WO2001030808A1 - Methods and compounds for modulating melanocortin receptor-ligand binding - Google Patents

Abstract

The present invention relates to methods and agonist/antagonist compounds for modulating melanocortin receptor-ligand binding. The invention includes a method for identifying residues comprising a ligand binding site for a melanocortin receptor of interest. Also included is a method of identifying agonists and/or antagonists that bind to a ligand binding site for a melanocortin receptor of interest. Agonists and antagonists of ligand binding to melanocortin receptors also are provided. The invention is exemplified by identification and manipulation of the C-terminus of the human agouti related protein, which binds melanocortin receptors 3 and 4. The methods can be applied to other melanocortin receptor agonists and antagonists.

Description

METHODS AND COMPOUNDS FOR MODULATING MELANOCORTIN RECEPTOR-

LIGAND BINDING

INTRODUCTION Technical Field The present invention relates to methods and compounds for modulating melanocortin receptor-ligand binding. Background

Recent biochemical investigations have identified agouti related protein ("AGRP") as playing a major role in the regulation of mammalian feeding behavior. The human AGRP is a 132 residue polypeptide (SEQ ID NO:l) that is a naturally occurring competitive antagonist of melanocortin receptors 3 and 4 ("MC3r" and "MC4r"), the overexpression of which results in adult onset obesity and diabetes in mice (Shutter, et al., Genes Dev. 11 :593-602 (1997); Huszar, et al., Cell 88:131-141 (1997); Hahn, et al., Nature Neurosci. 1 :271-171 (1997)). AGRP binding to MC4r in particular is the subject of intense interest since knockout mice that do not express MC4r exhibit the same phenotype as caused by overexpression of AGRP (Huszar, et al., supra). There is also evidence for the parallel expression of AGRP and neuropeptide Y in the arcuate nucleus of the hypothalamus, with neuropeptide Y known to stimulate feeding (Hahn, et al., supra). This region of the brain also expresses MC4r and is involved in energy homeostasis. Research has also focused on other melanocortin receptors, their antagonists and methods for modulating receptor activity. See for example, Wei, et al., WO9943709.

The growing body of evidence linking AGRP to weight control has yet to elucidate its exact mechanism of action. However, studies on AGRP do benefit from analogy to the much more widely studied agouti protein, as AGRP was originally identified through the homology of its C-terminal region with the same region of the agouti protein (Shutter, et al., supra). The agouti protein has been a focal point in obesity research for a number of years, since ectopic expression of the wild-type protein in mice leads to obesity and related disorders, i.e. the same symptoms as the overexpression of the more recently identified AGRP (Klebig, et al., Proc.

Natl. Acad. Sci. USA 92:4728-4732 (1995); Michaud, et al., J. Endocrinol 155:207-209 (1997).

However, unlike AGRP, agouti has distinct expression patterns in mice and humans, making in vivo work with mice less applicable to human obesity disorders. AGRP, like agouti, is selective for MC3r and MC4r but has approximately 100-fold greater binding affinity than agouti at these receptors (Fong, et al., Biochem. Res. Commun. 237:629 611 (1997)).

While full length agouti and AGRP are only 25% homologous, in their 46 residue Cys- rich C-terminal regions, nine of the 10 Cys residues are spatially conserved and there are a further 10 identical residues giving -40% sequence identity. Three consecutive, conserved residues RFF (111-113 in human AGRP) were determined to be essential to the biological activity of both agouti (Kiefer, et al., Biochemistry 36:2084-2090 (1997); Kiefer, et al., Biochemistry 37:991-997 (1998)) and AGRP (Tota, et al., Biochemistry 38:897-904 (1999)). Two recent investigations have shown that the chemically synthesized C-terminal region of AGRP competitively antagonizes α-melanocyte stimulating hormone (α-MSH) at melanocortin receptors with equal or greater potency than the full proteins (Quillan, et al., FEBS Lett. 428:59- 62 (1998); Yang, et al., Mol. Endocrinol. 13:148-155 (1999)), consistent with similar findings for agouti (Willard, et al., Biochemistry 34:12341-12346 (1995)). Thus the Cys-rich C-terminal region of AGRP, referred to as minimized agouti related protein ("MARP") is a prime candidate for structural studies:

CVRLH₅ESCLG₁₀QQVPC₁₅CDPCA₂₀TCYCR₂₅ FFNAF₃₀CYCRK₃₅LGTAM₄₀NPCSR₄₅T (SEQ ID NO:2) The covalent structure of MARP exhibits five disulfide bonds, which exist between the following ten Cys residues (Bures, et al., Biochemistry 37:12172-12177 (1998)): Cysi and Cysj₆; Cys₈ and Cys₂ ; Cys₁₅ and Cys₃₃; Cys^ and Cys₄₃; Cys₂₄ and Cys₃ι.

Despite the important biological activities of AGRP, no experimental 3D structure has been available for this protein. The inhibitor cystine knot ("ICK") family of proteins are also disulfide-rich and the structures of these invertebrate toxins have been used to suggest possible structures for the agouti and AGRP C-terminal regions (Kiefer, et al., Biochemistry 37:991-997 (1998); Tota, et al., supra). Indeed, the recently reported disulfide map for AGRP and a construct containing the C-terminal domain demonstrate ICK-like pairings (Norton, et al., Toxicon 36:1573-1583 (1998)) of the 10 Cys residues: 1-16, 8-22, 15-33, 19-43, 24-31 (using MARP numbering, Bures, et al., supra). Beyond such modeling, the only structural data published on either agouti or AGRP are circular dichroism (CD) spectra which suggest that both proteins have little regular secondary structure, although there may be some β-sheet structure, consistent with ICK structural characteristics (Willard, et al., supra; Rosenfeld, et al., Biochemistry 37:16041-16052 (1998)). Accordingly, there exists a need for a better understanding of the structure of AGRP and related proteins such as MARP. More particularly, a need exists for the identification and characterization of the receptor binding regions of AGRP, MARP and other ligands for melanocortin receptors such as MC3r and MC4r, and molecules that affect their interaction with agouti and AGRP proteins. This would provide a major new target for iterative drug design, synthesis, and selection. It also would be advantageous to devise methods and compositions for reducing the time required to discover compounds that target the ligand binding site of melanocortin receptors and administer them to organisms, and mammals in particular, to modulate physiological processes regulated by melanocortin receptors. The instant invention meets that need in describing the 3D structure in solution of the human AGRP Cys-rich C-terminal region.

SUMMARY OF THE INVENTION The present invention relates to the receptor binding region of the human agouti related protein ("AGRP"), which binds to melanocortin receptors 3 and 4 ("MC3r" and "MC4r"). Information about this region permits design of compounds that bind to the ligand binding site of melanocortin receptors and modulate ligand binding to the receptor. The compounds include agonists and antagonists that modulate melanocortin receptor activity by promoting (agonists) or blocking (antagonists) ligand binding to the receptor, particularly antagonists. The compounds of the invention can be receptor-, cell- and/or tissue-specific. The present invention also includes an NMR structure of the human AGRP C-terminus

("MARP"), which binds to the ligand binding site of melanocortin receptors. The NMR structure provides a means to obtain atomic modeling information of the specific amino acids and their atoms forming the receptor binding region and that interact with molecules located at the ligand binding site. The present invention further provides methods for identifying and designing small molecules that bind to the ligand binding site using atomic models of MARP. The method involves modeling test compounds that mimic or match the 3D conformation of MARP and therefore are expected to fit spatially into a melanocortin receptor ligand binding site of interest, using an atomic structural model comprising the MARP receptor binding region or portion thereof, screening the test compounds in a biological assay characterized by binding of a test compound to a melanocortin receptor ligand binding site, and identifying a test compound that modulates ligand binding to the melanocortin receptor. The invention also includes compositions and methods for identifying ligand binding sites of melanocortin receptors. The methods involve examining the MARP surface to identify residues that modulate ligand binding. The residues can be identified by homology to the receptor binding region of MARP, as described herein. Overlays and superpositioning with a three dimensional model of the MARP receptor binding region, or a portion thereof that contains a receptor binding region, also can be used for this purpose. Additionally, alignment and/or modeling can be used as a guide for the placement of mutations on the receptor binding region surface to characterize the nature of the ligand binding site on melanocortin receptors in the context of a cell. Also provided is a method of modulating the activity of a melanocortin receptor. The method can be in vitro or in vivo. The method comprises administering, in vitro or in vivo, a sufficient amount of a compound that binds to the ligand binding site. Preferred compounds bind to the site with greater affinity than ligand proteins found in a cell of interest.

The invention further includes a method for identifying an agonist or antagonist of ligand binding to a melanocortin receptor. The method comprises providing the atomic coordinates comprising a melanocortin receptor binding region or portion thereof to a computerized modeling system; modeling compounds which match or mimic the receptor binding region and thus fit spatially into the melanocortin receptor ligand binding site; and identifying in an assay for melanocortin receptor activity a compound that increases or decreases activity of the melanocortin receptor through binding the ligand binding site. The melanocortin receptor binding region is preferably the MARP receptor binding region or portion thereof.

Also provided is a machine-readable data storage medium with information for constructing and manipulating an atomic model comprising a receptor binding region or portion thereof. The medium comprises a data storage material encoded with machine readable data which, when using a machine programmed with instructions for using said data, is capable of displaying a graphical three-dimensional representation of a molecule that binds a melanocortin receptor.

Also provided is a method of identifying a compound that selectively modulates the activity of one type of melanocortin receptor compared to other melanocortin receptors. The method is exemplified by modeling test compounds that fit spatially and preferentially into a melanocortin receptor ligand binding site of interest using an atomic structural model of the

MARP receptor binding region, selecting a compound that mimics or matches one or more residues of the receptor binding region unique in the context of that region, and identifying in an assay for ligand binding activity a compound that selectively binds to the ligand binding site compared to other melanocortin receptors.

The invention finds use in the selection and characterization of peptide, peptidomimetic, as well as other small molecule compounds, such as small organic molecules, identified by the methods of the invention, particularly new lead compounds useful in treating melanocortin receptor-based disorders. The invention also includes a group of polypeptides or peptide-related compounds comprised of sequences from MARP. Members of this group may be made synthetically and can be modified in myriad ways.

The invention includes a method of treating a disease state in mammals by treatment with a polypeptide comprised of sequences from MARP. A preferred embodiment would be a method of treating a wasting syndrome, such as HIV wasting syndrome, cachexia, or anorexia. The invention further includes a method of screening for a compound that is capable of inhibiting binding of a melanocortin agonist or antagonist to a melanocortin receptor. This method can be used to screen for compounds that inhibit binding at specific melanocortin receptors such as MC3R or MC4R. Further, this method can be used to identify compounds that interfere with either agonist or antagonist binding at the melanocortin receptor. Examples of antagonists that interfere with agonist binding include, but are not limited to, MARP and AGRP.

The invention also includes methods of screening for binding of compound to a melanocortin receptor. Such methods can be used to screen for compounds that bind specific melanocortin receptors such as MC3R and MC4R, and can be used for high throughput screening.

The invention also pertains to a polypeptide molecule that acts as a strong antagonist of melanocortin receptor types 3 and 4, and is useful for the treatment of eating disorders and obesity. DESCRIPTION OF THE DRAWINGS

Fig. 1 shows the C_α backbone of the MARP minimized average structure. The N- terminal loop, central loop and C-terminal loop are indicated. Disulfide bonds are represented by dashed lines. Spheres represent residues with amides protected from HX for more than 12 hours, more than 24 hours and more than 8 days. Fig. 2 depicts the heavy atom (non-hydrogen) backbone representation of MARP for 14

NMR structures with residues 1-34 fit to the minimized average structure (Root Mean Square Deviation, "RMSD" 1.49A). Only the minimized average structure (thick cylinder) is shown for the more disordered C-terminal loop. Fig. 3 shows the backbone atoms for residues 24-31 of the family of 20 structures with residues 24-31 fit to the minimized average structure. The side chain heavy atoms of residues 25, 26 and 27, essential for activity, are shown.

DESCRIPTION OF SPECIFIC EMBODIMENTS The agouti related protein ("AGRP") is a mammalian signaling molecule, involved in weight homeostasis, that causes adult onset obesity when overexpressed in mice. AGRP was originally identified by homology to the agouti protein, another potent signaling molecule involved in obesity disorders in mice. While AGRP's exact mechanism of action is unknown, it has been identified as a competitive antagonist of melanocortin receptors 3 and 4 ("MC3r" and "MC4r"). MC4r in particular is implicated in the hypothalamic control of feeding behavior. Full length agouti and AGRP are only 25% homologous, however, their active C-terminal regions are ~40% homologous, with nine out of the 10 Cys residues spatially conserved. Until now, 3D structures have not been available for either agouti, AGRP or their C-terminal regions.

This invention relates to the 3D NMR structure in solution of the human AGRP Cys-rich C-terminal region as determined by H NMR using a protein prepared by total chemical synthesis. As used herein the term "the NMR structure" is understood to refer to the minimized average of the family of NMR structures. Because biochemical investigations demonstrate that this minimal region retains full biological activity, this protein is referred to herein as minimized agouti related protein ("MARP"). MARP residues 1-46 (SEQ ID NO:2), correspond to human AGRP residues 87-132 (residues 87-132 of SEQ ID NO:l). Thus, human AGRP numbering is obtained by adding 86 to MARP numbering.

MARP's topology is characterized by three large loops (referred to herein as the N- terminal loop, the central loop and the C-terminal loop), with four of the five disulfide bridges at the base of the structure, and an absence of canonical secondary structure such as helices or sheets. Two of the three loops are structurally well characterized by the NMR data as indicated by low RMSDs. The region of MARP containing the RFF triplet (Tota, et al., supra) (residues 25-27 in MARP) necessary for function is located in one of the best defined regions of the protein. While previously reported structural models of the C-terminal region of AGRP were attempted based on Cys homology between AGRP and certain toxin proteins, Cys spacing is not sufficient to correctly determine the 3D fold of the molecule. Despite the conservation of Cys spacing and the disulfide map between MARP and other small disulfide-rich proteins from the ICK family, it is apparent from the structure described herein that MARP does not adopt an ICK-like fold.

The 3D structure of MARP presents a basis for the development of methods and compositions for identifying compounds that modulate melanocortin receptor activity, in particular the activity of MC3r and MC4r. In a preferred embodiment, the 3D structure presents a basis for compounds that preferentially modulate MC4r activity. The compounds can be melanocortin receptor agonists or antagonists that bind to the ligand binding site (and that act as mimetics to the ligand in this regard), and promote (agonists) or block (antagonists) binding of the ligand to the target melanocortin receptor. Compounds that bind to the ligand binding site also are provided. The compounds can be natural or synthetic. Preferred compounds are small organic molecules, peptides and peptidomimetics (e.g., cyclic peptides, peptide analogs, or constrained peptides).

Accordingly, one aspect of the invention involves methods for identifying and designing small molecules that bind to the ligand binding site using atomic models of MARP. In particular, the invention provides for a method of identifying a compound that modulates ligand binding to a melanocortin receptor. The method involves modeling test compounds that mimic or match the 3D conformation of MARP and therefore are expected to fit spatially into a melanocortin receptor ligand binding site of interest, using an atomic structural model of a melanocortin receptor binding region or portion thereof, preferably comprising the MARP receptor binding region or portion thereof. The test compounds can fit spatially into the ligand binding site of interest based upon a geometric fit of its three-dimensional structure or based upon the spatial arrangement of atoms presenting specific chemical properties such as charge and hydrophobicity. The test compounds are then screened in an assay, such as a biological assay, characterized by binding of a test compound to a melanocortin receptor ligand binding site, and identifying a test compound that modulates ligand binding to the melanocortin receptor. Details of the atomic structural model are described in detail below.

As noted above, the 3D structure of MARP is characterized by three loops held together at the base by an apparent scaffold of four disulfide bonds 1-16, 8-22, 15-33 and 19-43. The fifth disulfide bond, 24-31, further stabilizes the base of the active loop which presents the RFF triplet on the protein surface. There is no identifiable canonical helical or sheet structure. It is clear from biochemical data that the RFF triplet is critical for the activity of MARP as a competitive antagonist of α-MSH stimulated activation of MC4r signaling. The structure described herein shows that MARP is structured to present the side chains of the RFF triplet on the surface of the protein and to the surrounding solvent. Recent work demonstrates that MARP is much more active than smaller AGRP derived peptides containing the RFF triplet (Tota, et al., Biochemistry 38:897-904 (1999)). Thus, the detailed fold of the central loop and perhaps the presence of the N- and C-terminal loops are critical for AGRP function. In addition, based upon work with chimeras of melanocortin receptors (I. Gantz, unpublished data), it is possible that the N- and C-terminal loops may confer receptor subtype specificity.

The previous absence of structural data on both AGRP and agouti encouraged the modeling of the C-terminal regions of these proteins onto the ICK family (Norton, et al., supra)) which is characterized by homologous Cys spacing (Kiefer, et al., Biochemistry 37:991-997 (1998); Tota, et al., supra). The ICK family of proteins primarily consists of small (<60 residues) disulfide-rich (three or four disulfides) toxin proteins from the venom of spiders and cone snails, which function as ion channel antagonists (Norton, et al., supra). The coincidence between the function of the majority of these toxins and the recent description of part of the agouti protein's mechanism of action being calcium dependent (Kim, et al., FASEB J. 10:1646- 1652 (1996); Kim, et al., Am. J. Physiol. 272:E379-384 (1997); Jones, et al., Am. J. Physiol.

270 :E 192- 196 (1996)) further encouraged these homology modeling efforts. The ICK motif in particular is characterized by the topology of the three disulfide bonds corresponding to 1-16, 8- 22 and 15-33 in MARP. In the ICK motif the first two disulfide bonds with their intervening main chain atoms form a topological circle through which the third disulfide bond passes, forming the cystine knot (Norton, et al., supra). The motif is further characterized by the identification of an irregular triple stranded antiparallel β-sheet, roughly corresponding to residues 6-8, 20-24 and 31-34 in MARP. The remaining two disulfide bonds in MARP each occur in individual ICK proteins as separate examples of potential "non-motif disulfide bonds, although no examples of ICK motif proteins with five disulfide bonds have been observed. Despite these apparent similarities, the experimental structure of MARP shows that this protein does not satisfy the criteria required for inclusion in the ICK family. While the first two disulfide bonds in MARP 1-16 and 8-22, together with the polypeptide backbone form a topological circle, none of the remaining disulfides passes through the circle to form a cystine knot. Instead, disulfide bond 15-33 is positioned adjacent to the circle with all of the fold on one side of this circle. In addition, MARP lacks the β-sheet found in ICK family proteins. The experimental determination of the distinctive 3D structure of MARP described herein suggests that Cys spacing and even the disulfide map of small Cys-rich proteins may not always be sufficient to accurately predict protein folds. These results speak to the potential limitations of "homology modeling" of protein structures, and may have important implications for the emerging field of genomic structural biology. Use of atomic modeling, alone or in combination with homology modeling may serve to remedy this. As described in the Examples, ligand binding studies, and analysis of atomic models derived from the MARP NMR structure reveal for the first time a previously unknown structure for MARP and its receptor binding region. By "receptor binding region" is intended a structural segment or segments of melanocortin receptor ligands, and MARP in particular, folded in such a way so as to give the proper geometry and amino acid residue conformation for binding to a melanocortin receptor. By "ligand binding site" is intended a structural segment or segments of melanocortin receptor polypeptide chain folded in such a way so as to give the proper geometry and amino acid residue conformation for binding a ligand. These are the physical arrangement of protein atoms in three-dimensional space forming a receptor binding region or a ligand binding site pocket or cavity. The MARP structure has three major loops: the N-terminal loop, residues 1-18 (residues

1-18 of SEQ ID NO:2), the central loop, residues 19-34 (residues 19-34 of SEQ ID NO:2) and the C-terminal loop, residues 35-46 (residues 35-46 of SEQ ID NO:2). Residues forming the receptor binding region are amino acids corresponding to (i.e., the same as or equivalent to) residues 24-31 of the central loop (residues 24-31 of SEQ ID NO:2), referred to herein as the "active" loop. In particular, residues 25, 26 and 27 (the "RFF" triplet) of the active loop are critical for activity. It has been found that a polypeptide comprising the central loop, preferably at least a portion of the N-terminal loop, is desirable for optimal biological activity. As used herein, the term "at least a portion of the N-terminal loop" is intended to mean a sequence that corresponds to (i.e., the same as or equivalent to), at least residues 15 to 18 of the N-terminal loop (residues 15-18 of SEQ ID NO:2), preferably at least residues 8 to 18 of the N-terminal loop (residues 8-18 of SEQ ID NO:2), and is also intended to include all of the N-terminal loop, i.e., residues 1 to 18 of the N-terminal loop (residues 1-18 of SEQ ID NO:2). The term is also intended to mean a sequence that corresponds to at least 20%, preferably at least 60%, and more preferably at least 90%, of the contiguous or non-contiguous amino acid residues or their atoms selected from amino acid residues 1 to 18 of the N-terminal loop (residues 1-18 of SEQ ID NO:2).

The invention also includes compositions and methods for identifying receptor binding regions of melanocortin receptor agonist and antagonists, along with ligand binding sites of melanocortin receptors. The methods involve examining the surface of a polypeptide of interest to identify residues that modulate ligand binding. The residues can be identified by homology to the receptor binding region of MARP described herein. A preferred method is alignment with the residues of any polypeptide corresponding to (i.e., the same as or equivalent to) residues 1-18 of the N-terminal loop (residues 1-18 of SEQ ID NO:2), residues 19-34 of the central loop

(residues 19-34 of SEQ ID NO:2) and residues 35-46 of the C-terminal loop (residues 35-46 of SEQ ID NO:2). Overlays and superpositioning with a three-dimensional model of the MARP receptor binding region, or a portion thereof that contains a receptor binding region, also can be used for this purpose. For example, melanocortin receptor agonists and antagonists identifiable by homology alignment include naturally occurring compounds or compounds structurally related to such naturally occurring compounds found in humans, along with synthetic compounds.

Alignment and/or modeling also can be used as a guide for the placement of mutations on the receptor binding region surface to characterize the nature of the ligand binding site on melanocortin receptors in the context of a cell. To destroy the ligand binding interaction, preferred mutations are to charged residues (e.g., Arg, Lys, or Glu) on the basis that bulky, surface charged residues might disrupt ligand binding, yet preserve the overall ligand structure and solubility. Mutants can be tested for ligand binding as well as the relative change in strength of the binding interaction. Ligand-dependent ligand interaction assays also can be tested for this purpose, such as those described herein.

Compounds that bind to the ligand binding site of melanocortin receptors can be identified by computational modeling and/or screening. For example, ligand agonists or antagonists can be identified by providing atomic coordinates comprising the MARP receptor binding region or portion thereof to a computerized modeling system, modeling them, and identifying compounds that mimic or match the receptor binding region and thus would be expected to fit spatially into the ligand binding site. By a "portion thereof is intended the atomic coordinates corresponding to a sufficient number of residues or their atoms of the receptor binding region that interact with a melanocortin receptor capable of binding the region. As another example, an atomic structural model utilized for computational modeling and/or screening of compounds that mimic or match the receptor binding region and thus would be expected to fit spatially into the ligand binding site, may include a portion of atomic coordinates of amino acid residues corresponding to the region composed of residues 24-31 of the central loop (residues 24-31 of SEQ ID NO:2), or their structural and functional equivalents. An atomic model can also be designed that includes residues 19-34 of the central loop (residues 19-34 of SEQ ID NO:2), and a model can further be designed that includes residues 19-34 of the central loop (residues 19-34 of SEQ ID NO:2) and some or all of residues 1-18 of the N-terminal loop (residues 1-18 of SEQ ID NO:2). Thus, for example, the atomic coordinates provided to the modeling system can contain atoms of MARP, all or part of the receptor binding region or a subset of atoms useful in the modeling and design of compounds that mimic or match the receptor binding region.

The atomic coordinates of MARP and its receptor binding region, can be used for modeling to identify other compounds or fragments that bind melanocortin receptors. By "modeling" is intended quantitative and qualitative analysis of molecular structure/function based on atomic structural information and receptor-ligand agonists/antagonists interaction models. This includes conventional numeric-based molecular dynamic and energy minimization models, interactive computer graphic models, modified molecular mechanics models, distance geometry and other structure-based constraint models. Modeling is preferably performed using a computer and may be further optimized using known methods. By "fits spatially" is intended that the three-dimensional structure of a compound is accommodated geometrically by a cavity or pocket of a melanocortin receptor ligand binding site or by a pattern of charge or hydrophobicity.

Compounds of particular interest fit spatially and preferentially into the ligand binding site. By "fits spatially and preferentially" is intended that a compound possesses a three- dimensional structure and conformation for selectively interacting with a melanocortin receptor ligand binding site. Compounds that fit spatially and preferentially into the ligand binding site interact with amino acid residues forming the ligand binding site. More specifically, these compounds mimic or match the MARP receptor binding region. The present invention also includes a method for identifying a compound capable of selectively modulating ligand binding to different melanocortin receptors. The method comprises the steps of modeling test compounds that fit spatially and preferentially into the ligand binding site of a melanocortin receptor of interest using an atomic structural model of a MARP, screening the test compounds in a biological assay for melanocortin receptor activity characterized by preferential binding of a test compound to the ligand binding site of a melanocortin receptor, and identifying a test compound that selectively modulates the activity of a melanocortin receptor. Such receptor- specific compounds are selected that exploit differences between the ligand binding sites of one type of receptor versus a second type of receptor. The invention also is applicable to generating new compounds that distinguish melanocortin receptor isoforms. This can facilitate generation of either tissue-specific or function-specific compounds.

The receptor-specific compounds of the invention preferably interact with conformationally constrained residues of the ligand binding site that are conserved among one type of receptor compared to a second type of receptor. "Conformationally constrained" is intended to refer to the three-dimensional structure of a chemical or moiety thereof having certain rotations about its bonds fixed by various local geometric and physical-chemical constraints. Conformationally constrained structural features of a ligand binding site include residues that have their natural flexible conformations fixed by various geometric and physical- chemical constraints, such as local backbone, local side chain, and topological constraints. These types of constraints are exploited to restrict positioning of atoms involved in receptor- ligand recognition and binding.

As described in the Examples, residues 25, 26 and 27 of the MARP receptor binding region are shown to be critical for activity. These three residues, along with the overall structure and composition of the N-terminus and central loop, appear to be necessary for optimal biological activity. Accordingly, modification to these residues can be exploited in the identification and design of compounds that modulate ligand binding to one melanocortin receptor compared to another. For modeling, docking algorithms and computer programs that employ them can be used to identify compounds that match or mimic the MARP receptor binding region. For example, docking programs can be used to predict how a small molecule of interest can interact with the melanocortin receptor ligand binding site. Fragment-based docking also can be used in building molecules de novo inside the ligand binding site, by placing chemical fragments that complement the site to optimize intermolecular interactions. The techniques can be used to optimize the geometry of the binding interactions. This design approach has been made possible by identification of the receptor binding region structure thus, the principles of molecular recognition can now be used to design a compound which matches the structure of this region. Compounds that structurally match or mimic the MARP receptor binding region serve as a starting point for an iterative design, synthesis and test cycle in which new compounds are selected and optimized for desired properties including affinity, efficacy, and selectivity. For example, the compounds can be subjected to addition modification, such as replacement and/or addition of R-group substituents of a core structure identified for a particular class of binding compounds, modeling and/or activity screening if desired, and then subjected to additional rounds of testing.

Computationally small molecule databases can be screened for chemical entities or compounds that can bind in whole, or in part, to a melanocortin receptor ligand binding site of interest. In this screening, the quality of fit of such entities or compounds to the binding site may be judged either by shape complementarity (DesJalais et al., J Med. Chem. (1988) 31:722- 729) or by estimated interaction energy (Meng et al., J Comp. Chem. (1992) 75:505-524). The molecule databases include any virtual or physical database, such as electronic and physical compound library databases, and are preferably used in developing compounds that modulate ligand binding.

Compounds can be designed intelligently by exploiting available structural and functional information by gaining an understanding of the quantitative structure-activity relationship (QSAR), using that understanding to design new compound libraries, particularly focused libraries having chemical diversity of one or more particular groups of a core structure, and incorporating any structural data into that iterative design process. For example, one skilled in the art may use one of several methods to screen chemical entities or fragments to compare them to the 3D structure of the AGRP C-terminus and thus, their ability to associate with the ligand binding site of a melanocortin receptor of interest. This process may begin by visual inspection of, for example, the receptor binding region on the computer screen. Selected fragments or chemical entities may then be positioned into all or part of the region. Docking may be accomplished using software such as Quanta and Sybyl, followed by energy minimization and molecular dynamics with standard molecular mechanics force-fields, such as CHARMM and AMBER.

Residues comprising a receptor binding region can be modeled to look for energetic contributions and interaction with the bound chemical entity. For example, a compound or fragment can be designed to contain hydrophobic groups that interact with hydrophobic residues of the ligand binding site.

Specialized computer programs may also assist in the process of selecting chemical entity fragments or whole compounds. These include: GRID (Goodford, J Med. Chem. (1985) 2S:849-857; available from Oxford University, Oxford, UK); MCSS (Miranker et al., Proteins:

Structure, Function and Genetics, (1991) 77:29-34; available from Molecular Simulations,

Burlington, MA); AUTODOCK (Goodsell et al., Proteins: Structure, Function and Genetics

(1990) 5:195-202; available from Scripps Research Institute, La Jolla, CA); and DOCK (Kuntz et al, J. Mol. Biol. (1982) 161:269-288; available from University of California, San Francisco, CA).

Additional commercially available computer databases for small molecular compounds include Cambridge Structural Database and Fine Chemical Database (Rusinko, Chem. Des. Auto. News (1993) 5:44-47).

Once suitable chemical entities or fragments have been selected, they can be assembled into a single compound. Assembly may be proceeded by visual inspection of the relationship of the fragments to each other on the three-dimensional image displayed on a computer screen in relation to the structure coordinates of a melanocortin receptor. This can be followed by manual model building using software such as Quanta or Sybyl.

Useful programs to aid one of skill in the art in connecting the individual chemical entities or fragments include: CAVEAT (Bartlett et al., "CAVEAT: A Program to Facilitate the Structure-Derived Design of Biologically Active Molecules", In: Molecular Recognition in Chemical and Biological Problems" , Special Pub., Royal Chem. Soc. (1989) 75:182-196; CAVEAT is available from the University of California, Berkeley, CA); 3D Database systems such as MACCS-3D (MDL Information Systems, San Leandro, CA; reviewed in Martin, J Med. Chem. (1992) 55:2145-2154); and HOOK (available from Molecular Simulations, Burlington, MA).

In addition to building a compound in a step-wise fashion, one fragment or chemical entity at a time as described above, compounds that bind to a ligand binding site of interest also may be designed as a whole or de novo using some portion(s) of the AGRP C-terminus, which is a molecule known to bind to the site. These methods include: LUDI (Bohm, J. Comp. Aid. Molec. Design (1992) (5:61-78; LUDI is available from Biosym Technologies, San Diego, CA); LEGEND (Nishibata et al., Tetrahedron (1991) 7:8985; LEGEND is available from Molecular Simulations, Burlington, MA); and LeapFrog (available from Tripos Associates, St. Louis, MO). Other molecular modeling techniques may also be employed in accordance with this invention. See, for example, Cohen et al., J Med. Chem. (1990) 55:883-894); Navia et al., Curr. Opin. Struct. Biol. (1992) 2:202-210). For example, where the structures of test compounds are known, a model of the test compound may be superimposed over the model of the structure of the invention. Numerous methods and techniques are known in the art for performing this step, any of which may be used. See, for example, Farmer, "Drug Design " Ariens, E.J., ed., 10:119-

143 (Academic Press, New York, 1980); U.S. Patent No. 5,331,573; U.S. Patent No. 5,500,807;

Verlinde, Structure, (1994) 2:577-587); and Kuntz et al., Science, (1992) 257:1078-1082). The model building techniques and computer evaluation systems described herein are not a limitation on the present invention.

Using these computer modeling systems a large number of compounds may be quickly and easily examined and expensive and lengthy biochemical testing avoided. Moreover, the need for actual synthesis of many compounds can be substantially reduced and/or effectively eliminated.

Compounds identified through modeling can be screened in an assay characterized by binding of the compound to a ligand binding site of interest for ligand binding activity, such as a biologically based assay. Screening can be in vitro and/or in vivo. Preferred assays include cell- free competition assays and cell culture based assays. The biological screening preferably centers on activity-based response models, binding assays (which measure how well a compound binds to the receptor), and bacterial, yeast and animal cell lines (which measure the biological effect of a compound in a cell). The assays can be automated for high capacity - high throughput screening (HTS) in which large numbers of compounds can be tested to identify compounds with the desired activity.

As an example, in vitro binding assays can be performed in which compounds are tested for their ability to block the binding of a ligand protein, fragment, fusion or peptide thereof, to a ligand binding site of interest. For cell and tissue culture assays, they may be performed to assess a compound's ability to block function of cellular ligands, such as competitively inhibiting [Nle⁴, D-Phe⁷]α-MSH ("NDP-MSH"), as described in the examples and the references cited therein, which are incorporated by reference.

In a preferred embodiment, compounds of the invention bind to a melanocortin receptor ligand binding site with greater affinity than the cellular ligand proteins. Tissue profiling and appropriate animal models also can be used to select compounds. Different cell types and tissues also can be used for these biological screening assays. Suitable assays for such screening are described herein and in the Examples and the references cited therein, which references are incorporated herein in their entirety by reference. For example, ligands or binding fragments thereof can be expressed and/or assayed for their ability to inhibit cAMP generation in cells transfected with MC4r (see Example 1), as described in the examples and the references cited therein, which are incorporated by reference.

The compounds selected can have agonist and/or antagonistic properties. The compounds also include those that exhibit new properties with varying mixtures of agonist and antagonist activities, depending on the effects of altering ligand binding in the context of different activities of melanocortin receptors which are mediated by proteins other than ligands, and which interact with the receptors at locations other than the ligand binding site. The compounds also include those, which through their binding to receptor locations that are conformationally sensitive to ligand binding, have allosteric effects on the receptor by stabilizing or destabilizing the ligand-bound conformation of the receptor, or by directly inducing the same, similar, or different conformational changes induced in the receptor by ligand binding.

Of particular interest is use of such compounds in a method of modulating melanocortin receptor activity in a mammal by administering to a mammal in need thereof a sufficient amount of a compound that fits spatially and preferentially into a ligand binding site of a melanocortin receptor of interest. By "modulating" is intended increasing or decreasing activity of a melanocortin receptor. For example, pre-clinical candidate compounds can be tested in appropriate animal models in order to measure efficacy, absorption, pharmacokinetics and toxicity following standard techniques known in the art. Compounds exhibiting desired properties are then tested in clinical trials for use in treatment of various melanocortin receptor- based disorders. These include feeding disorders, including wasting syndromes, obesity, and other disorders related to hypothalamic control of feeding. A wasting syndrome is an illness characterized by significant weight loss accompanied by other indicia of poor health, including poor appetite, gut disorder, or increased metabolic rate. Wasting syndromes include, but are not limited to, the wasting syndrome afflicting some patients diagnosed with Acquired Immune Deficiency Syndrome (AIDS) and various cancers. As methods of treating other symptoms of diseases such as AIDS progress, the incidence of wasting syndrome as the cause of death increases. Improved prophylaxis and treatment for HIV wasting syndrome is required (Kravick, et al., Arch. Intern. Med. 157:2069-2073, 1997). Anorexia and cachexia are well-known results of cancer that contribute to morbidity and mortality (Simons, et al, Cancer 82:553-560, 1998; Andrassy & Chwals, Nutrition 14:124-129, 1998). The reasons for the significant weight loss are multiple and may be directly related to the tumor, such as increased metabolic rate, but also include decreased intake due to poor appetite or gut involvement. Further, excessive leptin-like signaling may contribute to the pathogenesis of wasting illness (Schwartz, et al., Pro. Nutr. Soc. 56:785-791, 1997). The invention also provides for the NMR structure of the human AGRP C-terminus, preferably embodied in a computer readable form. Synthesis of MARP and concentrated solutions adequate for NMR spectroscopy are described in the examples. After the NMR structure of MARP is determined, the structural information can be used in computational methods to design synthetic agonist and antagonist compounds for the melanocortin receptors, and further structure-activity relationships can be determined through routine testing using the assays described herein and known in the art. It is expected that the structure coordinates of the NMR structure of MARP, as provided in Appendix 1, will be particularly useful for solving the NMR structure of other melanocortin receptor antagonists or agonists. The coordinates in

Appendix 1 represent the minimized average of the coordinates of a family of NMR structures. The minimized average was determined by (1) calculating an average from approximately 20 NMR structures found to meet the experimental NMR distance restraints and (2) subjecting this calculated average to a final round of energy minimization. One method that may be employed for this purpose is molecular replacement. In this method, the unknown NMR structure, may be determined using the structure coordinates of this invention as provided in Appendix 1. The Appendix 1 coordinates for the human AGRP C- terminus have been deposited with the Brookhaven National Laboratory Protein Data Bank, and have been assigned Brookhaven Protein Data Bank Accession Number lqu8. This method will provide an accurate structural form for the unknown NMR structure more quickly and efficiently than attempting to determine such information ab initio.

One aspect of the invention is an NMR structure of the minimized agouti related protein, embodied in a computer readable media. Atomic coordinate information gleaned from the NMR structure of the invention is preferably stored and provided in the form of a machine-readable data storage medium. This medium contains information for constructing and/or manipulating an atomic model of a receptor binding region or portion thereof. For example, the machine readable data for the receptor binding region may comprise structure coordinates of amino acids corresponding to (i.e., the same as or equivalent to) residues 1-18 of the N-terminal loop, (residues 1-18 of SEQ ID NO:2), and/or residues 19-34 of the central loop (residues 19-34 of SEQ ID NO:2) and/or residues 35-46 of the C-terminal loop (residues 35-46 of SEQ ID NO:2), or a homologue of the molecule or molecular complex comprising the region. The machine readable data for the receptor binding region may comprise structure coordinates of amino acids corresponding to residues 1-18 of the N-terminal loop, (residues 1-18 of SEQ ID NO:2) and residues 19-34 of the central loop (residues 19-34 of SEQ ID NO:2). The machine readable data may also comprise residues 24-31 of the central loop (residues 24-31 of SEQ ID NO:2) and a portion of the N-terminal loop, for example residues 15 to 18 (residues 15-18 of SEQ ID NO:2), residues 7 to 18 (residues 7-18 of SEQ ID NO:2), or residues 1 to 18 (residues 1-18 of SEQ ID

NO:2). The homologues comprise a receptor binding region that has a root mean square deviation from the backbone atoms of the amino acids of not more than 2.54 A, preferably not more than 1.66 A.

The machine-readable data storage medium can be used for interactive drug design and molecular replacement studies. For example, a data storage material is encoded with a first set of machine-readable data that can be combined with a second set of machine-readable data. For molecular replacement, the first set of data can comprise a Fourier transform of at least a portion of the structural coordinates of the AGRP C-terminus or portion thereof of interest, and the second data set comprises an X-ray diffraction pattern of a molecule such as the melanocortin receptor of interest. Using a machine programmed with instructions for using the first and second data sets a portion or all of the structure coordinates corresponding to the second data can be determined.

Protein for determination of the NMR structure and the assays described herein can be produced using synthetic or recombinant expression techniques. For example, AGRP C- terminal portion (MARP) can be readily synthesized using techniques described in Yang, et al. supra. In addition, high level expression of melanocortin receptors can be obtained in suitable expression hosts such as E. coli, yeast and other eukaryotic expression systems can also be used. The receptors can be expressed alone, as fragments of the mature or full-length sequence, or as fusions to heterologous sequences.

Initially, purification of the melanocortin receptor can be obtained by conventional techniques, such as hydrophobic interaction chromatography (HPLC), ion exchange chromatography (HPLC), and heparin affinity chromatography. To achieve higher purification of melanocortin receptors, the receptors can be ligand-shift-purified using a column that separates the receptor according to charge, such as an ion exchange or hydrophobic interaction column, and then bind the eluted receptor with a ligand, especially an agonist. The ligand induces a change in the receptor's surface charge such that when re-chromatographed on the same column, ligand-bound receptor is separated from unliganded receptor. Usually saturating concentrations of ligand are used in the column and the protein can be preincubated with the ligand prior to passing it over the column. The structural studies detailed herein indicate the general applicability of this technique for obtaining super-pure melanocortin receptors for use in assays to assess the antagonist or agonist activity of candidate compounds.

There are many uses and advantages provided by the present invention. For example, the methods and compositions described herein are useful for identifying peptides, peptidomimetics or small natural or synthetic organic molecules that modulate melanocortin receptor activity. The compounds are useful in treating melanocortin receptor-based disorders. Methods and compositions of the invention also find use in characterizing structure/function relationships of natural and synthetic ligand compounds.

Another aspect of the invention also pertains to a polypeptide molecule that acts as a strong antagonist of melanocortin receptor types 3 and 4, and is useful for the treatment of eating disorders and obesity. As discussed above, research has demonstrated that the final 46 residues of the human AGRP sequence ("MARP"), possesses full receptor antagonist activity. The structure of MARP, solved by use of nuclear magnetic resonance and described herein, suggests that the final 13 residues of MARP are unstructured and thus may not play a role in receptor binding or antagonism. Thus, substantial further minimization of human AGRP may be achieved. Specifically, it is expected that the sequence (indicated by the single letter amino acid code):

CVRLH5ESCLG10QQVPC15CDPAA20TCYCR25FFNAF30CYC (SEQ ID NO:3) which is designated "MARP-33" (based upon its 33 amino acid length) will fold with proper native-like disulfide bonds and possess the full biological activity of MARP. This sequence is equivalent to the first 33 amino acids of MARP but contain a Cys— »Ala substitution at position 19 (indicated as "A") to avoid having a non-bridged thiol.

MARP-33 finds utility as a potential therapeutic for the treatment of eating disorders and obesity, and may be administered to a patient in need thereof, to increase eating and fat deposition. In addition, the MARP-33 sequence may be modified such that the modified

MARP-33 may have therapeutic utility as a competitive inhibitor of in vivo human AGRP. Such modifications can include changing the sequence to increase binding affinity, to increase the level of antagonism and to increase stability of the molecule.

Accordingly, one aspect of the invention pertains to a method of treating a disease state in mammals that is alleviated by treatment with a polypeptide having an amino acid sequence: CVRLHESCLGQQVPCCDPAATCYCRFFNAFCYC (SEQ ID NO:3) which method comprises administering to a mammal in need of such a treatment a therapeutically effective amount of the polypeptide, which can be administered, by way of illustration and not limitation, in a liquid formulations or a solid formulations, such as in the form of a pharmaceutically acceptable salt thereof. Such a disease state can be a wasting syndrome, in addition, the invention encompasses a pharmaceutical composition comprising a therapeutically effective amount of a polypeptide of this sequence (SEQ ID NO:3), in a liquid, solid or other suitable formulation, including pharmaceutically acceptable carriers, stabilizers, excipients and so forth, such as are well known in the state of the art.

In addition, the structure and sequence of MARP-33 is a useful tool in rational drug design for the design of peptide-based therapeutics. Further, the structure of this molecule will facilitate the development of non-peptide based therapeutics.

The following examples illustrate various aspects of this invention. These examples do not limit the scope of this invention.

EXAMPLES Abbreviations JHNα three bond αH-NH scalar coupling constant

AGRP/ ART Agouti Related Protein/Agouti Related Transcript

Conformational shift experimental chemical shift-random coil chemical shift DQF-COSY two-dimensional double-quantum filtered correlation spectroscopy

HX hydrogen-deuterium exchange ICK Inhibitor Cystine Knot

MARP Minimized human Agouti Related Protein, residues 87-132 of human

AGRP MC3r/MC4r Melanocortin Receptor ³A

MRE Mean Residue Ellipticity α-MSH α-Melanocyte Stimulating Hormone

NDP-MSH [Nle⁴, D-Phe⁷]α-MSH, a superpotent melanocortin agonist nOe melanocortin Overhauser enhancement

NOESY two-dimensional melanocortin Overhauser spectroscopy

TOCSY two-dimensional total correlation spectroscopy Methods and Materials

A. Chemical protein synthesis: V'-Acetyl-MARP (SEQ ID NO:2) and Λ^-acetyl- MARP(Arg25Ala) were synthesized, folded and purified to each give a protein containing five disulfide bonds, as reported in Yang, et al., supra.

B. CD experiments: Circular dichroism ("CD") spectra were recorded at 25°C on an Aviv 60DS spectropolarimeter in a rectangular 1 mm path length cuvet for concentrations up to

60 μM. For concentrations higher than this a round cell with a 0.1 mm path length was used. All CD samples were 50 mM potassium phosphate, pH 4.25. Concentration dependence was ruled out in the range 20 μM-1 mM. Temperature dependence was determined for 5-85°C. The spectra are superimposable from 5 to 45°C.

C. NMR sample preparation: The activity of MARP used for the NMR sample and that of a single mutant were assayed by measuring the inhibition of cAMP production in the presence of NDP-MSH (Shutter, et al., supra; Huszar, et al., supra) in HEK-293 cells transfected with human MC4r. Control experiments were performed with no MARP. The NMR samples were found to be native-like with complete activity. NMR samples contained approximately 1.9 mM MARP at pH 4.2 in 50 mM KH₂P0₄ buffer in 90% H₂O/10% D₂O. Additional samples for HX experiments were prepared by lyophilization of protonated samples followed by reconstitution in 700 μl D₂O.

D. NMR experiments: 1H 2D NMR spectra were principally acquired at 15°C on a Varian 500 Unity Plus spectrometer using inverse probes. NMR data were routinely acquired with a 6000 Hz spectral width, 4096 complex points in t₂, and 512 (TOCSY/DQF-COSY) or 700 (NOESY) increments in tj. All spectra were processed using the MNMR package (Carlsberg Laboratory, Department of Chemistry, Denmark) and analyzed using XEASY (Bartels, et al., J. Biomol. NMR 5:1-10 (1995)), with chemical shifts referenced to 1.4-dioxime at 3.743 ppm. Sequential assignments of all backbone and >90% of side chain protons were accomplished using standard methods (Wϋthrich, K. "NMR of Proteins and Nucleic Acids" (John Wiley and Sons, New York, 1986); Redfield, C. in "NMR of Macromolecules: A Practical Approach, pp. 71-99 (Roberts, G.K.C., Ed., IRL Press at Oxford University Press, Oxford, 1993)) for 50 ms

TOCSY, 150 ms NOESY and DQF-COSY data. Additional data sets were acquired at 25°C and 30°C to resolve ambiguities. Examination of the three Pro residues identified nOes consistent only with trans-Pro. Four additional peaks were identified in the αN region of the TOCSY spectrum, however, associated spin systems could not be identified and neither could nOes to the peaks in question.

NOESY data for distance restraints were collected at 15°C using the WET sequence (Smallcombe, et al., J. Magn. Reson. Ser. A 117:295-303 (1995)) for water suppression, 1.6 s recycle delay and a mixing time of 80 ms in both H O and D₂O. ³J_HN_coupling constants at 25°C were determined by both linear least squares fitting of the antiphase doublets in a DQF- COSY and also using the INFIT (Szyperski, et al., J. Magn. Reson. 99:552-560 (1992)) module of XEASY with 150 ms NOESY data. These methods agreed to within ±0.5 Hz for all of the measured coupling constants. At 15°C larger intrinsic linewidths precluded accurate measurement of JHNα-

For amide exchange experiments, the magnet was preshimmed on a 21 residue peptide sample at pH 4 in D O/phosphate buffer. The first TOCSY experiment was begun 23 minutes after reconstituting the protonated sample in D₂O. Four TOCSY experiments identical to those described above, except for the number of t_/ increments, were acquired back to back at 15°C over a period of 24 hours. The first three consisted of 150 tj increments and the final experiment 300 increments. NOESY and DQF-COSY spectra were also acquired as described above. E. Structure calculations: Final structure calculations included the covalent connectivity of the published disulfide map (Bures, et al., supra), and were based on a total of 414 interproton distance constraints derived from the 80 ms 2D NOESY spectra and 34 backbone Φ dihedral angle constraints derived from coupling constant measurements, giving a total of 448 total restraints, or 9.7 restraints per residue. The distance restraints can be broken down into 228 intraresidue (backbone to side chain only), 129 sequential, 20 medium range (l<_\i-j_\≤5) and 37 long range (_\i-j >5) restraints. These restraints were assigned as strong, medium or weak. The total numbers of restraints in each category were 95 strong, 246 medium, and 77 weak. All categories had a lower limit of 1.6A, with upper limits of 2.8, 3.5 and 5 A for the strong, medium and weak categories, respectively. Trial structures were generated using the simulated annealing protocol from CNS version 0.4a (anneal. inp) with SUM averaging for the nOe distances (Brunger, et al., Acta Crystallogr. Sect. D Biol. Crystallogr. 54:905-921 (1998); Nilges, et al., FEBS Lett. 239:129-136 (1988); Nilges, et al., Protein Eng. 2:27-38 (1988); Kuszewski, et al., J. Magn. Reson. Ser. B 112:79-81 (1996); Stein, et al., J. Magn. Reson. 124:154-164 (1997)). Twenty structures with no bond or nOe angle violations were used to represent the solution structure of MARP (see Table 1 for Root Mean Square Deviations, "RMSDs"). Structures were displayed using MOLMOL (Koradi, et al., Science 278:135-138 (1997)).

Example 1 Chemical synthesis and characterization of MARP The synthesis and biochemical characterization of MARP were previously reported in Yang, et al., supra. The N-terminal residue (Cys-1) of MARP corresponds to the first Cys (Cys- 87) of the Cys-rich region in full length, 132 residue human AGRP. The material used for the MARP NMR sample showed native-like activity as measured by its ability to competitively inhibit NDP-MSH at MC4r, as has been shown in previous studies (Yang, et al., supra; Ollmann, et al., Science 278:135-138 (1997)). Previous mutational studies of agouti and AGRP showed residues Arg-25, Phe-26, Phe-27 (the RFF triplet, Tota, et al., supra) and Asp- 17 (in agouti) to be determinants of receptor binding (Kiefer, et al., Biochemistry 36:2084-2090 (1997); Kiefer, et al., Biochemistry 37:991-997 (1998); Tota, et al., supra)).

This experiment measured inhibition of NDP-MSH stimulated cAMP generation in cells transfected with MC4r. A control experiment was conducted with no MARP present. The addition of MARP (5.0 x 10^"6 M) demonstrated competitive inhibition of NDP-MSH. The addition of MARP with Arg-25 to Ala substitution (5.0 x 10^"6 M) demonstrated loss of inhibition due to a mutation in the active loop. This experiment thus showed that the replacement of Arg- 25 by Ala results in a complete loss of inhibitory activity.

The far-UV CD spectrum of MARP was measured in 50 mM phosphate buffer at pH 4.25 as a function of temperature with spectra shown every 5°C from 5°C to 30°C and also at 45°C. The spectrum was similar to that reported for a similar C-terminal fragment (residues 85- 132) of AGRP (residues 85-132 of SEQ ID NO:l) reported by Rosenfeld, et al., supra, and is characterized by a negative maximum at approximately 198 nm and a slight negative maximum at 245 nm. There was little indication of canonical α-helix, however the spectrum did suggest some β character or turns. The near-UV CD spectrum, indicative of tertiary structure, showed a weak minimum at approximately 275 nm which could be attributed to the disulfide bonds and possibly restricted orientations of the side chains of the Tyr-23 and Tyr-32.

At 25°C between 20 μM and 1.0 mM there was no observable concentration dependence as measured by CD. Between 1 mM and 1.9 mM there were no concentration dependent changes in the NMR spectra (i.e. linewidths, chemical shifts, etc. all remain constant). The far- UV CD spectrum remained constant between 5°C and 45°C, a temperature range well beyond that of the present NMR experiments. Thus, by all indications, MARP existed as a monomer and did not exhibit temperature dependence under the conditions of the NMR experiments. The characteristics of the NMR spectra were indicative of a well folded protein with a single predominant conformer. J_HNα coupling constants and temperature coefficient also indicated a fully folded, non-random coil conformer as shown in Table 1 below, which shows the MARP coupling constants at 25°C and NH temperature coefficients in 50 min phosphate buffer at pH 4.2. Table 1

³JNH coupling NH temperature

Residue constant (Hz) coefficient (ppb/°C)

Cys-1 6.93

Val-2 8.79 3.63

Arg-3 7.00 6.37

Leu-4 5.64 3.47

His-5 7.50 2.58

Glu-6 9.34 2.90

Ser-7 5.86

Cys-8 9.92 3.84

Leu-9 4.40 2.43

Gly-10 7.06

Gin- 11 8.14 4.94

Gin- 12 8.00 4.38

Val-13 9.03 5.56

Pro- 14 0.00

Cys- 15 4.33 6.74

Cys-16 5.42 3.10

Asp- 17 4.81

Pro- 18 0.00

Cys- 19 8.81 3.84

Ala-20 9.64 3.23

Thr-21 9.36 4.04

Cys-22 5.76 7.56

Tyr-23 2.01

Cys-24 3.14

Arg-25 5.90 2.93

Phe-26 8.70 3.67

Phe-27 3.68 6.21

Asn-28 7.82 5.66

Ala-29 7.56 2.10

Phe-30 7.59 4.84

Cys-31 9.17 5.84

Tyr-32 9.49 4.43

Cys-33 1.13

Arg-34 9.34 2.74

Lys-35 7.25 8.80

Leu-36 7.74 6.43

Gly-37 7.00

Thr-38 8.07 2.07

Ala-39 4.48 7.67

Met-40 7.50 2.98

Asn-41 8.19 1.08

Pro-42 0.00

Cys-43 7.24 7.86

Ser-44 7.26 8.38 ³J_NHα coupling NH temperature

Residue constant (Hz) coefficient (ppb/°C)

Arg-45 7.54 4.47

Thr-46 4.61

Example 2 Structural description of MARP The minimized average NMR structure of MARP is shown in Fig. 1. Consistent with the far-UV CD spectrum, MARP showed little evidence of helix or sheet secondary structure. The disulfide bonds (1-16, 8-22, 15-33, 19-43 and 24-31) appeared to form a scaffold upon which the structure was apportioned into three major loops, which are referred to as the N-terminal loop (residues 1-18), the central loop (residues 19-34) and the C-terminal loop (residues 35-46), and are indicated in Fig. 1. RMSDs for the individual loops are reported in Table 2 below: Table 2

Summary of MARP backbone and heavy atom RMSDs

Region (residues) Backbone RMSD* (A) Heavy atom RMSD^a (A)

Global (1-46) 2.54 3.26

N- and active loops (1-34) 1.66 2.38

N-terminal loop (1-18) 1.31 2.03

Central loop ( 19-34) 1.51 2.22

Active loop (24-31) 0.69 1.53

C-terminal loop (35-46) 2.36 3.43

* Determined by fitting the family of 20 NMR structures to the minimized average structure The N-terminal and central loops were much better defined both within the loops and with respect to each other than the C-terminal loop. The backbone RMSD for the entire protein (2.54A) was of the same order as that of the C-terminal loop (2.36A), while the backbone RMSD for residues 1-34 (1.66A) was of the order of the individual N-terminal and central loops. To demonstrate limited backbone structure variability of the N-terminal and central loops, a superposition of 14 structures (selected for clarity) for residues 1-34 and the MARP minimized average structure (residues 1-46) is shown in Fig. 2.

Four of the five disulfide bonds are located at the base of the structure where they appear to pinch together the bottoms of the loops to form the "core" of the protein (Fig. 1). The exception is disulfide bond 24-31 which stabilizes the central loop. The central loop, residues 19-34, contains the RFF triplet determined to be critical for activity. This motif is situated within an even smaller, well defined loop bound by Cys-24 and Cys-31 which is referred to as the "active" loop. The side chain atoms of the RFF triplet residues are located at the surface of the protein as depicted in Fig. 3. Recent experiments further highlight the importance of this active loop. These studies demonstrate that short cyclic peptides corresponding to residues 24- 31 of human AGRP do in fact antagonize MC3r and MC4r (Tota, et al., supra).

Inspection of the family of NMR structures and consideration of the observed HX revealed a structure for the central loop that is best described as an irregular hairpin with a well defined loop from Cys-24 to Cys-31 (RMSD 0.6A, Fig. 3) and a stem region which is both twisted around and curved along its z-axis (Fig. 1). This characterization was supported by critical examination of the nOe, ³JHN_α and chemical shift data. As shown in Fig. 3, the active loop is highly constrained with the RFF triplet side chains exposed to solvent. Arg-25 and Phe- 27 point out into the solvent, while one face of the Phe-26 aromatic ring rests parallel against the surface of the protein. Though the active loop satisfies several of the determinants for an Ω-loop (Leszczynski, et al., Science 234:849-855 (1986)), the side chain orientation of Arg-25 and Phe- 27 precludes its definition as such since Ω-loop side chains generally pack within the loop of backbone atoms.

HX experiments demonstrated that the amide protons of residues Cys-8, Ala-20, Thr-1, Tyr-23, Tyr-2, Cys-3 and Arg-34 are protected from exchange with solvent. To explore whether these results were consistent with the average structure, the program DSSP (Kabsch, et al., Biopolymers 22:2577-2637 (1983)) was used to identify potential hydrogen bonds. DSSP identified the backbone amides of Ala-20, Thr-21, Tyr-23, and Arg-34 as potential hydrogen bond donors. In addition, solvent accessible surface area calculations showed that residues Cys- 8 and Cys-33 were completely buried from solvent, though in the D₂O spectrum the αN crosspeaks of these two residues overlap, thus their individual protection from exchange is uncertain. Tyr-32 had only 8% solvent accessible surface area at the Cβprotons.

The NMR structure gave a well resolved fold, however, as mentioned previously, canonical helices and β-sheets were not identified on the basis of nOes or other protocols including the chemical shift index (Wishart, et al., Biochemistry 31:1647-1651 (1992)) or ³J_HNα coupling constants (Wϋthrich, K., supra).

The guidelines for these protocols assigned secondary structure on the basis of four or more consecutive residues with similar conformational shifts or JHN - Helical structure was characterized by J_HN« ^<6 Hz and negative conformational shifts and β-sheet by JHNrAδ Hz and positive conformational shifts. Even in the active loop and stem region of the central loop, no regular secondary structure was identified by these criteria. However, the chemical shift index pointed towards a possible extended strand from residue 31 to residue 35. Table 3 sets forth the

1 ^

H chemical shifts at 15°C, J_NHCC, at 250°C, and the NH temperature coefficients.

Table 3 MARP 1H chemical shifts at 15°C in 50 mm phosphate buffer at pH 4.2

Residue NH (ppm) H„ (ppm) H_β (ppm) Other (ppm)

Cys- 1 8.00 5.00 3.12, 2.81

Val-2 8.92 4.18 1.94, 2.08 CH^γ ₃ 0.91, 0.80

Arg-3 8.91 4.01 1.67, 1.95 H^γ1.69, 1.82, H^δ3.24, 3.26, NH 7.31

Leu-4 8.13 3.72 1.34, 1.54 H^γ1.03, CH^δ ₃ 0.79

His-5 8.99 4.25 3.70, 1.42 H^δ7.22, ^Hε8.51

Glu-6 8.04 4.66 2.16 H^γ2.24

Ser-7 8.63 4.68 3.84

Cys-8 8.08 4.96 3.57, 3.13

Leu-9 7.92 4.08 1.57, 1.44 H^γ1.44, CH^δ ₃0.82

Gly-10 8.78 4.07, 3.69

Gin- 11 8.06 4.24 1.95 H^γ 2.28. H^ε26.86, 7.49

Gin- 12 8.68 4.27 2.20, 1.95 H^γ 2.30, 2.34, H^ε2 6.82, 7.45

Val- 13 7.48 4.47 2.05 CH^γ ₃ 0.79, 0.90

Pro- 14 8.81 4.55 2.25, 1.99 H^γ 1.84, 1.99, H^δ 3.65, 3.77

Cys- 15 4.92 3.35, 1.74

Cys- 16 9.58 4.22 2.62, 3.17

Asp- 17 8.19 4.78 2.63, 2.41

Pro- 18 8.88 4.50 2.35 H^γ 1.98, H^δ 3.89, 4.04

Cys- 19 4.72 2.94, 3.47

Ala-20 8.01 4.94 1.26

Thr-21 8.76 4.58 4.00 CH^γ ₃ 1.16

Cys-22 8.94 4.58 2.94, 3.02

Tyr-23 8.71 4.62 2.79 H^δ 6.93, H^ε 6.74

Cys-24 8.30 4.91 3.23, 2.60

Arg-25 8.29 3.84 1.83, 1.55 H^v1.56, 1.23, H^δ 3.06

Phe-26 7.91 4.74 2.80, 3.32 H^δ 7.26, H^ε 7.42

Phe-27 8.54 4.19 3.12, 3.02 H^δ 7.17, H^ε 7.33

Asn-28 8.48 4.20 2.39, 7.77 H^δ2 6.66, 7.30

Ala-29 7.75 4.38 1.25

Phe-30 8.38 4.21 3.32, 3.36

Cys-31 8.34 5.63 2.59, 3.03

Tyr-32 8.88 5.20 2.59, 2.81 H^δ 6.92, H^ε 6.66

Cys-33 8.19 4.96 3.21, 2.66

Arg-34 9.43 4.75 1.83, 1.70 H^γ1.57, 1.69, H^δ 2.62, 2.88, Residue NH (ppm) H„ (mm) H_β (ppm) Other (ppm) NH 7.08

Lys-35 9.09 4.47 1.82, 1.68 H^γ1.25, 1.43, H^δ 1.64, H^ε 2.89

Leu-36 8.77 4.3640 1.63 H^γ1.52, CH^δ ₃ 0.82, 0.70

Gly-37 8.48 4.09, 4.01

Thr-38 7.82 4.42 4.42 CH^γ ₃ 1.21

Ala-39 8.53 4.16 1.42

Met-40 8.06 4.40 1.95, 2.09 H^γ2.51, 2.62

Asn-41 7.74 5.00 2.62, 2.81 H^δ2 7.67, 6.95

Pro-42 8.68 4.47 2.29 H^γ1.94, H^δ 3.61, 3.67

Cys-43 4.59 3.22, 3.13

Ser-44 8.37 4.46 3.86

Arg-45 8.26 4.43 1.93 H^γl .66, H^δ 3.21, NH 7.25

Thr-46 7.86 4.15 4.23 CH^γ ₃1.15

All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

The invention now being fully described, it will be apparent to one of ordinary skill in the art that many changes and modifications can be made thereto without departing from the spirit or scope of the appended claims.

Appendix 1

Atomic Coordinates for MARP

ATOM 1 CA CYS 1 -6.900 2.269 -8.250 1.00 4.62

ATOM 2 HA CYS 1 -5.897 2.041 -7.920 1.00 4.59

ATOM 3 HB1 CYS 1 -7.160 4.143 -9.269 1.00 5.13

ATOM 4 HB2 CYS 1 -6.230 4.254 -7.780 1.00 4.63

ATOM 5 C CYS 1 -7.900 1.623 -7.293 1.00 3.97

ATOM 6 O CYS 1 -9.067 1.425 -7.636 1.00 4.16

ATOM 7 CB CYS 1 -7.088 3.796 -8.249 1.00 4.78

ATOM 8 SG CYS 1 -8.576 4.376 -7.358 1.00 5.12

ATOM 9 N CYS 1 -7.069 1.766 -9.642 1.00 5.38

ATOM 10 HT1 CYS 1 -8.026 2.021 -9.958 1.00 5.84

ATOM 11 HT2 CYS 1 -6.942 0.734 -9.624 1.00 5.52

ATOM 12 HT3 CYS 1 -6.346 2.222 -10.234 1.00 5.55

ATOM 13 N VAL 2 -7.445 1.356 -6.070 1.00 3.32

ATOM 14 HN VAL 2 -6.517 1.593 -5.848 1.00 3.27

ATOM 15 CA VAL 2 -8.315 0.801 -5.028 1.00 2.84

ATOM 16 HA VAL 2 -9.187 0.372 -5.505 1.00 3.11

ATOM 17 CB VAL 2 -7.624 -0.292 -4.151 1.00 2.58

ATOM 18 HB VAL 2 -6.927 0.197 -3.490 1.00 2.54

ATOM 19 CGI VAL 2 -8.637 -1.028 -3.287 1.00 2.26

ATOM 20 HG11 VAL 2 -9.533 -1.215 -3.860 1.00 2.67

ATOM 21 HG12 VAL 2 -8.879 -0.427 -2.423 1.00 2.47

ATOM 22 HG13 VAL 2 -8.214 -1.969 -2.962 1.00 2.24

ATOM 23 CG2 VAL 2 -6.851 -1.295 -5.000 1.00 3.27

ATOM 24 HG21 VAL 2 -6.240 -1.922 -4.350 1.00 3.49

ATOM 25 HG22 VAL 2 -6.214 -0.766 -5.692 1.00 3.65

ATOM 26 HG23 VAL 2 -7.544 -1.914 -5.548 1.00 3.65

ATOM 27 C VAL 2 -8.750 1.967 -4.145 1.00 2.69

ATOM 28 O VAL 2 -8.214 3.071 -4.285 1.00 3.12

ATOM 29 N ARG 3 -9.694 1.759 -3.238 1.00 2.64

ATOM 30 HN ARG 3 -10.100 0.873 -3.138 1.00 2.81

ATOM 31 CA ARG 3 -10.120 2.851 -2.380 1.00 2.90

ATOM 32 HA ARG 3 -10.214 3.738 -2.998 1.00 3.25

ATOM 33 CB ARG 3 -11.466 2.539 -1.720 1.00 3.25

ATOM 34 HB1 ARG 3 -11.575 3.157 -0.841 1.00 3.44

ATOM 35 HB2 ARG 3 -11.477 1.501 -1.422 1.00 3.38

ATOM 36 CG ARG 3 -12.662 2.785 -2.628 1.00 3.73

ATOM 37 HG1 ARG 3 -13.287 1.905 -2.628 1.00 4.00

ATOM 38 HG2 ARG 3 -12.307 2.976 -3.630 1.00 3.91

ATOM 39 CD ARG 3 -13.486 3.978 -2.164 1.00 4.23

ATOM 40 HD1 ARG 3 -13.348 4.788 -2.865 1.00 4.60

ATOM 41 HD2 ARG 3 -13.136 4.285 -1.189 1.00 4.30

ATOM 42 NE ARG 3 -14.913 3.662 -2.078 1.00 4.74

ATOM 43 HE ARG 3 -15.185 2.745 -2.294 1.00 4.90

ATOM 44 CZ ARG 3 -15.856 4.540 -1.726 1.00 5.32

ATOM 45 NH1 ARG 3 -15.532 5.796 -1.425 1.00 5.50

ATOM 46 HH11 ARG 3 -14.578 6.091 -1.459 1.00 5.28 ^ 4^ ) W to t

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ATOM 96 HB2 GLU 6 -10.392 1.513 1.227 1.00 2.71

ATOM 97 CG GLU 6 -10.825 -0.566 1.307 1.00 2.93

ATOM 98 HG1 GLU 6 -11.828 -0.167 1.356 1.00 3.17

ATOM 99 HG2 GLU 6 -10.645 -1.192 2.168 1.00 3.31

ATOM 100 CD GLU 6 -10.701 -1.416 0.055 1.00 3.19

ATOM 101 OE1 GLU 6 -11.236 -1.006 -0.997 1.00 3.57

ATOM 102 OE2 GLU 6 -10.068 -2.491 0.127 1.00 3.55

ATOM 103 C GLU 6 -7.955 -0.456 2.557 1.00 2.53

ATOM 104 O GLU 6 -8.269 -1.602 2.876 1.00 2.98

ATOM 105 N SER 7 -6.729 -0.115 2.130 1.00 2.02

ATOM 106 HN SER 7 -6.557 0.801 1.870 1.00 1.87

ATOM 107 CA SER 7 -5.656 -1.086 2.013 1.00 1.98

ATOM 108 HA SER 7 -4.753 -0.563 1.693 1.00 1.74

ATOM 109 CB SER 7 -5.409 -1.725 3.371 1.00 2.69

ATOM 110 HB1 SER 7 -5.970 -2.643 3.442 1.00 3.00

ATOM 111 HB2 SER 7 -5.742 -1.040 4.133 1.00 2.94

ATOM 112 OG SER 7 -4.036 -2.005 3.572 1.00 3.30

ATOM 113 HG SER 7 -3.939 -2.883 3.947 1.00 3.67

ATOM 114 C SER 7 -6.066 -2.147 0.984 1.00 2.01

ATOM 115 O SER 7 -7.207 -2.143 0.517 1.00 2.80

ATOM 116 N CYS 8 -5.180 -3.074 0.643 1.00 1.78

ATOM 117 HN CYS 8 -4.285 -3.074 1.044 1.00 1.85

ATOM 118 CA CYS 8 -5.558 -4.119 -0.301 1.00 2.24

ATOM 119 HA CYS 8 -6.476 -3.820 -0.745 1.00 2.23

ATOM 120 HB1 CYS 8 -3.606 -4.528 -1.025 1.00 2.70

ATOM 121 HB2 CYS 8 -4.503 -3.311 -1.949 1.00 2.24

ATOM 122 C CYS 8 -5.763 -5.451 0.432 1.00 2.87

ATOM 123 O CYS 8 -5.575 -6.522 -0.140 1.00 3.43

ATOM 124 CB CYS 8 -4.568 -4.261 -1.425 1.00 2.52

ATOM 125 SG CYS 8 -5.047 -5.510 -2.659 1.00 3.51

ATOM 126 N LEU 9 -6.155 -5.353 1.714 1.00 3.1 1

ATOM 127 HN LEU 9 -6.288 -4.468 2.091 1.00 3.01

ATOM 128 CA LEU 9 -6.400 -6.509 2.578 1.00 3.82

ATOM 129 HA LEU 9 -7.159 -7.116 2.111 1.00 4.03

ATOM 130 CB LEU 9 -5.122 -7.346 2.756 1.00 4.45

ATOM 131 HB1 LEU 9 -5.161 -7.820 3.726 1.00 4.87

ATOM 132 HB2 LEU 9 -4.275 -6.676 2.739 1.00 4.44

ATOM 133 CG LEU 9 -4.890 -8.438 1.701 1.00 4.92

ATOM 134 HG LEU 9 -4.327 -8.018 0.881 1.00 4.96

ATOM 135 CD1 LEU 9 -4.073 -9.583 2.281 1.00 5.65

ATOM 136 HD11 LEU 9 -3.633 -9.273 3.217 1.00 5.93

ATOM 137 HD12 LEU 9 -3.291 -9.852 1.587 1.00 5.76

ATOM 138 HD13 LEU 9 -4.715 -10.435 2.449 1.00 6.14

ATOM 139 CD2 LEU 9 -6.214 -8.951 1.149 1.00 5.23

ATOM 140 HD21 LEU 9 -6.026 -9.607 0.313 1.00 5.47

ATOM 141 HD22 LEU 9 -6.812 -8.111 0.821 1.00 5.42

ATOM 142 HD23 LEU 9 -6.743 -9.490 1.921 1.00 5.45

ATOM 143 C LEU 9 -6.919 -6.051 3.948 1.00 3.94

ATOM 144 O LEU 9 -7.894 -6.604 4.462 1.00 4.53 4^ -fc- > 1 J to to υ. © Λ © Uι © υ.

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ATOM 243 CG ASP 17 -2.132 10.754 -5.977 1.00 6.17

ATOM 244 OD1 ASP 17 -1.748 1 1.210 -7.075 1.00 6.53

ATOM 245 OD2 ASP 17 -1.668 11.129 -4.880 1.00 6.56

ATOM 246 C ASP 17 -1.283 8.093 -6.351 1.00 4.80

ATOM 247 O ASP 17 -0.465 8.102 -7.271 1.00 4.56

ATOM 248 N PRO 18 -0.908 7.839 -5.070 1.00 5.13

ATOM 249 CA PRO 18 0.485 7.553 -4.669 1.00 5.06

ATOM 250 HA PRO 18 1.165 8.301 -5.023 1.00 5.33

ATOM 251 CB PRO 18 0.438 7.600 -3.127 1.00 5.85

ATOM 252 HB1 PRO 18 1.243 8.221 -2.761 1.00 6.32

ATOM 253 HB2 PRO 18 0.542 6.601 -2.729 1.00 5.79

ATOM 254 CG PRO 18 -0.891 8.182 -2.786 1.00 6.39

ATOM 255 HG1 PRO 18 -0.814 9.257 -2.712 1.00 6.94

ATOM 256 HG2 PRO 18 -1.249 7.765 -1.857 1.00 6.75

ATOM 257 CD PRO 18 -1.800 7.803 -3.912 1.00 5.92

ATOM 258 HD1 PRO 18 -2.598 8.519 -4.012 1.00 6.35

ATOM 259 HD2 PRO 18 -2.194 6.810 -3.762 1.00 5.99

ATOM 260 C PRO 18 0.965 6.192 -5.162 1.00 4.35

ATOM 261 O PRO 18 0.484 5.690 -6.181 1.00 4.45

ATOM 262 N CYS 19 1.908 5.589 -4.438 1.00 4.00

ATOM 263 HN CYS 19 2.257 6.027 -3.634 1.00 4.34

ATOM 264 CA CYS 19 2.427 4.285 -4.822 1.00 3.50

ATOM 265 HA CYS 19 2.416 4.237 -5.903 1.00 3.76

ATOM 266 HB1 CYS 19 4.354 5.069 -4.280 1.00 4.02

ATOM 267 HB2 CYS 19 4.412 3.492 -5.049 1.00 4.20

ATOM 268 C CYS 19 1.501 3.201 -4.285 1.00 2.98

ATOM 269 O CYS 19 0.949 2.444 -5.067 1.00 2.87

ATOM 270 CB CYS 19 3.880 4.100 -4.333 1.00 3.68

ATOM 271 SG CYS 19 4.070 3.305 -2.701 1.00 3.27

ATOM 272 N ALA 20 1.301 3.192 -2.958 1.00 3.00

ATOM 273 HN ALA 20 1.746 3.891 -2.427 1.00 3.43

ATOM 274 CA ALA 20 0.381 2.259 -2.261 1.00 2.68

ATOM 275 HA ALA 20 0.867 1.965 -1.352 1.00 2.43

ATOM 276 CB ALA 20 -0.896 2.998 -1.882 1.00 2.88

ATOM 277 HB1 ALA 20 -1.747 2.351 -2.033 1.00 3.03

ATOM 278 HB2 ALA 20 -0.997 3.878 -2.499 1.00 3.21

ATOM 279 HB3 ALA 20 -0.847 3.293 -0.841 1.00 3.09

ATOM 280 C ALA 20 0.077 0.972 -3.049 1.00 2.88

ATOM 281 O ALA 20 -0.665 1.040 -3.996 1.00 3.63

ATOM 282 N THR 21 0.639 -0.179 -2.606 1.00 2.52

ATOM 283 HN THR 21 1.178 -0.127 -1.824 1.00 2.17

ATOM 284 CA THR 21 0.492 -1.519 -3.247 1.00 3.01

ATOM 285 HA THR 21 0.640 -1.411 -4.288 1.00 3.44

ATOM 286 CB THR 21 1.615 -2.430 -2.712 1.00 3.51

ATOM 287 HB THR 21 2.292 -1.827 -2.145 1.00 3.87

ATOM 288 OG1 THR 21 2.342 -3.018 -3.765 1.00 3.92

ATOM 289 HG1 THR 21 1.829 -3.731 -4.147 1.00 4.04

ATOM 290 CG2 THR 21 1.139 -3.534 -1.814 1.00 3.64

ATOM 291 HG21 THR 21 1.148 -4.469 -2.352 1.00 3.73 ATOM 292 HG22 THR 21 0.138 -3.302 -1.513 1.00 3.73

ATOM 293 HG23 THR 21 1.776 -3.606 -0.946 1.00 3.90

ATOM 294 C THR 21 -0.893 -2.141 -3.040 1.00 2.82

ATOM 295 O THR 21 -1.823 -1.429 -2.757 1.00 2.79

ATOM 296 N CYS 22 -1.047 -3.453 -3.225 1.00 3.23

ATOM 297 HN CYS 22 -0.284 -3.987 -3.488 1.00 3.75

ATOM 298 CA CYS 22 -2.361 -4.091 -3.092 1.00 3.29

ATOM 299 HA CYS 22 -2.941 -3.546 -2.350 1.00 2.73

ATOM 300 HB1 CYS 22 -2.576 -4.621 -5.160 1.00 4.32

ATOM 301 HB2 CYS 22 -3.066 -2.975 -4.783 1.00 4.25

ATOM 302 C CYS 22 -2.268 -5.563 -2.656 1.00 3.83

ATOM 303 O CYS 22 -2.573 -6.463 -3.444 1.00 4.63

ATOM 304 CB CYS 22 -3.092 -3.999 -4.442 1.00 3.86

ATOM 305 SG CYS 22 -4.841 -4.522 -4.418 1.00 3.79

ATOM 306 N TYR 23 -1.888 -5.785 -1.377 1.00 3.64

ATOM 307 HN TYR 23 -1.689 -5.024 -0.821 1.00 3.23

ATOM 308 CA TYR 23 -1.799 -7.133 -0.783 1.00 4.31

ATOM 309 HA TYR 23 -2.770 -7.368 -0.375 1.00 4.75

ATOM 310 CB TYR 23 -1.483 -8.137 -1.875 1.00 4.99

ATOM 311 HB1 TYR 23 -0.990 -7.599 -2.663 1.00 4.83

ATOM 312 HB2 TYR 23 -2.406 -8.540 -2.250 1.00 5.71

ATOM 313 CG TYR 23 -0.599 -9.294 -1.468 1.00 5.19

ATOM 314 CD1 TYR 23 -1.141 -10.531 -1.148 1.00 5.34

ATOM 315 HD1 TYR 23 -2.212 -10.660 -1.183 1.00 5.46

ATOM 316 CD2 TYR 23 0.776 -9.147 -1.418 1.00 5.53

ATOM 317 HD2 TYR 23 1.205 -8.183 -1.658 1.00 5.77

ATOM 318 CE1 TYR 23 -0.332 -11.592 -0.790 1.00 5.56

ATOM 319 HE1 TYR 23 -0.771 -12.547 -0.545 1.00 5.82

ATOM 320 CE2 TYR 23 1.595 -10.200 -1.059 1.00 5.81

ATOM 321 HE2 TYR 23 2.667 -10.065 -1.025 1.00 6.26

ATOM 322 CZ TYR 23 1.036 -11.421 -0.748 1.00 5.71

ATOM 323 OH TYR 23 1.846 -12.475 -0.395 1.00 6.01

ATOM 324 HH TYR 23 2.160 -12.917 -1.187 1.00 6.05

ATOM 325 C TYR 23 -0.764 -7.219 0.355 1.00 4.08

ATOM 326 O TYR 23 -0.942 -7.990 1.300 1.00 4.31

ATOM 327 N CYS 24 0.305 -6.427 0.252 1.00 3.99

ATOM 328 HN CYS 24 0.376 -5.836 -0.520 1.00 4.20

ATOM 329 CA CYS 24 1.379 -6.392 1.250 1.00 3.97

ATOM 330 HA CYS 24 2.008 -5.546 0.990 1.00 3.54

ATOM 331 HB1 CYS 24 0.849 -7.090 3.203 1.00 4.45

ATOM 332 HB2 CYS 24 -0.159 -5.779 2.608 1.00 4.72

ATOM 333 C CYS 24 2.230 -7.664 1.271 1.00 4.56

ATOM 334 O CYS 24 1.702 -8.775 1.300 1.00 4.95

ATOM 335 CB CYS 24 0.841 -6.167 2.655 1.00 4.21

ATOM 336 SG CYS 24 1.818 -4.991 3.578 1.00 3.97

ATOM 337 N ARG 25 3.553 -7.485 1.306 1.00 4.81

ATOM 338 HN ARG 25 3.910 -6.572 1.315 1.00 4.70

ATOM 339 CA ARG 25 4.485 -8.614 1.370 1.00 5.41

ATOM 340 HA ARG 25 4.148 -9.364 0.676 1.00 5.78 ATOM 341 CB ARG 25 5.903 -8.174 0.986 1.00 5.35

ATOM 342 HB1 ARG 25 6.586 -8.982 1.206 1.00 5.48

ATOM 343 HB2 ARG 25 6.173 -7.317 1.584 1.00 5.55

ATOM 344 CG ARG 25 6.065 -7.802 -0.478 1.00 5.19

ATOM 345 HG1 ARG 25 5.093 -7.787 -0.945 1.00 5.37

ATOM 346 HG2 ARG 25 6.687 -8.541 -0.961 1.00 5.36

ATOM 347 CD ARG 25 6.712 -6.433 -0.636 1.00 5.12

ATOM 348 HD1 ARG 25 6.606 -5.890 0.292 1.00 5.22

ATOM 349 HD2 ARG 25 6.201 -5.899 -1.423 1.00 4.97

ATOM 350 NE ARG 25 8.134 -6.527 -0.973 1.00 5.60

ATOM 351 HE ARG 25 8.367 -6.596 -1.923 1.00 5.89

ATOM 352 CZ ARG 25 9.125 -6.525 -0.074 1.00 5.97

ATOM 353 NH1 ARG 25 8.863 -6.428 1.227 1.00 5.99

ATOM 354 HH11 ARG 25 7.920 -6.356 1.546 1.00 5.75

ATOM 355 HH12 ARG 25 9.614 -6.428 1.888 1.00 6.45

ATOM 356 NH2 ARG 25 10.386 -6.622 -0.477 1.00 6.62

ATOM 357 HH21 ARG 25 10.595 -6.697 -1.451 1.00 6.89

ATOM 358 HH22 ARG 25 1 1.127 -6.620 0.194 1.00 6.99

ATOM 359 C ARG 25 4.514 -9.217 2.773 1.00 5.75

ATOM 360 O ARG 25 4.289 -10.415 2.952 1.00 6.36

ATOM 361 N PHE 26 4.802 -8.369 3.759 1.00 5.44

ATOM 362 HN PHE 26 4.967 -7.433 3.534 1.00 4.97

ATOM 363 CA PHE 26 4.879 -8.793 5.158 1.00 5.87

ATOM 364 HA PHE 26 4.990 -9.862 5.151 1.00 6.37

ATOM 365 CB PHE 26 6.121 -8.187 5.838 1.00 5.87

ATOM 366 HB1 PHE 26 6.993 -8.742 5.519 1.00 6.14

ATOM 367 HB2 PHE 26 6.026 -8.279 6.907 1.00 5.89

ATOM 368 CG PHE 26 6.361 -6.740 5.515 1.00 5.68

ATOM 369 CD1 PHE 26 6.033 -5.748 6.420 1.00 5.91

ATOM 370 HD1 PHE 26 5.595 -6.022 7.368 1.00 6.30

ATOM 371 CD2 PHE 26 6.920 -6.381 4.303 1.00 5.59

ATOM 372 HD2 PHE 26 7.172 -7.156 3.593 1.00 5.76

ATOM 373 CE1 PHE 26 6.259 -4.419 6.122 1.00 5.89

ATOM 374 HE1 PHE 26 5.999 -3.653 6.837 1.00 6.28

ATOM 375 CE2 PHE 26 7.150 -5.054 3.995 1.00 5.57

ATOM 376 HE2 PHE 26 7.587 -4.787 3.044 1.00 5.74

ATOM 377 CZ PHE 26 6.819 -4.071 4.907 1.00 5.65

ATOM 378 HZ PHE 26 6.997 -3.032 4.670 1.00 5.75

ATOM 379 C PHE 26 3.576 -8.452 5.906 1.00 5.78

ATOM 380 O PHE 26 2.491 -8.694 5.373 1.00 5.69

ATOM 381 N PHE 27 3.669 -7.902 7.131 1.00 5.97

ATOM 382 HN PHE 27 4.549 -7.733 7.517 1.00 6.18

ATOM 383 CA PHE 27 2.477 -7.552 7.910 1.00 6.04

ATOM 384 HA PHE 27 1.921 -8.463 8.076 1.00 6.43

ATOM 385 CB PHE 27 2.876 -6.962 9.273 1.00 6.49

ATOM 386 HB1 PHE 27 2.391 -6.006 9.402 1.00 6.27

ATOM 387 HB2 PHE 27 3.945 -6.822 9.299 1.00 6.51

ATOM 388 CG PHE 27 2.497 -7.831 10.440 1.00 7.32

ATOM 389 CD1 PHE 27 3.134 -9.042 10.657 1.00 7.85 ATOM 390 HD1 PHE 27 3.910 -9.363 9.977 1.00 7.76

ATOM 391 CD2 PHE 27 1.503 -7.433 11.321 1.00 7.78

ATOM 392 HD2 PHE 27 1.000 -6.491 11.161 1.00 7.65

ATOM 393 CE1 PHE 27 2.787 -9.840 11.731 1.00 8.65

ATOM 394 HE1 PHE 27 3.290 -10.783 11.889 1.00 9.17

ATOM 395 CE2 PHE 27 1.151 -8.227 12.395 1.00 8.58

ATOM 396 HE2 PHE 27 0.375 -7.905 13.074 1.00 9.03

ATOM 397 CZ PHE 27 1.794 -9.432 12.601 1.00 8.94

ATOM 398 HZ PHE 27 1.521 -10.054 13.440 1.00 9.59

ATOM 399 C PHE 27 1.587 -6.578 7.130 1.00 5.30

ATOM 400 O PHE 27 0.738 -7.010 6.348 1.00 5.50

ATOM 401 N ASN 28 1.790 -5.270 7.320 1.00 4.68

ATOM 402 HN ASN 28 2.489 -4.973 7.933 1.00 4.87

ATOM 403 CA ASN 28 1.011 -4.272 6.600 1.00 3.97

ATOM 404 HA ASN 28 0.924 -4.630 5.591 1.00 3.66

ATOM 405 CB ASN 28 -0.399 -4.127 7.180 1.00 4.05

ATOM 406 HB1 ASN 28 -0.734 -3.109 7.042 1.00 4.27

ATOM 407 HB2 ASN 28 -0.372 -4.353 8.235 1.00 4.30

ATOM 408 CG ASN 28 -1.411 -5.060 6.513 1.00 4.05

ATOM 409 OD1 ASN 28 -2.267 -5.631 7.187 1.00 4.69

ATOM 410 ND2 ASN 28 -1.320 -5.227 5.185 1.00 3.69

ATOM 411 HD21 ASN 28 -0.620 -4.749 4.698 1.00 3.34

ATOM 412 HD22 ASN 28 -1.961 -5.829 4.749 1.00 4.01

ATOM 413 C ASN 28 1.730 -2.924 6.548 1.00 3.77

ATOM 414 O ASN 28 1.560 -2.065 7.416 1.00 3.98

ATOM 415 N ALA 29 2.500 -2.756 5.480 1.00 3.54

ATOM 416 HN ALA 29 2.541 -3.464 4.828 1.00 3.55

ATOM 417 CA ALA 29 3.237 -1.556 5.187 1.00 3.45

ATOM 418 HA ALA 29 2.593 -0.713 5.396 1.00 3.44

ATOM 419 CB ALA 29 4.495 -1.408 5.996 1.00 3.85

ATOM 420 HB1 ALA 29 4.945 -0.462 5.737 1.00 4.00

ATOM 421 HB2 ALA 29 5.173 -2.212 5.761 1.00 3.97

ATOM 422 HB3 ALA 29 4.255 -1.422 7.047 1.00 4.27

ATOM 423 C ALA 29 3.563 -1.564 3.712 1.00 3.33

ATOM 424 O ALA 29 4.201 -2.484 3.193 1.00 4.12

ATOM 425 N PHE 30 3.068 -0.553 3.063 1.00 2.67

ATOM 426 HN PHE 30 2.558 0.083 3.580 1.00 2.45

ATOM 427 CA PHE 30 3.209 -0.378 1.613 1.00 2.74

ATOM 428 HA PHE 30 2.894 0.626 1.371 1.00 2.83

ATOM 429 CB PHE 30 4.669 -0.566 1.171 1.00 3.35

ATOM 430 HB1 PHE 30 4.796 -0.143 0.185 1.00 3.67

ATOM 431 HB2 PHE 30 4.891 -1.622 1.133 1.00 3.59

ATOM 432 CG PHE 30 5.673 0.083 2.088 1.00 3.86

ATOM 433 CD1 PHE 30 5.441 1.346 2.615 1.00 4.35

ATOM 434 HD1 PHE 30 4.529 1.867 2.361 1.00 4.45

ATOM 435 CD2 PHE 30 6.847 -0.573 2.424 1.00 4.38

ATOM 436 HD2 PHE 30 7.040 -1.557 2.020 1.00 4.49

ATOM 437 CE1 PHE 30 6.360 1.938 3.459 1.00 5.11

ATOM 438 HE1 PHE 30 6.167 2.922 3.862 1.00 5.69 ATOM 439 CE2 PHE 30 7.769 0.016 3.266 1.00 5.15

ATOM 440 HE2 PHE 30 8.681 -0.505 3.520 1.00 5.77

ATOM 441 CZ PHE 30 7.525 1.273 3.785 1.00 5.42

ATOM 442 HZ PHE 30 8.245 1.735 4.444 1.00 6.14

ATOM 443 C PHE 30 2.287 -1.369 0.894 1.00 2.53

ATOM 444 O PHE 30 2.704 -2.085 -0.011 1.00 3.13

ATOM 445 N CYS 31 1.034 -1.406 1.362 1.00 2.06

ATOM 446 HN CYS 31 0.818 -0.818 2.101 1.00 2.12

ATOM 447 CA CYS 31 -0.017 -2.306 0.854 1.00 1.97

ATOM 448 HA CYS 31 0.224 -2.580 -0.153 1.00 2.16

ATOM 449 HB1 CYS 31 0.505 -4.333 1.205 1.00 2.78

ATOM 450 HB2 CYS 31 -1.059 -3.866 1.863 1.00 2.85

ATOM 451 C CYS 31 -1.410 -1.651 0.917 1.00 1.56

ATOM 452 O CYS 31 -1.944 -1.466 2.01 1 1.00 2.04

ATOM 453 CB CYS 31 -0.037 -3.553 1.710 1.00 2.51

ATOM 454 SG CYS 31 0.725 -3.308 3.341 1.00 2.99

ATOM 455 N TYR 32 -1.997 -1.284 -0.236 1.00 1.35

ATOM 456 HN TYR 32 -1.539 -1.446 -1.075 1.00 1.77

ATOM 457 CA TYR 32 -3.327 -0.635 -0.238 1.00 1.25

ATOM 458 HA TYR 32 -3.903 -1.138 0.503 1.00 1.48

ATOM 459 CB TYR 32 -3.228 0.842 0.167 1.00 1.68

ATOM 460 HB1 TYR 32 -4.003 1.065 0.885 1.00 1.99

ATOM 461 HB2 TYR 32 -3.365 1.461 -0.707 1.00 2.16

ATOM 462 CG TYR 32 -1.909 1.201 0.781 1.00 2.00

ATOM 463 CD1 TYR 32 -0.740 1.028 0.066 1.00 2.67

ATOM 464 HD1 TYR 32 -0.802 0.652 -0.942 1.00 3.10

ATOM 465 CD2 TYR 32 -1.829 1.678 2.069 1.00 2.45

ATOM 466 HD2 TYR 32 -2.743 1.831 2.638 1.00 2.72

ATOM 467 CE1 TYR 32 0.484 1.317 0.605 1.00 3.29

ATOM 468 HE1 TYR 32 1.382 1.166 0.001 1.00 3.99

ATOM 469 CE2 TYR 32 -0.612 1.976 2.629 1.00 3.14

ATOM 470 HE2 TYR 32 -0.576 2.335 3.635 1.00 3.79

ATOM 471 CZ TYR 32 0.550 1.794 1.900 1.00 3.39

ATOM 472 OH TYR 32 1.768 2.097 2.465 1.00 4.22

ATOM 473 HH TYR 32 1.978 3.019 2.301 1.00 4.50

ATOM 474 C TYR 32 -4.105 -0.726 -1.553 1.00 1.55

ATOM 475 O TYR 32 -5.142 -1.384 -1.621 1.00 2.20

ATOM 476 N CYS 33 -3.660 0.019 -2.571 1.00 2.04

ATOM 477 HN CYS 33 -2.872 0.584 -2.442 1.00 2.31

ATOM 478 CA CYS 33 -4.394 0.077 -3.831 1.00 2.83

ATOM 479 HA CYS 33 -5.173 -0.658 -3.776 1.00 3.09

ATOM 480 HB1 CYS 33 -5.053 1.928 -2.929 1.00 3.26

ATOM 481 HB2 CYS 33 -6.070 1.345 -4.241 1.00 3.20

ATOM 482 C CYS 33 -3.578 -0.177 -5.137 1.00 3.55

ATOM 483 O CYS 33 -4.167 -0.120 -6.219 1.00 4.43

ATOM 484 CB CYS 33 -5.055 1.457 -3.915 1.00 3.01

ATOM 485 SG CYS 33 -4.215 2.580 -5.069 1.00 3.40

ATOM 486 N ARG 34 -2.255 -0.404 -5.084 1.00 3.34

ATOM 487 HN ARG 34 -1.777 -0.399 -4.203 1.00 2.80 ATOM 488 CA ARG 34 -1.467 -0.586 -6.333 1.00 4.11

ATOM 489 HA ARG 34 -2.019 -0.101 -7.125 1.00 4.69

ATOM 490 CB ARG 34 -0.137 0.169 -6.148 1.00 4.21

ATOM 491 HB1 ARG 34 0.270 -0.122 -5.193 1.00 4.15

ATOM 492 HB2 ARG 34 -0.360 1.220 -6.109 1.00 4.16

ATOM 493 CG ARG 34 0.964 -0.004 -7.166 1.00 4.98

ATOM 494 HG1 ARG 34 0.969 0.843 -7.835 1.00 5.57

ATOM 495 HG2 ARG 34 0.809 -0.914 -7.721 1.00 5.02

ATOM 496 CD ARG 34 2.301 -0.067 -6.425 1.00 5.32

ATOM 497 HD1 ARG 34 2.654 -1.087 -6.429 1.00 5.23

ATOM 498 HD2 ARG 34 2.143 0.253 -5.385 1.00 5.43

ATOM 499 NE ARG 34 3.314 0.793 -7.034 1.00 6.08

ATOM 500 HE ARG 34 3.149 1.112 -7.946 1.00 6.18

ATOM 501 CZ ARG 34 4.441 1.162 -6.420 1.00 6.88

ATOM 502 NH1 ARG 34 4.701 0.747 -5.183 1.00 7.12

ATOM 503 HH11 ARG 34 4.052 0.155 -4.705 1.00 6.71

ATOM 504 HH12 ARG 34 5.546 1.028 -4.728 1.00 7.87

ATOM 505 NH2 ARG 34 5.313 1.944 -7.048 1.00 7.69

ATOM 506 HH21 ARG 34 5.125 2.257 -7.979 1.00 7.76

ATOM 507 HH22 ARG 34 6.157 2.222 -6.589 1.00 8.36

ATOM 508 C ARG 34 -1.287 -2.074 -6.731 1.00 4.36

ATOM 509 O ARG 34 -2.259 -2.732 -7.106 1.00 4.73

ATOM 510 N LYS 35 -0.057 -2.590 -6.668 1.00 4.53

ATOM 511 HN LYS 35 0.674 -2.028 -6.364 1.00 4.61

ATOM 512 CA LYS 35 0.255 -3.975 -7.032 1.00 4.93

ATOM 513 HA LYS 35 -0.509 -4.613 -6.607 1.00 5.03

ATOM 514 CB LYS 35 0.293 -4.170 -8.558 1.00 5.89

ATOM 515 HB1 LYS 35 -0.714 -4.346 -8.907 1.00 6.19

ATOM 516 HB2 LYS 35 0.895 -5.039 -8.780 1.00 6.25

ATOM 517 CG LYS 35 0.863 -2.989 -9.331 1.00 6.31

ATOM 518 HG1 LYS 35 0.552 -2.075 -8.852 1.00 6.46

ATOM 519 HG2 LYS 35 0.479 -3.014 -10.339 1.00 6.32

ATOM 520 CD LYS 35 2.384 -3.025 -9.387 1.00 6.93

ATOM 521 HD1 LYS 35 2.689 -3.525 -10.295 1.00 7.08

ATOM 522 HD2 LYS 35 2.755 -3.570 -8.532 1.00 7.20

ATOM 523 CE LYS 35 2.972 -1.623 -9.373 1.00 7.41

ATOM 524 HE1 LYS 35 3.647 -1.538 -8.533 1.00 7.65

ATOM 525 HE2 LYS 35 2.169 -0.911 -9.260 1.00 7.56

ATOM 526 NZ LYS 35 3.716 -1.314 -10.626 1.00 7.75

ATOM 527 HZ1 LYS 35 3.082 -1.396 -11.447 1.00 7.81

ATOM 528 HZ2 LYS 35 4.509 -1.975 -10.746 1.00 8.01

ATOM 529 HZ3 LYS 35 4.091 -0.344 -10.588 1.00 7.95

ATOM 530 C LYS 35 1.596 -4.335 -6.418 1.00 4.53

ATOM 531 O LYS 35 2.432 -3.453 -6.214 1.00 4.71

ATOM 532 N LEU 36 1.805 -5.602 -6.086 1.00 4.30

ATOM 533 HN LEU 36 1.130 -6.257 -6.243 1.00 4.47

ATOM 534 CA LEU 36 3.036 -5.989 -5.453 1.00 4.13

ATOM 535 HA LEU 36 3.354 -5.105 -4.916 1.00 3.77

ATOM 536 CB LEU 36 2.843 -7.055 -4.416 1.00 4.20

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END

Claims

CLAIMS What is claimed is:

1. A method of identifying a compound that modulates ligand binding to a melanocortin receptor, said method comprising: modeling test compounds that fit spatially into a melanocortin receptor ligand binding site of interest using an atomic structural model of a melanocortin receptor binding region or portion thereof; screening said test compounds in an assay characterized by binding of a test compound to a melanocortin receptor ligand binding site; and identifying a test compound that modulates ligand binding to said melanocortin receptor.

2. The method of Claim 1 wherein said melanocortin receptor binding region comprises the minimized agouti related protein receptor binding region or portion thereof.

3. The method of Claim 1 wherein said atomic structural model comprises atomic coordinates of amino acid residues corresponding to residues 1-18 of the N-terminal loop of the minimized agouti related protein (residues 1-18 of SEQ ID NO:2), residues 19-13 of the central loop of the minimized agouti related protein (residues 19-34 of SEQ ID NO:2), and residues 35- 46 of the C-terminal loop of the minimized agouti related protein (residues 35-46 of SEQ ID NO:2).

4. The method of Claim 1 wherein said atomic structural model comprises atomic coordinates of amino acid residues corresponding to residues 19-34 of the central loop of the minimized agouti related protein (residues 19-34 of SEQ ID NO:2) and at least residues 15-18 of the N-terminal loop of the minimized agouti related protein (residues 15-18 of SEQ ID NO:2).

5. The method of Claim 1 wherein said atomic structural model comprises atomic coordinates of amino acid residues corresponding to residues 19-34 of the central loop of the minimized agouti related protein (residues 19-34 of SEQ ID NO:2) and at least 20% of the contiguous or non-contiguous residues or their atoms are selected from residues 1-18 of the N- terminal loop of the minimized agouti related protein (residues 1-18 of SEQ ID NO:2).

6. The method of Claim 1 wherein said atomic structural model comprises atomic coordinates of amino acid residues corresponding to residues 24-31 of the active loop of the minimized agouti related protein (residues 24-31 of SEQ ID NO:2).

7. The method of Claim 1 wherein said atomic structural model comprises atomic coordinates of amino acid residues corresponding to residues 25-27 of the active loop of the minimized agouti related protein (residues 25-27 of SEQ ID NO:2).

8. The method of Claim 1 wherein said screening is in vitro.

9. The method of Claim 8 wherein said screening is high throughput screening.

10. The method of Claim 1 wherein said assay is a biological assay.

11. The method of Claim 1 wherein said test compound is from a library of compounds.

12. The method of Claim 1 wherein said test compound is an agonist or antagonist of ligand binding.

13. The method of Claim 12 wherein said test compound is a small organic molecule, a peptide, or peptidomimetic.

14. A method for identifying an agonist or antagonist of ligand binding to a melanocortin receptor, said method comprising the steps of: providing the atomic coordinates of a melanocortin receptor binding region or portion thereof to a computerized modeling system; modeling compounds which match or mimic the receptor binding region and thus fit spatially into the melanocortin receptor ligand binding site; and identifying in an assay for melanocortin receptor activity a compound that increases or decreases the activity of said melanocortin receptor by binding the ligand binding site of said melanocortin receptor, whereby an agonist or antagonist of ligand binding is identified.

15. The method of Claim 14 wherein said melanocortin receptor binding region comprises the minimized agouti related protein receptor binding region or portion thereof.

16. A machine-readable data storage medium, comprising a data storage material encoded with machine readable data which, when using a machine programmed with instructions for using said data, is capable of displaying a graphical three-dimensional representation of a molecule that binds a melanocortin receptor comprising structure coordinates of amino acid residues corresponding to residues 1-18 of the N-terminal loop of the minimized agouti related protein (residues 1-18 of SEQ ID NO:2), residues 19-13 of the central loop of the minimized agouti related protein (residues 19-34 of SEQ ID NO:2), and residues 35-46 of the C-terminal loop of the minimized agouti related protein (residues 35-46 of SEQ ID NO:2), or a homologue of said molecule.

17. The machine readable storage medium of Claim 16 wherein said molecule is a melanocortin receptor agonist.

18. The machine readable storage medium of Claim 16 wherein said molecule is a melanocortin receptor antagonist.

19. The machine-readable data storage medium according to Claim 16 wherein said molecule is defined by the set of structure coordinates depicted in Appendix 1 , or a homologue of said molecule, said homologue having a root mean square deviation from the backbone atoms of said amino acids of not more than 2.54A.

20. A machine-readable data storage medium comprising a data storage material encoded with a first set of machine readable data which, when combined with a second set of machine readable data, using a machine programmed with instructions for using said first set of data and said second set of data, can determine at least a portion of the structure coordinates corresponding to the second set of machine readable data, wherein: said first set of data comprises a Fourier transform of at least a portion of the structural coordinates selected from the group consisting of coordinates depicted in Appendix 1 ; and said second set of data comprises an X-ray diffraction pattern of a molecule.

21. An NMR structure of the minimized agouti related protein, embodied in a computer readable media.

22. A compound identified according to the method of Claim 1.

23. A polypeptide comprising the amino acid sequence:

CVRLHESCLGQQVPCCDPAATCYCRFFNAFCYC (SEQ ID NO:3) or a modified form thereof.

24. The polypeptide of Claim 23 wherein the polypeptide is chemically synthesized.

25. A method of treating a disease state in mammals that is alleviated by treatment with a polypeptide having an amino acid sequence:

CVRLHESCLGQQVPCCDPAATCYCRFFNAFCYC (SEQ ID NO:3) which method comprises administering to a mammal in need of such a treatment a therapeutically effective amount of said polypeptide, or a pharmaceutically acceptable salt thereof.

26. The method of Claim 25wherein said disease state is a wasting syndrome.

27. A pharmaceutical composition comprising a therapeutically effective amount of a polypeptide of the sequence: CVRLHESCLGQQVPCCDPAATCYCRFFNAFCYC (SEQ ID NO:3) or a pharmaceutically acceptable salt thereof.