compounds having melanocortin receptor activities that relate to feeding behavior in animals. Specifically, the invention advantageously provides means and methods for identifying compounds having melanocortin receptor agonist and/or antagonist activity that have been associated with either stimulating or inhibiting feeding behavior when administered to an animal. The invention thus provides an economical first step in screening compounds for the capacity to affect feeding behavior, including synthetic, peptidomimetic or organomimetic derivatives of melanocortin receptor agonists or antagonists as disclosed herein or elsewhere.

Specific preferred embodiments of the present invention will become evident from the following more detailed description of certain preferred embodiments and the claims.

DESCRIPTION OF THE DRAWINGS Figures IA and IB illustrate the nucleotide (SEQ ID No.: 3) and amino acid (SEQ ID No.: 4) sequence of the mouse melanocyte stimulating hormone receptor gene.

Figures 2A and 2B illustrate the nucleotide (SEQ ID No.: 5) and amino acid (SEQ ID No.: 6) sequence of the human melanocyte stimulating hormone receptor gene.

20 Figures 3A and 3B illustrate the nucleotide (SEQ ID No.: 7) and amino acid (SEQ ID No.: 8) sequence of the human adrenocorticotropic stimulating hormone receptor gene.

Figures 4A and 4B illustrate the nucleotide (SEQ ID No.: 9) and amino acid .(SEQ ID No.: 10) sequence of the bovine adrenocorticotropic stimulating hormone receptor gene.

Figures 5A and 5B illustrate the nucleotide (SEQ ID No.: 11) and amino acid (SEQ ID No.: 12) sequence of the rat melanocortin-3 receptor gene.

Figures 6A and 6B illustrate the nucleotide (SEQ ID No.: 15) and amino acid (SEQ ID No.: 16) sequence of the human melanocortin-4 receptor gene.

Figures 7A and 7B illustrate the nucleotide (SEQ ID No.: 17) and amino acid (SEQ ID No.: 18) sequence of the mouse melanocortin-5 receptor gene.

Figure 8 shows a graph of intracellular cAMP accumulation resulting from melanocyte stimulating hormone receptor agonist binding in human 293 cells transfected with a MSH receptor-encoding recombinant expression construct, wherein represents binding ofNDP-MSH, represents binding of ACTH and represents binding of aMSH.

Figure 9 illustrates the cAMP response of mouse YI cells to binding of Smelanocortin peptides to human melanocortin-2 (ACTH) receptor, as measured by the P-galactosidase assay described in Example 4, wherein represents binding to wildtype ACTH-R and represents binding to an ACTH-R variant.

Figure 10 illustrates the results of competition binding experiments of melanocortin peptides to cells expressing a recombinant expression construct encoding the rat melanocortin-3 receptor, wherein represents binding of NDP-MSH. -Arepresents binding of yMSH. represents binding of aMSH, represents binding of ACTH 4 0 and represents binding of ORG2766.

15 Figures 1 lA through lC illustrate the results of experiments showing intracellular cAMP accumulation caused by receptor-ligand binding in human 293 cells expressing the MC-3 receptor. In Figure 1 IA, represents binding of aMSH, -U- S represents binding of y 2 -MSH, represents binding of des-acetyl aMSH and -orepresents binding of ACTH,.3,. In Figure 11B, represents binding ofy,-MSH, -E- 20 represents binding of y 2 -MSH and represents binding of des-acetyl 'Y -MSH. In Figure 11C, represents binding ofACTH 4 represents binding of NDP-MSH and represents binding of ORG2766.

Figure 12 shows a graph of intracellular cAMP accumulation resulting from peptide binding to human melanocortin-4 receptor agonist in human 293 cells transfected with a MC-4 receptor-encoding recombinant expression construct, wherein represents binding ofACTH,,,,, represents binding of ACTH,.3,, represents binding of NDP-MSH, represents binding of aMSH, represents binding of y.- MSH, and represents binding of des-acetyl aMSH.

Figure 13 illustrates the results of cAMP accumulation and cAMP-dependent Pgalactosidase assays of melanocortin peptide binding to a rat melanocortin-5 receptor, wherein represents binding of aMSH, represents binding of P-MSH, and -orepresents binding of y-MSH, each determined using the P-gal method, and wherein -Urepresents binding of aMSH, represents binding of P-MSH, and represents binding ofy-MSH, each determined using the cAMP method.

Figure 14 illustrates the structure of the pCRE/ P-gal plasmid.

Figure 15 illustrates the results of the P-galactosidase-coupled, colorimetric melanocortin receptor binding assay using cells expressing each of the MC- 1, MC-3, MC4 or MC-5 receptors and contacted with aMSH or a variety of aMSH analogues, wherein represents binding of aMSH, represents binding of NDP-MSH, represents binding of SHU9128 (para-FI substituted), represents binding of SHU9203 (p-Cl substituted), represents binding ofSHU8914 (p-I substituted), and represents binding of SHU9119.

Figures 16A through 16 D show the results of the P-galactosidase-coupled, colorimetric melanocortin receptor binding assay to determine antagonist activity of melanocortin analogues SHU9119 and SHU8914 in cells expressing each of the 15 melanocortin receptors MC-3 and MC-4. In Figure 16A, represents binding of aMSH, represents binding of 1OOnM SHU9119, represents binding of SHU9119, and represents binding of InM SHU9119. In Figure 16B, represents *binding of aMSH, represents binding of 1OOnM SHU9119, represents binding SHU9119, and represents binding of O1nM SHU9119. In Figure 16C, -nrepresents binding of aMSH, represents binding of 1000nM SHU8914, -Arepresents binding of 100nM SHU8914, and represents binding of lOnM SHU8614.

gi In Figure 16D, represents binding of aMSH, represents binding of 100nM SHU8914, represents binding of 50nM SHU8914, and represents binding of 1OnM SHU8614.

Figure 17 shows the results of classic competition binding assays using the melanocortin analogues SHU9119 and SHU8914 at the MC3-R and MC-4 R receptors.

wherein represents binding of NDP-MSH, represents binding of SHU8914 (p-I substituted), and represents binding of SHU9119.

Figures 18A and 18B shows the results of cAMP accumulation experiments (performed using the P-galactosidase assay of Example 4) for rat MC-3 receptor (Figure 18A) and for mouse MC-4 receptor (Figure 18B). In Figure 18A, represents 11 binding of NDP-MSH, represents binding of MTII and represents binding of forskolin. In Figure 18B, represents binding of MTII, represents binding of NDP-MSH and represents binding of forskolin.

Figures 19A through 19C show the effect on food intake of intracerebroventricular administration of melanocortin analogue SHU9119 in mice. In Figure 19A, represents administration of acsf and represents administration of 6nmol of SHU9119 In Figuie 19B, represents administration of acsf(n=6) and represents administration of 6nmol of SHU9119 In Figure 19C, represents administration of acsf(n=I 1) and represents administration of 6nmol of SHU9119 (n=12).

Figures 20A through 20C show the effect on food intake of intraccrcbroventricular administration of melanocortin analogue MTII in mice. In Figure 20A, represents administration of acsf(n=8), represents administration of 0. nmol MTII represents administration of Inmol MTII and -A- .9° 15 represents administration of 3nmol MTII In Figure 20B, represents administration of acsf(n=12), represents administration of 3nmol MTII and 6nmol SHU91 I9 and represents administration of 3nmol MTII Figure 20D shows the effect on locomotor activity of intracerebroventricular administration of melanocortin analogue MTII in mice, wherein represents 20 administration of vehicle alone and represents administration of 3nmol MTII Figures 21A through 21D show the effect on food intake of intracerebroventricular administration of melanocortin analogue MTII in mice. In Figure 21A, represents administration of acsf and represents 25 administration of 3nmol MTII In Figure 21B, open bars represent administration of acsf solid bars represents administration of 1.18nmol neuropeptide Y (NPY; n=6) and stipled bars represents administration of 3nmol MTII and 1.18nmol NPY In Figure 21C, represents administration of acsf and represents administration of 3nmol MTII In Figure 21 D, represents administration of 100nmol MTII and represents administration of vehicle alone 12- DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The term "melanocortin receptor" as used herein reference to proteins having the biological activity of any of the disclosed melanocortin receptors, including the MC-1 (SEQ ID Nos.: 3, 4, 5 and MC-2 (ACTH; SEQ ID Nos.: 7, 8, 9 and 10), MC-3 (SEQ ID Nos.: 11 and 12), MC-4 (SEQ ID Nos.: 15 and 16) or MC-5 (SEQ ID Nos.: 17 and S18) receptors, as well as naturally-occurring and genetically-engineered allelic variations in these sequences.

Cloned nucleic acid provided by the present invention may encode MC receptor protein of any species of origin, including, for example, mouse, rat, rabbit, cat, and human, but preferably the nucleic acid provided by the invention encodes MC receptors of mammalian, most preferably rodent and human, origin.

The production of proteins such as the MC receptors from cloned genes by genetic engineering means is well known in this art. The discussion which follows is accordingly intended as an overview of this field, and is not intended to reflect the full .15 state of the art.

DNA which encodes MC receptors may be obtained, in view of the instant disclosure, by chemical synthesis, by screening reverse transcripts of mRNA from appropriate cells or cell line cultures, by screening genomic libraries from appropriate cells, or by combinations of these procedures, as illustrated below. Screening of mRNA 20 or genomic DNA may be carried out with oligonucleotide probes generated from the MC receptor gene sequence information provided herein. Probes may be labeled with a S. detectable group such as a fluorescent group, a radioactive atom or a chemiluminescent group in accordance with know procedures and used in conventional hybridization assays, as described in greater detail in the Examples below. In the alternative, MC 25 receptor gene sequences may be obtained by use of the polymerase chain reaction (PCR) procedure, with the PCR oligonucleotide primers being produced from the MC receptor gene sequences provided herein. See U.S. Patent Nos. 4,683,195 to Mullis et al. and 4,683,202 to Mullis.

MC receptor proteins may be synthesized in host cells transformed with a recombinant expression construct comprising a nucleic acid encoding each of the receptors disclosed herein. Such a recombinant expression construct can also be comprised of a vector that is a replicable DNA construct. Vectors are used herein either to amplify DNA encoding an MC receptor and/or to express DNA which encodes an MC receptor. For the purposes of this invention, a recombinant expression construct is a replicable DNA construct in which a DNA sequence encoding an MC receptor is operably linked to suitable control sequences capable of effecting the expression of the receptor in a suitable host cell. The need for such control sequences will vary depending upon the host selected and the transformation method chosen. Generally, control sequences include a transcriptional promoter, an optional operator sequence to control transcription, a sequence encoding suitable mrRNA ribosomal binding sites, and sequences which control the termination of transcription and translation. Amplification vectors do not require expression control domains. All that is needed is the ability to replicate in a host, usually conferred by an origin of replication, and a selection gene to facilitate recognition of transformants. See. Sambrook et al., 1990, Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Press: New York).

Also specifically provided by the invention are reporter expression constructs o. 15 comprising a nucleic acid encoding a protein capable of expressing a detectable phenotype, such as the production of a detectable reporter molecule, in a cell expressing the construct. Such constructs can be used for producing recombinant mammalian cell lines in which the reporter construct is stably expressed. Most preferably, however, the reporter construct is provided and used to induce transient expression over an 20 experimental period of from about 18 to 96 hrs in which detection of the reporter protein-produced detectable metabolite comprises an assay. Such reporter expression constructs are also provided wherein induction of expression of the reporter construct is controlled by a responsive element operatively linked to the coding sequence of the reporter protein, so that expression is induced only upon proper stimulation of the 25 responsive element. Exemplary of such a responsive element is a cAMP responsive element (CRE), which induces expression of the reporter protein as a result of an increase in intracellular cAMP concentration. In the context of the present invention, such a stimulus is associated with melanocortin receptor binding, so that a reporter construct comprising one or more CREs is induced to express the reporter protein upon binding of a receptor agonist to a MC receptor in a recombinantly transformed mammalian cell. Production and use of such a reporter construct is illustrated below in Example 14 Vectors useful for practicing the present invention include plasmids, viruses (including phage), retroviruses, and integratable DNA fragments fragments integratable into the host genome by homologous recombination). The vector replicates and functions independently of the host genome, or may, in some instances, integrate into the genome itself. Suitable vectors will contain replicon and control sequences which are derived from species compatible with the intended expression host. A preferred vector is the plasmid pcDNA/neo I. Transformed host cells are cells which have been transformed or transfected with recombinant expression constructs made using recombinant DNA techniques and comprising mammalian MC receptor-encoding sequences. Preferred host cells arc human 293 cells. Preferred host cells for the MC-2 (ACTH) receptor are YI cells (subclone OS3 or Y6). Transformed host cells are chosen that ordinarily express functional MC receptor protein introduced using the recombinant expression construct. When expressed, the mammalian MC receptor protein will S..typically be located in the host cell membrane. See, Sambrook et al., ibid.

15 Cultures of cells derived from multicellular organisms are a desirable host for recombinant MC receptor protein synthesis. In pnncipal, any higher eukarvotic cell culture is workable, whether from vertebrate or invertebrate culture. However, mammalian cells are preferred, as illustrated in the Examples. Propagation of such cells in cell culture has become a routine procedure. See Tissue Culture, Academic Press, 20 Kruse Patterson, editors (1973). Examples of useful host cell lines are human 293 cells, VERO and HeLa cells, Chinese hamster ovary (CHO) cell lines, mouse YI (subclone OS3), and W1138, BHK. COS-7, CV, and MDCK cell lines. Human 293 *oo cells are preferred.

Cells expressing mammalian MC receptor proteins made from cloned genes in accordance with the present invention may be used for screening agonist and antagonist compounds for MC receptor activity. Competitive binding assays are well known in the art and are described in the Examples below. Such assays are useful for drug screening of MC receptor agonist and antagonist compounds, as detected in receptor binding assays as described below.

One particular use of such screening assays are for developing drugs and other compounds useful in modifying or changing feeding behavior in mammals. The invention provides an assay system, comprising a panel of recombinant mammalian cells, heterologously expressing each of the MC receptors disclosed herein, wherein the panel is constructed of at least one cell line expressing an MC receptor, and most preferably comprising cells expressing each of the MC receptors. The invention provides such panels also comprising a detection means for detecting receptor agonist or antagonist binding, such as the reporter expression constructs described herein, using direct binding and competition binding assays as described in the Examples below. In the use of this panel, each MC receptor is assayed for agonist or antagonist patterns of binding a test compound, and a characteristic pattern of binding for all MC receptors is thereby determined for each test compound. This pattern is then compared with known MC receptor agonists and antagonists to identify new compounds having a pattern of receptor binding activity associated with a particular behavioral or physiological effect.

For example, provided herein is experimental evidence that MC-3 or MC-4 receptor antagonists are capable ofstimulating feeding in hungry animals, and that MC-3 or MC-4 agonists are capable of inhibiting feeding in animals otherwise stimulated to 15 eat. The invention provides an in vitro assay to characterize MC-3 and MC-4 o agonists/antagonists as a preliminary and economical step towards developing feeding behavior-modulating drugs for use in vivo.

These results on feeding behavior in vivo have been obtained with certain MC receptor binding analogues, SHU9119 and MTI. These compounds have the following 20 chemical structure: 9 0 oC H2N 'I/We Lys H /N AZsp j )-T-p N 0 0 N

NHW

His N Ne(2') I Ar H N

I

AHc-N -Ap s-Hi -Nal(2')7. *-Tr p-L o SMU-8119 16-

NH

2 Ac-Me'-.Ap.-His'-.Pthe -AgTrp Ly 'ONH

MT-I

Generally, those skilled in the art will recognize that peptides as described 15 herein may be modified by a variety of chemical techniques to produce compounds having essentially the same activity as the unmodified peptide, and optionally having other desirable properties. For example, carboxylic acid groups of the peptide, whether carboxyl-terminal or sidechain, may be provided in the form of a salt of a pharmaceutically-acceptable cation or esterified to form a ester, or converted to an amide of formula NRR 2 wherein R, and R, are each independently H or C,-C, 1 alkyl, or combined to form a heterocyclic ring, such as 5- or 6-membered. Amino groups of the peptide, whether amino-terminal or sidechain, may be in the form of a pharmaceutically-acceptable acid addition salt, such as the HCI, HBr, acetic, benzoic, toluene sulfonic, maleic, tartaric and other organic salts, or may be modified to alkyl or dialkyl amino or further converted to an amide. Hydroxyl groups of the peptide sidechain may be converted to C,-C 16 alkoxy or to a ester using wellrecognized techniques. Phenyl and phenolic rings of the peptide sidechain may be substituted with one or more halogen atoms, such as fluorine, chlorine, bromine or iodine, or with alkyl, alkoxy, carboxylic acids and esters thereof, or amides of such carboxylic acids. Methylene groups of the peptide sidechains can be extended to homologous C 2

-C

4 alkylenes. Thiols can be protected with any one of a 17 number of well-recognized protecting groups, such as acetamide groups. Those skilled in the art will also recognize methods for introducing cyclic structures into the peptides of this invention to select and provide conformational constraints to the structure that result in enhanced binding and/or stability. For example, a carboxyl-terminal or arino-terminal cysteine residue can be added to the peptide, so that when oxidized the peptide will contain a disulfide bond, thereby generating a cyclic peptide. Other peptide cyclizing methods include the formation of thioethers and carboxyl- and amino-terminal amides and esters.

Peptidomimetic and organomimetic embodiments are also hereby explicitly declared to be within the scope of the present invention, whereby the threedimensional arrangement of the chemical constituents of such peptido- and organomimetics mimic the three-dimensional arrangement of the peptide backbone and component amino acid sidechains in the peptide, resulting in such peptido- and organomimetics of the peptides of this invention having substantial biological activity.

15 It is implied that a pharmacophore exists for the receptor agonist and antagonist properties of these and related MC receptor binding analogues. A pharmacophore is an idealized, three-dimensional definition of the structural requirements for biological activity. Peptido- and organomimetics can be designed to fit each pharmacophore with current computer modeling software (computer aided drug design). MC receptor binding analogues derived using such software and comprising peptido- and organomimetics of SHU9119 and MTII and related analogues are within the scope of the claimed invention.

The MC receptor binding analogues, in particular those analogues that are MC-3 or MC-4 receptor agonists or antagonists are provided to be used in methods of influencing, modifying or changing feeding behavior in mammals in vivo. Specific examples of uses for the MC receptor binding analogues of the invention include but are not limited to treatment of eating disorders such as anorexia and obesity, and other pathological weight and eating-related disorders. Other examples are failure to thrive disorders and disease-related cachexia, such as occurs in cancer patients. Also within the scope of the analogues of the invention is use for enhancing appearance, athletic ability, or adjuvant to other therapies to treat disorders such as high blood pressure, high 18serum cholesterol, vascular and heart disease, stroke, kidney disease, diabetes and other metabolic disorders.

The Examples which follow are illustrative of specific embodiments of the invention, and various uses thereof. They set forth for explanatory purposes only, and are not to be taken as limiting the invention.

EXAMPLE 1 Isolation of an cMSH Receptor Probe by Random PCR Amplification of Human Melanoma cDNA Using Degenerate Oligonucleotide Primers In order to clone novel G-protein coupled receptors, cDNA prepared from RNA from human melanoma cells was used as template for a polymerase chain reaction (PCR)-based random cloning experiment. PCR was performed using a pair of degenerate oligonucleotide primers corresponding to the putative third and sixth transmembrane regions of G-protein coupled receptors (Libert et al., 1989, Science 244: 569-72; Zhou et al., 1990, Nature 347: 76-80). The PCR products obtained in this experment were characterized by nucleotide sequencing. Two novel sequences representing novel G-protein-coupled receptors were identified.

20 PCR amplification was performed as follows. Total RNA was isolated from a human melanoma tumor sample by the guanidinium thiocyanate method (Chirgwin et al., 1979, Biochemistry 18: 5294-5299). Double-stranded cDNA was synthesized from total RNA with murine reverse transcriptase (BRL, Gaithersburg, MD) by oligo-dT o o *priming (Sambrook et al., ibid.). The melanoma cDNA mixture was then subjected to 45 cycles of PCR amplification using 500 picomoles of degenerate oligonucleotide primers having the following sequence: Primer III (sense): GAGTCGACCTGTG(C/T)G(C/T)(C/G)AT(C/T)(A/G)CIIT(Gn)GAC(C/A)G(C/G)TAC (SEQ ID NO:1) 30 and Primer VI (antisense):

CAGAATTCAG(T/A)AGGGCAICCAGCAGAI(G/C)(G/A)(T/C)GAA

(SEQ ID NO:2) 19in 100 Vl of a solution containing 50 mM Tris-HCI (pH 2.5 mM MgCI, 0.01% gelatin, 200 uM each dNTP, and 2.5 units of Taq polymerase (Saiki et al., 1988, Science 239: 487-491). These primers were commercially synthesized by Research Genetics Inc. (Huntsville, AL). Each PCR amplification cycle consisted of incubations at 94°C for 1 min (denaturation), 45 C for 2 min (annealing), and72 C for 2 min (extension).

Amplified products of the PCR reaction were extracted with phenol/chloroform and precipitated with ethanol. After digestion with EcoRI and Sall, the PCR products were separated on a 1.2% agarose gel. A slice of this gel, corresponding to PCR products of 300 basepairs (bp) in size, was cut out and purified using glass beads and sodium iodide, and the insert was then cloned into a pBKS cloning vector (Stratagene, LaJolla, CA).

A total of 172 of such pBKS clones containing inserts were sequenced using Sequenase Biochemical Corp., Cleveland, OH) by the dideoxynucleotide chain termination method (Sanger et al., 1977, Proc. Natl. Acad. Sci. USA 74: 5463-5467).

Two types of sequences homologous to other G-protein coupled receptors were identified.

9 :EXAMPLE 2A Isolation of a Mouse czMSH (MC-I) Receptor cDNA Probes isolated in Example 1 was used to screen a Cloudman melanoma cDNA library in order to isolate a full-length cDNA corresponding to the cloned probe. One clone was isolated from a library of 5 x 10" clones screened as described below in Example 2B. This clone contained an insert of 2.6 kilobases The nucleotide sequence of the complete coding region was determined (see co-owned U.S. Patent No.

5,532,347, incorporated by reference); a portion of this cDNA comprising the coding region was sequenced and is shown in Figures IA and IB (SEQ ID Nos: 3 4).

EXAMPLE 2B Isolation of a Human ceMSH (MC-1) Receptor cDNA In order to isolate a human counterpart of the murine melanocyte aMSH receptor gene disclosed in Example 2A and co-owned U.S. Patent No. 5,532,347, a 20 human genomic library was screened at high stringency (50% formamide, 42 0 C) using the human PCR fragments isolated as described in Example 1. A genomic clone was determined to encode an human MSH receptor (SEQ ID NO:5). The human MSH receptor has a predicted amino acid sequence (SEQ ID NO:6) that is 75% identical and colinear with the mouse aMSH receptor cDNA sequence (Figures 2A and 2B, represented as human MSH-R). The predicted molecular weight of the human MSHR is 34.7kD.

EXAMPLE 2C Isolation of a Human ACTH (MC-2) Receptor cDNA For cloning the ACTH receptor a human genomic library was screened at high stringency (50% formamide, IM NaCI, 50nM Tris-HCl, pH 0.1 sodium pyrophosphate, 0.2% sodium dodecyl sulfate, 100g/ml salmon sperm DNA, 10X Denhardt's solution, 42°C), using the human PCR fragments isolated as described in Example 1 herein and U.S. Patent No. 5,280,112. incorporated by reference. A genomic clone was isolated that encodes a highly related G-coupled receptor protein (SEQ ID NO:7 and Figures 3A and 3B). The predicted amino acid sequence (SEQ ID NO:8) of this clone is 39% identical and also colinear, excluding the third intracellular loop and carboxy-terminal tail, with the human MSH receptor gene product. The predicted molecular weight of this putative ACTHR is 33.9 kilodaltons This clone was identified as encoding an MC-2 receptor based on its high degree of homology to the murine and human MSH receptors, and the pattern of expression in different tissue types, as described in Example 3 in U.S. Patent 5,280,112.

EXAMPLE 2D Isolation of a Bovine ACTH (MC-2) Receptor cDNA A bovine genomic DNA clone encoding the bovine counterpart of the MC-2 (ACTH) receptor was isolated from a bovine genomic library, essentially as described in Example 2C above, and its nucleotide sequence determined (as shown in Figures 4A and 4B; SEQ ID Nos:9 -21 22 EXAMPLE 2E Isolation of a Rat y-MSH (MC-3) Receptor cDNA The mouse aMSH receptor cDNA isolated as described in Example 2A and co-owned U.S. Patent No. 5,532,347 was used to screen a rat hypothalamus cDNA library at low s stringency (30% formamide, 5X SSC, 0.1% sodium pyrophosphate, 0.2% sodium dodecyl sulfate, 100ig/ml salmon sperm DNA, and 10% Denhardt's solution) at 42°C for 18h. A 1kb cDNA clone was isolated and sequenced as described in co-owned U.S. Patent No.

5,532,347, and this clone used to re-screen the rat hypothalamus cDNA library at high stringency (same conditions as above except that formamide was present at A cDNA clone approximately 2.0kb in length was isolated and analyzed as described in U.S. Patent No. 5,837,521, incorporated by reference; a portion of this cDNA comprising the coding region was sequenced and is shown in Figures 5A and 5B (SEQ ID Nos:l 1 12).

EXAMPLE 2F Isolation of a Human MC-4 Receptor DNA For cloning the MC-4 receptor, a human genomic library was screened at moderate stringency (40% formamide, 1M NaC1, 50mM Tris-HC1, pH 7.5, 0.1% sodium pyrophosphate, 0.2% sodium dodecyl sulfate, 100lg/ml salmon sperm DNA, Denhardt's solution, 42 0 using rat PCR fragments isolated as described in Example 1 herein, with the exception that the following primers were used for PCR: Primer II (sense):

GAGTCGACC(A/G)CCCATGTA(C/T)T(AGT)(C/T)TTCATCTG

(SEQ ID NO:13) and Primer VII (antisense):

CAGAATTCGGAA(A/G)GC(A/G)TA(G/T)ATGA(A/G)GGGGTC

(SEQ ID NO:14) A genomic clone was isolated that encodes a highly related G-coupled receptor protein (SEQ ID NO: 15 and Figures 6A and 6B) on a 1.9kb HindIII fragment. The predicted amino acid sequence (SEQ ID NO: 16) of this clone is 55-61% sequence a.

a.

identity with human MC-3 and MC-5 receptors, and 46-47% sequence identity with the human MC-I and MC-2 (ACTH) receptor.

EXAMPLE 2G Isolation of a Mouse MC-5 Receptor DNA One million clones from a mouse 129SVJ genomic library comprising 5,000,000 clones in the XFixlI vector (Stratagene) was screened at low stringency (hybridization in 40% formamide at 42°C, washing performed in 0.5X SSC at 60 0 C, as described above in Example 2E) using radiolabeled probed from the rat MC-3 and MC-4 receptors (as described in Examples 2E and 2F). Positively-hybridizing clones were isolated and sequenced, and the sequences obtained were compared to previously-isolated melanocortin receptor clones. One clone, comprising a previously-unknown sequence, was determined to encode the MC-5 melanocortin receptor. The nucleotide and amino acid sequences of this receptor are shown in Figures 7A and 7B (SEQ ID No.: 17 18).

EXAMPLE 3 Construction of a Recombinant Expression Construct, DNA Transfection and Functional Expression of the MCR Gene Products In order to produce recombinant mammalian cells expressing each of the melanocortin receptors of Example 2, cDNA from each receptor was cloned into a mammalian expression construct, the resulting recombinant expression construct transfected into human 293 cells, and cell lines generated that expressed the melanocortin receptor proteins in cellular membranes at the cell surface.

25 The mouse cMSH receptor was cloned by excising the entire coding region of the caMSH (MC-1) cDNA insert comprising a 2.1kb fragment and subcloning this fragment into the BamHI/Xhol sites of pcDNAI/neo expression vector (Invitrogen, San Diego, CA). The resulting plasmid was prepared in large-scale through one cycle of CsCI gradient ultracentrifugation, and 20 Ag of the plasmid transfected into each 100mm dish of 293 cells using the calcium phosphate method (see Chen Okayama, 1987, 7: 2745-2752). After transfection, cells were cultured in DMEM media supplemented with 10% calf serum in a 3% CO, atmosphere at 37°C. Selection was -23 performed with neomycin (G418; GIBCO) at a concentration of 1000 kg/ml; selection was started 72 hr after transfection and continued for 3 weeks.

The aMSHR is known to couple to G-proteins and thereby activate adenyl cyclase, increasing intracellular levels of cAMP (see Buckley Ramachandran, 1981, Proc. Nail. Acad. Sci. USA 78: 7431-7435; Grahame-Smith et al., 1967, J. Biol.

Chem. 242: 5535-5541; Mertz Catt, 1991, Proc. Natl. Acad. Sci. USA 88: 8525- 8529; Pawalek el al., 1976, Invest. Dermatol. 66: 200-209). This property of cells expressing the aMSH receptor was used analyze expression of the aMSH receptor in cell colonies transfected with the expression vectors described herein as follows. Cells (-lx10 6 were plated in 6-well dishes, washed once with DMEM containing 1% bovine serum albumin (BSA) and 0.5mM IBMX (a phosphodiesterase inhibitor), then incubated for 45 minutes at 37°C with varying concentrations of the melanotropic peptides aMSH, PMSH, yMSH, the MSH peptide analogues Nle 4 D-Phe 7 -aMSH (NDP-MSH), and ACTH. Following hormone treatment, the cells were washed twice 15 with phosphate buffered saline and intracellular cAMP extracted by lysing the cells with Iml of 60% ethanol. Intracellular cAMP concentrations were determined using an assay (Amersham) which measures the ability of cAMP to displace [8- 3 Hj cAMP from a high affinity cAMP binding protein (see Gilman, 1970, Proc. Natl. Acad. Sci.

USA 67: 305-312).

The results of these experiments are shown in Figure 8. The abscissa indicates the concentration of each hormone and the ordinate indicates the percentage of basal intracellular cAMP concentration achieved by each treatment. Points indicate the mean of duplicate incubations; the standard error did not exceed 15% for any data point. None of the peptides tested induced any change in intracellular cAMP in cells containing the vector alone. Cells expressing the murine aMSH receptor responded to melanotropic peptides with a 2-3 fold elevation of intracellular cAMP, similar to levels of cAMP induced by these peptides in the Cloudman cell line (see Pawalek, 1985, Yale J. Biol. Med. 58: 571-578). The ECso values determined for aMSH (2.0x10O-M), ACTH (8.0x1ld M) and the superpotent MSH analogue NDP-MSH (2.8x10 correspond closely to reported values (see Tatro et al., 1990, Cancer Res. 50: 1237-1242). As expected, the PMSH peptide had an ECso value comparable 24 to aMSH, while yMSH had little or no activity (see Slominski et al.,1992, Life Sci. 1103-1108), confirming the identity of this receptor as a melanocyte aMSH receptor.

A similar series of experiments were performed using mouse Y1 cells (subclone OS3; Schimmer et al., 1995, J. Cell. Physiol. 163: 164-171) expressing the human and bovine MC-2 (ACTH) receptor clones of Examples 2C and 2D. These results are shown in Figure 9, where the extent of cAMP responsive element-linked P-galactosidase activity (see Example 4, below) is shown with increasing concentrations of ACTH.

The entire coding region of the MC-3 receptor cDNA insert, obtained as described above in U.S. Patent No. 5,837,521, was contained in a 2.0kb restriction enzyme digestion fragment and was cloned into the BamHI/XhoI sites of pcDNA/neo I expression vector (Invitrogen, San Diego, CA). The resulting plasmid was prepared in large-scale through one cycle of CsCl gradient ultracentrifugation and 20p.g pcDNA/MC-3 receptor DNA were transfected into each 100mm dish of 293 cells using the calcium phosphate method (see Chen Okayama, 1987, Mol. Cell. Biol. 7: 2745-2752). After transfection, cells were cultured in DMEM media supplemented with 10% calf serum in a 3% CO 2 atmosphere at 37°C. Selection was performed with neomycin (G418; GIBCO) at a concentration of 1000 pg/ml; selection was started 72h after transfection and continued for 3 weeks.

Specific binding of melanocortin peptides to cells expressing the MC-3 receptor was demonstrated by competition experiments using 1 25 I-labeled Nle 4 -D-Phe -a-MSH (NDP- MSH, as described in Tatro et al., 1990, Cancer Res. 50: 1237-1242). Suspended cells (2x 105) were incubated at 37°C with 500,000 cpm of labeled peptide for 10min in binding buffer (Ham's F10 media plus 10mM HEPES, pH 7.2, 0.25% bovine serum albumin, 500K SI. IU/ml aprotinin, 100 pg/ml bacitracin and ImM 1,10-phenanthroline) in the presence or 'absence of the indicated concentrations of peptides. Maximum labeling was achieved within 25 The results of these experiments are shown in Figure 10. Labeled NDP-MSH binding to cells expressing the MC-3 receptor, produced as described above, is inhibited by competition with unlabeled peptides known to be melanocortin receptor agonists, having a relative order of potency as follows: o° NDP-MSH y-MSH a-MSH ACTH 4 0 ORG2766.

Approximate 1K values derived from this experiment are as shown in Table I: TABLE 1 Agonist (apErox) NDP-MSH 2 x y-MSH 5 x a-MSH 1 x 10 7 ACTH4-, 8 x cAMP production assays as described above were also used to analyze expression of MC3-R in cells transfected with the expression vectors described herein as follows. Cells (-5x106) were plated in 6-well dishes, washed once with DMEM containing 1% bovine serum albumin (BSA) and 0.5mM IBMX (a phosphodiesterase inhibitor), then incubated for h at 37 0 C with varying concentrations of the melanotropic :i peptides aMSH, y 3 MSH, yMSH, the MSH peptide analogues NIe 4 -D-Phe 7 -aMSH NDP-MSH), ACTH 4 0 and ACTH,.

3 Following hormone treatment, the cells were washed twice with phosphate buffered saline and intracellular cAMP extracted by lysing 20 the cells with Iml of 60% ethanol. Intracellular cAMP concentrations were determined using an assay which measures the ability of cAMP to displace cAMP from a high affinity cAMP binding protein (see Gilman, 1979, Proc. Natl. Acad. Sci. USA 67: 305-312).

The results of these experiments are shown in Figures 1 1A through I 1C. The 25 abscissa indicates the concentration of each hormone and the ordinate indicates the percentage of basal intracellular cAMP concentration achieved by each treatment. Points indicate the mean of duplicate incubations; the standard error did not exceed 15% for any data point. Figure 11 A depicts the results of experiments using peptides found in vivo; Figure 11B depicts results found with y-MSH variants; and Figure 11C shows results of synthetic melanocortin analogues. None of the peptides tested induced any change in intracellular cAMP in cells containing the vector alone. Cells expressing rat MC3-R responded strongly to every melanotropic peptide containing the MSH sequence -26 His-Phe-Arg-Trp, with up to a 60-fold elevation of intracellular cAMP levels. EC,, values ranged from 1-50 nM. The most potent ligand and the one having the lowest ECso was found to be yMSH. The order of potency for the naturally occurring melanocortins was found to be: y 2 -MSH= yMSH aMSH ACTH,.3 y 3 -MSH des-acetyl-aMSH ACTH,.

Ecs values for these compounds are shown in Table II: TABLE II onist -EC- NDP-MSH I x 10- 9 y,-MSH 3 x y,-MSH 3 x a-MSH 4 x

ACTH,

39 4 x y 3 -MSH 6 x 10 9 desacetyl-oMSH 8 x ACTH 0 1 x 0- 7 Additionally, a synthetic melanocortin peptide (ORG2766), known to have the greatest activity in vivo in stimulation of retention of learned behavior and in stimulation of neural regeneration, was unable to stimulate MC3-R-mediated cAMP production, and was also inactive as an antagonist. The results strongly indicate that this peptide does not bind to MC3-R protein.

25 The MC-4 receptor was cloned in a 1.9kb HindIl genomic DNA fragment after PCR amplification of a lambda phage clone into pcDNAI/Neo (Invitrogen). This plasmid was stably introduced into human 293 cells by calcium phosphate coprecipitation using standard techniques, and plasmid-containing cells selected in G418 containing media. Specificity of receptor-hormone binding was assayed using adenylate cylcase activity as described above. The MC-4 receptor was found to couple to adenylate cyclase activity having the following pattern of agonist affinity: NDP-MSH des-acetyl-a-MSH ACTHI,3 c-MSH y2-MSH ACTHo, 27whereas the synthetic ACTH4., analogue ORG2766 showed no detectable binding to the MC-4 receptor. The results ofadenylate cyclase activity assays are shown in Figure 12.

ECo, values for each of the tested MC-4 receptor agonists are as shown in Table Il1: TABLE III gonist Ec- NDP-MSH 1.1 x lO-"M desacetyl-aMSH 4.9 x ACTH,.3 6.8 x a-MSH 1.5 x y 2 -MSH 107-M ACTH4- A 1.6kb Apal-HindIIl fragment comprising the entire coding sequence of the mouse MC-5 melanocortin receptor disclosed in Example 2G above was cloned into the pcDNA/neo expression vector (Invitrogen) after PCR amplification of the lambda phage clone. This plasmid was stably introduced into human 293 cells by calcium phosphate co-precipitation using standard techniques, and plasmid-containing cells selected in 20 G418 containing media. Specificity of receptor-hormone binding was assayed using adenylate cylcase activity as described above. The MC-5 receptor was found to couple to adenylate cyclase activity having the following pattern of agonist affinity: a-MSH PMSH y-MSH The results of adenylate cyclase activity assays are shown in Figure 13. ECs 0 values for 25 each of the tested MC-5 receptor agonists are: a-MSH=1.7 x 10' 9 M and IMSH 5 x EXAMPLE 4 Melanocortin Analogue Binding to Mammalian Melanocortin Receptors Recombinant cells prepared as described above in Example 3 were used to characterize receptor binding of two melanocortin analogues comprising cyclic lactam heptapeptides.

28 The melanocortin receptor analogue SHIJ9I 19 has the following chemical structure: 0z

NH

0-k 000.

000* 0 00 0 0 Ac-Nle'-cyclo(Asp 5 D-Nal(2) 7 Lys" 0 aMSH-(4- I 0)-arnide The melanoco-in receptor analogue MTIl has the following chemical structure: N

H

N ,NH

NH

2 His Q2Phe f AV~

NH

2 Ac-Nle 4 _cyc lo(Asp', His', D-Phe', Mg 8, Trp 9 Lys" 0 aMSH-(4. I 0)-amide -29 These analogues were prepared as described in Hruby et al. (1995, Med. Chem.

38: 3454-3461).

These analogues were tested for melanocortin receptor binding using a colonmetric assay system developed by some of the instant inventors (Chen et al., 1995, Aialyt. Biochem. 226: 349-354) as follows. A series of concatamers of the synthetic oligonucleotide: 5'-GAATTCGACGTCACAGTATGACGGCCATGG-3' (SEQ ID No:19) was produced by self-annealing and ligation and a tandem tetramer obtained. This fragment was cloned upstream of a fragment of the human vasoactive intestinal peptide (-93-+152; SEQ ID No.: 13; see Fink et al., 1988, Proc. Natl. Acad. Sci. USA 85: 6662- 6666). This promoter was then cloned upstream of the P-galactosidase gene from E.

coli. The resulting plasmid construct is shown in Figure 14.

Transient transfection of the pCRE/P-gal plasmid described above was performed as follows. Cells grown to between 40-60% confluency (corresponding to :about 1.5 million cells/6cm tissue culture plate) were incubated with Opti-MEM (GIBCO-BRL, Long Island, NY) and then contacted with a pCRE/p-gal-lipofectin complex which was prepared as follows. 3pg plasmid DNA and 20pL lipofectin reagent 20 (GIBCO) were each diluted into 0.5mL Opti-MEM media and then mixed together. This mixture was incubated at room temperature for 15-20 min., and then the mixture (ImL) added to each 6cm plate. Transfected plates were incubated at 37°C for 5-24h, after which the plates were washed and incubated with DMEM media (GIBCO) and the cells split equally into a 96-well culture plate.

25 To assay melanocortin receptor analogue binding, human 293 cells expressing each of the melanocortin receptors MC-1, MC-3, MC-4 and MC-5, and mouse YI cells expressing the MC-2 receptor, were transiently transfected with pCRE/P-gal as described above and assayed as follows. Two days after transfection, cells were stimulated with hormones specific for each receptor or hormone analogue by incubation for 6h at 37°C with a mixture comprising 10'12 10"M) hormone or analogue, 0.1mg/mL bovine serum albumin and 0.1mM isobutylmethylxanthine in DMEM. The effect of hormone or analogue binding was determined by p-galactosidase assay according to the method of Feigner et al. (1994, J Biol. Chem. 269: 2550-2561). Briefly, media was aspirated from culture wells and 50pL lysis buffer (0.25M Tris-HCI, pH 8/0.1% Triton-X 100) added to each well. Cell lysis was enhanced by one round of freezing and thawing the cell/lysis buffer mixture. 10[L aliquots were sampled from each well for protein determination using a commercially-available assay (BioRad, Hercules, CA). The remaining -from each well was diluted with 401.L phosphate buffered saline/0.5% BSA and 150uL substrate buffer (60mM sodium phosphate/ ImM MgC 2 10mM KCI/ 5mM Pmercaptoethanol/ 2mg/mL o-nitrophenyl-P-D-galactopyranoside) added. Plates were incubated at 37 0 C for Ih and then absorbance at 405nm determined using a 96-well plate reader (Molecular Devices, Sunnyvale, CA). A series of two-fold dilutions from of purified P-galactosidase protein (Sigma Chemical Co, St. Louis, MO) were assayed in parallel in each experiment to enable conversion of OD 40 to known quantity of 1galactosidase protein.

The results of these experiments are shown in Figure 15. This Figure shows the results of the P-galactosidase assay described above using cells expressing each of the MC-1, MC-3, MC-4 or MC-5 receptors and contacted with aMSH or a variety of cMSH analogues, including SHU9119. These results showed that SHU9119 had relatively weak agonist activity for both the human MC-3 and MC-4 receptors.

These results demonstrated the development of a colorimetric assay for cAMP accumulation as the result of melanocortin receptor binding to agonists and antagonists.

The action of MTII, SHU9119, and the endogenous mouse agouti peptide as agonists or antagonists of rodent MC receptors was first determined by examining their ability to elevate intracellular cAMP in 293 cell lines expressing the rat MC3-R or mouse MC4-R (expressed as ICso values representing ligand concentration required for ~half-maximal inhibition of binding of (I-125)-(Nle 4 D-Phe )a-MSH tracer).

Agonist/antagonist activity was also shown by demonstrating inhibition of cAMP elevation by the potent a-MSH analogue [Nle 4 D-Phe']a-MSH. using either a cAMPresponsive P-galactosidase reporter construct as described above, or by direct adenyl cyclase assay as described in Example 3 (wherein ECs 0 values represent ligand concentration required for half-maximal activation of a cAMP-responsivc Pgalactosidase reporter). Competition binding experiments were determined as the amount of radioactivity bound in the presence of 5xl0 6 M unlabeled [Nle 4 D-Phe]a- MSH, and was typically 3-5% of total counts bound.

31 In these experiments, murine agouti peptide was produced using a baculovirus system as descnbed by Lu etal. (1994, Nature 321: 799-802), with the modification that the agouti peptide was purified from baculovirus supematants by 0.6M NaCI step elution from an EconoS cation exchange column (BioRad). Agouti peptide used in these assays was approximately 60% pure.

Competition binding assays were performed to determine whether SHU9119 had antagonist activity towards aMSH binding to either the MC-3-or MC-4 receptors. These assays were performed as follows. Human 293 cells (100,000 cells/well in 24-well plates) expressing either the MC-3 or MC-4 receptors prepared as described above were incubated with a solution of Img/mL BSA in PBS containing ]00,000cpm (3.1 x 2 I](Nle 4 D-Phe')aMSH and varying concentrations of aMSH, (Nle 4 D-Phe 7

)MSH

or SHU9119. Cells were incubated for 30min at 37 0 C, washed twice with PBS-BSA, lysed with 0.5mL 0.5N NaOH, and counted using a y-counter to quantitate the amount of bound 2 D-Phe')aMSH. Control experiments showed non-specific binding to occur at about 3-5% levels, and this was taken into account when analyzing the Sexperimental results.

The SHU9119 analogue was found to be a potent antagonist of both the human MC-3 and MC-4 receptors, as shown in Figure 16. These assays showed pA, values of 9 8.3 and 9.3 for the human MC-3 and MC-4 receptors, respectively, as determined using the method of Schild (1947, Brit. J. Pharmacol. 2: 189-206). In contrast, no significant alteration in ICo, values was detected in binding experiments using this analogue with either the MC-3 or MC-4 receptors (Figure 17).

The activity of the MTII analogue was also assayed for melanocortin receptor ~agonist activity. These results are shown in Figures 18A and 18B, and confirmed that the MTII analogue is a specific agonist of the MC-3 and MC-4 receptors.

Specific competition of [Nle 4 ,D-Phe')a-MSH binding to rat MC-3 receptor by agouti peptide was observed, although accurate ICs 0 values could not be determined because the peptide preparation was not homogenous (results not shown). Specific competition of a-MSH activation of human MC4-R by agouti was previously disclosed (Lu et al., 1994, Nature 371: 799-802).

EXAMPLE Feeding Behavior Effect of Melanocortin Analogue Binding in Brain The results shown in Example 4 above suggested a role in the regulation of feeding behavior in mammalian brain for MC receptor agonists and antagonists, in view of the antagonist properties of the agouti peptide at the MC-3 and MC-4 receptors. The agouti peptide was known to cause obesity when expressed ectopically in the mouse, and has been found to be a high affinity antagonist of the melanocyte stimulating hormone receptor (MC1-R) and of the hypothalamic MC-4 receptor (see Lu et al., ibid.). The former activity explained the inhibitory effect of the agouti peptide on eumelanin pigment synthesis. Similarly, it was hypothesized by the inventors that agouti causes obesity in mice by antagonizing hypothalamic MC-4 receptors. The cyclic-melanocortin analogue, SHU91 19, having been shown herein and elsewhere (Hruby et al.) to be a specific, high affinity antagonist of the central MC-3 and MC-4 receptors, was tested for the effect of direct administration to mouse brain on feeding behavior in the animals.

Intracerebroventricular (ICV) administration of SHU9119 was performed to avoid any complications caused by inhibition of peptide traverse of the blood-brain barrier.

Briefly, male C57B I/6J mice (18-29g) were maintained on a normal 12hr/12hr light dark cycle with food (Purina mouse chow) and water ad libitum. Animals were housed individually for 24 hrs, distributed into experimental and control groups, avoiding any bias as a function of prior weight, then injected with vehicle or vehicle plus drug just prior to the onset of a 12hr light or dark cycle. Fasted animals were deprived of food from 18:00 to 10:30 hrs to stimulate feeding during the daytime experimental period. Animals were lightly anesthetized with halothane, and administered into one lateral ventricle 2 pL of a solution of artificial cerebrospinal fluid alone (acsf, 25 comprising 130mM NaCI, 27mM NaHCO,, 1.2mM Na HPQ, 0.3mM NaHI P Na 2

SO

4 1.0mM CaCI 2 1.OmM MgCl, and 2.5mM KCI), or 6nmol SHU9119 in acsf. Freehand injections were performed as described by Laursen and Belknap (1986, J Pharmacol. Methods I6: 355-357) with some modifications. A lOp luertip syringe (Hamilton 701LT) was fitted with a 0.5 inch 27 gauge needle. Stiff tygon tubing was slipped over the needle to expose 3mM of the needle tip. The syringe was held at a 450 angle from the front of the skull with the bevel facing up. The coronal suture was found by lightly rubbing the needle over the skull. Maintaining the 45 angle, the needle -33was then inserted 1-2mm lateral to the midline, using only mild pressure to insert and remove the needle. The compounds indicated in a 2pl volume of acsf were administered slowly over approximately 15 seconds, and the needle removed after 35 seconds.

Animals were allowed to recover from anesthesia and placed into a cage containing a premeasured quantity of food pellets in a spill-free cup. Moribund animals were not included in the study.

Stimulation of feeding by intracerebroventricular administration of the melanocortin antagonist SHU9119 is shown in Figures 19A through 19C. Curves show cumulative food intake as a function of time following administration of the substances shown. Figure 19A shows stimulation of feeding by administration of SHU9119 just prior to lights off(19:00 hrs) to C57B1/6J mice fed ad libitum. Figure 19B, in contrast, shows no effect of morning (10:00 hrs) SHU91 19 administration in C57BI/6J mice fed ad libitum. Figure 19C illustrates stimulation of daytime feeding by SHU9119 administration in fasted C57B1/6J mice. In deriving the data points comprising these 15 Figures, food remaining was briefly removed and weighted at the time intervals indicated. Data points indicate the mean and bars indicate standard error. Significance of the effect over time was determined by ANOVA with repeated measures.

Significance of drug effects at individual time points was determined by two-way ANOVA, and is indicated in each Figure These results demonstrated that ICV administration of SHU9119 into one lateral ventricle of the C57B1/6J mouse just prior to lights out led to a mean 60% increase in o food intake over 12 hrs (Figure 19A; P<0.005). In contrast, daytime food intake in animals fed ad libitum was not stimulated by administration of SHU9119 (Figure 19B).

SHU9119-treatment did, however, significantly stimulate daytime food intake in animals fasted for 16 hrs prior to the experiment (Figure 19C; P<0.001). Stimulation of feeding was evident at approximately two hrs post-treatment, and continued for 12 hrs, to produce a mean 34% in food intake relative to vehicle-injected controls.

These results supported the hypothesis that agouti and/or SHU9119 stimulate feeding by antagonizing MC receptors in the central nervous system. To further test this hypothesis, a series of experiments were performed wherein MC receptor agonists were administered to animals primed by fasting to eat, to determine whether feeding in such animals could be inhibited by the MC receptor agonists. Animals were induced to feed by food deprivation for 16h prior to ICV administration of the non-specific melanocortin agonist MTII. In these experiments, ICV injections in male C57B 1/6J mice (20-30g) and the measurement of food intake were performed as described above.

Results of these experiments are shown in Figures 20A through 20C. In comparison to vehicle-injected animals, MTII was found to produce a potent inhibition of feeding within one hour after administration (Figure 20A) in a dose-responsive manner. Food intake was significantly inhibited for up to four hours following administration (P<0.001) at the highest dose administered (3nmol), and decreased food intake continued for the next four hours with normal rates of food intake resuming at about 8 hours after treatment. This dose-responsive inhibition of feeding had an IC 50 at the two hour time point of approximately 0.5nmol (Figure 20B). However, inhibition of feeding with 3nmol MTII was completely blocked by co-administration of 6nmol SHU9119 (Figure 20C; P<0.001), demonstrating that the effect results specifically from agonist binding to the MC-4 and/or MC-3 receptor.

15 Locomotor assays were performed to determine whether the effects on feeding behavior observed in these mice were secondary to generalized behavioral effects caused by administration of these melanocortin analogues. The effects of MTII on locomotor activity were tested by placing vehicle or MTII-treated mice in sound and light-proof cages containing multiple light beam detectors. These assays were performed by first injecting 3nmol MTII or.acsf as described above. At three hours (2:45-3:25) postinjection, 12 mice were placed into 12 separate boxes containing multiple infrared light sources and photodetectors. The boxes were contained within separate ventilated light and sound attenuating chambers (Coulboum model E10-20). Disruption of the infrared beams, with a 10msec resolution, was tallied independently for each one minute time period in each cage. The results of these assays are shown in Figure 20D. Data points indicate the mean total activity of light breaks) for 6 animals in each experimental group. Four way ANOVA statistical analysis was used to analyze the data, and indicated an absence of a significant difference among the two groups.

Inhibition of feeding by MTII could not be explained by any apparent behavioral abnormalities, or any effect on arousal or locomotor activity. MTII-treated animals appeared alert and exhibited no unusual behavior relative to controls. At approximately three hours after ICV administration, MTII-treated animals exhibited locomotor activity 35 that was indistinguishable from vehicle-treated animals (Figure 20D). The higher ini"al activity, indicative of exploratory behavior, and continued locomotion over a 15 i 'm period was indistinguishable between the two groups, indicating that the inhibition of feeding was not due to decreased locomotion or decreased arousal.

The administration of MTII also inhibited food intake in three other models of hyperphagia: the C57B1/6J-Lepob mouse, a neuropeptide Y (NPY)-injected C57BI/6J mouse and a C57B1/6J-A mouse. Figure 21A shows inhibition of feeding by intraccrebroventricular administration of MTII in A' mice (females, 19-28gms). Figure 21B shows inhibition of feeding by intracerebroventricular administration of MTII in C57BI/6J mice (females, 21-25gm) stimulated to feed by co-administration of NPY.

Figure 21C shows inhibition of feeding by intracerebroventricular administration of the MTII in ob/ob mice (females, 48-69 gms). Figure 21D shows inhibition of feeding in ob/ob mice by intraperitoneal administration of MTII (females, 40-45 gms). ICV injections and measurement of food intake were performed as described above, with the 15 exception of NPY treated animals, which were not fasted prior to experimentation.

Animals treated intraperitoneally received 100pl ofa ImM solution of MTII in saline, and vehicle injections consisted of the same amount of saline alone. Significance indicated for individual time points, determined as described above, was for 3nmol MTII vs. acsf(Figure 21A), 1.18 nmol NPY vs. 1.18 nmol NPY 3 nmol MTII (Figure 21B), 3nmol MTII vs. acsf (Figure 21C), and 100 nmol MTII vs. saline (Figure 21D).

The hyperphagia in these models can be clearly seen by comparing the 12 hr food intake following a fast in vehicle-injected C57B1/6J (2.4g, Figure 19A), C57B1/6J-A (3.7g, Figure 21A) and C57B1/6J-Lepo b (3.7g. Figure 21C) animals. As expected, MTII treatment inhibited food intake following a 16 hr fast in the C57B1/6J-A mouse (Figure 21A; P<0.05). Interestingly, while food intake for the first four hours is significantly inhibited relative to vehicle-injected animals, it is also significantly less inhibited in the C57B 1/6J-A animal than in the C57BI/6J animal given the same 3nmol dose (compare, Figure 20A versus Figure 21A, 1-4 hrs; P<0.001). The decreased effectiveness of the agonist in the presence of the A r allele is consistent with the proposal that this allele results in chronic expression of agouti peptide melanocortin antagonist in the brain.

MTII, upon co-administration, also significantly inhibited the profound stimulation of feeding induced by NPY, measured over a three hr period (Figure 21C; P<0.005). Co-administration of an approximately two-fold molar excess of MTII produced a 74% inhibition of NPY-stimulated food intake at the three hour time point.

Finally, MTII also inhibited hyperphagia due to absence of leptin in the C57B /6J-Lepb mouse (Figure 21C; P<0.001). MTII potently blocked feeding (Figure 20A) in these animals, in contrast to the less potent inhibition described above for the C57B 1/6J-A mouse.

The C57B/6J-Lepb animal was also used to test the ability of MTII to regulate feeding when administered peripherally. Moderate doses (100nmol) of MTII inhibited feeding in the C57Bl/6J-Lepob mouse (P<0.001) while low doses 1nmol) did not (date not shown). The kinetics were similar to those seen with ICV administration, with a potent inhibition of feeding for the first four hours. The 100-fold higher dose required peripherally, as well as the similar kinetics, suggest a primarily central nervous system -based mechanism of action of MTII.

These data show that melanocortinergic neurons exert a tonic inhibition of 15 feeding behavior, and that disruption of this signal leads to hyperphagia. With regard to the recently-discovered leptin hormone made by adipocytes, which is generally expressed at elevated levels in obese humans and rodents (such as the C57B1/6J-Lepb animal), the regulatory defect is understood to be an incapacity to respond properly to the leptin hormone signal. The instant results indicate that the melanocortins act independently, and physiologically "downstream," from the leptin hormone/receptor interaction, because it has been shown herein that melanocortin receptor agonists can potently inhibit feeding in the C57B 1/6J-Lepb animal.

These results suggest that MC receptor agonists and antagonists can affect mammalian feeding behavior, and provide a means for determining candidate compounds for the development of effective pharmacological products directed towards alleviating such human ailments as obesity, anorexia and cachexia.

9 EXAMPLE 6 Use of MC Receptor-Expressing Recombinant Cells for Screening Compounds that Affect Feeding Behavior in Mammals The results obtained in Example 5 indicated that cells expressing a variety of mammalian melanocortin receptors are useful for characterizing compounds as a first 37 step towards developing MC receptor agonists and antagonists for controlling feeding behavior in mammals, particularly obesity and overweight disorders in general, as well as anorexia, cachexia and other failure-to-thrive disorders.

A panel of mammalian melanocortin receptor-expressing recombinant cells are provided as described above in Example 3, wherein each member of the panel comprises appropriate mammalian cells, such as human 293 cells, comprising a recombinant expression construct encoding the MC-I, MC-2 (ACTH),--MC-3, MC-4 or receptor, the panel constructed to comprise cells functionally expressing each of these MC receptor proteins.

The panel is used as follows. Receptor agonist activity is assayed by transient or stable expression of a protein which produces a metabolite reporter molecule in response to receptor binding by any of the MC receptor proteins. An example of such a reporter system is the recombinant expression construct described in Example 4, wherein cAMP responsive elements (CREs) are operatively linked to a bacterially- 15 derived P-galactosidase (P-gal) gene. In the event of receptor binding, cAMP is produced in the mammalian cell, and the CRE induces P-gal expression. When coincubated with a colorless substrate for P-gal, receptor binding results in conversion of the colorless substrate to a blue-colored product, which can be easily scored visually or spectrophotometrically. Alternative reporter genes, such a luciferase, can also be used 20 as reporter systems, provided that expression of the reporter molecule-producing protein is functionally linked to receptor binding of a test compound. Alternatively, cAMP production resulting from MC receptor binding can also be measured directly.

Assay panels are arranged so that agonist activity can be identified, quantitated and correlated with expression of each MC receptor. Automated assays using such panels are also envisioned, whereby the qualitative and quantitative detection of a reporter metabolite is detected in an array (such as a 96-well tissue culture plate) and the data collected and assembled into a computer data-base or other analytical program.

Antagonist activity is detected by a modification of the above assay. In this assay, the inhibition of cAMP production by a standardized amount of a known receptor agonist, specific for each receptor, is assayed in the presence of a putative antagonist compound. Production of metabolite reporter molecules and their qualitative and quantitative detection is achieved as described above, and the specificity and potency of 38 each antagonist compound characterized with regard to the degree of inhibition achieved for each receptor.

In view of the instant disclosure, MC-3/MC-4 receptor antagonists are expected to be useful to inhibit food intake in a hungry animal, and MC-3/MC-4 receptor agonists are expected to be useful to increase food intake in an animal. Alternative patterns of feeding behavior associated with different patterns of MC receptor agonist/antagonist activity can be determined using this assay.

Compounds having agonist or antagonist activity with the MC-3 or MC-4 receptors detected using this assay are further screened in vivo to determine whether the observed receptor binding activity results in modification of feeding behavior when administered to an animal. In these assays, the MC receptor binding- compounds detected using the assay are administered intracraniovcntricularly as described above in Example 5 to animals after an overnight fast, to waking animals, or to animals that are not otherwise pnmed to be hungry. Feeding and locomotor activity is monitored in these 15 animals, and compounds affecting eating behavior (either by inhibiting feeding in otherwise hungry animals or stimulating feeding in otherwise sated animals) are selected for further development.

In addition, systemic administration of compounds found to be active by ICV :administration assays is used to screen such compounds for the ability to cross the bloodbrain barrier. Such compounds are also useful as templates for modifications aimed at ~increasing the availability of these compounds in the brain after systemic administration, ~for increasing bioactivity, or both.

It should be understood that the foregoing disclosure emphasizes certain specific embodiments of the invention and that all modifications or alternatives equivalent thereto are within the spirit and scope of the invention as set forth in the appended claims.

39- SEQUENCE LISTING GENERAL INFORMATION:

APPLICANT:

NAME: Oregon Health Sciences University STREET: 3181 S.W. Sam Jackson Park Road CITY: Portland STATE: Oregon COUNTRY: USA POSTAL CODE (ZIP): 97201 TELEPHONE: 503-494-8200 TELEFAX: 503-494-4729 (ii) TITLE OF INVENTION: Methods and Reagents for Discovering and Using Mammalian Melanocortin Receptor Agonists and Antagonists To Modulate Feeding Behavior in Animals (iii) NUMBER OF SEQUENCES: 19 (iv) COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.25 CURRENT APPLICATION DATA: APPLICATION NUMBER: INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: o LENGTH: 35 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE: NAME/KEY: mics feature LOCATION: 1..35 oo OTHER INFORMATION: /function "Degenerate oligonucleotide primer (sense)" /note= "The residue at positions 24 and 24 are inosine" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: GAGTCGACCT GTGYGYSATY RCNNTKGACM GSTAC INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 32 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE: NAME/KEY: mics feature LOCATION: 1..32 OTHER INFORMATION: /function "Degenerate oligonucleotide primer (antisense)" /note= "The residue at position 18 is inosine" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: CAGAATTCAG WAGGGCANCC AGCAGASRYG AA 32 INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 1260 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA to mRNA (ix) FEATURE: NAME/KEY: LOCATION: 1..14 (ix) FEATURE: NAME/KEY: CDS LOCATION: 15..959 (ix) FEATURE: NAME/KEY: 3'UTR LOCATION: 960..1260 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: TTCCTGACAA GACT ATG TCC ACT AG GAG CCC CAG AAG ACT CTT CTG OCT Met Ser Thr Gin Glu Pro Gin Lys Ser Leu Leu Gly TCT CTC AAC Ser Leu Asn.

.is TCA GAG CCT Ser Giu Pro TCC AAT GCC ACC TCT CAC Ser Asn Ala Thr Ser His CTT GGA CTG CCC ACC AAC CAG Leu Cly Leu Ala Thr Asn Gin TGO TOC CTG Trp Cys Leu GTG TCC ATC CCA Val Ser Ile Pro GGC CTC TTC CTC Oly Leu Phe Leu CTA GGC CTG OTO Leu Oly Leu Val CTG GTG GAG AAT Leu Val Ciu Asn CTG GTT GTG ATA Leu Vai Val Ile ATC ACC AAA AAC Ile Thr Lys Asn

CGC

A rg 6S AAC CTO CAC TO Asn Leu IHis Ser ATG TAT TAC TTC Met Tyr Tyr Phe ATC TC Ile Cys 7S TOO CTC GCC Cys Leu Ala ACT ACT ATC Thr Thr Ile TCT GAC CTO ATO Ser Asp Leu Met AGT GTC AGC ATC Ser Val Ser Ile GTG CTC GAG.

Val Leu Giu CCC AGA GTO Ala Arg Vai ATC CTO CTO CTG Ile Leu Leu Leu GTG CCC ATC CTG Val Giy lie Leu GCT TTC Ala Leu 110 GTG CAG CAG CTO Vai Gin Gin Leu AAC CTC ATT CAC Asn Leu Ilie Asp CTC ATC TOT GCC Leu Ile Cys Gly

TCC

Ser 125 ATG GTO TCC ACT Met Val Ser Ser TGC TTC CTG GCC Cys Phe Leu Cly ATT OCT ATA CAC Ile Ala Ile Asp

CC

Arg 140 TAC ATC TCC ATC TTC TAT GCG CTO COT Tyr Ile Ser Ile Phe Tyr Ala Leu Arg 145 CAC AOC ATC OTO His Ser Ile Val ACO CTO Thr Leu 155 CCC AGA OCA Pro Arg Ala TCC ACC ACC Ser Ser Thr 175 CCC GCT OTO OTO Arg Ala Val Val ATC TCC ATO Ile Trp Met CTC ACC ATC GTC Val Ser Ile Val 170 CTC TTT ATC ACC Leu Phe Ile Thr TAC AAO CAC ACA 0CC OTT CTO CTC Tyr Lys His Thr Ala Val Leu Leu 185 TGC CTC GTC ACT TTC TTT CTA CCC ATG CTG GCA CTC ATG GC ATT CTG Cys Leu Val Thr Phe Phe Leu Ala Met Leu Ala Leu Met Ala Ile Leu 190 195 200 626 TAT GCC CAC ATG Tyr Ala His Met 205 TTC ACC AGA GCC TGC CAG Phe Thr Ary Ala Cys Gin 210 CAC OTC His Val 215 CAG GGC ATT GCC Gin Gly Ile Ala 220 G74 CAG CTC CAC AAA AGO COG COG TCC ATC CCC CAA GGC TTC TGC CTC AAG Gin Leu His Lys Arg 225 Arg Arg Ser Ile Gin Gly Phe Cys Leu Lys 235 GGT OCT GCC Cly Ala Ala CCC CCC TTC Gly Pro Phe CTT ACT ATC CTT Leu Thr Ile Leu CCC ATT TTC TTC Giy Ile Phe Phe CTG TCC TGC Leu Cys Trp 250 TTC CTG CAT CTC Phe Leu His Leu TTC CTC ATC Leu Leu Ile 260 OTC CTC TOC CCT CAC CAC Vai Leu Cys Pro Gin His 265 CCC ACC Pro Thr 270 TCC ACC TCC ATC Cys Ser Cys Ile

TTC

Phe 27S AAG AAC TTC AAC Lys Asn Phe Asn CTC TTC Leu Phe 280 CTC CTC CTC Leu Leu Leu a a a.

ATC CTC CTC Ile Vai Leu 285 ACC TCC ACT Ser Ser Thr 290 GTT CAC CCC CTC Vai Asp Pro Leu TAT GCT TTC CC Tyr Ala Phe Arg CAG GAG CTC CCC ATC ACA CTC AAG GAG CTG CTC CTG TGC TCC TOO Gin Ciii Leu Arg Met Thr Leu Lys Ciii Val Leu Leu Cys Ser Trp 305 310 315 TGATCACAGG GCGCTGGGCA GAGGGTCACA GTGATATCCA GTGGCCTCCA TCTGTCAGAC CACACCTACT CATCCCTTCC TGATCTCCAT TTCTCTAAGG CTCCACACGA TGACCTTTAA AATAGAAACC CAGAGTCCCT GCGGCCAGGA GAAAGGGTAA CTCTGACTCC AGGCCTCACC CAGGGCAGCT ACGCAAGTG GAGGAGACAG GGATGGGAAC TCTAGCCCTG AGCAAGGGTC AGACCACAGG CTCCTGAAGA GCTTCACCTC TCCCCACCTA CAGGCAACTC CTGCTCA.AGC

C

914 959 1019 1079 1139 1199 1259 1260 INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 315 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: Met 1 Asn cys Val Arg Ser Leu Gin Ser Phe 145 Arg Phe Phe I Phe 1 Arg P 225 Se Al Le Sez

SC

Asr Asp Leu Leu Leu 130 ryr kla Ile The hr ~rg r Thr Thr Tyr Leu Leu Leu Leu Asp 115 Cys Ala I Val Thr T 1 Leu A 195 Arg A Arg S Gil Sel 2C Val Val His Met Glu 100 Asn Phe Leu Ial 'yr .80 la la er G1~ Hi~ Sei Glu Ser Val Val Leu Leu Arg Gly 165 Tyr Met Cys Ile u Pro s Leu Ile Asn Pro 70 Ser Gly Ile Gly Tyr 150 Ile Lys Leu Gin 1- 2 Arg C 230 Gin Gly Pro Val 55 Met Val Ile Asp Ile 135 [is Lrp b iis I \la L is !15 ;in C Lys Leu Asp 40 Leu Ser Ala Gly Val Leu 10 Thr Leu Val Gly Gin Leu Ala Ser Ser Ser Ile Tyr Tyr Phe Ile Cys Cys Leu Ala Leu Ser Leu Va1 120 Ile Ser let Thr .eu !00 Pal ;ly Ile Val 105 Leu Ala Ile Val Ala 185 Met Din Phe Val 90 Ala Ile Ile Val Ser 170 Val Ala Gly Cys Leu 250 75 Leu Arg Cys Asp Thr 155 Ile Leu Ile I Ile Leu I 235 Cys Glu Va1 Gly Arg 140 Leu Val Leu Leu Ala 220 Lys Crp Thr Ala Ser 125 Tyr Pro Ser Cys Tvr 205 Gin Gly Gly Thr Leu 110 Met lie Arg Ser Leu 190 Ala Leu Ala Pro Ile Val Va1 Ser Ala Thr 175 Val His His Ala Phe 255 Ile Gin Ser Ile Arg 160 Leu Thr Met Lys Thr 240 Phe Asn Ser Pro Trp Gly Leu Lys Asn Leu Thr Ile Leu Gly Ile Phe Phe Leu His Leu Leu 260 Cys Ile Phe Lys 275 Ser Thr Val Asp 290 Met Thr Leu Lys 305 Leu lie Val Leu Cys 265 Asn Phe Asn Leu Phe 280 Pro Leu Ile Tyr Ala 295 Glu Val Leu Leu Cys 310 Pro Gin His Pro Thr Cys Ser 270 Leu Leu Leu Ile Val Leu Ser 285 Phe Arg Ser Gln Glu Leu Arg 300 Ser Trp 315 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 1633 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA to mRNA (ix) FEATURE:

NAME/KEY:

LOCATION:

(ix) FEATURE:

NAME/KEY:

LOCATION:

(ix) FEATURE:

NAME/KEY:

LOCATION:

1..461

CDS

462..1415 3 'UTR 1416..1633 9 (xi) SEQUENCE DESCRIPTION: SEQ ID CCCGCATGTG GCCGCCCTCA ATGGAGGGCT CTGAGAACGA CTTTTAAAAC GCAGAGAAAA AGCTCCATTC TTCCCAGACC TCAGCGCAGC CCTGGCCCAG GAAGGGAGGA GACAGAGGCC AGGACGGTCC AGAGGTGTCG AAATGTCCTG GGAACCTGAG CAGCAGCCAC CAGGGAAGAG GCAGGGAGGG AGCTGAGGAC CAGGCTTGGT TGTGAGAATC CCTGAGCCCA GGCGGTTGAT GCCAGGAGGT GTCTGGACTG GCTGGGCCAT GCCTGGGCTG ACCTGTCCAG CCAGGGAGAG GGTGTGAOOG CAGATCTOOG GGTGCCCAOA TGGAAGGAOG CAGGCATGGO GACACCCAAG GCCCCCTGG C AGCACCATOA ACTAAGCAGG ACACCTGGAG GGGAAGAACT GTGGGGACCT 360 GGAGGCCTCC ALACGACTCCT TCCTGCTTCC TGGACAOGAC T ATG OCT OTG CAG Met Ala Val Gin 1 TCC ACC CCC ACA GCC Ser Thr Pro Thr Ala 473 OGA TCC CAG AGA AGA Gly Ser Gin Arg Arg

CTT

Leu CTG GGC TCC CTC AAC Leu Gly Ser Leu Asn 15 ATC CCC CAG CTG Ile Pro Gin Leu OGG CTG Gly Leu 2S OCT GCC AAC CAG ACA OGA GCC Ala Ala Asn Gin Thr Gly Ala 30 GAG GTG TCC ATC TCT GAC 000 CTC TTC CTC AGC CTO 000 Oiu Vai Ser Ile Ser Asp Gly Leu Phe Leu Ser Leu Giy CGO TGC CTO Arg Cys Leu CTG OTO AGC Leu Val Ser s0 AAC CGG AAC ASn Arg Asn TTG OTO GAO Leu Val Glu AAC OCC CTO OTG Asn Ala Leu Val CCC ACC ATC 0CC Ala Thr Ile Ala 665

S

S S

S

S 0*

S

S

S

CTO CAC Leu His TCA CCC ATG TAC Ser Pro Met Tyr TTC ATC TOC TOC Phe Ile Cys Cys GCC TTO TCO GAC Ala beu Ser Asp CTG GTG AGC GGO Leu Val Ser Glv

ACG

Th r AAC GTG CTG GAG Asn Val Leu Giu 0CC GTC ATC CTC Ala Val Ile Leu CTO GAG GCC GOT Leu Oiu Ala Gly CTO OTO 0CC COO Leu Val Aia Arg

OCT

Ala 110 OCG GTG CTO CAO CAG CTG Ala Val Leu Gin Gin Leu 115 GAC AAT OTC Asp Asn Val TOC TTC CTG Cys Phe Leu 13S GAC GTO ATC ACC Asp Val Ile Thr AOC TCC ATO CTO TCC AOC CTC Ser Ser Met Leu Ser Ser Leu 130 857 GOC 0CC ATC 0CC Gly Ala Ile Ala GAC COC TAC ATC Asp Arg Tyr Ile

TCC

Ser 145 ATC TTC TAC Ile Phe Tyr GCA CTG Ala Leu 150 CGC TAC CAC AOC ATC OTO ACC CTG CCG Arg Tyr His Ser Ile Vai Thr Leu Pro 155

CG

Arg 160 GCG CCG COA 0CC Ala Pro Arg Ala

OTT

Val 165 0CC 0CC ATC TG Ala Ala Ile Trp 0CC ACT 0TC GTC Ala Ser Val Val AOC ACO CTC Ser Thr Leu TTC ATC Phe Ile 180 1001 GGC TAC TAC GAC CAC GTG GCC GTC CTG CTG TOC CTC GTG GTC TTC TTC 14 1049 Gly Tyr Tyr Asp His Val Ala Val Leu Leu Cys Leu Val Val Phe Phe 195 CTG GCT ATG CTG Leu Ala Met Leu 200 CGG GCC TGC CAG Arg Ala Cys Gin 215 GTG CTC ATG CCC GTG CTG GAC GTC Val Leu Met Ala Val Leu Asp Val 205 CAC GCC CAG His Ala Gin

GCC

Gly 220 ATC GCC CGG CTC Ile Ala Arg Leu CAC ATG CTG GCC His Met Leu Ala 210 CAC AAG AGG CAG His Lys Arg Gin 225 GTC ACC CTC ACC Val Thr Leu Thr 1097 1145 CGC CCG Arg Pro 230 GTC CAC CAG GGC Val His Gin Gly GGC CTT AAA GGC Gly Leu Lys Gly 1193 1241 CTG CTG GGC ATT Leu Leu Gly Ile TTC CTC TGC TGG Phe Leu Cys Trp

GGC

Gly 255 CCC TTC TTC CTG Pro Phe Phe Leu CTC ACA CTC ATC Leu Thr Leu Ilie CTC TGC CCC GAG Leu Cys Pro Giu

CAC

His 270 CCC ACG TGC GGC Pro Thr Cys Gly TGC ATC Cys Ile 275 1289 0 0Oe* 0000 *060 OS 00 S S

S

TTC AAG AAC Phe Lys Asn ATC GAC CCC Ile Asp Pro 295 AAC CTC TTT CTC Asn Leu Phe Leu CTC ATC ATC TGC Leu Ile Ilie Cys AAT GCC ATC Asn Ala Ile 290 1337 CTC ATC TAC GCC TTC CAC AGC CAG GAG CTC CGC AGO ACG Leu Ile Tyr Ala Phe His Ser Gin Giu Leu Arg Arg Thr 1385 CTC AAG Leu Lys 310 GAG GTG CTG ACA Giu Val Leu Thr

TGC

Cys 315 TCC TGG TGA Ser Trp OCGCGGTGCA CGCGCTTTA-A 1435 *a 0 S

S*

*058

S

OTGTGCTGGG CAGAGGGAGG TGGTGATATT GTGGTCTGGr TCCTGTGTGA CCCTGGGCAG TTCCTTACCT CCCTOGTCCC CGTTTGTCAA. AGAGGATGGA CTA-AATGATC TCTGAAAGTC TTGAAGCGCG GACCCTTCTG GGCAGGGAGG GGTCCTGCALA AACTCCAGGC AGGACTTCTC ACCAGCAGTC GTGGGAAC INFORMATION FOR SEQ ID NO:6: SEQUENCE CHARACTERISTICS: LENGTH: 317 amino acids TYPE: amino acid TOPOLOGY: linear 1495 1555 1615 1633 (ii) MOLECULE (xi) SEQUENCE TYPE: protein DESCRIPTION: SEQ ID NO:6: Met Ala Val Gin Gly Ser Gin Arg Arg Leu Leu Gly Ser Leu Asn Ser

S

I

Th r Ala Gly Ly s 6S Ala Val Le u Leu Ser 145 Ala Thr Val His His 225 Val I P rc A rc Leu Asn Be u Ile Gin Ser 130 Ile P ro Leu /al1 let Ys rhr Thr Cys Val Arg Ser Le u Gin 115 Ser Ph e Arg Phe Phe 195 Le u Arg Leu *Ala Leu Ser Asn Asp Leu 100 Le u Leu Ty r Ala Ile 180 Ph e Ala Gin Thr Slie *Giu Be u Leu Leu Le u Asp Cy s Ala Val 165 Gly Leu Arg Arg Ile 24S Pro Val Val1 His 70 Leu Giu As n Phe Leu 150 Ala Tyr Alia Ala Pro 230 Be u Gir Ser Giu 55 Ser Val Ala Val Leu 135 A rg Ala Tyr Met Cys 215 Val Leu Leu Slie 40 *Asn Pro Se r Gly Ilie 120 Gly Ty r Ile Asp Le u 200 Gin His Gly Giy 25 Ser Ala Met Gly Ala 105 Asp Ala His Trp His 185 Val His Gin Ile 10 Beu Asp Le u Tyr Th r 90 Leu ValI Ile Ser Val 170 Val1 Leu Ala Gly Phe 250 Ala Gi'y Val1 Cy s 75 Asn Val1 Ile Ala Ile 155 Ala Ala Met Gin Phe 235 Phe Ala Leu Vai Ph e ValI Al a Th r Val 140 Val Ser Val Al a Gly 220 Gly Leu Asn Pae Al a Ile Le u Arg Cys 125 Asp Th r Val Leu Val 205 Ile Leu Cys Gin Leu Th r Cys Glu Ala 110 Ser Arg Leu Val1 Leu 190 Be u Aa Lys I rp is Thr Ser Ile Cy s Th r Ala Ser Ty r Pro Phe 175 Cys Asp Arg Gly Gly 255 Gly Be u Ala Beu Al a Val Met Ile Arg 160 Ser Le u la 1 ,e u kla ?ro Phe Phe Leu His Leu Thr Leu Ile Val 260 265 C'ss Gly Cys Ile Phe Lys Asn Phe Asn 275 280 Cys Asn Ala Ile Ile Asp Pro Leu Ile 290 295 Leu Arg Arg Thr Leu Lys Glu Val Leu 305 310 Leu Cys Pro Glu His Pro Thr 270 Leu Phe Leu Ala Leu Ile Ile 285 Tyr Ala Phe His Ser Gin Glu 300 Thr Cys Ser Trp 315 a INFORMATION FOR SEQ ID NO:7: SEQUENCE CHARACTERISTICS: LENGTH: 2012 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA to mRNA (ix) FEATURE: NAME/KEY: LOCATION: 1. .693 (ix) FEATURE: NAME/KEY: CDS LOCATION: 694. .1587 (ix) FEATURE: NAME/KEY: 3'UTR LOCATION: 1588. .2012 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: ACAACACTTT ATATATATTT TTATAAATGT AAGGGGTACA GGATATACCG TGTAGTGGTG AAGCCTGGGC TI1=TAGTGTA CGTGTTACCC ATAGGAATTT CTCATCACCC GCCCCCTCCA CATTCCACAC TCTATATCCA CGTGTATGCA TATAGCTCCA GTATTTGACT TCCTCTTTCT GAGTTATTTC ACTTTGATAA OTTGCTGCA. AAGACATGAC CTTATTCTTT TTGATAGCTG ATGTACCACA TTTCTTTATC CATTCACCCA TTGAGAACAC

AAGGTGCCAT

TCTGTCATCA

CCCTTCGAGT

CATATAAGTG

TGGCCTCCAC

GGGAGTACTC

TTAGTTGATT

TTTGTTACAT

GAATAACATA

CTCCALATGTC

AGAACATGTA

TTCCATCCAT

CATTGTGTAT

CCATATCTTT

GCTATTGTCA CTAGTGCTGC AATAAACATA CATGTGCAGG CTCCTTCTAA TATACTGATT TATATTTTAT GGAGAGAGAT AGAGTTCTTA GCGAGTGTGC TGTTTATTTC TAGTGTACTT GCAACTAATA TTCTGTATAC TCCCTTTAGG TGATTGGAGA TTTAACTTAG ATCTCCAGCA AGTGCTACAA GAACAAAAGA TCCTGAAGAA TCAATCAAGT TTCCGTGAAG TCAAGTCCAA GTAACATCCC CGCCTTAACC ACAAGCAGGA GAA ATG AAG CAC ATT ATC AAC TCG Met Lys His Ile Ile Asn Ser 480 TAT GAA AAC Tyr Giu Asn ATC AAC AAC ACA GCA AGA AAT AAT TCC GAC TGT CCT CGT Ile Asn Asn Thr Ala Arg Asn Asn Ser Asp Cys Pro Arg TGT OTT TTG CCG GAG GAG ATA TTT TTC ACA ATT TCC ATT OTT GGA OTT Cys Val Leu Pro Giu Giu Ile Phe Phe Thr Ile Ser lie Val Cly Val 810

TTG

Leu GAG AAT CTG ATC Giu Asn Leu Ile CTG CTG OCT CTG Leu Leu Ala Val TTC AAG AAT AAG Phe Lys Asn Lys TTG GCC ATA TCT Leu Ala Ile Ser

AAT

Asn CAG GCA CCC ATC Gin Ala Pro Met TTT TTC ATC TGT Phe Phe Ile Cys CAT ATG Asp Met 0* 0 0 000* CTG CCC AGC Leu Cly Ser AAC ATG GCC Asn Met Gly TAT AAG ATC TTG Tyr Lys Ile Leu A.AT ATC CTG ATC Asn Ile Leu Ile ATA TTC AGA Ile Leu Ary ACA GCC CAT Thr Ala His 954 1002 ATA CTC AAG CCA Ile Leu Lys Pro CCC AGT TTT GAA Cly Ser Phe Giu GAC ATC Asp Ile 10S ATC GAC TCC CTC Ile Asp Ser Leu

TTT

Phe 110 CTC CTC TCC COT Leu Leu Ser Arg

CTT

Leu 115 CCC TCC ATC TTC Cly Ser Ile Phe 1050 1098 CAC Asp 120 CTG CTC CTC ATT Leu Leu Val Ile GCC GAC CGC TAC Ala Asp Arg Tyr ACC ATC TTC CAC Thr Ile Phe His CTC COG TAC CAC AGC ATC GTC ACC ATC Leu Arg Tyr His Ser Ile Val Thr Met 140

CGC

Arg 145 CCC ACT GTC CTG CTG CTT Arg Thr Val Val Val Leu 150 1146 ACC OTC ATC Thr Val Ile ACG TTC TGC ACC Thr Phe Cys Thr ACT GGC ATC ACC Thr Cly Ile Thr ATC GTG ATC Met Vai Ile 165 1194 TTC TCC CAT CAT GTG CCC CAC GTG ATC ACC TTC ACG TCG CTG TTC CCG Phe Ser His His Val Pro His Val Ile Thr Phe Thr Ser Leu Phe Pro 170 175 180 CTG ATG CTG GTC TTC ATC CTG TGC CTC TAT GTG CAC ATG TTC CTG CTG Leu Met Leu Val Phe Ile Leu Cys Leu Tyr Val His Met Phe Leu Leu 185 190 195 GCT tGA TGG CAC ACC AGO AAG ATC TCC ACC CTC CCC AGA GCC AAC ATG Ala Arg Trp His Thr Arg Lys Ile Ser Thr Leu Pro Arcs Ala Asn Met 200

AAA

Lys GGG CCC ATG Gly Ala Met 205

CTG

Leu 210

GG

Gly

ACA

Tb r 220 ACC ATC CTG Thr Ilie Leu GTC TTC ATC Val Phe Ile 21S

C

Cy s TOG CCC CCC TTT OTG CTT CAT GTC CTC TTG ATO ACA TTC TGC CCA ACT Trp Ala Pro Phe Val Leu His Val Leu Leu Met Thr Phe Cys Pro Ser 235 240 245 AAC CCC TAC TOC CCC TGC TAC ATG TCT CTC TTC CAG GTG AAC GGC ATO Asn Pro Tyr Cys Ala Cys Tyr Met Ser Leu Phe Gin Val Asn Gly met 250 255 260 TTG ATC ATO 7CC AAT CCC GTC ATT GAC CCC 770 ATA TAT GCC TTC COO Leu Ilie Met Cys Asn Ala Val Ile Asp Pro Phe Ile Tyr Ala Phe Arg 265 270 275 AGC CCA GAG CTC AOG CAC OCA TTC AAA AAG ATC ATC TTC TGC AGC AGO Ser Pro Glu Leu Arg Asp Ala Phe Lys Lys Met Ile Phe Cys Ser Arg 280 285 290 2195 TAC TOG TAG AATGOCTGAT CCCTOOTTTT AOAATCCATC

OGAATAACGT

Tyr Trp 1242 1290 1338 1386 1434 1482 1530 1578 1627 TOCCAA TO C

TTCCCTAATG

CAGTCTATTO

OCATAOAATA

CATTACTTTT

CAAAGACTTG

AGO CCATTO C

CAGALATAOTO

GATOCALAGCA

TAOOOOCAAC

CAAAGTATTA

GCACCAATCT

CTTTCCTOTG

AGCCAATTTC

TAACATTCCA

TGACCCACCA

TCTATTTGTG

GGTACAAAAG

AGTAAAACAG

CACATTAACA

AGAGT

ACAAATGCCA

GCTAOTGTTT

ACTOCACACA

TAATTAGCTT

CAATAAAAAT

AGCCAGTTCT

CTGCTCCTCA

CTOCCCTTCC

CTGAATACTA

TGGCCAGGAA

TAAAACGTGT

ACTAAAACAI

TCCATTACTT

ATOACAAATG

TCAAGGGCTT

TGGGCTAAGG

CACOCGOCCT TTCCACGTOO 1687 1747 1807 1867 1927 1987 2012 INFORMATION FOPR SEQ ID NO:8: SEQUENCE CHARACTERISTICS: LENGTH: 297 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein Met

I

Asn Thr Val Ser Asn Ser Ser Ty r Arg 145 Thr Thr Tyr Thr (xi) SEQUENCE Lys His Ile Ile 5 Asn Ser Asp Cys Ile Ser Ile Val Phe Lys Asn Lys Leu Ala Ile Ser Ile Leu Ile Ile Phe Glu Thr Thr 100 Arg Leu Gly Ser 115 Ile Thr Ile Phe 130 Ary Thr Val Val Gly Ile Thr Met 165 Phe Thr Ser Leu 180 Val His Met Phe 195 Leu Pro Arg Ala 210 Pro Gly Asn Asp 70 Le u Al a Ile His Val 150 Val1 Phe Leu Asn Arg Val1 Leu 55 Met Arg His Phe Ala 135 Leu Ile Pro Le u Met Cys Le u 40 Gin Le u Asn Asp Asp 120 Le u Thr Phe Leu Ala 200 Ly s Val1 25 Giu Ala Gly Met Ile 105 Le u Arg Val Ser Met 185 Arg Gly Le u As n Pro Ser Gly 90 Ilie Le u Tyr Ile His 170 Le u T rp Ala Pro Le u Met Leu 75 Ile Asp Val1 His Trp 155 His Val- His Met Glu ile Tyr Ty r Le u Ser Ile Ser 140 Th r Val Phe Th r Thr Glu ValI Phe Lys Ly s Le u Ala 125 Ile Phe Pro Ile Arg Leu Ile Leu Phe Ile Pro Phe 110 Ala Val Cy s His Leu 190 Lys Th r Phe Leu Ile Le u Arg Leu Asp Th r Th r Val1 175 Cys Ile Ile Phe Ala Cys Glu Gly Leu Arg Met Gly 160 Ile Leu Ser Leu DESCRIPTION: SEQ ID NO:8: Asn Ser Tyr Glu Asn Ile Asn Asn Thr Ala Arg 10 220 Leu Gly Val 225 Phe Ile Phe 230 Cys Trp Leu Met Leu Phe Thr Phe Cys 245 Gin Val Asn 260 Pro Ser Asn Gly Met Leu Ala Pro Phe Val Leu His Val Leu 235 240 Pro Tyr Cys Ala Cys Tyr met Ser 250 255 Ile Met Cys Asn Ala Val Ile Asp 265 270 Pro Glu Leu Arg Asp Ala Phe Lys 285 Pro Phe Ile 275 Tyr Ala Phe Arg Ser 280 Lys Met 290 Ile Phe Cys Ser Arg Tyr Trp 295 S INFORMATION FOR SEQ ID NO: 9: SEQUENCE CHARACTERISTICS: LENGTH: 1108 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA to mR-NA (ix) FEATURE: NAME/KEY: LOCATION: 1. .132 (ix) FEATURE: NAME/KEY: CDS LOCATION: 133..1026 (ix) FEATURE: NAME/KEY: 3'UTR LOCATION: 1027. .1106

S

S

5555 (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: GGGGCCAGAA AGTTCCTGCT TCAGAGCAGA AGATCTTICAG CAAGAACTAC AAAGAAGAAA AGATTCTGGA GAATCAATCA AGTTTCCTGT CAAGTTCCAO TAACGTTTCT GTCTTAACTG CACACAGGAA AG ATG AAA CAC ATT CTC AAT CTG TAT GAA AAC CTC AAC Met Lys His Ile Leu Asn Leu Tyr Glu Asn Leu Asn 1 5 ACT ACA GCA AGA AAT AAC TCA CAC TCT CCT GCT GTG ATTTTCAGA21 TTG CCA GAA 216 Ser Thr Ala Arg Asn Asn Ser Asp Cys 20 GAG ATA 777 TTC ACA GTA TCC ATT CTT Pro Ala Vail CCC CTT TTG Cly Val Leu Ile Leu Pro Clu GAG AAC CTC ATG Ciu Asn Leu Met 264 Clu Ile Phe Phe Thr Vai Ser Ile Val 35 CTC CTt CTC CCT C7C CCC AAC AAT AAC ATC Val Leu Leu Ala Val Ala Lys Asn Lys Met 50 CTT CAC Leu Cmn 7CC CCC ATC Ser Pro Met TTT TTC ATC Phe Phe Ile AAC ATT TTC Lys Ile Leu GAG CCT CCA Clu Pro Arg 7CC AC Cys Ser CAA AAC Giu Ass TTC GCT ATT 7CC Leu Ala Ile Ser CAT ATC Asp Met 70 CTC GCC AC Leu Cly Ser ATC TAC Met Tyr CTT CTC ATC Val Leu Ile TTC A.AA AAC ATC Phe Lys Asn Met CCT TAC CTC Cly Tyr Leu CTC CAC 7CC Val Asp Ser CCC ACT TTT CAA Cly Ser Phe Clu ACA CCA CAT GAT Thr Ala Asp Asp CTC TTrC Leu Phe 110 ATC CTC 7CC CTT CTC CCC 7CC ATC TC Ile Leu Ser Leu Leu Cly Ser Ile Cys CTC TCT CTC ATT Leu Ser Val Ile 4*a* CCT CAC CCC TAC Ala Asp Arg Tyr ACA A7C TTC CAC Thr Ile Phe His CTC CAC TAC CAC Leu Gin Tyr His ATC ATC ACC CCC Ile Met Thr Pro

CCA

Ala 145 CCC 7CC CCT CCT Pro Cys Pro Arg CTC ACC CTC CTC Leu Thr Val Leu TCC CGA Trp Arg 155 CCC 7CC ACA Gly Cys Thr CCC ACA CTC Pro Thr Val 175

GC

Cly 160 ACT CCC ATT ACC Ser Cly Ile Thr CTC ACC TTC TCC Val Thr Phe Ser CAT CAC CTC His His Vai 170 CTC CCC TTC Leu Ala Phe ATC CCC TTC ACA Ile Ala Phe Thr CTC TTC CCC CTG Leu Phe Pro Leu ATC CTC Ile Leu 190 7CC C7C TAC CTG Cys Leu Tyr Val ATG TTC CTC CTC Met Phe Leu Leu CCC TCC CAC ACC Ary Ser His Thr 744

ACC

Arg 205 ACC ACC CCC 7CC Arg Thr Pro Ser

CTT

Leu 210 CCC AAA CCC AAC Pro Lys Ala Asn

ATC

Met 215 AGA CCC CCC CTC Arg Cly Ala Val 792 CTG ACT GTC CTG CTC GGG GTC TTC ATT TTC TGT TGG GCA CCC TTT GTC Leu Thr Val Leu Leu Gly Val Phe Ile Phe Cys Trp Ala Pro Phe Val 225 230 235 CTT CAT GTC CTC TTG ATG ACA TTC TGC CCA GCT GAC CCC TAC TGT CC Leu His Val Leu Leu Met Thr Phe Cys Pro Ala Asp Pro Tyr Cys Ala 240 245 250 TGC TAC ATG TCC CTC TTC CAG GTG AAT GGT GTG TTG ATC ATG TGT AAT Cys Tyr Met Ser Leu Phe Gin Val Asn Gly Val Leu Ile Met Cys Asn 255 260 265 GCC ATC ATC GAC CCC TTC ATA TAT GCC TTT CGG AGC CCA GAG CTC AGG Ala Ilie Ile Asp Pro Phe Ile Tyr Ala Phe Arg Ser Pro Giu Leu Arg 270 275 280 GTC GCA TTC AAA AAG ATG GTT ATC TOC AAC TGT TAC CAG TAG Val Ala Phe Lys Lys Met Val Ile Cys Asn Cys Tyr Gln 285 290 295 AATGATTGGT CCCTGATTTT AGGAGCCACA OGGATATACT GTCAGGGACA GAGTAGCGTG ACAGACCAAC AACACTAGGA CT INFORMATION FOR SEQ ID NO:iO: SEQUENCE CHAR-ACTERISTICS: LENGTH: 297 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:lO: Met Lys His Ilie Leu Asn Leu Tyr Glu Asn Leu Asn Ser Thr Ala Arg 1 5 10 Asn Asn Ser Asp Cys Pro Ala Val Ile Leu Pro Glu Glu Ile Phe Phe 25 Thr Val Ser Ile Val Gly Val Leu Glu As n Leu Met Val Leu Leu Ala 40 Val Ala Lys Asn Lys Met Leu Gin Sec Pro Met Tyr Phe Phe Ile Cys 55 Ser Leu Ala Ile Ser Asp Met Leu Gly Ser Met Tyr Lys Ile Leu Glu 70 75 840 888 936 984 1026 1086 1108

S

S

Asn Val Leu Ile AsnValLeulieMet Phe Lys Asn Met Gly Tyr Leu Glu Pro Arg Gly Ser Ser Tyr Ala 145 Ser Ala Tyr Se r Leu 225 Leu Leu Pro PhE Leu Th: 130 Pro Giy Phe Val Le u 210 Gly Met Phe Phe Giu Leu 115 *Thr *Cys Ilie Thr His 195 Pro Val Th r Gin Ile Se r 100 Gly Ile Pro Th r Ala 180 Met Lys Phe Phe Val 260 Ty r Thr Ser The Arg Ile 165 Le u Phe Al a Ile Cys 245 Asn Ala Ala Ile His His 150 Val Ph e Leu Asn Phe 230 Pro Gly *Asp Cys Al a 135 Le u Th r Pro Leu Met 215 Cys Ala Val1 Asp Val 105 Ser Leu 120 Leu Gin Thr Val Phe Ser Leu Met 185 Ala Arg 200 Arg Gly TrD Ala Asp Pro Leu IleI Val Ser Tyr Le u His 170 Leu Ser Ala Pro Ty r 250 M1et Asp Val His1 Trp 155 His Ala His Val Phe 235 Cys Cys Se r Ile Arg 140 Arg Val Phe Thr Th r 220 Val Ala Asn *Leu Ala 12S Ile Gly Pro Ile Arg 205 Leu Le u Cys Ala *Phe 110 Ala Met Cys Th r Leu 190 Arg Th r His Tyr Ile- 270 Ile As p Th r Th r Val 175 Cy s Th r Val1 Val1 M1et 255 Ile Leu Arg Pro Gly 160 Ile Leu Pro Leu Le u 240 Ser Asp 9 9

S.

9 9 265 The Arg Ser Pro Glu Leu Arg Val Ala Phe Lys 275 280 285 Lys Met 290 Val Ile Cys Asn Cys Tyr Gin 295 INFORMATION FOR SEQ ID NO:l1: SEQUENCE CHARLACTERISTICS: LENGTH: 1338 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: CDNA to MRNA (ix) FEATURE: NAME/KEY: LOCATION: 1. .297 (ix) FEATURE: NAME/KEY: CDS LOCATION: 298. .1269 (ix) FEATURE: NAME/KEY: 3'UTR LOCATION: 1270.. 1338 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l1: CGCTGTAACT GTACCAACCC GTGTTCGGTC GCGATGAGAA~ GAGACCACAG AGAGAGAGCOG TCAGACCGAC AGGGGATGAC ACAGGCTGGT CACAGTCTCC ACTGATTCTT CCAGACGCAA 120 ACCAAAGTTT TTTCTATGTC TCCAACCTCC CCCTCCTCCC CCCTTTCTCT CTGGAGAAAC 180 TAAAATCTAG ACTGGACAGC ATCCACAACA CAAGCACCTA GAAGAACATT TTTTTTTCCC 240 ACCAGCTTGC TCACCACCCT CCACCAGCTC CAGCCGGAAC TGGTCCCGCC CATAACC 297 ATC AAC TCT TCC TGC TGC CCC TCC TCC TCT TAT CCG ACC CTC CCT ALAC34 Met Asn Ser Ser Cys Cys Pro Ser Ser Ser Tyr Pro Thr Leu Pro Asn34 1 5 10 *CTC TCC CAG CAC CCT CCA CCC CCC TCT CCC ACC A.AC CCC ACT G AGT39 ::*Leu Ser Cmn His Pro Ala Ala Pro Ser Ala Ser Asn Arg Ser Cly Ser 25s CCC~oGG TTC TGC GAC CAG CTT TTC ATC ALAG CCA GAG CTC TTC CTC CCA CTC 441 Cly Phe Cys Clu Gin Val Phe Ile Lys Pro Giu Val Phe Leu Ala Leu 35 40 CCC ATC CTC ACT CTG ATC GAA AAC ATC CTC GTC ATC CTC CCT CTC GTC 489 Ile Val Ser Leu Met Clu Asn Ile Leu Vai Ile Leu Ala Val Val 55 AGC AAC CCC AAC CTG CAC TCC CCC ATG TAC TTC TTC CTC CTC ACC CTC 537 Arg Asn Cly Asn Leu His Ser Pro Met Tyr Phe Phe Leu Leu Ser Leu V 65 70 75 *CTC CAG CCC GAC CTG CTC CTG ACC CTG TCC AAC TCC CTG GAG ACC ATC 8 Leu Gin Ala Asp Leu Leu Val Ser Leu Ser Asn Ser Leu Giu Thr Ile 90 ATG ATC CTG GTT ATC AAC AGC GAO TCC CTG ACC TTG GAG GAO CAA TTC Met Ile Val ATO CAG CAC Ile Gin His 115 GTG GCC TCC Val Ala Ser 130 Ile Asn Ser Asp Ser Leu Thr Leu Giu Asp Gin Phe ATG GAO A.AC ATC Met Asp Asn Ile TTO GAC TCT ATO ATO TGC ATO TCO CTO Phe Asp Ser Met Ile Cys Ile Ser Leu 120 125 CTO GOC ATC GCC GTG GAO AGG TAC GTC Leu Ala Ile Ala Val Asp Arg Tyr Val 140 ATC TGC A-AC Ile Cys A-sn

ACC

Th r 145 ATC TTC TAT GCC Ile Phe Tyr Ala CT TAC CAC AGC Arg Tyr His Ser ATC ACG GTT AGG Met Thr Val Arg CCC OTO TOO TTG Ala Leu Ser Leu GTC GOC ATO TG Vai Ala Ile Trp TCC TGT CCC A-TO Cys Cys Cly Ile TOO GC Cys Oly 175 GTG ATO TTO Val Met Phe A-TO ACC A-TO Ilie Thr Met 195 GTC TAO TOO GAO Val Tyr Ser Ciu A-AG ATC OTO ATO LvS; Met Val Ile OTO TOO OTO Val Cys Leu 190 OTO TAO ATO Leu Tyr Ile 873 TTC TTC 0CC ATO Phe Phe Ala Met OTO CTO ATC 000 Leu Leu Met Cly CAC ATG His Met 210 TTO OTO TTC GCC Phe Leu Phe Ala

AGO

Arg 215 OTO CAC OTO OAG Leu His Val Gin ATO 000 GCA OTG Ile Ala Ala Levi

OCA

Pro 225 OCT GOT GAO CG Pro Ala Asp Gly

CTA

Leu 230 GCC CCG OAO CAG Ala Pro Cmn Gin TOG TOO ATO A-AG, Ser Cys Met Lys 1017 000 CTC ACC A-TO Ala Val Thr Ilie

ACC

Tb r 245 ATO OTG OTG 000 Ile Levi Levi Gly TTO ATO TTO TOO TOG CG Phe Ile Phe Cys Trp Ala 255 1065 COT TTC T Pro Phe Phe TAO TOO ATO Tyr Cys Ile 275

OTO

Le u 260 CA-C OTG GTO OTO His Leu Val Levi

ATO

Ile 265 ATO ACC TOO 000 Ile Thr Cys Pro A-CO AA-C CCC Thr Asn Pro 270 OTT OTO ATO Val Lei Ile 1113 1161 TOO TAO ACO 000 Cys Tyr Thr Ala TTO AAC A-CO TAO Phe Asn Thr Tyr A-TO TOO Met Cys 290 A-AC TOT GC ATO A-sn Ser Val Ile COO OTO A-TO TAO GCC TTC CCC AC OTO Pro Leu Ilie Tyr Ala Phe Arg Ser Leu 300 1209 GAG CTG CGA AAC ACC TTC AAG GAG ATT CTC TGC GGT TGC AAT GGC ATG Glu Leu Arg Asn Thr Phe Lys Glu Ile Leu Cys Gly Cys Asn Gly Met 305 310 315 320 AAC GTG GGC TAG GAACCCCCGA GGAGGTGTTC CACGGCTAGC CA.AGAGAGAA Asn Val Gly AAGCAATGCT CAGGTGAGAC ACAGAAGGG 1257 1309 1338

G

A

L

Mi

I.

T1 INFORMATION FOR SEQ ID NO:12: SEQUENCE CHARACTERISTICS: LENGTH: 323 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID I 'let Asn Ser Ser Cys Cys Pro Ser Ser Ser T 1 5 ~eu Ser Gin His Pro Ala Ala Pro Ser Ala S 25 1y Phe Cys Glu Gin Val Phe Ile Lys Pro G 40 ly Ile Val Ser Leu Met Giu Asn Ile Leu V~ 50 55 rg Asn Gly Asn Leu His Ser Pro Met Tyr PJ 65 70 eu Gin Ala Asp Leu Leu Val Ser Leu Ser A~ 90 et Ilie Val Val Ile Asn Ser Asp Ser Leu Ti 100 105 le Gin His Met Asp Asn Ile Phe Asp Ser Me 115 120 ii Ala Ser Ile Cys Asn Leu Leu Ala Ilie Al 130 135 ~r Ile Phe Tyr Ala Leu Arg Tyr His Ser Il 150 15 59 10: 12: 'yr Pro er Asn iu Val al Ile he Phe 75 sn Ser ir Leu t Ile a Val 140 e Met ~I 5 Th Arg Phe Leu Leu Leu G1u 125 \sp ~hr rLeu Ser Leu Ala Le u Glu Asp 110 Ile Arg Val Prc *Gl Ala Val Ser Th r Gin Ser Ty r Arg :Asn 'Ser Leu Val1 Leu Ile Phe Leu Val Ly s 160 Ala Len Ser Leu Ilie Val Ala Ilie Trp Val Cys Cys Gly Ile Cys Gly Val1 Ile His Pro 225 Ala Pro Ty r Met Met Phe Thr Met 195 Met Phe 210 Pro Ala Val Thr Phe Phe Cys Ile 275 Cys Asn Ile 180 Phe Leu Asp Ilie Len 260 Cys Ser 165 Val Ph e Phe Gly Th r 245 His Ty r Val 175 Tyr Ser Ala Met Ala Arg 215 Len Ala 230 Ile Len Leu Val Thr Ala Ilie Asp Gin Val1 200 Leu Pro Len Leu His 280 Pro Ser 185 Len His Gin Gly Ile 265 Phe Len Ly s Len Val1 Gin Val1 250 Ile Asn Ile Met Val Met Gly Gin Arg 220 His Ser 235 Phe Ilie Thr Cys Thr Tyr Tyr Ala 300 Ilie Thr 205 Ile Cys Phe Pro Len 285 Phe Cy s Ty r Ala Lys Trp 255 Asn Len Ser Len Ile Len Gly 240 Ala Pro Ilie Len 290 Gin Len Arg Asn Thr Phe Lys Gin Ile Len Cys Gly Cys Asn Gly Met 305 Asn Val Gly 310 315 320 INFORMATION FOR SEQ ID NO:13: SEQUENCE CHARACTERISTICS: LENGTH: 30 base pairs TYPE: nucleic acid STRAkNDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE: NAME/KEY: mics feature LOCATION: 1- OTHER INFORMATION: /function ="Degenerate oligonucleotide primer (sense)" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: GAGTCGACCR CCCATGTAYT DYTTCATCTG INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 1671 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA to rRNA (ix) FEATURE: NAME/KEY: LOCATION: 1..393 (ix) FEATURE: NAME/KEY: CDS LOCATION: 394..1389 (ix) FEATURE: NAME/KEY: 3'UTR LOCATION: 1390..1671 (xi) SEQUENCE DESCRIPTION: SEQ ID AGCTTCCGAG AGGCAGCCGA TGTGAGCATG TGCGCACAGA TrCGTCTCCC AATGGCATGG CAGCTTCAAG GAAAATTATT TTGAACAGAC TTGAATGCAT AAGATTAAAG TTAAAGCAGA 120 AGTGAGAACA AGAAAGCAAA GAGCAGACTC TTTCAACTGA GAATGAATAT TTTGAAGCCC 180 AAGATTTTAA CGTGATGATG ATTAGAGTCG TACCTAAAAG AGACTAAAAA CTCCATGTCA 240 AGCTCTGGAC TTGTGACATT TACTCACAGC AGGCATGGCA ATTTTAGCCT CACAACTTTC 300 AGACAGATAA AGACTTGGAG GAAATAACTG AGACGACTCC CTGACCCAGG AGGTTAAATC 360 AATTCAGGGG GACACTGGAA TTCTCCTGCC AGC ATO GTG AAC TCC ACC CAC COT 414 Met Val Asn Ser Thr His Arg 1 GGG ATG CAC ACT TCT CTG CAC CTC TGG AAC CGC AGC ACT TAC AGA CTG 462 Gly Met His Thr Ser Leu His Leu Trp Asn Ary Ser Ser Tyr Arg Leu 15 CAC AGC AAT GCC AGT GAG TCC CTT GGA AAA GGC TAC TCT GAT GGA GGG 510 His Ser Asn Ala Ser Glu Ser Leu Gly Lys Gly Tyr Ser Asp Gly Gly 30

TGC

Cys TAC GCG CAA CTT Tyr Ala Gin Leu TTT GTC TCT CCT Phe Val Ser Pro GAG AAT ATC TTA Gin Asn Ile Leu GAG GTG TTT GTG ACT CTG Giu Val Phe Val Thr Leu GTG ATC AGC TTG Val Ilie Ser Leu

TTG

Len ATT GTG GCA ATA Ile Val Ala Ile GCC AAG Ala Lys PAC AAC PAT Asn Lys Asn GTO GCT GAT Val Ala Asp CAT TCA CCC ATG His Ser Pro Met TTT TTC ATC TGC Phe Phe Ile Cys AGC TTG GCT Ser Len Aia ACC ATT ATC Thr Ile Ile ATG CTC GTG AGC Met Leu Val Ser TCA PAT GGA TCA Ser Asn Gly Ser ATC ACC Ile Thr 105 CTA TTA PAC CGT Leu Len Asn Arg GAT ACG GAT GCA Asp Thr Asp Ala AGT TTC ACA GTG Ser Phe Thr Val

PAT

As n 120 ATT GAT AAT CTC ATT GAC TCG GTG ATC le Asp Asn Val Ile Asp Ser Val Ile AGC TCC TTG CTT Ser Ser Len Len TCC ATT TGC AGC CTG CTT TCA ATT GCA Ser Ile Cys Ser Leu Len Ser Ile Ala GAC AGG TAC TT Asp Arg Tyr Phe ACT ATC Thr Ile 150 a. a a.

a a a TTC TAT GCT Phe Tyr Ala ATC AGC ATA Ile Ser Ilie 170

CTC

Len 155 CAG TAC CAT AAC Gin Tyr His Asn ATG ACA GTT PAG Met Thr Val Lys CGG GTT GGG Arg Val Gly 165 GGT AT? TTC Gly Ile Len ACT TGT ATC TGC Ser Cys Ile Trp GCT TGC ACG GTT Ala Cys Thr Val TTC ATC Phe Ile 185 ATT TAC TCA CAT Ile Tyr Ser Asp ACT CCT GTC ATC Ser Ala Vai Ile

ATC

Ile 195 TGC CTC ATC ACC Cys Len Ile Thr 990 a.

a

A-G

met 200 TTC TTC ACC ATG Phe Phe Thr met GCT CTC ATG GCT Ala Len Met Ala CTC TAT GTC CAC Len Tyr Val His 1038 TTC CTC ATCG CC Phe Len Met Ala CT? CAC ATT AAG ACG AT? GCT CTC CTC Len His Ile Lys Arg Ile Ala Val Len CCC GC Pro Gly 230 1086 1134 ACT GCT CC Thr Gly Aia CGC CPA CCT CC Arg Gin Gly Ala ATC AAG CCA CC Met Lys Cly Ala ATT ACC TTC Ile Thr Len 245 ACC ATC CTO ATT CCC GTC TTT OTT GTC TGC TOO GCC CCA TTC TTC CTC Thr Ile Leu Ile Cly Val Phe VJal Val Cys Trp Ala Pro Phe Phe Leu 250 2S5 260 CAC TTA ATA TTC TAC ATC TOT TOT CCT CAG AAT CCA TAT TGT OTG TGC His Leu Ile Phe Tyr Ile Ser Cys Pro Gin Asn Pro Tyr Cys Val Cys 265 270 275 TTC ATG TCT CAC TTT AAC TTC TAT CTC ATA CTG ATC ATG TOT AAT TCA Phe Met Ser His Phe Asn LeU Tyr Leu lie Leu Ile Met Cys Asn Ser 280 285 290 295 ATC ATC CAT CCT CTC ATT TAT OCA CTC CGG ACT CAA OAA CTC AGC AAA Ile Ile Asp Pro Leu Ile Tyr Ala Leu Arg Ser Gin Olu Leu Arg Lys 300 305 310 ACC TTC AAA GAC ATC ATC TCT TCC TAT CCC CTC OGA CCC CTT TOT CAC Thr Phe Lys Glu Ilie Ile Ser Ser Tyr Pro Leu Oly Oly Leu Cys Asp 315 320 325 TTO TCT AGC AGA TAT TAAATOCCCA CAGACCACOC AATATACCAA CATCCATAAO Leu Ser Ser Arg Tyr 330 AGACTTTTTC ACTCTTACCC TACCTCAATA TTCTACTTCT OCAACACCTT TCTCTTCOCT GTACCOTACT GOTTOAGATA TCCATTGTCT AAATTTAACC CTATGATTTT TAATCACAAA AAATGCCCAC TCTCTCTATT ATTTCCAATC TCATCCTACT TTTTTCOCCA TAAAATATGA ATCTATOTTA TACOTTOTAC OCACTOTOCA TTTACAAAAA GAAAACTCCT TATTAAAAGA

TT

INFORMATION FOR SEQ ID NO:16: SEQUENCE CHARACTERISTICS: LENGTH: 332 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: Met Val Asn Ser Thr His Arg Cly Met His Thr Ser Leu His Leu Trp 5 10 is Asn Arg Ser Ser Tyr Arg Leu His Ser Asn Ala Ser Glu Ser Leu Cly 25 1182 1230 1278 1326 1374 1429 1489 1549 1609 1669 1671

S

.5.5 *5 S S

S

S.

S S 5 *4

S*

SOS*

Lys Gly Tyr Ser Asp Gly Gly Cys Tyr Ala Gin Leu Phe Val Ser Pro Glu Glu Phe Asn Asp Ile Val 145 Met Cys Val Ala Arg 225 Met Cy Gin Va1 Ile Phe Gly Ala Cy 130 Asp Thr Thr Ile Ser 210 Ile Lys Trp Asn Phe Val Ile Ser Gin 115 Ser Arg Val Val Ile 195 Leu Ala Gly Ala Pro 275 Ile Val Ala Cys Glu 100 Ser Ser Tyr Lys Ser 180 Cys Tyr Val Ala Pro 260 Tyr Thr Ile Ser Thr Phe Leu Phe Arg 165 Gly Leu Val Leu Ile 245 Phe Cys Leu Ala 70 Leu Ile Thr Leu Thr 150 Val Ile Ile His Pro 230 Thr Phe Val SS 0 000 see.

rose .400 0000 0 *s

S.

C *5*S *3 ego.

0@

OSS*

S

Gly 55 Lys Ala Ile Val Ala 135 Ile Gly Leu Thr Leu 215 Gly Leu Leu Cys Ser 295 Val Asn Val Ile Asn 120 Ser Phe Ile Phe Met 200 Phe Thr Thr His Phe 280 Ile Ile Lys Ala Thr 105 Ile Ile Tvr Ser Ile 185 Phe Leu Gly Ile Leu 265 Met Ser Asn Asp 90 Leu Asp C s Ala Ile 170 Ile Phe Met Ala Leu 250 le Ser Leu Leu 75 Met Leu Asn Ser Leu 155 Ser Tyr Thr Ala Ile 235 Ile Phe His Leu His Leu Asn Val Leu 140 Gin Cvs Ser Met Arg 220 Arg Gly Tyr Phe Leu 300 Glu Ser Va1 Arg Ile 125 Leu Ty r Ile Asp Leu 205 Leu Gin Val Ile Asn 285 Ile Asn Pro Ser Thr 110 Asp Ser His Trp Ser 190 Ala His Gly Phe Ser 270 Leu Ile Met Val Asp Ser Ile Asn Ala 175 Ser Leu Ile a Val 255 :ys Tyr Leu Tyr Ser Thr Val Ala Ile 160 Ala Ala Met Lys Asn 240 Val Pro Leu Ile Leu 290 Met Cys Asn Ile Asp Pro Tyr Ala Leu Arg Ser Gin Giu Leu Arg Lys Thr Phe Lys Giu Ile Ile Ser Ser Tyr 305 310 315 320 Pro Leu Gly Gly Leu Cys Asp Leu Ser Ser Ary Tyr 325 330 INFORMATION FOR SEQ ID NO:17: SEQUENCE CHAR-ACTERISTICS: LENGTH: 978 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE: NA.ME/K-EY: CDS LOCATION: 1. .975 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: ATG AAC TCC TCC TCC ACC CTG ACT GTA TTG AAT CTT ACC CTG Met Asn Ser Ser Ser Thr Leu Thr Val Leu Asn Leu Thr Leu AAC GCC Asn Ala TCA GAG CAT GGC ATT TTA GGA TCA AAT GTC Ser Giu Asp Giy Ilie 20 Leu Gly Ser Val AAG AAC AAG Lys Asn Lys TCT TTG GCC Ser Leu Ala CTG GGT CTC Leu Gly Leu TGT GA.A GAA ATO Cys Giu Glu Met CCC ATT GCC Gly Ile Ala GAG CTC TTC CTG Glu Val Phe Leu GTC AGC CTC TTA GAG AAC ATC CTG GTC ATT Val Ser Leu Leu Glu Asn Ile Leu Val Ile GGG GCC Gly Ala GTG GGC Val Gly 7 ATA GTA AAG AAC Ile Val Lys Asn

A-AA

Lys AAC CTC CAC TCA Asn Leu His Ser ATG TAC TIC TT Met Tyr Phe Phe AGC TTA GCC Ser Leu Ala CCC GAC ATO CTG Ala Asp Met Leu TAC TTG CTA AAT Tyr Leu Leu Asn 100 AGC ATG TCC A.AT Ser Met Ser Asn TGG GAG ACT GTC ACC ATA Trp Glu Thr Vai Thr Ile GCC GAC ACC TTT GTG CGA Ala Asp Thr Phe Val Arg 110 AAT AAA CAC CTG GTG ATA Asn Lys His Leu Val Ile 105 CAC ATC CAC A.AC GTG TTC CAC TCC ATG ATC TGC ATC TCT GTG GTG GCC His Ile Asp 115 Asn Val Phe Asp Met Ile Cys Ile Val Val Ala TCG ATO Ser Met 130 TCC ACT TTC CTC Cys Ser Leu Leu ATT GC OTO GAT AGO TAC ATC ACC ATC Ile Ala Val Asp Arg Tyr Ile Thr Ile TTC TAt1 CCC TTG CGC TAC CAC CAC ATC ATC ACC GCG AGO CCC TCG CCC The Tyr Ala Leu Arg Tyr His His Ile Met Thr Ala Arg Arg Ser Gly 145 150 155 160 GTG ATC ATC GCC Val Ile Ile Ala ATT TGC ACC TTC Ile Trp Thr Phe ATA AOC TOC GCC Ile Ser Cys Gly ATT OTT Ile Val 175 528 TTC ATC ATC Phe Ile Ile ATG TTC TTC Met Phe Phe 195 TAT GAO TCC AAG Tyr Giu Ser Lys OTC ATC ATT TC Val Ile Ile Cys CTC ATC TCC Leu Ile Ser 190 ATA CAC ATO Ilie His Met ACC ATO CTO TTC Thr Met Leu Phe ATO CTC TCT CTC Met Val Ser Leu 624 TTC CTC Phe Leu 210 CTC CCC CCC AAC Leu Ala Arg Asn

CAT

His 215 CTC AAC COO ATA Val- Lys Ar Ile OCT TCC CCC ACA Ala Ser Pro Arg AAC TCC CTC AG Asn Ser Val Arg

CAA

Gin 230 AGO ACC AGC ATO Arg Thr Ser Met CCC OCT ATT ACC Gly Ala Ile Thr '72 0 ACC ATO CTA CTO Thr Met Leu Leu

CG

Cly 245 ATT TTC ATT GTC Ile Phe Ile Val TGO TCT CCC TTC Trp Ser Pro Phe TTT CTT Phe Leu 255 CAC CTT ATC His Leu Ilie TTT ATO TCT Phe Met Ser 275

TTA

Leu 260 ATO ATC TCC TOC Met Ile Ser Cys

CCT

Pro 265 CAC A.AC OTC TAC Cmn Asn Vai Tyr TGC TCT TGC Cys Ser Cys 270 TOC AAC TCC Cys Asn Ser 816 TAC TTC AAC ATO Tyr Phe Asn Met CTT ATA CTC ATC Leu Ilie Leu Ile CTC ATC Val Ilie 290 CAT CCT CTC ATC Asp Pro Leu Ilie CCC CTC CCC AGC Ala Leu Arg Ser GAO ATG CCC AGO Clu Met Arg Arg

ACC

Thr TTT AAO GAO ATC Phe Lys Ciu Ilie TOT TOT CAC GGA TTC COO COA CCT TOT Cys Cys His Oly Phe Arg Arg Pro Cys 315 AGO 960 Arg 320 CTC CTT GGC GGG TAT TAA Leu Leu Gly Gly Tyr 325 INFORMATION FOR SEQ ID NO:18: SEQUENCE CHARACTERISTICS: LENGTH: 325 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: 978 Ser Va 1 -vs Ala His Ser T he -145 Asr Glu Glu Ser 50 Asn Asp Le u Ile Met 130 Ty r Ser Asp Glu Le u Le u Met Le u Asp 115 Cys Ala Sei Gly Met Le u His Leu Asn 100 Asn Ser Leu rSer Ile Gly Glu Ser Val 85 Asn Val1 Leu Arg Cys 165 Th r Ser ValI Asn eu Asn Alh er Leu Alz Ile Ala Val Glu Val 40 Asn Ilie Leu Val Ile 5 Pro Met Tyr Phe Phe 70 Ser Met Ser Asn Ala 90 Lys His Leu Val Ile 105 Phe Asp Ser Met Ile 120 Leu Ala Ile Ala Val 135 Tyr His His Ilie Met 150 Phe Leu Thr Leu Gly Leu Gly Val1 75 Trp Ala Cys Asp Th r 155 Ile Ala Ile Gly Ser Glu Thr Asp Thr Ile Ser 125 Arg Tyr 140 Ala Arg Ser Cys Val1 Le u Val1 Phe 110 Val1 Ile Arg Gly Lys Ala Th r Val Val1 Th r Ser Ile 175 As n Val Ile Arg Al a Ile Gly 160 Val Val Ile Ile Ala Ile Trp Thr Phe Phe Ile Ile Tyr Tyr Giu Ser Lys Tyr Val Ile Ile Cys Leu Ile Ser 180 185 190 Met Phe Phe Thr Met Leu Phe Phe Met Val Ser Leu Tyr Ile His Met 195 200 205 Phe Leu Leu Ala Arg Asn His Val Lys Arg Ile Ala Ala Ser Pro Arg 210 215 220 Tyr Asn Ser Vai Arg Gin Arg Thr Ser Met Lys Gly Ala Ile Thr Leu 225 230 235 240 Thr Met Leu Leu Gly Ilie Phe Ile Val Cys Trp Ser Pro Phe Phe Leu 245 250 255 His Leu Ile Leu Met Ile Ser Cys Pro Gin Asn Val Tyr Cys Ser Cys 260 265 270 Phe Met Ser Tyr Phe Asn Met Tyr Leu Ile Leu Ile Met Cys Asn Ser 275 280 285 Val Ile Asp Pro Leu Ile Tyr Ala Leu Arg Ser Gin Glu Met Arg Arg 290 295 300 Thr Phe Lys Glu Ile Val Cys Cys His Gly Phe Arg Arg Pro Cys Ary 305 310 315 320 Leu Leu Gly Gly Tyr 325 INFORMATION FOR SEQ ID NO:19: Ci) SEQUENCE CHARACTERISTICS: LENGTH: 30 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (geflomic) (ix) FEATURE: NAME/KEY: misc feature LOCATION: 1. .32 CD) OTHER INFORMATION: /function "Degenerate oligonucleotide primer (antisense)', (xi) SEQUENCE DESCRIPTION: SEQ ID NO:i9: GAATTCGACG TCACAGTATG ACGGCCATGG

Claims (114)

1. A mammalian melanocortin MC-3 or MC-4 receptor agonist having the general formula: A-B-C-D-E-F-G-amide wherein A is Leu, Ile, Met, or substituted analogues thereof; B is Asp, Glu, or substituted analogues thereof; C is His or substituted analogues thereof; D is D-Phe, D-Tyr or substituted analogues thereof; E is Arg, Lys, homoArg, homoLys, or substituted analogues thereof; F is Trp or substituted analogues thereof; G is Lys, homoLys or substituted analogues thereof; and wherein the peptide is cyclised by the formation of an amide bond between the side chain carboxyl group of the Asp or Glu residue at position B in the peptide, and the side chain amino group of the Lys or homoLys residue at position G.

2. A mammalian melanocortin MC-3 or MC-4 receptor agonist according to claim 1 comprising the amino acid sequence A-Asp-His-D-Phe-Arg-Trp-Lys, or derivatives thereof, wherein A is Leu, Ile, Met, or substituted analogues thereof.

3. A mammalian melanocortin MC-3 or MC-4 receptor agonist according to claim 1 or claim 2, wherein one or more carboxylic acid groups of the peptide is/are in the form of a salt of a pharmaceutically acceptable cation.

4. A mammalian melanocortin MC-3 or MC-4 receptor agonist according to claim 1 or claim 2, wherein one or more carboxylic acid groups of the peptide is/are esterified to form a C 1 -C 6 ester. A mammalian melanocortin MC-3 or MC-4 receptor agonist according to claim 1 or claim 2, wherein one or more carboxylic acid groups of the peptide is/are converted to an amide of formula NRiR 2 wherein RI and RI are each independently H or CI-C 1 6 alkyl.

6. A mammalian melanocortin MC-3 or MC-4 receptor agonist according to claim 1 or claim 2, wherein carboxylic acid groups of the peptide are combined to form a heterocyclic ring. 30 7. A mammalian melanocortin MC-3 or MC-4 receptor agonist according to claim 0 6, wherein the heterocyclic ring is 5- or 6- membered.

8. A mammalian melanocortin MC-3 or MC-4 receptor agonist according to any of claims 1 to 7, wherein one or more amino groups of the peptide is/are in the form of a pharmaceutically acceptable acid addition salt. S 35 9. A mammalian melanocortin MC-3 or MC-4 receptor agonist according to claim 8, wherein said acid is selected from the group comprising HC1, HBr, acetic, benzoic, *o toluene sulfonic, maleic, and tartaric. A04420 A mammalian melanocortin MC-3 or MC-4 receptor agonist according to any one of claims 1 to 7, wherein one or more amino groups of the peptide is/are modified to C 1 C 1 6 alkyl or dialkyl amino.

11. A mammalian melanocortin MC-3 or MC-4 receptor agonist according to any one of claims 1 to 7, wherein one or more amino groups of the peptide is/are converted to an amide.

12. A mammalian melanocortin MC-3 or MC-4 receptor agonist according to any one of claims 1 to 11, wherein one or more hydroxyl groups of the peptide sidechain is/are converted to C 1 -C 1 6 alkoxy or to a C 1 -C 6 ester.

13. A mammalian melanocortin MC-3 or MC-4 receptor agonist according to any one of claims 1 to 12, wherein one or more phenyl/phenolic rings of the peptide sidechain is/are substituted with one or more halogen atoms.

14. A mammalian melanocortin MC-3 or MC-4 receptor agonist according to any one of claims 1 to 12, wherein one or more phenyl/phenolic rings of the peptide sidechain is/are substituted with one or more groups selected from C 1 -C 16 alkyl, C 1 -C 16 alkoxy, carboxylic acids or amides or esters of carboxylic acids. A mammalian melanocortin MC-3 or MC-4 receptor agonist according to any one of claims 1 to 12, wherein one or more methylene groups of the peptide sidechain is/are extended to C 2 -C 4 alkylenes.

16. A mammalian melanocortin MC-3 or MC-4 receptor agonist according to any one of claims 1 to 15, wherein said peptide is cyclised by addition of a C-terminal or N- terminal cysteine residue which forms a disulfide bond on oxidation, thereby generating a cyclic peptide.

17. A mammalian melanocortin MC-3 or MC-4 receptor agonist according to any one of claims 1 to 15, wherein said peptide is cyclised by formation of a thioether, C- terminal amide, N-terminal amide, C-terminal ester, or N-terminal ester.

18. A process for preparing a mammalian melanocortin MC-3 or MC-4 receptor agonist having the general formula: A-B-C-D-E-F-G 30 wherein A is Leu, Ile, Met, or substituted analogues thereof; B is Asp, Glu, or substituted analogues thereof B is Asp, Glu, or substituted analogues thereof; C is His or substituted analogues thereof; D is D-Phe, D-Tyr or substituted analogues thereof; E is Arg, Lys, homoArg, homoLys, or substituted analogues thereof; 35 F is Trp or substituted analogues thereof; G is Lys, homoLys or substituted analogues thereof; substantially as hereinbefore described.

19. A mammalian melanocortin MC-3 or MC-4 receptor agonist prepared by a RA4/ process according to claim 18. A04420 71 A mammalian melanocortin MC-3 or MC-4 receptor agonist according to claim 1 or claim 19 having the formula Ac-A 4 -cyclo(Asp 5 -His6-D-Phe 7 -Arg-Trp 9 -Lysl o )aMSH- (4-10)-amide, wherein A is Leu, Ile, Met, or substituted analogues thereof.

21. A mammalian melanocortin MC-3 or MC-4 receptor agonist as defined in claim 1, substantially as hereinbefore described.

22. A mammalian melanocortin MC-3 or MC-4 receptor antagonist having the general formula: A-B-C-D-E-F-G-amide wherein A is Leu, lie, Met, or substituted analogues thereof; B is Asp, Glu, or substituted analogues thereof; C is His or substituted analogues thereof; D is D-Nal or substituted analogues thereof; E is Arg, Lys, homoArg, homoLys, or substituted analogues thereof; F is Trp or substituted analogues thereof; G is Lys, homoLys or substituted analogues thereof; and wherein the peptide is cyclised by the formation of an amide bond between the side chain carboxyl group of the Asp or Glu residue at position B in the peptide, and the side chain amino group of the Lys or homoLys residue at position G.

23. A mammalian melanocortin MC-3 or MC-4 receptor antagonist according to claim 22 comprising the amino acid sequence A-Asp-His-D-Nal-Arg-Trp-Lys, or derivatives thereof, wherein A is Leu, Ile, Met, or substituted analogues thereof.

24. A mammalian melanocortin MC-3 or MC-4 receptor antagonist according to claim 22 or claim 23, wherein one or more carboxylic acid groups of the peptide is/are in the form of a salt of a pharmaceutically acceptable cation.

25. A mammalian melanocortin MC-3 or MC-4 receptor antagonist according to claim 22 or claim 23, wherein one or more carboxylic acid groups of the peptide is/are esterified to form a CI-C1 6 ester.

26. A mammalian melanocortin MC-3 or MC-4 receptor antagonist according to claim 22 or claim 23, wherein one or more carboxylic acid groups of the peptide is/are S: 30 converted to an amide of formula NR 1 R 2 wherein R, and R, are each independently H or Ci-C 6 alkyl.

27. A mammalian melanocortin MC-3 or MC-4 receptor antagonist according to claim 22 or claim 23, wherein carboxylic acid groups of the peptide are combined to form a heterocyclic ring. 35 28. A mammalian melanocortin MC-3 or MC-4 receptor antagonist according to claim 27, wherein the heterocyclic ring is 5- or 6- membered.

29. A mammalian melanocortin MC-3 or MC-4 receptor antagonist according to any one of claims 22 to 28, wherein one or more amino groups of the peptide is/are in the form of a pharmaceutically acceptable acid addition salt. Uj' A04420 m 72 A mammalian melanocortin MC-3 or MC-4 receptor antagonist according to claim 29, wherein said acid is selected from the group comprising HC1, HBr, acetic, benzoic, toluene sulfonic, maleic, and tartaric.

31. A mammalian melanocortin MC-3 or MC-4 receptor antagonist according to any one of claims 22 to 28, wherein one or more amino groups of the peptide is/are modified to Ci-C 16 alkyl or dialkyl amino.

32. A mammalian melanocortin MC-3 or MC-4 receptor antagonist according to any one of claims 22 to 28, wherein one or more amino groups of the peptide is/are converted to an amide.

33. A mammalian melanocortin MC-3 or MC-4 receptor antagonist according to any one of claims 22 to 32, wherein one or more hydroxyl groups of the peptide sidechain is/are converted to C 1 -C 6 alkoxy or to a C 1 -C 1 6 ester.

34. A mammalian melanocortin MC-3 or MC-4 receptor antagonist according to any one of claims 22 to 33, wherein one or more phenyl/phenolic rings of the peptide sidechain is/are substituted with one or more halogen atoms. A mammalian melanocortin MC-3 or MC-4 receptor antagonist according to any one of claims 22 to 33, wherein one or more phenyl/phenolic rings of the peptide sidechain is/are substituted with one or more groups selected from CI-C 1 6 alkyl, C 1 -C 1 6 alkoxy, carboxylic acids or amides or esters of carboxylic acids.

36. A mammalian melanocortin MC-3 or MC-4 receptor antagonist according to any one of claims 22 to 33, wherein one or more methylene groups of the peptide sidechain is/are extended to C 2 -C 4 alkylenes.

37. A mammalian melanocortin MC-3 or MC-4 receptor antagonist according to any one of claims 22 to 36, wherein said peptide is cyclised by addition of a C-terminal or N-terminal cysteine residue which forms a disulfide bond on oxidation, thereby generating a cyclic peptide.

38. A mammalian melanocortin MC-3 or MC-4 receptor antagonist according to any one of claims 22 to 36, wherein said peptide is cyclised by formation of a thioether, C- terminal amide, N-terminal amide, C-terminal ester, or N-terminal ester. 30 39. A process for preparing a mammalian melanocortin MC-3 or MC-4 receptor antagonist having the general formula: A-B-C-D-E-F-G wherein A is Leu, Ile, Met, or substituted analogues thereof; B is Asp, Glu, or substituted analogues thereof; 35 C is His or substituted analogues thereof; D is D-Nal or substituted analogues thereof; E is Arg, Lys, homoArg, homoLys, or substituted analogues thereof; F is Trp or substituted analogues thereof; S 14\ G is Lys, homoLys or substituted analogues thereof; A04420 73 substantially as hereinbefore described. A mammalian melanocortin MC-3 or MC-4 receptor antagonist prepared by a process according to claim 39.

41. A mammalian melanocortin MC-3 or MC-4 receptor antagonist according to claim 22 or claim 40 having the formula Ac-A 4 -cyclo(Asp 5 -His 6 -D-Nal(2) 7 -Arg 8 -Trp 9 Lys'o)aMSH-(4- 10)-amide, wherein A is Leu, Ile, Met, or substituted analogues thereof.

42. A mammalian melanocortin MC-3 or MC-4 receptor antagonist as defined in claim 22, substantially as hereinbefore described.

43. Use of a mammalian melanocortin MC-3 or MC-4 receptor agonist having the general formula: A-B-C-D-E-F-G-amide wherein A is Leu, Ile, Nle, Met, or substituted analogues thereof; B is Asp, Glu, or substituted analogues thereof; C is His or substituted analogues thereof; D is D-Phe, D-Tyr or substituted analogues thereof; E is Arg, Lys, homoArg, homoLys, or substituted analogues thereof; F is Trp or substituted analogues thereof; G is Lys, homoLys or substituted analogues thereof; and wherein the peptide is cyclised by the formation of an amide bond between the side chain carboxyl group of the Asp or Glu residue at position B in the peptide, and the side chain amino group of the Lys or homoLys residue at position G; for the manufacture of a medicament for inhibiting feeding behaviour in an animal.

44. A use according to claim 43, wherein said agonist has the formula Ac-Nle 4 cyclo(Asp 5 -His 6 -D-Phe 7 -Arg 8 -Trp 9 -Lys' o )aMSH-(4-10)-amide.

45. A use according to claim 43, wherein said agonist is an agonist according to any one of claims 1 to 17 or 19 to 21.

46. A use according to claim 43, wherein said agonist is an agonist according to claim

47. A medicament for inhibiting feeding behaviour in an animal comprising a 30 mammalian melanocortin MC-3 or MC-4 receptor agonist having the general formula: A-B-C-D-E-F-G-amide wherein A is Leu, Ile, Nle, Met, or substituted analogues thereof; B is Asp, Glu, or substituted analogues thereof; C is His or substituted analogues thereof; D is D-Phe, D-Tyr or substituted analogues thereof; E is Arg, Lys, homoArg, homoLys, or substituted analogues thereof; 0 F is Trp or substituted analogues thereof; G is Lys, homoLys or substituted analogues thereof; A04420 74 and wherein the peptide is cyclised by the formation of an amide bond between the side chain carboxyl group of the Asp or Glu residue at position B in the peptide, and the side chain amino group of the Lys or homoLys residue at position G, and a pharmaceutically or veterinary acceptable carrier.

48. A medicament according to claim 47, wherein said agonist has the formula Ac- Nle 4 -cyclo(Asp 5 -His 6 D-Phe 7 -ArgS-Trp 9 -LysL)aMSH-(4-10)-amide.

49. A medicament according to claim 47, wherein said agonist is an agonist according to any one of claims 1 to 17 or 19 to 21. A medicament according to claim 47, wherein said agonist is an agonist 0o according to claim

51. A method of inhibiting feeding behaviour in an animal, the method comprising administering to said animal an effective amount of a mammalian melanocortin MC-3 or MC-4 receptor agonist having the general formula: A-B-C-D-E-F-G-amide wherein A is Leu, Ile, Nle, Met, or substituted analogues thereof; B is Asp, Glu, or substituted analogues thereof; C is His or substituted analogues thereof; D is D-Phe, D-Tyr or substituted analogues thereof; E is Arg, Lys, homoArg, homoLys, or substituted analogues thereof; F is Trp or substituted analogues thereof; G is Lys, homoLys or substituted analogues thereof; and wherein the peptide is cyclised by the formation of an amide bond between the side chain carboxyl group of the Asp or Glu residue at position B in the peptide, and the side chain amino group of the Lys or homoLys residue at position G; 25 or of a medicament according to any one of claims 47 to

52. A method according to claim 51, wherein said agonist has the formula Ac-Nle 4 cyclo(Asp -His6-D-Phe 7 -Args-Trp 9 -Lys'l)aMSH-(4-10)-amide or said medicament is a medicament according to claim 48.

53. A method according to claim 51, wherein said agonist is an agonist according to 30 any one of claims 1 to 17 or 19 to 21 or said medicament is a medicament according to claim 49.

54. A method according to claim 51, wherein said agonist is an agonist according to claim 20 or said medicament is a medicament according to claim

55. A mammalian melanocortin MC-3 or MC-4 receptor agonist having the general 35 formula: A-B-C-D-E-F-G-amide wherein A is Leu, Ile, Nle, Met, or substituted analogues thereof; B is Asp, Glu, or substituted analogues thereof; SC is His or substituted analogues thereof; 9 9 9 9 9 9999 9 9. A04420 D is D-Phe, D-Tyr or substituted analogues thereof; E is Arg, Lys, homoArg, homoLys, or substituted analogues thereof; F is Trp or substituted analogues thereof; G is Lys, homoLys or substituted analogues thereof; and wherein the peptide is cyclised by the formation of an amide bond between the side chain carboxyl group of the Asp or Glu residue at position B in the peptide, and the side chain amino group of the Lys or homoLys residue at position G; or a medicament according to any one of claims 47 to when used for inhibiting feeding behaviour in an animal.

56. A mammalian melanocortin MC-3 or MC-4 receptor agonist or medicament when used according to claim 55, wherein said agonist has the formula Ac-Nle 4 -cyclo(Asp 5 His 6 -D-Phe 7 -Arg -Trp 9 -LyslI)aMSH-(4-10)-amide or said medicament is a medicament according to claim 48.

57. A mammalian melanocortin MC-3 or MC-4 receptor agonist or medicament when used according to claim 55, wherein said agonist is an agonist according to any one of claims 1 to 17 or 19 to 21 or said medicament is a medicament according to claim 49.

58. A mammalian melanocortin MC-3 or MC-4 receptor agonist or medicament when used according to claim 55, wherein said agonist is an agonist according to claim or said medicament is a medicament according to claim

59. Use of a mammalian melanocortin MC-3 or MC-4 receptor antagonist having the general formula: A-B-C-D-E-F-G-amide wherein A is Leu, Ile, Nle, Met, or substituted analogues thereof; B is Asp, Glu, or substituted analogues thereof; C is His or substituted analogues thereof; D is D-Nal or substituted analogues thereof; E is Arg, Lys, homoArg, homoLys, or substituted analogues thereof; F is Trp or substituted analogues thereof; G is Lys, homoLys or substituted analogues thereof; 30 and wherein the peptide is cyclised by the formation of an amide bond between the side chain carboxyl group of the Asp or Glu residue at position B in the peptide, and the side chain amino group of the Lys or homoLys residue at position G; "for the manufacture of a medicament for stimulating feeding behaviour in an animal.

60. A use according to claim 59, wherein said antagonist has the formula Ac-Nle 4 35 cyclo(Asps-His 6 -D-Nal(2) -Arg 8 -Trp 9 -Lys o )aMSH-(4-10)-amide.

61. A use according to claim 59, wherein said antagonist is an antagonist according to any one of claims 22 to 38 or 40 to 42.

62. A use according to claim 59, wherein said antagonist is an antagonist according to claim 41. A04420 76

63. A medicament for stimulating feeding behaviour in an animal comprising a mammalian melanocortin MC-3 or MC-4 receptor antagonist having the general formula: A-B-C-D-E-F-G-amide wherein A is Leu, Ile, Nle, Met, or substituted analogues thereof; B is Asp, Glu, or substituted analogues thereof; C is His or substituted analogues thereof; D is D-Nal or substituted analogues thereof; E is Arg, Lys, homoArg, homoLys, or substituted analogues thereof; F is Trp or substituted analogues thereof; G is Lys, homoLys or substituted analogues thereof; and wherein the peptide is cyclised by the formation of an amide bond between the side chain carboxyl group of the Asp or Glu residue at position B in the peptide, and the side chain amino group of the Lys or homoLys residue at position G, and a pharmaceutically or veterinary acceptable carrier.

64. A medicament according to claim 63, wherein said antagonist has the formula Ac-Nle 4 -cyclo(Asp 5 -His 6 -D-Nal(2) 7 -Arg 8 -Trp 9 -Lys 1 o)aMSH-(4-10)-amide. A medicament according to claim 63, wherein said antagonist is an antagonist according to any one of claims 22 to 38 or 40 to 42.

66. A medicament according to claim 63, wherein said antagonist is an antagonist according to claim 41.

67. A method of stimulating feeding behaviour in an animal, the method comprising administering to said animal an effective amount of a mammalian melanocortin MC-3 or MC-4 receptor antagonist having the general formula: A-B-C-D-E-F-G-amide wherein A is Leu, Ile, Nle, Met, or substituted analogues thereof; B is Asp, Glu, or substituted analogues thereof; C is His or substituted analogues thereof; D is D-Nal or substituted analogues thereof; E is Arg, Lys, homoArg, homoLys, or substituted analogues thereof; S 30 F is Trp or substituted analogues thereof; G is Lys, homoLys or substituted analogues thereof; and wherein the peptide is cyclised by the formation of an amide bond between the side chain carboxyl group of the Asp or Glu residue at position B in the peptide, and the side chain amino group of the Lys or homoLys residue at position G; S 35 or of a medicament according to any one of claims 63 to 66.

68. A method according to claim 67, wherein said antagonist has the formula Ac- *ee Nle 4 -cyclo(Asp 5 -His 6 -D-Nal(2) 7 -Arg-Trp 9 -Lys'o)aMSH-(4-10)-amide or said medicament is a medicament according to claim 64. A04420 77

69. A method according to claim 67, wherein said antagonist is an antagonist according to any one of claims 22 to 38 or 40 to 42 or said medicament is a medicament according to claim A method according to claim 67, wherein said antagonist is an antagonist according to claim 41 or said medicament is a medicament according to claim 66.

71. A mammalian melanocortin MC-3 or MC-4 receptor antagonist having the general formula: A-B-C-D-E-F-G-amide wherein A is Leu, Ile, Nle, Met, or substituted analogues thereof; B is Asp, Glu, or substituted analogues thereof; C is His or substituted analogues thereof; D is D-Nal or substituted analogues thereof; E is Arg, Lys, homoArg, homoLys, or substituted analogues thereof; F is Trp or substituted analogues thereof; G is Lys, homoLys or substituted analogues thereof; and wherein the peptide is cyclised by the formation of an amide bond between the side chain carboxyl group of the Asp or Glu residue at position B in the peptide, and the side chain amino group of the Lys or homoLys residue at position G; or a medicament according to any one of claims 63 to 66, when used for stimulating feeding behaviour in an animal.

72. A mammalian melanocortin MC-3 or MC-4 receptor antagonist or medicament when used according to claim 71, wherein said antagonist has the formula Ac-Nle 4 cyclo(Asp-His 6 -D-Nal(2) 7 -Arg 8 -Trp9-Lys'o)ocMSH-(4-10)-amide or said medicament is a medicament according to claim 64.

73. A mammalian melanocortin MC-3 or MC-4 receptor antagonist or medicament when used according to claim 71, wherein said antagonist is an antagonist according to any S one of claims 22 to 38 or 40 to 42 or said medicament is a medicament according to claim SO

74. A mammalian melanocortin MC-3 or MC-4 receptor antagonist or medicament S: 30 when used according to claim 71, wherein said antagonist is an antagonist according to claim 41 or said medicament is a medicament according to claim 66.

75. A peptidomimetic or organomimetic compound having mammalian melanocortin MC-3 or MC-4 receptor agonist activity, and which mimics the three- dimensional arrangement of the chemical constituents and sidechains of a peptide having 35 the general formula: A-B-C-D-E-F-G-amide s.. wherein A is Leu, Ile, Nle, Met, or substituted analogues thereof; A' B is Asp, Glu, or substituted analogues thereof; C is His or substituted analogues thereof; A04420 78 D is D-Phe, D-Tyr or substituted analogues thereof; E is Arg, Lys, homoArg, homoLys, or substituted analogues thereof; F is Trp or substituted analogues thereof; G is Lys, homoLys or substituted analogues thereof; and wherein the peptide is cyclised by the formation of an amide bond between the side chain carboxyl group of the Asp or Glu residue at position B in the peptide, and the side chain amino group of the Lys or homoLys residue at position G.

76. A peptidomimetic or organomimetic compound according to claim 75, wherein said peptide has the formula Ac-Nle 4 -cyclo(Asp-His 6 -D-Phe 7 -Arg 8 -Trp 9 -Lyslo)MSH-(4-

77. A peptidomimetic or organomimetic compound according to claim 75, wherein said peptide is a mammalian melanocortin MC-3 or MC-4 receptor agonist according to any one of claims 1 to 17 or 19 to 21.

78. A peptidomimetic or organomimetic compound according to claim 75, wherein said peptide is a mammalian melanocortin MC-3 or MC-4 receptor agonist according to claim

79. A method of designing a peptidomimetic or organomimetic compound having mammalian melanocortin MC-3 or MC-4 receptor agonist activity, said method comprising ascertaining the pharmacophore of a peptide having the general formula: A-B-C-D-E-F-G-amide wherein A is Leu, Ile, Nle, Met, or substituted analogues thereof; B is Asp, Glu, or substituted analogues thereof; C is His or substituted analogues thereof; D is D-Phe, D-Tyr or substituted analogues thereof; 25 E is Arg, Lys, homoArg, homoLys, or substituted analogues thereof; F is Trp or substituted analogues thereof; G is Lys, homoLys or substituted analogues thereof; and wherein the peptide is cyclised by the formation of an amide bond between the side chain carboxyl group of the Asp or Glu residue at position B in the peptide, and the side chain amino group of the Lys or homoLys residue at position G, and fitting a combination of suitable groups to said pharmacophore using computer aided drug design. A method according to claim 79, wherein said peptide has the formula Ac-Nle 4 cyclo(Asp 5 -His 6 -D-Phe 7 -Arg 8 -Trp 9 -Lys ')aMSH-(4-10)-amide.

81. A method according to claim 79, wherein said peptide is a mammalian S 35 melanocortin MC-3 or MC-4 receptor agonist according to any one of claims 1 to 17 or 19 to 21.

82. A method according to claim 79, wherein said peptide is a mammalian melanocortin MC-3 or MC-4 receptor agonist according to claim A04420 79

83. A peptidomimetic or organomimetic compound having mammalian melanocortin MC-3 or MC-4 receptor agonist activity, designed by a method according to claim 79.

84. A peptidomimetic or organomimetic compound according to claim 83, wherein said peptide has the formula Ac-Nle 4 -cyclo(Asp-His 6 -D-Phe 7 -ArgS-Trp 9 -Lys'I)aMSH-(4- A peptidomimetic or organomimetic compound according to claim 83, wherein said peptide is a mammalian melanocortin MC-3 or MC-4 receptor agonist according to any one of claims 1 to 17 or 19 to 21.

86. A peptidomimetic or organomimetic compound according to claim 83, wherein said peptide is a mammalian melanocortin MC-3 or MC-4 receptor agonist according to claim

87. A peptidomimetic or organomimetic compound having mammalian melanocortin MC-3 or MC-4 receptor antagonist activity, and which mimics the three- dimensional arrangement of the chemical constituents and sidechains of a peptide having the general formula: A-B-C-D-E-F-G-amide wherein A is Leu, Ile, Nle, Met, or substituted analogues thereof; B is Asp, Glu, or substituted analogues thereof; C is His or substituted analogues thereof; D is D-Nal or substituted analogues thereof; E is Arg, Lys, homoArg, homoLys, or substituted analogues thereof; F is Trp or substituted analogues thereof; G is Lys, homoLys or substituted analogues thereof; 25 and wherein the peptide is cyclised by the formationof an amide bond between the side chain carboxyl group of the Asp or Glu residue at position B in the peptide, and the side S' chain amino group of the Lys or homoLys residue at position G.

88. A peptidomimetic or organomimetic compound according to claim 87, wherein said peptide has the formula Ac-Nle 4 -cyclo(Asp-His 6 -D-Nal(2) 7 -Arg 8 -Trp 9 -Lys o)c)MSH- (4-10)-amide.

89. A peptidomimetic or organomimetic compound according to claim 87, wherein said peptide is a mammalian melanocortin MC-3 or MC-4 receptor antagonist according to any one of claims 22 to 38 or 40 to 42. A peptidomimetic or organomimetic compound according to claim 87, wherein 35 said peptide is a mammalian melanocortin MC-3 or MC-4 receptor antagonist according to claim 41.

91. A method of designing a peptidomimetic or organomimetic compound having mammalian melanocortin MC-3 or MC-4 receptor antagonist activity, said method SAL scomprising ascertaining the pharmacophore of a peptide having the general formula: A-B-C-D-E-F-G-amide A04420 wherein A is Leu, Ile, Nle, Met, or substituted analogues thereof; B is Asp, Glu, or substituted analogues thereof; C is His or substituted analogues thereof; D is D-Nal or substituted analogues thereof; E is Arg, Lys, homoArg, homoLys, or substituted analogues thereof; F is Trp or substituted analogues thereof; G is Lys, homoLys or substituted analogues thereof; and wherein the peptide is cyclised by the formation of an amide bond between the side chain carboxyl group of the Asp or Glu residue at position B in the peptide, and the side chain amino group of the Lys or homoLys residue at position G, and fitting a combination of suitable groups to said pharmacophore using computer aided drug design.

92. A method according to claim 91, wherein said peptide has the formula Ac-Nle 4 6 -D-Nal(2) 7 -Arg 8 -Trp 9 -Lys')OcMSH-(4-10)-amide.

93. A method according to claim 91, wherein said peptide is a mammalian melanocortin MC-3 or MC-4 receptor antagonist according to any one of claims 22 to 38 or to 42.

94. A method according to claim 91, wherein said peptide is a mammalian melanocortin MC-3 or MC-4 receptor antagonist according to claim 41. A peptidomimetic or organomimetic compound having mammalian melanocortin MC-3 or MC-4 receptor antagonist activity, designed by a method according to claim 91.

96. A peptidomimetic or organomimetic compound according to claim 95, wherein said peptide has the formula Ac-Nle 4 -cyclo(AspS-His 6 -D-Nal(2) 7 -Arg 8 -Trp 9 -Lys' o )aMSH- (4-10)-amide. S 25 97. A peptidomimetic or organomimetic compound according to claim 95, wherein said peptide is a mammalian melanocortin MC-3 or MC-4 receptor antagonist according to any one of claims 22 to 38 or 40 to 42.

98. A peptidomimetic or organomimetic compound according to claim 95, wherein said peptide is a mammalian melanocortin MC-3 or MC-4 receptor antagonist according to claim 41.

99. Use of a peptidomimetic or organomimetic compound according to any one of claims 75 to 78 or 83 to 86 for the manufacture of a medicament for inhibiting feeding behaviour in an animal.

100. A use according to claim 99, wherein said compound is a mimetic of a peptide 35 which has the formula Ac-Nle 4 -cyclo(Asp 5 -His6-D-Phe7-Arg8-Trp9-Lys' o )aMSH-(4-10)- amide.

101. A use according to claim 99, wherein said compound is a mimetic of a mammalian melanocortin MC-3 or MC-4 receptor agonist according to any one of claims 1 4, o 17 or 19 to 21. A04420 81

102. A use according to claim 99, wherein said compound is a mimetic of a mammalian melanocortin MC-3 or MC-4 receptor agonist according to claim

103. A medicament for inhibiting feeding behaviour in an animal, comprising a peptidomimetic or organomimetic compound according to any one of claims 75 to 78 or 83 to 86, and a pharmaceutically or veterinary acceptable carrier.

104. A medicament according to claim 103, wherein said compound is a mimetic of a peptide which has the formula Ac-Nle 4 -cyclo(Asp-His6-D-Phe -Arg-Trp 9 -Lys)caMSH-(4-

105. A medicament according to claim 103, wherein said compound is a mimetic of a 0o mammalian melanocortin MC-3 or MC-4 receptor agonist according to any one of claims 1 to 17 or 19 to 21.

106. A medicament according to claim 103, wherein said compound is a mimetic of a mammalian melanocortin MC-3 or MC-4 receptor agonist according to claim

107. A method of inhibiting feeding behaviour in an animal, the method comprising administering to said animal an effective amount of a peptidomimetic or organomimetic compound according to any one of claims 75 to 78 or 83 to 86 or a medicament according to any one of claims 103 to 106.

108. A method according to claim 107, wherein said compound is a mimetic of a peptide which has the formula Ac-Nle 4 -cyclo(Asp 5 -His 6 -D-Phe 7 -Arg 8 -Trp 9 -Lyslo)aMSH-(4- 10)-amide, or said medicament comprises said compound.

109. A method according to claim 107, wherein said compound is a mimetic of a mammalian melanocortin MC-3 or MC-4 receptor agonist according to any one of claims 1 to 17 or 19 to 21, or said medicament comprises said compound.

110. A method according to claim 107, wherein said compound is a mimetic of a 25 mammalian melanocortin MC-3 or MC-4 receptor agonist according to claim 20, or said *.medicament comprises said compound.

111. A peptidomimetic or organomimetic compound according to any one of claims 75 to 78 or 83 to 86 or a medicament according to any one of claims 103 to 106, when used for inhibiting feeding behaviour in an animal.

112. A peptidomimetic or organomimetic compound when used according to claim 111, wherein said compound is a mimetic of a peptide which has the formula Ac-Nle 4 cyclo(Asp -His6-D-Phe 7 -Arg-Trp9-Lysl )aMSH-(4-10)-amide, or said medicament comprises said compound.

113. A peptidomimetic or organomimetic compound when used according to claim S. 35 111, wherein said compound is a mimetic of a mammalian melanocortin MC-3 or MC-4 receptor agonist according to any one of claims 1 to 17 or 19 to 21, or said medicament comprises said compound. A04420 82

114. A peptidomimetic or organomimetic compound when used according to claim 111, wherein said compound is a mimetic of a mammalian melanocortin MC-3 or MC-4 receptor agonist according to claim 20, or said medicament comprises said compound.

115. Use of a peptidomimetic or organomimetic compound according to any one of s claims 87 to 90 or 95 to 98 for the manufacture of a medicament for stimulating feeding behaviour in an animal.

116. A use according to claim 115, wherein said compound is a mimetic of a peptide which has the formula Ac-Nle 4 -cyclo(AspS-His 6 -D-Nal(2) 7 -Arg 8 -Trp 9 -Lys )o)MSH-(4-10)- amide.

117. A use according to claim 115, wherein said compound is a mimetic of a mammalian melanocortin MC-3 or MC-4 receptor antagonist according to any one of claims 22 to 38 or 40 to 42.

118. A use according to claim 115, wherein said compound is a mimetic of a mammalian melanocortin MC-3 or MC-4 receptor antagonist according to claim 41.

119. A medicament for stimulating feeding behaviour in an animal, comprising a peptidomimetic or organomimetic compound according to any one of claims 87 to 90 or to 98, and a pharmaceutically or veterinary acceptable carrier.

120. A medicament according to claim 119, wherein said compound is a mimetic of a peptide which has the formula Ac-Nle 4 -cyclo(Asp-His6-D-Nal(2) 7 -Arg-Trp 9 -Lys')aMSH- (4-10)-amide.

121. A medicament according to claim 119, wherein said compound is a mimetic of a mammalian melanocortin MC-3 or MC-4 receptor antagonist according to any one of claims 22 to 38 or 40 to 42.

122. A medicament according to claim 119, wherein said compound is a mimetic of a 25 mammalian melanocortin MC-3 or MC-4 receptor antagonist according to claim 41.

123. A method of stimulating feeding behaviour in an animal, the method comprising administering to said animal an effective amount of a peptidomimetic or organomimetic compound according to any one of claims 87 to 90 or 95 to 98 or a medicament according Sto any one of claims 119 to 120.

124. A method according to claim 123, wherein said compound is a mimetic of a peptide which has the formula Ac-Nle4-cyclo(Asp-His6-D-Nal(2)7-ArgZ-Trp9-Lys'o)aMSH- '(4-10)-amide, or said medicament comprises said compound.

125. A method according to claim 123, wherein said compound is a mimetic of a mammalian melanocortin MC-3 or MC-4 receptor antagonist according to any one of claims S 35 22 to 38 or 40 to 42, or said medicament comprises said compound.

126. A method according to claim 123, wherein said compound is a mimetic of a mammalian melanocortin MC-3 or MC-4 receptor antagonist according to claim 41, or said medicament comprises said compound. A04420 83

127. A peptidomimetic or organomimetic compound according to according to any one of claims 87 to 90 or 95 to 98 or a medicament according to any one of claims 119 to 122, when used for stimulating feeding behaviour in an animal.

128. A peptidomimetic or organomimetic compound when used according to claim 127, wherein said compound is a mimetic of a peptide which has the formula Ac-Nle 4 cyclo(Asp 5 -His 6 -D-Nal(2) 7 -Arg 8 -Trp 9 -Lys' 0 )otMSH-(4-10)-amide, or said medicament comprises said compound.

129. A peptidomimetic or organomimetic compound when used according to claim 127, wherein said compound is a mimetic of a mammalian melanocortin MC-3 or MC-4 receptor antagonist according to any one of claims 22 to 38 or 40 to 42, or said medicament comprises said compound.

130. A peptidomimetic or organomimetic compound when used according to claim 127, wherein said compound is a mimetic of a mammalian melanocortin MC-3 or MC-4 receptor antagonist according to claim 41, or said medicament comprises said compound.

131. A method for characterising a compound as a mammalian melanocortin MC-3 or MC-4 receptor agonist that inhibits feeding behaviour in an animal, the method comprising: providing food to an animal that has been deprived of food for at least 12 hours with or without administering to the animal a candidate mammalian melanocortin MC-3 or MC-4 receptor agonist; and comparing the amount of food eaten by the animal with and without administration of the mammalian melanocortin MC-3 or MC-4 receptor agonist.

132. A method as defined in claim 131, substantially as hereinbefore described with reference to any one of the examples. 25 133. A method for characterising a compound as a mammalian melanocortin MC-3 or MC-4 receptor antagonist that stimulates feeding behaviour in an animal, the method comprising: providing food to an animal that has been deprived of food for at least 12 hours with or without administering to the animal a candidate mammalian melanocortin 30 MC-3 or MC-4 receptor antagonist; and comparing the amount of food eaten by the animal with and without administration of the mammalian melanocortin MC-3 or MC-4 receptor antagonist.

134. A method as defined in claim 133, substantially as hereinbefore described with reference to any one of the examples. 35 Dated 26 March, 2002 Oregon Health and Sciences University o* Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON A04420