WO2001085930A2 - Methods and compounds for modulating melanocortin receptor ligand binding and activity - Google Patents

Abstract

This invention relates to methods and agonist/antagonist compounds for modulating melanocortin receptor-ligand binding. 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, and the prodution of AGRP peptidomimetics that are melanocortin receptor ligands. The methods can be applied to other melanocortin receptor agonists and antagonists.

Description

METHODS AND COMPOUNDS FOR MODULATING MELANOCORTIN RECEPTOR LIGAND BINDING AND ACTIVITY

CROSS-REFERENCE TO RELATED APPLICATIONS

This claims benefit of and priority to U.S. provisional application no: 60/203,071, filed on May 9, 2000, and U.S. provisional application no: 60/226,047, filed on August 16, 2000, both of which are incorporated herein by reference, in their entirety, for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT This invention was made with Government support under Grant Nos.

GM46870, and DK58606 awarded by the National Institutes of Health. The Government of the United States of America may have certain rights in this invention.

FIELD OF THE INVENTION

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

BACKGROUND OF THE INVENTION

The melanocortin (MC) receptors are a group of cell surface proteins that mediate a variety of physiological effects, including: regulation of adrenal gland function, such as production of glucocorticoid, cortisol and aldosterone; control of melanocyte growth and pigment production; hemoregulation; immunomodulation; and analgesia. Five distinct MC receptors have been cloned and are expressed in a variety of tissues, including melanocytes, adrenal cortex, brain, gut, placenta, skeletal muscle, lung, spleen, thymus, bone marrow, pituitary, gonads and adipose tissue. Two MC eceptors, MC3R and MC4R, are expressed in brain tissue, while MClr is found in the skin. A variety of ligands termed melanocortins function as agonists that stimulate the activity of MC receptors. The melanocortins include melanocyte-stimulating hormones (MSH) such as alph-MSH, beta- MSH and gamma-MSH, as well as adrenocorticotropic hormone. Another MCR ligand that has been discovered is the Agouti Related Protein (AGRP), which is an antagonist. AGRP is a natural antagonist of melanocortin receptors 3 and 4 (MC3R and MC4R). Recent biochemical investigations have identified 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. (1997) Genes Dev. 11:593-602; Huszar et al. (1997) Cell 88:131-141; Hahn, et al. (1997) Nature Neurosci. 1:271-171).

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, (1995) Proc. Natl. Acad. Set USA 92:4728-4732; Michaud, et al. (1997) J. Endocrinol 155:207-209. 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. (1997) Biochem. Res. Commun. 237:629 611).

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 ( iefer, et al. (1997) Biochemistry 36:2084-2090; Kiefer, et al. (1998) Biochemistry 37: 991-997) and AGRP (Tota et al. (1999) Biochemistry 38:897- 904). Two recent investigations have shown that the chemically synthesized C-terminal region of AGRP competitively antagonizes -melanocyte stimulating hormone (α-MSH) at melanocortin receptors with equal or greater potency than the full proteins (Quillan, et al. (1998) FEBS Lett. 428:59-62; Yang, et al. (1999) Mol. EndocrinoL 13: 148-155.), consistent with similar findings for agouti (Willard, et al. (1995) Biochemistry 34:12341-12346). Thus the Cys-rich C-terminal region of AGRP, is referred to as minimized agouti related protein ("MARP", CVRLH₅ESCLG₁₀QQVPC₁₅CDPCA₂oTCYCR₂₅FFNA F₃₀CYCR K^LGTAM_MNPCSR^T, SEQ ID NO:2, where subscripts denote amino acid position). The covalent structure of MARP exhibits five disulfide bonds, which exist between the following ten Cys residues (Bures, et al. (1988) Biochemistry 2>1 ^r: 12172- 12177): Cysi and Cys₁₆; Cys₈ and Cys₂₂; Cys₁₅ and Cys₃₃; Cys^ and Cys ₃; 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. (1988) Biochemistry 37:991-997; 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. (1998) Toxicon 36:1573-1583) 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, it is believed that 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. (1998) Biochemistry 37:16041-16052).

U.S. Patents of interest in this area include: 5,994,087; 5,932,779; 5,869,257; 5,843,652; 5,817,787; 5,766,877; 5,731,408; 5,703,220; and 5,622,860. Also of interest are: WO 99/64002; WO 99/57148; WO 99/55679; WO 99/55832; WO 99/54358; WO 99/50295; WO 99/43709; WO 99/31508; WO 99/21571; WO 98/56914; WO 98/10068; and WO 97/43412. SUMMARY OF THE INVENTION

This invention provides a detailed description of the three-dimensional (3D) structure (NMR structure) in solution of the human AGRP Cys-rich C-terminal region. In addition, further minimization of human AGRP is achieved and a minimal sequence (designated herein as MARP-33) is identified that provides both activity and receptor specificity against 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 and/or by activating the receptor themselves. In particular, using information provided herein, a class of peptidomimeti.es (e.g. non-peptide ligands) are provided that are ligands for the melanocortin receptor and that can block and/or modulate activity of the melanocortin receptors.

In one preferred embodiment, the peptides this emmbodiments provides a melanocortin receptor ligand that is a polypeptide comprising a peptide sequence having the formula: CX¹X²X³X⁴X⁵X⁶CX⁷X⁸X⁹X¹⁰X^πX¹²CCDPX¹³ATCYCX¹⁴X¹⁵X¹⁶NAFCYCR_n (Formula I), where X¹, X², X³, X⁴, X⁵, X⁶, X⁷, X⁸, X⁹, X¹⁰, X¹¹, X¹², X¹³, X¹⁴, X¹⁵, and X¹⁶ are independently selected amino acids, and n is zero or one. The amino acids include native amino acids, both D- and L-form amino acids, and modified or derivatized amino acids or amino acid analogues. In certain particularly preferred embodiments, X¹, X², X³, X⁴, X⁵, X⁶, X⁷, X⁸, X⁹, X¹⁰, X¹¹, X¹², X¹³, X¹⁴, X¹⁵, and X¹⁶ are independently selected from the group consisting of alanine, asparagine, arginine,aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In certain embodimetns, the polypeptide consists of a polypeptide having the formula shown above (Formula I). In certain preferred embodiments, the peptide is not AGRP and said polypeptide is not MARP. In certain preferred embodiments, the polypeptide excludes one or more of the final 13 residues of MARP (residues 34-46 of MARP). In certain embodiments, the polypeptide is not CVRLHESCLGQQVPCCDPAATCYCRFFNAFCYC (SEQ ID NO:3) or does not comprise this sequence.

In certain embodiments, X^^X^X⁶ is VRLHES, or conservative substitutions thereof, and/or X⁷X⁸X⁹X¹⁰X^πX¹² is LGQQVP, or conservative substitutions thereof, and/or X¹⁴X¹⁵X¹⁶ is RFF or conservative substitutions thereof. In certain embodiments, X is not a cysteine and in particularly preferred embodiments, X is A.

In addition to the peptide melanocortin ligands described above, this invention provides a non-peptide melanocortin receptor ligand of the structural formula of Formula I shown herein in which B, Ui, U₂, R, Ri and R₂ are independently selected from the group consisting of: hydrogen, alkyl, derivatized alkyl, cycloalkyl, derivatized cycloalkyl, halocycloalkyl, aloxycycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl, and heteroarylalkyl; J is selected from the group consisting of carbon, nitrogen, silicon, and sulfur; X is selected from the group consisting of hydrogen, carbon, nitrogen, oxygen, silicon, and sulfur; and Z is selected from the group consisting of a continuing peptide bond, a hydroxyl; -NH₂-, -NH-(n), and -N-(n,n'), and -O-(y), where where n and n' are independently selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, or a derivatized form thereof, and y is selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, aryl, arylalkyl, heteroaryl heteroarylalkyl, or a derivatized form thereof. In preferred embodiments, the ligand is a ligand for a MC3r and/or an MC4r melanocortin receptor. Particularly preferred non- peptide ligands have a molecular weight ranging from about 200 to 1000 daltons. In preferred embodiments, the ligand has a structure that mimics the backbone of the AGRP active loop. Preferred ligands comprise a terminal gaunidino moiety and or at least one methylbenzyl moiety. Particularly preferred ligands include the ligands of Formulas III, IV, V, and VI, shown herein.

In another embodiment, this invention also provides libraries for screening for modulators of a melanocortin receptor. Preferred libraries comprise a plurality of polypeptide and/or non-polypeptide members that are ligands (e.g. as described above) that bind to a melanocortin receptor (e.g. MC3r, MC4r, etc.). Preferred libraries comprise at least 10, more preferably at least 100, and most preferably at least 1000 different members. In certain embodiments, the libraries are provided in multi-well plates.

Also provided are pharmaceutical compositions comprising one or more of the melanocortin-binding peptide and/or non-peptide ligands of this invention (e.g. as described above). The pharmaceutical compositions optionally further comprise a pharmaceutically acceptable excipient.

In certain embodiments, this invention provides methods of modulating the activity of a melanocortin receptor (e.g. MC3r, MC4r, etc.) and/or methods of modulating a melanocortin receptor mediated physiological process. The methods involve contacting the receptor with a peptide ligand and or with a non-peptide ligand of this invention (e.g. as described above). In preferred embodiments, the contacting blockage of the receptor site and/or upregulation or downregulation of melanocortin receptor activity (e.g. as measured by cAMP assays). In certain embodiments the ligand is a melanocortin agonist. In certain embodiments, the ligand is a melanocortin receptor antagonist.

This invention also provides methods of prescreening for a modulator of a melanocortin receptor (e.g. MC3r, MC4r, etc.). The methods involve contacting a melanocortin receptor with one or more of the melanocortin receptor peptide ligands and/or non-peptide ligands of this invention (e.g. the ligands described above), and detecting binding of the peptide to the melanocortin receptor wherein specific binding of the peptide to the melanocortin receptor indicates that said peptide is a potential modulator of the melanocortin receptor.

In still another embodiment, this invention provides methods of screening for a modulator of melanocortin receptor activity. The methods involve contacting a melanocortin receptor (e.g. MC3r, MC4r, etc.) with a peptide ligand or a non-peptide ligand of this invention and detecting activity of the melanocortin receptor wherein a difference in activity of the receptor, as compared to a control, indicates that the ligand is a modulator of melanocortin receptor activity. In preferred embodiments, the control is a negative control comprising the same assay without the ligand.

This invention also provides a method of identifying a compound that modulates ligand binding to a melanocortin receptor, where the method involves 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 the 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. In preferred embodiments, the melanocortin receptor binding region comprises the minimized agouti related protein receptor binding region (mini- AGRP) or a portion thereof. In preferred embodiments, the 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). In certain preferred embodiments, the 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). In certain preferred embodiments, the 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 noncontiguous 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). The atomic structural model can 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). In certain embodiments, the 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). The screening is preferably in vitro. In certain embodiments, the screening is high throughput screening (HTS). Preferred assays include, but are not limited to biological assays. The test compound can be provided from a library of compounds and preferred test compounds are agonists or antagonists of ligand binding. Particularly preferred test compounds include a small organic molecule, a peptide, or peptidomimetic.

In another embodiment this invention provides methods 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. In preferred embodiments, the melanocortin receptor binding region comprises the minimized agouti related protein receptor binding region or portion thereof.

Also provided is 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. In certain embodiments, the molecule is a melanocortin receptor agonist or antagonist. In particularly preferred embodiments, the molecule is defined by the set of structure coordinates depicted in Table 4 or Table 5, or a homologue of the molecule, the homologue having a root mean square deviation from the backbone atoms of said amino acids of not more than 10, preferably no more than 5 and most preferably no more than 2.54A.

A machine-readable data storage medium is also provided that comprises a data storage material encoded with a first set of machine readable data that, 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 the 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 Table 4 or Table 5; and the second set of data comprises an X-ray diffraction pattern of a molecule.

In still another embodiment this invention provides an NMR structure of a minimized agouti related protein (mini- AGRP), embodied in a computer readable media.

This invention also provides a polypeptide comprising the amino acid sequence: CVRLHESCLGQQVPCCDPAATCYCRFFNAFCYC (SEQ ID NO:3) or a modified form thereof, where the polypeptide is not a full-length AGRP and the polypeptide is not a MARP. Preferred such polypeptides are chemically synthesized.

Also provided is a method of treating a disease state in mammals that is alleviated by treatment with a polypeptide having an amino acid sequence: CVRLHESCLGQQVP CCDPAATCYCRFFNAFCYC (SEQ ID NO:3) where the 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. In certain embodiments, the disease state is a wasting syndrome. This invention also includes a pharmaceutical composition comprising a therapeutically effective amount of a polypeptide of the sequence: CVRLHESCLGQQVPC CDPAATCYCRFFNAFCYC (SEQ ID NO:3) or a pharmaceutically acceptable salt thereof.

DEFINITIONS

The terms "residue" or "amino acid" as used herein refers to natural, synthetic, or modified amino acids (amino acid analogues). Such amino acids include both "D" and "L" forms. Various amino acid analogues include, but are not limited to, 2- aminoadipic acid, 3-aminoadipic acid, beta-alanine, beta-aminopropionic acid, 2- aminobutyric acid, 4-aminobutyric acid, piperidinic acid, 6-aminocaproic acid, 2- aminoheptanoic acid, 2-aminoisobutyric acid, 3-aminoisobutyric acid, 2-aminopimelic acid, 2,4- diaminobutyric acid, desmosine, 2,2'-diaminopimelic acid, 2,3-diaminopropionic acid, N-ethylglycine, N-ethylasparagine, hydroxylysine, allo-hydroxylysine, 3-hydroxyproline, 4- hydroxyproline, isodesmosine, allo-isoleucine, N-methylglycine, sarcosine, N- methylisoleucine, 6-N-methyllysine, norvaline, norleucine, ornithine, etc.

The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. The term also includes variants on the traditional peptide linkage joining the amino acids making up the polypeptide. The term "peptidomimetic" as used herein refers to a molecule that structurally and chemically resembles a peptide of two or more amino acids with respect to the features critical for a particular desired activity (e.g. receptor specificity, activity, etc.). The term "peptidomimetic" is includes peptide analogs that serve as appropriate substitutes for peptides in interactions with e.g., receptors and enzymes. Peptidomimetics can be "peptides" incorporating modified residues and/or backbones, e.g. that may have improved pharmacokinetic properties as a result of proteolytic stability, or unique structural and/or hydrogen bonding motifs, etc. Peptidomimetics also include organic molecules that are capapble of mimicing one more properties (e.g. binding specificity, affinity, etc.) of a peptide. Such organic molecule typically comprise a "scaffold" that mimics part or all of the 3 -dimensional structure of the subject peptide and places appropriate functional groups in a spatial orientation sufficient to achieve the desired functional properties. Particularly preferred peptidomimetics include small organic molecules. The term "small organic molecule" refers to a molecule of a size comparable to those organic molecules generally used in pharmaceuticals. The term excludes biological macromolecules (e.g., proteins, nucleic acids, etc.). Preferred small organic molecules range in size up to about 5000 Da, more preferably up to 2000 Da, and most preferably up to about 1000 Da.

The term database refers to a means for recording and retrieving information. In preferred embodiments the database also provides means for sorting and/or searching the stored information. The database can comprise any convenient media including, but not limited to, paper systems, card systems, mechanical systems, electronic systems, optical systems, magnetic systems or combinations thereof. Preferred databases include electronic (e.g. computer-based) databases. Computer systems for use in storage and manipulation of databases are well known to those of skill in the art and include, but are not limited to "personal computer systems", mainframe systems, distributed nodes on an inter- or intranet, data or databases stored in specialized hardware (e.g. in microchips), and the like. The term "conservative substitution" is used in reference to proteins, peptides, etc. to reflect amino acid substitutions that do not substantially alter the activity (specificity or binding affinity) of the molecule. Typically, conservative amino acid substitutions involve substitution of one amino acid for another amino acid with similar chemical properties (e.g. charge or hydrophobicity). The following six groups each contain amino acids that are typical conservative substitutions for one another: 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

The terms "binding partner", or "capture agent", or a member of a "binding pair" refers to molecules that specifically bind other molecules to form a binding complex such as antibody-antigen, lectin-carbohydrate, nucleic acid-nucleic acid, biotin-avidin, etc.

The term "specifically binds", as used herein, when referring to a biomolecule (e.g., protein, nucleic acid, antibody, receptor, etc.), refers to a binding reaction which is determinative of the presence biomolecule in heterogeneous population of molecules (e.g., proteins and other biologies). Thus, under designated conditions (e.g. imrnunoassay conditions in the case of an antibody or stringent hybridization conditions in the case of a nucleic acid), the specified ligand or antibody binds to its particular "target" molecule and does not bind in a significant amount to other molecules present in the sample.

"Alkyl" means a linear saturated monovalent hydrocarbon radical of one to six carbon atoms or a branched saturated monovalent hydrocarbon radical of three to six carbon atoms, e.g., methyl, ethyl, propyl, 2-propyl, butyl, pentyl, and the like.

"Alkenyl" means a linear monovalent hydrocarbon radical of two to six carbon atoms or a branched monovalent hydrocarbon radical of three to six carbon atoms containing at least one double bond, e.g., ethenyl, 2-propenyl, and the like.

"Alkynyl" means a linear monovalent hydrocarbon radical of two to six carbon atoms or a branched monovalent hydrocarbon radical of three to six carbon atoms containing at least one triple bond, e.g., ethynyl, propynyl, butynyl, and the like.

"Cycloalkyl" means a cyclic saturated monovalent hydrocarbon radical of three to seven carbon atoms, e.g., cyclopropyl, cyclohexyl, and the like.

"Halo" means fluoro, chloro, bromo, and iodo. "Haloalkyl" means alkyl substituted with one or more halogen atoms, including those substituted with different halogens, e. .,-CH₂Cl, ~CF₃, ~CH₂CF₃, ~ CF₂CF₃, ~CH₂CC1₃, and the like.

"Alkoxy", "alkenyloxy", "cycloalkyloxy", or "haloalkyloxy" means a radical -OR where R is alkyl, alkenyl, cycloalkyl, or haloalkyl respectively as defined above-, e.g., methoxy, ethoxy, propoxy, 2-propoxy, ethenyloxy, cyclopropyloxy, cyclobutyloxy, ~OCH₂ Cl, -OCF₃, and the like.

"Alkylthio" or "cycloalkylthio" means a radical --SR where R is alkyl or cycloalkyl respectively as defined above, e.g., methylthio, butylthio, cyclopropylthio, and the like. "Acyl" means a radical ~C(O)R where R is hydrogen, alkyl, or haloalkyl as defined above, e.g., formyl, acetyl, trifluoroacetyl, butanoyl, and the like.

"Amino" means a radical ~NH₂

"Monosubstituted amino" means a radical --NHR where R is alkyl or acyl, e.g., methylamino, (l-methylethyl)amino, and the like. "Disubstituted amino" means a radical — NRR' where R and R' are independently alkyl or acyl, e.g.,dimethylamino, methylethylamino, di(l- methylethyl)amino, and the like. "Hydroxyalkyl" means a linear monovalent hydrocarbon radical of two to six carbon atoms or a branched monovalent hydrocarbon radical of three to six carbons substituted with one or two hydroxy groups, provided that if two hydroxy groups are present they are not both on the same carbon atom. Representative examples include, but are not limited to, 2-hydroxyethyl, 2-hydroxypropyl, 3-hydroxypropyl, l-(hydroxymethyl)- 2-methylpropyl, 2-hydroxybutyl, 3-hydroxybutyl, 4hydroxybutyl, 2,3-dihydroxypropyl, 1- (hydroxymethyl)-2-hydroxyethyl, 2,3-dihydroxybutyl, 3,4dihydroxybutyl and 2- (hydroxymethyl)-3-hydroxypropyl, 2-hydroxyethyl, 2,3-dihydroxypropyl, and 1- (hydroxymethyl)-2-hydroxyethyl. "Alkoxyalkyl" means a linear monovalent hydrocarbon radical of one to six carbon atoms or a branched monovalent hydrocarbon radical of three to six carbons substituted with at least one alkoxy group as defined above, e.g., 2-methoxyethyl, 2- methoxypropyl, and the like.

"Hydroxyalkyloxy" or "alkoxyalkyloxy" means a radical-OR where R is hydroxyalkyl or alkoxyalkyl respectively as defined above, e.g., 2-hydroxyethyloxy, 2- methoxyethyloxy, and the like.

"Aminoalkyl" means a linear monovalent hydrocarbon radical of two to six carbon atoms or a branched monovalent hydrocarbon radical of three to six carbons substituted with at least one — NRR' where R and R' are independently selected from hydrogen, alkyl, or acyl, e.g., 2-aminoethyl, 2-N,N-diethylaminopropyl, 2-N- acetylaminoethyl, and the like.

"Aryl" means a monovalent monocyclic or bicyclic aromatic hydrocarbon radical of 6 to 12 ring atoms, and optionally substituted independently with one or more substituents selected from alkyl, haloalkyl, cycloalkyl, alkoxy, alkylthio, halo, nitro, acyl, cyano, amino, monosubstituted amino, disubstituted amino, -hydroxy, carboxy, or alkoxycarbonyl. Representative examples include, but are not limited to, phenyl, biphenyl, 1-naphthyl, and 2-naphthyl and the derivatives thereof.

"Heteroaryl" means a monovalent monocyclic or bicyclic aromatic radical of 5 to 10 ring atoms containing one or more, sometimes one or two ring heteroatoms selected from N, O, or S, the remaining ring atoms being C. The heteroaryl ring is optionally substituted independently with one or more substituents, sometimes one or two substituents, selected from alkyl, haloalkyl, cycloalkyl, alkoxy, alkylthio, halo, nitro, acyl, cyano, amino, monosubstituted amino, disubstituted amino, hydroxy, carboxy, or alkoxycarbonyl. Specifically the term heteroaryl includes, but is not limited to, pyridyl, pyrrolyl, thienyl, furanyl, indolyl, quinolyl, benzopyranyl, and thiazolyl, and the derivatives thereof.

"Heterocycloamino" means a saturated monovalent cyclic group of 3 to 8 ring atoms, wherein at least one ring atom is N and optionally contains a second ring heteroatom selected from the group consisting of N, O, or S(O) _n (where n is an integer from 0 to 2), the remaining ring atoms being C. The heterocycloamino ring may be optionally fused to a benzene ring or it may be optionally substituted independently with one or more substituents, sometimes one or two substituents, selected from alkyl, haloalkyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl, heteroaryl, heteroaralkyl, halo, cyano, acyl, amino, monosubstituted amino, disubstituted amino, carboxy, or alkoxycarbonyl. More specifically the term heterocycloamino includes, but is not limited to, pyrrohdino, piperidino, morpholino, piperazino, indolino, and thiomorpholino, and the derivatives thereof.

"Heterocyclo" means a saturated monovalent cyclic group of 3 to 8 ring atoms in which one or two ring atoms are heteroatoms selected from N, O, or S(O)_n, where n is an integer from 0 to 2, the remaining ring atoms being C. The heterocyclo ring may be optionally fused to a benzene ring or it may be optionally substituted independently with one or more substituents, sometimes one or two substituents, selected from alkyl, haloalkyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl, heteroaralkyl, halo, cyano, acyl, monosubstituted amino, disubstituted amino, carboxy, or alkoxycarbonyl. More specifically the term heterocyclo includes, but is not limited to, pyrrolidino, piperidino, morpholino, piperazino, tetrahydropyranyl, and thiomorpholino, and the derivatives thereof.

"Cycloalkylalkyl" means a radical -R^aR^b where R^a is an alkylene group and R^b is a cycloalkyl group as defined above e.g., cyclopropylmethyl, cyclohexylpropyl, 3- cyclohexyl-2-methylpropyl, and the like. "Cycloalkylalkyloxy" means a radical —OR where R is a cycloalkylalkyl group as defined above e.g., cyclopropylmethyloxy, 3-cyclohexylpropyloxy, and the like. "Aralkyl" means a radical - R^aR ^b where R^a is an alkylene group and R ^b is an aryl group as defined above e.g., benzyl, phenylethyl, 3-(3-chlorophenyl)-2- methylpentyl, and the like. "Heteroaralkyl" means a radical - R^aR^b where R^a is an alkylene group and R^b is a heteroaryl group as defined above e.g., 2-,3-, or 4-pyridylmethyl, furan-2-ylmethyl and the like. "Heterocycloalkyl" means a radical - R^aR^b where R^a is an alkylene group and R^b is a heterocyclo group as defined above e.g., morpholin-4-ylethyl, tetrahydrofuran-2- ylmethyl and the like.

A "pro-drug" is a compound that releases an active drug (e.g. a mini- AGRP, a peptidomimetic as described herein) when such prodrug is administered to a mammalian subject. In certain embodiments, prodrugs are prepared by modifying functional groups present in the peptidomimetics of this invention or by modifyng mini- AGRP polypeptides in such a way that the modifications may be cleaved in vivo to release the active compound. Peptidomimetic prodrugs include the peptideomimetics described herein wherein a hydroxy, amino, or sulfhydryl group in compound is bonded to any group that may be cleaved in vivo to regenerate the free hydroxyl, amino, or sulfhydryl group, respectively. Examples of prodrugs include, but are not limited to esters (e.g., acetate, formate, and benzoate derivatives), carbamates (e.g., N,N-dimethylaminocarbonyl) of hydroxy functional groups in compounds of formula (I), and the like. "Optional" or "optionally" means that the subsequently described event or circumstance may, but need not, occur, and that the description includes instances where the event or circumstance occurs and instances in which it does not. For example, "heterocyclo group optionally mono- or di- substituted with an alkyl group" means that the alkyl may, but need not, be present, and the description includes situations where the heterocyclo group is mono- or disubstituted with an alkyl group and situations where the heterocyclo group is not substituted with the alkyl group.

Compounds that have the same molecular formula but differ in the nature or sequence of bonding of their atoms or the arrangement of their atoms in space are termed "isomers." Isomers that differ in the arrangement of their atoms in space are termed "stereoisomers." Stereoisomers that are not mirror images of one another are termed "diastereomers" and those that are non-superimposable mirror images of each other are termed "enantiomers." When a compound has an asymmetric center, for example, it is bonded to four different groups, a pair of enantiomers is possible. An enantiomer can be characterized by the absolute configuration of its asymmetric center and is described by the R- and S-sequencing rules of Cahn and Prelog, or by the manner in which the molecule rotates the plane of polarized light and designated as dextrorotatory or levorotatory (i.e., as (+) or (-)-isomers respectively). A chiral compound can exist as either individual enantiomer or as a mixture thereof. A mixture containing equal proportions of the enantiomers is called a "racemic mixture."

The compounds of this invention may possess one or more asymmetric centers; such compounds can therefore be produced as individual (R)- or (S)- stereoisomers or as mixtures thereof. Unless indicated otherwise, the description or naming of a particular compound in the specification and claims is intended to include both individual enantiomers and mixtures, racemic or otherwise, thereof. The methods for the determination of stereochemistry and the separation of stereoisomers are well-known in the art (see discussion in Chapter 4 of "Advanced Organic Chemistry", 4th edition J. March, John Wiley and Sons, New York, 1992).

A "pharmaceutically acceptable excipient" means an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic and neither biologically nor otherwise undesirable, and includes an excipient that is acceptable for veterinary use as well as human pharmaceutical use. "A pharmaceutically acceptable excipient" as used in the specification and claims includes both one and more than one such excipient.

A "pharmaceutically acceptable salt" of a compound means a salt that is pharmaceutically acceptable and that possesses the desired pharmacological activity of the parent compound. Such salts include: (1) acid addition salts, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or formed with organic acids such as acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4- hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4- toluenesulfonic acid, camphorsulfonic acid, 4methylbicyclo>2.2.2!oct-2-ene-l-carboxylic acid, glucoheptonic acid, 4,4'-methylenebis-(3-hydroxy-2-ene-l-carboxylic acid), 3- phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, and the like; or (2) salts formed when an acidic proton present in the parent compound either is replaced by a metal ion, e.g., an alkali metal ion, an alkaline earth ion, or an aluminum ion; or coordinates with an organic base such as ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, and the like.

"Treating" or "treatment" of a disease includes: (1) preventing the disease, i.e. causing the clinical symptoms of the disease not to develop in a mammal that may be exposed to or predisposed to the disease but does not yet experience or display symptoms of the disease, (2) inhibiting the disease, i.e., arresting or reducing the development of the disease or its clinical symptoms, or (3) relieving the disease, i.e., causing regression of the disease or its clinical symptoms.

A "therapeutically effective amount" means the amount of a compound that, when administered to a mammal for treating a disease, is sufficient to effect such treatment for the disease. The "therapeutically effective amount" will vary depending on the compound, the disease and its severity and the age, weight, etc., of the mammal to be treated.

The term database refers to a means for recording and retrieving information. In preferred embodiments the database also provides means for sorting and/or searching the stored information. The database can comprise any convenient media including, but not limited to, paper systems, card systems, mechanical systems, electronic systems, optical systems, magnetic systems or combinations thereof. Preferred databases include electronic (e.g. computer-based) databases. Computer systems for use in storage and manipulation of databases are well known to those of skill in the art and include, but are not limited to

"personal computer systems", mainframe systems, distributed nodes on an inter- or intranet, data or databases stored in specialized hardware (e.g. in microchips), and the like

BRIEF DESCRIPTION OF THE DRAWINGS

Figures 1 A and IB show the Cα backbone of the MARP minized average structure determined at 500 mHz and 800 mHz, respectively. Figure 1 A: 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. Figure IB shows a C(alpha) backbone representation of the lowest energy AGRP(87-132) structure of the family of 40 determined from 800 MHz NOESY data and deposited in the PDB (code: IHYK). The N-terminal loop is show in light grey at the lower left,the central loop is dark grey (top middle), and the C- terminal loop is shown at the lower right. The disulfide bonds are included as thin dashed white lines. The spheres highlight residues the amides of which were protected from HX: medium gray > 12 hours, light gray > 24 hours and dark gray > 8 days.

Figure 2A 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. Figure 2B shows an all atom backbone representation of AGRP(87-132) for the 40 structures deposited in the PDB calculated from 800 MHz NOESY data. Only the backbone atoms of 1-34 have been aligned (RMSD = 0.34 angstroms). The disulfide-bound residues are indicated with straight lines interconnecting regions of the backbone, and are included for all members of the family.

Figure 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. Figure 4 illustrates a reaction scheme for the synthesis of peptidomimetics of this invention.

DETAILED DESCRIPTION

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.

In one embodiment, this invention provides a detailed three-dimensional (3D) NMR structure in solution of the human AGRP Cys-rich C-terminal region as determined by 1H 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 was not sufficient to correctly determine the 3D fold of the molecule. It was initially thought, based on 500 mHz data that MARP did not adopt an ICK-like fold, however, new data obtained at 800 mHZ (Table 5 herein) shows that MARP does adopt the ICK-like fold.

The 3D structure of MARP provided herein 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. The 3D structure presents precise structural information that permits the rational design of compounds that preferentially modulate MC4r or MC3r activity.

Based on an analysis of this structure, a minimal AGRP domain is identified (designated as a mini- AGRP) that shows both melanocortin activity and melanocortin specificity. Residues are identified that can be systematically or randomly altered to produce a large number of mini-AGRPs having differing receptor specificity and/or binding affinity.

Collections of such mini-AGRPs provide convenient libraries that can be screened to identify mini-AGRPs having particular activity/specificity profiles. THese AGRPs can be used to modulate melanocortin receptor activity (in vivo or in vitro) or they can be used as "lead compounds" for the design of peptido mimetics.

In addition a class of peptidomimetics is identified herein that are specific ligands to melanocortin receptors (e.g. MC3r and/or MC4r). In certain embodiments, the peptidomimetics are used to modulate melanocortin receptor activity in vivo or in vitro. In other embodiments, they are provided as libraries that can be screened for peptidomimetics having particular desired specificity/activity profiles.

I. Structure of MARP

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 structure. The 800 mHz data reveal a small beta sheet structure. The RFF triplet is critical for the activity of MARP as a competitive antagonist of -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. (1999) Biochemistry 38:897-904). Thus, the detailed fold of the central loop and perhaps the presence of the N- and C-terminal loops are important for AGRP function. In addition, based upon work with chimeras of melanocortin receptors, we believe the N- and C-terminal loops 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:E192-196 (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 determined at 500 mHz, suggested that this protein did 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. However, based on the 800 mHz NMR data, we have not assigned MARP to the ICK class.

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 noncontiguous 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).

II. Structure of mini-AGRP Another aspect of the invention also pertains to the identification of

"minimal" polypeptides (mini-AGRPs) that show specific binding and/or activity at melanocortin receptors. show. Certain preferred mini-AGRPs are strong antagonists of melanocortin receptor types 3 and 4, and are useful for antagonizing melanocortin receptor in a variety of contexts, e.g. for the treatment of eating disorders and obesity. As indicated 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, indicated that the final 13 residues of MARP are unstructured are unlikely to play a significant role in receptor binding or antagonism. Thus, substantial further minimization of human AGRP was achieved. Specifically, the sequence (indicated by the single letter amino acid code):

CVRLHsESCLG^QQVPCisCDPAAaoTCYCR^FFNAFgoCYC (SEQ ID NO:3) designated "MARP-33" (based upon its 33 amino acid length) is believed to fold with proper nativelike disulfide bonds and possesses the full biological activity of MARP. This sequence is equivalent to the first 33 amino acids of MARP but contain a Cys to Ala substitution at position 19 (indicated as "A") to avoid having a non-bridged thiol. Another mini-AGRP was made having the sequence: CVRLHESCLGQQVPCCDPAATCYCRFFNAFCYCR (SEQ ID NO:4). This sequence is equivalent to the first 34 amino acids of MARP, but contain a Cys to Ala substitution at position 19 (indicated as "A") to avoid having a non-bridged thiol. The additional terminal residue (R) facilitates solid state synthesis. This mini-AGRP was shown to specifically bind an antagonize melanocortin receptors. In addition, the mini-ARGPs can be modified to improve binding specificity, and/or to alter activity (e.g. agonist, antagonist, compeitive inhibitor, etc.). Such modifications include, but are not limited to changing the sequence to increase binding affinity, to increase the level of antagonism and to increase stability of the molecule. In particularly preferred embodiments, the amino acid substitutions are made in the RFF residues (residues 25-27 of SEQ ID NO:3) and/or in the N-terminal loop (e.g. one or more of residues 2-7 and/or residues 9-14 of SEQ ID NO:3).

In particularly preferred embodiments, mini-ARGPs are represented by formula I:

CX¹X²X³X⁴X⁵X⁶CX⁷X⁸X⁹X¹⁰X¹¹X¹²CCDPX¹³ATCYCX¹⁴X¹⁵X¹⁶NAFCYCR_n r

where X¹, X², X³, X⁴, X⁵, X⁶, X⁷, X⁸, X⁹, X¹⁰, X¹¹, X¹², X¹³, X¹⁴, X¹⁵, and X¹⁶ are independently selected amino acids (including natural, synthetic, or modified amino acids); and n is zero or one. In certain embodiments, in each of the varied domains, one or more of the native residues can be preserved. Thus, for example, X^^X^X⁶ includes, but is not limited to VX²X³X⁴X⁵X⁶, X¹RX³X⁴X⁵X⁶, X^LX⁴^⁶, X^^FD^X⁶, X^!X²X³X⁴X⁵S, VRX³X⁴X⁵X⁶, VX²LX⁴X⁵X⁶, VX²X³HX⁵X⁶, VX²X³X⁴EX⁶, VX²X³X⁴X⁵S, ^RLX^X⁶, X¹RX³HX⁵X⁶, X¹RX³X⁴EX⁶, X^!RX³X⁴X⁵S, X^!X²LHX⁵X⁶, X'xlxW, X^lX²LX⁴EX⁶, X^LX^S, X^^HEX⁶, X¹X²X³HX⁵S, X^lX²X³X⁴ES, VRLX⁴X⁵X⁶, VX²LHX⁵X⁶, VRLHES and the like. Similar permutations are available for

X⁷X⁸X⁹X¹⁰X^πX¹² (e.g. LGQQVP, LX⁸X⁹X¹⁰X^πX¹², X⁷GX⁹X¹⁰XⁿX¹², X⁷X⁸QX¹⁰X^πX¹², X⁷X⁸X⁹QX^πX¹², X⁷X⁸X⁹X¹⁰VX¹², X⁷X⁸X⁹X¹⁰XⁿP, LGX⁹X¹⁰X^πX¹², LX⁸QX¹⁰X^πX¹², LX⁸X⁹QX^πX¹², LX⁸X⁹X¹⁰VX¹², LX⁸X⁹X¹⁰X^πP, LGQX¹⁰X^πX¹², and the like).

Similarly, the "RFF" domain can be fully mutated or can retain one or more of the native residues. Thus, for example, X¹⁴X¹⁵X¹⁶ includes RFF, R¹⁵X¹⁵X¹⁶, X¹⁴FX¹⁶, X¹⁴X¹⁵F, RFX¹⁶, RX¹⁵F, X¹⁵FF. In certain preferred embodiments, X¹³ is not cysteine. The mini-AGRPs form a class of melanocortin receptor binding and/or modulating agents and thus find utility as modulators (e.g. upregulators, downregulators, competitive inhibitors) of melanocortin receptors, particular MC3r and/or MC4r. The compounds can be used individually or combined into a library suitable for screening for members having particular activities, binding affinities, and the like.

Certain mini-AGRPs are good potential therapeutics for the treatment of eating disorders and obesity, or can be used as therapeutic lead compounds for the development of therapeutics (e.g. as models for peptidomimetics, etc.). Where the compounds are used as therapetics, they can be administered to a patient (human or non- human mammal) in need thereof, to increase eating and fat deposition.

III. Preparation of mini-AGRP's and mini-AGRP libraries.

In certain embodiments, this invention provides libraries of mini-AGRPs. The libraries typically comprise a plurality of

Preferred libraries comprise at least 20, preferably at least 50, more preferably at least 100, and most preferably at least 10,000, 50,000, 100,000, or even at least 1,000,000 different members.

Using the mini-AGRP sequence information provided herein, such libraries can be routinely prepared using methods well known to those of skill in the art. Such methods include, but are not limited to "traditional" chemical syntheses methods, light- directed chemical syntheses, and recombinant expression.

Solid phase peptide synthesis in which the C-terminal amino acid of the sequence is attached to an insoluble support followed by sequential addition of the remaining amino acids in the sequence is the preferred method for preparing the peptide compounds of the present invention. Techniques for solid phase synthesis are well known t those of skill in the art (see, e.g., Barany and Merrifield, Solid-Phase Peptide Synthesis; pp. 3-284 in The Peptides: Analysis, Synthesis, Biology. Vol. 2: Special Methods in Peptide Synthesis, Part A., Merrifield, et al. J. Am. Chem. Soc. 85, 2149-2156 (1963), and Gross and Meienhofer, eds. Academic press, N. Y., 1980 and Stewart et al., Solid Phase Peptide Synthesis, 2nd ed. Pierce Chem. Co., Rockford, Ul. (1984)). Solid phase synthesis is most easily accomplished with commercially available peptide synthesizers utilizing FMOC or TBOC chemistry. In particularly preferred embodiments, peptide synthesis is performed using Fmoc synthesis. For example, AGRP C-terminal portion (MARP) can be readily synthesized using techniques described in Yang, et al. supra. Each desired peptide can be individually synthesized. Where large numbers of different peptides are desired (e.g. for a library) combinatorial synthesis methods are available. Combinatorial peptide synthesis can be performed according to the methods of Furka et al, (1991) Int. J. Pept. Protein Res. 37 487-493. At the desired positions that are to be altered the synthesis resins are separated coupled with the desired amino acid(s) and then pooled for the next coupling reaction. Such methods are easily accomplished using multiple peptide synthesizers. After synthesis, the proteins are purified if necessary (e.g. via HPCL), refolded and disulfide bonds are formed. Formation of intrapeptide disulfide bonds is often achieved by oxidation of the free thiol or sulfur-protected precursors under varying reaction conditions (see, e.g., Andreu et al. (1994) Pages 91-169 In Peptide Synthesis Protocols; Pennington, M. W., Bunn, B. M., Ed.; Humana Press: New Jersey; Moroder et al (1996) Biopolymers 40: 207-234; Annis and Barany (1997) Meth. Enzymol, 289: 198-221; Tam et al. (1991) J. Am. Chem. Soc. Ill:, 6657-6662; Munson and Barany (1993) J. Am. Chem. Soc. 115: 10203-10216; Shik (1993) J. Org. Chem. 58: 3003-3008; Annis and Barany (1998) J. Am. Chem. Soc, 120: 7226-7238; Shi and Rabenstein (1999) J. Org. Chem. 64: 4590-4595). Suitable oxidants include, but are not limited to iodine, thallium(IU) trifluroacetate (Fujii et α/.(1987) Chem. Pharm. Bull, 35: 2339-2347), and the like.

Alternatively, recombinant expression methods can be used to produce the peptides of this invention. In this approach, a nucleic acid encoding the desired polypeptide (and optionally a purification tag, e.g. His₆) is provided in an appropirate vector. A cell (e.g. E. coli, SF-3 cell, etc.) is transfected with the nucleic acid and, under appropriate conditions, transcribes and translates the desired protein. The protein is then recovered, refolded, if necessary and cross-linked using standard methods well known to those of skill in the art.

Molecular cloning techniques to achieve these ends are known in the art. A wide variety of cloning and in vitro amplification methods are suitable for the construction of recombinant nucleic acids. Examples of these techniques and instructions sufficient to direct persons of skill through many cloning exercises are found, e.g., in Berger and Kimmel, (1989) Guide to Molecular Cloning Techniques, Methods in Enzymology volume 152 Academic Press, Inc., San Diego, CA; Sambrook et al (1989) Molecular Cloning - A Laboratory Manual (2nd ed.) Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor Press, NY; and Ausubel et al. (1994) Current Protocols in Molecular Biology, Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc.,

IV. Use of molecular models to design small molecules that bind the melanocortin receptor ligand binding site.

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 methodsof 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 herein.

The atomic coordinates of MARP and its receptor binding region, provided herein, 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) 31:122-129) or by estimated interaction energy (Meng et al., J. Comp. Chem. (1992) 73:505-524). 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) 28:849-857; 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) 8: 195-202; available from Scripps Research Institute, La Jolla, CA); and DOCK (Kuntz et al, J. Mol. Biol. (1982) 767:269-288; 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) 8:44-47).

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) 78:182-196; 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) 35:2145-2154); 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) 47:8985;

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) 33:883-894);

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," Aliens, 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.

V. AGRP peptidomimetics.

Using the three-dimensional model of AGRP provided herein, a class of non- peptide melanocortin receptor ligands (peptidomimetics) were designed. The subject ligands are low molecular weight compounds that structurally mimic the AGRP active loop backbone. The subject non-peptide compounds find use in a variety of different applications, including the modulation of melanocortin receptor mediated physiological processes. By ligands of melanocortin receptors (MCR) is meant that the subject non- peptide compounds bind to melanocortin receptors. In many embodiments, the subject compounds preferentially bind to the following melanocortin receptors: MC3R, as described in U.S. Patent No. 5,837,521, the disclosure of which is herein incorporated by reference; and MC4R, as described in U.S. Patent No. 5,703,220, the disclosure of which is herein incorporated by reference. As the subject non-peptide compounds are MCR ligands, the subject compounds bind to an MCR with an affinity corresponding to a K_d of about 50 mM or lower, preferably of about 5 mM or lower, more preferably of about 500 μM or lower, and most preferably about 50 μM, 5 μM, or 1 μM or lower. In many embodiments, the affinity of the subject non-peptide compounds for an MCR, usually either MC3r or MC4r, ranges from about ranges from about 100 μM to 0.1 pM, and more usually about 10 μM to 0.1 nM As the subject compounds are non-peptide compounds, they are protease resistant. By protease resistant is meant that the subject compounds are resistant to proteolytic cleavage, at least by the proteases described in Miller et al. (1994), Bioorg Med Chem Let 4: 2657-2662. The subject non-peptide compounds are low molecular weight compounds. By low molecular weight is meant that the subject compounds have a molecular weight that ranges from about 200 to 1100 g/mol, usually from about 300 to 900 g/mol and more usually from about 400 to 800 g/mol.

A feature of the subject non-peptide compounds is that they structurally mimic the active loop 3-D conformation when bound by the receptor. By active loop is meant residues 111-116 or Arg-Phe-Phe-Asn-Ala-Phe (SEQ ID NO: ) of the Agouti

Related Protein. More specifically, the subject non-peptide compounds are characterized by substantially structurally mimicking the backbone phi angle of amino acid 113 in AGRP, i.e. Phell3 phi = -55.4°, and the U U interatomic distance (see structure below). As the subject compounds substantially structurally mimic the active loop, in 9 of 10 lowest energy structures calculated with distance geometry the following requirements should be met.

The phi angle mimicking amino acid 113 of AGRP, any deviation from the above angles should not exceed about -90° to -10° or 10° to 90°, usually about -85° to -20° or 20° to 85°, and more usually about -80° to -30° or 30° to 80° in phi space. The interatomic U]_.-U₂ distance should not exceed about 6.5 A, usually about 5.7 A, and more usually about about 5.5 A. In many preferred embodiments, the subject non-peptide ligands have the formula of Formula II:

where: each B, U_1; U , R, Ri and R₂ is independently selected from the group consisting of: hydrogen; alkyl; derivatized alkyl, e.g. halo alkyl, alkoxyalkyl, heteroalkyl, etc.; cycloalkyl; derivatized cycloalkyl, e.g. halocycloalkyl, aloxycycloalkyl, heterocycloalkyl; aryl; arylalkyl; heteroaryl; or heteroarylalkyl; J is carbon, nitrogen, silicon, or sulfur; X is hydrogen, carbon, nitrogen, oxygen, silicon, or sulfur; Z is a continuing peptide bond; hydroxyl; amide of the form -NH₂-, -NH-(n) or -N-(n,n'), where n or n' can be any alkyl, alkenyl, alkynyl, cycloalkyl, aryl, arylalkyl, heteroaryl or heteroarylalkyl, or derivatized form thereof; or ester of the form -O-(y) where y can be any alkyl, alkenyl, alkynyl, cycloalkyl, aryl, arylalkyl, heteroaryl or heteroarylalkyl, or derivatized form thereof. In certain prefered embodiments, B comprises a heteroalkyl moiety, where in certain preferred embodiments the heteroalkyl moiety comprises a guanidino moiety. In certain preferred embodiments, each U is a cycloalkyl, preferably arylalkyl, and more preferably methylbenzyl. In many preferred embodiments, the subject compounds are compounds in which B, R, Ri and R₂ are as described above.

Specific non-peptide compounds of interest include the compounds of Formulas III, IV, V, and VI, shown below:

<

The subject compounds may be synthesized using any convenient protocol. In one representative protocol, the peptoid portion of the subject molecules is synthesized using a submonomer approach, where a representative submonomer synthesis protocol that may be adapted to synthesize the subject compounds is described in U.S. Patent Nos. 5,977,301 and 5,831,005; the disclosures of which are herein incorporated by reference. Following production of the peptoid portion, the terminal "B" group as described above is added to the terminus of the peptoid portion of the compound. A representative sheme for preparation of the subject compounds is provided in the Experimental Section, infra.

VII. Uses of melanocortin receptor ligands.

A) Use as melanocortin receptor probes.

The melanocortin receptor ligands of this invention are useful in a wide variety of contexts. Because the ligands specifically bind the melanocortin receptor, they can be used as probes to specifically detect/localize melanocortin receptors (in vivo, in vitro, in cell culture, etc.). Thus, the ligands can be used to detect the presence or absence or to quantify the expression level of a melanocortin receptor. In such applications, the receptor ligands are preferably labeled with a detectable label.

Detectable labels suitable for use in the present invention include any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Useful labels in the present invention include, but are not limited to biotin for staining with labeled streptavidin conjugate, magnetic beads (e.g., DynabeadsTM), fluorescent dyes (e.g., fluorescein, texas red, rhodamine), fluorescent proteins (e.g., green fluorescent protein (GFP), red fluorescent protein (RFP), and the like), radiolabels (e.g., ³H, ¹²⁵1, ³⁵S, ¹⁴C, or ³²P), enzymes (e.g., horse radish peroxidase, alkaline phosphatase and others commonly used in an ELISA), chemiluminescent labels (e.g. luciferins), and colorimetric labels such as colloidal gold (e.g., gold particles in the 40 -80 nm diameter size range scatter green light with high efficiency) or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads. Other suitable labels include spin labels, e.g., provided by reporter molecules with an unpaired electron spin which can be detected by electron spin resonance (ESR) spectroscopy. Exemplary spin labels include organic free radicals, transitional metal complexes, particularly vanadium, copper, iron, and manganese, and the like.

It will be recognized that fluorescent labels are not to be limited to single species organic molecules, but include inorganic molecules, multi-molecular mixtures of organic and/or inorganic molecules, crystals, heteropolymers, and the like. Thus, for example, CdSe-CdS core-shell nanocrystals enclosed in a silica shell can be easily derivatized for coupling to a biological molecule (Bruchez et al. (1998) Science, 281: 2013- 2016). Similarly, highly fluorescent quantum dots (zinc sulfide-capped cadmium selenide) have been covalently coupled to biomolecules for use in ultrasensitive biological detection (Warren and Nie (1998) Science, 281: 2016-2018).

Patents teaching the use of labels include U.S. Patent Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241.

The label may be added to the melanocortin receptor ligand prior to, or after the ligand is contacted to the sample. So called "direct labels" are detectable labels that are directly attached to or incorporated into the ligand prior application of the ligand to the sample. In contrast, so called "indirect labels" are joined to the bound ligand after it has contacted the sample. Often, the indirect label becomes attached to a binding moiety present on the ligand before it is contacted to the sample. Thus, for example, the ligand may be biotinylated before it is used in the assay. After the ligand is bound to melanocortin receptor(s) in the sample, an avidin-conjugated fluorophore will bind the biotin on the ligand thereby providing a label that is easily detected.

The label is attached to the ligand directly or through a linker moiety. In certain embodiments, the label can be conveniently attached to an amino or carboxyl terminus of the ligand or to the R group of any amino acid(s) comprising the ligand as long as it does not interfere with specific binding of the ligand.

The label is detected using a method appropriate to the nature of the label. Thus, for example, enzymatic labels are detected by providing the appropriate substrate and reaction conditions for the enzyme and detecting loss of substrate or increase of reaction product. Radioactive labels are detected, e.g. via scintillography. Fluorescent labels and/or colorimetric labels are detected using optical methods (e.g. fluorometry, image analysis, etc).

B) Use in altering melanocortin receptor activity. In certain preferred embodiments, the melanocortin receptor ligands of this invention are used to alter (modulate) melanocortin receptor activity. The ligand can act as a simple competitive inhibitor blocking access to the receptor by its native ligand. Alternatively the ligands of this invention can act as receptor agonists or antagonists. The melanocortin receptor can be modulated in vivo, or in vitro. 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. The subject compounds (e.g. mini-AGRPs and/or non-peptide ligands) find use in a variety of different applications, and are particularly suited for use in modulating MC3R and/or MC4R mediated physiological processess. As such, the subject compounds find use in modulating feeding behaviour, and treating disease conditions associated with feeding behaviour. For example, MCR agonists or AGRP binding antagonists of the subject methods find use inhibiting appetite, where such compounds find use in the treatment of disease conditions associated with excessive caloric intake, e.g. obesity and the like. Conversely, MCR antagonists of the subject invention find use in treating disease conditions where increasing appetite is attractive, e.g. anorexia, cachexia and the like. Other disease conditions in which the subject compounds may find use include those described in: WO 99/21517; WO 98/10068; WO 99/54358; WO 99/31508; WO 99/43709; WO 99/55679; and WO 99/57148. Other representative uses for the subject ligands, e.g. screening assays for

MCR ligands, etc., are also disclosed in these listed patent applications, the disclosures of which are herein incorporated by reference). Labeled, i.e. radio, fluorescent, biotin, antigen, etc., non-peptide ligands of melanocortin receptors find use as biological tracers for receptor identification in vivo. The compounds of the subject invention find use in a variety of different hosts. Generally such hosts are "mammals" or "mammalian," where these terms are used broadly to describe organisms which are within the class mammalia, including the orders carnivore (e.g., dogs and cats), rodentia (e.g., mice, guinea pigs, and rats), and primates (e.g., humans, chimpanzees, and monkeys). In many embodiments, the hosts will be humans.

The compouds of this invention are also useful as "lead compounds" for the development of therapeutics. 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 ADDS 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).

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: CX¹X²X³X⁴X⁵X⁶CX⁷X⁸X⁹X¹⁰X¹¹X¹²CCDPX¹³ATCYCX¹⁴X¹⁵X¹⁶N AFC YCR_n (SEQ ID NO:_), wherein X¹, X², X³, X⁴, X⁵, X⁶, X⁷, X⁸, X⁹, X¹⁰, X¹¹, X¹², X¹³, X¹⁴, X¹⁵, and n is 0 or 1, 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. In one preferred embodiment, the polypeptide has the amino acid sequence: CVRLHESCLGQQVPCC DPAATCYCRFFNAFCYC (SEQ ID NO:3). In certain embodiments, such a disease state can be a wasting syndrome. There are many other 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

VIII. Screening of peptidomimetics and/or mini-AGRPs.

A) Binding and activity assays.

The compounds of this invention (e.g. mini-AGRPs or peptidomimetics) or libraries thereof can be screened to identify those having a particular receptor specificity, and/or avidity, and/or activity (e.g. agonist, antagonist, etc.). Such screening methods can readily identify "lead compounds" displaying the desired properties. Once lead compounds are identified, a variety of homologs and analogs can be prepared, e.g. to facilitate an understanding of the relationship between chemical structure and biological or functional activity. These studies define structure activity relationships which are then used to design drugs with improved potency, selectivity and pharmacokinetic properties. Combinatorial chemistry can also be used to rapidly generate a variety of structures for lead optimization. In preferred embodiments, such assays include binding assays and/or activity assays. Binding assays typically measure the ability of the ligand(s) of interest to bind a melanocortin receptor (e.g. MC3r, MC4r, etc.). The assays can be qualitative (positive or negative) or quantitative, e.g. provide a measure of binding affinity. Suitable binding assays are well known to those of skill in the art. In a preferred embodiment, the binding assay is a competitive assay that measures the ability of the test composition to displace/compete a reference ligand from a melanocortin receptor. In one preferred approach specific binding of ligands of this invention to cells expressing

1 the MC-3 receptor is determined by competition experiments using labeled (e.g. I- labeled) Nle⁴-D-Phe⁷- -MSH (NDP-MSH), as described in Tatro et al. (1990) Cancer Res. 50: 1237-1242).

In various embodiments, screening can be in vitro and/or in vivo. Certain preferred assays include cell-free competition assays and cell culture based assays.

Activity assays measure the ability of the compounds of this invention to activate (agonize) or inhibit (antagonize) activity at one or more melanocortin receptors or to block or augment the activity of known antagonists or antagonists. Activity assays for melanocortin receptors are well known to those of skill in the art.

Preferred assays measure, directly, or indirrectly, melanocortin receptor induced changes in intracellular cAMP concentrations. One such assay measures the ability of cAMP to displace (8-³H) cAMP from a high affinity cAMP binding protein (see Gilman (1979) Proc. Natl. Acad. Set, USA, 67: 305-312). Briefly, test cells are exposed to the ligands to be screened. Following treatment, the cells are washed twice with phosphate buffered saline and intracellular cAMP extracted by lysing the cells with 1 mL of 60% ethanol. The assay is then run as described by Gilman, supra.). Another convenient assay system is described in U.S. patent 6,100,048. This patent discloses recombinant expression constructs comprising nucleic acid encoding mammalian melanocortin receptors, and mammalian cells into which the recombinant expression constructs have been introduced that express functional mammalian melanocortin receptors. A panel is provided of such transformed mammalian cells expressing melanocortin receptors for screening compounds for receptor agonist and antagonist activity. One typical panel includes MClr, MC2r, MC3r, and MC4r. In various embodiments, each of the cells of the panel of mammalian cells expressing mammalian melanocortin receptors further comprises a recombinant expression construct encoding a cyclic AMP responsive element (CRE) transcription factor binding site that is operatively linked to a nucleic acid sequence encoding a protein capable of producing a detectable metabolite. Expression of the protein that produces the detectable metabolite is dependent on binding of the test compound to the melanocortin receptor expressed by each cell in the panel and the intracellular production of cAMP as a result. Detection of the metabolite thereby provides a measure of the agonist or antagonist activity of the ligand(s) in question.

In a preferred embodiment, compounds of the invention bind to a melanocortin receptor ligand binding site with greater affinity than the native cellular ligand proteins. Preferred compounds show at least a 1.25 fold greater affinity, preferably at least a 1.5 fold greater affinity, more preferably at least a 2 fold greater affinity, and most preferably at least a 5 fold or 10 fold greater affinity than the native cellular ligand proteins.

Preferred compounds can additionally or alternatively, show a different receptor specificity, e.g. specificity for MC4r rather than specificity for MC3r and MC4r, etc. 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. The assays described herein are meant to be illustrative and not limiting.

Using the teaching provided herein, other suitable assays will be apparent to those of skill in the art.

In certain preferred embodiments, the compounds that s

B) High throughput screening The activity or binding assays of this invention are also amenable to "high- throughput" modalities. As described above preferred ligands of this invention bind to melanocortin receptors and thereby modulate receptor activity. Preferred assays detect MC receptor binding and/or activity. High throughput assays for the presence, absence, or quantification of particular products are well known to those of skill in the art. Similarly, binding assays are similarly well known. Thus, for example, U.S. Patent 5,559,410 and 5,585,639 discloses high throughput binding assays. In addition, high throughput screening systems are commercially available

(see, e.g., Zymark Corp., Hopkinton, MA; Air Technical Industries, Mentor, OH; Beckman Instruments, Inc. Fullerton, CA; Precision Systems, Inc., Natick, MA, etc.). These systems typically automate entire procedures including all sample and reagent pipetting, liquid dispensing, timed incubations, and final readings of the microplate in detector(s) appropriate for the assay. These configurable systems provide high throughput and rapid start up as well as a high degree of flexibility and customization. The manufacturers of such systems provide detailed protocols the various high throughput. Thus, for example, Zymark Corp. provides technical bulletins describing screening systems for detecting the modulation of gene transcription, ligand binding, and the like. It is noted that HTS can be efficiently accomplished by screening combinations of ligands in a single assay. Thus, for example, a 680 well microtitre plate could contain 100 different ligands per well providing 68,000 different ligands per plate. Those wells that show a positive score for a particular assay are then sequentially subsampled to identify the particular ligand(s) providing the positive signal.

C) Databases of melanocortin receptor modulators.

In certain embodiments, the agents (ligands) that score positively in the assays described herein (e.g. show an ability to bind and/or modulate a melanocortin receptor) can be entered into a database of putative and/or actual modulators of Socs2 expression. Alternatively or additionally, a specificity fingerprint of a putative or acctual modulator can be entered into the database.

The term database refers to a means for recording and retrieving information. In preferred embodiments, the database also provides means for sorting and/or searching the stored information. The database can comprise any convenient media including, but not limited to, paper systems, card systems, mechanical systems, electronic systems, optical systems, magnetic systems or combinations thereof. Preferred databases include electronic (e.g. computer-based) databases. Computer systems for use in storage and manipulation of databases are well known to those of skill in the art and include, but are not limited to "personal computer systems", mainframe systems, distributed nodes on an inter- or intranet, data or databases stored in specialized hardware (e.g. in microchips), and the like.

IX. Methods for identifying receptor binding regions of melanocortin receptor agonist and antagonists. 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 DD 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.

X. Pharmaceutical Preparations

Also provided are pharmaceutical preparations of the subject non-peptide compounds. The subject compounds can be incorporated into a variety of formulations for therapeutic administration. More particularly, the compounds of the present invention can be formulated into pharmaceutical compositions by combination with appropriate, pharmaceutically acceptable carriers or diluents, and may be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants and aerosols. The formulations may be designed for administration via a number of different routes, including oral, buccal, rectal, parenteral, intraperitoneal, intradermal, transdermal, intracheal, etc., administration. In pharmaceutical dosage forms, the compounds may be administered in the form of their pharmaceutically acceptable salts, or they may also be used alone or in appropriate association, as well as in combination, with other pharmaceutically active compounds. The following methods and excipients are merely exemplary and are in no way limiting.

For oral preparations, the compounds can be used alone or in combination with appropriate additives to make tablets, powders, granules or capsules, for example, with conventional additives, such as lactose, mannitol, corn starch or potato starch; with binders, such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins; with disintegrators, such as corn starch, potato starch or sodium carboxymethylcellulose; with lubricants, such as talc or magnesium stearate; and if desired, with diluents, buffering agents, moistening agents, preservatives and flavoring agents.

The compounds can be formulated into preparations for injection by dissolving, suspending or emulsifying them in an aqueous or nonaqueous solvent, such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol; and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives.

The compounds can be utilized in aerosol formulation to be administered via inhalation. The compounds of the present invention can be formulated into pressurized acceptable propellants such as dichlorodifluoromethane, propane, nitrogen and the like.

Furthermore, the compounds can be made into suppositories by mixing with a variety of bases such as emulsifying bases or water-soluble bases. The compounds of the present invention can be administered rectally via a suppository. The suppository can include vehicles such as cocoa butter, carbowaxes and polyethylene glycols, which melt at body temperature, yet are solidified at room temperature.

Unlike typical peptide formulations, the peptides of this invention comprising D-form amino acids can be administered, even orally, without protection against proteolysis by stomach acid, etc. Nevertheless, in certain embodiments, peptide delivery can be enhanced by the use of protective excipients. This is typically accomplished either by complexing the polypeptide with a composition to render it resistant to acidic and enzymatic hydrolysis or by packaging the polypeptide in an appropriately resistant carrier such as a liposome. Means of protecting polypeptides for oral delivery are well known in the art (see, e.g., U.S. Patent 5,391,377 describing lipid compositions for oral delivery of therapeutic agents).

Where the ligands of this invention are polypeptides for oral administration, it is desirable to protect the polypeptide from digestion. This is readily accomplished by a variety of encapsulation technologies. One preferred encapsulation system is a "sustained- release system".

Such sustained release systems are well known to those of skill in the art and can often maintain elevated serum half -life. In one preferred embodiment, the ProLease biodegradable microsphere delivery system for proteins and peptides (Tracy (1998) Biotechnol Prog. 14: 108; Johnson et al. (1996) Nature Med. 2: 795; Herbert et al. (1998), Pharmaceut. Res. 15, 357) a dry powder composed of biodegradable polymeric microspheres containing the protein in a polymer matrix that can be compounded as a dry formulation with or without other agents.

The ProLease microsphere fabrication process was specifically designed to achieve a high protein encapsulation efficiency while maintaining protein integrity. The process consists of (i) preparation of freeze-dried protein particles from bulk protein by spray freeze-drying the drug solution with stabilizing excipients, (ii) preparation of a drug- polymer suspension followed by sonication or homogenization to reduce the drug particle size, (iii) production of frozen drug-polymer microspheres by atomization into liquid nitrogen, (iv) extraction of the polymer solvent with ethanol, and (v) filtration and vacuum drying to produce the final dry-powder product. The resulting powder contains the solid form of the protein, which is homogeneously and rigidly dispersed within porous polymer particles. The polymer most commonly used in the process, poly(lactide-co-glycolide) (PLG), is both biocompatible and biodegradable.

Encapsulation can be achieved at low temperatures (e.g., -40°C). During encapsulation, the protein is maintained in the solid state in the absence of water, thus minimizing water-induced conformational mobility of the protein, preventing protein degradation reactions that include water as a reactant, and avoiding organic-aqueous interfaces where proteins may undergo denaturation. A preferred process uses solvents in which most proteins are insoluble, thus yielding high encapsulation efficiencies (e.g., greater than 95%).

Unit dosage forms for oral or rectal administration such as syrups, elixirs, and suspensions may be provided wherein each dosage unit, for example, teaspoonful, tablespoonful, tablet or suppository, contains a predetermined amount of the composition containing one or more inhibitors. Similarly, unit dosage forms for injection or intravenous administration may comprise the inhibitor(s) in a composition as a solution in sterile water, normal saline or another pharmaceutically acceptable carrier. The term "unit dosage form," as used herein, refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of compounds of the present invention calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier or vehicle. The specifications for the novel unit dosage forms of the present ' invention depend on the particular compound employed and the effect to be achieved, and the pharmacodynamics associated with each compound in the host.

The pharmaceutically acceptable excipients, such as vehicles, adjuvants, carriers or diluents, are readily available to the public. Moreover, pharmaceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents and the like, are readily available to the public.

In therapeutic applications, the compositions of this invention are administered to a patient suffering from one or more symptoms of a wasting syndromes, obesity, and other disorders related to hypothalamic control of feeding. Such conditions include, but are not limited to wasting syndrome accompanying ADDS and various cancers. The compounds are administered in an amount sufficient to cure or at least partially prevent or arrest one or more symptoms of the the disease and/or its complications. An amount adequate to accomplish this is defined as a "therapeutically effective dose." Amounts effective for this use will depend upon the severity of the disease and the general state of the patient's health. Single or multiple administrations of the compositions may be administered depending on the dosage and frequency as required and tolerated by the patient. In any event, the composition should provide a sufficient quantity of the active agents of the formulations of this invention to effectively treat (ameliorate one or more symptoms) the patient.

The concentration of ligand (polypeptide or peptiodmimetic) can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight and the like in accordance with the particular mode of administration selected and the patient's needs. Concentrations, however, will typically be selected to provide dosages ranging from about 0.1 mg/kg/day to about 1000 mg/kg/day, preferably from about 1 mg/kg/day to about 100 mg/kg/day, more preferably from about 5 mg/kg/day to about 50 mg/kg/day. It will be appreciated that such dosages may be varied to optimize a therapeutic regimen in a particular subject or group of subjects.

XI. Kits. In still another embodiment, this invention provides kits for practice of the methods described herein. In certain embodiments the kits comprise a container containing one or more of the melanocortin ligands of this invention. The ligands can be labeled or unlabeled, and/or, optionally provided in a unit dosage form, and/or optionally, provided with or in a pharmacological acceptable excipient. The kits can optionally additionally include one or more detectable labels for labeling the ligand(s).

Certain preferred kits provide libraries of the ligands of this invention as described herein.

The kits can optionally include any reagents and/or apparatus to facilitate practice of the methods described herein. Such reagents and apparatus include, but are not limited to buffers, instrumentation, devices for administering the ligand(s) (e.g. syringes, etc.), microtiter plates, labeling reagents streptavidin or biotin conjugated substrates, PAGE gels, blotting membranes, reagents for detecting a signal, and the like.

In addition, the kits may include instructional materials containing directions (i.e., protocols) for the practice of the methods of this invention. Preferred instructional materials provide protocols for utilizing the kit contents for modulating melanocortin receptor activity and/or for screening for particular melanocortin receptors, and/or for treating a disease or pathological state. While the instructional materials typically comprise written or printed materials they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this invention. Such media include, but are not limited to electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. Such media may include addresses to internet sites that provide such instructional materials.

XII. Computer-based methods.

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 Tables 4 and 5, will be particularly useful for solving the NMR structure of other melanocortin receptor antagonists or agonists. The coordinates in Table 4 and Table 5 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 Table 4 and Table 5. The coordinates in Table 4 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. The coordinates for Table 5 have been deposited with the Brookhaven National Laboratory Protein Data Bank, and have been assigned Brookhaven Protein Data Bank Accession Number IHYK. 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 DD 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 DD NO:2) and residues 19-34 of the central loop (residues 19-34 of SEQ DD 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 DD NO:2), residues 7 to 18 (residues 7-18 of SEQ DD NO:2), or residues 1 to 18 (residues 1-18 of SEQ DD 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 2.54A, 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.

EXAMPLES

The following examples are offered to illustrate, but not to limit the claimed invention.

Example 1 Chemical synthesis and characterization of MARP

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 %

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:

Λ^-Acetyl-MARP (SEQ DD NO:2) and 7V^α-acetyl-MARP(Arg25 Ala) 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. Initial NMR data were routinely acquired with a 6000 Hz spectral width, 4096 complex points in t₂, and 512 (TOCS Y/DQF-COS Y) or 700 (NOESY) increments in tr. All spectra were processed using the MNMR package (Carlsberg Laboratory, Department of Chemistry, Denmark) and analyzed using XEAS Y (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. The same experiments were repeated at 800 mHz.

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., /. 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 H₂O and D₂O. J_HNαCOupling constants at 25°C were determined by both linear least squares fitting of the antiphase doublets in a DQF-COSY and also using the UNFIT (Szyperski, et al., /. 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 J_HNα-

For amide exchange experiments, the magnet was preshimmed on a 21 residue peptide sample at pH 4 in D₂O/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 tj 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<ιz- 7i<5) and 37 long range (ιz-jι>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 1.6 A, 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., Ada 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, /. 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)).

Results and Discussion.

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 DD 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. ³J_HN 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. ³J_{H α} coupling constants and temperature coefficients.

Residue ³Jm_a coupling constant (Hz) NH temperature 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

Gln-11 8.14 4.94

Gln-12 8.00 4.38

Val-13 9.03 5.56 Residue ³J_NHα coupling constant (Hz) NH temperature coefficient

(ppb/°C)

Pro-i4 σoδ

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

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 Figure 1. Consistent with the far-UV CD spectrum, MARP showed little evidence of helical or regular 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 Figure 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) 2^5 326

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 Figure 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 (Figure 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 Figure 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 0.6 A, Figure 3) and a stem region which is both twisted around and curved along its z-axis (Figure 1). This characterization was supported by critical examination of the nOe, mx_α and chemical shift data. As shown in Figure 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. (1986) Science 234:849-855), 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 D O 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 Cgprotons. The NMR structure (Table 4) 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 /_HN_OC 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 J_HNα- Helical structure was characterized by ³J_HNα<6 Hz and negative conformational shifts and β-sheet by JHN_α>8 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 1H chemical shifts at 15°C, ³J_NHα, 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) Hβ (ppm) Other (ppm^")

Cys- 1 8.00 5.00 3.12, 2.81

Val-2 8.92 4.18 1.94, 2.08 CH 0.91, 0.80

Arg-3 8.91 4.01 1.67, 1.95 1^1.69, 1.82, ^3.24, 3.26, NH 7.31

Leu-4 8.13 3.72 1.34, 1.54 1371.03, 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 H71.44, CH^δ ₃0.82

Gly-10 8.78 4.07, 3.69

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

Gin- 12 8.68 4.27 2.20, 1.95 H 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 IF^" 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 H7 1.98, 11* 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 CΕC₃ 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 1371.56, 1.23, H⁸3.06

Phe-26 7.91 4.74 2.80, 3.32 H³7.26, 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^δ26.66, 7.30

Ala-29 7.75 4.38 1.25 Residue NH (ppm) Hv (ppm) Hβ (ppm) Other (ppm)

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 1371.57, 1.69, 13* 2.62, 2.88, NH 7.08

Lys-35 9.09 4.47 1.82, 1.68 H71.25, 1.43, H* 1.64, H⁶2.89

Leu-36 8.77 4.3640 1.63 H7l.52, CH*3 0.82, 0.70

Gly-37 8.48 4.09, 4.01

Thr-38 7.82 4.42 4.42 CH 1.21

Ala-39 8.53 4.16 1.42

Met-40 8.06 4.40 1.95, 2.09 H72.51, 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 H7l.94, H* 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 H71.66. H* 3.21, NH 7.25

Thr-46 7.86 4.15 4.23 CH7₃1.15

Table 4. MARP atomic coordinates in 3 dimensional space determine by NMR at 500 mHz.

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 HGll 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 HHll ARG 3 -14.578 6.091 -1.459 1.00 5.28

ATOM 47 HH12 ARG 3 -16.246 6.446 -1.162 1.00 6.06

ATOM 48 NH2 ARG 3 -17.129 4.160 -1.677 1.00 6.01

ATOM 49 HH21 ARG 3 -17.379 3.219 -1.903 1.00 6.19

ATOM 50 HH22 ARG 3 -17.837 4.816 -1.413 1.00 6.50

ATOM 51 C ARG 3 -9.053 3.103 -1.329 1.00 2.75

ATOM 52 O ARG 3 -8.519 2.163 -0.738 1.00 2.50

ATOM 53 N LEU 4 -8.725 4.372 -1.111 1.00 3.24

ATOM 54 HN LEU 4 -9.167 5.082 -1.615 1.00 3.68

ATOM 55 CA LEU 4 -7.708 4.726 -0.146 1.00 3.40

ATOM 56 HA LEU 4 -6.847 4.143 -0.371 1.00 3.11 ATOM 57 CB LEU 4 -7.351 6.196 -0.264 1.00 4.25

ATOM 58 HB1 LEU 4 -7.379 6.479 -1.304 1.00 4.60

ATOM 59 HB2 LEU 4 -6.353 6.339 0.110 1.00 4.61

ATOM 60 CG LEU 4 -8.285 7.097 0.500 1.00 4.65

ATOM 61 HG LEU 4 -8.344 6.741 1.509 1.00 4.56

ATOM 62 GDI LEU 4 -7.773 8.520 0.517 1.00 5.17

ATOM 63 HDll LEU 4 -7.232 8.714 -0.396 1.00 5.39

ATOM 64 HD12 LEU 4 -7.113 8.647 1.363 1.00 5.60

ATOM 65 HD13 LEU 4 -8.605 9.203 0.599 1.00 5.27

ATOM 66 CD2 LEU 4 -9.659 6.991 -0.103 1.00 5.22

ATOM 61 HD21 LEU 4 -10.392 7.382 0.584 1.00 5.44

ATOM 68 HD22 LEU 4 -9.857 5.945 -0.295 1.00 5.42

ATOM 69 HD23 LEU 4 -9.690 7.541 -1.030 1.00 5.58

ATOM 70 C LEU 4 -8.186 4.418 1.274 1.00 3.37

ATOM 71 O LEU 4 -9.386 4.241 1.507 1.00 3.67

ATOM 72 N HIS 5 -7.244 4.346 2.217 1.00 3.48

ATOM 73 HN HIS 5 -6.311 4.491 1.965 1.00 3.68

ATOM 74 CA HIS 5 -7.564 4.044 3.618 1.00 3.77

ATOM 75 HA ms 5 -6.629 3.939 4.150 1.00 3.80

ATOM 76 CB HIS 5 -8.361 5.193 4.240 1.00 4.60

ATOM 77 HB1 HIS 5 -8.651 4.923 5.245 1.00 4.82

ATOM 78 HB2 HIS 5 -9.248 5.373 3.650 1.00 4.71

ATOM 79 CG HIS 5 -7.583 6.467 4.309 1.00 5.19

ATOM 80 ND1 HIS 5 -8.108 7.657 4.768 1.00 5.95

ATOM 81 HD1 HIS 5 -9.021 7.792 5.094 1.00 6.16

ATOM 82 CD2 HIS 5 -6.300 6.730 3.966 1.00 5.27

ATOM 83 HD2 HIS 5 -5.586 6.017 3.564 1.00 4.82

ATOM 84 CE1 HIS 5 -7.182 8.597 4.702 1.00 6.46

ATOM 85 HE1 HIS 5 -7.307 9.629 4.993 1.00 7.11

ATOM 86 NE2 HIS 5 -6.076 8.060 4.221 1.00 6.10

ATOM 87 HE2 HIS 5 -5.204 8.506 4.184 1.00 6.44

ATOM 88 C HIS 5 -8.338 2.726 3.736 1.00 3.44

ATOM 89 O HIS 5 -8.921 2.423 4.779 1.00 3.81

ATOM 90 N GLU 6 -8.318 1.949 2.656 1.00 2.90

ATOM 91 HN GLU 6 -7.824 2.247 1.869 1.00 2.77

ATOM 92 CA GLU 6 -8.984 0.664 2.592 1.00 2.75

ATOM 93 HA GLU 6 -9.613 0.554 3.462 1.00 3.21

ATOM 94 CB GLU 6 -9.838 0.592 1.327 1.00 2.53

ATOM 95 HB1 GLU 6 -9.174 0.488 0.478 1.00 2.28 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 3.11

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 HDll 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

ATOM 145 N GLY 10 -6.261 -5.033 4.530 1.00 3.63

ATOM 146 HN GLY 10 -5.501 -4.627 4.073 1.00 3.32

ATOM 147 CA GLY 10 -6.681 -4.513 5.824 1.00 4.02

ATOM 148 HA1 GLY 10 -5.848 -4.575 6.507 1.00 4.32

ATOM 149 HA2 GLY 10 -7.490 -5.120 6.203 1.00 4.54

ATOM 150 C GLY 10 -7.146 -3.066 5.745 1.00 3.77

ATOM 151 O GLY 10 -8.151 -2.771 5.104 1.00 4.10

ATOM 152 N GLN 11 -6.406 -2.169 6.402 1.00 3.61

ATOM 153 HN GLN 11 -5.613 -2.477 6.886 1.00 3.76

ATOM 154 CA GLN 11 -6.727 -0.736 6.407 1.00 3.67

ATOM 155 HA GLN 11 -6.984 -0.452 5.397 1.00 3.70

ATOM 156 CB GLN 11 -7.923 -0.451 7.325 1.00 4.51

ATOM 157 HB1 GLN 11 -7.690 0.399 7.949 1.00 4.89

ATOM 158 HB2 GLN 11 -8.094 -1.312 7.954 1.00 4.88

ATOM 159 CG GLN 11 -9.210 -0.148 6.570 1.00 4.88

ATOM 160 HG1 GLN 11 -9.043 -0.320 5.517 1.00 5.09

ATOM 161 HG2 GLN 11 -9.467 0.889 6.727 1.00 4.85

ATOM 162 CD GLN 11 -10.376 -1.008 7.023 1.00 5.60

ATOM 163 OE1 GLN 11 -10.540 -2.140 6.568 1.00 6.07

ATOM 164 NE2 GLN 11 -11.195 -0.474 7.924 1.00 6.05

ATOM 165 HE21 GLN 11 -11.006 0.433 8.244 1.00 5.94

ATOM 166 HE22 GLN 11 -11.958 -1.009 8.229 1.00 6.67

ATOM 167 C GLN 11 -5.510 0.082 6.852 1.00 3.45

ATOM 168 O GLN 11 -5.279 0.248 8.052 1.00 4.02

ATOM 169 N GLN 12 -4.718 0.570 5.893 1.00 2.94

ATOM 170 HN GLN 12 -4.930 0.397 4.955 1.00 2.77

ATOM 171 CA GLN 12 -3.520 1.338 6.219 1.00 3.05

ATOM 172 HA GLN 12 -3.399 1.297 7.292 1.00 3.57

ATOM 173 CB GLN 12 -2.294 0.679 5.573 1.00 3.41 ATOM 174 HB1 GLN 12 -1.434 1.314 5.730 1.00 3.57

ATOM 175 HB2 GLN 12 -2.471 0.583 4.512 1.00 3.50

ATOM 176 CG GLN 12 -1.976 -0.700 6.130 1.00 4.12

ATOM 177 HG1 GLN 12 -1.131 -1.105 5.591 1.00 4.31

ATOM 178 HG2 GLN 12 -2.834 -1.340 5.984 1.00 4.40

ATOM 179 CD GLN 12 -1.639 -0.671 7.609 1.00 4.78

ATOM 180 OE1 GLN 12 -0.584 -0.178 8.007 1.00 5.27

ATOM 181 NE2 GLN 12 -2.538 -1.199 8.436 1.00 5.19

ATOM 182 HE21 GLN 12 -3.359 -1.574 8.052 1.00 5.11

ATOM 183 HE22 GLN 12 -2.342 -1.191 9.396 1.00 5.76

ATOM 184 C GLN 12 -3.640 2.820 5.813 1.00 2.91

ATOM 185 O GLN 12 -4.438 3.561 6.388 1.00 3.44

ATOM 186 N VAL 13 -2.821 3.248 4.843 1.00 2.75

ATOM 187 HN VAL 13 -2.199 2.625 4.444 1.00 2.86

ATOM 188 CA VAL 13 -2.795 4.629 4.377 1.00 3.05

ATOM 189 HA VAL 13 -3.226 5.246 5.153 1.00 3.57

ATOM 190 CB VAL 13 -1.331 5.091 4.154 1.00 3.46

ATOM 191 HB VAL 13 -1.291 6.137 4.363 1.00 3.85

ATOM 192 CGI VAL 13 -0.380 4.402 5.127 1.00 3.80

ATOM 193 HGll VAL 13 -0.395 3.337 4.956 1.00 3.78

ATOM 194 HG12 VAL 13 -0.691 4.607 6.140 1.00 4.15

ATOM 195 HG13 VAL 13 0.622 4.776 4.976 1.00 4.28

ATOM 196 CG2 VAL 13 -0.878 4.877 2.709 1.00 3.89

ATOM 197 HG21 VAL 13 -0.918 5.815 2.176 1.00 4.21

ATOM 198 HG22 VAL 13 -1.528 4.163 2.224 1.00 4.18

ATOM 199 HG23 VAL 13 0.135 4.503 2.700 1.00 4.14

ATOM 200 C VAL 13 -3.615 4.813 3.090 1.00 3.04

ATOM 201 O VAL 13 -4.211 3.857 2.589 1.00 2.95

ATOM 202 N PRO 14 -3.671 6.053 2.539 1.00 3.66

ATOM 203 CA PRO 14 -4.438 6.341 1.318 1.00 4.09

ATOM 204 HA PRO 14 -5.448 5.975 1.401 1.00 4.39

ATOM 205 CB PRO 14 -4.454 7.870 1.263 1.00 5.04

ATOM 206 HB1 PRO 14 -5.342 8.242 1.750 1.00 5.62

ATOM 207 HB2 PRO 14 -4.437 8.197 0.234 1.00 5.18

ATOM 208 CG PRO 14 -3.224 8.289 1.986 1.00 5.32

ATOM 209 HG1 PRO 14 -3.368 9.268 2.418 1.00 5.91

ATOM 210 HG2 PRO 14 -2.384 8.296 1.308 1.00 5.77

ATOM 211 CD PRO 14 -3.010 7.267 3.068 1.00 4.46

ATOM 212 HD1 PRO 14 -3.474 7.590 3.989 1.00 4.68 ATOM 213 HD2 PRO 14 -1.956 7.102 3.216 1.00 4.53

ATOM 214 C PRO 14 -3.795 5.765 0.053 1.00 3.77

ATOM 215 O PRO 14 -2.821 5.013 0.129 1.00 4.07

ATOM 216 N CYS 15 -4.341 6.138 -1.114 1.00 3.60

ATOM 217 HN CYS 15 -5.104 6.751 -1.110 1.00 3.75

ATOM 218 CA CYS 15 -3.814 5.676 -2.394 1.00 3.70

ATOM 219 HA CYS 15 -2.780 5.986 -2.427 1.00 3.90

ATOM 220 HB1 CYS 15 -4.871 3.825 -2.642 1.00 4.62

ATOM 221 HB2 CYS 15 -3.464 3.720 -1.587 1.00 4.34

ATOM 222 C CYS 15 -4.533 6.333 -3.587 1.00 3.85

ATOM 223 O CYS 15 -4.584 7.561 -3.661 1.00 4.33

ATOM 224 CB CYS 15 -3.851 4.149 -2.500 1.00 4.05

ATOM 225 SG CYS 15 -2.856 3.502 -3.887 1.00 4.02

ATOM 226 N CYS 16 -5.057 5.523 -4.536 1.00 3.70

ATOM 227 HN CYS 16 -4.985 4.556 -4.441 1.00 3.50

ATOM 228 CA CYS 16 -5.710 6.043 -5.736 1.00 4.08

ATOM _, 229 HA CYS 16 -5.636 5.275 -6.496 1.00 3.93

ATOM 230 HB1 CYS 16 -7.294 7.293 -4.999 1.00 4.46

ATOM 231 HB2 CYS 16 -7.597 5.564 -4.810 1.00 4.09

ATOM 232 C CYS 16 -4.979 7.301 -6.249 1.00 4.59

ATOM 233 O CYS 16 -5.610 8.273 -6.672 1.00 5.05

ATOM 234 CB CYS 16 -7.196 6.327 -5.471 1.00 4.36

ATOM 235 SG CYS 16 -8.216 6.341 -6.984 1.00 5.13

ATOM 236 N ASP 17 -3.630 7.259 -6.197 1.00 4.60

ATOM 237 HN ASP 17 -3.198 6.451 -5.847 1.00 4.30

ATOM 238 CA ASP 17 -2.777 8.374 -6.639 1.00 5.13

ATOM 239 HA ASP 17 -2.901 8.469 -7.708 1.00 5.59

ATOM 240 CB ASP 17 -3.219 9.692 -5.975 1.00 5.62

ATOM 241 HB1 ASP 17 -3.503 9.496 -4.953 1.00 5.76

ATOM 242 HB2 ASP 17 -4.072 10.083 -6.508 1.00 5.74

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

ATOM 244 OD1 ASP 17 -1.748 11.210 -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 GDI 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 HHll 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 11.127 -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 HEl 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 HEl 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 GDI 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 HEl 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 2.011 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 GDI 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 HEl 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 HHll 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 HEl 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

ATOM 537 HB1 LEU 36 3.786 -7.540 -4.232 1.00 4.50

ATOM 538 HB2 LEU 36 2.120 -7.776 -4.770 1.00 4.66

ATOM 539 CG LEU 36 2.360 -6.422 -3.135 1.00 3.78

ATOM 540 HG LEU 36 2.622 -5.367 -3.189 1.00 3.57

ATOM 541 CD1 LEU 36 0.849 -6.524 -3.055 1.00 4.07

ATOM 542 HDll LEU 36 0.535 -6.468 -2.028 1.00 4.35

ATOM 543 HD12 LEU 36 0.528 -7.464 -3.481 1.00 4.26

ATOM 544 HD13 LEU 36 0.405 -5.712 -3.610 1.00 4.30

ATOM 545 CD2 LEU 36 3.040 -7.016 -1.907 1.00 4.09

ATOM 546 HD21 LEU 36 4.020 -6.576 -1.793 1.00 4.26

ATOM 547 HD22 LEU 36 3.138 -8.084 -2.027 1.00 4.48

ATOM 548 HD23 LEU 36 2.447 -6.802 -1.026 1.00 4.28

ATOM 549 C LEU 36 4.137 -6.293 -6.405 1.00 4.76

ATOM 550 O LEU 36 4.441 -7.429 -6.775 1.00 5.31

ATOM 551 N GLY 37 4.752 -5.198 -6.707 1.00 4.97

ATOM 552 HN GLY 37 4.412 -4.392 -6.286 1.00 4;82

ATOM 553 CA GLY 37 5.915 -5.144 -7.545 1.00 5.70

ATOM 554 HA1 GLY 37 5.776 -4.401 -8.313 1.00 6.04

ATOM 555 HA2 GLY 37 6.065 -6.105 -7.993 1.00 6.19

ATOM 556 C GLY 37 7.120 -4.776 -6.704 1.00 5.54

ATOM 557 O GLY 37 7.963 -3.978 -7.115 1.00 6.08

ATOM 558 N THR 38 7.161 -5.356 -5.495 1.00 5.00

ATOM 559 HN THR 38 6.446 -5.959 -5.236 1.00 4.79

ATOM 560 CA THR 38 8.202 -5.119 -4.526 1.00 4.96

ATOM 561 HA THR 38 8.007 -5.786 -3.699 1.00 5.16

ATOM 562 CB THR 38 9.591 -5.445 -5.075 1.00 5.78

ATOM 563 HB THR 38 10.154 -4.531 -5.184 1.00 6.05 ATOM 564 OG1 THR 38 9.526 -6.095 -6.337 1.00 6.18

ATOM 565 HG1 THR 38 9.079 -6.941 -6.244 1.00 6.46

ATOM 566 CG2 THR 38 10.345 -6.343 -4.136 1.00 6.38

ATOM 567 HG21 THR 38 9.756 -7.232 -3.961 1.00 6.46

ATOM 568 HG22 THR 38 10.500 -5.827 -3.199 1.00 6.55

ATOM 569 HG23 THR 38 11.296 -6.614 -4.570 1.00 6.89

ATOM 570 C THR 38 8.149 -3.700 -3.987 1.00 4.17

ATOM 571 O THR 38 8.747 -2.781 -4.553 1.00 4.31

ATOM 572 N ALA 39 7.460 -3.542 -2.852 1.00 3.68

ATOM 573 HN ALA 39 7.044 -4.326 -2.437 1.00 3.92

ATOM 574 CA ALA 39 7.370 -2.242 -2.190 1.00 3.19

ATOM 575 HA ALA 39 6.953 -1.533 -2.893 1.00 3.39

ATOM 576 CB ALA 39 6.459 -2.305 -0.952 1.00 2.97

ATOM 577 HB1 ALA 39 5.453 -1.989 -1.214 1.00 3.17

ATOM 578 HB2 ALA 39 6.845 -1.652 -0.184 1.00 3.05

ATOM 579 HB3 ALA 39 6.429 -3.317 -0.578 1.00 3.21

ATOM 580 C ALA 39 8.789 -1.809 -1.820 1.00 3.22

ATOM 581 O ALA 39 9.116 -0.625 -1.846 1.00 3.39

ATOM 582 N MET 40 9.628 -2.825 -1.539 1.00 3.43

ATOM 583 HN MET 40 9.270 -3.743 -1.595 1.00 3.65

ATOM 584 CA MET 40 11.056 -2.668 -1.215 1.00 3.68

ATOM 585 HA MET 40 11.340 -3.533 -0.635 1.00 3.98

ATOM 586 CB MET 40 11.888 -2.677 -2.505 1.00 4.33

ATOM 587 HB1 MET 40 12.568 -1.837 -2.491 1.00 4.41

ATOM 588 HB2 MET 40 11.222 -2.575 -3.350 1.00 4.46

ATOM 589 CG MET 40 12.704 -3.945 -2.692 1.00 5.16

ATOM 590 HG1 MET 40 12.841 -4.118 -3.751 1.00 5.45

ATOM 591 HG2 MET 40 12.162 -4.774 -2.262 1.00 5.31

ATOM 592 SD MET 40 14.325 -3.847 -1.907 1.00 6.00

ATOM 593 CE MET 40 15.309 -3.173 -3.244 1.00 6.60

ATOM 594 HEl MET 40 15.124 -3.736 -4.146 1.00 6.92

ATOM 595 HE2 MET 40 16.356 -3.239 -2.987 1.00 6.56

ATOM 596 HE3 MET 40 15.041 -2.139 -3.404 1.00 7.01

ATOM 597 C MET 40 11.386 -1.423 -0.381 1.00 3.37

ATOM 598 O MET 40 11.559 -1.513 0.835 1.00 3.68

ATOM 599 N ASN 41 11.510 -0.272 -1.048 1.00 3.35

ATOM 600 HN ASN 41 11.386 -0.269 -2.021 1.00 3.59

ATOM 601 CA ASN 41 11.864 0.977 -0.377 1.00 3.57

ATOM 602 HA ASN 41 12.586 0.745 0.390 1.00 4.02 ATOM 603 CB ASN 41 12.503 1.948 -1.377 1.00 4.36

ATOM 604 HB1 ASN 41 12.506 2.941 -0.952 1.00 4.61

ATOM 605 HB2 ASN 41 11.920 1.953 -2.286 1.00 4.37

ATOM 606 CG ASN 41 13.932 1.569 -1.727 1.00 5.30

ATOM 607 OD1 ASN 41 14.191 0.473 -2.226 1.00 5.84

ATOM 608 ND2 ASN 41 14.868 2.477 -1.470 1.00 5.81

ATOM 609 HD21 ASN 41 14.592 3.331 -1.074 1.00 5.65

ATOM 610 HD22 ASN 41 15.798 2.256 -1.685 1.00 6.50

ATOM 611 C ASN 41 10.651 1.636 0.277 1.00 3.02

ATOM 612 O ASN 41 9.507 1.390 -0.108 1.00 3.02

ATOM 613 N PRO 42 10.898 2.490 1.291 1.00 3.12

ATOM 614 CA PRO 42 9.833 3.192 2.018 1.00 3.15

ATOM 615 HA PRO 42 9.154 2.496 2.484 1.00 3.54

ATOM 616 CB PRO 42 10.588 3.974 3.102 1.00 4.06

ATOM 617 HB1 PRO 42 10.553 3.428 4.033 1.00 4.67

ATOM 618 HB2 PRO 42 10.133 4.945 3.230 1.00 4.13

ATOM 619 CG PRO 42 11.983 4.089 2.593 1.00 4.46

ATOM 620 HG1 PRO 42 12.673 4.160 3.421 1.00 5.17

ATOM 621 HG2 PRO 42 12.072 4.956 1.954 1.00 4.81

ATOM 622 CD PRO 42 12.235 2.834 1.810 1.00 3.83

ATOM 623 HD1 PRO 42 12.611 2.055 2.456 1.00 4.33

ATOM 624 HD2 PRO 42 12.927 3.025 1.002 1.00 3.84

ATOM 625 C PRO 42 9.043 4.145 1.125 1.00 2.55

ATOM 626 O PRO 42 9.610 5.065 0.531 1.00 2.68

ATOM 627 N CYS 43 7.731 3.923 1.041 1.00 2.61

ATOM 628 HN CYS 43 7.340 3.178 1.541 1.00 3.08

ATOM 629 CA CYS 43 6.862 4.768 0.230 1.00 2.66

ATOM 630 HA CYS 43 7.382 5.025 -0.623 1.00 3.10

ATOM 631 HB1 CYS 43 4.942 3.935 0.709 1.00 2.52

ATOM 632 HB2 CYS 43 5.840 3.031 -0.503 1.00 2.27

ATOM 633 C CYS 43 6.535 6.042 0.962 1.00 3.31

ATOM 634 O CYS 43 6.796 7.152 0.494 1.00 3.75

ATOM 635 CB CYS 43 5.579 4.021 -0.159 1.00 2.41

ATOM 636 SG CYS 43 4.616 4.831 -1.478 1.00 3.14

ATOM 637 N SER 44 5.994 5.829 2.121 1.00 3.89

ATOM 638 HN SER 44 5.876 4.902 2.362 1.00 3.89

ATOM 639 CA SER 44 5.605 6.891 3.055 1.00 4.87

ATOM 640 HA SER 44 4.903 6.474 3.757 1.00 5.17

ATOM 641 CB SER 44 6.842 7.366 3.832 1.00 5.33 ATOM 642 HB1 SER 44 7.327 6.513 4.284 1.00 5.78

ATOM 643 HB2 SER 44 6.539 8.054 4.606 1.00 5.38

ATOM 644 OG SER 44 7.771 8.016 2.981 1.00 5.71

ATOM 645 HG SER 44 8.433 7.384 2.689 1.00 5.91

ATOM 646 C SER 44 4.916 8.060 2.338 1.00 5.60

ATOM 647 O SER 44 4.324 7.887 1.270 1.00 5.82

ATOM 648 N ARG 45 4.992 9.244 2.946 1.00 6.31

ATOM 649 HN ARG 45 5.466 9.302 3.798 1.00 6.38

ATOM 650 CA ARG 45 4.385 10.456 2.392 1.00 7.28

ATOM 651 HA ARG 45 4.713 11.288 2.997 1.00 7.53 ^•

ATOM 652 CB ARG 45 4.845 10.690 0.944 1.00 7.96

ATOM 653 HB1 ARG 45 4.706 11.733 0.701 1.00 8.31

ATOM 654 HB2 ARG 45 4.231 10.094 0.285 1.00 7.94

ATOM 655 CG ARG 45 6.301 10.331 0.684 1.00 8.60

ATOM 656 HG1 ARG 45 6.471 10.321 -0.383 1.00 8.78

ATOM 657 HG2 ARG 45 6.498 9.351 1.089 1.00 8.86

ATOM 658 CD ARG 45 7.254 11.328 1.326 1.00 9.04

ATOM 659 HD1 ARG 45 6.712 11.904 2.062 1.00 9.20

ATOM 660 HD2 ARG 45 7.632 11.990 0.560 1.00 8.91

ATOM 661 NE ARG 45 8.384 10.664 1.981 1.00 9.74

ATOM 662 HE ARG 45 8.404 10.675 2.960 1.00 9.93

ATOM 663 CZ ARG 45 9.375 10.047 1.328 1.00 10.31

ATOM 664 NH1 ARG 45 9.388 10.005 -0.002 1.00 10.31

ATOM 665 HHll ARG 45 8.653 10.435 -0.525 1.00 9.86

ATOM 666 HH12 ARG 45 10.134 9.541 -0.479 1.00 10.88

ATOM 667 NH2 ARG 45 10.357 9.466 2.011 1.00 11.06

ATOM 668 HH21 ARG 45 10.355 9.490 3.011 1.00 11.21

ATOM 669 HH22 ARG 45 11.100 9.004 1.525 1.00 11.58

ATOM 670 C ARG 45 2.858 10.387 2.457 1.00 7.69

ATOM 671 O ARG 45 2.168 10.651 1.468 1.00 8.34

ATOM 672 N THR 46 2.336 10.036 3.633 1.00 7.60

ATOM 673 HN THR 46 2.935 9.842 4.379 1.00 7.26

ATOM 674 CA THR 46 0.895 9.934 3.836 1.00 8.32

ATOM 675 HA THR 46 0.488 9.396 2.997 1.00 8.41

ATOM 676 CB THR 46 0.582 9.157 5.122 1.00 8.61

ATOM 677 HB THR 46 -0.480 9.219 5.314 1.00 8.70

ATOM 678 OG1 THR 46 1.266 9.717 6.233 1.00 8.93

ATOM 679 HG1 THR 46 2.175 9.410 6.243 1.00 9.05

ATOM 680 CG2 THR 46 0.951 7.688 5.037 1.00 8.89 ATOM 681 HG21 , THR 46 0.433 7.232 4.206 1.00 9.07

ATOM 682 HG22 THR 46 0.668 7.191 5.954 1.00 9.06

ATOM 683 HG2 i THR 46 2.017 7.593 4.891 1.00 9.01

ATOM 684 C THR 46 0.249 11.319 3.889 1.00 9.05

ATOM 685 OT1 THR 46 0.759 12.187 4.632 1.00 9.34

ATOM 686 OT2 THR 46 -0.761 11.525 3.184 1.00 9.53

Table 5. MARP atomic coordinates in 3 dimensional space determine by NMR at 800 mHz.

ATOM 1 N CYS A 1 -12.726 -3.631 3.103 1.00 0.00 N

ATOM 2 CA CYS A 1 -11.311 -3.175 3.172 1.00 0.00 C

ATOM 3 C CYS A 1 -10.813 -2.734 1.797 1.00 0.00 C

ATOM 4 O CYS A 1 -11.608 -2.465 0.895 1.00 0.00 O

ATOM 5 CB CYS A 1 -10.421 -4.311 3.710 1.00 0.00 C

ATOM 6 SG CYS A 1 -11.306 -5.794 4.301 1.00 0.00 s

ATOM 7 H CYS A 1 -13.283 -2.870 2.668 1.00 0.00 H

ATOM 8 HA CYS A 1 -11.258 -2.333 3.848 1.00 0.00 H

ATOM 9 1HB CYS A 1 -9.754 -4.626 2.925 1.00 0.00 H

ATOM 10 2HB CYS A 1 -9.837 -3.932 4.534 1.00 0.00 H

ATOM 11 N VAL A 2 -9.494 -2.666 1.644 1.00 0.00 N

ATOM 12 CA VAL A 2 -8.887 -2.267 0.382 1.00 0.00 C

ATOM 13 C VAL A 2 -8.548 -3.483 -0.461 1.00 0.00 C

ATOM 14 O VAL A 2 -8.532 -4.609 0.036 1.00 0.00 O

ATOM 15 CB VAL A 2 -7.610 -1.429 0.600 1.00 0.00 C

ATOM 16 CGI VAL A 2 -7.264 -0.642 -0.655 1.00 0.00 C

ATOM 17 CG2 VAL A 2 -7.772 -0.495 1.792 1.00 0.00 C

ATOM 18 H VAL A 2 -8.913 -2.896 2.399 1.00 0.00 H

ATOM 19 HA VAL A 2 -9.602 -1.668 -0.161 1.00 0.00 H

ATOM 20 HB VAL A 2 -6.794 -2.106 0.807 1.00 0.00 H

ATOM 21 1HG1 VAL A 2 -6.616 0.182 -0.397 1.00 0.00 H

ATOM 22 2HG1 VAL A 2 -8.171 -0.259 -1.100 1.00 0.00 H

ATOM 23 3HG1 VAL A 2 -6.763 -1.289 -1.359 1.00 0.00 H

ATOM 24 1HG2 VAL A 2 -8.466 0.293 1.540 1.00 0.00 H

ATOM 25 2HG2 VAL A 2 -6.814 -0.065 2.043 1.00 0.00 H

ATOM 26 3HG2 VAL A 2 -8.150 -1.052 2.637 1.00 0.00 H

ATOM 27 N ARG A 3 -8.292 -3.256 -1.740 1.00 0.00 N

ATOM 28 CA ARG A 3 -7.971 -4.344 -2.645 1.00 0.00 C ATOM 29 C ARG A 3 -6.520 -4.785 -2.479 1.00 0.00 c

ATOM 30 O ARG A 3 -5.866 -4.443 -1.495 1.00 0.00 O

ATOM 31 CB ARG A 3 -8.238 -3.931 -4.093 1.00 0.00 c

ATOM 32 CG ARG A 3 -9.563 -3.209 -4.286 1.00 0.00 c

ATOM 33 CD ARG A 3 -10.155 -3.490 -5.657 1.00 0.00 c

ATOM 34 NE ARG A 3 -11.422 -2.790 -5.860 1.00 0.00 N

ATOM 35 CZ ARG A 3 -11.970 -2.581 -7.054 1.00 0.00 c

ATOM 36 NH1 ARG A 3 -11.368 -3.017 -8.154 1.00 0.00 N

ATOM 37 NH2 ARG A 3 -13.123 -1.934 -7.151 1.00 0.00 N

ATOM 38 H ARG A 3 -8.331 -2.339 -2.086 1.00 0.00 H

ATOM 39 HA ARG A 3 -8.617 -5.168 -2.389 1.00 0.00 H

ATOM 40 1HB ARG A 3 -7.445 -3.274 -4.421 1.00 0.00 H

ATOM 41 2HB ARG A 3 -8.241 -4.815 -4.714 1.00 0.00 H

ATOM 42 1HG ARG A 3 -10.256 -3.545 -3.529 1.00 0.00 H

ATOM 43 2HG ARG A 3 -9.401 -2.148 -4.183 1.00 0.00 H

ATOM 44 1HD ARG A 3 -9.454 -3.168 -6.411 1.00 0.00 H

ATOM 45 2HD ARG A ^' 3 -10.323 -4.553 -5.752 1.00 0.00 H

ATOM 46 HE ARG A 3 -11.888 -2.458 -5.064 1.00 0.00 H

ATOM 47 IHHl ARG A 3 -10.497 -3.506 -8.088 1.00 0.00 H

ATOM 48 2HH1 ARG A 3 -11.784 -2.858 -9.049 1.00 0.00 H

ATOM 49 1HH2 ARG A 3 -13.582 -1.605 -6.326 1.00 0.00 H

ATOM 50 2HH2 ARG A 3 -13.534 -1.777 -8.049 1.00 0.00 H

ATOM 51 N LEU A 4 -6.023 -5.552 -3.445 1.00 0.00 N

ATOM 52 CA LEU A 4 -4.657 -6.049 -3.403 1.00 0.00 c

ATOM 53 C LEU A 4 -3.655 -4.912 -3.579 1.00 0.00 c

ATOM 54 O LEU A 4 -2.627 -4.869 -2.904 1.00 0.00 O

ATOM 55 CB LEU A 4 -4.453 -7.095 -4.498 1.00 0.00 c

ATOM 56 CG LEU A 4 -3.030 -7.636 -4.611 1.00 0.00 c

ATOM 57 CD1 LEU A 4 -2.800 -8.754 -3.607 1.00 0.00 c

ATOM 58 CD2 LEU A 4 -2.756 -8.121 -6.027 1.00 0.00 c

ATOM 59 H LEU A 4 -6.593 -5.796 -4.201 1.00 0.00 H

ATOM 60 HA LEU A 4 -4.498 -6.509 -2.441 1.00 0.00 H

ATOM 61 1HB LEU A 4 -5.119 -7.921 -4.303 1.00 0.00 H

ATOM 62 2HB LEU A 4 -4.722 -6.653 -5.444 1.00 0.00 H

ATOM 63 HG LEU A 4 -2.339 -6.840 -4.390 1.00 0.00 H

ATOM 64 1HD1 LEU A 4 -1.768 -9.067 -3.646 1.00 0.00 H

ATOM 65 2HD1 LEU A 4 -3.439 -9.590 -3.847 1.00 0.00 H

ATOM 66 3HD1 LEU A 4 -3.031 -8.398 -2.613 1.00 0.00 H ATOM 67 1HD2 LEU A 4 -3.092 -9.142 -6.129 1.00 0.00 H

ATOM 68 2HD2 LEU A 4 -1.694 -8.068 -6.226 1.00 0.00 H

ATOM 69 3HD2 LEU A 4 -3.284 -7.495 -6.731 1.00 0.00 H

ATOM 70 N HIS A 5 -3.963 -3.995 -4.487 1.00 0.00 N

ATOM 71 CA HIS A 5 -3.096 -2.863 -4.747 1.00 0.00 C

ATOM 72 C HIS A 5 -3.884 -1.696 -5.334 1.00 0.00 C

ATOM 73 O HIS A 5 -3.679 -1.308 -6.484 1.00 0.00 O

ATOM 74 CB HIS A 5 -1.958 -3.263 -5.689 1.00 0.00 C

ATOM 75 CG HIS A 5 -2.420 -4.027 -6.891 1.00 0.00 C

ATOM 76 ND1 HIS A 5 -1.567 -4.754 -7.694 1.00 0.00 N

ATOM 77 CD2 HIS A 5 -3.655 -4.175 -7.426 1.00 0.00 C

ATOM 78 CE1 HIS A 5 -2.255 -5.317 -8.670 1.00 0.00 C

ATOM 79 NE2 HIS A 5 -3.524 -4.982 -8.530 1.00 0.00 N

ATOM 80 H HIS A 5 -4.790 -4.078 -4.985 1.00 0.00 H

ATOM 81 HA HIS A 5 -2.685 -2.559 -3.808 1.00 0.00 H

ATOM 82 1HB HIS A 5 -1.457 -2.372 -6.034 1.00 0.00 H

ATOM 83 2HB HIS A 5 -1.256 -3.881 -5.149 1.00 0.00 H

ATOM 84 HD1 HIS A 5 -0.598 -4.844 -7.567 1.00 0.00 H

ATOM 85 HD2 HIS A 5 -4.572 -3.740 -7.054 1.00 0.00 H

ATOM 86 HEl HIS A 5 -1.850 -5.946 -9.449 1.00 0.00 H

ATOM 87 HE2 HIS A 5 -4.265 -5.332 -9.068 1.00 0.00 H

ATOM 88 N GLU A 6 -4.784 -1.141 -4.532 1.00 0.00 N

ATOM 89 CA GLU A 6 -5.605 -0.015 -4.963 1.00 0.00 C

ATOM 90 C GLU A 6 -5.739 1.011 -3.845 1.00 0.00 C

ATOM 91 O GLU A 6 -5.364 0.752 -2.701 1.00 0.00 O

ATOM 92 CB GLU A 6 -6.988 -0.503 -5.397 1.00 0.00 C

ATOM 93 CG GLU A 6 -7.710 0.464 -6.322 1.00 0.00 C

ATOM 94 CD GLU A 6 -8.816 -0.205 -7.114 1.00 0.00 C

ATOM 95 OE1 GLU A 6 -8.635 -1.372 -7.517 1.00 0.00 O

ATOM 96 OE2 GLU A 6 -9.863 0.441 -7.332 1.00 0.00 O

ATOM 97 H GLU A 6 -4.899 -1.494 -3.625 1.00 0.00 H

ATOM 98 HA GLU A 6 -5.113 0.449 -5.804 1.00 0.00 H

ATOM 99 1HB GLU A 6 -6.879 -1.445 -5.911 1.00 0.00 H

ATOM 100 2HB GLU A 6 -7.597 -0.651 -4.518 1.00 0.00 H

ATOM 101 IHG GLU A 6 -8.142 1.256 -5.727 1.00 0.00 H

ATOM 102 2HG GLU A 6 -6.993 0.884 -7.013 1.00 0.00 H

ATOM 103 N SER A 7 -6.278 2.179 -4.180 1.00 0.00 N

ATOM 104 CA SER A 7 -6.462 3.245 -3.202 1.00 0.00 C ATOM 105 C SER A 7 -7.396 2.797 -2.083 1.00 0.00 C

ATOM 106 O SER A 7 -8.345 2.047 -2.316 1.00 0.00 O

ATOM 107 CB SER A 7 -7.018 4.497 -3.880 1.00 0.00 C

ATOM 108 OG SER A 7 -6.070 5.060 -4.769 1.00 0.00 O

ATOM 109 H SER A 7 -6.559 2.327 -5.108 1.00 0.00 H

ATOM 110 HA SER A 7 -5.496 3.473 -2.778 1.00 0.00 H

ATOM 111 1HB SER A 7 -7.905 4.237 -4.439 1.00 0.00 H

ATOM 112 2HB SER A 7 -7.269 5.230 -3.128 1.00 0.00 H

ATOM 113 HG SER A 7 -6.144 4.637 -5.629 1.00 0.00 H

ATOM 114 N CYS A 8 -7.124 3.260 -0.867 1.00 0.00 N

ATOM 115 CA CYS A 8 -7.941 2.904 0.286 1.00 0.00 C

ATOM 116 C CYS A 8 -8.771 4.094 0.762 1.00 0.00 C

ATOM 117 O CYS A 8 -9.807 3.916 1.401 1.00 0.00 O

ATOM 118 CB CYS A 8 -7.058 2.390 1.427 1.00 0.00 C

ATOM 119 SG CYS A 8 -5.978 3.656 2.170 1.00 0.00 s

ATOM 120 H CYS A 8 -6.353 3.853 -0.744 1.00 0.00 H

ATOM 121 HA CYS A 8 -8.611 2.117 -0.017 1.00 0.00 H

ATOM 122 1HB CYS A 8 -7.688 1.998 2.211 1.00 0.00 H

ATOM 123 2HB CYS A 8 -6.426 1.599 1.051 1.00 0.00 H

ATOM 124 N LEU A 9 -8.302 5.303 0.439 1.00 0.00 N

ATOM 125 CA LEU A 9 -8.981 6.552 0.818 1.00 0.00 C

ATOM 126 C LEU A 9 -9.886 6.374 2.037 1.00 0.00 C

ATOM 127 O LEU A 9 -11.044 5.977 1.911 1.00 0.00 O

ATOM 128 CB LEU A 9 -9.802 7.079 -0.360 1.00 0.00 C

ATOM 129 CG LEU A 9 -10.634 6.025 -1.093 1.00 0.00 C

ATOM 130 CD1 LEU A 9 -11.893 6.651 -1.675 1.00 0.00 C

ATOM 131 CD2 LEU A 9 -9.811 5.362 -2.186 1.00 0.00 C

ATOM 132 H LEU A 9 -7.471 5.361 -0.072 1.00 0.00 H

ATOM 133 HA LEU A 9 -8.219 7.277 1.059 1.00 0.00 H

ATOM 134 1HB LEU A 9 -10.469 7.845 0.009 1.00 0.00 H

ATOM 135 2HB LEU A 9 -9.124 7.528 -1.071 1.00 0.00 H

ATOM 136 HG LEU A 9 -10.935 5.263 -0.391 1.00 0.00 H

ATOM 137 1HD1 LEU A 9 -11.708 7.693 -1.894 1.00 0.00 H

ATOM 138 2HD1 LEU A 9 -12.698 6.571 -0.961 1.00 0.00 H

ATOM 139 3HD1 LEU A 9 -12.164 6.134 -2.583 1.00 0.00 H

ATOM 140 1HD2 LEU A 9 -8.792 5.249 -1.850 1.00 0.00 H

ATOM 141 2HD2 LEU A 9 -9.830 5.978 -3.074 1.00 0.00 H

ATOM 142 3HD2 LEU A 9 -10.227 4.393 -2.413 1.00 0.00 H ATOM 143 N GLY A 10 -9.348 6.673 3.215 1.00 0.00 N

ATOM 144 CA GLY A 10 -10.121 6.541 4.436 1.00 0.00 C

ATOM 145 C GLY A 10 -9.254 6.529 5.682 1.00 0.00 C

ATOM 146 O GLY A 10 -9.723 6.856 6.773 1.00 0.00 O

ATOM 147 H GLY A 10 -8.422 6.989 3.254 1.00 0.00 H

ATOM 148 IHA GLY A 10 -10.812 7.369 4.502 1.00 0.00 H

ATOM 149 2HA GLY A 10 -10.685 5.622 4.393 1.00 0.00 H

ATOM 150 N GLN A 11 -7.987 6.152 5.523 1.00 0.00 N

ATOM 151 CA GLN A 11 -7.049 6.098 6.646 1.00 0.00 C

ATOM 152 C GLN A 11 -7.329 4.901 7.553 1.00 0.00 C

ATOM 153 O GLN A 11 -6.435 4.097 7.819 1.00 0.00 O

ATOM 154 CB GLN A 11 -7.109 7.391 7.462 1.00 0.00 C

ATOM 155 CG GLN A 11 -7.285 8.643 6.616 1.00 0.00 C

ATOM 156 CD GLN A 11 -6.672 9.870 7.259 1.00 0.00 C

ATOM 157 OE1 GLN A 11 -5.574 9.813 7.813 1.00 0.00 O

ATOM 158 NE2 GLN A 11 -7.382 10.990 7.193 1.00 0.00 N

ATOM 159 H GLN A 11 -7.672 5.903 4.630 1.00 0.00 H

ATOM 160 HA GLN A 11 -6.059 5.995 6.238 1.00 0.00 H

ATOM 161 1HB GLN A 11 -7.936 7.326 8.146 1.00 0.00 H

ATOM 162 2HB GLN A 11 -6.193 7.488 8.025 1.00 0.00 H

ATOM 163 IHG GLN A 11 -6.813 8.483 5.658 1.00 0.00 H

ATOM 164 2HG GLN A 11 -8.341 8.817 6.471 1.00 0.00 H

ATOM 165 1HE2 GLN A 11 -8.249 10.961 6.738 1.00 0.00 H

ATOM 166 2HE2 GLN A 11 -7.010 11.799 7.601 1.00 0.00 H

ATOM 167 N GLN A 12 -8.564 4.785 8.029 1.00 0.00 N

ATOM 168 CA GLN A 12 -8.941 3.684 8.907 1.00 0.00 C

ATOM 169 C GLN A 12 -9.394 2.459 8.111 1.00 0.00 C

ATOM 170 O GLN A 12 -9.943 1.514 8.677 1.00 0.00 O

ATOM 171 CB GLN A 12 -10.056 4.121 9.865 1.00 0.00 C

ATOM 172 CG GLN A 12 -11.051 5.103 9.266 1.00 0.00 C

ATOM 173 CD GLN A 12 -11.598 4.640 7.930 1.00 0.00 C

ATOM 174 OE1 GLN A 12 -10.903 4.677 6.915 1.00 0.00 O

ATOM 175 NE2 GLN A 12 -12.851 4.199 7.924 1.00 0.00 N

ATOM 176 H GLN A 12 -9.236 5.452 7.790 1.00 0.00 H

ATOM 177 HA GLN A 12 -8.072 3.415 9.485 1.00 0.00 H

ATOM 178 1HB GLN A 12 -10.599 3.248 10.177 1.00 0.00 H

ATOM 179 2HB GLN A 12 -9.606 4.582 10.733 1.00 0.00 H

ATOM 180 IHG GLN A 12 -11.877 5.223 9.952 1.00 0.00 H ATOM 181 2HG GLN A 12 -10.559 6.055 9.126 1.00 0.00 H

ATOM 182 1HE2 GLN A 12 -13.347 4.199 8.771 1.00 0.00 H

ATOM 183 2HE2 GLN A 12 -13.231 3.892 7.074 1.00 0.00 H

ATOM 184 N VAL A 13 -9.160 2.473 6.800 1.00 0.00 N

ATOM 185 CA VAL A 13 -9.542 1.354 5.946 1.00 0.00 c

ATOM 186 C VAL A 13 -8.322 0.528 5.546 1.00 0.00 c

ATOM 187 O VAL A 13 -7.729 0.753 4.492 1.00 0.00 O

ATOM 188 CB VAL A 13 -10.263 1.836 4.669 1.00 0.00 c

ATOM 189 CGI VAL A 13 -10.752 0.652 3.847 1.00 0.00 c

ATOM 190 CG2 VAL A 13 -11.419 2.758 5.027 1.00 0.00 c

ATOM 191 H VAL A 13 -8.718 3.248 6.399 1.00 0.00 H

ATOM 192 HA VAL A 13 -10.221 0.725 6.502 1.00 0.00 H

ATOM 193 HB VAL A 13 -9.557 2.393 4.070 1.00 0.00 H

ATOM 194 IHGl VAL A 13 -11.816 0.529 3.990 1.00 0.00 H

ATOM 195 2HG1 VAL A 13 -10.241 -0.244 4.164 1.00 0.00 H

ATOM 196 3HG1 VAL A 13 -10.548 0.831 2.801 1.00 0.00 H

ATOM 197 1HG2 VAL A 13 -11.116 3.785 4.886 1.00 0.00 H

ATOM 198 2HG2 VAL A 13 -11.696 2.604 6.059 1.00 0.00 H

ATOM 199 3HG2 VAL A 13 -12.264 2.543 4.391 1.00 0.00 H

ATOM 200 N PRO A 14 -7.930 -0.446 6.386 1.00 0.00 N

ATOM 201 CA PRO A 14 -6.777 -1.305 6.110 1.00 0.00 c

ATOM 202 C PRO A 14 -7.034 -2.258 4.950 1.00 0.00 c

ATOM 203 O PRO A 14 -8.181 -2.502 4.578 1.00 0.00 O

ATOM 204 CB PRO A 14 -6.591 -2.086 7.411 1.00 0.00 c

ATOM 205 CG PRO A 14 -7.935 -2.085 8.053 1.00 0.00 c

ATOM 206 CD PRO A 14 -8.582 -0.784 7.665 1.00 0.00 c

ATOM 207 HA PRO A 14 -5.889 -0.723 5.908 1.00 0.00 H

ATOM 208 1HB PRO A 14 -6.261 -3.090 7.187 1.00 0.00 H

ATOM 209 2HB PRO A 14 -5.859 -1.592 8.030 1.00 0.00 H

ATOM 210 IHG PRO A 14 -8.518 -2.917 7.685 1.00 0.00 H

ATOM 211 2HG PRO A 14 -7.831 -2.146 9.126 1.00 0.00 H

ATOM 212 1HD PRO A 14 -9.646 -0.916 7.532 1.00 0.00 H

ATOM 213 2HD PRO A 14 -8.384 -0.027 8.409 1.00 0.00 H

ATOM 214 N CYS A 15 -5.961 -2.791 4.378 1.00 0.00 N

ATOM 215 CA CYS A 15 -6.075 -3.717 3.259 1.00 0.00 c

ATOM 216 C CYS A 15 -6.799 -4.995 3.672 1.00 0.00 c

ATOM 217 O CYS A 15 -6.585 -5.518 4.766 1.00 0.00 O

ATOM 218 CB CYS A 15 -4.694 -4.058 2.701 1.00 0.00 c ATOM 219 SG CYS A 15 -4.736 -4.924 1.101 1.00 0.00 s

ATOM 220 H CYS A 15 -5.072 -2.557 4.720 1.00 0.00 H

ATOM 221 HA CYS A 15 -6.652 -3.229 2.487 1.00 0.00 H

ATOM 222 1HB CYS A 15 -4.133 -3.147 2.567 1.00 0.00 H

ATOM 223 2HB CYS A 15 -4.177 -4.692 3.407 1.00 0.00 H

ATOM 224 N CYS A 16 -7.654 -5.495 2.785 1.00 0.00 N

ATOM 225 CA CYS A 16 -8.412 -6.714 3.043 1.00 0.00 C

ATOM 226 C CYS A 16 -7.515 -7.955 2.994 1.00 0.00 C

ATOM 227 O CYS A 16 -7.967 -9.063 3.287 1.00 0.00 O

ATOM 228 CB CYS A 16 -9.544 -6.852 2.023 1.00 0.00 C

ATOM 229 SG CYS A 16 -11.200 -7.104 2.750 1.00 0.00 s

ATOM 230 H CYS A 16 -7.774 -5.030 1.929 1.00 0.00 H

ATOM 231 HA CYS A 16 -8.838 -6.639 4.025 1.00 0.00 H

ATOM 232 1HB CYS A 16 -9.585 -5.961 1.417 1.00 0.00 H

ATOM 233 2HB CYS A 16 -9.334 -7.698 1.391 1.00 0.00 H

ATOM 234 N ASP A 17 -6.252 -7.772 2.614 1.00 0.00 N

ATOM 235 CA ASP A 17 -5.316 -8.888 2.521 1.00 0.00 C

ATOM 236 C ASP A 17 -4.478 -9.026 3.793 1.00 0.00 C

ATOM 237 O ASP A 17 -4.012 -8.032 4.356 1.00 0.00 O

ATOM 238 CB ASP A 17 -4.398 -8.705 1.311 1.00 0.00 C

ATOM 239 CG ASP A 17 -5.169 -8.577 0.014 1.00 0.00 C

ATOM 240 ODl ASP A 17 -6.127 -7.776 -0.030 1.00 0.00 O

ATOM 241 OD2 ASP A 17 -4.818 -9.278 -0.959 1.00 0.00 O

ATOM 242 H ASP A 17 -5.944 -6.872 2.383 1.00 0.00 H

ATOM 243 HA ASP A 17 -5.895 -9.789 2.384 1.00 0.00 H

ATOM 244 1HB ASP A 17 -3.807 -7.811 1.448 1.00 0.00 H

ATOM 245 2HB ASP A 17 -3.739 -9.559 1.236 1.00 0.00 H

ATOM 246 N PRO A 18 -4.267 -10.271 4.261 1.00 0.00 N

ATOM 247 CA PRO A 18 -3.475 -10.536 5.463 1.00 0.00 C

ATOM 248 C PRO A 18 -2.086 -9.917 5.375 1.00 0.00 C

ATOM 249 O PRO A 18 -1.417 -10.011 4.345 1.00 0.00 O

ATOM 250 CB PRO A 18 -3.381 -12.068 5.519 1.00 0.00 C

ATOM 251 CG PRO A 18 -3.806 -12.542 4.170 1.00 0.00 C

ATOM 252 CD PRO A 18 -4.767 -11.513 3.654 1.00 0.00 C

ATOM 253 HA PRO A 18 -3.973 -10.170 6.350 1.00 0.00 H

ATOM 254 1HB PRO A 18 -2.363 -12.360 5.737 1.00 0.00 H

ATOM 255 2HB PRO A 18 -4.038 -12.441 6.291 1.00 0.00 H

ATOM 256 IHG PRO A 18 -2.947 -12.613 3.519 1.00 0.00 H ATOM 257 2HG PRO A 18 -4.293 -13.501 4.254 1.00 0.00 H

ATOM 258 IHD PRO A 18 -4.726 -11.462 2.575 1.00 0.00 H

ATOM 259 2HD PRO A 18 -5.770 -11.730 3.988 1.00 0.00 H

ATOM 260 N CYS A 19 -1.661 -9.276 6.456 1.00 0.00 N

ATOM 261 CA CYS A 19 -0.355 -8.631 6.501 1.00 0.00 c

ATOM 262 C CYS A 19 -0.270 -7.482 5.495 1.00 0.00 c

ATOM 263 O CYS A 19 0.816 -6.977 5.211 1.00 0.00 O

ATOM 264 CB CYS A 19 0.753 -9.652 6.222 1.00 0.00 c

ATOM 265 SG CYS A 19 2.439 -9.011 6.483 1.00 0.00 s

ATOM 266 H CYS A 19 -2.242 -9.230 7.243 1.00 0.00 H

ATOM 267 HA CYS A 19 -0.218 -8.232 7.495 1.00 0.00 H

ATOM 268 1HB CYS A 19 0.620 -10.502 6.875 1.00 0.00 H

ATOM 269 2HB CYS A 19 0.679 -9.980 5.195 1.00 0.00 H

ATOM 270 N ALA A 20 -1.418 -7.065 4.959 1.00 0.00 N

ATOM 271 CA ALA A 20 -1.450 -5.971 3.995 1.00 0.00 c

ATOM 272 C ALA A 20 -2.007 -4.701 4.631 1.00 0.00 c

ATOM 273 O ALA A 20 -3.025 -4.738 5.322 1.00 0.00 O

ATOM 274 CB ALA A 20 -2.271 -6.363 2.777 1.00 0.00 c

ATOM 275 H ALA A 20 -2.259 -7.496 5.218 1.00 0.00 H

ATOM 276 HA ALA A 20 -0.435 -5.784 3.671 1.00 0.00 H

ATOM 277 1HB ALA A 20 -3.318 -6.393 3.041 1.00 0.00 H

ATOM 278 2HB ALA A 20 -1.960 -7.339 2.431 1.00 0.00 H

ATOM 279 3HB ALA A 20 -2.119 -5.638 1.992 1.00 0.00 H

ATOM 280 N THR A 21 -1.332 -3.580 4.395 1.00 0.00 N

ATOM 281 CA THR A 21 -1.761 -2.300 4.948 1.00 0.00 c

ATOM 282 C THR A 21 -1.668 -1.197 3.900 1.00 0.00 c

ATOM 283 O THR A 21 -0.790 -1.223 3.038 1.00 0.00 O

ATOM 284 CB THR A 21 -0.909 -1.938 6.166 1.00 0.00 c

ATOM 285 OG1 THR A 21 -1.401 -0.766 6.792 1.00 0.00 O

ATOM 286 CG2 THR A 21 0.547 -1.702 5.830 1.00 0.00 c

ATOM 287 H THR A 21 -0.526 -3.615 3.837 1.00 0.00 H

ATOM 288 HA THR A 21 -2.791 -2.400 5.257 1.00 0.00 H

ATOM 289 HB THR A 21 -0.957 -2.749 6.879 1.00 0.00 H

ATOM 290 HG1 THR A 21 -1.514 -0.074 6.135 1.00 0.00 H

ATOM 291 1HG2 THR A 21 0.824 -2.313 4.984 1.00 0.00 H

ATOM 292 2HG2 THR A 21 1.160 -1.965 6.680 1.00 0.00 H

ATOM 293 3HG2 THR A 21 0.697 -0.660 5.588 1.00 0.00 H

ATOM 294 N CYS A 22 -2.578 -0.231 3.975 1.00 0.00 N ATOM 295 CA CYS A 22 -2.593 0.876 3.026 1.00 0.00 C

ATOM 296 C CYS A 22 -1.412 1.813 3.253 1.00 0.00 C

ATOM 297 O CYS A 22 -1.294 2.439 4.306 1.00 0.00 O

ATOM 298 CB CYS A 22 -3.901 1.658 3.132 1.00 0.00 C

ATOM 299 SG CYS A 22 -4.104 2.941 1.855 1.00 0.00 s

ATOM 300 H CYS A 22 -3.254 -0.263 4.684 1.00 0.00 H

ATOM 301 HA CYS A 22 -2.519 0.461 2.035 1.00 0.00 H

ATOM 302 1HB CYS A 22 -4.731 0.972 3.045 1.00 0.00 H

ATOM 303 2HB CYS A 22 -3.940 2.141 4.095 1.00 0.00 H

ATOM 304 N TYR A 23 -0.542 1.906 2.253 1.00 0.00 N

ATOM 305 CA TYR A 23 0.632 2.766 2.333 1.00 0.00 C

ATOM 306 C TYR A 23 0.475 3.975 1.414 1.00 0.00 C

ATOM 307 O TYR A 23 -0.119 3.879 0.340 1.00 0.00 O

ATOM 308 CB TYR A 23 1.891 1.978 1.958 1.00 0.00 C

ATOM 309 CG TYR A 23 3.143 2.827 1.888 1.00 0.00 C

ATOM 310 CD1 TYR A 23 3.810 3.214 3.043 1.00 0.00 C

ATOM 311 CD2 TYR A 23 3.656 3.238 0.664 1.00 0.00 C

ATOM 312 CE1 TYR A 23 4.953 3.988 2.982 1.00 0.00 C

ATOM 313 CE2 TYR A 23 4.799 4.012 0.595 1.00 0.00 C

ATOM 314 CZ TYR A 23 5.444 4.383 1.756 1.00 0.00 C

ATOM 315 OH TYR A 23 6.582 5.155 1.691 1.00 0.00 O

ATOM 316 H TYR A 23 -0.695 1.383 1.439 1.00 0.00 H ATOM 317 HA TYR A 23 0.721 3.110 3.352 1.00 0.00 H

ATOM 318 1HB TYR A 23 2.056 1.207 2.695 1.00 0.00 H

ATOM 319 2HB TYR A 23 1.745 1.521 0.990 1.00 0.00 H

ATOM 320 HDl TYR A 23 3.423 2.901 4.001 1.00 0.00 H

ATOM 321 HD2 TYR A 23 3.150 2.947 -0.244 1.00 0.00 H

ATOM 322 HEl TYR A 23 5.457 4.279 3.891 1.00 0.00 H

ATOM 323 HE2 TYR A 23 5.184 4.323 -0.367 1.00 0.00 H

ATOM 324 HH TYR A 23 7.126 4.862 0.957 1.00 0.00 H

ATOM 325 N CYS A 24 1.016 5.111 1.846 1.00 0.00 N

ATOM 326 CA CYS A 24 0.939 6.340 1.066 1.00 0.00 C

ATOM 327 C CYS A 24 2.304 6.702 0.490 1.00 0.00 C

ATOM 328 O CYS A 24 3.292 6.786 1.220 1.00 0.00 O

ATOM 329 CB CYS A 24 0.418 7.487 1.932 1.00 0.00 C

ATOM 330 SG CYS A 24 -1.207 7.168 2.691 1.00 0.00 s

ATOM 331 H CYS A 24 1.476 5.122 2.711 1.00 0.00 H

ATOM 332 HA CYS A 24 0.249 6.173 0.251 1.00 0.00 H ATOM 333 1HB CYS A 24 1.121 7.673 2.730 1.00 0.00 H

ATOM 334 2HB CYS A 24 0.329 8.375 1.325 1.00 0.00 H

ATOM 335 N ARG A 25 2.356 6.913 -0.822 1.00 0.00 N

ATOM 336 CA ARG A 25 3.604 7.265 -1.486 1.00 0.00 C

ATOM 337 C ARG A 25 4.021 8.698 -1.170 1.00 0.00 C

ATOM 338 O ARG A 25 5.153 9.100 -1.439 1.00 0.00 O

ATOM 339 CB ARG A 25 3.483 7.070 -2.992 1.00 0.00 C

ATOM 340 CG ARG A 25 4.456 6.045 -3.554 1.00 0.00 C

ATOM 341 CD ARG A 25 5.307 6.628 -4.673 1.00 0.00 C

ATOM 342 NE ARG A 25 4.492 7.237 -5.725 1.00 0.00 N

ATOM 343 CZ ARG A 25 4.271 8.546 -5.838 1.00 0.00 C

ATOM 344 NH1 ARG A 25 4.785 9.398 -4.957 1.00 0.00 N

ATOM 345 NH2 ARG A 25 3.530 9.007 -6.836 1.00 0.00 N

ATOM 346 H ARG A 25 1.537 6.832 -1.354 1.00 0.00 H

ATOM 347 HA ARG A 25 4.361 6.607 -1.118 1.00 0.00 H

ATOM 348 1HB ARG A 25 2.482 6.744 -3.217 1.00 0.00 H

ATOM 349 2HB ARG A 25 3.664 8.013 -3.479 1.00 0.00 H

ATOM 350 IHG ARG A 25 5.108 5.711 -2.761 1.00 0.00 H

ATOM 351 2HG ARG A 25 3.896 5.206 -3.940 1.00 0.00 H

ATOM 352 IHD ARG A 25 5.964 7.376 -4.258 1.00 0.00 H

ATOM 353 2HD ARG A 25 5.899 5.834 -5.105 1.00 0.00 H

ATOM 354 HE ARG A 25 4.092 6.637 -6.388 1.00 0.00 H

ATOM 355 IHHl ARG A 25 5.340 9.063 -4.199 1.00 0.00 H

ATOM 356 2HH1 ARG A 25 4.612 10.378 -5.054 1.00 0.00 H

ATOM 357 1HH2 ARG A 25 3.138 8.372 -7.502 1.00 0.00 H

ATOM 358 2HH2 ARG A 25 3.363 9.989 -6.923 1.00 0.00 H

ATOM 359 N PHE A 26 3.103 9.457 -0.593 1.00 0.00 N

ATOM 360 CA PHE A 26 3.368 10.838 -0.231 1.00 0.00 C

ATOM 361 C PHE A 26 2.610 11.196 1.043 1.00 0.00 C

ATOM 362 O PHE A 26 2.227 10.315 1.814 1.00 0.00 O

ATOM 363 CB PHE A 26 2.964 11.769 -1.380 1.00 0.00 C

ATOM 364 CG PHE A 26 4.046 12.730 -1.783 1.00 0.00 C

ATOM 365 CD1 PHE A 26 4.284 13.002 -3.120 1.00 0.00 C

ATOM 366 CD2 PHE A 26 4.824 13.363 -0.826 1.00 0.00 C

ATOM 367 CE1 PHE A 26 5.279 13.884 -3.496 1.00 0.00 C

ATOM 368 CE2 PHE A 26 5.820 14.246 -1.195 1.00 0.00 C

ATOM 369 CZ PHE A 26 6.047 14.508 -2.532 1.00 0.00 C

ATOM 370 H PHE A 26 2.225 9.077 -0.401 1.00 0.00 H ATOM 371 HA PHE A 26 4.427 10.938 -0.049 1.00 0.00 H

ATOM 372 1HB PHE A 26 2.711 11.173 -2.243 1.00 0.00 H

ATOM 373 2HB PHE A 26 2.099 12.346 -1.083 1.00 0.00 H

ATOM 374 HDl PHE A 26 3.684 12.516 -3.875 1.00 0.00 H

ATOM 375 HD2 PHE A 26 4.647 13.158 0.221 1.00 0.00 H

ATOM 376 HEl PHE A 26 5.457 14.087 -4.542 1.00 0.00 H

ATOM 377 HE2 PHE A 26 6.419 14.732 -0.439 1.00 0.00 H

ATOM 378 HZ PHE A 26 6.825 15.198 -2.822 1.00 0.00 H

ATOM 379 N PHE A 27 2.389 12.484 1.258 1.00 0.00 N

ATOM 380 CA PHE A 27 1.674 12.953 2.425 1.00 0.00 C

ATOM 381 C PHE A 27 0.292 12.313 2.499 1.00 0.00 C

ATOM 382 O PHE A 27 0.004 11.530 3.404 1.00 0.00 O

ATOM 383 CB PHE A 27 1.552 14.479 2.388 1.00 0.00 C

ATOM 384 CG PHE A 27 2.188 15.144 1.197 1.00 0.00 C

ATOM 385 CD1 PHE A 27 3.479 15.642 1.273 1.00 0.00 C

ATOM 386 CD2 PHE A 27 1.495 15.270 0.004 1.00 0.00 C

ATOM 387 CE1 PHE A 27 4.067 16.253 0.182 1.00 0.00 C

ATOM 388 CE2 PHE A 27 2.077 15.878 -1.092 1.00 0.00 C

ATOM 389 CZ PHE A 27 3.364 16.372 -1.002 1.00 0.00 C

ATOM 390 H PHE A 27 2.704 13.137 0.616 1.00 0.00 H

ATOM 391 HA PHE A 27 2.240 12.666 3.299 1.00 0.00 H

ATOM 392 1HB PHE A 27 0.513 14.742 2.382 1.00 0.00 H

ATOM 393 2HB PHE A 27 2.015 14.880 3.269 1.00 0.00 H

ATOM 394 HDl PHE A 27 4.030 15.550 2.199 1.00 0.00 H

ATOM 395 HD2 PHE A 27 0.489 14.884 -0.068 1.00 0.00 H

ATOM 396 HEl PHE A 27 5.073 16.637 0.254 1.00 0.00 H

ATOM 397 HE2 PHE A 27 1.527 15.970 -2.015 1.00 0.00 H

ATOM 398 HZ PHE A 27 3.822 16.848 -1.857 1.00 0.00 H

ATOM 399 N ASN A 28 -0.555 12.648 1.532 1.00 0.00 N

ATOM 400 CA ASN A 28 -1.907 12.105 1.474 1.00 0.00 C

ATOM 401 C ASN A 28 -2.437 12.127 0.043 1.00 0.00 C

ATOM 402 O ASN A 28 -3.223 13.002 -0.322 1.00 0.00 O

ATOM 403 CB ASN A 28 -2.840 12.901 2.391 1.00 0.00 C

ATOM 404 CG ASN A 28 -2.653 14.399 2.249 1.00 0.00 C

ATOM 405 ODl ASN A 28 -1.863 15.009 2.970 1.00 0.00 O

ATOM 406 ND2 ASN A 28 -3.381 15.001 1.317 1.00 0.00 N

ATOM 407 H ASN A 28 -0.262 13.273 0.836 1.00 0.00 H

ATOM 408 HA ASN A 28 -1.868 11.081 1.815 1.00 0.00 H ATOM 409 1HB ASN A 28 -3.864 12.661 2.146 1.00 0.00 H

ATOM 410 2HB ASN A 28 -2.646 12.626 3.417 1.00 0.00 H

ATOM 411 1HD2 ASN A 28 -3.990 14.452 0.779 1.00 0.00 H

ATOM 412 2HD2 ASN A 28 -3.280 15.969 1.203 1.00 0.00 H

ATOM 413 N ALA A 29 -2.002 11.166 -0.766 1.00 0.00 N

ATOM 414 CA ALA A 29 -2.439 11.092 -2.155 1.00 0.00 C

ATOM 415 C ALA A 29 -2.410 9.657 -2.673 1.00 0.00 C

ATOM 416 O ALA A 29 -3.456 9.057 -2.917 1.00 0.00 O

ATOM 417 CB ALA A 29 -1.571 11.985 -3.028 1.00 0.00 C

ATOM 418 H ALA A 29 -1.370 10.496 -0.422 1.00 0.00 H

ATOM 419 HA ALA A 29 -3.453 11.460 -2.205 1.00 0.00 H

ATOM 420 1HB ALA A 29 -1.817 11.822 -4.068 1.00 0.00 H

ATOM 421 2HB ALA A 29 -0.530 11.747 -2.865 1.00 0.00 H

ATOM 422 3HB ALA A 29 -1.748 13.020 -2.773 1.00 0.00 H

ATOM 423 N PHE A 30 -1.210 9.116 -2.842 1.00 0.00 N

ATOM 424 CA PHE A 30 -1.053 7.753 -3.336 1.00 0.00 C

ATOM 425 C PHE A 30 -1.161 6.744 -2.196 1.00 0.00 C

ATOM 426 O PHE A 30 -0.212 6.017 -1.903 1.00 0.00 O

ATOM 427 CB PHE A 30 0.293 7.598 -4.048 1.00 0.00 C

ATOM 428 CG PHE A 30 0.239 7.930 -5.513 1.00 0.00 C

ATOM 429 GDI PHE A 30 -0.238 9.157 -5.942 1.00 0.00 C

ATOM 430 CD2 PHE A 30 0.667 7.012 -6.459 1.00 0.00 C

ATOM 431 CE1 PHE A 30 -0.288 9.465 -7.289 1.00 0.00 C

ATOM 432 CE2 PHE A 30 0.621 7.315 -7.807 1.00 0.00 C

ATOM 433 CZ PHE A 30 0.142 8.542 -8.222 1.00 0.00 C

ATOM 434 H PHE A 30 -0.411 9.645 -2.633 1.00 0.00 H

ATOM 435 HA PHE A 30 -1.848 7.564 -4.042 1.00 0.00 H

ATOM 436 1HB PHE A 30 1.015 8.254 -3.586 1.00 0.00 H

ATOM 437 2HB PHE A 30 0.627 6.575 -3.950 1.00 0.00 H

ATOM 438 HDl PHE A 30 -0.575 9.880 -5.213 1.00 0.00 H

ATOM 439 HD2 PHE A 30 1.041 6.052 -6.136 1.00 0.00 H

ATOM 440 HEl PHE A 30 -0.662 10.426 -7.610 1.00 0.00 H

ATOM 441 HE2 PHE A 30 0.956 6.591 -8.535 1.00 0.00 H

ATOM 442 HZ PHE A 30 0.105 8.780 -9.276 1.00 0.00 H

ATOM 443 N CYS A 31 -2.327 6.707 -1.557 1.00 0.00 N

ATOM 444 CA CYS A 31 -2.563 5.790 -0.448 1.00 0.00 C

ATOM 445 C CYS A 31 -3.314 4.548 -0.921 1.00 0.00 C

ATOM 446 O CYS A 31 -4.529 4.582 -1.107 1.00 0.00 O ATOM 447 CB CYS A 31 -3.354 6.487 0.658 1.00 0.00 C

ATOM 448 SG CYS A 31 -2.525 7.950 1.359 1.00 0.00 s

ATOM 449 H CYS A 31 -3.045 7.312 -1.839 1.00 0.00 H

ATOM 450 HA CYS A 31 -1.602 5.488 -0.058 1.00 0.00 H

ATOM 451 1HB CYS A 31 -4.306 6.809 0.261 1.00 0.00 H

ATOM 452 2HB CYS A 31 -3.525 5.788 1.463 1.00 0.00 H

ATOM 453 N TYR A 32 -2.581 3.455 -1.109 1.00 0.00 N

ATOM 454 CA TYR A 32 -3.182 2.203 -1.560 1.00 0.00 C

ATOM 455 C TYR A 32 -2.630 1.021 -0.770 1.00 0.00 C

ATOM 456 O TYR A 32 -1.607 1.136 -0.097 1.00 0.00 O

ATOM 457 CB TYR A 32 -2.933 1.994 -3.056 1.00 0.00 C

ATOM 458 CG TYR A 32 -1.568 2.454 -3.520 1.00 0.00 c

ATOM 459 CD1 TYR A 32 -0.413 2.005 -2.892 1.00 0.00 c

ATOM 460 CD2 TYR A 32 -1.437 3.336 -4.584 1.00 0.00 c

ATOM 461 CE1 TYR A 32 0.837 2.423 -3.314 1.00 0.00 c

ATOM 462 CE2 TYR A 32 -0.192 3.757 -5.011 1.00 0.00 c

ATOM 463 CZ TYR A 32 0.940 3.299 -4.374 1.00 0.00 c

ATOM 464 OH TYR A 32 2.182 3.717 -4.797 1.00 0.00 O

ATOM 465 H TYR A 32 -1.616 3.491 -0.942 1.00 0.00 H

ATOM 466 HA TYR A 32 -4.246 2.268 -1.389 1.00 0.00 H

ATOM 467 1HB TYR A 32 -3.022 0.942 -3.285 1.00 0.00 H

ATOM 468 2HB TYR A 32 -3.676 2.542 -3.616 1.00 0.00 H

ATOM 469 HDl TYR A 32 -0.497 1.318 -2.062 1.00 0.00 H

ATOM 470 HD2 TYR A 32 -2.326 3.694 -5.082 1.00 0.00 H

ATOM 471 HEl TYR A 32 1.723 2.062 -2.814 1.00 0.00 H

ATOM 472 HE2 TYR A 32 -0.112 4.445 -5.842 1.00 0.00 H

ATOM 473 HH TYR A 32 2193 4.675 -4.859 1.00 0.00 H

ATOM 474 N CYS A 33 -3.317 -0.116 -0.854 1.00 0.00 N

ATOM 475 CA CYS A 33 -2.895 -1.319 -0.141 1.00 0.00 c

ATOM 476 C CYS A 33 -1.444 -1.671 -0.460 1.00 0.00 c

ATOM 477 O CYS A 33 -0.976 -1.477 -1.582 1.00 0.00 O

ATOM 478 CB CYS A 33 -3.810 -2.497 -0.489 1.00 0.00 c

ATOM 479 SG CYS A 33 -3.186 -4.122 0.064 1.00 0.00 s

ATOM 480 H CYS A 33 -4.127 -0.145 -1.406 1.00 0.00 H

ATOM 481 HA CYS A 33 -2.975 -1.118 0.918 1.00 0.00 H

ATOM 482 1HB CYS A 33 -4.773 -2.344 -0.029 1.00 0.00 H

ATOM 483 2HB CYS A 33 -3.933 -2.542 -1.562 1.00 0.00 H

ATOM 484 N ARG A 34 -0.747 -2.197 0.540 1.00 0.00 N ♦

ATOM 485 CA ARG A 34 0.647 -2.591 0.386 1.00 0.00 C

ATOM 486 C ARG A 34 0.907 -3.912 1.103 1.00 0.00 C

ATOM 487 O ARG A 34 0.720 -4.018 2.316 1.00 0.00 O

ATOM 488 CB ARG A 34 1.573 -1.506 0.936 1.00 0.00 C

ATOM 489 CG ARG A 34 3.050 -1.838 0.796 1.00 0.00 C

ATOM 490 CD ARG A 34 3.891 -0.582 0.635 1.00 0.00 C

ATOM 491 NE ARG A 34 5.318 -0.855 0.790 1.00 0.00 N

ATOM 492 CZ ARG A 34 6.078 -1.392 -0.162 1.00 0.00 C

ATOM 493 NH1 ARG A 34 5.551 -1.715 -1.337 1.00 0.00 N

ATOM 494 NH2 ARG A 34 7.366 -1.606 0.062 1.00 0.00 N

ATOM 495 H ARG A 34 -1.183 -2.327 1.407 1.00 0.00 H

ATOM 496 HA ARG A 34 0.842 -2.721 -0.669 1.00 0.00 H

ATOM 497 1HB ARG A 34 1.382 -0.584 0.408 1.00 0.00 H

ATOM 498 2HB ARG A 34 1.357 -1.361 1.984 1.00 0.00 H

ATOM 499 IHG ARG A 34 3.375 -2.365 1.681 1.00 0.00 H

ATOM 500 2HG ARG A 34 3.187 -2.466 -0.072 1.00 0.00 H

ATOM 501 IHD ARG A 34 3.717 -0.172 -0.349 1.00 0.00 H

ATOM 502 2HD ARG A 34 3.587 0.137 1.381 1.00 0.00 H

ATOM 503 HE ARG A 34 5.731 -0.626 1.648 1.00 0.00 H

ATOM 504 IHHl ARG A 34 4.580 -1.557 -1.513 1.00 0.00 H

ATOM 505 2HH1 ARG A 34 6.128 -2.118 -2.048 1.00 0.00 H

ATOM 506 1HH2 ARG A 34 7.767 -1.363 0.946 1.00 0.00 H

ATOM 507 2HH2 ARG A 34 7.937 -2.009 -0.653 1.00 0.00 H

ATOM 508 N LYS A 35 1.330 -4.919 0.345 1.00 0.00 N

ATOM 509 CA LYS A 35 1.609 -6.237 0.908 1.00 0.00 C

ATOM 510 C LYS A 35 3.021 -6.317 1.486 1.00 0.00 C

ATOM 511 O LYS A 35 3.507 -7.404 1.797 1.00 0.00 O

ATOM 512 CB LYS A 35 1.423 -7.316 -0.160 1.00 0.00 C

ATOM 513 CG LYS A 35 0.015 -7.886 -0.206 1.00 0.00 C

ATOM 514 CD LYS A 35 -0.268 -8.568 -1.535 1.00 0.00 C

ATOM 515 CE LYS A 35 0.516 -9.866 -1.674 1.00 0.00 C

ATOM 516 NZ LYS A 35 -0.380 -11.052 -1.750 1.00 0.00 N

ATOM 517 H LYS A 35 1.454 -4.775 -0.617 1.00 0.00 H

ATOM 518 HA LYS A 35 0.899 -6.410 1.703 1.00 0.00 H

ATOM 519 lHB LYS A 35 1.649 -6.893 -1.127 1.00 0.00 H

ATOM 520 2HB LYS A 35 2110 -8.125 0.039 1.00 0.00 H

ATOM 521 IHG LYS A 35 -0.098 -8.608 0.589 1.00 0.00 H

ATOM 522 2HG LYS A 35 -0.694 -7.082 -0.067 1.00 0.00 H ATOM 523 IHD LYS A 35 -1.322 -8.790 -1.598 1.00 0.00 H

ATOM 524 2HD LYS A 35 0.012 -7.902 -2.338 1.00 0.00 H

ATOM 525 1HE LYS A 35 1.109 -9.817 -2.576 1.00 0.00 H

ATOM 526 2HE LYS A 35 1.169 -9.973 -0.820 1.00 0.00 H

ATOM 527 1HZ LYS A 35 -0.019 -11.814 -1.140 1.00 0.00 H

ATOM 528 2HZ LYS A 35 -0.430 -11.401 -2.728 1.00 0.00 H

ATOM 529 3HZ LYS A ^' 35 -1.340 -10.797 -1.436 1.00 0.00 H

ATOM 530 N LEU A 36 3.679 -5.167 1.633 1.00 0.00 N

ATOM 531 CA LEU A 36 5.025 -5128 2.175 1.00 0.00 C

ATOM 532 C LEU A 36 5.990 -5.915 1.305 1.00 0.00 C

ATOM 533 O LEU A 36 5.918 -7.141 1.223 1.00 0.00 O

ATOM 534 CB LEU A 36 5.039 -5.664 3.604 1.00 0.00 C

ATOM 535 CG LEU A 36 4.725 -4.611 4.663 1.00 0.00 C

ATOM 536 GDI LEU A 36 3.342 -4.840 5.256 1.00 0.00 C

ATOM 537 CD2 LEU A 36 5.783 -4.613 5.756 1.00 0.00 C

ATOM 538 H LEU A 36 3.251 -4.329 1.374 1.00 0.00 H

ATOM 539 HA LEU A 36 5.339 -4.096 2.190 1.00 0.00 H

ATOM 540 1HB LEU A 36 4.308 -6.458 3.678 1.00 0.00 H

ATOM 541 2HB LEU A 36 6.017 -6.072 3.807 1.00 0.00 H

ATOM 542 HG LEU A 36 4.730 -3.640 4.190 1.00 0.00 H

ATOM 543 1HD1 LEU A 36 2.594 -4.694 4.490 1.00 0.00 H

ATOM 544 2HD1 LEU A 36 3.176 -4.138 6.060 1.00 0.00 H

ATOM 545 3HD1 LEU A 36 3.276 -5.848 5.638 1.00 0.00 H

ATOM 546 1HD2 LEU A 36 5.340 -4.294 6.687 1.00 0.00 H

ATOM 547 2HD2 LEU A 36 6.581 -3.934 5.486 1.00 0.00 H

ATOM 548 3HD2 LEU A 36 6.183 -5.609 5.870 1.00 0.00 H

ATOM 549 N GLY A 37 6.890 -5.193 0.658 1.00 0.00 N

ATOM 550 CA GLY A 37 7.871 -5.823 -0.206 1.00 0.00 C

ATOM 551 C GLY A 37 9.003 -6.472 0.567 1.00 0.00 C

ATOM 552 O GLY A 37 10.174 -6191 0.315 1.00 0.00 O

ATOM 553 H GLY A 37 6.888 -4.219 0.770 1.00 0.00 H

ATOM 554 IHA GLY A 37 7.376 -6.578 -0.800 1.00 0.00 H

ATOM 555 2HA GLY A 37 8.284 -5.076 -0.868 1.00 0.00 H

ATOM 556 N THR A 38 8.654 -7.347 1.506 1.00 0.00 N

ATOM 557 CA THR A 38 9.649 -8.042 2.312 1.00 0.00 C

ATOM 558 C THR A 38 10.046 -9.357 1.655 1.00 0.00 C

ATOM 559 O THR A 38 9.603 -10.429 2.070 1.00 0.00 O

ATOM 560 CB THR A 38 9.110 -8.298 3.720 1.00 0.00 C ATOM 561 OG1 THR A 38 7.895 -9.024 3.669 1.00 0.00 O

ATOM 562 CG2 THR A 38 8.854 -7.029 4.505 1.00 0.00 C

ATOM 563 H THR A 38 7.707 -7.534 1.658 1.00 0.00 H

ATOM 564 HA THR A 38 10.522 -7.408 2.379 1.00 0.00 H

ATOM 565 HB THR A 38 9.832 -8.885 4.269 1.00 0.00 H

ATOM 566 HG1 THR A 38 7.196 -8.459 3.332 1.00 0.00 H

ATOM 567 1HG2 THR A 38 8.717 -6.205 3.820 1.00 0.00 H

ATOM 568 2HG2 THR A 38 9.698 -6.827 5.147 1.00 0.00 H

ATOM 569 3HG2 THR A 38 7.965 -7.150 5.106 1.00 0.00 H

ATOM 570 N ALA A 39 10.878 -9.267 0.625 1.00 0.00 N

ATOM 571 CA ALA A 39 11.335 -10.449 -0.099 1.00 0.00 C

ATOM 572 C ALA A 39 12.107 -11.402 0.810 1.00 0.00 C

ATOM 573 O ALA A 39 12.308 -12.568 0.469 1.00 0.00 O

ATOM 574 CB ALA A 39 12.191 -10.040 -1.288 1.00 0.00 C

ATOM 575 H ALA A 39 11.190 -8.381 0.342 1.00 0.00 H

ATOM 576 HA ALA A 39 10.464 -10.959 -0.474 1.00 0.00 H

ATOM 577 1HB ALA A 39 11.591 -9.475 -1.985 1.00 0.00 H

ATOM 578 2HB ALA A 39 12.577 -10.922 -1.775 1.00 0.00 H

ATOM 579 3HB ALA A 39 13.014 -9.429 -0.946 1.00 0.00 H

ATOM 580 N MET A 40 12.536 -10.904 1.963 1.00 0.00 N

ATOM 581 CA MET A 40 13.283 -11.718 2.914 1.00 0.00 C

ATOM 582 C MET A 40 12.334 -12.525 3.796 1.00 0.00 C

ATOM 583 O MET A 40 12.618 -13.671 4.143 1.00 0.00 O

ATOM 584 CB MET A 40 14.178 -10.833 3.783 1.00 0.00 C

ATOM 585 CG MET A 40 15.537 -11.448 4.081 1.00 0.00 C

ATOM 586 SD MET A 40 16.877 -10.242 4.016 1.00 0.00 s

ATOM 587 CE MET A 40 16.815 -9.759 2.292 1.00 0.00 C

ATOM 588 H MET A 40 12.347 -9.970 2.179 1.00 0.00 H

ATOM 589 HA MET A 40 13.902 -12.400 2.352 1.00 0.00 H

ATOM 590 1HB MET A 40 14.336 -9.892 3.277 1.00 0.00 H

ATOM 591 2HB MET A 40 13.679 -10.645 4.722 1.00 0.00 H

ATOM 592 IHG MET A 40 15.512 -11.882 5.069 1.00 0.00 H

ATOM 593 2HG MET A 40 15.733 -12.222 3.353 1.00 0.00 H

ATOM 594 1HE MET A 40 16.197 -8.880 2.186 1.00 0.00 H

ATOM 595 2HE MET A 40 16.398 -10.566 1.708 1.00 0.00 H

ATOM 596 3HE MET A 40 17.814 -9.541 1.943 1.00 0.00 H

ATOM 597 N ASN A 41 11.206 -11.920 4.149 1.00 0.00 N

ATOM 598 CA ASN A 41 10.211 -12.581 4.986 1.00 0.00 C ATOM 599 C ASN A 41 8.806 -12.072 4.664 1.00 0.00 C

ATOM 600 O ASN A 41 8.174 -11.402 5.481 1.00 0.00 O

ATOM 601 CB ASN A 41 10.529 -12.356 6.467 1.00 0.00 C

ATOM 602 CG ASN A 41 11.147 -13.578 7.117 1.00 0.00 C

ATOM 603 ODl ASN A 41 11.058 -14.688 6.591 1.00 0.00 O

ATOM 604 ND2 ASN A 41 11.780 -13.382 8.268 1.00 0.00 N

ATOM 605 H ASN A 41 11.035 -11.007 3.837 1.00 0.00 H

ATOM 606 HA ASN A 41 10.253 -13.639 4.775 1.00 0.00 H

ATOM 607 1HB ASN A 41 11.223 -11.534 6.559 1.00 0.00 H

ATOM 608 2HB ASN A 41 9.618 -12.114 6.994 1.00 0.00 H

ATOM 609 1HD2 ASN A 41 11.812 -12.471 8.629 1.00 0.00 H

ATOM 610 2HD2 ASN A 41 12.189 -14.156 8.710 1.00 0.00 H

ATOM 611 N PRO A 42 8.299 -12.384 3.459 1.00 0.00 N

ATOM 612 CA PRO A 42 6.966 -11.956 3.028 1.00 0.00 C

ATOM 613 C PRO A 42 5.855 -12.784 3.665 1.00 0.00 C

ATOM 614 O PRO A 42 6.027 -13.974 3.927 1.00 0.00 O

ATOM 615 CB PRO A 42 7.002 -12.182 1.518 1.00 0.00 C

ATOM 616 CG PRO A 42 7.954 -13.313 1.332 1.00 0.00 C

ATOM 617 CD PRO A 42 8.987 -13.177 2.420 1.00 0.00 C

ATOM 618 HA PRO A 42 6.800 -10.909 3.235 1.00 0.00 H

ATOM 619 1HB PRO A 42 6.013 -12.435 1.165 1.00 0.00 H

ATOM 620 2HB PRO A 42 7.351 -11.289 1.023 1.00 0.00 H

ATOM 621 IHG PRO A 42 7.431 -14.252 1.428 1.00 0.00 H

ATOM 622 2HG PRO A 42 8.422 -13.243 0.362 1.00 0.00 H

ATOM 623 IHD PRO A 42 9.265 -14.149 2.800 1.00 0.00 H

ATOM 624 2HD PRO A 42 9.857 -12.655 2.051 1.00 0.00 H

ATOM 625 N CYS A 43 4.715 -12.147 3.909 1.00 0.00 N

ATOM 626 CA CYS A 43 3.582 -12.799 4.505 1.00 0.00 C

ATOM 627 C CYS A 43 2.788 -13.563 3.458 1.00 0.00 C

ATOM 628 O CYS A 43 3.287 -14.505 2.841 1.00 0.00 O

ATOM 629 CB CYS A 43 2.717 -11.741 5.173 1.00 0.00 C

ATOM 630 SG CYS A 43 3.620 -10.655 6.325 1.00 0.00 s

ATOM 631 H CYS A 43 4.627 -11.204 3.684 1.00 0.00 H

ATOM 632 HA CYS A 43 3.923 -13.485 5.246 1.00 0.00 H

ATOM 633 1HB CYS A 43 2.274 -11.119 4.409 1.00 0.00 H

ATOM 634 2HB CYS A 43 1.942 -12.227 5.718 1.00 0.00 H

ATOM 635 N SER A 44 1.554 -13.151 3.270 1.00 0.00 N

ATOM 636 CA SER A 44 0.670 -13.781 2.308 1.00 0.00 C ATOM 637 C SER A 44 0.936 -13.263 0.900 1.00 0.00 c

ATOM 638 O SER A 44 0.285 -12.326 0.439 1.00 0.00 O

ATOM 639 CB SER A 44 -0.791 -13.539 2.688 1.00 0.00 c

ATOM 640 OG SER A 44 -1.145 -14.269 3.851 1.00 0.00 O

ATOM 641 H SER A 44 1.231 -12.404 3.801 1.00 0.00 H

ATOM 642 HA SER A 44 0.869 -14.835 2.337 1.00 0.00 H

ATOM 643 1HB SER A 44 -0.943 -12.488 2.879 1.00 0.00 H

ATOM 644 2HB SER A 44 -1.429 -13.852 1.874 1.00 0.00 H

ATOM 645 HG SER A 44 -1.908 -14.821 3.663 1.00 0.00 H

ATOM 646 N ARG A 45 1.898 -13.880 0.219 1.00 0.00 N

ATOM 647 CA ARG A 45 2.250 -13.481 -1.138 1.00 0.00 c

ATOM 648 C ARG A 45 2.882 -14.639 -1.904 1.00 0.00 c

ATOM 649 O ARG A 45 3.804 -14.443 -2.697 1.00 0.00 O

ATOM 650 CB ARG A 45 3.211 -12.291 -1.108 1.00 0.00 c

ATOM 651 CG ARG A 45 3.124 -11.405 -2.341 1.00 0.00 c

ATOM 652 CD ARG A 45 4.407 -11.447 -3.153 1.00 0.00 c

ATOM 653 NE ARG A 45 5.470 -10.655 -2.540 1.00 0.00 N

ATOM 654 CZ ARG A 45 6.607 -10.337 -3.156 1.00 0.00 c

ATOM 655 NH1 ARG A 45 6.833 -10.743 -4.399 1.00 0.00 N

ATOM 656 NH2 ARG A 45 7.520 -9.611 -2.526 1.00 0.00 N

ATOM 657 H ARG A 45 2.382 -14.621 0.639 1.00 0.00 H

ATOM 658 HA ARG A 45 1.343 -13.184 -1.643 1.00 0.00 H

ATOM 659 1HB ARG A 45 2.988 -11.685 -0.241 1.00 0.00 H

ATOM 660 2HB ARG A 45 4.222 -12.661 -1.027 1.00 0.00 H

ATOM 661 IHG ARG A 45 2.307 -11.746 -2.960 1.00 0.00 H

ATOM 662 2HG ARG A 45 2.939 -10.387 -2.028 1.00 0.00 H

ATOM 663 IHD ARG A 45 4.737 -12.473 -3.230 1.00 0.00 H

ATOM 664 2HD ARG A 45 4.206 -11.061 -4.142 1.00 0.00 H

ATOM 665 HE ARG A 45 5.330 -10.342 -1.622 1.00 0.00 H

ATOM 666 IHHl ARG A 45 6.148 -11.291 -4.880 1.00 0.00 H

ATOM 667 2HH1 ARG A 45 7.688 -10.501 -4.856 1.00 0.00 H

ATOM 668 1HH2 ARG A 45 7.356 -9.303 -1.590 1.00 0.00 H

ATOM 669 2HH2 ARG A 45 8.375 -9.373 -2.989 1.00 0.00 H

ATOM 670 N THR A 46 2.382 -15.846 -1.661 1.00 0.00 N

ATOM 671 CA THR A 46 2.899 -17.036 -2.328 1.00 0.00 c

ATOM 672 C THR A 46 2.368 -17.133 -3.754 1.00 0.00 c

ATOM 673 O THR A 46 2.909 -17.946 -4.533 1.00 0.00 O

ATOM 674 CB THR A 46 2.522 -18.293 -1.543 1.00 0.00 c ATOM 675 OG1 THR A 46 2.802 -18.127 -0.165 1.00 0.00 O

ATOM 676 CG2 THR A 46 3.250 -19.534 -2.014 1.00 0.00 c

ATOM 677 OXT THR A 46 1.413 -16.397 -4.081 1.00 0.00 O

ATOM 678 H THR A 46 1.648 -15.940 -1.019 1.00 0.00 H

ATOM 679 HA THR A 46 3.976 -16.954 -2.363 1.00 0.00 H

ATOM 680 HB THR A 46 1.461 -18.470 -1.656 1.00 0.00 H

ATOM 681 HG1 THR A 46 2.011 -17.825 0.289 1.00 0.00 H

ATOM 682 1HG2 THR A 46 3.933 -19.271 -2.809 1.00 0.00 H

ATOM 683 2HG2 THR A 46 2.534 -20.255 -2.378 1.00 0.00 H

ATOM 684 3HG2 THR A 46 3.803 -19.960 -1.191 1.00 0.00 H

TER 685 THR A 46

Example 3 Screening of mini-AGRPs for melanocortin Receptor activity. The mini-AGRP, given by the sequence: Ac-

CVRLHESCLGQQVPCCDPAATCYCRFFNAFCYCR-NH₂ (SEQ DD NO: >) was synthesized using Fmoc solid phase chemistry (where Ac is an acetyl group and -NH₂ denotes a C-terminal amide). The peptide was prepared as a single chain and then partially purified by HPLC. The peptide was then folded under oxidizing conditions to form the disulfide bonds, and then repurified. HPLC indicated a uniform product and mass spectrometry demonstrated loss of 8.0 AMUs consistent with formation of four disulfide bonds. The folding yield was greater than 80% as determined by HPLC.

Amino acid analysis verified the correct omposition and allowed for the preparation of stock solutions of known mini-AGRP concentration (to within 5%). The mini-AGRP binding was tested by examining its ability to displace

NDP-MSH, a well established high affinity MCR agonist. To within experimental error, the mini-AGRP displaced NDP-MSH at each of the melanocortin receptors (MC1R, MC3R, MC4R and MC5R) with potency equivalent to that of AGRP(87-132) (MARP). Antagonist activity was then determined using a cAMP assay. With both brain receptors MC3R and MC4R, mini-AGRP exhibited potent activity as determined by its ability to suppress - MSH stimulated cAMP production. Taken together, these studies demonstrate that this mini-AGRP is readily synthesized, folded to a uniform product, and exhibits MCR binding, selectivity and antagonist activity comparable to that of the AGRP C-terminal domain (AGRP(87-132); MARP ).

Example 4 Synthesis and Assay of Melanocortin Receptor Binding Ligands (Peptidomimetics)

A) Synthesis of non-peptide Compounds

A synthesis scheme for the peptidomimetics is illustrated in Figure 4. To 20% Piperidine/DMF 2 x 5min treated and DMF washed Rink Amide-MBHA resin (Novabiochem, Switzerland), 5 equivalents of Fmoc-Aminoisobutyric acid (Fmoc-Aib) or 7 of Bromoacetic acid (Aldrich, USA) in DMF, activated as the symmetric anhydride by incubating 30 min. with Diisopropylcarbodiimide (DIC), is reacted for 20 min., then washed with DMF. The washed resin, in the case of Fmoc-Aib treated with 20% Piperidine/DMF 2 x 5 min. and DMF washed, is reacted with 20-40 equivalents, concentration 2-4 M, of primary or secondary amine or bromide of choice in DMSO or DMF with the appropriate tertiary amine (note: methylamine is only available as a 2M solution in various solvents). This is known as the submonomer approach (Zuckermann et al., JAmer Chem Soc 1992). The process is repeated 1-2 times, until yielding a complete molecule, or intermediate. To the intermediate, 7 equivalents of bromoacetic acid or 5-8 equivalents of a carboxylic acid of choice, activated as the symmetric anhydride by incubating 30 min. with Diisopropylcarbodiimide (DIC) or as the assymetric anhydride, in the case of an enantiomeric carboxylic acid, is reacted for 0.5-2 hr., then washed with DMF. If the intermidiate is brominated then reacted with 20 equivalents of the thiol or 2-4 M of the primary or secondary amine of choice, if the thiol or amine contains one or more accessible amine then the carboxylic acid of choice is coupled or the amine is guanylated. If the intermediate has an accessible amine then one of the following may happen: 1) alkylation by the primary or secondary bromide of choice 2) guanylation 3) a carboxylic acid of choice is coupled. The reaction of amines with 3,5-Dimethylpyrazole-l-carboxamidine nitrate or O-rnethylisourea is well known in the art. The resulting non-peptides are cleaved, usually, but not limited to, by exposing the resin bound molecules to 95:5 TFA:H₂O. The cleavage from Resin is done in 95:2.5:2.5 TFA:TIS: water. B) Activity Assay

HEK 293 cells stably expressing the human melanocortin receptors (hMCR's) 1-5 are grown in media. After removal from media, cells are washed twice with MEM (Life Technologies, Gaithersberg, MD) and then preincubated with AGRP (87-132) in 0.5 ml MEM 0.2% BSA for 30 min. before incubation with ¹²⁵I-NDP-MSH or 125 I- AGRP (87-132). Concentrations of cold ligand are added from 100 mM - 1 nM. Binding reactions are terminated by washing cells twice with MEM 0.2% BSA. The cells are lysed with 1 N NaOH 1% Triton X, and radioactivity of the lysate is quantified with a gamma- analytical counter. AGRP (87-132) hMC4R IC₅₀ = 9 nM ± 1.7 nM. IC₅₀ < 100 mM is considered a specific binder

It is apparent from the above results and discussion that the subject invention provides an important new class of MCR ligands, where the ligands have broad ranging applicability as either MCR antagonists and agonists and therefore find use in a variety of therapeutic and related applications. It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

Claims

CLAIMSWhat is claimed is;

1. A method of modulating the activity of a melanocortin receptor, said method comprising contacting said receptor with a peptide having the formula: CX¹X²X³X⁴X⁵X⁶CX⁷X⁸X⁹X¹⁰X¹¹X¹²CCDPX¹³ATCYCX¹ X¹⁵X¹⁶NAFCYCR_rι wherein

X¹, X², X³, X⁴, X⁵, X⁶, X⁷, X⁸, X⁹, X¹⁰, X¹¹, X¹², X¹³, X¹⁴, X¹⁵, and X¹⁶ are independently selected amino acids, and n is zero or one.

2. The method of claim 1, wherein X¹, X², X³, X⁴, X⁵, X⁶, X⁷, X⁸, X⁹,

X¹⁰, X¹¹, X¹², X¹³, X¹⁴, X¹⁵, and X¹⁶ are independently selected from the group consisting of alanine, asparagine, arginine,aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

3. The method of claim 1, wherein said peptide is not

CVRLHESCLGQQVPCCDPAATCYCRFFNAFCYC (SEQ ID NO:3).

4. The method of claim 1 , wherein X^x x is VRLHES or conservative substitutions thereof.

5. The method of claim 4, wherein X^^x x⁶ is VRLHES.

6. The method of claim 1 , wherein X⁷X⁸X⁹X¹⁰XⁿX¹² is LGQQVP or conservative substitutions thereof.

7. The method of claim 6, wherein X⁷X⁸X⁹X¹⁰X^πX¹² is LGQQVP.

8. The method of claim 7, wherein X⁷X⁸X⁹X¹⁰X^πX¹² is LGQQVP or conservative substitutions thereof.

9. The method of claim 8, wherein X⁷X⁸X⁹X¹⁰X^{1 l}Xⁿ is LGQQVP.

10. The method of claim 1, wherein X¹³ is not a cysteine.

11. The method of claim 1 , wherein X¹³ is A.

12. The method of claim 1, wherein X¹⁴X¹⁵X¹⁶ is RFF or conservative substitutions thereof.

13. The method of claim 4, wherein X¹⁴X¹⁵X¹⁶ is RFF or conservative substitutions thereof.

14. The method of claim 8, wherein X¹⁴X¹⁵X¹⁶ is RFF or conservative substitutions thereof.

15. The method of claim 1, wherein said receptor is in a cell culture.

16. The method of claim 1, wherein said receptor is in vivo culture.

17. The method of claim 1, wherein said receptor is an MC3 receptor.

18. The method of claim 1, wherein said receptor is an MC4 receptor.

19. A library for screening for modulators of a melanocortin receptor, said library comprising a plurality of polypeptide members wherein said members have the formula:

CX¹X²X³X⁴X⁵X⁶CX⁷X⁸X⁹X¹⁰X¹¹X¹²CCDPX¹³ATCYCX¹⁴X¹⁵X¹⁶NAFCYCR_n wherein

X¹, X², X³, X⁴, X⁵, X⁶, X⁷, X⁸, X⁹, X¹⁰, X¹¹, X¹², X¹³, X¹⁴, X¹⁵, and

X¹⁶ are independently selected amino acids, and n is zero or one.

20. The library of claim 19, wherein X¹, X², X³, X⁴, X⁵, X⁶, X⁷, X⁸, X⁹, X¹⁰, X¹¹, X¹², X¹³, X¹⁴, X¹⁵, and X¹⁶ are independently selected from the group consisting of aspartic acid, alanine, asparagine, arginine,cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

21. The method of claim 19, wherein X^x x^⁶ is VRLHES or conservative substitutions thereof.

22. The method of claim 21, wherein X^ x⁴^ is VRLHES.

23. The method of claim 19, wherein X⁷X⁸X⁹X¹⁰X^πX¹² is LGQQVP or conservative substitutions thereof.

24. The method of claim 23, wherein X⁷X⁸X⁹X¹⁰X^πX¹² is LGQQVP.

25. The method of claim 24, wherein X⁷X⁸X⁹X¹⁰X^πX¹² is LGQQVP or conservative substitutions thereof.

26. The method of claim 25, wherein X⁷X⁸X⁹X¹⁰X^πX¹² is LGQQVP.

27. The method of claim 19, wherein X¹³ is not a cysteine.

28. The method of claim 19, wherein X¹³ is A.

29. The method of claim 19, wherein X¹⁴X¹⁵X¹⁶ is RFF or conservative substitutions thereof.

30. The method of claim 21 , wherein X¹⁴X¹⁵X¹⁶ is RFF or conservative substitutions thereof.

31. The method of claim 25, wherein X¹⁴X¹⁵X¹⁶ is RFF or conservative substitutions thereof.

32. A method of prescreening for a modulator of a melanocortin receptor, said method comprising: i) contacting a melanocortin receptor a peptide having the formula:

CX¹X²X³X⁴X⁵X⁶CX⁷X⁸X⁹X¹⁰X¹¹X¹²CCDPX¹³ATCYCX¹⁴X¹⁵X¹⁶NAFCYCR_n wherein

X¹, X², X³, X⁴, X⁵, X⁶, X⁷, X⁸, X⁹, X¹⁰, X¹¹, X¹², X¹³, X¹⁴, X¹⁵, and X¹⁶ are independently selected amino acids, and n is zero or one; and ii) detecting binding of said peptide to said melanocortin receptor wherein specific binding of said peptide to said melanocortin receptor indicates that said peptide is a potential modulator of said melanocortin receptor.

33. The method of claim 32, wherein said peptide is not CVRLHESCLGQQVPCCDPAATCYCRFFNAFCYC (SEQ ID NO:3).

34. The method of claim 32, wherein said melanocortin receptor is selected from the group consisting of MC3r, and MC4r.

35. A method of screening for a modulator of melanocortin receptor activity, said method comprising: i) contacting a melanocortin receptor with a peptide having the formula:

CX¹X²X³X⁴X⁵X⁶CX⁷X⁸X⁹X¹⁰X¹¹X¹²CCDPX¹³ATCYCX¹⁴X¹⁵X¹⁶NAFCYCR_n wherein

X¹, X², X³, X⁴, X⁵, X⁶, X⁷, X⁸, X⁹, X¹⁰, X", X¹², X¹³, X¹⁴, X¹⁵, and X¹⁶ are independently selected amino acids, and n is zero or one; and ii) detecting activity of said melanocortin receptor wherein a difference in activity of said receptor, as compared to a control, indicates that said peptide is a modulator of melanocortin receptor activity.

36. The method of claim 35, wherein said control is a negative control comprising the same assay without said peptide.

37. The method of claim 35, wherein said peptide is not CVRLHESCLGQQVPCCDPAATCYCRFFNAFCYC (SEQ DD NO:3).

38. The method of claim 35, wherein said melanocortin receptor is selected from the group consisting of MC3r, and MC4r.

39. A polypeptide comprising a peptide sequence having the formula 35wherein said polypeptide is not AGRP and said polypeptide is not MARP.

40. The polypeptide of claim 39, wherein said polypeptide excludes one or more of the final 13 residues of MARP (residues 34-46 of MARP).

41. The polypeptide of claim 39, wherein said polyypeptide has the formula: CX¹X²X³X⁴X⁵X⁶CX⁷X⁸X⁹X¹⁰X¹¹X¹²CCDPX¹³ATCYCX¹⁴X^:L5X¹⁶NAFCYCR_n wherein

X¹, X², X³, X⁴, X⁵, X⁶, X⁷, X⁸, X⁹, X¹⁰, X¹¹, X¹², X¹³, X¹⁴, X¹⁵, and X¹⁶ are independently selected amino acids, and n is zero or one.

42. The polypeptide of claim 41, wherein said polypeptide is not

CVRLHESCLGQQVPCCDPAATCYCRFFNAFCYC (SEQ DD NO:3).

43. A pharmaceutical composition comprising: a pharmaceutically acceptable excipient; and a polypeptide having the formula: CX¹X X³X⁴X⁵X⁶CX⁷X⁸X⁹X¹⁰XⁿX¹²CCDPX¹³ATCYCX¹⁴X¹⁵X¹⁶NAFCYCR_n wherein

X¹, X², X³, X⁴, X⁵, X⁶, X⁷, X⁸, X⁹, X¹⁰, X¹¹, X¹², X¹³, X¹⁴, X¹⁵, and X are independently selected amino acids, and n is zero or one.

44. The composition of claim 43, wherein said polypeptide is not

CVRLHESCLGQQVPCCDPAATCYCRFFNAFCYC (SEQ DD NO:3).

45. A non-peptide melanocortin receptor ligand of the stractural formula:

wherein B, Ui, U₂, R, Ri, and R₂ are independently selected from the group consisting of: hydrogen, alkyl, derivatized alkyl, cycloalkyl, derivatized cycloalkyl, halocycloalkyl, aloxycycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl, and heteroarylalkyl; J is selected from the group consisting of carbon, nitrogen, silicon, and sulfur;

X is selected from the group consisting of hydrogen, carbon, nitrogen, oxygen, silicon, and sulfur; and

Z is selected from the group consisting of a continuing peptide bond, a hydroxyl; -NH₂-, -NH-(n), and -N-(n,n'), and -O-(y), where where n and n' are independently selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, and a derivatized form thereof, and y is selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, aryl, arylalkyl, heteroaryl heteroarylalkyl, and a derivatized form thereof.

46. The non-peptide melanocortin receptor ligand according to claim 45, wherein said ligand is a ligand for a melanocortin receptor selected from the group consisting of MC3r and MC4r.

47. ^• The non-peptide melanocortin receptor ligand according to claim 45, wherein said ligand has a molecular weight ranging from about 200 to 1000 daltons.

48. The non-peptide melanocortin receptor ligand according to claim 45, wherein said ligand has a structure that mimics the backbone of the AGRP active loop.

49. The non-peptide melanocortin receptor ligand according to claim 45, wherein said ligand comprises a terminal gaunidino moiety.

50. The non-peptide melanocortin receptor ligand according to claim 45, wherein said ligand comprises at least one methylbenzyl moiety.

51. The non-peptide melanocortin receptor ligand according to claim 45, wherein said ligand has the structural formula:

52. The non-peptide melanocortin receptor ligand according to claim 45, wherein said ligand has the structural formula:

53. The non-peptide melanocortin receptor ligand according to claim 45, wherein said ligand has the structural formula:

54. The non-peptide melanocortin receptor ligand according to claim 45, wherein said ligand has the structural formula:

55. A pharmaceutical preparation of a non-peptide melanocortin receptor ligand according to claim 45.

56. The pharmaceutical preparation according to claim 55, wherein said ligand is a melanocortin receptor antagonist.

57. The pharmaceutical preparation according to claim 56, wherein said ligand is a melanocortin receptor agonist.

58. A method for modulating a melanocortin receptor mediated physiological process, said method comprising: contacting said melanocortin receptor with a non-peptide melanocortin receptor ligand according to claim 45.

59. The method according to claim 58, wherein said ligand is a melanocortin receptor agonist.

60. The method according to claim 58, wherein said ligand is a melanocortin receptor antagonist.