WO2008015276A1

WO2008015276A1 - Prostaglandin-binding pocket of ppar protein and use thereof

Info

Publication number: WO2008015276A1
Application number: PCT/EP2007/058076
Authority: WO
Inventors: Anders Svensson; Michel Fodje
Original assignee: Novo Nordisk AS
Current assignee: Novo Nordisk AS
Priority date: 2006-08-03
Filing date: 2007-08-03
Publication date: 2008-02-07
Anticipated expiration: 2009-02-03

Abstract

Described are methods for evaluating the ability of a chemical entity to associate with a prostaglandin-binding pocket or part of a prostaglandin-binding pocket of a PPAR, PPARγ or PPARγ-like protein using structure coordinates of the prostaglandin-binding pocket as described herein, as well as methods for identifying, selecting, designing and/or optimizing chemical entities binding PPAR, PPARγ or PPARγ-like protein using such coordinates, as well as computer systems comprising data storage media comprising data defining such coordinates.

Description

PROSTAGLANDIN-BINDING POCKET OF PPAR PROTEIN AND USE THEREOF

FIELD OF THE INVENTION

The present invention relates to methods of using the structure coordinates of a pros- taglandin-binding pocket in PPARγ. The invention provides a three-dimensional model of the prostaglandin-binding pocket and means for identifying, designing, screening, evaluating, selecting and/or optimising of chemical entities by rational drug design.

BACKGROUND OF THE INVENTION

The peroxisome proliferator-activated receptors, PPARα, PPARy, and PPARδ, are examples of orphan nuclear receptors (ONR) involved in transcriptional regulation of metabolic pathways. The nuclear receptor PPARγ is a central regulator of adipose tissue development and an important modulator of gene expression in a number of specialized cell types including adipocytes, epithelial cells, and macrophages. PPARγ signaling pathways impact both cellular and systemic lipid metabolism and have links to obesity, diabetes, and cardiovascular disease (Walczak et al., 2002; Willson et al., 2000). In addition, a dependence between PPARγ activation by 15-Deoxy Δ¹²'¹⁴-prostaglandin J₂, a natural ligand to PPARγ, and a reduction in cancer development (Nosjean and Boutin, 2002; Sawai et al., 2006; Coyle et al., 2005) have been shown. Thiazolidinediones (TZDs) like Rosiglitazone, originally discovered to possess the ability to lower blood glucose and used in treatment of type 2 diabetes, also may inhibit growth and invasiveness of cancer cell lines (Ferruzzi et al., 2005; Han and Roman, 2006).

The crystals structure of the ligand binding domain (LBD) of PPARγ in complex with

Rosiglitazone has been reported (Nolte et al., 1998) and coordinates, accession number 2PRG, have been deposited in the Protein Data Bank (PDB, www.rcsb.org/pdb; Berman et al., 2000). In the same paper (Nolte et al., 1998) the apo structure of the LBD PPARγ is described and the coordinates deposited in the PDB with the accession number IPRG.

SUMMARY OF THE INVENTION

The present invention provides methods for evaluating the ability of a chemical entity to associate with a prostaglandin-binding pocket or part of a prostaglandin-binding pocket of a PPARγ or PPARγ-like protein using structure coordinates of the prostaglandin-binding pocket as described herein, as well as methods for identifying, selecting, designing and/or optimizing chemical entities binding PPARγ or PPARγ-like protein using such coordinates, as well as computer systems comprising data storage media comprising data defining such coordinates.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 lists the atomic structure coordinates of one of the crystallographic independent molecules of the prostaglandin B₂-complex with LBD PPARy (chains P and A, respectively) as derived by X-ray diffraction. A residue type of "E2P" indicates the prostaglandin B₂ molecule while that of "D" indicates a dimethyl sulfoxide (DMSO) molecule. The following abbreviations are used in Figure 1 : "Atom type "refers to the element whose coordinates are measured. The first letter in the column defines the element. The "#"sign is an abbreviation for "number" in a numbering of atoms and residues. Residues further have a chain name indicated by a letter in front of the digits as part of the residue numbering. "Resid" refers to the amino acid residue identity in the molecular model. "X, Y, Z" define the atomic position of the element measured. "B" is a thermal factor that measures movement of the atom around its atomic center. "Occ" is an occupancy factor that refers to the fraction of the molecules in which each atom occu- pies the position specified by the coordinates. A value of "1" indicates that the atom has the same position in all symmetry related molecules of the crystal.

Figure 2 lists the atomic structure coordinates of the second crystallographic independent molecule of the prostaglandin B₂ and LBD PPARy-complex as (chains P and B, respectively) derived by X-ray diffraction. A residue type of "E2P" indicates the prostaglandin B₂ molecule. The abbreviations are the same as those in Figure 1.

Figure 3 shows a detailed representation of the prostaglandin B₂-binding part of the PPARγ prostaglandin-binding pocket, in complex with prostaglandin B₂. In black; the prostaglandin B₂ molecule, labelled PGB₂, binding to the PPARγ A molecule (Figure 1). In light and dark gray; the C_α-trace of the PPARγ A molecule. Dark gray colored residues were, by the CONTACT soft- ware program, calculated to be within 5 A distance from the bound Prostaglandin B₂ molecule, and are labelled with their residue type and number according to Figure 1. Light gray coloured residues are more than 5 A away from the bound prostaglandin B₂ molecule and are not labelled.

DETAILED DESCRIPTION OF THE INVENTION

In order that the invention described herein may be more fully understood, the following detailed description is set forth. The present invention is based, in part, on the discovery of a prostaglandin-binding pocket in the PPARY structure. Briefly, as described in Example 1, from re-refinement of the so called apo-LBD-PPARγ structure (Nolte et al., 1998; deposited under accession number IPRG in the Protein Data Bank), it was discovered that the PPARγ protein molecule was not in an apo state, but actually bound a ligand. The ligand was recognized to be a prostaglandin molecule; prostaglandin B₂, which is closely related to the natural PPARγ ligand, 15-Deoxy Δ¹²'¹⁴- prostaglandin J₂. Further, based on the close contact between atom 012 of the prostaglandin B2 molecule and the main-chain oxygen of the LEU 228 residue of the PPARy molecule (Figure 3), it was discovered that the enol form of the prostaglandin B₂ molecule could make a hydro- gen bond to the main chain oxygen of LEU 228. It was also found that the keto form could form a hydrogen-bond to the main-chain amide hydrogen of LEU 228. As shown in Figure 3, adjacent residues also involved in the interaction with the central ring part of the Prostaglandin B2 molecule included PHE 226, PRO 227, and MET 329. Without being limited to theory, Leu-228-based interactions could be mechanisms by which other prostaglandin such as, e.g., 15-deoxy-Δ¹²'¹⁴-Prostaglandin J₂, or novel prostaglandin-like chemical entities could bind to the PPAR ligand-binding domain (LBD) described herein.

The binding site of the prostaglandin molecule was found to differ from the Rosiglitazone binding site (Nolte et al., 1998), and only a few atoms of one of the hydrocarbon tails of the prostaglandin molecule were found to reach the Rosiglitazone binding site. Simplified, the complete binding pocket could be described as T-shaped (Nolte et al., 1998), with Rosiglitazone situated in the vertical part of the T-shaped pocket and prostaglandin B₂ situated in the one of side bars of the horizontal part of the T-shape.

The prostaglandin-binding pocket of PPARy was also discovered to comprise parts not immediately occupied by prostaglandin B2, comprising residues ILE 296, ILE 325, THR 328, ALA 331, PHE 374, ASN 375, ALA 376, LEU 377, GLU 378, LEU 379, ASP 380 and LEU 435. The residues defining these parts could be useful in designing novel chemical entities targeting the prostaglandin-binding pocket described herein, or the LBD of PPARy or a PPARy-like protein.

The following abbreviations are used throughout the application: A = Ala = Alanine; T = Thr = Threonine; V = VaI = Valine ; C = Cys = Cysteine; L = Leu = Leucine ; Y = Tyr = Tyrosine ; I

= He = Isoleucine ; N = Asn = Asparagine P = Pro = Proline; Q = GIn = Glutamine; F = Phe

= Phenylalanine; D = Asp = Aspartic Acid; W = Trp = Tryptophan; E = GIu = Glutamic Acid;

M = Met = Methionine; K = Lys = Lysine; G = GIy = Glycine; R = Arg = Arginine; S = Ser =

Serine; and H = His = Histidine. As used herein, the following definitions shall apply unless otherwise indicated.

Throughout the specification, the word "comprise", or variations such as "comprises" or "comprising" will be understood to imply the inclusion of a stated integer or groups of integers but not exclusion of any other integer or groups of integers.

All residue numbers of the PPARγ structure described in the present patent specification use the numbering scheme as in Figure 1 and SEQ ID NO: 1). SEQ ID NO: 1 is the full-length sequence of mature PPARγ, corresponding to SwissProt entry UNIPROT: P37231, identifier PPARG_HUMAN, Version 87 without the 28-amino acid signal sequence. SEQ ID NO:2 is the LBD PPARγ sequence of Chain A, also shown by structure coordinates in Figure 1. SEQ ID NO: 3 is the LBD PPARγ sequence of Chain B, as also shown by structure coordinates in Figure 2. SEQ ID NO:2 (Chain A) and 3 (Chain B) have the same amino acid sequence. The first amino acid residue of SEQ ID NOS:2 and 3, a GIu residue, corresponds to residue number 207 in SEQ ID NO: 1, which also is seen in Figure 1. The last amino acid of SEQ ID NOS:2 and 3 corresponds to residue 476 of SEQ ID NO: lThe term "about" when used in the context of RMSD values takes into consideration the standard error of the RMSD value, which is ±0.1 A.

The term "associating with" refers to a condition of proximity between a chemical entity or compound, or portions thereof, and a prostaglandin-binding pocket or binding site on a protein. The association may be non-covalent wherein the juxtaposition is energetically favoured by hydrogen bonding or by van der Waals or electrostatic interactions or it may be covalent.

The term "to quantify" refers in the present context to compile calculated values from the computational calculations of said fitting operation between the chemical entity and the prostaglandin-binding pocket.

As examples of quantification of association mention can be made of the two computer programmes FlexX (A fast flexible docking method using an incremental construction algorithm. M. Rarey, B. Kramer, T. Lengauer, and G. Klebe. Journal of Molecular Biology, 261(3) :470- 489, 1996) and GOLD (Development and Validation of a Genetic Algorithm for Flexible Docking. G. Jones, P. Willett, R. C. Glen, A. R. Leach and R. Taylor, Journal of Molecular Biology, 267, 727-748, 1997). FlexX calculates a binding energy (expressed in kJ/mol with smaller, more negative energies being more favourable, -10 kJ/mol or less being a typical value for an association between a chemical entity and a binding pocket), whereas GOLD calculates only a binding score (expressed in unitless numbers, with higher numbers being more favourable). The term "PPAR protein" refers to orphan nuclear receptors (ONR) from the orphan nuclear receptor family. Examples of this family of orphan nuclear receptors include but are not limited to PPARα, PPARy, and PPARδ.

"Prostaglandin-binding pocket" refers to a binding pocket of a molecule or molecular complex defined by the structure coordinates of a certain set of amino acid residues present in the

PPARγ or PPARγ-like protein structure, as described below. In general, the ligand for the prostaglandin-binding pocket is a lipid such as prostaglandin. In one aspect of the invention, the prostaglandin-binding pocket is located in the ligand binding domain (LBD) of the PPARγ molecule. In the protein molecule, PPARγ, the prostaglandin-binding pocket is buried between the following structure elements (numbering as in Nolte et al, 1998 incorporated herein by reference) : The loop between helix 1 and 2, the loop between strand 3 and 4, and between helices 3, 5, 8 and 10. The LBD of the PPARγ protein comprises amino acid residues 207-476 of SEQ ID NO: 1, corresponding to SEQ ID NO:2 and 3.

The term "prostaglandin" designates a member of a group of lipid compounds, prostaglandins, that are derived from fatty acids. Prostaglandins contain 20 carbon atoms of which 5 forms a ring structure which may also contain double bonds, a ketone, or alcohol groups. They are also unsaturated carboxylic acids. Prostaglandins are mediators with strong physiological effects.

The term "PPARγ-like" refers to all or a portion of a molecule or molecular complex that has a commonality of shape and/or sequence identity to all or a portion of the PPARγ protein. Typically, a PPARy-like protein comprises a sequence segment which is at least 65% identical to the LBD of PPARy, i.e., residues 207-476 of SEQ ID NO: 1. In specific and separate embodiments, the sequence identity between a sequence segment of a PPARy-like protein and the LBD of PPARy is at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%. In the segment or portion of the PPARy-like protein aligned with the LBD of PPARy, at least 3, alternatively all four, of PHE 226, PRO 227, LEU 228, and MET 329 may also be conserved. Additionally, in a PPARγ-like prostaglandin-binding pocket, the commonality of shape may be defined by a root mean square deviation of the structure coordinates of the backbone atoms between the amino acids in a PPARγ-like prostaglandin-binding pocket and the amino acids in the PPARγ prostaglandin-binding pocket (as set forth in Figures 1 or 2). Compared to an amino acid in the PPARγ prostaglandin-binding pocket, the corresponding amino acids in the PPARγ-like prostaglandin-binding pocket may or may not be identical. Depending on the PPARγ amino acid residues that define the PPARγ prostaglandin binding pocket, one skilled in the art would be able to locate the corresponding amino acid residues that define a PPARγ-like- prostaglandin binding pocket in a protein based upon sequence and structural homology. In one aspect of the invention, the PPARγ-like protein is a PPARγ homologue.

The term "PPARγ protein complex" or "PPARγ homologue complex" refers to a molecular complex formed by associating the PPARγ protein or PPARγ homologue with a chemical entity.

The term "binding pocket" refers to a region of a molecule or molecular complex that, as a result of its shape, electrostatic complementarity and hydrophobicity, favourably associates with another chemical entity or compound. The term "pocket" includes, but is not limited to, cleft, channel or site. PPAR, PPARγ or PPARγ-like molecules may have binding pockets which include, but are not limited to, peptide or substrate binding, lipid-binding, like the pros- taglandin-binding pocket and antibody binding sites.

The term "ligand" refers to a chemical entity that, as a result of its shape, electrostatic complementarity and hydrophobicity, favourably associates with another chemical entity or compound. The term "ligand" includes, but is not limited to, a lipid, a substrate, an agonist, an antagonist, an inhibitor, an antibody, a drug, a peptide, a protein, DNA, RNA, PNA or a com- pound.

The term "chemical entity" refers to a chemical compound, a complex of at least two chemical compounds, and a fragment of such a compound or complex. The chemical entity may be, for example, a ligand, a lipid, a substrate, an agonist, an antagonist, an inhibitor, an antibody, a drug, a peptide, a protein, DNA, RNA, PNA or a compound. In one aspect, the chemical entity is selected from the group consisting of prostaglandins, a prostaglandin analogue and a ligand to the prostaglandin-binding pocket.

"Conservative substitutions" refers to residues that are physically or functionally similar to the corresponding reference residues. That is, a conservative substitution and its reference residue have similar size, shape, electric charge, chemical properties including the ability to form covalent or hydrogen bonds, or the like. Preferred conservative substitutions are those fulfilling the criteria defined for an accepted point mutation in Dayhoff et al., Atlas of Protein Sequence and Structure, 5, pp. 345-352 (1978 & Supp.), which is incorporated herein by reference. Examples of conservative substitutions are substitutions including but not limited to the following groups: (a) valine, glycine; (b) glycine, alanine; (c) valine, isoleucine, leucine; (d) aspartic acid, glutamic acid; (e) asparagine, glutamine; (f) serine, threonine; (g) lysine, ar- ginine, methionine; and (h) phenylalanine, tyrosine, tryptophan. The term "corresponding amino acid" or "residue which corresponds to" refers to a particular amino acid or analogue thereof in a PPARγ protein or PPARγ-like protein molecule such as a PPARγ homologue that is identical or functionally equivalent to an amino acid in PPARγ according to SEQ ID NOS: 1 or 2/3.

Methods for identifying a corresponding amino acid are known in the art and are based upon sequence, structural alignment, its functional position or a combination thereof as compared to the PPARγ protein molecule.

For example, corresponding amino acids may be identified by superimposing the backbone atoms of the amino acids in PPARγ and the PPARγ homologue using well known software appli- cations, such as QUANTA (Accelrys Inc., San Diego, ^®2001, 2002). The corresponding amino acids may also be identified using sequence alignment programs such as the "bestfit" program available from the Genetics Computer Group which uses the local homology algorithm described by Smith and Waterman in Advances in Applied Mathematics 2,482 (1981), which is incorporated herein by reference.

The term "domain" refers to a portion of the PPARγ protein or PPARγ-like protein that can be separated based on its biological function, for example, ligand binding, DNA binding or Zinc fingers. The domain may comprise a binding pocket, a sequence or a structural motif.

The term "fitting operation" refers to an operation that utilizes the structure coordinates of a chemical entity, binding pocket, molecule or molecular complex, or portion thereof, to associ- ate the chemical entity with the binding pocket, molecule or molecular complex, or portion thereof. This may be achieved by positioning, rotating or translating the chemical entity in the binding pocket to match the shape and electrostatic complementarity of the binding pocket. Covalent interactions, non-covalent interactions such as hydrogen bond, electrostatic, hydrophobic, van der Waals interactions, and non-complementary electrostatic interactions such as repulsive charge-charge, dipole-dipole and charge-dipole interactions may be optimized. Alternatively, one may minimize the deformation energy of binding of the chemical entity to the binding pocket.

The term "generating a three-dimensional structure" or "generating a three-dimensional representation" refers to converting the lists of structure coordinates into structural models or graphical representation in three-dimensional space. This can be achieved through commercially or publicly available software. The three-dimensional structure may be displayed or used to perform computer modelling or fitting operations. In addition, the structure coordinates themselves may be used to perform computer and fitting operations.

The term "homology model" refers to a structural model derived from known three- dimensional structure (s). Generation of the homology model, termed "homology modelling", can include sequence alignment, residue replacement, residue conformation adjustment through energy minimization, or a combination thereof.

The term "homologue of PPARγ" or "PPARγ homologue" refers to a molecule that is homologous to PPARγ by structure or sequence. Examples of homologues include but are not limited to human PPARγ and PPARγ from another species with conservative substitutions, additions, deletions or a combination thereof; or another member of the PPAR family including, but not limited to, PPARα and PPARδ, with conservative substitutions, additions, deletions or a combination thereof.

The term "homologue of PPARγ ligand binding domain (LBD)" or " PPARγ LBD homologues" refer to a molecule having amino acids which correspond to the amino acids in the PPARγ LBD. Examples of homologues include but are not limited to the LB domain of human PPARγ and

PPARγ from another species with conservative substitutions; or the LBD of another member of the PPAR family including, but not limited to, PPARα and PPARδ, or with conservative substitutions.

The term "molecular complex" or "complex" refers to a molecule associated with at least one chemical entity.

The term "part of a binding pocket" refers to less than all of the amino acid residues that define the binding pocket. For example, the structure coordinates of residues that constitute part of a binding pocket may be specific for defining the chemical environment of the binding pocket, or useful in designing fragments of an inhibitor that may interact with those residues. For example, the portion of residues may be key residues that play a role in ligand binding, or may be residues that are spatially related and define a three-dimensional compartment of the binding pocket. The residues may be contiguous or non-contiguous in primary sequence.

The term "part of a PPARγ ligand binding domain (LBD)" or "part of a PPARγ-like ligand binding domain (LBD)" refers to less than all of the PPARγ or PPARγ-like LBD, respectively. The struc- ture coordinates of residues that constitute part of a PPARγ or PPARγ-LBD may be specific for defining the chemical environment of the domain, or useful in designing fragments of an in- hibitor that interact with those residues. For example, the portion of residues may be residues that play a role in ligand binding, or may be residues that are spatially related and define a three-dimensional compartment of the domain. The residues may be contiguous or noncontiguous in primary sequence. For example, part of a PPARγ-LBD can be the ligand binding site, the co-activator site, the lipid binding pocket, the prostaglandin-binding pocket, the AF-2 helix.

The term "part of a PPARγ protein" or "part of a PPARγ homologue" refers to less than all of the amino acid residues of a PPARγ protein or homologue. In one aspect, part of a PPARγ protein or homologue defines the binding pockets, domains, and motifs of the protein or homo- logue. The structure coordinates of residues that constitute part of a PPARγ protein or homologue may be specific for defining the chemical environment of the protein, or useful in designing fragments of an inhibitor that may interact with those residues. The portion of residues may also be residues that are spatially related and define a three-dimensional compartment of a binding pocket, motif or domain. The residues may be contiguous or non- contiguous in primary sequence. For example, the portion of residues may be key residues that play a role in ligand or substrate binding, peptide binding, co-activator binding, DNA binding, metal ion binding, antibody binding, catalysis, structural stabilization or degradation.

The term "root mean square deviation" or "RMSD" means the square root of the arithmetic mean of the squares of the deviations from the mean. It is a way to express the deviation or variation from a trend or object. For purposes of this invention, the "root mean square deviation" defines the variation in the backbone of a protein from the backbone of the PPARγ protein molecule, a binding pocket, a motif, a domain, or portion thereof, as defined by the structure coordinates of PPARγ protein molecule described herein. It would be apparent to the skilled worker that the calculation of RMSD involves a standard error.

The term "structure coordinates" refers to Cartesian coordinates derived from mathematical equations related to the patterns obtained on diffraction of a monochromatic beam of X-rays by the atoms (scattering centers) of a protein or protein complex in crystal form. The diffraction data are used to calculate an electron density map of the repeating unit of the crystal. The electron density maps are then used to establish the positions of the individual atoms of the molecule or molecular complex.

Once a chemical entity has been designed or selected according to the invention, the efficiency with which that chemical entity may bind to the PPARγ or PPARγ-like prostaglandin- binding pocket as defined herein may be tested and optimized by computational evaluation. For example, an effective PPARγ prostaglandin-binding pocket ligand must preferably demonstrate a relatively small difference in energy between its bound and free states (i. e., a small deformation energy of binding). Thus, the most efficient PPARγ prostaglandin-binding pocket ligands should preferably be designed with a deformation energy of binding of not greater than about 10 kcal/mole, more preferably, not greater than 7 kcal/mole. PPARγ prostaglandin- binding pocket inhibitors may interact with the prostaglandin-binding pocket in more than one conformation that is similar in overall binding energy. In those cases, the deformation energy of binding is taken to be the difference between the energy of the free chemical entity and the average energy of the conformations observed when the inhibitor binds to the protein.

The term "sufficiently homologous to PPARγ" refers to a protein that has a sequence homology of at least 20% compared to PPARγ protein. In one aspect, the sequence homology is at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99%.

The term "three-dimensional structural information" refers to information obtained from the structure coordinates. Structural information generated can include the three-dimensional structure or graphical representation of the structure. Structural information can also be generated when subtracting distances between atoms in the structure coordinates, calculating chemical energies for a PPARγ molecule or molecular complex or homologues thereof, calculating or minimizing energies for an association of a PPARγ molecule or molecular complex or homologues thereof to a chemical entity.

The PPARγ protein or its homologue may be produced by any well-known method, including synthetic methods, such as solid phase, liquid phase and combination solid phase/liquid phase syntheses; recombinant DNA methods, including cloning, optionally combined with site directed mutagenesis; and/or purification of the natural products. In a preferred aspect, the protein is overexpressed in a baculovirus system, an E. coli system, or a Pichia pastoris sys- tern,

Prostaαlandin-bindinα Pocket of PPARγ Protein, Protein Complexes or Homologues thereof

As disclosed above, applicants have provided the three-dimensional X-ray crystal structures of a PPARγ prostaglandin complex. The invention may in one aspect be useful for inhibitor or activator design for novel drugs to be used in the treatment of cancer or other prostaglandin- and/or PPARy-related diseases or conditions, and to study the role of PPARγ in cell signalling. The atomic coordinate data is presented in Figures 1-2. In order to use the structure coordinates generated for the binding pocket it is often necessary to convert the structure coordinates into a three-dimensional shape. This is achieved through the use of commercially available software that is capable of generating three- dimensional graphical representations of molecules or portions thereof from a set of structure coordinates.

Binding pockets, also referred to as binding sites in the present invention, are of significant utility in fields such as drug discovery. The association of natural ligands or substrates with the binding pockets of their corresponding receptors or enzymes is the basis of many biological mechanisms of action. Similarly, many drugs exert their biological effects through associa- tion with the binding pockets of receptors and enzymes. Such associations may occur with all or part of the binding pocket. An understanding of such associations will help lead to the design of drugs having more favorable associations with their target receptor or enzyme, and thus, improved biological effects. Therefore, this information is valuable in designing potential inhibitors of the binding pockets of biologically important targets. The prostaglandin-binding pocket of this invention may be important for drug design.

In one aspect, the prostaglandin-binding pocket is defined by three-dimensional structure coordinates of a set of amino acids that comprises amino acid residues corresponding to four or more selected from the group consisting of TYR 222, PHE 226, PRO 227, LEU 228, THR 229, LYS 230, CYS 285, ARG 288, SER 289, ALA 292, GLU 295, ILE 296, ILE 325, ILE 326, TYR 327, THR 328, MET 329, LEU 330, ALA 331, SER 332, LEU 333, MET 364, PHE 374, ASN 375, ALA 376, LEU 377, GLU 378, LEU 379, ASP 380, ASP 381, LEU 384, and LEU 435 of SEQ ID NO: 1

In another aspect, the PPARγor PPARγ-like protein molecule comprises an amino acid sequence at least 65% identical to residues 207-476 of SEQ ID NO: 1, and the prostaglandin- binding pocket is defined by three-dimensional structure coordinates of a set of amino acids that comprises (i) an amino acid residue corresponding to LEU 228, and (ii) three or more amino acids corresponding to amino acid residues selected from the group consisting of TYR 222, PHE 226, PRO 227, THR 229, LYS 230, CYS 285, ARG 288, SER 289, ALA 292, GLU 295, ILE 296, ILE 325, ILE 326, TYR 327, THR 328, MET 329, LEU 330, ALA 331, SER 332, LEU 333, MET 364, PHE 374, ASN 375, ALA 376, LEU 377, GLU 378, LEU 379, ASP 380, ASP 381, LEU 384, and LEU 435 of SEQ ID NO: 1.

In one embodiment of any of the preceding aspects, the PPARγor PPARγ-like protein molecule comprises an amino acid sequence at least 65% identical to residues 207-476 of SEQ ID NO: 1, and the prostaglandin-binding pocket is defined by three-dimensional structure coordinates of a set of amino acids comprising an amino acid residue corresponding to LEU 228 and six or more corresponding to amino acid residues selected from the group consisting of TYR 222, PHE 226, PRO 227, THR 229, LYS 230, CYS 285, ARG 288, SER 289, ALA 292, GLU 295, ILE 326, TYR 327, MET 329, LEU 330, SER 332, LEU 333, MET 364, ASP 381, and LEU 384.

In an additional or alternative embodiment, the prostaglandin-binding pocket is defined by three-dimensional structure coordinates of a set of amino acids comprising an amino acid residue corresponding to LEU 228 and ten or more corresponding to amino acid residues selected from the group consisting of TYR 222, PHE 226, PRO 227, THR 229, LYS 230, CYS 285, ARG 288, SER 289, ALA 292, GLU 295, ILE 326, TYR 327, MET 329, LEU 330, SER 332, LEU 333, MET 364, ASP 381, and LEU 384.

In another additional or alternative embodiment, the prostaglandin-binding pocket is defined by three-dimensional structure coordinates of a set of amino acids comprises amino acid residues corresponding to PHE 226, PRO 227, LEU 228 and MET 329.

In another additional or alternative embodiment, the prostaglandin-binding pocket is defined by three-dimensional structure coordinates of a set of amino acids comprising amino acid residues corresponding to TYR 222, PHE 226, PRO 227, LEU 228, THR 229, LYS 230, ALA 292, GLU 295, MET 329, SER 332, LEU 333, ASP 381, and LEU 384.

In another additional or alternative embodiment, the prostaglandin-binding pocket is defined by three-dimensional structure coordinates of a set of amino acids comprising amino acid residues corresponding to one or more selected from the group consisting of CYS 285, ARG 288, SER 289, ILE 326, TYR 327, LEU 330 and MET 364.

In another additional or alternative embodiment, the prostaglandin-binding pocket is defined by three-dimensional structure coordinates of a set of amino acids comprising amino acid residues corresponding to TYR 222, PHE 226, PRO 227, LEU 228, THR 229, LYS 230, CYS 285, ARG 288, SER 289, ALA 292, GLU 295, ILE 326, TYR 327, MET 329, LEU 330, SER 332, LEU 333, MET 364, ASP 381, and LEU 384.

In another additional or alternative embodiment, the prostaglandin-binding pocket is defined by three-dimensional structure coordinates of a set of amino acids comprising amino acid residues corresponding to one or more selected from the group consisting of ILE 296, ILE 325, THR 328, ALA 331, PHE 374, ASN 375, ALA 376, LEU 377, GLU 378, LEU 379, ASP 380 and LEU 435.

In another additional or alternative embodiment, the prostaglandin-binding pocket is defined by three-dimensional structure coordinates of a set of amino acids comprising amino acid residues corresponding to ILE 296, ILE 325, THR 328, ALA 331, PHE 374, ASN 375, ALA 376, LEU 377, GLU 378, LEU 379, ASP 380 and LEU 435.

In another additional or alternative embodiment, the prostaglandin-binding pocket is defined by three-dimensional structure coordinates of a set of amino acids comprising amino acid residues corresponding to TYR 222, PHE 226, PRO 227, LEU 228, THR 229, LYS 230, CYS 285, ARG 288, SER 289, ALA 292, GLU 295, ILE 296, ILE 325, ILE 326, TYR 327, THR 328, MET 329, LEU 330, ALA 331, SER 332, LEU 333, MET 364, PHE 374, ASN 375, ALA 376, LEU 377, GLU 378, LEU 379, ASP 380, ASP 381, LEU 384, and LEU 435.

In another aspect, the prostaglandin-binding pocket is defined by three-dimensional structure coordinates of a set of amino acids that correspond to four or more PPARγ amino acid residues selected from the group consisting of TYR 222, PHE 226, PRO 227, LEU 228, THR 229, LYS 230, ALA 292, GLU 295, ILE 296, ILE 325, THR 328, MET 329, ALA 331, SER 332, LEU 333, PHE 374, ASN 375, ALA 376, LEU 377, GLU 378, LEU 379, ASP 380, ASP 381, LEU 384, and LEU 435 and optionally one or more selected from the group consisting of CYS 285, ARG 288, SER 289, ILE 326, TYR 327, LEU 330 and MET 364 according to figure 1, wherein the root mean square deviation of the backbone atoms between said amino acid residues and said PPARγ residues is not greater than about 3 A. In one aspect, the RMSD is not greater than about 2.0. In one aspect, the RMSD is not greater than about 1.5. In one aspect, the RMSD is not greater than about 1.0. In one aspect, the RMSD is not greater than about 0.8. In one aspect, the RMSD is not greater than about 0.5. In one aspect, the RMSD is not greater than about 0.3. In one aspect, the RMSD is not greater than about 0.2.

In another aspect, the prostaglandin-binding pocket is defined by three-dimensional structure coordinates of a set of amino acids that correspond to PPARγ amino acid residues selected from the group consisting of PHE 226, PRO 227, LEU 228 and MET 329 according to figure 1, wherein the root mean square deviation of the backbone atoms between said amino acid resi- dues and said PPARγ residues is not greater than about 3 A. In one aspect, the RMSD is not greater than about 2.0. In one aspect, the RMSD is not greater than about 1.5. In one aspect, the RMSD is not greater than about 1.0. In one aspect, the RMSD is not greater than about 0.8. In one aspect, the RMSD is not greater than about 0.5. In one aspect, the RMSD is not greater than about 0.3. In one aspect, the RMSD is not greater than about 0.2.

In another aspect, the prostaglandin-binding pocket is defined by three-dimensional structure coordinates of a set of amino acids that correspond to four or more PPARγ amino acid residues selected from the group consisting of TYR 222, PHE 226, PRO 227, LEU 228, THR 229, LYS 230, ALA 292, GLU 295, MET 329, SER 332, LEU 333, ASP 381, and LEU 384 and optionally one or more selected from the group consisting of CYS 285, ARG 288, SER 289, ILE 326, TYR 327, LEU 330 and MET 364 according to figure 1, wherein the root mean square deviation of the backbone atoms between said amino acid residues and said PPARγ residues is not greater than about 3 A. In one aspect, the RMSD is not greater than about 2.0. In one aspect, the RMSD is not greater than about 1.5. In one aspect, the RMSD is not greater than about 1.0. In one aspect, the RMSD is not greater than about 0.8. In one aspect, the RMSD is not greater than about 0.5. In one aspect, the RMSD is not greater than about 0.3. In one aspect, the RMSD is not greater than about 0.2.

In another aspect, the prostaglandin-binding pocket is defined by three-dimensional structure coordinates of a set of amino acids that correspond to PPARγ amino acid residues PHE 226, PRO 227, LEU 228 and MET 329 and optionally one or more selected from the group consisting Of TYR 222, THR 229, LYS 230, ALA 292, GLU 295, ILE 296, ILE 325, THR 328, ALA 331, SER 332, LEU 333, PHE 374, ASN 375, ALA 376, LEU 377, GLU 378, LEU 379, ASP 380, ASP 381, LEU 384, and LEU 435 and optionally one or more selected from the group consisting of CYS 285, ARG 288, SER 289, ILE 326, TYR 327, LEU 330 and MET 364 according to figure 1, wherein the root mean square deviation of the backbone atoms between said amino acid residues and said PPARγ residues is not greater than about 3 A. In one aspect, the RMSD is not greater than about 2.0. In one aspect, the RMSD is not greater than about 1.5. In one aspect, the RMSD is not greater than about 1.0. In one aspect, the RMSD is not greater than about 0.8. In one aspect, the RMSD is not greater than about 0.5. In one aspect, the RMSD is not greater than about 0.3 In one aspect, the RMSD is not greater than about 0.2.

In another aspect, the prostaglandin-binding pocket is defined by three-dimensional structure coordinates of a set of amino acids that correspond to PPARγ amino acid residues PHE 226, PRO 227, LEU 228 and MET 329 and optionally one or more selected from the group consisting Of TYR 222, THR 229, LYS 230, ALA 292, GLU 295, SER 332, LEU 333, ASP 381, and LEU 384 and optionally one or more selected from the group consisting of CYS 285, ARG 288, SER 289, ILE 326, TYR 327, LEU 330 and MET 364 according to figure 1, wherein the root mean square deviation of the backbone atoms between said amino acid residues and said PPARγ residues is not greater than about 3 A. In one aspect, the RMSD is not greater than about 2.0. In one aspect, the RMSD is not greater than about 1.5. In one aspect, the RMSD is not greater than about 1.0. In one aspect, the RMSD is not greater than about 0.8. In one aspect, the RMSD is not greater than about 0.5. In one aspect, the RMSD is not greater than about 0.3. In one aspect, the RMSD is not greater than about 0.2.

In another aspect, the prostaglandin-binding pocket is defined by three-dimensional structure coordinates of a set of amino acids that correspond to PPARγ amino acid residues TYR 222, PHE 226, PRO 227, LEU 228, THR 229, LYS 230, ALA 292, GLU 295, MET 329, SER 332, LEU 333, ASP 381, and LEU 384 and optionally one or more selected from the group consisting of CYS 285, ARG 288, SER 289, ILE 326, TYR 327, LEU 330 and MET 364 according to figure 1, wherein the root mean square deviation of the backbone atoms between said amino acid residues and said PPARγ residues is not greater than about 3 A. In one aspect, the RMSD is not greater than about 2.0. In one aspect, the RMSD is not greater than about 1.5. In one aspect, the RMSD is not greater than about 1.0. In one aspect, the RMSD is not greater than about 0.8. In one aspect, the RMSD is not greater than about 0.5. In one aspect, the RMSD is not greater than about 0.3. In one aspect, the RMSD is not greater than about 0.2.

In another aspect, the prostaglandin-binding pocket is defined by three-dimensional structure coordinates of a set of amino acids that correspond to PPARγ amino acid residues TYR 222, PHE 226, PRO 227, LEU 228, THR 229, LYS 230, ALA 292, GLU 295, ILE 296, ILE 325, THR 328, MET 329, ALA 331, SER 332, LEU 333, PHE 374, ASN 375, ALA 376, LEU 377, GLU 378, LEU 379, ASP 380, ASP 381, LEU 384, and LEU 435 and optionally one or more selected from the group consisting of CYS 285, ARG 288, SER 289, ILE 326, TYR 327, LEU 330 and MET 364 according to figure 1, wherein the root mean square deviation of the backbone atoms between said amino acid residues and said PPARγ residues is not greater than about 3 A. In one aspect, the RMSD is not greater than about 2.0. In one aspect, the RMSD is not greater than about 1.5. In one aspect, the RMSD is not greater than about 1.0. In one aspect, the RMSD is not greater than about 0.8. In one aspect, the RMSD is not greater than about 0.5. In one aspect, the RMSD is not greater than about 0.3. In one aspect, the RMSD is not greater than about 0.2.

In another aspect, the prostaglandin-binding pocket is defined by three-dimensional structure coordinates of a set of amino acids that correspond to PPARγ amino acid residues TYR 222, PHE 226, PRO 227, LEU 228, THR 229, LYS 230, CYS 285, ARG 288, SER 289, ALA 292, GLU 295, ILE 326, TYR 327, MET 329, LEU 330, SER 332, LEU 333, MET 364, ASP 381, and LEU 384 according to figure 1, wherein the root mean square deviation of the backbone atoms be- tween said amino acid residues and said PPARγ residues is not greater than about 3 A. In one aspect, the RMSD is not greater than about 2.0. In one aspect, the RMSD is not greater than about 1.5. In one aspect, the RMSD is not greater than about 1.0. In one aspect, the RMSD is not greater than about 0.8. In one aspect, the RMSD is not greater than about 0.5. In one aspect, the RMSD is not greater than about 0.3. In one aspect, the RMSD is not greater than about 0.2.

In another aspect, the prostaglandin-binding pocket is defined by three-dimensional structure coordinates of a set of amino acids that correspond to PPARγ amino acid residues TYR 222, PHE 226, PRO 227, LEU 228, THR 229, LYS 230, CYS 285, ARG 288, SER 289, ALA 292, GLU 295, ILE 296, ILE 325, ILE 326, TYR 327, THR 328, MET 329, LEU 330, ALA 331, SER 332, LEU 333, MET 364, PHE 374, ASN 375, ALA 376, LEU 377, GLU 378, LEU 379, ASP 380, ASP 381, LEU 384, and LEU 435 according to figure 1, wherein the root mean square deviation of the backbone atoms between said amino acid residues and said PPARγ residues is not greater than about 3 A. In one aspect, the RMSD is not greater than about 2.0. In one aspect, the RMSD is not greater than about 1.5. In one aspect, the RMSD is not greater than about 1.0. In one aspect, the RMSD is not greater than about 0.8. In one aspect, the RMSD is not greater than about 0.5. In one aspect, the RMSD is not greater than about 0.3. In one aspect, the RMSD is not greater than about 0.2.

In another aspect, the prostaglandin-binding pocket is defined by three-dimensional structure coordinates of a set of amino acids that correspond to PPARγ amino acid residues TYR 222, PHE 226, PRO 227, LEU 228, THR 229, LYS 230, CYS 285, ARG 288, SER 289, ALA 292, GLU

295, ILE 326, TYR 327, MET 329, LEU 330, SER 332, LEU 333, MET 364, ASP 381, and LEU 384 according to the structure of PPARγ-complex in Figure 1. These above-identified amino acid residues was found to be within 5 A distance of the ligand bound in the prostaglandin- binding pocket of PPARγ identified using the program QUANTA (Accelrys Inc., San Diego, ^®2001, 2002) and the CONTACT software program of the CCP4 program package (Bailey, 1994), Table 1 and Table 2, which allow the display and output of all residues within 5 A from the inhibitor.

Further, the LBD of the PPARγ molecule, with or without any bound ligands, can be run through the VOIDOO software program (Kleywegt and Jones, 1994). From the output of the program voids, pockets within the PPARγ molecule not filled with atoms, around the position for the prostaglandin B₂ site can predict residues which could be reached and utilized in a drug design process. The amino acid residues which were found using this method was ILE

296, ILE 325, THR 328, ALA 331, PHE 374, ASN 375, ALA 376, LEU 377, GLU 378, LEU 379, ASP 380 and LEU 435. These residues were situated more than 5 A from the prostaglandin B2 molecule, but were found to form part of the prostaglandin-binding pocket.

It will be readily apparent to those of skill in the art that the numbering of amino acids in other homologues of PPARγ may be different than that set forth for PPARγ. Corresponding amino acids in homologues of PPARγ are easily identified by visual inspection of the amino acid sequences or by using commercially available sequence homology, structural homology or structure superimposition software programs.

In one aspect of the invention, the PPARγ or PPARγ-like protein comprises the amino acid sequence of SEQ ID NO:2 or a homologous sequence.

In another aspect of the invention, the PPARγ protein comprises the amino acid sequence of SEQ ID NO: 1.

Those of skill in the art understand that a set of structure coordinates for a molecule or a molecular-complex or a portion thereof, is a relative set of points that define a shape in three dimensions. Thus, it is possible that an entirely different set of coordinates could define a similar or identical shape. Moreover, slight variations in the individual coordinates will have little effect on overall shape. In terms of binding pockets, these variations would not be expected to significantly alter the nature of ligands that could associate with those pockets.

The variations in coordinates discussed above may be generated as a result of mathematical manipulations of the PPARγ structure coordinates. For example, the structure coordinates set forth in Figure 1 or 2 could be manipulated by crystallographic permutations of the structure coordinates, integer additions or subtractions to sets of the structure coordinates, translation or rotation of the structure coordinates or any combination of the above.

Alternatively, modifications in the crystal structure due to mutations, additions, substitutions, and/or deletions of amino acids, or other changes in any of the components that make up the crystal could also account for variations in structure coordinates. If such variations are within a certain root mean square deviation as compared to the original coordinates, the resulting three-dimensional shape is considered encompassed by this invention. Thus, for example, a ligand that binds to the prostaglandin-binding pocket of PPARγ would also be expected to bind to another binding pocket whose structure coordinates define a shape that falls within the ac- ceptable root mean square deviation. Various computational analyses may be necessary to determine whether a prostaglandin- binding pocket of a molecule or molecular complex is sufficiently similar to the prostaglandin- binding pocket of PPARγ. Such analyses may be carried out using well known software applications, such as ProFit (A. C. R. Martin, SciTech Software, ProFit version 2.5, University CoI- lege London, http://www. bioinf. org. uk/software), Swiss-Pdb Viewer (Guex et al., 18, pp. 2714-2723 (1997)), the Molecular Similarity application of QUANTA (Accelrys Inc., San Diego, ^®2001, 2002) and as described in the accompanying User's Guide, which are incorporated herein by reference.

The above programs permit comparisons between different structures, different conformations of the same structure, and different parts of the same structure.

The procedure used in QUANTA (Accelrys Inc., San Diego, ^®2001, 2002) and Swiss-Pdb Viewer to compare structures is divided into four steps: 1) load the structures to be compared; 2) define the atom equivalences in these structures; 3) perform a fitting operation on the structures; and 4) analyze the results.

The procedure used in ProFit to compare structures includes the following steps load the structures to be compared; 2) specify selected residues of interest; 3) define the atom equivalences in the selected residues; 4) perform a fitting operation on the selected residues; and 5) analyze the results.

Each structure in the comparison is identified by a name. One structure is identified as the target (i. e., the fixed structure); all remaining structures are working structures (i. e., moving structures). Since atom equivalency within the above programs is defined by user input, for the purpose of this invention we will define equivalent atoms as protein backbone atoms (N, C and for PPARγ amino acids and corresponding amino acids in the structures being compared.

The corresponding amino acids may be identified by sequence alignment programs such as the "bestfit" program available from the Genetics Computer Group which uses the local homology algorithm described by Smith and Waterman in Advances in Applied Mathematics 2,482 (1981), which is incorporated herein by reference. A suitable amino acid sequence alignment will require that the proteins being aligned share minimum percentage of identical amino acids. Generally, a first protein being aligned with a second protein should share in excess of about 35% identical amino acids [Hanks et al., Science, 241,42 (1988); Hanks and Quinn, Methods in Enzymology, 200,38 (1991) ]. The identification of equivalent residues can also be assisted by secondary structure alignment, for example, aligning the α-helices, β- sheets in the structure. The program Swiss-Pdb Viewer has its own best fit algorithm that is based on secondary sequence alignment.

When a rigid fitting method is used, the working structure is translated and rotated to obtain an optimum fit with the target structure. The fitting operation uses an algorithm that computes the optimum translation and rotation to be applied to the moving structure, such that the root mean square difference of the fit over the specified pairs of equivalent atom is an absolute minimum. This number, given in angstroms, is reported by the above programs. The Swiss-Pdb Viewer program sets an RMSD cutoff for eliminating pairs of equivalent atoms that have high RMSD values. An RMSD cutoff value can be used to exclude pairs of equivalent atoms with extreme individual RMSD values. In the program ProFit, the RMSD cutoff value can be specified by the user.

The RMSD values are averages of individual RMSD values calculated for the backbone atoms (C, N and Ca) of all residues in the LBD or prostaglandin-binding pocket between the refer- ence structure and the other PPARγ-inhibitor complex structures.

Computer Systems

According to another aspect, this invention provides a machine-readable data storage medium, comprising a data storage material encoded with machine-readable data, wherein said data defines the above-mentioned prostaglandin-binding pockets by comprising the structure coordinates of said amino acid residues according to any one of Figures 1-2.

In one aspect of the invention, a computer comprising : (a) a machine-readable data storage medium, comprising a data storage material encoded with machine-readable data, wherein said data defines a prostaglandin-binding pocket of a PPAR protein molecule, which prostaglandin-binding pocket is defined by three-dimensional structure coordinates of a set of amino acids that comprises amino acid residues corresponding to four or more selected from the group consisting of TYR 222, PHE 226, PRO 227, LEU 228, THR 229, LYS 230, CYS 285, ARG 288, SER 289, ALA 292, GLU 295, ILE 296, ILE 325, ILE 326, TYR 327, THR 328, MET 329, LEU 330, ALA 331, SER 332, LEU 333, MET 364, PHE 374, ASN 375, ALA 376, LEU 377, GLU 378, LEU 379, ASP 380, ASP 381, LEU 384, and LEU 435 of SEQ ID NO: 1; (b) a working memory for storing instructions for processing said machine-readable data; (c) a central processing unit coupled to said working memory and to said machine-readable data storage medium for processing said machine-readable data and means for generating three- dimensional structural information of said binding pocket; and (d) output hardware coupled to said central processing unit for outputting three-dimensional structural information of said binding pocket, or information produced using said three-dimensional structural information of said binding pocket, is provided.

In another aspect of the invention, a computer comprising : (a) a machine-readable data storage medium, comprising a data storage material encoded with machine-readable data, wherein said data defines a prostaglandin-binding pocket of a PPARγor PPARγ-like protein molecule comprising an amino acid sequence at least 65% identical to residues 207-476 of SEQ ID NO: 1, which prostaglandin-binding pocket is defined by three-dimensional structure coordinates of a set of amino acids that comprises (i) an amino acid residue corresponding to LEU 228, and (ii) three or more amino acids corresponding to amino acid residues selected from the group consisting of TYR 222, PHE 226, PRO 227, THR 229, LYS 230, CYS 285, ARG 288, SER 289, ALA 292, GLU 295, ILE 296, ILE 325, ILE 326, TYR 327, THR 328, MET 329, LEU 330, ALA 331, SER 332, LEU 333, MET 364, PHE 374, ASN 375, ALA 376, LEU 377, GLU 378, LEU 379, ASP 380, ASP 381, LEU 384, and LEU 435 of SEQ ID NO: 1; (b) a working memory for storing instructions for processing said machine-readable data; (c) a central processing unit coupled to said working memory and to said machine-readable data storage medium for processing said machine-readable data and means for generating three- dimensional structural information of said binding pocket; and (d) output hardware coupled to said central processing unit for outputting three-dimensional structural information of said binding pocket, or information produced using said three-dimensional structural information of said binding pocket, is provided. In another aspect of the invention, a computer comprising : (a) a machine-readable data storage medium, comprising a data storage material encoded with machine-readable data, wherein said data defines a prostaglandin-binding pocket of a PPARγ or PPARγ-like protein molecule, which prostaglandin-binding pocket is defined by three- dimensional structure coordinates of a set of amino acids that correspond to four or more PPARγ amino acid residues selected from the group consisting of TYR 222, PHE 226, PRO 227, LEU 228, THR 229, LYS 230, ALA 292, GLU 295, ILE 296, ILE 325, THR 328, MET 329, ALA 331, SER 332, LEU 333, PHE 374, ASN 375, ALA 376, LEU 377, GLU 378, LEU 379, ASP 380, ASP 381, LEU 384, and LEU 435 and optionally one or more selected from the group consisting of CYS 285, ARG 288, SER 289, ILE 326, TYR 327, LEU 330 and MET 364 according to figure 1, wherein the root mean square deviation of the backbone atoms between amino acid residues of said molecule and said PPARγ residues is not greater than about 3 A; (b) a working memory for storing instructions for processing said machine-readable data; (c) a central processing unit coupled to said working memory and to said machine-readable data storage medium for processing said machine-readable data and means for generating three- dimensional structural information of said binding pocket; and (d) output hardware coupled to said central processing unit for outputting three-dimensional structural information of said binding pocket, or information produced using said three-dimensional structural information of said binding pocket, is provided.

To use the structure coordinates generated for PPARγ, homologues thereof, or its pros- taglandin-binding pocket, it is at times necessary to convert them into a three-dimensional shape. This is achieved through the use of commercially or publicly available software that is capable of generating a three-dimensional structure of molecules or portions thereof from a set of structure coordinates. The three-dimensional structure may be displayed as a graphical representation.

Therefore, according to another aspect, this invention provides a machine-readable data storage medium comprising a data storage material encoded with machine readable data. In one aspect, a machine programmed with instructions for using said data, is capable of generating a three-dimensional structure of any of the prostaglandin-binding pockets that are defined herein.

This invention also provides a computer comprising : (a) a machine-readable data storage medium comprising a data storage material encoded with machine-readable data, wherein said data defines any one of the above defined prostaglandin-binding pockets; (b) a working memory for storing instructions for processing said machine-readable data; (c) a central processing unit (CPU) coupled to said working memory and to said machine-readable data storage medium for processing said machine readable data and means for generating three- dimensional structural information of said binding pockets; and (d) output hardware coupled to said central processing unit for outputting three-dimensional structural information of said prostaglandin-binding pockets, or information produced using said three-dimensional struc- tural information of said prostaglandin-binding pockets.

Three-dimensional data generation may be provided by an instruction or set of instructions such as a computer program or commands for generating a three-dimensional structure or graphical representation from structure coordinates, or by calculating distances between atoms, calculating chemical energies for a PPARγ molecule or molecular complex or homologues thereof, or calculating or minimizing energies for an association of a PPARγ molecule or molecular complex or homologues thereof to a chemical entity. The graphical representation can be generated or displayed by commercially available software programs. Examples of software programs include but are not limited to QUANTA (Accelrys Inc., San Diego, ^®2001, 2002), O (Jones et al. Acta A47, pp. 110-119 (1991)) and RIBBONS (Carson, Appl. Crystal- logr., 24, pp. 9589-961 (1991)), which are incorporated herein by reference. Certain software programs may imbue this representation with physico-chemical attributes which are known from the chemical composition of the molecule, such as residue charge, hydrophobicity, tor- sional and rotational degrees of freedom for the residue or segment, etc. Examples of software programs for calculating chemical energies are described in the Rational Drug Design section.

In one aspect, the computer is executing instruction(s) such as a computer program for three-dimensional data generation.

Information of said binding pocket or information produced by using said binding pocket can be outputted through display terminals, touchscreens, facsimile machines, modems, CD- ROMs, printers or disk drives. The information can be in graphical or alphanumeric form.

The "Computer system" includes in one aspect of the invention a computer comprising a central processing unit ("CPU"), a working memory which may be, e. g., RAM (random-access memory) or "core" memory, mass storage memory (such as one or more disk drives, DVD or CD-ROM drives), one or more cathode-ray tube ("CRT") displays or liquid-crystal-display (LCDs) terminals, one or more keyboards, one or more input lines using e.g. Ethernet cable, USB or Firewire cables, RSR232, parallel cables or so called wire-less connection, and one or more output lines using e.g. Ethernet cable, USB or Firewire cables, RSR232, parallel cables or so called wire-less connection, all of which are interconnected by a conventional bidirectional system bus.

Input hardware, coupled to computer by input lines, may be implemented in a variety of ways. Machine-readable data of this invention may be inputted via the use of a modem or modems connected by a telephone line dedicated data line or so called wire-less connection.

Alternatively or additionally, the input hardware may comprise CD-ROM drives, DVD drives, disk drives or various sorts of flash memory drives. In conjunction with display terminal, keyboard mouse or pen tablets may also be used as an input device.

Output hardware, coupled to computer by output lines, may similarly be implemented by conventional devices. By way of example, output hardware may include CRT display or LCD ter- minal for displaying a graphical representation of a prostaglandin-binding pocket of this invention using a program such as QUANTA as described herein. Output hardware may also include a printer, so that hard copy output may be produced, or a disk drive, to store system output for later use. Output hardware may also include a CD or DVD recorder, ZIP, USB drives, various sorts of flash memory or other machine-readable data storage device.

In operation, CPU coordinates the use of the various input and output devices, coordinates data accesses from mass storage and accesses to and from working memory, and determines the sequence of data processing steps. A number of programs may be used to process the machine-readable data of this invention. Such programs are discussed in reference to the computational methods of drug discovery as described herein. Specific references to components of the hardware system are included as appropriate throughout the following descrip- tion of the data storage medium.

A cross section of a magnetic data storage medium which can be encoded with machine- readable data that can be carried out by a system such as e.g. a layered medium. Medium can be a conventional floppy diskette or hard disk, having a suitable substrate, which may be conventional, and a suitable coating, which may be conventional, on one or both sides, con- taining magnetic domains (not visible) whose polarity or orientation can be altered magnetically. Medium may also have an opening (not shown) for receiving the spindle of a disk drive or other data storage device.

The magnetic domains of coating of medium are polarized or oriented so as to encode in manner which may be conventional, machine readable data such as that described herein, for execution by a system such.

A cross section of an optically-readable data storage medium which also can be encoded with such a machine-readable data, or set of instructions, which can be carried out by a system such as a system described above. Medium can be a conventional compact disk read only memory (CD-ROM) or a rewritable medium such as a magneto-optical disk which is optically readable and magneto-optically writeable. Medium preferably has a suitable substrate, which may be conventional, and a suitable coating, which may be conventional, usually of one side of substrate.

In the case of CD-ROM, as is well known, coating is reflective and is impressed with a plurality of pits to encode the machine-readable data. The arrangement of pits is read by reflecting laser light off the surface of coating. A protective coating, which preferably is substantially transparent, is provided on top of coating. In the case of a magneto-optical disk, as is well known, coating has no pits, but has a plurality of magnetic domains whose polarity or orientation can be changed magnetically when heated above a certain temperature, as by a laser. The orientation of the domains can be read by measuring the polarization of laser light reflected from coating. The arrangement of the domains encodes the data as described above.

In one aspect, the structure coordinates of PPARγ- or PPARγ-like molecules are produced by homology modelling (SaIi, A. "Modeling mutations and homologous proteins," Curr. Opin. Biotech., 6, 437-451 (1995)) of at least a portion of the structure coordinates of Figure 1 or 2. Homology modelling can be used to generate structural models of PPARγ homologues or other homologous proteins based on the known structure of PPARγ. This can be achieved by performing one or more of the following steps: performing sequence alignment between the amino acid sequence of an unknown molecule against the amino acid sequence of PPARγ; identifying conserved and variable regions by sequence or structure; generating structure coordinates for structurally conserved residues of the unknown structure from those of PPARγ; generating conformations for the structurally variable residues in the unknown structure; replacing the non-conserved residues of PPARγ with residues in the unknown structure; building side chain conformations; and refining and/or evaluating the unknown structure.

For example, since the protein sequence of the ligand binding domain of PPARy and PPARα or PPARδ can be aligned relative to each other, it is possible to construct models of the struc- tures of PPARα or PPARδ, particularly in the regions of the binding pockets, using the PPARγ structure, or the opposite way, making PPARγ models using PPARα or PPARδ molecular structures. Software programs that are useful in homology modelling include XALIGN (Wishart, D. S. et al., Comput. Appl. Biosci., 10, pp. 687-88 (1994)) and CLUSTAL W Alignment Tool (Hig- gins D. G. et al., Methods Enzymol., 266, pp. 383-402 (1996)). See also, U. S. Patent No. 5,884, 230. These references are incorporated herein by reference.

To perform the sequence alignment, programs such as available from the Genetics Computer Group (Waterman in Advances in Applied Mathematics 2, 482 (1981), which is incorporated herein by reference) and CLUSTAL W Alignment Tool (Higgins D. G. et al., Methods Enzymol., 266, pp. 383-402 (1996), which is incorporated by reference) can be used. To model the amino acid side chains of PPARα or PPARδ, the amino acid residues in PPARγ can be replaced, using a computer graphics program such as "O" (Jones et al, (1991) Acta Cryst. Sect. A, 47: 110-119), by those of the homologous protein, where they differ. The same orientation or a different orientation of the amino acid can be used. Insertions and deletions of amino acid residues may be necessary where gaps occur in the sequence alignment. Homology modelling can be performed using, for example, the computer programs SWISS- MODEL available through Glaxo Wellcome Experimental Research in Geneva, Switzerland; WHATIF available on EMBL servers ; Schnare et al., J. MoI. Biol, 256: 701-719 (1996); Blun- dell et al., Nature 326: 347-352 (1987); Fetrow and Bryant, Bio/Technology 11 : 479-484 (1993); Greer, Methods in Enzymology 202: 239-252 (1991); and Johnson et Crit. Rev. Bio- chem. MoI Biol. 29: 1-68 (1994). An example of homology modelling can be found, for example, in Szklarz G. D., Life Sci. 61 : 2507-2520 (1997). These references are incorporated herein by reference.

Thus, in accordance with the present invention, data capable of generating the three- dimensional structure of the above prostaglandin-binding pockets, can be stored in a machine-readable storage medium, which is capable of displaying a graphical three-dimensional representation of the structure.

Rational Drug Design

The PPARγ structure coordinates as described above or the three-dimensional graphical repre- sentation generated from these coordinates may be used in conjunction with a computer for a variety of purposes, including drug discovery.

For example, the structure encoded by the data may be computationally evaluated for its ability to associate with chemical entities. Chemical entities that associate with PPARγ may inhibit or activate PPARγ or its homologues, and are potential drug candidates. Alternatively, the structure encoded by the data may be displayed in a graphical three-dimensional representation on a computer screen. This allows visual inspection of the structure, as well as visual inspection of the structure's association with chemical entities.

In a further aspect of the invention, a method for screening for chemical entities useful for the treatment of prostaglandin and/or PPAR related diseases such as cancer (especially in lung, skin, breast, colon, pancreas, prostate, liposarcoma), inflammation, skin and hair disorders, diabetes, obesity, hypertension, and impaired glucose tolerance, is provided. In one embodiment, the invention provides a method for screening for chemical entities useful for the treatment of cancer (especially in lung, skin, breast, colon, pancreas, prostate, and/or liposarcoma).

In one aspect, the invention thus provides a method for evaluating the ability of a chemical entity to associate with a prostaglandin-binding pocket of a PPAR protein molecule, which prostaglandin-binding pocket is defined by three-dimensional structure coordinates of a set of amino acids that comprises amino acid residues corresponding to four or more selected from the group consisting of TYR 222, PHE 226, PRO 227, LEU 228, THR 229, LYS 230, CYS 285, ARG 288, SER 289, ALA 292, GLU 295, ILE 296, ILE 325, ILE 326, TYR 327, THR 328, MET 329, LEU 330, ALA 331, SER 332, LEU 333, MET 364, PHE 374, ASN 375, ALA 376, LEU 377, GLU 378, LEU 379, ASP 380, ASP 381, LEU 384, and LEU 435 of SEQ ID NO: 1, comprising the steps of: (a) providing the structure coordinates of said prostaglandin-binding pocket on a computer comprising the means for generating three-dimensional structural information from said structure coordinates; (b) employing computational means to perform a fitting operation between the chemical entity and the prostaglandin-binding pocket; and (c) analyzing the results of said fitting operation to evaluate the association between the chemical entity and the prostaglandin-binding pocket. In a further aspect of the invention, step (c) further comprises that the association is quantified. In another aspect, the invention provides a method for evaluating the ability of a chemical entity to associate with prostaglandin-binding pocket of a PPARγor PPARγ-like protein molecule comprising an amino acid sequence at least 65% identical to residues 207-476 of SEQ ID NO: 1, which prostaglandin-binding pocket is defined by three-dimensional structure coordinates of a set of amino acids that comprises (i) an amino acid residue corresponding to LEU 228, and (ii) three or more amino acids corresponding to amino acid residues selected from the group consisting of TYR 222, PHE 226, PRO 227, THR 229, LYS 230, CYS 285, ARG 288, SER 289, ALA 292, GLU 295, ILE 296, ILE 325, ILE 326, TYR 327, THR 328, MET 329, LEU 330, ALA 331, SER 332, LEU 333, MET 364, PHE 374, ASN 375, ALA 376, LEU 377, GLU 378, LEU 379, ASP 380, ASP 381, LEU 384, and LEU 435 of SEQ ID NO: 1, comprising the steps of: (a) providing the structure coordinates of said prostaglandin-binding pocket on a computer comprising the means for generating three- dimensional structural information from said structure coordinates; (b) employing computational means to perform a fitting operation between the chemical entity and the prostaglandin-binding pocket; and (c) analyzing the results of said fitting operation to evaluate the association between the chemical entity and the prostaglandin-binding pocket. In a further aspect of the invention, step (c) further comprises that the association is quantified.

In another aspect, the invention provides a method for evaluating the ability of a chemical entity to associate with a prostaglandin-binding pocket of a PPARγ or PPARγ-like protein molecule, which prostaglandin-binding pocket is defined by three-dimensional structure coordinates of a set of amino acids that correspond to four or more PPARγ amino acid residues selected from the group consisting of TYR 222, PHE 226, PRO 227, LEU 228, THR 229, LYS 230, ALA 292, GLU 295, ILE 296, ILE 325, THR 328, MET 329, ALA 331, SER 332, LEU 333, PHE 374, ASN 375, ALA 376, LEU 377, GLU 378, LEU 379, ASP 380, ASP 381, LEU 384, and LEU 435 and optionally one or more selected from the group consisting of CYS 285, ARG 288, SER 289, ILE 326, TYR 327, LEU 330 and MET 364 according to figure 1, wherein the root mean square deviation of the backbone atoms between amino acid residues of said molecule and said PPARγ residues is not greater than about 3 A, comprising the steps of: (a) providing the structure coordinates of said prostaglandin-binding pocket on a computer comprising the means for generating three-dimensional structural information from said structure coordinates; (b) employing computational means to perform a fitting operation between the chemical entity and the prostaglandin-binding pocket; and (c) analyzing the results of said fitting operation to evaluate the association between the chemical entity and the prostaglandin- binding pocket. In a further aspect of the invention, step (c) further comprises that the association is quantified.

In a further aspect, the above method further comprises generating a three-dimensional graphical representation of the prostaglandin-binding pocket prior to step (b).

In above aspect of the invention and in the following aspects the prostaglandin-binding pocket can be further defined as follows:

In one aspect of the invention, the prostaglandin-binding pocket is defined by structure coordinates of a set of amino acids that correspond to four or more PPARγ amino acid residues selected from the group consisting of TYR 222, PHE 226, PRO 227, LEU 228, THR 229, LYS 230, ALA 292, GLU 295, MET 329, SER 332, LEU 333, ASP 381, and LEU 384.

In a further aspect of the invention, the prostaglandin-binding pocket is defined by structure coordinates of a set of amino acids that correspond to PPARγ amino acid residues PHE 226, PRO 227, LEU 228 and MET 329 and optionally one or more selected from the group consisting Of TYR 222, THR 229, LYS 230, ALA 292, GLU 295, ILE 296, ILE 325, THR 328, ALA 331, SER 332, LEU 333, PHE 374, ASN 375, ALA 376, LEU 377, GLU 378, LEU 379, ASP 380, ASP 381, LEU 384, and LEU 435.

In a further aspect of the invention, the prostaglandin-binding pocket is defined by structure coordinates of a set of amino acids that correspond to PPARγ amino acid residues PHE 226, PRO 227, LEU 228 and MET 329 and optionally one or more selected from the group consisting Of TYR 222, THR 229, LYS 230, ALA 292, GLU 295, SER 332, LEU 333, ASP 381, and LEU 384. In a further aspect of the invention, the prostaglandin-binding pocket is defined by structure coordinates of a set of amino acids that correspond to PPARγ amino acid residues TYR 222, PHE 226, PRO 227, LEU 228, THR 229, LYS 230, ALA 292, GLU 295, MET 329, SER 332, LEU 333, ASP 381, and LEU 384.

In a further aspect of the invention, the prostaglandin-binding pocket is defined by structure coordinates of a set of amino acids that correspond to PPARγ amino acid residues TYR 222, PHE 226, PRO 227, LEU 228, THR 229, LYS 230, ALA 292, GLU 295, ILE 296, ILE 325, THR

328, MET 329, ALA 331, SER 332, LEU 333, PHE 374, ASN 375, ALA 376, LEU 377, GLU 378, LEU 379, ASP 380, ASP 381, LEU 384, and LEU 435.

In a further aspect of the invention, the prostaglandin-binding pocket is defined by structure coordinates of a set of above amino acids and further comprises one or more amino acids that correspond to PPARγ amino acid residues selected from the group consisting of CYS 285, ARG 288, SER 289, ILE 326, TYR 327, LEU 330 and MET 364.

In a further aspect of the invention, the prostaglandin-binding pocket is defined by structure coordinates of a set of amino acids that correspond to PPARγ amino acid residues TYR 222, PHE 226, PRO 227, LEU 228, THR 229, LYS 230, CYS 285, ARG 288, SER 289, ALA 292, GLU 295, ILE 326, TYR 327, MET 329, LEU 330, SER 332, LEU 333, MET 364, ASP 381, and LEU 384.

In a further aspect of the invention, the prostaglandin-binding pocket is defined by structure coordinates of a set of amino acids that correspond to PPARγ amino acid residues TYR 222, PHE 226, PRO 227, LEU 228, THR 229, LYS 230, CYS 285, ARG 288, SER 289, ALA 292, GLU 295, ILE 296, ILE 325, ILE 326, TYR 327, THR 328, MET 329, LEU 330, ALA 331, SER 332, LEU 333, MET 364, PHE 374, ASN 375, ALA 376, LEU 377, GLU 378, LEU 379, ASP 380, ASP 381, LEU 384, and LEU 435.

In another aspect, the invention provides a method of designing a compound or complex that associates with a prostaglandin-binding pocket of a PPAR protein molecule, which prostaglandin-binding pocket is defined by three-dimensional structure coordinates of a set of amino acids that comprises amino acid residues corresponding to four or more selected from the group consisting of TYR 222, PHE 226, PRO 227, LEU 228, THR 229, LYS 230, CYS 285, ARG 288, SER 289, ALA 292, GLU 295, ILE 296, ILE 325, ILE 326, TYR 327, THR 328, MET

329, LEU 330, ALA 331, SER 332, LEU 333, MET 364, PHE 374, ASN 375, ALA 376, LEU 377, GLU 378, LEU 379, ASP 380, ASP 381, LEU 384, and LEU 435 of SEQ ID NO: 1, comprising the steps of: (a) providing the structure coordinates of said prostaglandin-binding pocket on a computer comprising the means for generating three- dimensional structural information from said structure coordinates; and (b) using the computer to perform a fitting operation to associate a first chemical entity with the prostaglandin-binding pocket; (c) performing a fitting op- eration to associate at least a second chemical entity with the prostaglandin-binding pocket; (d) quantifying the association between the first and second chemical entity and the prostaglandin-binding pocket; (e) optionally repeating steps b) to d) with another first and second chemical entity, selecting a first and a second chemical entity based on said quantified association of all of said first and second chemical entity; (f) optionally, visually inspecting the re- lationship of the first and second chemical entity to each other in relation to the prostaglandin-binding pocket on a computer screen using the three-dimensional graphical representation of the prostaglandin-binding pocket and said first and second chemical entity; and (g) assembling the first and second chemical entity into a compound or complex that associates with said prostaglandin-binding pocket by model building.

In another aspect, the invention provides a method of designing a compound or complex that associates with a prostaglandin-binding pocket of a PPARγor PPARγ-like protein molecule comprising an amino acid sequence at least 65% identical to residues 207-476 of SEQ ID NO: 1, which prostaglandin-binding pocket is defined by three-dimensional structure coordinates of a set of amino acids that comprises (i) an amino acid residue corresponding to LEU 228, and (ii) three or more amino acids corresponding to amino acid residues selected from the group consisting of TYR 222, PHE 226, PRO 227, THR 229, LYS 230, CYS 285, ARG 288, SER 289, ALA 292, GLU 295, ILE 296, ILE 325, ILE 326, TYR 327, THR 328, MET 329, LEU 330, ALA 331, SER 332, LEU 333, MET 364, PHE 374, ASN 375, ALA 376, LEU 377, GLU 378, LEU 379, ASP 380, ASP 381, LEU 384, and LEU 435 of SEQ ID NO: 1, comprising the steps of: (a) providing the structure coordinates of said prostaglandin-binding pocket on a computer comprising the means for generating three- dimensional structural information from said structure coordinates; and (b) using the computer to perform a fitting operation to associate a first chemical entity with the prostaglandin-binding pocket; (c) performing a fitting operation to associate at least a second chemical entity with the prostaglandin-binding pocket; (d) quantifying the association between the first and second chemical entity and the prostaglandin-binding pocket; (e) optionally repeating steps b) to d) with another first and second chemical entity, selecting a first and a second chemical entity based on said quantified association of all of said first and second chemical entity; (f) optionally, visually inspecting the relationship of the first and second chemical entity to each other in relation to the pros- taglandin-binding pocket on a computer screen using the three-dimensional graphical representation of the prostaglandin-binding pocket and said first and second chemical entity; and (g) assembling the first and second chemical entity into a compound or complex that associates with said prostaglandin-binding pocket by model building.

In another aspect, the invention provides a method of designing a compound or complex that associates with a prostaglandin-binding pocket of a PPARγ or PPARγ-like protein molecule, which prostaglandin-binding pocket is defined by three-dimensional structure coordinates of a set of amino acids that correspond to four or more PPARγ amino acid residues selected from the group consisting of TYR 222, PHE 226, PRO 227, LEU 228, THR 229, LYS 230, ALA 292, GLU 295, ILE 296, ILE 325, THR 328, MET 329, ALA 331, SER 332, LEU 333, PHE 374, ASN 375, ALA 376, LEU 377, GLU 378, LEU 379, ASP 380, ASP 381, LEU 384, and LEU 435 and optionally one or more selected from the group consisting of CYS 285, ARG 288, SER 289, ILE

326, TYR 327, LEU 330 and MET 364 according to figure 1, wherein the root mean square deviation of the backbone atoms between amino acid residues of said molecule and said PPARγ residues is not greater than about 3 A, comprising the steps of: (a) providing the structure coordinates of said prostaglandin-binding pocket on a computer comprising the means for generating three- dimensional structural information from said structure coordinates; and (b) using the computer to perform a fitting operation to associate a first chemical entity with the prostaglandin-binding pocket; (c) performing a fitting operation to associate at least a second chemical entity with the prostaglandin-binding pocket; (d) quantifying the association between the first and second chemical entity and the prostaglandin-binding pocket; (e) option- ally repeating steps b) to d) with another first and second chemical entity, selecting a first and a second chemical entity based on said quantified association of all of said first and second chemical entity; (f) optionally, visually inspecting the relationship of the first and second chemical entity to each other in relation to the prostaglandin-binding pocket on a computer screen using the three-dimensional graphical representation of the prostaglandin-binding pocket and said first and second chemical entity; and (g) assembling the first and second chemical entity into a compound or complex that associates with said prostaglandin-binding pocket by model building.

According to another aspect, the invention provides a method for designing, selecting and/or optimizing a chemical entity that binds a prostaglandin-binding pocket of a PPAR protein molecule, which prostaglandin-binding pocket is defined by three-dimensional structure coordinates of a set of amino acids that comprises amino acid residues corresponding to four or more selected from the group consisting of TYR 222, PHE 226, PRO 227, LEU 228, THR 229, LYS 230, CYS 285, ARG 288, SER 289, ALA 292, GLU 295, ILE 296, ILE 325, ILE 326, TYR

327, THR 328, MET 329, LEU 330, ALA 331, SER 332, LEU 333, MET 364, PHE 374, ASN 375, ALA 376, LEU 377, GLU 378, LEU 379, ASP 380, ASP 381, LEU 384, and LEU 435 of SEQ ID NO: 1, comprising the steps of: (a) providing the structure coordinates of said prostaglandin- binding pocket on a computer comprising the means for generating three-dimensional structural information from said structure coordinates; and (b) designing, selecting and/or optimizing said chemical entity by performing a fitting operation between said chemical entity and said three-dimensional structural information of said prostaglandin-binding pocket.

According to another aspect, the invention provides a method for designing, selecting and/or optimizing a chemical entity that binds a prostaglandin-binding pocket of a PPARγor PPARγ- like protein molecule comprising an amino acid sequence at least 65% identical to residues 207-476 of SEQ ID NO: 1, which prostaglandin-binding pocket is defined by three-dimensional structure coordinates of a set of amino acids that comprises (i) an amino acid residue corresponding to LEU 228, and (ii) three or more amino acids corresponding to amino acid residues selected from the group consisting of TYR 222, PHE 226, PRO 227, THR 229, LYS 230, CYS 285, ARG 288, SER 289, ALA 292, GLU 295, ILE 296, ILE 325, ILE 326, TYR 327, THR 328, MET 329, LEU 330, ALA 331, SER 332, LEU 333, MET 364, PHE 374, ASN 375, ALA 376, LEU 377, GLU 378, LEU 379, ASP 380, ASP 381, LEU 384, and LEU 435 of SEQ ID NO: 1, comprising the steps of: (a) providing the structure coordinates of said prostaglandin-binding pocket on a computer comprising the means for generating three-dimensional structural information from said structure coordinates; and (b) designing, selecting and/or optimizing said chemical entity by performing a fitting operation between said chemical entity and said three- dimensional structural information of said prostaglandin-binding pocket.

According to another aspect, the invention provides a method for designing, selecting and/or optimizing a chemical entity that binds the prostaglandin-binding pocket of a PPARγ or PPARγ- like protein molecule, which prostaglandin-binding pocket is defined by three-dimensional structure coordinates of a set of amino acids that correspond to four or more PPARγ amino acid residues selected from the group consisting of TYR 222, PHE 226, PRO 227, LEU 228, THR 229, LYS 230, ALA 292, GLU 295, ILE 296, ILE 325, THR 328, MET 329, ALA 331, SER 332, LEU 333, PHE 374, ASN 375, ALA 376, LEU 377, GLU 378, LEU 379, ASP 380, ASP 381, LEU 384, and LEU 435 and optionally one or more selected from the group consisting of CYS 285, ARG 288, SER 289, ILE 326, TYR 327, LEU 330 and MET 364 according to figure 1, wherein the root mean square deviation of the backbone atoms between amino acid residues of said molecule and said PPARγ residues is not greater than about 3 A comprising the steps of: (a) providing the structure coordinates of said prostaglandin-binding pocket on a computer comprising the means for generating three-dimensional structural information from said structure coordinates; and (b) designing, selecting and/or optimizing said chemical entity by performing a fitting operation between said chemical entity and said three-dimensional structural information of said prostaglandin-binding pocket.

In one aspect, the above methods for designing, selecting, and/or optimizing a chemical entity further comprises the following steps before step (a) : (c) producing a crystal of a mole- cule or molecular complex comprising PPARγ or homologue thereof; (d) determining the three-dimensional structure coordinates of the molecule or molecular complex by X-ray diffraction of the crystal; and (e) identifying the prostaglandin-binding pocket.

Three-dimensional structural information in step (a) may be generated by instructions such as a computer program or commands that can generate a three-dimensional structure or graphi- cal representation; subtract distances between atoms; calculate chemical energies for a PPARγ molecule, molecular complex or homologues thereof; or calculate or minimize energies of an association of PPARγ molecule, molecular complex or homologues thereof to a chemical entity. These types of computer programs are known in the art. The graphical representation can be generated or displayed by commercially available software programs. Examples of software programs include but are not limited to QUANTA (Accelrys Inc., San Diego, ^®2001, 2002), O (Jones et al., Acta A47, pp. 110-119 (1991)) and RIBBONS (Carson, J. Appl. 24, pp. 9589- 961 (1991)), which are incorporated herein by reference. Certain software programs may imbue this representation with physico-chemical attributes which are known from the chemical composition of the molecule, such as residue charge, hydrophobicity, torsional and rotational degrees of freedom for the residue or segment, etc. Examples of software programs for calculating chemical energies are described below.

In one aspect, the method is for evaluating the ability of a chemical entity to associate with a prostaglandin-binding pocket as defined above.

This method comprises the steps of: (a) employing computational means to perform a fitting operation between the chemical entity and the prostaglandin-binding pocket as described before; (b) analyzing the results of said fitting operation to quantify the association between the chemical entity and the prostaglandin-binding pocket; and optionally (c) outputting said quantified association to a suitable output hardware, such as a CRT display terminal or LCD monitor, a CD or DVD recorder, a disk drive, or other machine-readable data storage device, as described previously. The method may further comprise generating a three-dimensional structure, graphical representation thereof, or both of all or part of the molecule or molecular complex prior to step (a). In another aspect, the invention provides a method for screening a plurality of chemical entities to associate more strongly than a given threshold value, such as a deformation energy of binding of less than 7 kcal/mol, with said prostaglandin-binding pocket:

(a) employing computational means, which utilize said structure coordinates to perform a fit- ting operation between one of said chemical entities from the plurality of chemical entities and said prostaglandin-binding pocket;

(b) quantifying the binding between the chemical entity and the prostaglandin-binding pocket; (c) repeating steps (a) and (b) for each remaining chemical entity; and (d) outputting to a suitable output hardware a set of chemical entities that associate with the prostaglandin- binding pocket more strongly than a chosen threshold value, optionally defined as an upper quantile yielding the desired fraction of chemical entities.

In another aspect, the method comprises the steps of: (a) constructing a computer model of a prostaglandin-binding pocket; b) selecting a chemical entity to be evaluated by a method selected from the group consisting of assembling said chemical entity; selecting a chemical en- tity from a small molecule database; de novo ligand design of said chemical entity; and modifying a known agonist or inhibitor, or a portion thereof, of an PPARγ protein or homologue thereof; (c) employing computational means to perform a fitting operation between computer models of said chemical entity to be evaluated and said prostaglandin-binding pocket in order to provide an energy-minimized configuration of said chemical entity in the prostaglandin- binding pocket; and (d) evaluating the results of said fitting operation to quantify the association between said chemical entity and the prostaglandin-binding pocket model, whereby evaluating the ability of said chemical entity to associate with said prostaglandin-binding pocket.

In another aspect, the invention provides a method of using a computer for evaluating the ability of a chemical entity to associate with a prostaglandin-binding pocket of a PPARγ or

PPARγ-like protein molecule comprising an amino acid sequence at least 65% identical to residues 207-476 of SEQ ID NO: 1, which prostaglandin-binding pocket is defined by three- dimensional structure coordinates of a set of amino acids that comprises (i) an amino acid residue corresponding to LEU 228, and (ii) three or more amino acids corresponding to amino acid residues selected from the group consisting of TYR 222, PHE 226, PRO 227, THR 229, LYS 230, CYS 285, ARG 288, SER 289, ALA 292, GLU 295, ILE 296, ILE 325, ILE 326, TYR 327, THR 328, MET 329, LEU 330, ALA 331, SER 332, LEU 333, MET 364, PHE 374, ASN 375, ALA 376, LEU 377, GLU 378, LEU 379, ASP 380, ASP 381, LEU 384, and LEU 435 of SEQ ID NO: 1, wherein said computer comprises a machine-readable data storage medium comprising a data storage material encoded with said structure coordinates defining said prostaglandin- binding pocket and means for generating a three-dimensional graphical representation of the prostaglandin-binding pocket, and wherein said method comprises the steps of: (a) position- ing a first chemical entity within said prostaglandin-binding pocket using a graphical three- dimensional representation of the structure of the chemical entity and the prostaglandin- binding pocket; (b) performing a fitting operation between said chemical entity and said prostaglandin-binding pocket by employing computational means; and (c) analyzing the results of said fitting operation to quantify the association between said chemical entity and the pros- taglandin-binding pocket and optionally (d) outputting said quantified association to a suitable output hardware.

In another aspect, the invention provides a method of using a computer for evaluating the ability of a chemical entity to associate with a prostaglandin-binding pocket of a PPARγ or PPARγ-like protein molecule, which prostaglandin-binding pocket is defined by three- dimensional structure coordinates of a set of amino acids that correspond to four or more

PPARγ amino acid residues selected from the group consisting of TYR 222, PHE 226, PRO 227, LEU 228, THR 229, LYS 230, ALA 292, GLU 295, ILE 296, ILE 325, THR 328, MET 329, ALA 331, SER 332, LEU 333, PHE 374, ASN 375, ALA 376, LEU 377, GLU 378, LEU 379, ASP 380, ASP 381, LEU 384, and LEU 435 and optionally one or more selected from the group consist- ing of CYS 285, ARG 288, SER 289, ILE 326, TYR 327, LEU 330 and MET 364 according to figure 1, wherein the root mean square deviation of the backbone atoms between amino acid residues of said molecule and said PPARγ residues is not greater than about 3 A, wherein said computer comprises a machine-readable data storage medium comprising a data storage material encoded with said structure coordinates defining said prostaglandin-binding pocket and means for generating a three-dimensional graphical representation of the prostaglandin- binding pocket, and wherein said method comprises the steps of: (a) positioning a first chemical entity within said prostaglandin-binding pocket using a graphical three-dimensional representation of the structure of the chemical entity and the prostaglandin-binding pocket; (b) performing a fitting operation between said chemical entity and said prostaglandin-binding pocket by employing computational means; and (c) analyzing the results of said fitting operation to quantify the association between said chemical entity and the prostaglandin-binding pocket and optionally (d) outputting said quantified association to a suitable output hardware.

The above methods may further comprise the steps of: (e) repeating steps (a) through (d) with a second chemical entity; and (f) selecting at least one of said first or second chemical entity that associates with said prostaglandin-binding pocket based on said quantified association of said first or second chemical entity.

In yet another aspect of the invention, a method for identifying a ligand of a prostaglandin- binding pocket of a PPAR protein molecule, which prostaglandin-binding pocket is defined by three-dimensional structure coordinates of a set of amino acids that comprises amino acid residues corresponding to four or more selected from the group consisting of TYR 222, PHE 226, PRO 227, LEU 228, THR 229, LYS 230, CYS 285, ARG 288, SER 289, ALA 292, GLU 295, ILE 296, ILE 325, ILE 326, TYR 327, THR 328, MET 329, LEU 330, ALA 331, SER 332, LEU 333, MET 364, PHE 374, ASN 375, ALA 376, LEU 377, GLU 378, LEU 379, ASP 380, ASP 381, LEU 384, and LEU 435 of SEQ ID NO: 1, comprising the steps of: (a) using a three- dimensional structure of the prostaglandin-binding pocket to design, select and/or optimize a chemical entity; (b) contacting the chemical entity with the molecule; and (c) identifying any chemical entity capable of binding the molecule as a ligand, is provided.

In another aspect of the invention, a method for identifying a ligand of a prostaglandin- binding pocket of a PPARγ or PPARγ-like protein molecule comprising an amino acid sequence at least 65% identical to residues 207-476 of SEQ ID NO: 1, which prostaglandin-binding pocket is defined by three-dimensional structure coordinates of a set of amino acids that comprises (i) an amino acid residue corresponding to LEU 228, and (ii) three or more amino acids corresponding to amino acid residues selected from the group consisting of TYR 222, PHE 226, PRO 227, THR 229, LYS 230, CYS 285, ARG 288, SER 289, ALA 292, GLU 295, ILE 296, ILE 325, ILE 326, TYR 327, THR 328, MET 329, LEU 330, ALA 331, SER 332, LEU 333, MET 364, PHE 374, ASN 375, ALA 376, LEU 377, GLU 378, LEU 379, ASP 380, ASP 381, LEU 384, and LEU 435 of SEQ ID NO: 1, comprising the steps of: (a) using a three-dimensional structure of the prostaglandin-binding pocket to design, select and/or optimize a chemical en- tity; (b) contacting the chemical entity with the molecule; and (c) identifying any chemical entity capable of binding the molecule as a ligand, is provided.

In another aspect of the invention, a method for identifying a ligand of a prostaglandin- binding pocket of a PPARγ or PPARγ-like protein molecule, which prostaglandin-binding pocket is defined by three-dimensional structure coordinates of a set of amino acids that correspond to four or more PPARγ amino acid residues selected from the group consisting of TYR 222, PHE 226, PRO 227, LEU 228, THR 229, LYS 230, ALA 292, GLU 295, ILE 296, ILE 325, THR 328, MET 329, ALA 331, SER 332, LEU 333, PHE 374, ASN 375, ALA 376, LEU 377, GLU 378, LEU 379, ASP 380, ASP 381, LEU 384, and LEU 435 and optionally one or more selected from the group consisting of CYS 285, ARG 288, SER 289, ILE 326, TYR 327, LEU 330 and MET 364 according to figure 1, wherein the root mean square deviation of the backbone atoms between amino acid residues of said molecule and said PPARγ residues is not greater than about 3 A, comprising the steps of: (a) using a three-dimensional structure of the prostaglandin- binding pocket to design, select and/or optimize a chemical entity; (b) contacting the chemi- cal entity with the molecule; and (c) identifying any chemical entity capable of binding the molecule as a ligand, is provided.

Alternatively, the structure coordinates of the prostaglandin-binding pocket as defined above may be utilized in a method for identifying an agonist or antagonist of a molecule comprising a prostaglandin-binding pocket of PPARγ. This method comprises the steps of: (a) using a three-dimensional structure of the prostaglandin-binding pocket to design, select and/or optimize a chemical entity; (b) contacting the chemical entity with the molecule or molecular complex; (c) monitoring the activity of the molecule or molecular complex; and (d) classifying the chemical entity as an agonist or antagonist based on the effect of the chemical entity on the activity of the molecule or molecular complex.

In one aspect, step (a) is using a three-dimensional structure of the prostaglandin-binding pocket of the molecule or molecular complex. In another aspect, the three-dimensional structure is displayed as a graphical representation.

In another aspect, the method comprises the steps of: (a) constructing a computer model of a prostaglandin-binding pocket of the molecule or molecular complex; (b) selecting a chemical entity to be evaluated by a method selected from the group consisting of assembling said chemical entity; selecting a chemical entity from a small molecule database; de novo ligand design of said chemical entity; and modifying a known agonist or inhibitor, or a portion thereof, of an PPARγ protein or homologue thereof; (c) employing computational means to perform a fitting operation between computer models of said chemical entity to be evaluated and said prostaglandin-binding pocket in order to provide an energy-minimized configuration of said chemical entity in the prostaglandin-binding pocket; and (d) evaluating the results of said fitting operation to quantify the association between said chemical entity and the prostaglandin-binding pocket model, whereby evaluating the ability of said chemical entity to associate with said prostaglandin-binding pocket; (e) synthesizing said chemical entity; and (f) contacting said chemical entity with said molecule or molecular complex to determine the ability of said compound to activate or inhibit said molecule.

In one aspect, the invention provides a method of designing a compound or complex that associates with the prostaglandin-binding pocket comprising the steps of: (a) providing the structure coordinates of said prostaglandin-binding pocket or protein on a computer comprising the means for generating three-dimensional structural information from said structure coordinates; and (b) using the computer to perform a fitting operation to associate a first chemical entity with the prostaglandin-binding pocket; (c) performing a fitting operation to associate at least a second chemical entity with all or, part of the prostaglandin-binding pocket; (d) quantifying the association between the first and second chemical entity and the prostaglandin-binding pocket; (e) optionally repeating steps (b) to (d) with another first and second chemical entity, selecting a first and a second chemical entity based on said quantified association of all of said first and second chemical entity; (f) optionally, visually inspecting the relationship of the first and second chemical entity to each other in relation to the prostaglandin-binding pocket on a computer screen using the three-dimensional graphical representation of the prostaglandin-binding pocket and said first and second chemical entity; and (g) assembling the first and second chemical entity into a compound or complex that associates with said prostaglandin-binding pocket by model building.

In one aspect, the present invention permits the use of molecular design techniques to identify, select and design chemical entities, including inhibitory compounds, capable of binding to PPAR, PPARγ or PPARγ-like prostaglandin-binding pockets.

The present elucidation of the prostaglandin-binding pocket on PPARγ can provide the necessary information for designing new chemical entities and compounds that may interact with PPARγ substrate or prostaglandin-binding pockets or PPARγ-like substrate or prostaglandin- binding pockets, in whole or in part. Due to the homology in the binding pocket core between PPARγ, PPARα and PPARδ, compounds that inhibit or activate PPARγ are also expected to inhibit PPARα and PPARδ, especially those compounds that bind to the prostaglandin-binding pocket.

Throughout this section, discussions about the ability of a chemical entity to bind to, associate with or inhibit PPARγ prostaglandin-binding pockets refer to features of the entity alone.

Assays to determine if a compound binds to a PPAR protein, PPARγ, or a PPARy-like protein are well known in the art and are for example as described below as assay method 1 and assay method 2.

Assay method 1: PPARγ receptor binding assay: This assay can be used to, with use of a suitable ligand toward the prostaglandin-binding pocket, hereafter called the "Start-ligand", identify chemical entities such as, e.g., low molecular weight compounds, which displace the Start-ligand from the Ligand-binding-domain (LBD) of the Peroxisome proliferator-activated receptor γ (PPARγ). Exemplary "Start-ligands" include Prostaglandin J₂, Prostaglandin B₂, 15-Deoxy Δ¹²'¹⁴-prostaglandin J₂, as well as other suitable ligands known in the art. When new ligands are designed, these may serve as new Start-ligands for further drug testing and development.

The method is a ligand binding assay based upon IPA (imaging proximity assay) particles and is based on the assay by Nichols et al. (Nichols et al., 1998a; Nichols et al. ,1998b).

The method can be described as follows: The Start-ligand is marked with ³H, while GST-PPARγ-LBD is marked with biotin and the scintillation proximity assay SPA particles are coated with Streptavidin (SA). The receptor is coupled to Glutathiontransferase (GST); GST is a tag that is used to purify the receptor from homogenized cells. When the SPA particles are added they bind to the biotin residues on the GST-PPARγ-LBD. ³H-Start-ligand binds to GST-PPARγ-LBD, and the proximity between the radioactive tritium and the SPA particle results in emission of light from the SPA particles. The amount of light emitted is proportional to the amount of ³H-Start-ligand bound to the binding protein. When a compound that displaces the Start-ligand is present it results in a decrease in the amount of light emitted. Following the binding constant (K_d) can be determined. Compounds with a Kd value < 1 mM is regarded as a "Hit" and can be used further in the drug design process. The Hit is preferably tested also by assay method 2.

Assay method 2: A cell based transfection assay:

With this PPAR response element (PPRE) reporter assay, the selective transactivation of PPARγ in chosen cancer cell line after treatment by small molecule compounds, like ligand Hits from assay 1, can be evaluated. Differences in transactivation between different ligands for a cho- sen cell lines is screened. The assay is based on the Allred and Kilgore published assay (Allred and Kilgore, 2005) as described in the following.

The PPRE reporter plasmid : A reporter construct, 3XPPRE-TK-pGL3, contains three copies of a PPRE sequence (AGGACAAAGGTCA) upstream of the mTK promoter between the Xhol and Hinόlll restriction enzyme sites of the pGL3 basic vector (Promega, Madison, WI). BamHI and BgIIl is used to release a 2.2 kb fragment containing the 3XPPRE-mTK-Luciferase. This fragment is ligated into the BamH I receptor site of pRL-TK plasmid (Promega) completing the new reporter which contains both Luciferase and Renilla in a single expression plasmid. Renilla expression is used as a transfection efficiency control.

The transfection assay: Cells are transiently transfected with 5 μg of PPRE reporter plasmid per 12-well plate. Chosen cancer cells are transfected with ESCORT transfection reagent (Sigma-Aldrich) for 4 h. Cells are subsequently treated with the substance to be tested in about micro-molar concentration for 18 h. PPARγ ligand concentrations for each compound used are those shown to be maximally effective following dose reponse studies. Proper vehicle controls including ethanol, DMSO, and methyl acetate are run for each treatment group. Following treatment, cells are lysed in 50 μl passive lysis buffer and treated according to manu- facturer's instructions (Promega dual luciferase assay kit). Luminometry are performed and data are calculated as raw Luciferase Units (RLUs) divided by raw Renilla units. Mean fold induction is obtained by dividing the RLU data from each treatment well by the mean values of the vehicle control appropriate for each treatment. Each set of treatments are performed in replicates of six in three separate experiments. Showing more than 1.05 fold induction change such a compound is regarded as a "hit" and can be used further in the drug design process.

Other methods are readily known by and available to the skilled person in the field.

The design of compounds that bind to or inhibit a PPAR, PPARγ or PPARy-like prostaglandin- binding pocket according to this invention generally involves consideration of two factors. First, the chemical entity must be capable of physically and structurally associating with parts or all of the prostaglandin-binding pocket. Non-covalent molecular interactions important in this association include hydrogen bonding, van der Waals' interactions, hydrophobic interactions and electrostatic interactions.

Second, the chemical entity must be able to assume a conformation that allows it to associate with the PPAR, PPARγ or PPARy-like prostaglandin-binding pocket directly. Although certain portions of the chemical entity will not directly participate in these associations, those portions of the chemical entity may still influence the overall conformation of the molecule. This, in turn, may have a significant impact on potency. Such conformational requirements include the overall three-dimensional structure and orientation of the chemical entity in relation to all or a portion of the prostaglandin-binding pocket, or the spacing between functional groups of a chemical entity comprising several chemical entities that directly interact with the PPAR, PPARγ or PPARγ -like prostaglandin-binding pockets. The potential inhibitory or binding effect of a chemical entity on a prostaglandin-binding pocket may be analyzed prior to its actual synthesis and testing by the use of computer modelling techniques. If the theoretical structure of the given entity suggests insufficient interaction and association between it and the prostaglandin-binding pocket, testing of the entity is obviated.

However, if computer modelling indicates a strong interaction, the molecule may then be synthesized and tested for its ability to bind to a prostaglandin-binding pocket. This may be achieved by testing the ability of the molecule to bind and/or inhibit/activate a PPAR protein such as PPARγ or a PPARy-like protein using the assays described above. In this manner, syn- thesis of inoperative compounds may be avoided.

A potential inhibitor of a prostaglandin-binding pocket of, e.g., PPARγ, may be computationally evaluated by means of a series of steps in which chemical entities or fragments are screened and selected for their ability to associate with the PPARγ prostaglandin-binding pocket.

One skilled in the art may use one of several methods to screen chemical entities or frag- ments for their ability to associate with a prostaglandin-binding pocket of, e.g., PPARγ. This process may begin by visual inspection of, for example, a PPARγ prostaglandin-binding pocket on the computer screen based on the PPARγ structure coordinates in any of Figures 1-2 or other coordinates which define a similar shape generated from the machine-readable storage medium. Selected fragments or chemical entities may then be positioned in a variety of orien- tations, or docked, within that prostaglandin-binding pocket as defined supra. Docking may be accomplished using software such as QUANTA (Accelrys Inc., San Diego, ^®2001, 2002) and Sybyl (Tripos Associates, St. Louis, MO), followed by energy minimization and molecular dynamics with standard molecular mechanics force fields, such as CHARMM and AMBER.

Specialized computer programs may also assist in the process of selecting fragments or chemical entities. These include:

1. GRID (P. J. Goodford, "A Computational Procedure for Determining Energetically Favorable Binding Sites on Biologically Important Macromolecules", Med. Chem., 28, pp. 849-857 (1985) ). GRID is available from Oxford University, Oxford, UK.

2. MCSS (A. Miranker et al., "Functionality Maps of Binding Sites: A Multiple Copy Simultane- ous Search: Structure, Function and Genetics, 11, pp. 29-34 (1991) ). MCSS is available from

Molecular Simulations, San Diego, CA. 3. AUTODOCK (D. S. Goodsell et al., "Automated Docking of Substrates to Proteins by Simulated Annealing", Proteins: Structure, Function, and Genetics, 8, pp. 195-202 (1990) ). AUTODOCK is available from Scripps Research Institute, La JoIIa, CA.

4. DOCK (I. D. Kuntz et al., "A Geometric Approach to Macromolecule-Ligand Interactions", J. MoI. 161, pp. 269-288 (1982) ). DOCK is available from University of California, San Francisco, CA.

Once suitable chemical entities or fragments have been selected, they can be assembled into a single compound or complex. Assembly may be preceded by visual inspection of the relationship of the fragments to each other on the three-dimensional image displayed on a com- puter screen in relation to the structure coordinates of PPARγ. This would be followed by manual model building using software such as QUANTA (Accelrys Inc., San Diego, ^®2001, 2002) or Sybyl (Tripos Associates, St. Louis, MO).

Useful programs to aid one of skill in the art in connecting the individual chemical entities or fragments include:

1. CAVEAT (P. A. 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, 78, pp. 182-196 (1989); G. Lauri and P. A. Bartlett, "CAVEAT: a Program to Facilitate the Design of Organic Molecules", Comput. Aided MoI. Des., 8, pp. 51-66 (1994) ). CAVEAT is available from the University of California, Berkeley, CA.

2. 3D Database systems such as ISIS (MDL Information Systems, San Leandro, CA). This area is reviewed in Y. C. Martin, "3D Database Searching in Drug Design", J. Med. Chem., 35, pp. 2145-2154 (1992).

3. HOOK (M. B. Eisen et al.,"HOOK : A Program for Finding Novel Molecular Architectures that Satisfy the Chemical and Steric Requirements of a Macromolecule Binding Site", Proteins:

Struct., Funct, Genet., 19, pp. 199-221 (1994) ). HOOK is available from Molecular Simulations, San Diego, CA.

Instead of proceeding to build an inhibitor of a PPARγ prostaglandin-binding pocket in a stepwise fashion one fragment or chemical entity at a time as described above, inhibitory or other PPARγ binding compounds may be designed as a whole or "de novo" using either an empty binding pocket or optionally including some portion (s) of a known inhibitor (s). There are many de novo ligand design methods including:

1. LUDI (H. -J. Computer Program LUDI: A New Method for the De Novo Design of Enzyme Inhibitors", J. Comp. Aid. Molec. Design, 6, pp. 61-78 (1992)). LUDI is available from Molecu- lar Simulations Incorporated, San Diego, CA.

2. LEGEND (Y. Nishibata et al., Tetrahedron, 47, p. 8985 (1991)). LEGEND is available from Molecular Simulations Incorporated, San Diego, CA.

3. LeapFrog (available from Tripos Associates, St. Louis, MO).

4. SPROUT (V. Gillet et al., "SPROUT : A Program for Structure Generation)", Comput. Aided MoI. Design, pp. 127-153 (1993)). SPROUT is available from the University of Leeds, UK.

Other molecular modelling techniques may also be employed in accordance with this invention (see, e. g., N. C. Cohen et al., "Molecular Modelling Software and Methods for Medicinal Chemistry, J. Med. Chem., 33, pp. 883-894 (1990); see also, M. A. Navia and M. A. Murcko, "The Use of Structural Information in Drug Design", Current Opinions in Structural Biology, 2, pp. 202-210 (1992); L. M. Balbes et al.,"A Perspective of Modern Methods in Computer-Aided Drug Design", Reviews in Computational Chemistry, Vol. 5, K. B. Lipkowitz and D. B. Boyd, Eds., VCH, New York, pp. 337-380 (1994); see also, W. C. Guida, "Software For Structure- Based Drug Design", Curr. Opin. Struct. Biology, 4, pp. 777-781 (1994)).

Once a chemical entity has been designed or selected by the above methods, the efficiency with which that chemical entity may bind to the prostaglandin-binding pocket of, e.g., PPARγ, may be tested and optimized by computational evaluation. For example, an effective prostaglandin-binding pocket inhibitor must preferably demonstrate a relatively small difference in energy between its bound and free states (i. e., a small deformation energy of binding). Thus, the most efficient prostaglandin-binding pocket inhibitors should preferably be designed with a deformation energy of binding of not greater than about 10 kcal/mole, more preferably, not greater than 7 kcal/mole. PPARγ prostaglandin-binding pocket inhibitors may interact with the prostaglandin-binding pocket in more than one conformation that is similar in overall binding energy. In those cases, the deformation energy of binding is taken to be the difference between the energy of the free chemical entity and the average energy of the conformations observed when the inhibitor binds to the protein. A chemical entity designed or selected as binding to a prostaglandin-binding pocket of, e.g., PPARγ, may be further computationally optimized so that in its bound state it would preferably lack repulsive electrostatic interaction with the target enzyme and with the surrounding water molecules. Such non-complementary electrostatic interactions include repulsive charge- charge, dipole-dipole and charge-dipole interactions.

Specific computer software is available in the art to evaluate compound deformation energy and electrostatic interactions. Examples of programs designed for such uses include: Gaussian 94, revision C (M. J. Frisch, Gaussian, Inc., Pittsburgh, PA ^®1995 ; AMBER, version 4.1 (P. A. Kollman, University of California at San ; QUANTA/CHARMM (Accelrys Inc., San Diego, ^®2001, 2002); Insight II/Discover (Molecular Simulations, Inc., San Diego, CA ^®1998; DelPhi (Molecular Simulations, Inc., San Diego, CA^®1998 ; and AMSOL (Quantum Chemistry Program Exchange, Indiana University). These programs may be implemented, for instance, using a Silicon Graphics workstation such as an Indigo2 with "IMPACT" graphics. Other hardware systems and software packages will be known to those skilled in the art.

Another approach enabled by this invention, is the computational screening of small molecule databases for chemical entities or compounds that can bind to the prostaglandin-binding pocket of, e.g., PPARγ. In this screening, the quality of fit of such entities to the prostaglandin-binding pocket may be judged either by shape complementarity or by estimated interaction energy (E. C. Meng et al., J. Comp. Chem., 13, pp. 505-524 (1992)).

According to another aspect, the invention provides compounds which associate with the prostaglandin-binding pocket of, e.g., PPARγ, produced or identified by the method set forth above.

Another particularly useful drug design technique enabled by this invention is iterative drug design. Iterative drug design is a method for optimizing associations between a protein and a compound by determining and evaluating the three-dimensional structures of successive sets of protein/compound complexes.

In iterative drug design, crystals of a series of protein or protein complexes are obtained and then the three-dimensional structure of each crystal is solved.

Such an approach provides insight into the association between the proteins and compounds of each complex. This is accomplished by selecting compounds with inhibitory activity, obtaining crystals of this new protein/compound complex, solving the three-dimensional structure of the complex, and comparing the associations between the new protein/compound complex and previously solved protein/compound complexes. By observing how changes in the compound affected the protein/compound associations, these associations may be optimized.

In some cases, iterative drug design is carried out by forming successive protein-compound complexes and then crystallizing each new complex. High throughput crystallization assays may be used to find a new crystallization condition or to optimize the original protein or complex crystallization condition for the new complex. Alternatively, a pre-formed protein crystal may be soaked in the presence of an inhibitor, thereby forming a protein/compound complex and obviating the need to crystallize each individual protein/compound complex.

Structure Determination of Other Molecules

The structure coordinates set forth in Figures 1-2 can also be used to aid in obtaining structural information about other crystallized molecules or molecular complexes. This may be achieved by any of a number of well-known techniques, including molecular replacement.

According to an alternate aspect, the machine-readable data storage medium comprises a data storage material encoded with a first set of machine readable data which comprises the Fourier transform of at least a portion of the structure coordinates set forth in Figures 1-2 or homology model thereof, and which, when using a machine programmed with instructions for using said data, can be combined with a second set of machine readable data comprising the X-ray diffraction pattern of a molecule or molecular complex to determine at least a portion of the structure coordinates corresponding to the second set of machine readable data.

In another aspect, the invention provides a computer for determining at least a portion of the structure coordinates corresponding to X-ray diffraction data obtained from a molecule or molecular complex, wherein said computer comprises: (a) a machine-readable data storage medium comprising a data storage material encoded with machine-readable data, wherein said data comprises at least a portion of the structure coordinates of PPARγ according to any one of Figures 1-2 or homology model thereof; (b) a machine-readable data storage medium comprising a data storage material encoded with machine-readable data, wherein said data comprises X-ray diffraction data obtained from said molecule or molecular complex; and

(c) instructions for performing a Fourier transform of the machine-readable data of (a) and for processing said machine-readable data of (b) into structure coordinates. For example, the Fourier transform of at least a portion of the structure coordinates set forth in any one of Figures 1-2 or homology model thereof may be used to determine at least a portion of the structure coordinates of PPARγ homologues. In one aspect, the molecule is a PPARγ homologue. In another aspect, the molecular complex is selected from the group con- sisting of PPARγ complex and PPARγ homologue complex.

Therefore, in another aspect this invention provides a method of utilizing molecular replacement to obtain structural information about a molecule or a molecular complex of unknown structure wherein the molecule or molecular complex is sufficiently homologous to PPARγ, comprising the steps of: (a) crystallizing said molecule or molecular complex of unknown structure; (b) generating an X-ray diffraction pattern from said crystallized molecule or molecular complex; (c) applying at least a portion of the PPARγ structure coordinates set forth in one of Figures 1-2 or a homology model thereof to the X-ray diffraction pattern to generate a three-dimensional electron density map of at least a portion of the molecule or molecular complex whose structure is unknown; and (d) generating a structural model of the molecule or molecular complex from the three-dimensional electron density map.

In one aspect, the method is performed using a computer. In another aspect, the molecule is selected from the group consisting of PPARγ and PPARγ homologues. In another aspect, the molecule is a PPAR molecular complex or homologue thereof.

By using molecular replacement, all or part of the structure coordinates of the PPARγ as pro- vided by this invention or homology model thereof (and set forth in any one of Figures 1-2) can be used to determine the structure of a crystallized molecule or molecular complex whose structure is unknown more quickly and efficiently than attempting to determine such information ab initio.

Molecular replacement provides an accurate estimation of the phases for an unknown struc- ture. Phases are a factor in equations used to solve crystal structures that can not be determined directly. Obtaining accurate values for the phases, by methods other than molecular replacement, is a time-consuming process that involves iterative cycles of approximations and refinements and greatly hinders the solution of crystal structures. However, when the crystal structure of a protein containing at least a homologous portion has been solved, the phases from the known structure may provide a satisfactory estimate of the phases for the unknown structure. Thus, this method involves generating a preliminary model of a molecule or molecular complex whose structure coordinates are unknown, by orienting and positioning the relevant portion of the PPARγ according to any one of Figures 1-2 within the unit cell of the crystal of the unknown molecule or molecular complex so as best to account for the observed X-ray diffrac- tion pattern of the crystal of the molecule or molecular complex whose structure is unknown. Phases can then be calculated from this model and combined with the observed X-ray diffraction pattern amplitudes to generate an electron density map of the structure whose coordinates are unknown. This, in turn, can be subjected to any well-known model building and structure refinement techniques to provide a final, accurate structure of the unknown crystal- lized molecule or molecular complex (E. Lattman, "Use of the Rotation and Translation Functions", in Meth. 115, pp. 55-77 (1985); M. G. Rossmann, ed., "The Molecular Replacement Method", Int. Sci. Rev. Sen, No. 13, Gordon & Breach, New York (1972)).

The structure of any portion of any crystallized molecule or molecular complex that is sufficiently homologous to any portion of the PPARγ can be resolved by this method.

In one aspect, the method of molecular replacement is utilized to obtain structural information about a PPARγ homologue. The structure coordinates of PPARγ as provided by this invention are particularly useful in solving the structure of PPARγ complexes that are bound by ligands, substrates and inhibitors.

Furthermore, the structure coordinates of PPARγ as provided by this invention are useful in solving the structure of PPARγ proteins that have amino acid substitutions, additions and/or deletions (referred to collectively as "PPARγ mutants", as compared to naturally occurring PPARγ). These PPARγ mutants may optionally be crystallized in co-complex with a chemical entity, such as a non-hydrolyzable prostaglandin analogue or a suicide substrate. The crystal structures of a series of such complexes may then be solved by molecular replacement and compared with that of wild-type PPARγ. Potential sites for modification within the various binding pockets of the enzyme may thus be identified. This information provides an additional tool for determining the most efficient binding interactions, for example, increased hydrophobic interactions, between PPARγ and a chemical entity or compound.

The structure coordinates are also particularly useful in solving the structure of crystals of PPARγ or PPARγ homologues co-complexed with a variety of chemical entities. This approach enables the determination of the optimal sites for interaction between chemical entities, including candidate PPARγ inhibitors. For example, high resolution X-ray diffraction data collected from crystals exposed to different types of solvent allows the determination of where each type of solvent molecule resides. Small molecules that bind tightly to those sites can then be designed and synthesized and tested for their PPARγ inhibition activity.

All of the complexes referred to above may be studied using well-known X-ray diffraction techniques and may be refined using 1.5-3. 4 resolution X-ray data to an R value of about 0.30 or less using computer software, such as X-PLOR (Yale distributed by Molecular Simulations, Inc.; see, e. g., Blundell & Johnson, supra; Meth. Enzymol., vol. 114 & 115, H. W. Wy- ckoff et al., Academic Press (1985) ) or CNS (Brunger et al., Acta Cryst., D54, pp. 905-921, (1998)).

The features disclosed in the foregoing description may, both separately and in any combina- tion thereof, be material for realising the invention in diverse forms thereof.

The following formulation examples are illustrative only and are not intended to limit the scope of the invention in any way.

EXAMPLES

EXAMPLE 1 - Re-Refinement of PPARv Structure

Crystallographic coordinates of the in the Protein Data Bank (Berman et al., 2000) accession code IPRG deposited Peroxisome Proliferator- Activated Receptor-γ (PPARγ), ligand binding domain, (Nolte et al., 1998) were downloaded along with deposited structure factors, accession code rlprgsf. The structure was reported as an Apo structure of PPARγ, a structure with- out any bound ligand. To check this, REFMAC5 of the CCP4 crystallographic program package (Bailey, 1994) was used to re-refine the structure. CCP4 was run using the CCP4i interface (Liz Potterton (CCP4 Newsletter 34, September 1997) http://www.ccp4.ac.uk/newslet- ters.php). The IPRG coordinates were stripped of any water molecules and 50 cycles of maximum likelihood restrained refinement was performed using the deposited structure fac- tors with data between 45.3 and 2.0 A resolution. Bulk solvent correction was used. The crystallographic space group number 5 was used for refinement. The refinement ended in with an R-value of 0.264 and with a root-mean-square deviation (RMSD) from ideal bond lengths of 0.020 A. SigmaA weighted electron density maps, 2Fo-Fc and Fo-Fc, were generated after the last cycle. The crystallographic refinement was followed by computer graphics inspection of the electron density maps using the Coot program (Emsley and Cowtan, 2004). The Coot command "Validate/Unmodelled blobs", using 3 sigma cut-off on the Fo-Fc map, gave a list of three "blobs", unmodelled electron densities larger than water molecules in size. The first and second "blob" in the list pointed to the same location of the crystallographically independent molecules A and B, respectively, the first "blob" showing a highest sigma value of 5 in the Fo- Fc map and 2.5 in the 2Fo-Fc map. The second "blob" was showing a 5.6 sigma high peak in the Fo-Fc map and 2.7 sigma in the 2Fo-Fc map.

The shape of the blobs, a central disk-shape with arms protruding on opposite sides of the disk and pointing away from each other, reminded very much of the shape of a prostaglandin molecule, a natural ligand of PPARγ. Different types of prostaglandin molecules were therefore modeled into the difference electron density, among those the natural PPARγ ligand 15-Deoxy Δ¹²'¹⁴-prostaglandin J₂, using the Coot program followed by REFMAC5 refinement, an then model and map inspection by the Coot program. It was concluded that Prostaglandin B₂ fitted the electron density best. Prostaglandin B₂ is a non-native prostaglandin commonly used as an internal standard for HPLC-traced extractions and chromatograms which makes it probable to have been used during the purification of the PPARγ molecule. Despite its non-native na- ture, the prostaglandin B₂ molecule is similar enough to most likely occupy the same binding site, with slight deviations, as would 15-Deoxy Δ¹²'¹⁴-prostaglandin J₂. The prostaglandin B₂ molecule fits very well with the electron density with the exception of the two last carbons of the hydrocarbon arm containing the carboxyl group. For those two moieties there is no electron density seen.

The third "blob" found by the Coot program was situated in a mostly hydrophobic pocket on the surface of the PPARγ A molecule formed by residues like Leu237, Val248, Pro246 and Phe347. It was interpreted as DMSO (dimethyl sulfoxide) molecule and inserted by the Coot program and refined in REFMAC5. Moreover, a disordered part of the main-chain, amino acid 239 to 244, of the A molecule, was modelled in two discrete conformations. Thereafter water molecules were added by the ARP_WARP program software (Perrakis et al., 1999) implemented by the CCP4i procedure of REFMAC5. For statistics from the final refinement, see Table 1.

The amino acid residues within a 5 A radius of the prostaglandin B₂ molecule was calculated with the help of the CONTACT software program of the CCP4 program package, Table 2 and 3, and in QUANTA. From the close contact between P 012 and Leu 228 O, Table 2 and Table 3, it is clear that it has to be the enol form of the prostaglandin B₂ molecule making a hydrogen bond to the main chain oxygen of PPARγ. The LBD PPARγ amino acid residues within 5 A distance from the bound prostaglandin B₂ molecule were TYR 222, PHE 226, PRO 227, LEU 228, THR 229, LYS 230, CYS 285, ARG 288, SER 289, ALA 292, GLU 295, ILE 326, TYR 327, MET 329, LEU 330, SER 332, LEU 333, MET 364, ASP 381, and LEU 384.

Further, the PPARγ molecule, without any bound ligands, was run through the VOIDOO software program (Kleywegt and Jones, 1994). From the output of the program voids, pockets within the PPARγ molecule not filled with atoms, around the position for the prostaglandin B₂ site was used to predict residues which could be reached and utilized in a drug design process. The amino acid residues which were added, thus extending the pocket filled by the prostaglandin B₂ molecule, were ILE 296, ILE 325, THR 328, ALA 331, PHE 374, ASN 375, ALA 376, LEU 377, GLU 378, LEU 379, ASP 380 and LEU 435.

Table 1 : DATA USED IN REFINEMENT.

RESOLUTION RANGE HIGH (ANGSTROMS) : 2.00

RESOLUTION RANGE LOW (ANGSTROMS) : 115.47

DATA CUTOFF (SIGMA(F)) : NONE

COMPLETENESS FOR RANGE (%) : 76.06 NUMBER OF REFLECTIONS : 31793

FIT TO DATA USED IN REFINEMENT. CROSS-VALIDATION METHOD THROUGHOUT FREE R VALUE TEST SET SELECTION RANDOM R VALUE (WORKING + TEST SET) 0.24317 R VALUE (WORKING SET) 0.24007

FREE R VALUE 0.30111

FREE R VALUE TEST SET SIZE (%) 5.1 FREE R VALUE TEST SET COUNT 1693

FIT IN THE HIGHEST RESOLUTION BIN. TOTAL NUMBER OF BINS USED 20 BIN RESOLUTION RANGE HIGH 2.001 BIN RESOLUTION RANGE LOW 2.053 REFLECTION IN BIN (WORKING SET) 1219 BIN COMPLETENESS (WORKING+TEST) (%) 39.27 BIN R VALUE (WORKING SET) 0.330

BIN FREE R VALUE SET COUNT 62 BIN FREE R VALUE 0.365 RMS DEVIATIONS FROM IDEAL VALUES COUNT RMS WEIGHT

BOND LENGTHS REFINED ATOMS (A): 4312 ; 0.017 ; 0.022

BOND ANGLES REFINED ATOMS (DEGREES): 5801 ; 1.775 ; 1.999

TORSION ANGLES, PERIOD 1 (DEGREES): 522 ; 6.838 ; 5.000

Table 2:

PPARγ - prostaglandin B₂ interactions of the A and P residue chain, respectively. A cut-off of 5.0 A was used. The contacts were identified by the CONTACT computer program of the CCP4 suite. In the last column "***" indicates a strong possibility for a hydrogen bond at this contact (distance < 3.3 A) as calculated by CONTACT, " *" indicates a weak possibility (distance > 3.3 A). Blank indicates that the program considered there to be no possibility of a hydrogen bond.

Table 3:

PPARγ - prostaglandin B₂ interactions of the B and P residue chain, respectively. A cut-off of 5.0 A was used. The contacts were identified by the CONTACT computer program of the CCP4 suite. In the last column "***" indicates a strong possibility for a hydrogen bond at this contact (distance < 3.3 A) as calculated by CONTACT, " *" indicates a weak possibility (distance > 3.3 A). Blank indicates that the program considered there to be no possibility of a hydrogen bond.

Table 4:

PPARg-LBD residues within 5 A radius of prostaglandin B2 molecule 1. See also Figure 3.

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EXEMPLARY EMBODIMENTS

The following are exemplary embodiments of the invention.

1. A prostaglandin-binding pocket of a PPAR protein molecule, which prostaglandin-binding pocket is defined by three-dimensional structure coordinates of a set of amino acids that comprises amino acids corresponding to four or more selected from the group consisting Of TYR 222, PHE 226, PRO 227, LEU 228, THR 229, LYS 230, CYS 285, ARG 288, SER 289, ALA 292, GLU 295, ILE 296, ILE 325, ILE 326, TYR 327, THR 328, MET 329, LEU 330, ALA

331, SER 332, LEU 333, MET 364, PHE 374, ASN 375, ALA 376, LEU 377, GLU 378, LEU 379, ASP 380, ASP 381, LEU 384, and LEU 435 of SEQ ID NO: 1.

2. A prostaglandin-binding pocket of a PPARγ or PPARγ-like protein molecule comprising an amino acid sequence at least 65% identical to residues 207-476 of SEQ ID NO: 1, which prostaglandin-binding pocket is defined by three-dimensional structure coordinates of a set of amino acids that comprises (i) an amino acid corresponding to LEU 228, and (ii) amino acids corresponding to three or more selected from the group consisting of TYR 222, PHE 226, PRO 227, THR 229, LYS 230, CYS 285, ARG 288, SER 289, ALA 292, GLU 295, ILE 296, ILE 325, ILE 326, TYR 327, THR 328, MET 329, LEU 330, ALA 331, SER 332, LEU 333, MET 364, PHE 374, ASN 375, ALA 376, LEU 377, GLU 378, LEU 379, ASP 380, ASP 381, LEU 384, and LEU 435 of SEQ ID NO: 1.

3. A prostaglandin-binding pocket according to any one of embodiments 1-2, wherein the set of amino acids comprises an amino acid corresponding to LEU 228 and amino acids corresponding to six or more selected from the group consisting of TYR 222, PHE 226, PRO 227, THR 229, LYS 230, CYS 285, ARG 288, SER 289, ALA 292, GLU 295, ILE 326, TYR 327, MET 329, LEU 330, SER 332, LEU 333, MET 364, ASP 381, and LEU 384.

4. A prostaglandin-binding pocket according to any one of embodiments 1-3, wherein the set of amino acids comprises an amino acid corresponding to LEU 228 and amino acids corresponding to ten or more selected from the group consisting of TYR 222, PHE 226, PRO

227, THR 229, LYS 230, CYS 285, ARG 288, SER 289, ALA 292, GLU 295, ILE 326, TYR 327, MET 329, LEU 330, SER 332, LEU 333, MET 364, ASP 381, and LEU 384.

5. A prostaglandin-binding pocket according to any one of embodiments 1-4, wherein the set of amino acids comprises amino acids corresponding to PHE 226, PRO 227, LEU 228 and MET 329.

6. A prostaglandin-binding pocket according to any one of embodiments 1-5, wherein the set of amino acids comprises amino acids corresponding to TYR 222, PHE 226, PRO 227, LEU 228, THR 229, LYS 230, ALA 292, GLU 295, MET 329, SER 332, LEU 333, ASP 381, and

LEU 384.

7. A prostaglandin-binding pocket according to any one of embodiments 1-6, wherein the set of amino acids comprises amino acids corresponding to one or more selected from the group consisting of CYS 285, ARG 288, SER 289, ILE 326, TYR 327, LEU 330 and MET 364.

8. A prostaglandin-binding pocket according to any one of embodiments 1-7, wherein the set of amino acids comprises amino acids corresponding to TYR 222, PHE 226, PRO 227, LEU

228, THR 229, LYS 230, CYS 285, ARG 288, SER 289, ALA 292, GLU 295, ILE 326, TYR 327, MET 329, LEU 330, SER 332, LEU 333, MET 364, ASP 381, and LEU 384. 9. A prostaglandin-binding pocket according to any one of embodiments 1-8, wherein the set of amino acids comprises amino acids corresponding to one or more selected from the group consisting of ILE 296, ILE 325, THR 328, ALA 331, PHE 374, ASN 375, ALA 376, LEU 377, GLU 378, LEU 379, ASP 380 and LEU 435.

10. A prostaglandin-binding pocket according to any one of embodiments 1-9, wherein the set of amino acids comprises amino acids corresponding to ILE 296, ILE 325, THR 328, ALA 331, PHE 374, ASN 375, ALA 376, LEU 377, GLU 378, LEU 379, ASP 380 and LEU 435.

11. A prostaglandin-binding pocket according to any one of embodiments 1-10, wherein the set of amino acids comprises amino acids corresponding to TYR 222, PHE 226, PRO 227, LEU 228, THR 229, LYS 230, CYS 285, ARG 288, SER 289, ALA 292, GLU 295, ILE 296,

ILE 325, ILE 326, TYR 327, THR 328, MET 329, LEU 330, ALA 331, SER 332, LEU 333, MET 364, PHE 374, ASN 375, ALA 376, LEU 377, GLU 378, LEU 379, ASP 380, ASP 381, LEU 384, and LEU 435.

12. A method for evaluating the ability of a chemical entity to associate with the pros- taglandin-binding pocket of any of embodiments 1-11, comprising the steps of: (a) providing the structure coordinates of said prostaglandin-binding pocket on a computer comprising the means for generating three-dimensional structural information from said structure coordinates; (b) employing computational means to perform a fitting operation between the chemical entity and the prostaglandin-binding pocket; and (c) analyzing the re- suits of said fitting operation to evaluate the association between the chemical entity and the prostaglandin-binding pocket.

13. The method according to embodiment 12, further comprising in step (c) to quantify the association between the chemical entity and the prostaglandin-binding pocket.

14. A method of using a computer for evaluating the ability of a chemical entity to associate with a prostaglandin-binding pocket according to any of embodiments 1-11, wherein said computer comprises a machine-readable data storage medium comprising a data storage material encoded with said structure coordinates defining said prostaglandin-binding pocket and means for generating a three-dimensional graphical representation of the prostaglandin-binding pocket, and wherein said method comprises the steps of: (a) posi- tioning a first chemical entity within said prostaglandin-binding pocket using a graphical three-dimensional representation of the structure of the chemical entity and the prostaglandin-binding pocket ; (b) performing a fitting operation between said chemical entity and said prostaglandin-binding pocket by employing computational means; and (c) analyzing the results of said fitting operation to quantify the association between said chemical entity and the prostaglandin-binding pocket.

15. The method according to embodiment 14, further comprising the steps of: (d) repeating steps (a) through (c) with a second chemical entity; and (e) selecting at least one of said first or second chemical entity that associates with said prostaglandin-binding pocket based on said quantified association of said first or second chemical entity.

16. A method for identifying a ligand of the prostaglandin-binding pocket of any of embodiments 1-11, comprising the steps of: (a) using a three-dimensional structure of the pros- taglandin-binding pocket to design, select and/or optimize a chemical entity; (b) contacting the chemical entity with the molecule; and (c) identifying any chemical entity capable of binding the molecule as a ligand.

17. A method for designing, selecting and/or optimizing a chemical entity that binds to the prostaglandin-binding pocket of any of embodiments 1-11, comprising the steps of: (a) providing the structure coordinates of said prostaglandin-binding pocket on a computer comprising the means for generating three-dimensional structural information from said structure coordinates; and (b) designing, selecting and/or optimizing said chemical entity by performing a fitting operation between said chemical entity and said three-dimensional structural information of said prostaglandin-binding pocket.

18. A method of designing a compound or complex that associates with the prostaglandin- binding pocket of any of embodiments 1-11, comprising the steps of: (a) providing the structure coordinates of said prostaglandin-binding pocket on a computer comprising the means for generating three-dimensional structural information from said structure coordinates; and (b) using the computer to perform a fitting operation to associate a first chemical entity with the prostaglandin-binding pocket; (c) performing a fitting operation to associate at least a second chemical entity with the prostaglandin-binding pocket; (d) quantifying the association between the first and second chemical entity and the prostaglandin-binding pocket; (e) optionally repeating steps b) to d) with another first and second chemical entity, selecting a first and a second chemical entity based on said quan- tified association of all of said first and second chemical entity; (f) optionally, visually inspecting the relationship of the first and second chemical entity to each other in relation to the prostaglandin-binding pocket on a computer screen using the three-dimensional graphical representation of the prostaglandin-binding pocket and said first and second chemical entity; and (g) assembling the first and second chemical entity into a compound or complex that associates with said prostaglandin-binding pocket by model building.

19. The method according to any one of embodiments 1-18 for screening for chemical entities useful for the treatment of prostaglandin- and/or PPAR-related diseases.

20. The method according to embodiment 19, wherein the prostaglandin- and/or PPAR-related diseases is cancer, inflammation, skin and hair disorders, diabetes, obesity, hypertension, and impaired glucose tolerance.

21. The method according to embodiment 20, wherein the prostaglandin- and/or PPAR-related diseases is cancer.

22. A computer comprising : (a) a machine-readable data storage medium, comprising a data storage material encoded with machine-readable data, wherein said data defines the prostaglandin-binding pocket of any of embodiments 1-11; (b) a working memory for storing instructions for processing said machine-readable data; (c) a central processing unit coupled to said working memory and to said machine-readable data storage medium for processing said machine-readable data and means for generating three-dimensional structural information of said prostaglandin-binding pocket; and (d) output hardware coupled to said central processing unit for outputting three-dimensional structural information of said prostaglandin-binding pocket, or information produced using said three-dimensional structural information of said prostaglandin-binding pocket.

23. The computer according to embodiment 22, wherein said means for generating three- dimensional structural information is provided by means for generating a three- dimensional graphical representation of said prostaglandin-binding pocket.

24. The computer according to any one of embodiments 22-23, wherein said output hardware is a display terminal, a printer, CD or DID recorder, ZIP or JAZ drive, USB drive, a disk drive, memory stick or other machine-readable data storage device.

25. The prostaglandin-binding pocket, method, or computer according to any one of embodiments 1-24, wherein the root mean square deviation of the backbone atoms between amino acids of said molecule and said PPARγ residues is not greater than about 3 A. 26. The prostaglandin-binding pocket, method, or computer according to any one of embodiments 1-24, wherein the root mean square deviation of the backbone atoms between amino acids of said molecule and said PPARγ residues is not greater than about 1.5 A.

27. The prostaglandin-binding pocket, method, or computer according to any one of embodi- ments 1-24, wherein the PPARγ-like protein molecule comprises an amino acid sequence at least 80% identical to SEQ ID NO: 2.

28. The prostaglandin-binding pocket, method, or computer according to any one of embodiments 1-24, wherein the PPARγ-like protein molecule comprises an amino acid sequence at least 90% identical to SEQ ID NO: 2.

29. The prostaglandin-binding pocket, method, or computer according to any one of embodiments 1-24, wherein the PPARγ-like protein molecule comprises an amino acid sequence at least 95% identical to SEQ ID NO: 2.

30. The prostaglandin-binding pocket, method, or computer according to any one of embodiments 1-24, wherein the PPARγ protein molecule comprises the amino acid sequence of SEQ ID NO: 2.

31. The prostaglandin-binding pocket, method, or computer according to any one of embodiments 1-24, wherein the PPARγ protein molecule comprises the amino acid sequence of SEQ ID NO: 1.

All references, including publications, patent applications and patents, cited herein are hereby incorporated by reference to the same extent as if each reference was individually and specifically indicated to be incorporated by reference and was set forth in its entirety herein.

All headings and sub-headings are used herein for convenience only and should not be construed as limiting the invention in any way,

Any combination of the above-described elements in all possible variations thereof is encom- passed by the invention unless otherwise indicated herein or otherwise clearly con-tradicted by context. The terms "a" and "an" and "the" and similar referents as used in the context of de-scribing the invention are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless other-wise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. Unless otherwise stated, all exact values provided herein are representative of corresponding approximate values (e.g., all exact exemplary values provided with respect to a particular factor or measurement can be considered to also pro-vide a corre- sponding approximate measurement, modified by "about," where appropriate).

All methods described herein can be performed in any suitable order unless other-wise indicated herein or otherwise clearly contradicted by context.

The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise indicated. No language in the specification should be construed as indicating any element is essential to the practice of the invention unless as much is explicitly stated.

The citation and incorporation of patent documents herein is done for convenience only and does not reflect any view of the validity, patentability and/or enforceability of such patent documents,

The description herein of any aspect or embodiment of the invention using terms such as "comprising", "having", "including" or "containing" with reference to an element or elements is intended to provide support for a similar aspect or embodiment of the invention that "consists of", "consists essentially of", or "substantially comprises" that particular element or elements, unless otherwise stated or clearly contradicted by context (e.g., a composition described herein as comprising a particular element should be understood as also describing a composition consisting of that element, unless otherwise stated or clearly contradicted by context).

This invention includes all modifications and equivalents of the subject matter recited in the aspects or claims presented herein to the maximum extent permitted by applicable law.

Claims

1. A method for evaluating the ability of a chemical entity to associate with a prostaglandin- binding pocket of a PPARγor PPARγ-like protein molecule comprising an amino acid sequence at least 65% identical to residues 207-476 of SEQ ID NO: 1, which prostaglandin- binding pocket is defined by three-dimensional structure coordinates of a set of amino acids that comprises (i) an amino acid corresponding to LEU 228, and (ii) amino acids corresponding to three or more selected from the group consisting of TYR 222, PHE 226, PRO 227, THR 229, LYS 230, CYS 285, ARG 288, SER 289, ALA 292, GLU 295, ILE 296, ILE 325, ILE 326, TYR 327, THR 328, MET 329, LEU 330, ALA 331, SER 332, LEU 333, MET 364, PHE 374, ASN 375, ALA 376, LEU 377, GLU 378, LEU 379, ASP 380, ASP 381, LEU

384, and LEU 435 of SEQ ID NO: 1, the method comprising the steps of: (a) providing the structure coordinates of said prostaglandin-binding pocket on a computer comprising the means for generating three-dimensional structural information from said structure coordinates; (b) employing computational means to perform a fitting operation between the chemical entity and the prostaglandin-binding pocket; and (c) analyzing the results of said fitting operation to evaluate the association between the chemical entity and the prostaglandin-binding pocket.

2. The method according to claim 1, wherein the set of amino acids comprises an amino acid corresponding to LEU 228 and amino acids corresponding to six or more selected from the group consisting of TYR 222, PHE 226, PRO 227, THR 229, LYS 230, CYS 285, ARG 288,

SER 289, ALA 292, GLU 295, ILE 326, TYR 327, MET 329, LEU 330, SER 332, LEU 333, MET 364, ASP 381, and LEU 384.

3. The method according to any one of claims 1-2, wherein the set of amino acids comprises an amino acid corresponding to LEU 228 and amino acids corresponding to ten or more selected from the group consisting of TYR 222, PHE 226, PRO 227, THR 229, LYS 230,

CYS 285, ARG 288, SER 289, ALA 292, GLU 295, ILE 326, TYR 327, MET 329, LEU 330, SER 332, LEU 333, MET 364, ASP 381, and LEU 384.

4. The method according to any one of claims 1-3, wherein the set of amino acids comprises amino acids corresponding to PHE 226, PRO 227, LEU 228 and MET 329.

5. The method according to any one of claims 1-4, wherein the set of amino acids comprises amino acids corresponding to TYR 222, PHE 226, PRO 227, LEU 228, THR 229, LYS 230, ALA 292, GLU 295, MET 329, SER 332, LEU 333, ASP 381, and LEU 384.

6. The method according to any one of claims 1-5, wherein the set of amino acids comprises amino acids corresponding to one or more selected from the group consisting of CYS 285, ARG 288, SER 289, ILE 326, TYR 327, LEU 330 and MET 364.

7. The method according to any one of claims 1-6, wherein the set of amino acids comprises amino acids corresponding to TYR 222, PHE 226, PRO 227, LEU 228, THR 229, LYS 230,

8. The method according to any one of claims 1-7, wherein the set of amino acids comprises amino acids corresponding to one or more selected from the group consisting of ILE 296, ILE 325, THR 328, ALA 331, PHE 374, ASN 375, ALA 376, LEU 377, GLU 378, LEU 379,

ASP 380 and LEU 435.

9. The method according to any one of claims 1-8, wherein the set of amino acids comprises amino acids corresponding to ILE 296, ILE 325, THR 328, ALA 331, PHE 374, ASN 375, ALA 376, LEU 377, GLU 378, LEU 379, ASP 380 and LEU 435.

10. The method according to any one of claims 1-9, wherein the set of amino acids comprises amino acids corresponding to TYR 222, PHE 226, PRO 227, LEU 228, THR 229, LYS 230, CYS 285, ARG 288, SER 289, ALA 292, GLU 295, ILE 296, ILE 325, ILE 326, TYR 327, THR 328, MET 329, LEU 330, ALA 331, SER 332, LEU 333, MET 364, PHE 374, ASN 375, ALA 376, LEU 377, GLU 378, LEU 379, ASP 380, ASP 381, LEU 384, and LEU 435.

11. The method according to any one of claims 1-10, further comprising in step (c) to quantify the association between the chemical entity and the prostaglandin-binding pocket.

12. The method according to any one of claims 1-11, wherein the PPARγ protein molecule comprises the amino acid sequence of SEQ ID NO: 2.

13. The method according to any one of claims 1-12, wherein the PPARγ protein molecule comprises the amino acid sequence of SEQ ID NO: 1.

14. The method according to any one of claims 1-13 for use in the designing of, or screening for, chemical entities useful for the treatment of a prostaglandin- and/or PPAR-related disease.

15. The method according to claim 14, wherein the disease is cancer.