FIELD OF THE INVENTION

The present invention generally relates to a binding site, termed Site II, in nuclear hormone receptors. The present invention relates to: machine-readable data storage media comprising structure coordinates of Site II; computer systems capableof producing three-dimensional representations of all or any part of Site II; methods used in the design and identification of ligands of Site II and of modulators of nuclear hormone receptors (NHRs); ligands of Site II; modulators of NHRs; methods ofmodulating NHRs; pharmaceutical compositions comprising modulators of NHRs; methods of treating diseases by administering modulators of an NHR; methods of designing mutants; mutant NHRs or portions of mutant NHRs; methods of measuring the binding of atest molecule to Site II; and models of Site II.

BACKGROUND OF THE INVENTION

The nuclear hormone receptor (NHR) family of transcription factors bind low molecular weight ligands and either stimulate or repress transcription. (in The Nuclear Receptor Facts Book, V. Laudet and H. Gronemeyer, Academic Press, p 345, 2002). NHRs stimulate transcription by binding to DNA and inducing transcription of specific genes. NHRs may also stimulate transcription by not binding to DNA itself, rather they may modulate the activity of other DNA binding proteins (Stocklin, E., et al.,Nature (1996) 383:726-8). The process of stimulation of transcription is called transactivation. NHRs repress transcription by interacting with other transcription factors or coactivators and inhibiting the ability of these other transcription factorsor coactivators to induce transcription of specific genes. The process of repression of transcription is called transrepression. (for a review see The Nuclear Receptor Factsbook, V. Laudet and H. Gronemeyer, Academic Press, p 42, 2002). For example,the glucocorticoid receptor, estrogen receptor, androgen receptor and peroxisome proliferator activated receptors .alpha. and .gamma. have been shown to repress the activity of the transcription factors AP-1 and NF-.kappa.B (Jonat, C., et al., Cell,62, p 1189-1204, (1990) Kallio, P. J., et al., Mol. Endocrinol., 9, p 017-1028 (1995), Keller, E. T., et al., J. Biol. Chem., 271, p 26267-26275 (1996), Jones, D. C., et al., J. Biol. Chem., 277 (9), p 6838-6845, (2002), Ricote, M., et al., Nature,391, p 79-82, (1998), Valentine, J. E., et al, J. Biol. Chem., 275, p 25322-25329, (2000).

The nuclear hormone receptor family includes the glucocorticoid receptor (Hollenberg, S. M. et al. (1985) Nature, 318, p 635), progesterone receptor (Misrahi, M. et al. (1987) Biochem. Biophys. Res. Commun. 143, p 740), androgen receptor(Lubahn D. B., et al (1988), estrogen receptors (Green, S., et al. (1986) Nature 320, p 134), mineralocorticoid receptor (Arriza, J. L., et al., (1987) Science 237, p 268), retinoid receptors (RXRs and RARs) (Mangelsdorf, et al. (1990) Nature, 345, p 224and Petkovich M., et al (1987) Nature 330, p 444), Vitamin D receptor, thyroid receptor (TR) (Nakai, A. et al., (1988) Mol. Endocrinol. 2, p 1087), peroxisome proliferator activated receptor (PPAR) (Greene, M. E., et al. (1995) Gene Expression 4, p281), orphan nuclear receptors and others. Glucocorticoid receptor, progesterone receptor, androgen receptor, estrogen receptor, and mineralocorticoid receptor are steroid hormone receptors (SHRs).

Although the sequences vary amongst the various nuclear hormone receptors, they can be divided into functional domains including an N-terminal transactivation domain, a central DNA binding domain and a C-terminal ligand and dimerization domain. The ligands which bind these receptors act in a ligand, cell type, and promoter dependent fashion and include: glucocorticoids, progestins, retinoids, mineralocorticoids, and others. In addition to steroids, recent studies have shown that non-steroidscan bind to nuclear hormone receptors and induce a biologic response (Coghlan, M J, et al, J. Med. Chem. 44, p 2879, 2001). Ligand cross-talk can occur between the receptors, for example, progesterone can bind not only the progesterone receptor but theglucocorticoid receptor as well (Zhang, S., Mol. Endocrinology 10, p 24, 1996).

Three-dimensional structures of some of the nuclear hormone receptors have been elucidated through crystallization or homology modeling. A homology model of the glucocorticoid receptor is disclosed in WO 00/52050, published Sep. 8, 2000.

Recent publications by the same research group: Bledsoe, et. al., Cell, online publication by Cell Press, Jul. 1, 2002, DOI: 10.1016/S0092867402008176; Cell, Vol 110, 93-105, 12 Jul. 2002; and Apolito, et. al., in WO 03/015692 A2, publishedFeb. 27, 2003; describe the successful crystallization and xray structural elucidation of the glucocorticoid receptor LBD as the dimer. X-ray structure coordinates were provided in WO 03/015692. Disruption of the dimeric structure was found to occurupon mutation of selected residues at the dimerization interface. Despite structural similarity to other steroid receptors, the GR LBD dimer represents a unique dimer configuration. The GR LBD used for this crystalization was a mutant (F602S) designedto provide a more soluble LBD construct.

Also recently, Kauppi et. al. published the stucture of the GR LBD bound to an antagonist, RU-486, in: the Journal of Biological Chemistry Online, JBC Papers In Press as DOI:10.1074/JBC.M212711200, Apr. 9, 2003; and in J. Biol. Chem., Vol. 278,Issue 25, 22748-22754, Jun. 20, 2003. In this structure, the GR LBD exhibits a significant displacement of helix 12, typical of antagonist action. In addition to the antagonist-bound LBD, a dimer structure similar to that reported by Bledsoe, et. al.was also described. The structure of the GR LBD-RU-486 complex was deposited in with the RCSB (1nhz.pdb)

Three dimensional structures of other nuclear hormone receptors are disclosed as follows, with RCSB (Research Collaboratory for Structural Bioinformatics, pdb file format) references in parentheses: RXRalpha (1lbd) Bourguet, W., Ruff, M.,Chambon, P., Gronemeyer, H., Moras, D. Nature 375 pp. 377 (1995); PPAR-gamma (2prg) Nolte, R. T., Wisely, G. B., Westin, S., Cobb, J. E., Lambert, M. H., Kurokawa, R., Rosenfeld, M. G., Willson, T. M., Glass, C. K., Milburn, M. V. Nature 395 pp. 137(1998); RARgamma (2lbd) Renaud, J. P., Rochel, N., Ruff, M., Vivat, V., Chambon, P., Gronemeyer, H., Moras, D. Nature 378 pp. 681 (1995); PR (1a28) Williams, S. P., Sigler, P. B. Nature 393 pp. 392 (1998); VitDR (1db1) Rochel, N., Wurtz, J. M.,Mitschler, A., Klaholz, B., Moras, D. Mol. Cell 5 pp. 173 (2000); AR (1e3g) Matias, P. M., Donner, P., Coelho, R., Thomaz, M., Peixoto, C., Macedo, S., Otto, N., Joschko, S., Scholz, P., Wegg, A., Basler, S., Schafer, M., Egner, U., Carrondo, M. A. J.Biol. Chem. 275 pp. 26164 (2000); ERalpha (1a52) Tanenbaum, D. M., Wang, Y., Williams, S. P., Sigler, P. B. Proc Natl Acad Sci USA 95 pp. 5998 (1998); ERbeta (1l2j) Shiau, A. K., Barstad, D., Radek, J. T., Meyers, M. J., Nettles, K. W.,Katzenellenbogen, B. S., Katzenellenbogen, J. A., Agard, D. A., Greene, G. L. Nat. Struct. Biol. 9 pp. 359 (2002). It is generally thought that all steroid ligands bind to nuclear hormone receptors at the classical ligand binding site, which we termsite I (Evans, R. M. Science 240, p 889, 1988). Limited proteolysis studies and cell transfection/mutagenesis studies have delineated the functional domains of nuclear hormone receptors which include a DNA binding domain, ligand binding domain and atransactivation domain. These studies provided the evidence that hormones bind to the ligand binding domain. Mutagenesis of GR has defined the dexamethasone interacting surface, defined as Site I, which includes amino acids Met-560, Met-639, Gln-642and Thr-739 (Lind, U., et al. J. Biol. Chem. 275, p 19041, 2000).

Recently, a second ligand binding site in ER-.alpha. and ER-.beta. has been reported based on computational analysis and docking experiments with steroids. (van Horn, W. J. Med. Chem. 45, p 584, 2002). This second binding site is notcompletely delineated. It is reported to have no obvious function, to be an evolutionary remnant, and to be absent in other nuclear receptors such as RAR.gamma.. Furthermore, there is no discussion of transrepression whatsoever. In addition, EndocrineSociety Meeting June 2003, presentation OR34-1, Wang, Y., Chirgadze, N Y, Briggs, S L, Khan, S., Jensen, E V., Burris, T P., A second binding site for hydroxytamoxifen with the ligand binding domain of estrogen receptor beta, describes the crystalstructure of estrogen receptor bound with 4-hydroxytamoxifen, in which the ligand is found in two locations: the usual steroid binding pocket and a second site located along the hydrophobic groove near the cofactor binding region. This second site isremote from the Site II location described in this application.

The glucocorticoid receptor (GR) is a member of the nuclear hormone receptor family of transcription factors, and a member of the steroid hormone family of transcription factors. Affinity labeling of the glucocorticoid receptor protein allowedthe production of antibodies against the receptor which facilitated cloning the human (Weinberger, et al. Science 228, p 740-742, 1985, Weinberger, et al. Nature, 318, P635-641, 1985) and rat (Miesfeld, R. Nature, 312, p 779-781, 1985) glucocorticoidreceptors. Subsequently, glucocorticoid receptors from other species were cloned including mouse (Danielson, M. et al. EMBO J., 5, 2513), sheep (Yang, K., et al. J. Mol. Endocrinol. 8, p 173-180, 1992), and marmoset (Brandon, D. D., et al, J. Mol.Endocrinol. 7, p 89-96, 1991). There is also a C-terminally distinct isoform of GR termed GR-beta. This isoform is identical to GR up to amino acid 727 and then diverges in the last C-terminal 15 amino acids. GR-beta is not known to bindglucocorticoids, is unable to transactivate, but does bind DNA (Hollenberg, S M. et al. Nature, 318, p 635, 1985, Bamberger, C. M. et al. J. Clin Invest. 95, p 2435, 1995). It is possible that GR-beta binds compounds other than the typicalglucocorticoids.

Glucocorticoids which interact with GR have been used for over 50 years to treat inflammatory diseases. It has been clearly shown that glucocorticoids exert their anti-inflammatory activity via the inhibition by GR of the transcription factorsNF-.kappa.B and AP-1. This inhibition is termed transrepression. It has been shown that the primary mechanism for inhibition of these transcription factors by GR is via a direct physical interaction. This interaction alters the transcription factorcomplex and inhibits the ability of NF-.kappa.B and AP-1 to stimulate transcription (Jonat, C., et al. Cell, 62, p 1189, 1990, Yang-Yen, H. F., et al. Cell 62, p 1205, 1990, Diamond, M. I. et al. Science 249, p 1266, 1990, Caldenhoven, E. et al., Mol.Endocrinol. 9, p 401, 1995). Other mechanisms such as sequestration of co-activators by GR have also been proposed (Kamer Y, et al., Cell 85, p 403, 1996, Chakravarti, D. et al., Nature 383, p 99, 1996). NF-.kappa.B and AP-1 play key roles in theinitiation and perpetuation of inflammatory and immunological disorders (Baldwin, A S, Journal of Clin. Investigation 107, p 3, 2001, Firestein, G. S., and Manning, A. M. Arthritis and Rheumatism, 42, p 609, 1999, Peltz, G., Curr. Opin, in Biotech. 8,p 467, 1997). NF-.kappa.B and AP-1 are involved in regulating the expression of a number of important inflammatory and immunomodulatory genes including: TNF-alpha, IL-1, IL-2, IL-5, adhesion molecules (such as E-selectin), chemokines (such as Eoxtaxinand Rantes), Cox-2, and others.

Although glucocorticoids are very effective anti-inflammatory agents, their systemic use is limited by their side effects which include diabetes, osteoporosis, glaucoma, Cushingoid syndrome, muscle loss, facial swelling, personality changes, andothers. (Stanbury, R M, and Graham, E M, Br. J. Opthalmology 82, p 704, 1998, Da Silva, J A P., Bijlsma, J. Rheumatic Disease Clinics of North America, 26, p 859, 2000)

In addition to leading to transrepression, the interaction of a glucocorticoid with GR can lead to stimulation by GR of transcription of certain genes. This stimulation of transcription is termed transactivation. Transactivation requiresdimerization of GR and binding to a glucocorticoid response element (GRE). DNA binding is mediated via Zn fingers in the DNA binding domain (Giguere, V. et al Cell 46, p 645, 1986; Rusconi, S. and Yamamoto, K. R. EMBO J., 6, p 1309, 1987). DNA sequencespecific interactions are determined by the C-terminal part of the first Zn finger (Danielsen, M., et al. Cell 57, p 1131, 1989). Several GR target genes have been identified including MMTV, metallothionein, and tyrosine amino transferase (Ringold, G Met al, Cell 6, p 299,1975; Scheidereit, C., et al Nature, 304, p 749, 1983; Hager, L J, Palmiter R D, Nature 291, 340, 1981; Grange, T., et al. Oncogene, 20, p 3028, 2001). Transrepression, as opposed to transactivation, can occur in the absence ofdimerization, and as mentioned above is believed to involve the direct interaction of GR with AP-1 and NF-.kappa.B.

Recent studies using a transgenic GR dimerization defective mouse which cannot bind DNA have shown that the transactivation (DNA binding) activities of GR could be separated from the transrepressive (non-DNA binding) effect of GR. These studiesalso indicate that many of the side effects of glucocorticoid therapy are due to the ability of GR to induce transcription of various genes involved in metabolism, whereas, transrepression, which does not require DNA binding leads to suppression ofinflammation. (Tuckermann, J. et al. Cell 93, p 531, 1998; Reichardt, H M. EMBO J., 20, p 7168, 2001).

Compounds which can induce transrepression of GR with none to minimal induction of transactivation have been termed "dissociated steroids" (Vayassiere, B M, et al., Mol. Endocrinology, 11, p 1249, 1997). Such "dissociated" compounds would beuseful to treat inflammatory diseases. See FIG. 1 for a graphical description of transactivation mediated by GR dimers versus transrepression mediated by GR monomers. It is possible that these "dissociated" compounds bind to GR without inducingdimerization yet allow the monomer to transrepress AP-1 and NF-.kappa.B. Another plausible explanation is that "dissociated" compounds may alter the conformation of GR to enable transrepression without inducing a DNA binding conformation.

There are several examples in the literature of compounds that possess dissociated activity as defined by the ratio of the effective concentration required to induce DNA binding in a cellular assay relative to the effective concentration requiredto transrepress, or inhibit AP-1 or NF-.kappa.B activity. The first report of a "dissociated steroid" published by Vayssiere, et al. Molecular Endocrinology, 11, p 1245, 1997, showed that a derivative of dexamethasone had potent in vitro and in vivoanti-inflammatory activity with minimal induction of DNA binding. Subsequent studies (Coghlan, M J, et al., J. Med. Chem. 44, p 4481, 2001) have shown that non-steroidal compounds can bind to GR and elicit transrepressive activity with moderatetransactivation activity. It is believed that each of the compounds described above act via the dexamethasone binding site.

Ursodeoxycholic acid (UDCA) has recently been shown to repress NF-.kappa.B activity via a GR mediated pathway. The compound appears to be "dissociated" as it does not induce DNA binding in a cellular assay. This compound, although acting in aGR dependent fashion, does not compete for dexamethasone binding to GR. Although direct binding of UDCA to GR has not been demonstrated, mutagenesis studies suggest that the ligand binding domain (LBD) of GR is required for activity. (Miura, T., J.Biol. Chem. 276, p 47371, 2001). However, these studies did not delineate the specific amino acids which are involved in UDCA activity.

The art is in need of modulators of NHRs. A modulator of an NHR may be useful in treating NHR-associated diseases, that is diseases associated with the expression products of genes whose transcription is stimulated or repressed by NHRs. Forinstance, the art is in need of modulators of NHR that induce inhibition of AP-1 and NF-.kappa.B, as such modulators would be useful in the treatment of inflammatory and immune associated diseases and disorders, such as osteoarthritis, rheumatoidarthritis, multiple sclerosis, asthma, inflammatory bowel disease, transplant rejection, and graft vs. host disease.

The art is in need of compounds that possess dissociated activity, as such compounds would be useful in treating inflammatory and immune associated diseases and disorders without exhibiting unwanted side effects. For instance, in the case of GR,although glucocorticoids are potent anti-inflammatory agents, their systemic use is limited by side effects. A dissociated compound that retained the anti-inflammatory efficacy of glucocorticoids while minimizing the side effects such as diabetes,osteoporosis and glaucoma would be of great benefit to a very large number of patients with inflammatory diseases.

The art is in need of compounds that antagonize transactivation. For instance, in the case of GR, such compounds may be useful in treating metabolic diseases associated with increased levels of glucocorticoid, such as diabetes, osteoporosis andglaucoma.

The art is in need of compounds that induce transactivation. For instance, in the case of GR, such compounds may be useful in treating metabolic diseases associated with a deficiency in glucocorticoid. Such diseases include Addison's disease.

In order to design compounds that modulate an NHR in specific ways, one needs to understand how ligands bind to an NHR and modulate the activity of the NHR.

SUMMARY OF THE INVENTION

We have identified a second binding site in the ligand binding domain of nuclear hormone receptors (NHRs). We refer to this second binding site as Site II.

Site II is a structure defined by structure coordinates that describe conserved residue backbone atoms having a root mean square deviation of not more than 2.0 .ANG. from the conserved residue backbone atoms described by the structurecoordinates of amino acids E537-V543, L566, G567, Q570-W577, S599-A607, W610, R611, R614, Q615, P625, Y663, L664 and K667 of SEQ ID NO:13 according to Table I. Table I is located under the heading for Example 21.

FIG. 2 shows the amino acids of Site II in various human NHRs. The structure coordinates of Site II in the NHRs of FIG. 2 are given in Table III, located under the heading for Example 22. Two sets of xray structure coordinates of Site II in GRare given in Table IV, located under the heading for Example 23, and Table V, located under the heading for Example 24. FIG. 6 shows the amino acids of Site II in the GR of various species.

We have found that ligands of Site II modulate NHRs. Ligands of Site II induce transrepression. Ligands of Site II possess dissociated activity. Ligands of Site II antagonize transactivation.

The invention provides machine-readable data storage media comprising data storage material encoded with machine readable data comprising all or any part of the structure coordinates of Site II. The invention provides computer systems comprisingthe machine-readable data storage media of the invention capable of producing three-dimensional representations of all or any part of Site II.

The invention provides methods used in the design and identification of ligands of Site II and modulators of NHRs. The invention provides: methods of docking a test molecule into all or any part of the cavity circumscribed by Site II; methodscomprising identifying structural and chemical features of all or any part of Site II; methods of designing a ligand of Site II comprising modeling all or any part of Site II and designing a chemical entity that has structural and chemicalcomplementarity with all or any part of Site II; methods of evaluating the potential of a chemical entity to bind to all or any part of Site II; methods for identifying a modulator of an NHR; and methods for identifying a ligand of Site II.

The invention provides ligands of Site II. The invention provides modulators of NHRs.

The invention provides methods of modulating an NHR.

The invention provides pharmaceutical compositions comprising modulators of NHRs.

The invention provides methods of treating diseases by administering a modulator of an NHR. Such diseases include NHR-associated diseases, diseases associated with NHR transactivation, diseases associated with NHR transrepression, diseasesassociated with AP-1-dependent gene expression, diseases associated with NF-.kappa.B-dependent gene expression, inflammatory or immune associated diseases and disorders, diseases treatable by inducing NHR transrepression, and diseases treatable byantagonizing NHR transactivation.

The invention provides methods of designing mutants comprising mutating Site II by making an amino acid substitution, deletion or insertion, and the resultant mutant NHRs, or portions of mutant NHRs, comprising a mutation in Site II.

The invention provides methods of measuring the binding of a test molecule to Site II.

The invention provides models of Site II.

All documents referred to herein, including but not limited to U.S. patent applications, are incorporated herein by reference in their entirety.

BRIEF DESCRIPTION OF THE FIGURES

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

FIG. 1. Graphical description of transactivation mediated by GR dimers versus transrepression mediated by GR monomers.

FIG. 2. Consensus alignments carried out using ICM (Molsoft LLC, La Jolla, Calif.) between human GR (glucocorticoid receptor) LBD and other human NHR LBDs, indicating by shading the residues of Site II, i.e. residues corresponding to residues ofGR Site II. Dots are spaceholders and do not represent amino acids. Numbers refer to the first residue in each line, are specific for each NHR and are based on the full-length NHR. For the NHRs listed below, with the exception of GR and MR, structuraldata was obtained from the RCSB references listed below, and the numbering system in the RCSB references was used. For GR and MR, structural data was obtained by homology modeling using the literature references below, and the numbering system in thoseliterature references was used. The RCSB references (in parentheses) and literature references for the various NHRs are as follows:

RXRalpha (SEQ ID NO:3) (1lbd) Bourguet, W., Ruff, M., Chambon, P., Gronemeyer, H., Moras, D. Nature 375 pp. 377 (1995); PPAR-gamma (SEQ ID NO:10) (2prg) Nolte, R. T., Wisely, G. B., Westin, S., Cobb, J. E., Lambert, M. H., Kurokawa, R.,Rosenfeld, M. G., Willson, T. M., Glass, C. K., Milburn, M. V. Nature 395 pp. 137 (1998); RARgamma (SEQ ID NO:4) (2lbd) Renaud, J. P., Rochel, N., Ruff, M., Vivat, V., Chambon, P., Gronemeyer, H., Moras, D. Nature 378 pp. 681 (1995); PR (SEQ ID NO:5)(1a28) Williams, S. P., Sigler, P. B. Nature 393 pp. 392 (1998); VitDR (SEQ ID NO:9) (1db1) Rochel, N., Wurtz, J. M., Mitschler, A., Klaholz, B., Moras, D. Mol. Cell 5 pp. 173 (2000); AR (SEQ ID NO:6) (1e3g) Matias, P. M., Donner, P., Coelho, R.,Thomaz, M., Peixoto, C., Macedo, S., Otto, N., Joschko, S., Scholz, P., Wegg, A., Basler, S., Schafer, M., Egner, U., Carrondo, M. A. J. Biol. Chem. 275 pp. 26164 (2000); ERalpha (SEQ ID NO:7) (1a52) Tanenbaum, D. M., Wang, Y., Williams, S. P., Sigler,P. B. Proc Natl Acad Sci USA 95 pp. 5998 (1998); ERbeta (SEQ ID NO:8) (1l2j) Shiau, A. K., Barstad, D., Radek, J. T., Meyers, M. J., Nettles, K. W., Katzenellenbogen, B. S., Katzenellenbogen, J. A., Agard, D. A., Greene, G. L. Nat. Struct. Biol. 9pp. 359 (2002); TRbeta (SEQ ID NO:12) (1bsx) Wagner, R. L., Darimont, B. D., Apriletti, J. W., Stallcup, M. R., Kushner, P. J., Baxter, J. D., Fletterick, R. J., Yamamoto, K. R. Genes Dev. 12 pp. 3343 (1998). MR and GR structural data were obtainedby homology modeling to PR using the sequences from the following references: GR (SEQ ID NO:13), PIR Accession Number QRHUGA, Hollenberg, S. M., Weinberger, C., Ong, E. S., Cerelli, G., Oro, A., Leba, R., Thompson, E. B., Rosenfeld, M. G., Evans, R. M.Nature (1985) 318: 635-641; MR (SEQ ID NO:11), PIR Accession Number A29613, Arriza, J. L.; Weinberger, C., Cerelli, G., Glaser, T. M., Handelin, B. L., Housman, D. E., Evans, R. M., Science (1987) 237: 268-275.

FIG. 3. GR homology model displayed in ribbon format with dexamethasone (green) and Compound 15 (violet) displayed as space-filling models docked in Site I and Site II, respectively. The location of Site I (dexamethasone site) represents theclassical steroid binding site (eg, consistent with the location of progesterone in PR, 1A28). The location of Site II (Compound 15 site) represents the novel binding site which is the subject of this invention.

FIG. 4. Twenty-seven analogues used in the correlation of observed AP-1 inhibition and calculated contact energy scores as derived from docking into the homology model of GR Site II. Compound numbers are given to the left of each compound.

FIG. 5. The relationship between calculated contact energies of a series of twenty-seven analogues of Compound 15 and their % AP-1 inhibition (at 10 .mu.M). Each analogue was modeled as the S-enantiomer and manually positioned in GR Site II ina manner consistent with the orientation depicted in FIG. 3. Energetics were calculated after geometry/energy minimization using Flo (Colin McMartin, Thistlesoft, Colebrook, Conn.).

FIG. 6. Sequence alignments of the GR from various species conducted using the program LOOK (Version 3.5.2, Molecular Applications Group, Palo Alto, Calif.). The sequence for each GR starts at residue 1. Alignments were made based on pair-wisesequence identity. Site II residues are shaded. Dots are spaceholders and do not represent amino acids. Numbers refer to the first residue in each line, are specific for each GR, and are based on the full-length GR. GR sequences were obtained fromthe following sources: Squirrel (SEQ ID NO:14) (Saimiri boliviensis boliviensis) (GenBank U87951) Reynolds, P. D., Pittler, S. J. and Scammell, J. G. J. Clin. Endocrinol. Metab. 82 (2), 465-472 (1997); Pig GR (SEQ ID NO:15) (GenBank AF141371) Gutscher,M., Eder, S., Mueller, M. and Claus, R. Submitted to GenBank (08-APR-1999) Institut fuer Tierhaltung und Tierzuechtung (470), FG Tierhaltung und Leistungsphysiologie, Universitaet Hohenheim, Garbenstr. 17, Stuttgart 70599, Germany; Guinea Pig (SEQ IDNO:16) (GenBank L13196) Keightley, M. C. and Fuller, P. J. Mol. Endocrinol. 8 (4), 431-439 (1994); Marmoset (SEQ ID NO:17) (GenBank U87953) Reynolds, P. D., Pittler, S. J. and Scammell, J. G. J. Clin. Endocrinol. Metab. 82 (2), 465-472 (1997); Ma'zMonkey (SEQ ID NO:18) (GenBank U87952) Reynolds, P. D., Pittler, S. J. and Scammell, J. G. J. Clin. Endocrinol. Metab. 82 (2), 465-472 (1997); rat (SEQ ID NO:19) (GenBank M14053) Miesfeld, R., Rusconi, S., Godowski, P. J., Maler, B. A., Okret, S.,Wikstrom, A. C., Gustafsson, J. A. and Yamamoto, K. R. Cell 46 (3), 389-399 (1986); mouse (SEQ ID NO:20) (GenBank X04435) Danielsen, M., Northrop, J. P. and Ringold, G. M. EMBO J. 5 (10), 2513-2522 (1986); Human (SEQ ID NO:21) (PIR Accession NumberQRHUGA) Hollenberg, S. M., Weinberger, C., Ong, E. S., Cerelli, G., Oro, A., Leba, R., Thompson, E. B., Rosenfeld, M. G., Evans, R. M., Nature (1985) 318: 635-641.

FIG. 7. Ribbon diagram of the LBDs of 11 NHRs detailed in FIG. 2, based on a consensus alignment paradigm (ICM, Molsoft LLC, La Jolla, Calif.). The glucocorticoid receptor (GR) homology model is represented by the blue ribbon.

FIG. 8. Graphic demonstration that in a highly sensitive, artificial assay, mutations in Site II inhibit the ability of Site II ligands to induce transactivation, whereas there was a minimal effect on the Site I compound dexamethasone. RLU onthe Y-axis is relative light units, a measurement of transactivation. Various mutants and the wild-type are given on the X-axis. Compound A, a Site II ligand, is indicated by the left, darker, solid bar in each pair of bars. Dexamethasone is indicatedby the right, lighter, hatched bar in each pair of bars.

FIG. 9. Graphic demonstration that the Site I antagonist RU486 inhibits dexamethasone-mediated repression of AP-1 activity, whereas Site II compounds, such as Compound A and Compound B, act in an additive fashion with dexamethasone to repressAP-1 activity. The Y-axis denotes % inhibition of AP-1 activity. The X-axis denotes concentration of dexarnethasone. Concentrations of RU486, Compound A, and Compound B are denoted by the indicated symbols.

FIG. 10. Graphic demonstration of an assay to indirectly measure the interaction of Site II ligands with GR showing that Site II ligands which do not inhibit dexamethasone on their own can displace other Site II ligands which do inhibitdexamethasone, thereby allowing dexamethasone to bind to GR. Compound D is a Site II ligand that does inhibit dexamethasone. Compound A, Compound B, and Compound C are Site II ligands that do not inhibit dexamethasone on their own. Compound A,Compound B, and Compound C are denoted by the indicated symbols and are the competitor compounds whose concentration is denoted on the X-axis. The Y-axis denotes % inhibition of dexamethasone binding.

FIGS. 11a and 11b. Graphic demonstrations that a Site II ligand inhibits AP-1-mediated transcription in a GR dependent fashion. The Y-axes denote relative light units (RLU), a measurement of AP-1 activity. On the X-axes, Compound A is a SiteII ligand, DEX is dexamethasone, and PMA is phorbol myristic acid. In FIG. 11a, AP-1 activity is measure without the presence of GR. In FIG. 11b, AP-1 activity is measured in the presence of GR.

DETAILED DESCRIPTION OF THE INVENTION

We have identified a second binding site in the ligand binding domain of nuclear hormone receptors (NHRs). We refer to this second binding site as Site II.

Site II is a structure defined by structure coordinates that describe conserved residue backbone atoms having a root mean square deviation of not more than 2.0 .ANG. from the conserved residue backbone atoms described by the structurecoordinates of amino acids E537-V543, L566, G567, Q570-W577, S599-A607, W610, R611, R614, Q615, P625, Y663, L664 and K667 of SEQ ID NO:13 according to Table I. That is, a Site II is any structure that falls within the given root mean square deviation. Table I is located under the heading for Example 21.

FIG. 2 shows the amino acids of Site II in various human NHRs. The structure coordinates of Site II in the NHRs of FIG. 2 are given in Table III, located under the heading for Example 22. Two sets of xray structure coordinates of Site II in GRare given in Table IV, located under the heading for Example 23, and Table V, located under the heading for Example 24. FIG. 6 shows the amino acids of Site II in the GR of various species.

We have found that ligands of Site II modulate NHRs. Ligands of Site II induce transrepression. Ligands of Site II possess dissociated activity. Ligands of Site II antagonize transactivation.

For all of the present inventions described further below, the following information on possible and preferable embodiments applies.

Said Site II preferably is a nuclear hormone receptor (NHR) Site II, more preferably steroid hormone receptor (SHR) Site II, most preferably a glucocorticoid receptor (GR) Site II.

Said NHR is preferably an SHR, more preferably a GR.

Preferably said NHR is selected from the group consisting of: RXR-alpha; RXR-beta; progesterone receptor (PR); androgen receptor (AR); estrogen receptor-alpha (ER-alpha); ER-beta; vitamin D receptor (VitDR); peroxisome proliferator activatedreceptor-gamma (PPAR-gamma); thyroid receptor-alpha (TR-alpha); TR-beta; mineralocorticoid receptor (MR); and glucocorticoid receptor (GR). More preferably, said NHR is selected from the group consisting of: RXR-alpha; RXR-beta; progesterone receptor(PR); androgen receptor (AR); vitamin D receptor (VitDR); peroxisome proliferator activated receptor-gamma (PPAR-gamma); thyroid receptor-alpha (TR-alpha); TR-beta; mineralocorticoid receptor (MR); and glucocorticoid receptor (GR). Most preferably, saidNHR is selected from the group consisting of: RXR-alpha; RXR-beta; androgen receptor (AR); vitamin D receptor (VitDR); peroxisome proliferator activated receptor-gamma (PPAR-gamma); thyroid receptor-alpha (TR-alpha); TR-beta; mineralocorticoid receptor(MR); and glucocorticoid receptor (GR).

Preferably said SHR is selected from the group consisting of: PR; AR; ER-alpha; ER-beta; MR; and GR. More preferably, said SHR is selected from the group consisting of: PR; AR; MR; and GR. Most preferably, said SHR is selected from the groupconsisting of: AR; MR; and GR.

Said RXR-alpha Site II preferably is composed of amino acids L236-P244, A272-A273, Q276-W283, G305-S313, H316-R317, A320-V321, T329, L368-G369, and R372 of SEQ ID NO:3 according to FIG. 2. Preferably said structure coordinates of said RXR-alphaSite II define the structure of amino acids L236-P244, A272-A273, Q276-W283, G305-S313, H316-R317, A320-V321, T329, L368-G369, and R372 of SEQ ID NO:3 according to Table III, or define the structure of the conserved residue backbone atoms according toTable III. By this it is meant that preferably said structure coordinates define the same shape as the structure of the amino acids according to Table III, or as the structure of the residue backbone atoms according to Table II, but not necessarily thatthe structure coordinates are identical to those of the amino acids or residue backbone atoms in the table, as the structure coordinates may be of a coordinate system other than Cartesian coordinates. More preferably, said structure coordinates of saidSite II are the structure coordinates of Site II amino acids L236-P244, A272-A273, Q276-W283, G305-S313, H316-R317, A320-V321, T329, L368-G369, and R372 of SEQ ID NO:3 according to Table III.

Said RAR-gamma Site II preferably is composed of amino acids S194-P202, L233-A234, C237-F244, A266-R274, T277-R278, T280-E282, D290, T328-G329 and S332 of SEQ ID NO:4 according to FIG. 2. Preferably said structure coordinates of said RAR-gammaSite II define the structure of amino acids S 194-P202, L233-A234, C237-F244, A266-R274, T277-R278, T280-E282, D290, T328-G329 and S332 of SEQ ID NO:4 according to Table III, or define the structure of the conserved residue backbone atoms according toTable III. More preferably, said structure coordinates of said Site II are the structure coordinates of Site II amino acids S194-P202, L233-A234, C237-F244, A266-R274, T277-R278, T280-E282, D290, T328-G329 and S332 of SEQ ID NO:4 according to Table III.

Said PR preferably is composed of amino acids M692-V698, L721-G722, Q725-W732, S754-G762, W765-R766, K769-H770, P780, F818-L819 and K822 of SEQ ID NO:5 according to FIG. 2. Preferably said structure coordinates of said PR Site II define thestructure of amino acids M692-V698, L721-G722, Q725-W732, S754-G762, W765-R766, K769-H770, P780, F818-L819 and K822 of SEQ ID NO:5 according to Table III, or define the structure of the conserved residue backbone atoms according to Table III. Morepreferably, said structure coordinates of said Site II are the structure coordinates of Site II amino acids M692-V698, L721-G722, Q725-W732, S754-G762, W765-R766, K769-H770, P780, F818-L819 and K822 of SEQ ID NO:5 according to Table III.

Said AR Site II preferably is composed of amino acids E678-V684, L708-G709, Q712-W719, S741-A749, W752-R753, T756-N757, P767, F805-L806 and K809 of SEQ ID NO:6 according to FIG. 2. Preferably said structure coordinates of said AR Site II definethe structure of amino acids E678-V684, L708-G709, Q712-W719, S741-A749, W752-R753, T756-N757, P767, F805-L806 and K809 of SEQ ID NO:6 according to Table III, or define the structure of the conserved residue backbone atoms according to Table III. Morepreferably, said structure coordinates of said Site II are the structure coordinates of Site II amino acids E678-V684, L708-G709, Q712-W719, S741-A749, W752-R753, T756-N757, P767, F805-L806 and K809 of SEQ ID NO:6 according to Table III.

Said ER-alpha Site II preferably is composed of amino acids L320-1326, L348-A349, E352-W359, A381-G389, W392-R393, E396, P405, F444-V445 and K448 of SEQ ID NO:7 according to FIG. 2. Preferably said structure coordinates of said ER-alpha Site IIdefine the structure of amino acids L320-1326, L348-A349, E352-W359, A381-G389, W392-R393, E396, P405, F444-V445 and K448 of SEQ ID NO:7 according to Table III, or define the structure of the conserved residue backbone atoms according to Table III. Morepreferably, said structure coordinates of said Site II are the structure coordinates of Site II amino acids L320-1326, L348-A349, E352-W359, A381-G389, W392-R393, E396, P405, F444-V445 and K448 of SEQ ID NO:7 according to Table III.

Said ER-beta Site II preferably is composed of amino acids L273-H279, L297-A298, E301-W308, C330-G338, W341-R342, D345, P354, Y393-L394 and K397 of SEQ ID NO:8 according to FIG. 2. Preferably said structure coordinates of said ER-beta Site IIdefine the structure of amino acids L273-H279, L297-A298, E301-W308, C330-G338, W341-R342, D345, P354, Y393-L394 and K397 of SEQ ID NO:8 according to Table III, or define the structure of the conserved residue backbone atoms according to Table III. Morepreferably, said structure coordinates of said Site II are the structure coordinates of Site II amino acids L273-H279, L297-A298, E301-W308, C330-G338, W341-R342, D345, P354, Y393-L394 and K397 of SEQ ID NO:8 according to Table III.

Said VitDR Site II preferably is composed of amino acids L136-D144, L182-VI 83, S186-F193, S215-R223, E226-S227, T229-D231, G238, H279-V280 and M283 of SEQ ID NO:9 according to FIG. 2. Preferably said structure coordinates of said VitDR Site IIdefine the structure of amino acids L136-D144, L182-V183, S186-F193, S215-R223, E226-S227, T229-D231, G238, H279-V280 and M283 of SEQ ID NO:9 according to Table III, or define the structure of the conserved residue backbone atoms according to Table III. More preferably, said structure coordinates of said Site II are the structure coordinates of Site II amino acids L136-D144, L182-V183, S186-F193, S215-R223, E226-S227, T229-D231, G238, H279-V280 and M283 of SEQ ID NO:9 according to Table III.

Said PPAR-gamma Site II preferably is composed of amino acids Y219-P227, R288-S289, A292-Y299, G321-M329, S332-L333, N335-K336, E343, L384-A385 and I388 of SEQ ID NO:10 according to FIG. 2. Preferably said structure coordinates of saidPPAR-gamma Site II define the structure of amino acids Y219-P227, R288-S289, A292-Y299, G321-M329, S332-L333, N335-K336, E343, L384-A385 and I388 of SEQ ID NO:10 according to Table III, or define the structure of the conserved residue backbone atomsaccording to Table III. More preferably, said structure coordinates of said Site II are the structure coordinates of Site II amino acids Y219-P227, R288-S289, A292-Y299, G321-M329, S332-L333, N335-K336, E343, L384-A385 and I388 of SEQ ID NO:10 accordingto Table III.

Said MR Site II preferably is composed of amino acids E743-1749, L772-A773, Q776-W783, S805-A813, W816-R817, K820-H821, P831, Y869-T870 and K873 of SEQ ID NO:11 according to FIG. 2. Preferably said structure coordinates of said MR Site II definethe structure of amino acids E743-1749, L772-A773, Q776-W783, S805-A813, W816-R817, K820-H821, P831, Y869-T870 and K873 of SEQ ID NO:11 according to Table III, or define the structure of the conserved residue backbone atoms according to Table III. Morepreferably, said structure coordinates of said Site II are the structure coordinates of Site II amino acids E743-1749, L772-A773, Q776-W783, S805-A813, W816-R817, K820-H821, P831, Y869-T870 and K873 of SEQ ID NO:11 according to Table III.

Said TR-beta Site II preferably is composed of amino acids T226-Q235, 1267-1268, A271-F278, C300-R308, V311-R312, D314-E316, G324, V362-A363 and Q366 of SEQ ID NO:12 according to FIG. 2. Preferably said structure coordinates of said TR-beta SiteII define the structure of amino acids T226-Q235, 1267-1268, A271-F278, C300-R308, V311-R312, D314-E316, G324, V362-A363 and Q366 of SEQ ID NO:12 according to Table III, or define the structure of the conserved residue backbone atoms according to TableIII. More preferably, said structure coordinates of said Site II are the structure coordinates of Site II amino acids T226-Q235, 1267-1268, A271-F278, C300-R308, V311-R312, D314-E316, G324, V362-A363 and Q366 of SEQ ID NO:12 according to Table III.

Said GR Site II preferably is composed of amino acids E537-V543, L566, G567, Q570-W577, S599-A607, W610, R611, R614, Q615, P625, Y663, L664 and K667 of SEQ ID NO:13 according to FIG. 2. Preferably said structure coordinates of said GR Site IIdefine the structure of amino acids E537-V543, L566, G567, Q570-W577, S599-A607, W610, R611, R614, Q615, P625, Y663, L664 and K667 of SEQ ID NO:13 according to Table I, Table III, Table IV or Table V, or define the structure of the aforementioned aminoacids according to the structure coordinates disclosed in Bledsoe, et. al., Cell, online publication by Cell Press, Jul. 1, 2002; DOI: 10.1016/S0092867402008176, or define the structure of the conserved residue backbone atoms according to any of theaforementioned. Preferably, said structure coordinates of said Site II are the structure coordinates of Site II amino acids E537-V543, L566, G567, Q570-W577, S599-A607, W610, R611, R614, Q615, P625, Y663, L664 and K667 of SEQ ID NO:13 according to TableI, Table III, Table IV or Table V.

Said GR Site II is preferably selected from the group consisting of: human GR Site II composed of amino acids E537-V543, L566, G567, Q570-W577, S599-A607, W610, R611, R614, Q615, P625, Y663, L664 and K667 of SEQ ID NO:19 according to FIG. 6;squirrel GR Site II composed of amino acids E537-V543, L566, G567, Q570-W577, S599-A607, W610, R611, R614, Q615, P625, Y663, L664 and K667 of SEQ ID NO:14 according to FIG. 6; pig GR Site II composed of amino acids E501-V507, L530, G531, Q534-W541,S563-A571, W574, R575, R578, Q579, P589, Y627, L628 and K631 of SEQ ID NO:15 according to FIG. 6; guinea pig GR Site II composed of amino acids E531-V537, L560, G561, Q564-W571, S593-A601, W604, R605, K608, Q609, P619, Y557, L558 and K561 of SEQ ID NO:16according to FIG. 6; marmoset GR Site II composed of amino acids E537-V543, L566, G567, Q570-W577, S599-A607, W610, R611, R614, Q615, P625, Y663, L664 and K667 of SEQ ID NO:17 according to FIG. 6; ma'z monkey GR Site II composed of amino acids E537-V543,L566, G567, Q570-W577, S599-A607, W610, R611, R614, Q615, P625, Y663, L664 and K667 of SEQ ID NO:18 according to FIG. 6; rat GR Site II E555-V561, L584, G585, Q588-W595, S617-A625, W628, R629, R632, Q633, P643, Y681, L682 and K685 of SEQ ID NO:20according to FIG. 6; and mouse GR Site II E543-V549, L572, G573, Q576-W583, S605-A613, W616, R617, R620, Q621, P631, Y669, L670 and K673 of SEQ ID NO:21 according to FIG. 6.

Said nuclear hormone receptor can be of any source, preferably human.

Said glucocorticoid receptor can be of any source, preferably human, rat, mouse, sheep, marmoset, squirrel, pig, guinea pig, or ma'z monkey. Most preferably said glucorticoid receptor is human.

Said NHR Site II may be native or mutant. Preferably said NHR Site II is a native NHR Site II. Said SHR Site II maybe native or mutant. Preferably said SHR Site II is a native SHR Site II. Said GR Site II may be native or mutant. Preferablysaid GR Site II is a native GR Site II.

Said Site II may be found on a protein of any source, including mammalian, fungal, bacterial and plant. Preferably said Site II is found on a mammalian protein, more preferably on a human protein.

Preferably the conserved residue backbone atoms of said Site II have a root mean square deviation of less than 1.9, 1.8, 1.7, 1.6 or 1.5 .ANG., more preferably of less than 1.4, 1.3, 1.2, 1.1, 1.03, 1.02, or 1.0 .ANG., yet more preferably of lessthan 0.93, 0.92, 0.9, 0.8, 0.7, 0.6 0.5, 0.4, 0.3, 0.2, or 0.1 .ANG., most preferably 1.02, 0.92 or 0.0 .ANG. from the conserved residue backbone atoms described by the structure coordinates of amino acids E537-V543, L566, G567, Q570-W577, S599-A607,W610, R611, R614, Q615, P625, Y663, L664 and K667 according to Table I.

Preferably the conserved residue backbone atoms of said Site II have a root mean square deviation of less than 2.0, 1.9, 1.8, 1.7, 1.6 or 1.5 .ANG., more preferably of less than 1.4, 1.3, 1.2, 1.1, 1.03, 1.02, or 1.0 .ANG., yet more preferably ofless than 0.93, 0.92, 0.9, 0.8, 0.7, 0.6 0.5, 0.4, 0.3, 0.2, or 0.1 .ANG., most preferably 1.02, 0.92 or 0.0 .ANG. from the conserved residue backbone atoms described by the structure coordinates of amino acids E537-V543, L566, G567, Q570-W577,S599-A607, W610, R611, R614, Q615, P625, Y663, L664 and K667 according to Table IV.

Preferably the conserved residue backbone atoms of said Site II have a root mean square deviation of less than 2.0, 1.9, 1.8, 1.7, 1.6 or 1.5 .ANG., more preferably of less than 1.4, 1.3, 1.2, 1.1, 1.03, 1.02, or 1.0 .ANG., yet more preferably ofless than 0.93, 0.92, 0.9, 0.8, 0.7, 0.6 0.5, 0.4, 0.3, 0.2, or 0.1 .ANG., most preferably 1.02, 0.92 or 0.0 .ANG. from the conserved residue backbone atoms described by the structure coordinates of amino acids E537-V543, L566, G567, Q570-W577,S599-A607, W610, R611, R614, Q615, P625, Y663, L664 and K667 according to Table V.

Said structure coordinates may be those determined for a Site II to which a ligand is bound or to which no ligand is bound. Said structure coordinates may be those determined for a Site II of a ligand binding domain in which a Site I ligand isbound to Site I. Said structure coordinates may be those determined for a Site II of an NHR that is in monomer, dimer, or other form.

As is illustrated in FIG. 3, the cavity circumscribed by Site II and the cavity circumscribed by Site I (in GR, the dexamethasone binding site) share a common wall section. That is, some amino acids are common to both Site II and Site I. Howeverthe cavity circumscribed by Site II is distinct from the cavity circumscribed by Site I, as the two cavities are on opposite sides of the common wall. We manually docked dexamethasone into GR Site I (see Example 10) and determined that the followingamino acid residues are in contact distance, i.e. within 2-3 Angstroms, of dexamethasone and thus make up GR Site I: M560, L563, N564, L566, G567, Q570, M601, M604, A605, L608, R611, F623, M639, Q642, M646, L732, Y735, C736, T739 and E748. The followingamino acid residues are common to both GR Site I and Site II: L566, G567, Q570, M601, M604, A605 and R611. The following amino acid residues are unique to GR Site II, i.e. they are not part of GR Site I: E537-V543, V571-W577, S599-W600, F602-L603,F606-A607, W610, R614, Q615, P625, Y663, L664 and K667. The following amino acid residues are unique to GR Site I, i.e. they are not part of GR Site II: M560, L563, N564, L608, F623, M639, Q642, M646, L732, Y735, C736, T739 and E748. The amino acids inother NHRs and non-human GR corresponding to the above-recited GR amino acids can be seen in FIGS. 2 and 6 respectively. We have identified Site II in NHRs as a binding site whose ligands modulate NHRs.

We defined Site II through use of structure coordinates of the ligand binding domain (LBD) of the glucocorticoid receptor (GR), which are provided in Table I. The structure coordinates of the LBD of GR were determined using homology modeling, andlater confirmed based on the xray structural elucidation of the GR LBD provided in Apolito, et. al., in WO 03/015692 A2, published Feb. 27, 2003, and Kauppi et. al. in the Journal of Biological Chemistry Online, JBC Papers In Press asDOI:10.1074/JBC.M212711200, Apr. 9, 2003. Thus, some description of homology models and structure coordinates is appropriate here.

Homology models are useful when there is no experimental information available on the three-dimensional structure of the protein of interest. A three dimensional model can be constructed on the basis of the known structure of a homologousprotein (Greer et. al., 1991, Lesk, et. al., 1992, Cardozo, et. al., 1995, Sali, et. al., 1995). Those of skill in the art will understand that a homology model is constructed on the basis of first identifying a template, or, protein of known structurewhich is similar to the protein without known structure. This can be accomplished through pairwise alignment of sequences using such programs as the MODELLER module found in InsightII (Accelrys, Inc., San Diego, Calif.).

Those of skill in the art will understand that a set of structure coordinates for a protein or part of a protein is a relative set of points that define a shape in three dimensions. For a number of reasons, including those that follow below, thestructure coordinates that define two identical or almost identical shapes may vary slightly. If variations are within an acceptable standard error as compared to the original coordinates, the resulting three-dimensional shape is considered to beequivalent. Thus, for example, a ligand that bound to the structure defined by the structure coordinates of amino acids E537-V543, L566, G567, Q570-W577, S599-A607, W610, R611, R614, Q615, P625, Y663, L664 and K667 according to Table I would also beexpected to bind to a site having a shape that fell within the acceptable error. Such sites with structures within the acceptable standard error are also within the scope of this invention.

Various computational analyses are therefore necessary to determine whether a molecule or a portion thereof is sufficiently similar to all or parts of the disclosed homology model to be considered equivalent. Such analyses may be carried out incurrent software applications, such as InsightII (Accelrys Inc., San Diego, Calif.) Version 2000 as described in the User's Guide, online or software applications available in the SYBYL software suite (Tripos Inc., St. Louis, Mo.).

Using the superimposition tool in the program InsightII, for instance, comparisons can be made between different structures and different conformations of the same structure. The procedure used in InsightII to compare structures is divided intofour steps: 1) load the structures to be compared; 2) define the atom equivalencies in these structures; 3) perform a fitting operation; and 4) analyze the results. Each structure is identified by a name. One structure is identified as the target(i.e., the fixed structure); the second structure (i.e., moving structure) is identified as the source structure. Since atom equivalency within InsightII is defined by user input, for the purpose of this invention we will define equivalent atoms asprotein backbone atoms, also known as residue backbone atoms, (N, C.alpha., C and O) for all residues between the two structures being compared. We will also consider only rigid fitting operations. When a rigid fitting method is used, the movingstructure 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 squaredifference of the fit over the specified pairs of equivalent atoms is an absolute minimum. This number, given in Angstroms (.ANG.), is reported by InsightII.

Three-dimensional coordinates give the location of the centers of all atoms in a protein molecule and are typically expressed as Cartesian coordinates (eg, distances in three directions, each perpendicular to the other), or polar coordinates (eg,sets of angle/distance pairs from a universal origin), or internal coordinates (eg, sets of angle/distance pairs from one atom center to the next). Thus, it is possible that an entirely different set of coordinates could define an identical or similarshape, depending on which coordinate system is used.

Slight variations in the individual coordinates, as emanate from generation of similar homology models using different alignment templates, and/or using different methods in generating the homology model, will have minor effects on the overallshape.

Variations in coordinates may also be generated because of mathematical manipulations of the structure coordinates. For example, the structure coordinates set forth in Table I could be manipulated by fractionalization of the structurecoordinates, integer additions or subtractions to sets of the structure coordinates, inversion of the structure coordinates or any combination of the above.

The structure coordinates of an actual xray structure of a protein would be expected to have some variation from the homology model of that very same protein. For example, the location of sidechains may vary to some extent. As examples, thehomology model GR Site II coordinates were compared to the GR Site II x-ray structure coordinates available from the disclosures in WO 03/015692 A2, Feb. 27, 2003 Apolito, et. al. and Kauppi et. al., in the Journal of Biological Chemistry Online, JBCPapers In Press as

DOI:10.1074/jbc.M212711200, Apr. 9, 2003, RCSB file: 1nhz.pdb (GR LBD bound to an antagonist, RU 486). When the backbone atoms of the homology model Site II residues, ie, E537-V543, L566, G567, Q570-W577, S599-A607, W610, R611, R614, Q615,P625, Y663, L664 and K667 of SEQ ID NO;13 according to Table I were compared, root mean square deviations (rmsds) of 0.92 and 1.02 .ANG. were obtained between the homology model of Table I Site II residues and the Apolito Site II residues, and betweenthe homology model of Table I Site II residues and the Kauppi Site II residues, respectively. These observations underscore the similarity of the Site II homology model structure to actual crystal structures.

Variations in structure coordinates can be due to mutations, additions, substitutions, and/or deletions of amino acids of a protein being studied.

Variations in structure coordinates can be due to variations in proteins whose shape is being described by the structure coordinates given. For instance, rigid fitting operations conducted (see Example 13) between the GR LBD homology model andseveral closely-related NHRs known to have similar structure and function (ie, progesterone receptor LBD, androgen receptor LBD, estrogen receptor alpha LBD and estrogen receptor beta LBD as examples) yielded Site II root mean square (rms) deviations inconserved residue backbone atom comparisons of 0.57-0.71 .ANG.. These Site II rms deviations could be greater if other variation factors described above were present in the calculations. GR LBDs from non-human species may also have slight variations inshape from that of human GR LBD defined by the structure coordinates in Table 1.

For the purpose of this invention, any structure that has a root mean square deviation of residue backbone atoms (N, C.alpha., C, O) of less than 2.0 Angstroms (.ANG.) when superimposed on the relevant residue backbone atoms described bystructure coordinates listed in Table I is considered to be equivalent. Preferably the root mean square deviation is less than 1.9, 1.8, 1.7, 1.6 or 1.5 .ANG., more preferably of less than 1.4, 1.3, 1.2, 1.1, 1.03, 1.02, or 1.0 .ANG., yet morepreferably of less than 0.93, 0.92, 0.9, 0.8, 0.7, 0.6 0.5, 0.4, 0.3, 0.2, or 0.1 .ANG., most preferably 1.02, 0.92 or 0.0 .ANG..

Within the context of the present invention, "conserved" refers to a portion of a protein backbone which is found in common between two proteins. That is, if portions of two proteins are aligned and compared using the three-dimensionalcoordinates of their residue backbone atoms for super-positioning, and comparison of the structure coordinates of the residue backbone atoms yields an rms of 2.0 .ANG. or less, then the residue backbone atoms are considered to be conserved between thetwo proteins.

We made the claimed inventions through a series of experiments described below in the Examples. To help in understanding the invention, that series of experiments is summarized here.

Twenty-seven compounds, which are analogues, were synthesized and shown to inhibit GR binding in GR Site I binding assays and to induce transrepression in AP-1 cellular transrespressional assays. Some of these compounds were tested in cellulartranscriptional assays and shown to induce none to minimal transactivation. Thus these compounds were shown to have dissociated activity.

Twelve analogues of the twenty-seven compounds (some of which are among the twenty-seven compounds) were synthesized and the racemic mixtures were separated into enantiomers. Each of these twenty-four enantiomers was tested in the GR bindingassay and the cellular transrepressional assay. It was observed that the S enantiomer of each pair induced AP-1 inhibitory activity when GR was present but did not inhibit well dexamethasone binding to GR, while the R enantiomer of each pair inducedminimal AP-1 inhibitory activity when GR was present and inhibited well dexamethasone binding to GR. Each enantiomer was also tested in the cellular transcriptional assay and induced none to minimal transactivation. This suggested that there is analternate site on GR to which these compounds bind that does not result in inhibition of dexamethasone binding to GR.

A homology model of the ligand binding domain (LBD) of GR was constructed using the known crystal structure of the progesterone receptor (PR). Site II in the LBD of GR was identified by the complementarity of three-dimensional shape andfunctional features between the Site II and compounds having AP-1 inhibitory activity. Manual docking of one such compound was performed and confirmed the identity of Site II and its role in transrepression. Binding energetics of the S enantomer of thetwenty-seven compounds to Site II were calculated and correlated with AP-1 inhibitory activity of these compounds. This positive correlation further confirmed the identity and role of Site II.

As binding energetics to Site II correlates to AP-1 inhibitory activity and all compounds that were tested for binding to Site II are dissociated compounds, Site II was determined to be a target for compounds that have AP-1 inhibitory activity aswell as compounds that have dissociated activity.

Additional studies were performed to elucidate the relationship between binding at Site II and binding at Site I. One S enantiomer and dexamethasone were used concurrently in cellular transrepressional assays and cellular transcriptional assays. In the cellular transrepressional assays, it was observed that the dissociated compound (i.e. the S enantiomer) and dexamethasone had an additive effect on AP-1 inhibitory activity. In the cellular transcriptional assays, it was observed that thepresence of a dissociated compound along with dexamethasone reduced transactivation as compared to dexamethasone alone.

The cellular transcriptional assay was performed with a titration of dexamethasone in the presence or absence of each of both enantiomers of a pair. Again, an additive effect on AP-1 inhibitory activity was seen with each of the enantiomers anddexamethasone. In contrast, the Site I antagonist RU 486 inhibited the ability of dexamethasone to induce transrepression.

Other studies performed have shown that mutations in Site II alter the ability of an S enantiomer to modulate GR function. In a highly sensitive, artificial assay system to measure transactivation, it was shown that mutations of residues 543 or607 prevented the compound from inducing transactivation, whereas, in the wild type protein transactivation was seen. Dexamethasone induced transactivation in both the mutants and the wild type protein.

A further study demonstrated that both an S enantiomer and dexamethasone act in a GR-dependent fashion.

The studies performed to date suggest that both enantiomers interact with Site II. Example 17 shows that both enantiomers R (Compound B) and S (Compound A) act in an additive fashion with saturating levels of dexamethasone to suppress AP-1activity. Since dexamethasone binds to Site I, it is most likely that the R and S enantiomers interact with Site II to allosterically enhance the repressive activity.

The following definitions are provided to more fully describe the present invention in its various aspects. The definitions are intended to be useful for guidance and elucidation, and are not intended to limit the disclosed invention and itsembodiments. Additional definitions may be provided elsewhere in the specification.

The terms "nuclear hormone receptor" and "NHR," as used herein, refer to a member of the nuclear hormone receptor family of transcription factors which bind low molecular weight ligands and stimulate or repress transcription. NHRs include, butare not limited to, glucocorticoid receptors (GRs), progesterone receptors (PRs), androgen receptors (ARs), estrogen receptors (ERs), mineralocorticoid receptors (MRs), retinoid receptors (RXRs and RARs), Vitamin D receptors (VitDRs), thyroid receptors(TRs), peroxisome proliferator activated receptors (PPARs), and orphan nuclear receptors (i.e. receptors for which the ligands are not yet identified) that bind nuclear hormones. "Nuclear hormone receptor" includes orphan nuclear receptors, which aregene products that embody structural features of nuclear hormone receptors and were identified without any prior knowledge of their association with a putative ligand.

The structural features that define a nuclear hormone receptor, including an orphan nuclear receptor, are the four following features (as disclosed in Giguere, V. (1999) Endocrine Reviews 20(5) p 689: 1. An NHR has a modulator domain includingthe AF-1 domain responsible in part for transcriptional activation function. Modulator domains can also include regions for promoters and cell-specific cofactors and can interact with steroid receptor co-activators (SRCs). 2. An NHR has a DNA bindingdomain (DBD) composed of two zinc finger modules composed of 60-70 amino acids and a carboxy-terminal extension (CTE) that providesprotein-protein and protein-DNA interactions upon homo- or heterodimer receptorbinding. 3. An NHR has a hinge region thatis the hinge between the DBD and the carboxy-terminal ligand binding domain. The hinge region is variable is primary structure and amino acid sequence length. 4. An NHR has a ligand binding domain (LBD) that contains the AF-2 motif (which correspondsto helix 12 of NHRs) and provides a structured region whereby AF-2 (helix 12) is packed closely to the LBD core forming an interface with at least 3 other helices of the core. The interface is involved with binding of co-activator or co-repressorpolypeptides.

"Nuclear hormone receptor" and "NHR," as used herein, refer to NHRs from any source, including but not limited to: glucocorticoid receptor as disclosed in Hollenberg, S. M., Weinberger, C., Ong, E. S., Cerelli, G., Oro, A., Leba, R., Thompson, E.B., Rosenfeld, M. G., Evans, R. M., Nature (1985) 318: 635-641 progesterone receptor as disclosed in Misrahi, M. et al. (1987) Biochem. Biophys. Res. Commun. 143, p 740; androgen receptor as disclosed in Lubahn D. B., et al (1988); estrogen receptorsas disclosed in Green, S., et al. (1986) Nature 320, p 134); mineralocorticoid receptor as disclosed in Arriza, J. L., et al., (1987) Science 237, p 268; retinoid receptors (RXRs and RARs) as disclosed in Mangelsdorf, et al. (1990) Nature, 345, p 224 andPetkovich M., et al (1987) Nature 330, p 444; Vitamin D receptor, thyroid receptor (TR) as disclosed in Nakai, A. et al., (1988) Mol. Endocrinol. 2, p 1087; peroxisome proliferator activated receptor (PPAR) as disclosed in Greene, M. E., et al. (1995)Gene Expression 4, p 281; RXRalpha (1lbd) as disclosed in Bourguet, W., Ruff, M., Chambon, P., Gronemeyer, H., Moras, D. Nature 375 pp. 377 (1995); PPARgamma (2prg) as disclosed in Nolte, R. T., Wisely, G. B., Westin, S., Cobb, J. E., Lambert, M. H.,Kurokawa, R., Rosenfeld, M. G., Willson, T. M., Glass, C. K., Milburn, M. V. Nature 395 pp. 137 (1998); RARgamma (2lbd) as disclosed in Renaud, J. P., Rochel, N., Ruff, M., Vivat, V., Chambon, P., Gronemeyer, H., Moras, D. Nature 378 pp. 681 (1995); PR(1a28) as disclosed in Williams, S. P., Sigler, P. B. Nature 393 pp. 392 (1998); VitDR (1db1) as disclosed in Rochel, N., Wurtz, J. M., Mitschler, A., Klaholz, B., Moras, D. Mol. Cell 5 pp. 173 (2000); AR (1e3g) as disclosed in Matias, P. M., Donner,P., Coelho, R., Thomaz, M., Peixoto, C., Macedo, S., Otto, N., Joschko, S., Scholz, P., Wegg, A., Basler, S., Schafer, M., Egner, U., Carrondo, M. A. J. Biol. Chem. 275 pp. 26164 (2000); ERalpha (1 as2) as disclosed in Tanenbaum, D. M., Wang, Y.,Williams, S. P., Sigler, P. B. Proc Natl Acad Sci USA 95 pp. 5998 (1998); ERbeta (1l2j) as disclosed in Shiau, A. K., Barstad, D., Radek, J. T., Meyers, M. J., Nettles, K. W., Katzenellenbogen, B. S., Katzenellenbogen, J. A., Agard, D. A., Greene, G. L.Nat. Struct. Biol. 9 pp. 359 (2002); TRbeta (1bsx) as disclosed in Wagner, R. L., Darimont, B. D., Apriletti, J. W., Stallcup, M. R., Kushner, P. J., Baxter, J. D., Fletterick, R. J., Yamamoto, K. R. Genes Dev. 12 pp. 3343 (1998); GR, PIR AccessionNumber QRHUGA, as dislcosed in Hollenberg, S. M., Weinberger, C., Ong, E. S.; Cerelli, G., Oro, A., Leba, R., Thompson, E. B., Rosenfeld, M. G., Evans, R. M., Nature (1985) 318: 635-641; MR, PIR Accession Number A29613, as disclosed in Arriza, J. L.;Weinberger, C., Cerelli, G., Glaser, T. M., Handelin, B. L., Housman, D. E., Evans, R. M., Science (1987) 237: 268-275. Orphan nuclear receptors include but are not limited to: Rev Erb (alpha) (1hlz) as disclosed in Sierk, M. L., et. al., Biochemistry(2001) 40: pp. 12833; Pxr (1ilh) as disclosed in Watkins, R. E., et. al., Science (2002) 292: pp. 2329; ERR3 (1 kv6) as disclosed in Greschik, H., et. al., Mol. Cell (2002) 9: pp. 303; Nurrl (1ovl) as disclosed in Wang, Z., et. al., Nature (2003) 423:pp. 555; ERR1 as disclosed in Guiguere, V., et al., Nature (1988) 331: 91-94. Other NHRs, including orphan nuclear receptors, include those disclosed in: The Nuclear Receptor Facts Book, V. Laudet and H. Gronemeyer, Academic Press, p 345, 2002; andFrancis et al, Annu. Rev. Physiol. 2003, 65:261-311.

The terms "steroid hormone receptor" and "SHR," as used herein, refer to a member of the nuclear hormone receptor family of transcription factors which bind steroids and stimulate or repress transcription. SHRs include, but are not limited to,glucocorticoid receptors (GRs), progesterone receptors (PRs), androgen receptors (ARs), estrogen receptors (ERs), mineralocorticoid receptors (MRs), and orphan receptors (i.e. receptors for which the ligands are not yet identified) that bind steroids. These terms, as used herein, refer to steroid hormone receptors from any source, including but not limited to human.

The terms "glucocorticoid receptor" and "GR," as used herein, refer to a member of the nuclear hormone receptor family of transcription factors which bind glucocorticoids and stimulate or repress transcription, and to the GR-beta isoform. Theseterms, as used herein, refer to glucocorticoid receptor from any source, including but not limited to: human glucocorticoid receptor as disclosed in Weinberger, et al. Science 228, p 740-742, 1985, and in Hollenberg, S. M., Weinberger, C., Ong, E. S.,Cerelli, G., Oro, A., Leba, R., Thompson, E. B., Rosenfeld, M. G., Evans, R. M.; Nature (1985) 318: 635-641; rat glucocorticoid receptor as disclosed in Miesfeld, R. Nature, 312, p 779-781, 1985; mouse glucocortoid receptor as disclosed in Danielson, M.et al. EMBO J., 5, 2513; sheep glucocorticoid receptor as disclosed in Yang, K., et al. J. Mol. Endocrinol. 8, p 173-180, 1992; marmoset glucocortoid receptor as disclosed in Brandon, D. D., et al, J. Mol. Endocrinol. 7, p 89-96, 1991; human GR-beta asdisclosed in Hollenberg, S M. et al. Nature, 318, p 635, 1985, Bamberger, C. M. et al. J. Clin Invest. 95, p 2435, 1995; Squirrel (Saimiri boliviensis boliviensis) (GenBank U87951) as disclosed in Reynolds, P. D., Pittler, S. J. and Scammell, J. G. J.Clin. Endocrinol. Metab. 82 (2), 465-472 (1997); Pig GR (GenBank AF141371) as disclosed in Gutscher, M., Eder, S., Mueller, M. and Claus, R. Submitted to GenBank (08-APR-1999) Institut fuer Tierhaltung und Tierzuechtung (470), FG Tierhaltung undLeistungsphysiologie, Universitaet Hohenheim, Garbenstr. 17, Stuttgart 70599, Germany; Guinea Pig (GenBank L13196) as disclosed in Keightley, M. C. and Fuller, P. J. Mol. Endocrinol. 8 (4), 431-439 (1994); Marmoset (GenBank U87953) as disclosed inReynolds, P. D., Pittler, S. J. and Scammell, J. G. J. Clin. Endocrinol. Metab. 82 (2), 465-472 (1997); Ma'z Monkey (GenBank U87952) as disclosed in Reynolds, P. D., Pittler, S. J. and Scammell, J. G. J. Clin. Endocrinol. Metab. 82 (2), 465-472 (1997);rat (GenBank M14053) as disclosed in Miesfeld, R., Rusconi, S., Godowski, P. J., Maler, B. A., Okret, S., Wikstrom, A. C., Gustafsson, J. A. and Yamamoto, K. R. Cell 46 (3), 389-399 (1986); mouse (GenBank X04435) as disclosed in Danielsen, M., Northrop,J. P. and Ringold, G. M. EMBO J. 5 (10), 2513-2522 (1986); Human (Protein Information Resource (PIR) Accession Number QRHUGA) as disclosed in Hollenberg, S. M., Weinberger, C., Ong, E. S., Cerelli, G., Oro, A., Leba, R., Thompson, E. B., Rosenfeld, M.G., Evans, R. M., Nature (1985) 318: 635-641.

The term "binding site," as used herein, refers to a region of a molecule or molecular complex that, as a result of its shape, favorably associates with, i.e. binds, another molecule, such other molecule being a ligand of the binding site. Abinding site, such as Site II, is analogous to a wall and circumscribes a space referred to as a "cavity" or "pocket." The ligand of the binding site situates in the cavity.

The terms "binds" in all its grammatical forms, as used herein, refers to a condition of proximity between or amongst molecules, chemical compounds or chemical entities. The association may be non-covalent (i.e. non-bonded or reversible),wherein the juxtaposition is energetically favored by hydrogen bonding or van der Waals or electrostatic interactions, or it may be covalent (i.e. bonded or irreversible).

The term "soaked," as used herein, refers to a process in which the protein crystal is transferred to a solution containing the compound of interest.

The terms "at least a portion of," "a portion of," "any part of," and "any portion of," in all their grammatical forms, as used herein when referring to Site II, or the structure coordinates of Site II, or the cavity circumscribed by Site II,refer to all or any part of Site II, or the structure coordinates of Site II, or the cavity circumscribed by Site II, wherein Site II is a structure defined by structure coordinates that describe conserved residue backbone atoms having a root mean squaredeviation of not more than 2.0 .ANG. from the conserved residue backbone atoms described by the structure coordinates of amino acids E537-V543, L566, G567, Q570-W577, S599-A607, W610, R611, R614, Q615, P625, Y663, L664 and K667 according to Table I.Preferably the terms relate to a sufficient number of residues or the corresponding structure coordinates so as to be useful in docking or modeling a ligand in the cavity circumscribed by Site II. Preferably, the terms comprise one or more of thefollowing residues or the corresponding structure coordinates: E537-V543, V571-W577, S599-W600, F602-L603, F606-A607, W610, R614, Q615, P625, Y663, L664 and K667. These are the residues of Site II that are not also part of Site I. More preferably, theterms comprise one or more of the following residues or the corresponding structure coordinates: E537-V543, V571-W577, S599-W600, F602-L603, F606-A607, W610, R614, Y663, L664 and K667. Preferably, the terms relate to at least four amino acid residues,more preferably at least five amino acids, more preferably at least eight amino acid residues, more preferably at least fifteen amino acid residues, more preferably at least twenty amino acid residues, more preferably at least twenty-five amino acidresidues, most preferably at least thirty amino acid residues.

The term "mutant," as used herein, refers to a protein, or portion of protein, having one or more amino acid deletions, insertions, inversions, repeats, or substitutions as compared to the relevant native protein or relevant portion of nativeprotein. A native protein is one occurring in nature. A mutant Site II falls within the scope of this invention so long as the rms deviation in conserved residue backbone atoms between such mutant Site II and the the Site II residues according to TableI falls within 2.0 Angstroms. A mutant may have the same, similar, or altered activity as compared to the native protein. Activity refers to transrepression, transactivation, and ligand binding. Preferred mutants have at least 25% sequence identity,more preferably at least 50% sequence identity, more preferably at least 75% sequence identity, and most preferably at least 95% sequence identity to the native protein or portion of native protein.

The term "root mean square deviation" 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 or portion of a protein from the relevant portion of the backbone of another protein, such as the LBD defined by the structure coordinates of Table I.

The term "structure coordinates," "structural coordinates," "atomic coordinates," or "atomic structure coordinates" refers to coordinates that specify the location of the centers of atoms in a protein molecule or molecular complex. The termsinclude, but are not limited to, Cartesian coordinates, polar coordinates, and internal coordinates. The structure coordinates may be generated by any means, including the building of a homology model or derivation from mathematical equations related tothe patterns obtained on diffraction of a monochromatic beam of X-rays by the atoms (scattering centers) of a molecule or molecular complex in crystal form. The diffraction data are used to calculate an electron density map of the repeating unit of thecrystal. The electron density maps are then used to establish the positions of the individual atoms of the molecule or molecular complex.

The term "molecule," as used herein, has the meaning generally used in the art and includes, but is not limited to, proteins, nucleic acids, and chemical compounds, including small organic compounds. "Small organic compounds" are also known as"small organic molecules" or "small molecules."

The term "complex" or "molecular complex," as used herein, refers to a covalent or non-covalent association of a molecule with its ligand.

The term "chemical entity," as used herein, refers to chemical compounds, complexes of at least two chemical compounds, and fragments of such chemical compounds or complexes. A modulator may be a chemical entity. A ligand may be a chemicalentity.

The term "compound," as used herein, refers to a chemical compound.

The term "test molecule," as used herein, refers to a molecule, preferably a chemical compound, that is being tested for specific characteristics.

The term "ligand," as used herein, refers to a molecule that binds to another molecule or portion of another molecule.

The term "modulator," as used herein, refers to a molecule whose presence induces an activity in the molecule that it modulates. A modulator can bind to the molecule that it modulates, i.e. be a ligand of the molecule it modulates. A preferredmodulator is a ligand of the molecule that it modulates. Modulators include, but are not limited to, small organic molecules, chemical compounds, peptides, peptidomimetics (eg., cyclic peptides, peptide analogs, or constrained peptides) and nucleicacids. Modulators can be natural or synthetic. Preferred modulators are small organic molecules.

The term "modeling" in all its grammatical forms, as used herein, refers to the development of a mathematical construct designed to mimic real molecular geometry and behavior in proteins and small molecules. These mathematical constructsinclude, but are not limited to: energy calculations for a given geometry of a molecule utilizing forcefields or ab initio methods known in the art; energy minimization using gradients of the energy calculated as atoms are shifted so as to produce alower energy; conformational searching, ie, locating local energy minima; molecular dynamics wherein a molecular system (single molecule or ligand/protein complex) is propagated forward through increments of time according to Newtonian mechanics usingtechniques known to the art; calculations of molecular properties such as electrostatic fields, hydrophobicity and lipophilicity; calculation of solvent-accessible or other molecular surfaces and rendition of the molecular properties on those surfaces;comparison of molecules using either atom-atom correspondences or other criteria such as surfaces and properties; quantitiative structure-activity relationships in which molecular features or properties dependent upon them are correlated with activity orbio-assay data.

The term "fits spatially" in all its grammatical forms, as used herein, refers to when the three-dimensional structure of a compound is accommodated geometrically by a cavity or pocket of a protein, such as the cavity circumscribed by Site II.

The terms "docking" and "performing a fitting operation," in all their grammatical forms, as used herein, refer to the computational placement of a chemical entity (eg. a potential ligand, preferably a small organic molecule) within a space(i.e. cavity) at least partially enclosed by the protein structure (i.e. binding site) so that structural and chemical feature complementarity (i.e. binding contacts) between chemical entity and binding site components can be assessed in terms ofinteractions typical of protein/ligand complexes. Specifically, the structural and chemical features may include both bonded and non-bonded interactions, and more generally, the non-bonded interactions which occur in the bulk of reversibleprotein/ligand complexes would include forces such as hydrogen-bonding, electrostatic or charge interactions, vander Waal's interactions, and hydrophobic interactions. Such placement could be conducted manually or automatically using software designedfor such purpose.

The term "transrepress" or "transrepression," in all their grammatical forms, is used herein to refer to the process in which an NHR represses transcription by inhibiting a transcription factor or coactivator from inducing transcription. Theterm is not limited to any specific mechanism of action, any specific transcription factor or coactivator, or any specific gene whose transcription is repressed. AP-1 and NF-.kappa.B are two transcription factors, among others, that can be inhibited byan NHR.

The term "transactivate" or "transactivation," in all their grammatical forms, is used herein to refer to the process in which an NHR stimulates transcription, either by binding to DNA and inducing transcription, or by modulating the activity ofanother DNA binding protein that induces transcription. The term is not limited to any specific mechanism of action or any specific gene whose transcription is stimulated.

The term, "NF-.kappa.B-dependent gene expression," as used herein, refers to the expression of those genes that are under the regulatory control of the NF-.kappa.B transcription factor. Such genes include, but are not limited to theimmune-related and inflammatory genes encoding TNF-alpha, IL-1, IL-2, IL-5, adhesion molecules (such as E-selectin), chemokines (such Eoxtaxin and Rantes), and Cox-2.

The term, "AP-1-dependent gene expression," as used herein, refers to the expression of genes that are under the regulatory control of the AP-1 transcription factor. Such genes include, but are not limited to the immune-related and inflammatorygenes encoding TNF-alpha, IL-1, IL-2, IL-5, adhesion molecules (such as E-selectin), chemokines (such Eoxtaxin and Rantes), and Cox-2.

The term "dissociated compound" is used herein to refer to a modulator of an NHR that induces transrepression and induces none to minimal transactivation. The term "dissociated activity" refers to the activity in which a dissociated compoundinduces transrepression and induces none to minimal transactivation.

The term "treat", "treating", or "treatment," in all grammatical forms, as used herein refers to the prevention, reduction, or amelioration, partial or complete alleviation, or cure of a disease, disorder, or condition.

The term "NHR-associated disease," as used herein, refers to a disease or disorder associated with the expression product of a gene whose transcription is stimulated or repressed by an NHR. Stimulation is through transactivation. Repression isthrough transrepression. Such diseases include, but are not limited to, inflammatory and immune associated diseases and disorders, diseases associated with AP-1-dependent gene expression, diseases associated with NF-.kappa.B-dependent gene expression,diseases associated with NHR transrepression, diseases associated with NHR transactivation, diseases treatable by inducing NHR transrepression, and diseases treatable by antagonizing NHR transactivation.

The term "SHR-associated disease," as used herein, refers to a disease or disorder associated with the expression