EP1144997A2 - Methodes et composes de modulation d'activite de recepteurs nucleaires - Google Patents

Methodes et composes de modulation d'activite de recepteurs nucleaires

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Publication number
EP1144997A2
EP1144997A2 EP99916206A EP99916206A EP1144997A2 EP 1144997 A2 EP1144997 A2 EP 1144997A2 EP 99916206 A EP99916206 A EP 99916206A EP 99916206 A EP99916206 A EP 99916206A EP 1144997 A2 EP1144997 A2 EP 1144997A2
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EP
European Patent Office
Prior art keywords
atom
leu
receptors
receptor
nuclear receptor
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EP99916206A
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German (de)
English (en)
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EP1144997A3 (fr
Inventor
Andrew Shiau
Peter J. Kushner
David A. Agard
Geoffrey L. Greene
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University of California
Arch Development Corp
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University of California
Arch Development Corp
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Publication of EP1144997A2 publication Critical patent/EP1144997A2/fr
Publication of EP1144997A3 publication Critical patent/EP1144997A3/fr
Withdrawn legal-status Critical Current

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6875—Nucleoproteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/08—Drugs for genital or sexual disorders; Contraceptives for gonadal disorders or for enhancing fertility, e.g. inducers of ovulation or of spermatogenesis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/14—Drugs for disorders of the endocrine system of the thyroid hormones, e.g. T3, T4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/06—Antiarrhythmics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
    • C07K14/721—Steroid/thyroid hormone superfamily, e.g. GR, EcR, androgen receptor, oestrogen receptor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • G01N33/78—Thyroid gland hormones, e.g. T3, T4, TBH, TBG or their receptors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/72—Assays involving receptors, cell surface antigens or cell surface determinants for hormones
    • G01N2333/723—Steroid/thyroid hormone superfamily, e.g. GR, EcR, androgen receptor, oestrogen receptor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/20—Screening for compounds of potential therapeutic value cell-free systems

Definitions

  • the present invention relates to improved methods and compounds for modulating nuclear receptor activity.
  • the present invention relates to improved methods and compounds for modulating estrogen receptor activity.
  • Ba ⁇ kgrQund Cells contain receptors that can elicit a biological response by binding various molecules including proteins, hormones and/or drugs.
  • Nuclear receptors represent a super family of proteins that are hormone/ligand-activated transcription factors that enhance or repress transcription in a cell type-, ligand- and promoter-dependent manner.
  • a classic nuclear receptor, the estrogen receptor ⁇ (ER ⁇ ) is a key factor in regulating the differentiation and maintenance of neural, skeletal, cardiovascular and reproductive tissues (Korach, Science 266:1524-1527 (1994); Smith, et al, New Engl. I Med- 331:1056-1061 (1994)).
  • the nuclear receptor family also includes receptors for glucocorticoids, androgens, mineralocorticoids, progestins, thyroid hormones, vitamin D, retinoids, peroxisome proliferators and eicosanoids.
  • a subset of the nuclear receptor family are the steroid receptors, which include the estrogen, glucocorticoid and progestin receptors.
  • Overall sequence conservation between nuclear receptors varies between different families of receptors; however, sequence conservation between functional regions, or modules, of the receptors is high. For example, nuclear receptors can be organized into functional modules comprising an N-terminal transcriptional activation domain, a central DNA binding domain (DBD), and a C-terminal ligand binding domain (LBD).
  • DBD central DNA binding domain
  • LBD C-terminal ligand binding domain
  • the LBD of nuclear receptors represents a hormone/ligand-dependent molecular switch, and recognizes a variety of compounds diverse in their size, shape and chemical properties. Accordingly, the estrogen hormones exert their physiological effects by binding to the estrogen receptor (Beato, et al., Cell 83(6):851-857 (1995); Tsai. et al.. Annu. Rev. Biochem. 63:451-86 (1994)). Binding of a hormone to a nuclear receptor's LBD also changes its ability to modulate transcription of DNA, although they may have transcription-independent actions..
  • ER ⁇ ligands bind exclusively to the C-terminal LBD. Some of these ligands. including the endogenous estrogen. 17 ⁇ -estradiol (E 2 ), and the synthetic nonsteroidal estrogen, diethylstilbestrol (DES), function as pure agonists whereas others such as ICI- 164,384 function as pure antagonists.
  • Synthetic ligands such as tamoxifen and raloxifene (RAL) belong to a growing class of molecules known as selective estrogen receptor modulators (SERMs), which function as antagonists in specific tissue and promoter contexts (Grese, et al.. Proc. Natl. Acad. Sci. USA 94:14105-10 (1997)).
  • Nuclear receptors also bind proteins, such as chaperone complexes, corepressors, or coactivators, that are involved in receptor function.
  • proteins such as chaperone complexes, corepressors, or coactivators
  • ligand-dependent activation of transcription by nuclear receptors is mediated by interactions with coactivators.
  • Receptor agonists promote coactivator binding and antagonists block coactivator binding.
  • Hormone binding by a nuclear receptor can increase or decrease binding affinity to these proteins, and can influence or mediate the multiple actions of the nuclear receptors on transcription.
  • AFs activation functions located within different domains of the protein
  • AF-1 in the N-terminus
  • AF- 2 in the LBD
  • AFs can act independently or cooperatively, depending on the cell type and the promoter context.
  • the activity of AF-1 is regulated by growth factors acting through the MAP kinase pathway (Kato. et al.. Science 270:1491-1494 (1995)) and is generally believed to be activated in a ligand-independent manner, while AF-2 activity (“transcriptional activity”) is responsive to ligand binding (Kumar, et al.. Cell 51(6):941-951 (1987)).
  • the binding of agonists triggers transcriptional activity whereas the binding of antagonists does not (Berry, et al., EMBO J. 9:281 1-8 (1990)).
  • coactivators mediate transcriptional activity.
  • SRC-1 Consistent with its proposed role in AF-2 directed transcriptional activation, SRC-1 possesses histone acetylase activity and the ability to interact not only with agonist-bound receptors but also with other coactivators and several general transcription factors (Kamei, et al., Cell 85(3):403-14 (1996); Onate, et al., supra; Spencer, et al., Nature 389: 194-8 (1997); Takeshita, et al., Endocrinology 137:3594-7 (1996)). SRC-1 and GRIPl also bind to the agonist-bound LBDs of both the human TR ⁇ and human ER ⁇ using the putative coactivator binding site (Feng, et al., supra).
  • LXXLL SEQ ID NO:l
  • NR box Ding, et al., Mol. Endocrinol. 12:302-313 (1998); Heery, et al., Nature 387:733-736 (1997); Le Douarin, et al., EMBO J. 15:6701-15 (1996); Torchia, et al., supra).
  • nuclear receptors The medical importance of nuclear receptors is significant. They have been implicated in breast cancer, prostate cancer, cardiac arrhythmia, infertility, osteoporosis, hyperthyroidism, hypercholesterolemia, obesity and other conditions. For example, compounds that modulate ER ⁇ transcriptional activity are currently being used to treat osteoporosis, cardiovascular disease and breast cancer (Gradishar. et al., J. Clin. Oncol. 15:840-52 (1997) and Jordan. J. Natl. Cancer Inst. 90:967-71 (1998)). A need continues to exist for further identification and characterization of the key residues within the ligand binding domains of the nuclear receptors, and molecules that affect the receptor by binding to these sites.
  • the present invention relates to the further identification and manipulation of the ligand binding domain (LBD) of nuclear receptors, which facilitates the design of compounds that bind to the LBD and modulate nuclear receptor activity, and the estrogen receptor in particular.
  • LBD ligand binding domain
  • the compounds include agonists and antagonists that modulate nuclear receptor activity, and can be receptor-, cell- and/or tissue-specific. In particular, the compounds modulate nuclear receptor activity by affecting coactivator-coactivator binding site interactions.
  • the present invention also includes protein cocrystals of the nuclear receptors with an agonist bound to the LBD and a peptide bound to the coactivator binding site and methods for making them.
  • the invention also includes protein cocrystals of the nuclear receptors with an antagonist bound to the LBD and methods for making them.
  • the cocrystals provide means to obtain atomic modeling information of the specific amino acids and their atoms forming the LBD and coactivator binding sites and that interact with molecules that bind to the sites.
  • the cocrystals also provide modeling information regarding the ligand uclear receptor and coactivator uclear receptor interactions, as well the structure of ligands bound thereto.
  • the present invention further provides methods for identifying and designing molecules that modulate ligand binding to a nuclear receptor using atomic models of nuclear receptors.
  • the method involves modeling test compounds that fit spacially into a nuclear receptor LBD using an atomic structural model comprising a nuclear receptor LBD or portion thereof. screening the test compounds in an assay, such as a biological assay, characterized by binding of a test compound to the nuclear receptor LBD. and identifying a test compound that modulates ligand binding to the receptor.
  • the invention also includes compositions and methods for identifying key residues within the LBDs of nuclear receptors.
  • the methods involve examining the surface of a nuclear receptor of interest to identify residues that modulate ligand and/or coactivator binding.
  • the residues can be identified by homology to the key residues within the LBD of human ER ⁇ described herein. Overlays and superpositioning with a three dimensional model of a nuclear receptor's LBD, and/or a portion thereof, also can be used for this purpose. Additionally, alignment and/or modeling can be used as a guide for the placement of mutations on the LBD surface to characterize the nature of the site in the context of a cell.
  • the method can be in vitro or in vivo.
  • the method comprises administering in vitro or in vivo a sufficient amount of a compound that binds to the ligand binding domain and acts either as an agonist or an antagonist.
  • Preferred compounds bind to the site with greater affinity than ligands found in a cell of interest.
  • the invention further includes a method for identifying an agonist or antagonist of ligand binding to a nuclear receptor.
  • the method comprises providing the atomic coordinates comprising a nuclear receptor ligand binding domain or portion thereof to a computerized modeling system; modeling compounds which fit spatially into the nuclear receptor ligand binding domain; and identifying in an assay, for example a biological assay, for nuclear receptor activity a compound that increases or decreases activity of the nuclear receptor through binding the ligand binding domain.
  • a machine-readable data storage medium with information for constructing and manipulating an atomic model comprising the ligand binding domain or portions thereof.
  • the medium comprises a data storage material encoded with machine readable data which, when using a machine programmed with instructions for using said data, is capable of displaying a graphical three-dimensional representation of a molecule or molecular complex for a nuclear receptor ligand binding domain.
  • the method is exemplified by modeling test compounds that fit spatially and preferentially into a nuclear receptor ligand binding domain of interest using an atomic structural model of a nuclear receptor LBD. selecting a compound that interacts with one or more residues of the LBD unique in the context of that site, and identifying in an assay, for example a biological assay, for ligand binding activity a compound that selectively binds to the LBD compared to other nuclear receptors.
  • the unique features involved in receptor-selective ligand binding can be identified by comparing atomic models of different nuclear receptors or isoforms of the same type of receptor.
  • the invention finds use in the selection and characterization of peptide. peptidomimetic. as well as other compounds, such as small organic molecules, identified by the methods of the invention, particularly new lead compounds useful in treating nuclear receptor-based disorders, in particular steroid receptor-based disorders, and more specifically estrogen receptor-based disorders.
  • Figure 1 provides a stereo view of the electron density of the complexes, where Figure 1A is a stereo view of the electron density of the DES-ER ⁇ LBD-GRIP1 NR Box II peptide complex and Figure IB is a stereo view of the electron density of the OHT-ER ⁇ LBD complex.
  • Figure 1 is a black and white graphical representation of a figure that was generated using BOBSCRIPT (Esnouf, J. Mol. Graph. Model. 15, 132-4, 1 12-3 (1997)) and rendered using Raster3D (Merritt, et al., Acta Crvstallogr. D 50:869-873 (1994)).
  • Figure 2 was generated using BOBSCRIPT and rendered using Raster3D as described above.
  • Figure 2A shows the overall structure of the DES-ER ⁇ LBD-GRIP1 NR Box II peptide complex in two orthogonal views.
  • Figure 2B shows the overall structure of the OHT-ER ⁇ LBD complex in two orthogonal views similar to those of the agonist complex in Figure 2A.
  • Figures 3A and 3B were generated using BOBSCRIPT and rendered using Raster3D as described above.
  • Figures 3C and 3D were created using GRASP (Nicholls. GRASP Manual (New York: Columbia University, 1992)).
  • Figure 3 A shows a close-up view of the coactivator peptide bound to the LBD, i.e., the NR Box II peptide/LBD interface. The regions of the LBD that do not interact with the peptide have been omitted for clarity.
  • Helices 3. 4 and 5 are labeled H3. H4 and H5 respectively.
  • the side chains of receptor residues which interact with the peptide are depicted, except for Lys 362 (blue) and Glu 542 (red), the side chains are colored by atom type (carbon and sulfur atoms are colored green, oxygen atoms are colored red and nitrogen atoms are colored blue).
  • Helix 12 is colored magenta.
  • the peptide. colored gold, is depicted as a C ⁇ worm; only the side chains of He 689 and the three motif leucines (Leu 690, Leu 693 and Leu 694) are drawn (Figure 3C).
  • Figure 3B shows the helix 12/LBD interface as a close-up view of the OHT-LBD complex showing helix 12 bound to part of the coactivator binding site.
  • the coactivator peptide is depicted as in Figure 3A and the view is equivalent to that in Figure 3 A.
  • the side chains of Leu 690 and Leu 694 are bound in a hydrophobic groove and those of He 689 and Leu 693 rest against the edge of this groove.
  • Figure 3D shows the electrostatic surface of the ER ⁇ LBD complexed with OHT, showing positive (blue) and negative (red) regions as in Figure 3C.
  • Residues 530-51 1 are depicted as in Figure 3B and the view is equivalent to that in Figure 3B.
  • the side chains of Leu 540 and Leu 544 are embedded in the hydrophobic groove, that of Met 543 lies along the edge of this groove.
  • Figure 4 was generated using LIGPLOT (Wallace, et al.. Protein Eng. 8:127-34 (1995)) and provides schematic diagrams illustrating the DES interactions with the LBD ( Figure 4A) and OHT interactions with the ligand binding pocket ( Figure 4B). Residues that interact with the ligands are drawn at approximately their true positions. The residues that form van der Waals contacts with ligand are depicted as labeled arcs with radial spokes that face towards the ligand atoms with which they interact. The residues that hydrogen bond to ligand are shown in ball- and-stick representation. Hydrogen bonds are represented as dashed cyan lines and the distance of each bond is given. The ligand rings and the individual ligand atoms are labeled.
  • Figure 5 was generated using BOBSCRIPT and rendered using Raster3D as described above, and shows a comparison of helix 12 from the OHT complex and the NR Box II peptide.
  • the hydrogen bonds between the ⁇ -amino group of Lys 362 and the backbone carbonyls of residues 543 and 544 of helix 12 are illustrated as dashed magenta lines.
  • the hydrogen bonds between the ⁇ -amino group of Lys 362 and the backbone carbonyls of residues 693 and 696 of the coactivator peptide are depicted as dashed orange lines.
  • L540 Leu 540.
  • M543 Met 543
  • L544 Leu 544.
  • Figures 6A and 6D were generated using BOBSCRIPT and rendered using Raster3D as described above.
  • Figures 6B and 6C were created using MidasPlus (Huang, et al.. J. Mol. Graph. 9:230-6. 242 (1991)).
  • Figure 6A shows that agonists and antagonists promote different LBD conformations, as ribbon representations of the DES complex (without the coactivator peptide), the OHT complex and the E 2 complex such as is described in Tanenbaum, et al., supra. The hormones are shown in space-filling representation. In each complex, helix 12 is colored magenta and the main chain of residues 339 to 341, 421 to 423, and 527 to 530 is indicated in red.
  • FIG. 6B shows DES bound in the ligand binding cavity.
  • the A' ring of DES (A'), Phe 404 (404), Met 421 (421 ) and Phe 425 (425) are labeled.
  • the carbon atoms of side chain of Met 421 are colored magenta, and the sulfur atom is colored yellow.
  • Figure 6C is a cross-section of a space-filling model of OHT (red) bound in the ligand binding pocket. The view is equivalent to that in Figure 6B.
  • the B ring of OHT (B), Phe 404 (404), Met 421 (421) and Phe 425 (425) are labeled.
  • the side chain of Met 421 is colored as in Figure 6B.
  • the conformation of the B ring forces Met 421 to adopt a different conformation than in the one it adopts in the DES complex (compare with Figure 6B).
  • Figure 6D provides a comparison of the ligand binding pocket bound to DES (green) and to OHT (red).
  • the structures of the OHT complex and the DES complex were overlapped as in Figure 5.
  • the A rings of both ligands point out of the page; the B ring of OHT and the A' ring of DES point into the page.
  • the LBD bound to OHT is colored blue and the LBD bound to DES is colored light blue-grey.
  • the side chains of some of the residues whose conformations are dramatically different between the two complexes are drawn; Met 342 (342), Met 343 (343), Phe 404 (404), Met 421 (421), He 424 (424), Phe 425 (425), His 524 (524), Leu 525 (525), Met 528 (528).
  • the sulfur atom of Met 421 is colored yellow in both structures.
  • Figure 7 illustrates a model of antagonist action. Agonist (white triangle) binding stabilizes a conformation of the LBD that promotes coactivator (yellow) binding.
  • Residues 527- 530 are part of helix 11 (blue) and the length of the interhelical loop prevents helix 12 (magenta) from binding to the static region of the surface involved in transcriptional activity. Antagonist (white cross) side chains preclude helix 12 from being positioned over the ligand binding pocket. Residues 527-530 (red) adopt an extended conformation as a result of antagonist-driven structural perturbations in and around the ligand binding pocket. The length of the loop between helices 1 1 and 12 allows helix 12 to bind the static region of this surface and inhibit coactivator recognition.
  • Figure 8 shows alignment of amino acid sequences (single letter amino acid designations) containing residues that form the coactivator binding sites of several nuclear receptors: human and recombinant thyroid hormones (hTR ⁇ and rTR ⁇ ) (SEQ ID NO:5 and 6 and SEQ ID NO:7 and 8), retinoids (hRAR ⁇ and hRXR ⁇ ) (SEQ ID NO:9 and 10 and SEQ ID NO:l 1 and 12), peroxisome (hPPAR ⁇ ) (SEQ ID NO: 13 and 14), vitamin D (hVDR) (SEQ ID NO: 15 and 16), estrogen (hER ⁇ ) (SEQ ID NO: 17 and 18), glucocorticoid (hGR) (SEQ ID NO: 19 and 20), progestin (hPR) (SEQ ID NO:21 and 22), mineralocorticoid (hMR) (SEQ ID NO:23 and 24) and androgen (hAR) (SEQ ID NO:25 and 26).
  • the boxes represent residues of alpha-helix (H3, H4, H5, H6 and HI 2); lower case letters “h” and "q” represent hydrophobic and polar residues, respectively.
  • the present invention provides methods and compositions for identifying compounds that modulate nuclear receptor activity, in particular steroid receptor activity, and more particularly estrogen receptor activity.
  • the compounds are nuclear receptor agonists or antagonists that bind to the ligand binding domain.
  • Compounds that bind to the LBD also are provided.
  • the compounds can be natural or synthetic.
  • Preferred compounds are small organic molecules, peptides and peptidomimetics (e.g., cyclic peptides, peptide analogs, or constrained peptides).
  • ER ⁇ Tyr537 residue plays a role in stabilizing the unliganded receptor so that helix 12 is free to interact with the coactivator binding site.
  • the ER is quite unique in having a tvrosine at this position as hTR ⁇ , rTR ⁇ , hRAR ⁇ , hGR.
  • hPR, hMR and hAR all have a proline residue.
  • hRXR ⁇ has as aspartic acid residue
  • hPPAR ⁇ has a histidine residue
  • hVDR has a threonine residue at positions corresponding to the Tyr 537 residue of hER ⁇ . Therefore, selective agonists and antagonists can be designed for the estrogen receptor that interact with Tyr 537.
  • the unwinding of helix 11 increases the length of the loop between helices 1 1 and 12, allowing helix 12 to move away from the ligand binding pocket and towards the coactivator binding site, where it occludes the coactivator recognition groove by mimicking the interactions of the coactivator, and thus inhibits coactivator recognition (see Figure 7).
  • Modifications to a ligand that interfere with binding or interaction with one or more of the amino acids positions indicated would cause receptor relaxation, affecting the receptor's secondary structure and cause the unwinding of helix 12. Compounds based upon such modified ligands would act as antagonists.
  • one aspect of the invention is a method of identifying a compound that modulates (i.e., increases or decreases) nuclear receptor activity, comprising: modeling test compounds that fit spatially into a nuclear receptor ligand binding domain of interest using an atomic structural model of the estrogen receptor ⁇ ligand binding domain or portion thereof, screening the test compounds in an assay, for example a biological assay, characterized by binding of a test compound to the ligand binding domain, and identifying a test compound that modulates nuclear receptor activity, wherein the atomic structural model comprises atomic coordinates of amino acid residues corresponding to residues of human estrogen receptor ⁇ Met343, Leu346. Ala350. Glu353. Leu384. Leu387. Leu391, Arg394, Phe404.
  • the nuclear receptor is the ER.
  • the test compound can be an agonist and nuclear receptor activity is measured by binding of a coactivator or a compound that mimics a coactivator, to the coactivator binding site, as defined below.
  • the test compound can be an antagonist and nuclear receptor activity is measured by the unwinding of helix 12 and/or the blocking of coactivator binding to the coactivator binding site. The screening is typically in vitro, and high throughput screening is preferable.
  • test compounds can be designed, as is described later, or can be obtained from a library of compounds, and include, by means of illustration and not limitation, small organic molecules, peptides and peptidomimetics.
  • the method described above may also include the step of providing the atomic coordinates of the estrogen receptor ⁇ ligand binding domain or portion thereof to a computerized modeling system, prior to said modeling step.
  • portion thereof is intended to mean the atomic coordinates corresponding to a sufficient number of residues or their atoms of the LBD that interact with a compound capable of binding to the site. This includes receptor residues having an atom within 4.5A of a bound compound or fragment thereof.
  • the atomic coordinates provided to the modeling system can contain atoms of the nuclear receptor LBD, part of the LBD such as atoms corresponding to the LBD or a subset of atoms useful in the modeling and design of compounds that bind to a LBD.
  • the atomic coordinates of a compound that fits into the ligand binding domain also can be used for modeling to identify compounds or fragments that bind the site.
  • modeling is intended quantitative and qualitative analysis of molecular structure/function based on atomic structural information and receptor-ligand agonists/antagonists interaction models. This includes conventional numeric-based molecular dynamic and energy minimization models, interactive computer graphic models, modified molecular mechanics models, distance geometry and other structure-based constraint models. Modeling is preferably performed using a computer and may be further optimized using known methods.
  • fit spatially is intended that the three- dimensional structure of a compound is accommodated geometrically by a cavity or pocket of a nuclear receptor LBD. It is expected that targeting the corresponding amino acids on other nuclear receptors will have the same effect.
  • one embodiment of the invention pertains to methods of designing antagonists that bind the LBD of a nuclear receptor but do not interact with one of more residues within the LBD that correspond to (i.e.. the same as or equivalent to) human ER ⁇ residues Met343.
  • another embodiment of the invention pertains to methods of designing agonists that bind the LBD of a nuclear receptor and have enhanced interaction with one or more residues within the LBD that correspond to the human ER ⁇ residues Met343, Leu346. Ala350. Glu353, Leu384. Leu387, Leu391, Arg394, Phe404. Met421 , Leu428, Gly521, His524, Leu525 and Met528, preferably Met343, Met421, His524, Leu525 and Met528.
  • An example of enhanced interaction is where the agonist has a greater binding affinity for one or more of said residues, as compared to an endogenous ligand. Such corresponding positions for other members of the nuclear receptor family are shown in Table 1.
  • corresponding amino acid residues of other nuclear receptors such as other estrogen receptors, thyroid receptors, retinoid receptors, glucocorticoid receptors, progestin receptors, mineralocorticoid receptors, androgen receptors, peroxisome receptors and vitamin D receptors, may also be used in the methods of the invention.
  • coactivator binding site is used herein to mean a structural segment or segments of the nuclear receptor polypeptide chain folded in such a way so as to give the proper geometry and amino acid residue conformation for binding coactivator. This is the physical arrangement of protein atoms in three-dimensional space forming a coactivator binding site
  • residues forming the coactivator binding site on nuclear receptors are amino acids that correspond to (i.e.. the same as or equivalent to) human TR residues of C-terminal helix 3 (Ile280. Thr281. Val283. Val284, Ala287. and Lys288), helix 4 (Phe293). helix 5 (Gln301, Ile302. Leu305. Lys306), helix 6 (Cys309). and helix 12 (Pro453. Leu454. Glu457, Val458 and Phe459), as shown in Figure 8.
  • the coactivator binding site is highly conserved among the nuclear receptor super family.
  • this site corresponds to a surprisingly small cluster of residues on the surface of the LBD that form a prominent hydrophobic cleft.
  • the hydrophobic cleft is formed by hydrophobic residues corresponding to human TR residues of C-terminal helix 3 (Ile280. Val283. Val284, and Ala287), helix 4 (Phe293), helix 5 (Ile302 and Leu305), helix 6 (Cys309), and helix 12 (Leu454, Val458 and Phe459).
  • This hydrophobic cleft of the coactivator binding site is also highly conserved among the nuclear receptor super family.
  • residues forming the coactivator binding site on the estrogen receptor were found to correspond to those positions described above for the human TR. Accordingly, the residues forming the coactivator binding site on ER ⁇ are the human ER ⁇ residues of C-terminal helix 3 (Leu354, Val355, Met357, Ile358. Ala361, and Lys362), helix 4 (Phe367), helix 5 (Gln375, Val376, Leu379, Glu380), helix 6 (Trp383), and helix 12 (Asp538, Leu539, Glu542, Met543 and Leu544), as shown in Figure 8.
  • this site corresponds to residues on the surface of the LBD that form a prominent hydrophobic cleft, formed by hydrophobic residues corresponding to human ER ⁇ residues of C-terminal helix 3 (Leu354, Met357, Ile358 and Ala361 ), helix 4 (Phe367), helix 5 (Val376, Leu379), helix 6 (Trp383), and helix 12 (Leu539. Met543 and Leu544).
  • This corroborates the data presented by Apriletti, et al., supra, for the nuclear receptor family.
  • coactivator mimics are peptides or polypeptides that mimic the coactivator binding site recognition area on the surface of a coactivator such that a "coactivator mimic” acts as a competitive inhibitor of coactivator binding to the coactivator binding site.
  • Coactivator mimics can be used in an assay to determine receptor activity and hence the agonist or antagonist nature of a test compound, in that an agonist will permit a coactivator mimic to bind to the coactivator binding site, while an antagonist will prevent such binding.
  • coactivator mimics may have therapeutic utility when administered in combination with an agonist compound of the invention.
  • Another embodiment of the invention pertains to a method of identifying a compound that modulates ligand binding to a nuclear receptor, typically by binding to the ligand binding domain.
  • This method comprises the steps of modeling test compounds that fit spatially into a nuclear receptor ligand binding domain of interest using an atomic structural model of the estrogen receptor ⁇ ligand binding domain or portion thereof, screening the test compounds in an assay characterized by binding of a test compound to the binding domain, and identifying a test compound that modulates ligand binding to said nuclear receptor, wherein said atomic structural model comprises atomic coordinates of amino acid residues corresponding to residues of human estrogen receptor ⁇ Met343, Leu346, Ala350, Glu353, Leu384, Leu387, Leu391, Arg394, Phe404, Met421, Leu428, Gly521, His524, Leu525 and Met528, preferably Met343.
  • the nuclear receptor is ER, TR, GR or PR.
  • the screening is typically in vitro such as by high throughput screening.
  • Suitable test compounds can be designed or obtained from a library of compounds, and include, by means of illustration and not limitation, small organic molecules, peptides and peptidomimetics.
  • the test compounds can be either agonists or antagonists of ligand binding.
  • the invention also includes compositions and methods for identifying key residues within the ligand binding domains of nuclear receptors. The methods involve examining the surface of a nuclear receptor of interest to identify residues that modulate ligand and/or coactivator binding.
  • the residues can be identified by homology to the key residues on the LBD of human ER ⁇ described herein.
  • a preferred method is alignment with the residues of any nuclear receptor corresponding to (i.e., equivalent to) human ER ⁇ residues of Met343, Leu346, Ala350, Glu353, Leu384, Leu387, Leu391, Arg394. Phe404, Met421, Leu428. Gly521. His524, Leu525 and Met528, preferably Met343, Met421. His524. Leu525 and Met528.
  • Overlays and superpositioning with a three-dimensional model of a nuclear receptor LBD, or a portion thereof that contains these or corresponding residues, also can be used for this purpose. For example.
  • nuclear receptors identifiable by homology alignment include normal nuclear receptors or proteins structurally related to nuclear receptors found in humans, natural mutants of nuclear receptors found in humans, normal or mutant receptors found in animals, as well as non- mammalian organisms such as pests or infectious organisms, or viruses.
  • Alignment and/or modeling also can be used as a guide for the placement of mutations on the LBD surface to characterize the nature of the site in the context of a cell.
  • Selected residues are mutated to preserve global receptor structure and solubility in the case of an agonist, or to disassemble such structure and permit helix 12 to unwind, as is the case with an antagonist.
  • Mutants can be tested for ligand binding as well as the relative change in strength of the binding interaction.
  • Ligand-dependent coactivator interaction assays also can be tested for this purpose, such as those described herein.
  • the present invention relates to the structural and functional effects on the estrogen receptor's LBD. of the binding of two chemically-related compounds, the agonist, diethylstilbestrol (DES).
  • DES diethylstilbestrol
  • ER ⁇ LBD human estrogen receptor ⁇ ligand binding domain
  • SEQ ID NO:4 GRIPl NR Box II peptide sequence
  • the Examples provide the 2.03 A resolution crystal structure of the hER ⁇ LBD bound to DES and the coactivator and the 1.9A x-ray crystal structure of the hER ⁇ LBD bound to OHT, i.e.. the crystals defract with at least 2.03A or 1.9A resolution, respectively.
  • compounds of interest are discovered, i.e., agonists or antagonists of ligand binding are identified, by a method for identifying an agonist or antagonist of ligand binding to a nuclear receptor.
  • the method comprises the steps of providing the atomic coordinates of the ER ⁇ LBD or portion thereof to a computerized modeling system, modeling compounds which fit spatially into the LBD, and identifying in an assay for nuclear receptor activity a compound which increases or decreases the activity of the nuclear receptor by binding the LBD of the nuclear receptor.
  • the atomic coordinates are of the amino acid residues corresponding to residues of human estrogen receptor ⁇ Met343. Leu346, Ala350. Glu353, Leu384. Leu387. Leu391. Arg394. Phe404. Met421. Leu428, Gly521, His524, Leu525 and Met528. preferably Met343. Met421, His524. Leu525 and Met528.
  • the present invention also includes a method for identifying a compound capable of selectively modulating nuclear receptor activity.
  • the method comprises the steps of modeling test compounds that fit spatially and preferentially into the LBD of a nuclear receptor of interest using an atomic structural model of a nuclear receptor, screening the test compounds in an assay for nuclear receptor activity characterized by preferential binding of a test compound to the LBD of a nuclear receptor, and identifying a test compound that selectively modulates the activity of a nuclear receptor.
  • Such receptor-specific compounds are selected that exploit differences between the LBDs of one type of nuclear receptor versus a second type of nuclear receptor.
  • the invention also is applicable to generating new compounds that distinguish nuclear receptor isoforms. This can facilitate generation of either tissue-specific or function-specific compounds.
  • GR subfamily members have usually one receptor encoded by a single gene, although there are exceptions.
  • PR isoforms A and B, translated from the same mRNA by alternate initiation from different AUG codons.
  • This method is especially applicable to the ER subfamily which usually has several receptors that are encoded by at least two (ER: ⁇ , ⁇ ) genes or have alternate RNA splicing.
  • the receptor-specific compounds of the invention preferably interact with conformationally constrained residues of the LBD that are conserved among one type of nuclear receptor compared to a second type of nuclear receptor.
  • Conformationally constrained is intended to refer to the three-dimensional structure of a chemical or moiety thereof having certain rotations about its bonds fixed by various local geometric and physical-chemical constraints.
  • Conformationally constrained structural features of a LBD include residues that have their natural flexible conformations fixed by various geometric and physical-chemical constraints, such as local backbone, local side chain, and topological constraints. These types of constraints are exploited to restrict positioning of atoms involved in receptor-coactivator recognition and binding.
  • the present invention also provides for a computational method using three dimensional models of nuclear receptors based on crystals of nuclear receptors.
  • the computational method of designing a nuclear receptor ligand determines which amino acid or amino acids of a nuclear receptor LBD interact with a chemical moiety (at least one) of the ligand using a three dimensional model of a crystallized protein comprising a nuclear receptor LBD with a bound ligand, and selecting a chemical modification (at least one) of the chemical moiety to produce a second chemical moiety with a structure that either decreases or increases an interaction between the interacting amino acid and the second chemical moiety compared to the interaction between the interacting amino acid and the chemical moiety.
  • crystal structures of the hER ⁇ with DES/peptide and with OHT have shown that amino acid residues that correspond to hER ⁇ Met343.
  • one embodiment of the invention is a computational method of designing a nuclear receptor ligand where at least one amino acid residue of a nuclear receptor LBD that corresponds to human estrogen receptor ⁇ Met343, Leu346, Ala350, Glu353, Leu384, Leu387, Leu391, Arg394. Phe404, Met421, Leu428, Gly521, His524, Leu525 and Met528, preferably Met343, Met421.
  • His524, Leu525 and Met528, interacts with at least one first chemical moiety of the ligand, comprising the step of selecting at least one chemical modification of the first chemical moiety to produce a second chemical moiety with a structure to either decrease or increase an interaction between the interacting amino acid and the second chemical moiety as compared to the interaction between the interacting amino acid and the first chemical moiety.
  • This computational method may further comprise determining a change in interaction between the interacting amino acid and the ligand after chemical modification of the first chemical moiety.
  • the chemical modification can either enhance or reduce hydrogen bonding interaction, charge interaction, hydrophobic interaction. Van Der Waals interaction or dipole interaction between the second chemical moiety and the interacting amino acid as compared to the interaction between the first chemical moiety and the interacting amino acid.
  • Chemical modifications will often enhance or reduce interactions an atom of a LBD amino acid and an atom of an LBD ligand. Steric hindrance will be a common means of changing the interaction of the LBD binding cavity with the activation domain.
  • Chemical modifications are preferably introduced at C-H, C- and C-OH position in ligands. where the carbon is part of the ligand structure which remains the same after modification is complete. In the case of C-H, C could have 1. 2 or 3 hydrogens, but usually only one hydrogen will be replace. The H or OH are removed after modification is complete and replaced with the desired chemical moiety. Such chemical modifications would preferably involve the addition of substituents.
  • substituents are hydrophobic groups, including by way of example and not limitation, alkyl groups such as ethyl, propyl, isopropyl, etc., and aromatic groups such as benzyl, etc.
  • a known ligand for the nuclear receptor of interest as the chemical backbone, upon which to base agonist/antagonist design.
  • the known ligand would be modified as described above.
  • 17 ⁇ -estradiol is an endogenous ligand for the hER ⁇ .
  • positions of interest are C6 ⁇ , C7 ⁇ , C 12 ⁇ , C 15 ⁇ , C 16 ⁇ and C 17 ⁇ .
  • Modifications at one or more of these free carbons on 17 ⁇ -estradiol's backbone would affect the ligand's interactions with one or more of the Met343, Leu346, Ala350, Glu353. Leu384, Leu387, Leu391, Arg394, Phe404, Met421, Leu428, Gly521, His524, Leu525 and Met528, preferably Met343, Met421, His524, Leu525 and Met528 residues, either providing for enhancing interaction, which would be the basis for agonist design, or reduced interaction, which would be the basis for antagonist design.
  • Known agonists include diethylstilbestrol (synthetic), moxestrol (synthetic), mesohexestrol (synthetic), coumestrol (clover), ⁇ 9 -THC (cannabis), o,p-DDT (insecticide), zearalenone (fungal) and kepone (insecticide).
  • Known estrogen receptor antagonists include the ICI series of modified steroids such as ICI 164,384 and EM800.
  • Known SERM's include tamoxifen. raloxifene and GW5638.
  • agonists could be positioned in the ligand binding pocket through computational or manual docking. Positions for substitution would then be selected based on the predicted binding orientation of these compounds.
  • hybrid molecules could be generated that also possessed side chains that prevented helix 12 from adopting the agonist- bound position. Novel SERMs can be produced by varying the strength of two different effects: the helix 12 displacement and the secondary structure disorganization.
  • docking algorithms and computer programs that employ them can be used to identify compounds that fit into the ligand binding domain.
  • docking programs can be used to predict how a molecule of interest can interact with the nuclear receptor LBD.
  • Fragment-based docking also can be used in building molecules de novo inside the LBD, by placing chemical fragments that complement the site to optimize intermolecular interactions. The techniques can be used to optimize the geometry of the binding interactions. This design approach has been made possible by identification of the LBD structure thus, the principles of molecular recognition can now be used to design a compound which is complementary to the structure of this site.
  • Compounds fitting the LBD serve as a starting point for an iterative design, synthesis and test cycle in which new compounds are selected and optimized for desired properties including affinity, efficacy, and selectivity.
  • the compounds can be subjected to addition modification, such as replacement and/or addition of R-group substituents of a core structure identified for a particular class of binding compounds, modeling and/or activity screening if desired, and then subjected to additional rounds of testing.
  • Computationally small molecule databases can be screened for chemical entities or compounds that can bind in whole, or in part, to a nuclear receptor ligand binding domain of interest. In this screening, the quality of fit of such entities or compounds to the binding site may be judged either by shape complementarity (DesJalais, et al., J. Med. Chem. 31 :722-729 (1988)) or by estimated interaction energy (Meng, et al., J. Comp. Chem. 13:505-524 (1992)).
  • the molecule databases include any virtual or physical database, such as electronic and physical compound library databases, and are preferably used in developing compounds that modulate coactivator binding.
  • Compounds can be designed intelligently by exploiting available structural and functional information by gaining an understanding of the quantitative structure-activity relationship (QSAR), using that understanding to design new compound libraries, particularly focused libraries having chemical diversity of one or more particular groups of a core structure. and incorporating any structural data into that iterative design process.
  • QSAR quantitative structure-activity relationship
  • one skilled in the art may use one of several methods to screen chemical entities or fragments for their ability to associate with the ligand binding domain of a nuclear receptor of interest. This process may begin by visual inspection of. for example, the LBD on the computer screen. Selected fragments or chemical entities may then be positioned into all or part of the site. Docking may be accomplished using software such as Quanta and Sybyl, followed by energy minimization and molecular dynamics with standard molecular mechanics force-fields, such as CHARMM and AMBER.
  • Specialized computer programs may also assist in the process of selecting chemical entity fragments or whole compounds. These include: GRID (Goodford. J. Med. Chem. 28:849-857 (1985), available from Oxford University, Oxford, UK); MCSS (Miranker. et al., "Proteins: Structure, Function and Genetics” 1 1 :29-34 (1991), available from Molecular Simulations, Burlington. MA); AUTODOCK (Goodsell, et al., "Proteins: Structure, Function and Genetics” 8:195-202 (1990), available from Scripps Research Institute, La Jolla, CA); and DOCK (Kuntz, et al, J. Mol. Biol. 161 :269-288 (1982), available from University of California, San Francisco, CA).
  • CAVEAT Bartlett, et al., "CAVEAT: A Program to Facilitate the Structure-Derived Design of Biologically Active Molecules", in Molecular Recognition in Chemical and Biological Problems, Special Pub., Royal Chem. Soc. 78:182-196 (1989), available from the University of California. Berkeley, CA); 3D Database systems such as MACCS-3D (MDL Information Systems. San Leandro. CA. reviewed in Martin. J. Med. Chem. 35:2145-2154 (1992)); and HOOK (available from Molecular Simulations. Burlington. MA).
  • CAVEAT Bartlett, et al., "CAVEAT: A Program to Facilitate the Structure-Derived Design of Biologically Active Molecules", in Molecular Recognition in Chemical and Biological Problems, Special Pub., Royal Chem. Soc. 78:182-196 (1989), available from the University of California. Berkeley, CA
  • 3D Database systems such as MACCS-3D (MDL Information Systems.
  • compounds that bind to a ligand binding domain of interest also may be designed as a whole or de novo using either an empty LBD or optionally including some portion(s) of a molecule known to bind to the site, such as a known ligand.
  • LUDI Bohm. J. Comp. Aid. Molec. Design 6:61-78 (1992), available from Biosm Technologies, San Diego, CA
  • LEGEND (Nishibata. et al., Tetrahedron 47:8985 (1991), available from Molecular Simulations, Burlington. MA); and LeapFrog (available from Tripos Associates, St. Louis, MO).
  • Compounds identified through modeling can be screened in assays such as are well known in the art.
  • assays which include biological assays, are characterized by binding of the compound to a ligand binding domain of interest for ligand binding activity. Screening can be, for example, in vitro, in cell culture, and/or in vivo.
  • Biological screening preferably centers on activity-based response models, binding assays (which measure how well a compound binds to the receptor), and bacterial, yeast and animal cell lines (which measure the biological effect of a compound in a cell).
  • the assays can be automated for high capacity-high throughput screening (HTS) in which large numbers of compounds can be tested to identify compounds with the desired activity.
  • HTS high capacity-high throughput screening
  • in vitro binding assays can be performed in which compounds are tested for their ability to block the binding of a ligand. fragment, fusion or peptide thereof, to a ligand binding domain of interest.
  • a compound's ability to block function of cellular coactivators such as members of the pi 60 family of coactivator proteins, such as SRC-1, AIB1, RAC3.
  • compounds of the invention bind to a ligand binding domain with greater affinity than the endogenous ligands.
  • Tissue profiling and appropriate animal models also can be used to select compounds. Different cell types and tissues also can be used for these biological screening assays. Suitable assays for such screening are described herein and in Shibata, et al.. Recent Prog. Horm. Res. 52: 141-164 (1997); Tagami, et al., Mol. Cell. Biol. 17(5):2642-2648 (1997); Zhu, et al., J. Biol- Chem. 272(14):9048-9054 (1997); Lin, et al., Mol- Cdl- Biol. 17(10):6131-6138 (1997); Kakizawa, et al., J. BjoJ- Chem.
  • the compounds selected can have agonist and/or antagonistic properties.
  • the compounds also include those that exhibit new properties with varying mixtures of agonist and antagonist activities, depending on the effects of altering ligand binding in the context of different activities of nuclear receptors, either hormone-dependent or hormone-independent, which are mediated by proteins other than coactivators, and which interact with the receptors at locations other than the coactivator binding site.
  • the compounds also include those, which through their binding to receptor locations that are conformationally sensitive to hormone binding, have allosteric effects on the receptor by stabilizing or destabilizing the hormone-bound conformation of the receptor, or by directly inducing the same, similar, or different conformational changes induced in the receptor by the binding of hormone.
  • a method of modulating nuclear receptor activity in a mammal by administering to a mammal in need thereof a sufficient amount of a compound that fits spatially and preferentially into a ligand binding domain of a nuclear receptor of interest.
  • modulating is intended increasing or decreasing activity of a nuclear receptor.
  • pre-clinical candidate compounds can be tested in appropriate animal models in order to measure efficacy, absorption, pharmacokinetics and toxicity following standard techniques known in the art. Compounds exhibiting desired properties are then tested in clinical trials for use in treatment of various nuclear receptor-based disorders.
  • ER-based disorders such as postmenopausal symptoms and cancer resulting from loss of estrogen production, and osteoporosis and cardiovascular disease stemming from traditional estrogen replacement therapy.
  • Others include GR-based disorders including Type II diabetes and inflammatory conditions such as rheumatic diseases.
  • a compound will have more than one of these traits, i.e., a compound will act as an agonist in one tissue, while acting as an antagonist in another tissue.
  • tissue-selective antagonism of SERMs such as OHT and RAL is the result of numerous factors (Grainger, et al.. Nature Medicine 2(4):381-385 (1996); Grese, et al., supra; and Jordan, J. Natl. Cancer Inst. 90:967-71 (1998))
  • dissection of the mechanisms of action of these ligands requires a comprehensive understanding of how they act on the LBD and regulate its interactions with other cellular factors.
  • the instant invention shows, unexpectedly, that ligand-mediated structural perturbations in and around the ligand binding pocket, and not simply side chain effects, contribute to receptor antagonism. Accordingly, by adjusting the balance between these two effects provides a novel strategy for the design of improved SERMs.
  • one aspect of the invention is a method of modulating nuclear receptor activity in a mammal by administering to a mammal in need thereof a sufficient amount of a ligand that fits spatially and preferentially into a ligand binding domain of a nuclear receptor of interest, wherein the ligand is designed by a computational method where at least one amino acid residue of a nuclear receptor LBD that corresponds to hER ⁇ Met343, Leu346.
  • Such a method involves selecting at least one chemical modification of the first chemical moiety to produce a second chemical moiety with a structure that either decreases or increases an interaction between the interacting amino acid and the second chemical moiety as compared to the interaction between the interacting amino acid and the first chemical moiety.
  • Compounds designed by this method can be either agonists or antagonists and the method of modulating nuclear receptor activity can comprise administering an antagonist alone, an agonist alone or an agonist in combination with a coactivator or a compound that mimics a coactivator by binding to the coactivator binding site.
  • the coactivator can be a known coactivator.
  • the coactivator mimic can be designed by a computational method where at least one amino acid residue of a nuclear receptor coactivator binding site that corresponds to hER ⁇ helix 3 residues Leu354, Val355, Met357, Ile358, Ala361 and Lys362, helix 4 residue Phe367, helix 5 residues Gln375, Val376, Leu379 and Glu380, helix 6 residue Trp383. and helix 12 residues Asp538, Leu539, Glu542, Met543 and Leu544, interacts with at least one first chemical moiety of the coactivator mimic.
  • the method involves selecting at least one chemical modification of the first chemical moiety to produce a second chemical moiety with a structure that either decreases or increases an interaction between the interacting amino acid and the second chemical moiety as compared to the interaction between the interacting amino acid and the first chemical moiety.
  • coactivator mimics can be designed to bind into the site involved in transcriptional activity only when helix- 12 is in its agonist bound state. If such coactivator mimics are specific for this site of a particular receptor, it is possible to selectively inhibit that receptor only in the presence of agonist. This could lead to novel, tissue specific antagonism based on the levels of endogenous agonists. Agonists designed by the methods of the instant invention could be used in assay to determine the specificity of coactivator mimics.
  • the effective levels in a given tissue could be modulated by giving known antagonists or antagonists designed by the methods of the instant invention.
  • the crystal structure of the LBD/DES/GRIP1 peptide complex, described herein, precisely defines the binding site that would need to be targeted.
  • ER LBDs are co-crystallized with a peptide molecule comprising a coactivator GRIPl NR Box II peptide sequence (SEQ ID NO:4) bound to the coactivator binding site and DES with the cocrystal structure refined to a resolution of 2.03 A and co-crystallized with OHT with the cocrystal structure refined to a resolution of 1.9A.
  • the invention also provides for cocrystals made from nuclear receptor ligand binding domains with a ligand bound to the ligand binding domain and a molecule bound to the coactivator binding site.
  • the cocrystal structure is refined to a resolution greater than 3.6A, i.e.. having a resolution value less than 3.6A. More preferably the cocrystal structure is refined to greater than 3.4A, 3.2A, 3.0A. 2.8A, 2.6A, 2.4A, 2.2A, even more preferably to a resolution greater than 2.03 A.
  • the invention further provides for cocrystals made from nuclear receptor ligand binding domains with a ligand bound to the ligand binding domain.
  • the cocrystal structure is refined to a resolution greater than 3.6A, i.e.. having a resolution value less than 3.6A. More preferably the cocrystal structure is refined to greater than 3.4A, 3.2A. 3.0A, 2.8A, 2.6A, 2.4A, 2.2A, 2.0A, even more preferably to a resolution greater than 1.9A.
  • Crystals are made from purified nuclear receptor LBDs that are usually expressed by a cell culture, such as E. coli. E. coli is often a preferred expression system. The thyroid receptor was successfully expressed in E. coli in Apriletti, et al., supra. However, it has long been believed that a human heat shock protein was required for successful recombinant expression of the estrogen receptor. Therefore, it was quite unexpected to find that the estrogen receptor could be expressed as an active protein in E. coli.
  • different crystals (cocrystals) for the same nuclear receptor are separately made using different coactivators-type molecules, such as protein fragments, fusions or small peptides.
  • the coactivator-type molecules preferably contain NR-box sequences necessary for binding to the coactivator binding site, or derivatives of NR-box sequences.
  • Other molecules can be used in co-crystallization, such as small organics that bind to the coactivator or hormone binding site(s).
  • Heavy atom substitutions can be included in the LBD and/or a co-crystallizing molecule.
  • the structural information can be used in computational methods to design synthetic compounds for the nuclear receptor, and further structure-activity relationships can be determined through routine testing using the assays described herein and known in the art.
  • nuclear receptor LBDs may crystallize in more than one crystal form the structure coordinates of such receptors or portions thereof, as provided in Appendices 1 and 2, are particularly useful to solve the structure of those other crystal forms of nuclear receptors. They may also be used to solve the structure of mutants or co-complexes of nuclear receptors having sufficient homology.
  • the unknown crystal structure may be determined using the structure coordinates of this invention as provided in Appendices 1 and 2.
  • the Appendix 1 coordinates for the DES-ER ⁇ LBD-GRIP1 NR Box II peptide complex and for the Appendix 2 coordinates for the OHT-ER ⁇ LBD complex have been deposited with the Brookhaven National Laboratory Protein Data
  • Atomic coordinate information gleaned from the crystals of the invention can be stored.
  • the information is provided in the form of a machine-readable data storage medium.
  • This medium contains information for constructing and/or manipulating an atomic model of a ligand binding domain or portion thereof.
  • the machine readable data for the ligand binding domain comprises structure coordinates of amino acids corresponding to hER ⁇ Met343, Leu346, Ala350, Glu353, Leu384, Leu387, Leu391, Arg394.
  • the machine-readable data storage medium can be used for interactive drug design and molecular replacement studies. For example, a data storage material is encoded with a first set of machine-readable data that can be combined with a second set of machine-readable data.
  • the first set of data can comprise a Fourier transform of at least a portion of the structural coordinates of the nuclear receptor or portion thereof of interest
  • the second data set comprises an X-ray diffraction pattern of the molecule or molecular complex of interest.
  • Protein for crystals and assays described herein can be produced using expression and purification techniques described herein and known in the art.
  • high level expression of nuclear receptor LBDs can be obtained in suitable expression hosts such as E. coli.
  • Yeast and other eukaryotic expression systems can be used with nuclear receptors that bind heat shock proteins as these nuclear receptors are generally more difficult to express in bacteria, with the exception of ER, which can be expressed in bacteria.
  • Representative nuclear receptors or their ligand binding domains have been cloned and sequenced: human ER (as described in Seielstad, et al., Molecular Endocrinology 9(6 :647-658 (1995), incorporated herein by reference), human GR, and human PR. The LBD for each of these receptors has been identified.
  • Coactivator proteins can be expressed using techniques known in the art, particularly members of the pi 60 family of coactivator proteins that have been cloned and/or expressed previously, such as SRC-1, AIB1, RAC3, p/CIP, and GRIPl and its homologues TIF 2 and NcoA-2.
  • a preferred method for expression of coactivator protein is to express a fragment that retains transcriptional activation activity using the "Song and Fields” method (also referred to as the "yeast 2-hybrid” method) as described in publications by Hong, et al., Mol. Cell. Biol. 17:2735-44 (1997) and Proc. Natl. Acad. Sri.
  • ER ⁇ can be expressed without any portion of the DBD or amino-terminal domain. Portions of the DBD or amino-terminus can be included if further structural information with amino acids adjacent the LBD is desired.
  • the LBD used for crystals will be less than 320 amino acids in length.
  • the ER ⁇ LBD will be at least 220 amino acids in length and most preferably at least 250 amino acids in length.
  • the LBD used for crystallization can comprise amino acids spanning from 297 to 554 of the ER ⁇ .
  • the LBDs are purified to homogeneity for crystallization. Purity of LBDs can be measured with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). mass spectrometry (MS) and hydrophobic high performance liquid chromatography (HPLC).
  • the purified LBD for crystallization should be at least 97.5 % pure, preferably at least 99.0% pure, and more preferably at least 99.5% pure.
  • the receptors can be ligand-shift-purified using a column that separates the receptor according to charge, such as an ion exchange or hydrophobic interaction column, and then bind the eluted receptor with a ligand. especially an agonist.
  • the ligand induces a change in the receptor's surface charge such that the liganded receptor elutes at a different position than the unliganded receptor.
  • Purification can also be accomplished by use of a purification handle or "tag,” such as with a histidine amino acid engineered to reside on the end of the protein, such as on the N- terminus, and then using a nickel or cobalt chelation column for purification. (Janknecht, Proc. Natl. Acad. Sci. USA. 88:8972-8976 (1991)) incorporated by reference.
  • a purification handle or "tag” such as with a histidine amino acid engineered to reside on the end of the protein, such as on the N- terminus
  • a nickel or cobalt chelation column for purification.
  • purified LBD such as ER ⁇ LBD
  • Ligand equilibration can be established between 2 and 37°C, although the receptor tends to be more stable in the 2-20°C range.
  • crystals are made with the hanging drop methods detailed herein. Regulated temperature control is desirable to improve crystal stability and quality. Temperatures between 4 and 25°C are generally used and it is often preferable to test crystallization over a range of temperatures. The crystals are then subjected to vapor diffusion and bombarded with x-rays to obtain x-ray diffraction pattern following standard procedures.
  • ligands and peptides containing a sequence that binds to a coactivator binding site of a nuclear receptor of interest can be used in microcrystallization trials, and the appropriate compounds selected for further crystallization.
  • Ligands and peptides can be assayed for binding to the ligand binding domain and coactivator binding sites of a nuclear receptor of interest by any number of techniques, including those assays described herein.
  • the hanging drop vapor diffusion method is preferred. Conditions of pH.
  • solvent and solute components and concentrations and temperature can be adjusted, for instance, as described in the Examples.
  • seeding of prepared drops with microcrystals of the complex can be used. Collection of structural information can be determined by molecular replacement using the structure of the ER ⁇ LBD determined herein. The structure is refined following standard techniques known in the art.
  • the methods and compositions described herein are useful for identifying peptides, peptidomimetics or small natural or synthetic organic molecules that modulate nuclear receptor activity.
  • the compounds are useful in treating nuclear receptor-based disorders.
  • Methods and compositions of the invention also find use in characterizing structure/function relationships of natural and synthetic ligands.
  • PPAR ⁇ /SRC-1 peptide complex (Nolte, et al., supra) are similar to those of the ER ⁇ /GRIPl NR box II peptide complex described herein, suggesting that the mechanisms of NR box recognition are conserved across the nuclear receptor family.
  • the hydrophobic face of the NR box helix is formed by the side chains of the three motif leucines and the isoleucine preceding the motif (He 689).
  • He 689 The functional importance of the conserved leucines in receptor binding has been demonstrated by numerous in vitro and in vivo studies. In contrast, the role of the residue preceding the motif in receptor binding has been poorly characterized. Both biochemical and structural data implicate He 689 as a key receptor binding determinant. In the crystal, only the side chains of the motif leucines and He 689 extensively contact the LBD in both noncrvstallographic symmetry related peptides.
  • the different amino acids in the position immediately preceding the LXXLL motif might allow some degree of adaptability to these distinct surfaces; however, there may be no NR box sequence that is capable of efficiently binding to all nuclear receptors. Multiple NR boxes may therefore provide coactivators the diversity of interfaces necessary to recognize a variety of targets.
  • ER ⁇ transcriptional activity is blocked by antagonists such as OHT and RAL.
  • the most striking feature of the structures of the OHT and RAL liganded ER ⁇ LBDs is that helix 12 is bound to the static region of the coactivator recognition groove ( Figure 3B and (Brzozowski. et al., supra).
  • a comparison of these two structures with the structure of the coactivator/LBD complex reveals that in the antagonist complexes, the region of helix 12 with an NR box-like sequence (LXXML versus LXXLL) (SEQ ID NO:2 versus SEQ ID NO:l) functions as an intramolecular mimic of the coactivator helix ( Figure 5 and Brzozowski. et al.. supra).
  • NO:l appears to be unique among nuclear receptors.
  • an intramolecular inhibitor with a suboptimal recognition sequence would compete for coactivator binding given its extremely high local concentration.
  • Helix 3 8 and 1 1 in the DES and E? complexes are between one to two turns longer than they are in the OHT complex ( Figure 6A and (Brzozowski, et al.. supra).
  • Helix 1 1 ends at Cys 530 in the DES and E 2 complexes and it ends at Tyr 526 in the OHT complex.
  • Helix 12 begins at Leu 536 in the OHT complex. This appears to be necessary; in the antagonist complex, Leu 536 forms a cooperative network of nonpolar contacts and hydrogen bonds with Glu 380 and Tyr 537 that stabilizes the N-terminus of helix 12 ( Figure IB).
  • the loop connecting helices 1 1 and 12 would be required to span ⁇ 17A over five residues. Although theoretically possible, this conformation would be highly strained and hence unlikely. In contrast, the longer loop connecting helices 1 1 and 12 in the OHT complex allows helix 12 to extend to the static region of the coactivator binding groove.
  • the secondary structure differences between the agonist complexes and the OHT complex arise from distinct arrangements of packing interactions induced by the different ligands.
  • a cooperative network of van der Waals contacts, organized around DES or E between various hydrophobic residues from helices 3, 7, 8 and 11 and the ⁇ hairpin appears to stabilize the longer helices in the agonist complexes ( Figure 4A and 6D).
  • the placement of the OHT B ring forces many of ligand binding pocket residues that surround it to adopt conformations that are dramatically different from those they adopt in either the DES or E 2 structures.
  • many of the interresidue packing interactions present in the DES and E 2 structures are either absent or altered in the OHT structure ( Figure 6D).
  • the human ER ⁇ -LBD 297-554 was overexpressed as described previously (Seielstad. et al., Mol. Endocrinol. 9:647-658 (1995)) in BL21(DE3)pLysS cells transformed with a modified pET-23d-ERG vector that contained the sequence Met-Asp-Pro fused to residues 297 through 554 of the hER ⁇ (provided by Paul Sigler of Yale University). Clarified bacterial lysates were adjusted to 3 M in urea and 0.7 M in NaCl and then applied to a 10-ml column of estradiol- Sepharose (Greene, et al., Proc. Natl. Acad. Sri.
  • the hER ⁇ -LBD-ligand complexes eluted at 150-200 mM NaCl. Pooled fractions were concentrated by ultrafiltration and analyzed by SDS-PAGE, native PAGE, and electrospray ionization mass spectrometry.
  • GST-pulldown Assays A fusion between glutathione-S-transferase (GST) and amino acids 282-595 of hER ⁇ was constructed by subcloning the EcoRI fragment from pSG5 ER ⁇ -LBD (Lopez et al., submitted manuscript) into pGEX-3X (Pharmacia).
  • the He 358-> Arg, Lys 362->Ala, and Leu 539->Arg mutations were introduced into the GST-LBD construct using the QuikChange Kit (Stratagene) according to the manufacturer's instructions.
  • the Val 376->Arg and Glu 542->Lys mutations were created in the GST-LBD construct by subcloning the Bsml/Hindlll fragments of derivatives of pSG5-ER-HEGO (Tora. et al.. EMBO J. 8:1981-6 (1989)) into which these mutations had already been introduced. All constructs were verified by automated sequencing (University of Chicago Cancer Research Center DNA Sequencing Facility).
  • the wild-type and mutant GST-LBDs were expressed in BL21(DE3) cells.
  • Total ligand binding activity was determined by a controlled pore glass bead assay (Greene, et al., Mol. Endocrinol. 2:714-726 (1988)) and protein levels were monitored by western blotting with a monoclonal antibody to hER ⁇ (H222).
  • Cleared extracts containing the GST-LBDs were incubated in buffer alone (50 mM Tris, pH 7.4, 150 mM NaCl, 2 mM EDTA, 1 mM DTT, 0.5% NP-40 and a protease inhibitor cocktail) or with 1 ⁇ M of either DES or OHT for 1 hour at 4°C.
  • Extract samples containing thirty pmol of GST-LBD were then incubated with 10 ⁇ l glutathione- Sepharose-4B beads (Pharmacia) for 1 hour at 4°C. Beads were washed five times with 20 mM HEPES, pH 7.4, 400 mM NaCl, and 0.05% NP-40. 35 S-labeled GRIPl was synthesized by in vitro transcription and translation using the TNT Coupled Reticulocyte Lysate System (Promega) according to the manufacturer's instructions and pSG5-GRIPl (provided by Michael Stallcup of the University of Southern California) as the template.
  • TNT Coupled Reticulocyte Lysate System Promega
  • Immobilized GST-LBDs were incubated for 2.5 hours with 2.5 ⁇ l aliquots of crude translation reaction mixture diluted in 300 ⁇ l of Tris- buffered saline (TBS). After five washes in TBS containing 0.05% NP-40. proteins were eluted by boiling the beads for 10 minutes in sample buffer. Bound j:, S-GRIPl was quantitated by fluorography following SDS-PAGE. Crystallization and Data Collection
  • Crystals of the DES-hER ⁇ LBD-GRIP1 NR Box II peptide complex were obtained by hanging drop vapor diffusion. Prior to crystallization, the DES-hER ⁇ LBD (residues 297-554) complex was incubated with a 2-4 fold molar excess of the GRIPl NR Box II peptide (SEQ ID NO:4) for 7-16 hr. Two ⁇ L samples of this solution were mixed with equal volume samples of reservoir buffer consisting of 25-27% (w/v) PEG 4000. 90 mM Tris (pH 8.75-9.0) and 180 mM Na Acetate and suspended over wells containing 800 ⁇ L of the reservoir buffer. After 4-7 days at 19-21°C. rod-like crystals were obtained.
  • Two molecules each of the DES-LBD and the coactivator peptide form the asymmetric unit.
  • a 200 ⁇ m x 40 ⁇ m x 40 ⁇ m crystal was transferred to a cryosolvent solution containing 25% (w/v) PEG 4000, 10% (w/v) ethylene glycol, 100 mM Tris (pH 8.5), 200 mM Na Acetate and 10 ⁇ M peptide and frozen in an N 2 stream at -170°C in a rayon loop. Diffraction data from this crystal were measured at - 170°C using a 300 mm MAR image plate at the Stanford Synchrotron Radiation Laboratory (SSRL) at beamline 7-1 at a wavelength of 1.08 A.
  • SSRL Stanford Synchrotron Radiation Laboratory
  • Crystals of the hER ⁇ LBD complexed to OHT were obtained by the hanging drop vapor diffusion method. Equal volume aliquots (2 ⁇ L) of a solution containing 3.9 mg/mL protein- ligand complex and the reservoir solution containing 9% (w/v) PEG 8000, 6% (w/v) ethylene glycol, 50 mM HEPES (pH 6.7) and 200 mM NaCl were mixed and suspended over 800 ⁇ L of the reservoir solution. Hexagonal plate-like crystals formed after 4-7 days at 21-23°C. Both crystal size and quality were improved through microseeding techniques.
  • the asymmetric unit consists of a single LBD monomer; the dimer axis lies along a crystallographic two-fold.
  • a single crystal 400 ⁇ m x 250 ⁇ m x 40 ⁇ m was briefly incubated in a cryoprotectant solution consisting of 10% (w/v) PEG 8000, 25% (w/v) ethylene glycol, 50 mM HEPES (pH 7.0) and 200 mM NaCl and then flash frozen in liquid N suspended in a rayon loop.
  • Diffraction data were measured at -170°C using a 345 mm MAR image plate at SSRL at beamline 9-1 and at a wavelength of 0.98A.
  • the images of both data sets were processed with DENZO and scaled with SCALEPACK (Otwinowski. et al.. Methods Enzvmol. 276:307-326 (1997)) using the default - 3 ⁇ cutoff.
  • the final model consisted of residues 306-551, the ligand and 79 waters. According to PROCHECK (CCP4, 1994), 91.6% of all residues in the model were in the core regions of the Ramachandran plot and none were in the disallowed regions.
  • the high resolution data set of the DES-LBD-GRIP1 NR Box II peptide complex became available when the R free of the OHT-LBD model was -31%. Both monomers in the asymmetric unit of the DES complex crystal were relocated using AMoRe and the incompletely refined OHT-LBD model (with helix 12 and the loop between helices 11 and 12 removed) as the search model.
  • the missing parts of the model were built and the rest of the model was corrected using MOLOC and two-fold averaged maps generated in DM. Initially, refinement was carried out with REFMAC using tight NCS restraints. At later stages, the model was refined without NCS restraints using the simulated annealing, positional and B-factor refinement protocols in X- PLOR and a maximum-likelihood target. All B-factors were refined isotropically and anisotropic scaling and a bulk solvent correction were used. The Rf ree set contained a random sample of 6.5% of all data. In refinement, all data between 27 and 2.03A (with no ⁇ cutoff) were used.
  • the final model was composed of residues 305-549 of monomer A, residues 305-461 and 470-554 of monomer B, residues 687-697 of peptide A, residues 686-696 of peptide B, two ligand molecules, 147 waters, two carboxymethyl groups and a chloride ion. According to PROCHECK, 93.7% of all residues in the model were in the core regions of the Ramachandran plot and none were in the disallowed regions.
  • Figure 1 A provides a view of a 2Fo-Fc electron density map calculated at 2.03A resolution and contoured at 1.0 ⁇ showing the GRIPl NR box II interaction with the LBD.
  • the GRIPl NR Box II peptide (SEQ ID NO:4) was omitted from the model prior to map calculation. He 689 from the peptide and two of the three receptor residues with which it interacts (Glu 542 and Leu 539) are labeled. Asp 538 has been omitted for clarity. The hydrogen bonds between the ⁇ -carboxylate of Glu 542 and the amides of residues 689 and 690 of the peptide are depicted as dashed orange bonds.
  • Figure IB provides a view of a 2Fo-Fc electron density map calculated at 1.90A resolution and contoured at 1.0 ⁇ showing the N-terminal region of helix 12.
  • the dashed orange bonds depict the water-mediated hydrogen bond network between the imidazole ring of His 377. the ⁇ -carboxylate of Glu 380. and the amide of Tyr 537.
  • the three labeled residues (Glu 380. Leu 536 and Tyr 537) interact with each other through van der Waals contacts and/or hydrogen bonds. Intriguingly, mutations in each these three residues dramatically increase the transcriptional activity of unliganded ER ⁇ LBD (Eng, et al., Mol. Cell. Biol.
  • GRIPl a mouse pi 60 coactivator, interacts both in vivo and in vitro with the ER ⁇ LBD bound to agonist (Ding, et al., supra), but not with the LBD bound to antagonist (Norris, et al., J.
  • the binding reactions were performed on ice for 45 minutes in 10 ⁇ l of buffer containing 20mM Tris, pH 8.1, lmM DTT, and 200mM NaCl and then subjected to 6% native PAGE. Gels were stained with GELCODE Blue Stain reagent (Pierce).
  • the asymmetric unit of the DES-LBD-GRIP1 NR Box II peptide complex crystals contains the same noncrystallographic dimer of LBDs that has been observed in the previously determined structures of the LBD bound to both E2 and RAL (Brzozowski. et al.. supra and
  • the two LBDs of the dimer adopt similar structures (r.m.s. d. 0.47A based on C ⁇ positions).
  • each LBD complexed with DES closely resembles that of the LBD bound to E 2 (Brzozowski. et al.. supra); each monomer is a wedge shaped molecule consisting of three layers of eleven to twelve helices and a single beta hairpin (Figure 2 A).
  • the hydrophobic face of helix 12 is packed against helices 3. 5/6 and 11 covering the ligand binding pocket ( Figure 2A).
  • One GRIPl NR Box II peptide is bound to each LBD in a hydrophobic cleft composed of residues from helices 3, 4, 5 and 12 and the turn between 3 and 4 ( Figures 2 A and 3A).
  • FIG. 2A The overall structures of the DES-ER ⁇ LBD-GRIP1 NR Box II peptide complex and the OHT-ER ⁇ LBD complex are illustrated in Figure 2.
  • the coactivator peptide and the LBD are shown as ribbon drawings.
  • the peptide is colored gold and helix 12 (residues 538- 546) is colored magenta.
  • Helices 3, 4 and 5 (labeled H3. H4 and H5 respectively) are colored blue.
  • DES colored green, is shown in space-filling representation.
  • Figure 2B the LBD is depicted as a ribbon drawing.
  • helix 12 (residues 536-544) is colored in magenta and helices 3. 4 and 5 are colored blue.
  • OHT. in red. is shown in space-filling representation.
  • the GRIPl NR Box II peptide binding site is a shallow groove composed of residues Leu 354. Val 355. He 358. Ala 361 and Lys 362 from helix 3; Phe 367 and Val 368 from helix 4; Leu 372 from the turn between helices 3 and 4; Gin 375, Val 376. Leu 379 and Glu 380 from helix 5: and Asp 538. Leu 539. Glu 542 and Met 543 from helix 12 ( Figure 3 A).
  • the floor and sides of this groove are completely nonpolar, but the ends of this groove are charged (Figure 3C).
  • the LBD interacts primarily with the hydrophobic face of the GRIPl NR Box II peptide ⁇ helix formed by the side chains of He 689 and the three LXXLL motif (SEQ ID NO:l) leucines (Leu 690, Leu 693 and Leu 694).
  • the side chain of Leu 690 is deeply embedded within the groove and forms van der Waals contacts with the side chains of He 358, Val 376, Leu 379, Glu 380 and Met 543 ( Figure 3 A and 3C).
  • the side chain of Leu 694 is similarly isolated within the groove and makes van der Waals contacts with the side chains of He 358, Lys 362, Leu 372, Gin 375, Val 376 and Leu 379 ( Figure 3 A and 3C).
  • Leu 693. rest against the rim of the groove ( Figure 3A and 3C).
  • the side chain of He 689 lies in a shallow depression formed by the side chains of Asp 538, Leu 539 and Glu 542.
  • the side chain of Leu 693 makes nonpolar contacts with the side chains of He 358 and Leu 539.
  • GST glutathione-S-transferase
  • DES (273 A 3 ) is accommodated within the same binding pocket that recognizes E 2 (252A 3 ).
  • DES is completely encased within the narrower half of the LBD in a predominantly hydrophobic cavity composed of residues from helices 3, 6, 7, 8. 1 1 , and 12 as well as the S1/S2 hairpin ( Figures 2A and 4A).
  • the interaction of DES with ER ⁇ resembles that of E 2 .
  • One of the phenolic rings of DES lies in the same position as the E 2 A ring near helices 3 and 6.
  • the DES A ring ( Figure 4A) is engaged by the side chains of Phe 404.
  • DES also forms contacts with the LBD that E 2 does not. There are unoccupied cavities adjacent to the ⁇ face of the B ring and the ⁇ face of the C ring of the E 2 (Brzozowski. et al.. supra and Tanenbaum, et al., supra).
  • the ethyl groups of DES which project perpendicularly from the plane of the phenolic rings, fit snugly into these spaces.
  • the resulting additional nonpolar contacts with the side chains of Ala 350, Leu 384, Phe 404, and Leu 428 (Figure 4A) may account for the higher affinity of DES for the receptor (Kuiper, et al., Endocrinology 138:863-70 (1997)).
  • Example 6 Structure of the OHT-LBD Complex The binding of OHT induces a conformation of the LBD that differs in both secondary and tertiary structural organization from that driven by DES binding.
  • the main chain from residues 339 to 341. 421 to 423. and 527 to 530 form parts of helices 3. 8 and 1 1 respectively In contrast, these regions adopt an extended conformation in the OHT complex
  • Helix 12 mimics the hydrophobic interactions of the NR box peptide with the static region of the groove with a stretch of residues (residues 540 to 544) that resembles an NR box (LLEML instead of LXXLL) (SEQ ID NO 3 instead of SEQ ID NO 1)
  • the side chains of Leu 540 and Met 543 e in approximately the same locations as those of the first and second motif leucines (Leu 690 and Leu 693) in the peptide complex ( Figure 5)
  • Leu 540 is inserted into the groove and makes van der Waals contacts with Leu 354, Val 376 and Glu 380 ( Figures 3B and 3D)
  • Met 543 lies along the edge of the groove and forms van der Waals contacts with the side chains of Leu 354, Val 355 and He 358 ( Figures 3B and 3D)
  • the side chain position of Leu 544 almost exactly overlaps that of the third NR box leucine, Leu 694 ( Figure 5) Deep within the groove, the
  • Lys 362 interacts with the C-terminal turn of helix 12 much as it does with the equivalent turn of the peptide helix ( Figures 3A and 3B)
  • the Lys 362 side chain packs against the C-terminal turn of the helix 12 with its ⁇ -amino group hydrogen bonding to the carbonyls of residues 543 and 544 ( Figure 5) G ⁇ en that the capping interaction at the N-terminal turn coactivator helix is formed b ⁇ a helix 12 residue (Glu 542). the N-terminal turn ot helix 12 in the antagonist complex is forced to interact with another residue.
  • Glu 380 Figures 3B and 3D).
  • Glu 380 ⁇ -carboxylate forms van der Waals contacts with Tyr 537 and interacts with the amide of Tyr 537 through a series of water-mediated hydrogen bonds ( Figure I B).
  • helix 12 In addition to forming these "NR box-like" interactions, helix 12 also forms van der Waals contacts with areas of the LBD outside of the coactivator recognition groove.
  • the side chain of Leu 536 forms van der Waals contacts with Glu 380 and Tip 383 and that of Tyr 537 forms van der Waals contacts with His 373, Val 376 and Glu 380 ( Figures IB, 3B and 3D).
  • helix 12 in the OHT complex buries more solvent accessible surface area
  • OHT is bound within the same pocket that recognizes DES, E 2 and RAL.
  • the orientation of OHT within the binding pocket appears to be dictated by the positioning of two structural features of this ligand, the phenolic A ring and the bulky side chain ( Figures 4B and 6C).
  • the A ring of OHT is bound in approximately the same location as the A ring of DES near helices 3 and 6 with its phenolic hydroxyl hydrogen bonding to a structurally conserved water and to the side chains of Glu 353 and Arg 394 ( Figure 4B).
  • the side chain of OHT exits the binding pocket between helices 3 and 1 1 ( Figures 2B and 4B).
  • the OHT C ring ( Figure 4B) forms van der Waals contacts with the side chains of Met 343, Leu 346, Thr 347, Ala 350, Tip 383. Leu 384, Leu 387 and Leu 525.
  • the positioning of the flexible dimethylaminoethyl region of the side chain is stabilized by van der Waals contacts with Thr 347, Ala 350 and Tip 383 and by a salt-bridge between the dimethylamino group of the side chain and the ⁇ -carboxylate of Asp 351, which lies 3.8 A away ( Figure 4B).
  • the structural effects of the placement of the B ring are not limited to the residues that contact the B ring; the conformations of these residues force other residues throughout the binding pocket to, in turn, adopt alternative conformations.
  • the conformation adopted by Met 421 in the OHT complex prevents the side chains of Phe 404 and Phe 425 from occupying the positions they take in the DES complex ( Figure 6B and 6C).
  • Phe 404 does not make van der Waals contacts with the OHT A ring as it does with the A rings of DES or E 2 ( Figure 6C).
  • Phe 404 only contacts the ethyl group of OHT ( Figures 6C and 6D).
  • the alternative conformations of the side chains of both the residues that directly contact the B ring and those that are indirectly affected by it force the main chain throughout the binding pocket to adopt a different conformation as well (Figure 6D).
  • ATOM 140 CD PRO A 324 21.774 -1.466 -6.935 1.00 31.01
  • ATOM 141 CA PRO A 324 21.935 -2.032 -9.290 1.00 30.29
  • ATOM 148 CA PRO A 325 21.125 0.242 -12.21 1 1.00 25.59
  • ATOM 236 CA PHE A 337 -3.055 7.821 - 15.582 1.00 46.61
  • ATOM 262 CA ALA A 340 -5.078 -0.158 -13.574 1.00 40.24
  • ATOM 309 CA THR A 347 4.436 -3.196 -10.674 1.00 23.91
  • ATOM 310 CB THR A 347 3.164 -4.058 -10.641 1.00 26.39
  • ATOM 345 CA ARG A 352 12.1 15 -5.787 -13.347 1.00 21.07
  • ATOM 390 CA MET A 357 17.837 -6.906 -6.610 1.00 21.51 ATOM 391 CB MET A 357 16.503 -6.668 -5.898 1.00 17.60
  • ATOM 405 N ASN A 359 20.207 -10.325 -7.897 1.00 27.91
  • ATOM 406 CA ASN A 359 21.601 -10.401 -8.293 1.00 29.16
  • ATOM 433 CA LYS A 362 24.530 -12.097 -4.047 1.00 33.33
  • ATOM 472 CA PRO A 365 29.231 -9.442 1.733 1.00 37.82
  • ATOM 478 CA GLY A 366 28.307 -12.635 3.554 1.00 38.27
  • ATOM 642 CA ILE A 386 14.685 -2.484 -0.388 1.00 15.01
  • ATOM 752 CA PRO A 399 1.243 17.709 -3.173 1.00 37.10
  • ATOM 762 CA LYS A 401 7.972 14.007 -5.966 1.00 30.75

Abstract

La présente invention concerne des méthodes et des composés d'agonistes/antagonistes de modulation de l'activité de récepteurs nucléaires, et de liaison aux ligands de récepteurs nucléaires. L'invention concerne une méthode d'identification de résidus comprenant un domaine de liaison aux ligands d'un récepteur nucléaire présentant un certain intérêt. L'invention concerne également une méthode d'identification d'agonistes et/ou d'antagonistes se liant au domaine de liaison aux ligands des récepteurs nucléaires, et du récepteur d'oestrogènes en particulier. L'identification et la manipulation du domaine de liaison aux ligands du récepteur d'oestrogènes et de composés se liant à ce site illustrent la présente invention. Les méthodes selon la présente invention peuvent s'appliquer à d'autres récepteurs nucléaires, y compris TR, GR, et PR.
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CA2324060A1 (fr) 1999-10-07
AU3457199A (en) 1999-10-18
EP1144997A3 (fr) 2002-08-28
JP2002516983A (ja) 2002-06-11
WO1999050658A3 (fr) 2001-08-16
KR20010042373A (ko) 2001-05-25
WO1999050658A2 (fr) 1999-10-07

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