EP1297175A4 - Procedes et composes pour moduler la fixation de coactivateurs a des recepteurs nucleaires - Google Patents

Procedes et composes pour moduler la fixation de coactivateurs a des recepteurs nucleaires

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Publication number
EP1297175A4
EP1297175A4 EP01950770A EP01950770A EP1297175A4 EP 1297175 A4 EP1297175 A4 EP 1297175A4 EP 01950770 A EP01950770 A EP 01950770A EP 01950770 A EP01950770 A EP 01950770A EP 1297175 A4 EP1297175 A4 EP 1297175A4
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European Patent Office
Prior art keywords
atom
peptide
coactivator
nuclear receptor
binding
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EP01950770A
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German (de)
English (en)
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EP1297175A2 (fr
Inventor
Rodney K Guy
John D Baxter
Beatrice Darimont
Weijun Feng
Robert J Fletterick
Peter J Kushner
Richard L Wagner
Brian L West
Keith R Yamamoto
Timothy R Geistlinger
James R Arnold
Irwin D Kuntz
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University of California
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University of California
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Publication of EP1297175A2 publication Critical patent/EP1297175A2/fr
Publication of EP1297175A4 publication Critical patent/EP1297175A4/fr
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    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B20/00—ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
    • G16B20/30—Detection of binding sites or motifs
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/06—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D209/56—Ring systems containing three or more rings
    • C07D209/80—[b, c]- or [b, d]-condensed
    • C07D209/82—Carbazoles; Hydrogenated carbazoles
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D209/56—Ring systems containing three or more rings
    • C07D209/80—[b, c]- or [b, d]-condensed
    • C07D209/82—Carbazoles; Hydrogenated carbazoles
    • C07D209/88—Carbazoles; Hydrogenated carbazoles with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to carbon atoms of the ring system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D225/00—Heterocyclic compounds containing rings of more than seven members having one nitrogen atom as the only ring hetero atom
    • C07D225/04—Heterocyclic compounds containing rings of more than seven members having one nitrogen atom as the only ring hetero atom condensed with carbocyclic rings or ring systems
    • C07D225/08—Heterocyclic compounds containing rings of more than seven members having one nitrogen atom as the only ring hetero atom condensed with carbocyclic rings or ring systems condensed with two six-membered rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
    • C07K1/047—Simultaneous synthesis of different peptide species; Peptide libraries
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/50—Cyclic peptides containing at least one abnormal peptide link
    • C07K7/54—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring
    • C07K7/56—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring the cyclisation not occurring through 2,4-diamino-butanoic acid
    • 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
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B15/00—ICT specially adapted for analysing two-dimensional or three-dimensional molecular structures, e.g. structural or functional relations or structure alignment
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B15/00—ICT specially adapted for analysing two-dimensional or three-dimensional molecular structures, e.g. structural or functional relations or structure alignment
    • G16B15/30—Drug targeting using structural data; Docking or binding prediction
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2299/00—Coordinates from 3D structures of peptides, e.g. proteins or enzymes
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B20/00—ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations

Definitions

  • the present invention relates to methods and compounds for modulating nuclear receptor coactivator binding.
  • 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.
  • the nuclear receptor family includes receptors for glucocorticoids (GRs), androgens (ARs), mineralocorticoids (MRs), progestins (PRs), estrogens (ERs), thyroid hormones (TRs), vitamin D (VDRs), retinoids (RARs and RXRs), peroxisomes (XPARs and PPARs) and icosanoids (IRs).
  • GRs glucocorticoids
  • ARs mineralocorticoids
  • PRs progestins
  • ERs estrogens
  • TRs vitamin D
  • VDRs vitamin D
  • RARs and RXRs retinoids
  • XPARs and PPARs peroxisomes
  • IRs icosanoids
  • 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.
  • nuclear receptors can stimulate transcription in response to hormone binding by recruiting coactivator proteins to promoters of responsive genes (Glass et al., Curr. Opin. Cell Biol. (1997) 9:222-32); and Horwitz et al, Mol. Endocrinol. (1996) 10:1167-77).
  • Coactivators of the pi 60 family mediate activity of a transcriptional activation domain, called AF2, that is part of the nuclear receptor's LBD.
  • AF2 transcriptional activation domain
  • a few receptor mutants deficient in coactivator-dependent activation have been isolated (TR: CoUingwood et al. Proc. Natl. Acad. Sci. (1997) 94:248-253; VDR: Jurutka et al., J. Biol. Chem. (1997) 227:14592-14599, Masayama et al., Mol. Endocrinol. (1997) 11: 1507-1517; ER and RAR: Henttu et al, Mol. Cell Biol. (1997) 7:1832-1839). While these studies support the physiological relevance of the observed interaction, the structural and functional nature of the site to which coactivators bind has not been defined.
  • the present invention relates to identification and manipulation of the coactivator binding site of nuclear receptors. Identification of this site permits design and obtention of compounds that bind to the coactivator binding site of nuclear receptors and modulate coactivator binding to the receptor.
  • the compounds include agonists and antagonists that modulate nuclear receptor activity by promoting (agonists) or blocking (antagonists) hormone-dependent coactivator binding to the receptor, particularly antagonists.
  • the compounds of the invention can be receptor-, cell- and/or tissue-specific.
  • the method involves modeling test compounds that fit spacially into a nuclear receptor coactivator binding site of interest using an atomic structural model comprising a nuclear receptor coactivator binding site or portion thereof, coactivator structure or portion thereof, screening the test compounds in a biological assay characterized by binding of a test compound to a nuclear receptor coactivator binding site, and identifying a test compound that modulates coactivator binding to the nuclear receptor.
  • the invention also includes compositions and methods for identifying coactivator binding sites of nuclear receptors.
  • the methods involve examining the surface of a nuclear receptor of interest to identify residues that modulate coactivator binding.
  • the residues can be identified by homology to the coactivator binding site of human TR described herein. Overlays and superpositioning with a three dimensional model of a nuclear receptor LBD, or a portion thereof that contains a coactivator binding site, 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 coactivator binding site.
  • Preferred compounds bind to the site with greater affinity than coactivator proteins found in a cell of interest. Binding at this site, the compound can compete for binding of coactivator proteins, thereby inhibiting gene transcription, or in some cases promoting it, even when hormone is or is not bound.
  • the structures of the bound coactivator NR box helices from GRIPl and SRC1 bound to various nuclear receptors, such as ER, TR, and PPAR, have been analyzed and have been shown to have similar backbone conformations. It is therefore believed that organic scaffolds that mimic this helix geometry could serve as a general basis for combinatorial libraries targeted to all NR:GRIP-1 complexes. These scaffolds and libraries would serve to affect the interactions of nuclear receptors and their coactivators.
  • the invention further includes a method for identifying an agonist or antagonist of coactivator binding to a nuclear receptor.
  • the method comprises providing the atomic coordinates comprising a nuclear receptor coactivator binding site or portion thereof to a computerized or mechanical modeling system; and/or providing the atomic coordinates comprising a molecule or molecules bound to the nuclear receptor coactivator binding site or portion thereof to a computerized or mechanical modeling system; modeling compounds or libraries of compounds which fit spatially into the nuclear receptor coactivator binding site; and identifying in an assay for nuclear receptor activity a compound or compounds that increases or decreases activity of the nuclear receptor through binding the coactivator binding site.
  • a machine-readable data storage medium with information for constructing and manipulating an atomic model comprising a coactivator binding site or portion thereof.
  • the medium comprises a data storage material encoded with machine readable data which, when using a machine programmed with instructions for using said data, is capable of displaying a graphical three-dimensional representation of a molecule or molecular complex for a nuclear receptor coactivator binding site.
  • the method is exemplified by modeling test compounds, combinatorial libraries, and probe molecules that have a spatial and/or electrostatic preference for a nuclear receptor coactivator binding site of interest using an atomic structural model of a nuclear receptor coactivator binding site, selecting a compound that interacts with one or more aspects of the coactivator binding site unique in the context of that site, and identifying in an assay for coactivator binding activity a compound that selectively binds to the coactivator binding site compared to other nuclear receptors.
  • the unique features involved in receptor-selective coactivator binding can be identified by comparing atomic models of different receptors or isoforms of the same type of receptor, and by comparing features of molecules that show preferential complementarity for different 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 small molecule compounds, such as small organic molecules, identified by the methods of the invention, particularly new lead compounds, scaffolds and combinatorial libraries useful in treating nuclear receptor-based disorders.
  • Figure 1 shows the specific effects of mutations on hTR ⁇ l transcriptional activation in HeLa cells and correlation with effects on binding to GST-GRIP 1.
  • T 3 dependent activation of transcription of a reporter gene expressed as the percentage of WT is plotted for each mutant.
  • GST-GRIPl binding analyzed by autoradiography after separation using 10% SDS-PAGE, was also expressed as the percentage of WT and plotted for each mutant.
  • the GST-GRIPl used included GRIPl amino acids 721-1121; the same results were obtained using a GST-GRDPl construct including GRIPl amino acids 563-1121 (data not shown).
  • FIG. 2 shows that overexpression of full-length GRIPl rescues loss of transcriptional activation by hTR ⁇ l mutants. Indicated amounts of the expression vector for full-length GRIPl, pSG5-GRD?l, is included in the cotransfections, which otherwise are performed as in Figure 1. The WT or different representative hTR ⁇ l mutants are indicated.
  • Figure 3 shows specific hER ⁇ surface mutants cause loss of transcriptional activation in HeLa cells in parallel with their loss of in vitro GRIPl binding.
  • the fold E activation expressed as the percentage of WT
  • the phosphorimager quantitation of in vitro binding of [ 35 S]-labeled hER ⁇ WT and mutants to GST-GRIPl (GRIPl amino acids 721-1121) also expressed as the percentage of WT is plotted for each mutant.
  • Figure 4 shows a plot of the fold E 2 activation observed when the indicated amounts of the full-length GRIPl expression vector, pSG5-GRIPl, are added to the co- transfection experiment, which otherwise is performed as for Figure 3.
  • the WT or different hER ⁇ mutants are indicated.
  • the data represent the averages of three independent experiments, with standard deviations less than 10%.
  • Figure 5 shows a CPK model of the TR ⁇ -LBD, indicating the LBD surface locations of mutations made in the full-length hTR ⁇ l. Mutated residues having no effect on GRIPl binding or effect on activation in HeLa cells are shaded gray. Mutated residues with diminished GRIPl and SRC-la binding and diminished activation in HeLa cells are colored to reflect chemical properties of the residues: red, blue (purple), and green indicate acidic, basic, and hydrophobic residues, respectively.
  • the main chain structures of the TR ⁇ - and TR ⁇ -LBDs are the same (data not shown).
  • Figure 7 shows binding affinity assays of GST-GRIPl constructs with NR- boxes 1, 2, and/or 3 and their interaction with TR LBD.
  • GRIP-1 NR boxes 1,2 and 3 interact differently with TR ⁇ LBD.
  • Single letter designations are used for the amino acids.
  • Figure 8 shows binding affinity assays of GST-GRIPl constructs with NR- boxes 1, 2, and/or 3 and their interaction with TR and GR LBDs. TR and GR differ in their interactions with GRIP-1.
  • Figure 9 shows binding affinity assays for NR-box 2- and 3-peptides and GRIPl and their interaction with TR LBD.
  • NR box 2- and 3 -containing peptides reproduce the affinity and specificity of the NR interaction domain.
  • Figure 10 shows binding affinity assays for NR-box 2- and 3-peptides and their interaction with TR LBD. Sequence adjacent to the (SEQ ID NO: 1) LxxLL motif modulate the affinity of NR-box-TR ⁇ LBD interactions.
  • Figure 11 shows binding affinity assays for mutant GRIPl and NR-box 2- and 3-peptides and their interaction with TR LBD.
  • the individual leucine residues of the (SEQ ID NO: 1) LxxLL motif are crucial for binding of the GRIP-1 NR interaction domain to TR ⁇ LBD.
  • Figure 12 shows the contents of the asymmetric unit of the crystallized hTR ⁇ LBD:GRIP1 NR-box 2 peptide complex.
  • the crystal lattice consists of a repeating unit containing a 2:2 complex of hTR LBD and GRIPl site 2 peptide. Positions of the two GRIPl site 2 peptides are boxed, in green (sitel), and red (site 2), with the peptides drawn as a C-alpha trace.
  • the two NCS related monomers of the hTR LBD are shown as a secondary structure ribbon drawing, with monomer 1 in light grey, and monomer 2 in dark grey.
  • the side chains of the hydrophobic residues 1689, L690, L693, L694 of the GRIPl NR-box 2 peptides are drawn to emphasize those interactions observed in both bound peptides.
  • Figure 13 shows a ribbon diagram depicting the interaction of the GRIPl NR-box 2 peptide with the hTR ⁇ LBD.
  • the GRIPl NR-box 2 peptide dark grey
  • Portions of the hTR ⁇ LBD, and the neighboring monomer, are omitted for clarity.
  • Figure 14 shows interface between the GRIPl NR-box 2 peptide and the hTR ⁇ LBD.
  • Side chains of residues of the hTR ⁇ LBD within 4.5A of the GRIP-1 NR-box 2 peptide are labeled.
  • the color of the individual side chains reflects the chemical nature of the residue: acidic residues are red, basic residue are blue, aliphatic residues are green, aromatic residues are brown, and polar residues are orange.
  • the peptide is depicted as a C- alpha trace with the side chains of (SEQ ID NO: 2) ILxxLL motif shown explicitly.
  • Figure 16 shows molecular surface of the hTR LBD.
  • the side chains of the leucines resides fit within a hydrophobic groove formed from helices H3, H5, and H12, while the side chain of the non-conserved isoleucine residue packs against the outside edge of the groove.
  • the remainder of the peptide is shown as main chain.
  • Figure 17 shows complementarity between the (SEQ ID NO: 1) LxxLL motif and the surface of the hTR LBD.
  • the side chains of the (SEQ ID NO: 2) ILxxLL motif are shown in a CPK representation, with the main chain of the peptide drawn as a C- alpha trace.
  • the three leucince residues fit into pockets of the coactivator binding site of the hTR ⁇ LBD, depicted as mesh, while the isoleucine residue rests on the edge of the site's cleft.
  • Figure 18 shows the coactivator binding site cleft, one side of which is formed by conformationally hormone-responsive residues.
  • On the left is a view of the TR- LBD molecular surface showing the concave surfaces in gray. The cavity is shown at the center of the figure.
  • On the right is shown a CPK model of the TR-LBD, overlaid with a molecular surface view, which is restricted to a 12A radius of the hydrophobic cavity.
  • Mutated residues of the coactivator binding site that are hormone-insensitive V284, K288, 1302 and K306) are located on one side of the cleft and are colored yellow.
  • Mutated CBS residues likely undergo a conformational change upon hormone binding (L454 and E457) are located on the opposite side of the cleft and are colored red.
  • Figure 19 shows alignment of amino acid sequences (single letter amino acid designations) containing residues that form the coactivator binding sites of several nuclear receptors.
  • the boxes represent residues of alpha-helix (H3, H4, H5, H6 and H12); lower case letters “h” and "q” represent hydrophobic and polar residues, respectively.
  • Figure 20 shows a table of sequences and circular dichroism data for 9 peptides designed to fit tightly to the coactivator binding site. These data reveal that TG17 is highly helical relative to the others.
  • Figure 21 shows the relative competitive abilities of the 9 peptides in blocking the binding of a GRIPl NR box 2 peptide to the binding site of hTRbeta.
  • Figure 24 shows the manual optimization of an exemplary docked scaffold to improve fit, atom economy, and alignment of substituents into sub-pockets within the coactivator binding site.
  • Figure 25 shows the amino acid sequence and chemical structure of native GRIPl.
  • an IC 50 value is shown, reflecting the ability of native GRIP 1 to competitively inhibit the interaction of hTR and GRIPl.
  • Figure 26 shows the amino acid sequence and chemical structure of TG 17.
  • an IC 50 value is shown, reflecting the ability of TG17 to competitively inhibit the interaction of hTR and GRIPl.
  • Figure 27 shows the amino acid sequence and chemical structure of TG 14.
  • an IC 50 value is shown, reflecting the ability of TG14 to competitively inhibit the interaction of hTR and GRIPl.
  • Figure 31 shows the amino acid sequence and chemical structure of TG 8.
  • an IC 50 value is shown, reflecting the ability of TG8 to competitively inhibit the interaction of hTR and GRIPl.
  • the present invention provides methods and compositions for identifying compounds that modulate nuclear receptor activity.
  • the compounds can be nuclear receptor agonists or antagonists that bind to the coactivator binding site (and that act as mimetics to the coactivator in this regard), and promote (agonists) or block (antagonists) binding of the coactivator to the target nuclear receptor.
  • Compounds that bind to the coactivator binding site also are provided.
  • the compounds can be natural or synthetic.
  • Preferred compounds are small organic molecules, peptides and peptidomimetics (e.g., cyclic peptides, peptide analogs, or constrained peptides).
  • coactivator binding site is intended a structural segment or segments of nuclear receptor polypeptide chain folded in such a way so as to give the proper geometry and amino acid residue conformation for binding a coactivator. This is the physical arrangement of protein atoms in three-dimensional space forming a coactivator binding site pocket or cavity.
  • Residues forming the site are amino acids corresponding 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, He302, Leu305, Lys306), helix 6 (Cys309), and helix 12 (Leu454, Glu457, Val458 and Phe459).
  • the coactivator binding site is highly conserved among the nuclear receptor super family (Figure 19). Thus, 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 (He280, Val283, Val284, and Ala287), helix 4 (Phe293), helix 5 (He302 and Leu305), helix 6 (Cys309), and helix 12 (Leu454, Val458 and Phe459).
  • the hydrophobic cleft of the coactivator binding site also is highly conserved among the nuclear receptor super family ( Figure 19).
  • the invention also includes compositions and methods for identifying coactivator binding sites of nuclear receptors.
  • the methods involve examining the surface of a nuclear receptor of interest to identify residues and contiguous peptide sequences that modulate coactivator binding.
  • the residues and contiguous peptide sequences can be identified by homology to the coactivator binding site or by alignment of consensus sequences found in human TR described herein.
  • a preferred method is alignment with the residues or conserved peptide sequences of any nuclear receptor corresponding to (i.e., equivalent to) human TR residues of the C-terminal helix 3 (Ue280, Thr281, Val283, Val284, Ala287, and Lys288), helix 4 (Phe293), helix 5 (Gln301, He302, Leu305, Lys306), helix 6 (Cys309), and helix 12 (Pro453, Leu454, Glu457, Val458 and Phe459).
  • Overlays and superpositioning with a three-dimensional model of a'nuclear receptor LBD, or a portion thereof that contains a coactivator binding site also can be used for this purpose.
  • 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.
  • preferred mutations are of hydrophobic residues to charged residues (e.g., Arg, Lys, or Glu) on the basis that bulky, surface charged residues might disrupt coactivator binding, yet preserve global receptor structure and solubility, and of hydrogen bonding residues to alanine on the basis that such a mutation will preserve the global fold and local binding site shape, but alter the electrostatic components of coactivator binding.
  • Mutants can be tested for coactivator 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.
  • coactivator agonists or antagonists can be identified by providing atomic coordinates comprising a nuclear receptor coactivator binding site or portion thereof to a computerized or mechanical modeling system, modeling them, and identifying compounds that fit spatially into the coactivator binding site.
  • a "portion thereof is intended the atomic coordinates corresponding to a sufficient number of residues or their atoms of the coactivator binding site 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.
  • human TR residues V284, Phe293, He302, Leu305 and Leu454 contain side chain atoms that are within 4.5A, and interact with, hydrophobic residues of a (SEQ ID NO: 1) LxxLL motif of an NR-box 2 coactivator peptide.
  • an atomic structural model utilized for modeling and/or screening of compounds that bind to the coactivator binding site may include a portion of atomic coordinates of amino acid residues corresponding to the site composed of residues of human thyroid receptor selected from Val284, Lys288, Ile302, Lys306, Leu454 and Glu457, or their structural and functional equivalents found in other receptors.
  • 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 coactivator binding site or a subset of atoms useful in the modeling and design of compounds that bind to a coactivator binding site. It is also possible to identify components of the coactivator that are important for binding to nuclear receptors and to design molecules that will match those criteria. Programs that users skilled in the art would employ to perform such de novo design include programs such as LUDI, Leapfrog, Invention, and Hook.
  • the atomic coordinates of a compound that fits into the coactivator binding site 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-coactivator 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, as well as mechanical modeling. 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 coactivator binding site
  • Compounds of particular interest fit spatially, preferentially, and/or electrostatically into the coactivator binding site.
  • fit spatially and preferentially it is intended that a compound possesses a three-dimensional structure and conformation for selectively interacting with a nuclear receptor coactivator binding site.
  • fit electrostatically it is intended that a compound possesses a three-dimensional structure and conformation that matches the electrostatics of the binding site.
  • Compounds that fit spatially and preferentially into the coactivator binding site interact with amino acid residues forming the primarily hydrophobic cleft of this site.
  • the primarily hydrophobic cleft of the coactivator binding site comprises a small cluster of hydrophobic residues.
  • the present invention also includes a method for identifying a compound capable of selectively modulating coactivator binding to different nuclear receptors.
  • the method comprises the steps of modeling test compounds that fit spatially and/or electrostatically and preferentially into the coactivator binding site of a particular nuclear receptor of interest using atomic structural models of the nuclear receptors, choosing compounds that exhibit preferential modeling scores to the receptor of interest, screening the test compounds in a biological assay for nuclear receptor activity characterized by preferential binding of a test compound to the coactivator binding site 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 coactivator binding sites of one type of receptor versus a second type of receptor, such as the differences depicted in Figure 19.
  • Coactivators bound to different nuclear receptors have been shown to have similar alpha-helical conformations in the bound and unbound states.
  • the recognition that the helical geometry of the coactivator NR box remains constant during binding even though each coactivator binding site of the nuclear receptors is different allows for the targeting of all nuclear receptors with the same scaffolds and libraries rather than designing a separate scaffold or library for each nuclear receptor.
  • the design of the compounds, including peptides, peptidomimetics and small molecules is based on this helical coactivator structure.
  • the individual compounds that will bind each nuclear receptor will vary, but the general structure (i.e., the scaffold) will remain the same.
  • Stabilized ⁇ -helical peptides often utilizing macrolactams formed by the side chains of lysine and glutamic acid or aspartic acid in an i to i+3, i to i+4, or i to i+1 relationship, have been applied with some success to dissect the function of peptide hormone receptors.
  • a series of macrolactam GRIP 1 NR box compounds were synthesized by solid phase peptide synthesis using the Fmoc synthesis strategy with orthogonal protection of the relevant lactam precursor side chains.
  • this 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 TR subfamily which usually has several receptors that are encoded by at least two (TR: ⁇ , ⁇ ) or three (RAR, RXR, and PPAR: ⁇ , ⁇ , ⁇ ) genes or have alternate RNA splicing.
  • comparison of sequences of the GR and TR coactivator interaction surface shows a highly negatively charged sequence at the C-terminal end of TR helix 12 (E460 and D461) that is neutral in the equivalent positions in GR helix 12 (GR residues T788 and N759, corresponding to TR residue positions 460 and 461, as depicted in Figure 19).
  • the cocrystal of the hTR ⁇ LBD complexed with the GRIPl NR-box 2 peptide shows that TR residues E460 and D461 interact with positively charged residues of the NR-box 2 peptide.
  • TR but not TR is able to interact with peptides containing the hydrophobic interaction motifs of ⁇ 53 (SEQ ID NO: 3; FxxLW) and VP16 (SEQ ID NO: 4; FxxAL).
  • TR exhibits preferential interaction with NR-box peptides comprising the (SEQ ID NO: 1) LxxLL motif, but GR does not discriminate and can bind peptides containing a generic amphipathic helix motif. Accordingly, these real differences among the various nuclear receptors can be exploited in the identification and design of compounds that modulate coactivator binding to one nuclear receptor compared to another.
  • docking algorithms and computer programs that employ them can be used to identify compounds that fit into the coactivator binding site.
  • docking programs can be used to predict how a small molecule of interest can interact with the nuclear receptor coactivator binding site and to rank these existing compounds by estimated binding interactions.
  • Molecules or scaffolds for combinatorial libraries that are potentially new chemical entities can also be built de novo to fit inside the coactivator binding site, by arranging chemical fragments that complement the coactivator binding site to optimize intermolecular interactions.
  • scaffolds is meant the portion of the compounds in a library that remains constant in all the compounds. It is the molecule that is used to build a library.
  • a substituent is a variable, pendant group that is attached to the scaffold to form a compound in the library.
  • combinatorial library is meant a collection of compounds built by variation of substituents on a common scaffold. The combinatorial libraries are useful in both the discovery phase (for identifying lead compounds) and in the development phase (for optimizing certain properties).
  • Scaffolds for combinatorial libraries can be designed to deliver substitutents to subpockets in the coactivator binding site.
  • Substituents at positions in a combinatorial library built from these scaffolds can be selected to optimize interaction with a binding site structure.
  • This design approach has been made possible by identification of the coactivator binding site structure.
  • 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 coactivator binding site 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 additional 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 coactivator binding site of interest. In this screening, the quality of fit of such entities or compounds to the binding site may be judged by shape complementarity (DesJalais et al., J. Med. Chem. (1988) 37:722-729) or by estimated interaction energy (Meng et al., J. Comp. Chem.
  • the molecule databases include any virtual or physical database, such as electronic and physical compound library databases, and are preferably used in developing compounds and virtual combinatorial libraries that modulate coactivator binding. Libraries can consist of, for example, information regarding small organic molecules, peptides or peptidomimetics.
  • human TR residues V284, Phe293, Ile302, Leu305 and Leu454 contain side chain atoms that are within 4.5A, and interact with, hydrophobic residues of a (SEQ ID NO: 1) LxxLL motif of an NR-box 2 coactivator peptide.
  • a (SEQ ID NO: 1) LxxLL motif of an NR-box 2 coactivator peptide can be constructed.
  • Small organic molecules that mimic one or more of these particular interactions also can be designed, for example, by including one or more R- groups that are hydrophobic and fit into the site.
  • Specialized computer programs may also assist in the process of selecting chemical entity fragments or whole compounds. These include: GRID (Goodford, J. Med. Chem. (1985) 25:849-857; available from Oxford University, Oxford, UK); MCSS (Miranker et al., Proteins: Structure, Function and Genetics, (1991) 77:29-34; available from Molecular Simulations, Burlington, MA); AUTODOCK (Goodsell et al, Proteins: Structure, Function and Genetics (1990) 5:195-202; available from Scripps Research Institute, La Jolla, CA); and DOCK (Kuntz et al, J. Mol. Biol (1982) 767:269-288; available from University of California, San Francisco, CA).
  • compounds that bind to a coactivator binding site of interest also may be designed as a whole or de novo using either an empty coactivator binding site or optionally including some portion(s) of a molecule known to binds to the site, such as an NR-box type peptide.
  • LUDI Bohm, J. Comp. Aid. Molec.
  • LUDI is available from Biosym Technologies, San Diego, CA
  • LEGEND Nabata et al., Tetrahedron (1991) 47:8985
  • LEGEND is available from Molecular Simulations, Burlington, MA
  • LeapFrog available from Tripos Associates, St. Louis, MO.
  • entities may be designed by selecting a core chemical entity, or scaffold, that possess the correct geometry to present pendent groups, or substituents, to subpockets within the coactivator binding site.
  • Scaffolds can be designed to match vectors, geometrical descriptors of the arrangement of chemical fragments in space within the coactivator binding site, drawn from central areas of the binding site into pockets. These vectors can be derived from atoms in a bound ligand such as the GRTPl peptide or from the position of the pockets. Databases can be searched for molecules matching these vectors using programs such as CAVEAT. Virtual libraries built from those scaffolds, by adding sets of substituents can be evaluated by the computational methods described to test for expected fit to the coactivator binding site.
  • this method was applied to generate the virtual combinatorial libraries represented by the structures in Figure 23.
  • Two molecules were designed as scaffolds by carrying out a CAVEAT search of available databases. One of these molecules, was manually redesigned after visual inspection of its best docked configuration (shown in the left panel of Figure 24). The substituent attachment points of the molecule were modified to achieve better delivery of R groups to the target pockets.
  • a six-membered ring was removed to improve the shape complementarity of the molecule to the TR binding site and reduce the average molecular weight of the compounds in the libraries designed from this scaffold.
  • a five-membered ring was changed to a six-membered ring to facilitate synthesis.
  • a quaternary methyl group was added to block aromatization of the indole system.
  • Virtual combinatorial libraries were created by adding all combinations of substituents from Figure X to the Rl, R2, and R3 positions of scaffolds 1 and 2 in Figure 23. Libraries were created by adding the substituents to the R positions through linking groups. The groups at R4 are exposed to solvent in the predicted binding conformation of the scaffolds. The R4 positions of scaffolds with the highest scoring linking groups were later varied to alter the calculated logP of the compounds to improve their predicted pharmacological properties.
  • scaffolds have been designed to fit these criteria.
  • a set of scaffolds are determined. For example, two scaffolds are shown in Figure 24. These scaffolds have been fully optimized using this computational methodology. Their structures have been analyzed to optimize their fit into the coactivator binding site, and to determine the ability to synthesize compounds comprising these scaffolds. By slight variations in the computational methods, many more scaffolds become available.
  • the scaffolds will be synthesized to determine whether any compounds have the desired function.
  • the function is determined by using one of the assays herein described.
  • the compounds are tested for their ability to affect the interaction of receptor and coactivator.
  • a matrix is generated that delineates the effects of each substituent in each position. This data is further analyzed to discern which substituents confer the desired effects upon library members.
  • This set of relationships between structure of compound and effect perceived by assay is described as a "structure activity relationship," or S AR.
  • a set of covalently conformationally constrained peptides representing analogs of the second NR box of GRIPl has been designed.
  • covalently conformationally constrained is meant the covalent joining of amino acid side chain functionalities to restrict the conformations available to the peptide.
  • These peptides were synthesized using known methods [Peptide Synthesis Protocol] and were shown to have the properties described in Figure 20.
  • TG-17 peptide number 9
  • TG17 exhibited the degree of helical character expected of a mimic of the coactivator peptide structure.
  • Figures 25-32 represent examples of designed compounds. For each compound, the amino acid, chemical structure and IC 50 value are shown. The IC 50 value reflects the ability of the compound to competitively inhibit the interaction of hTR and GRIPl.
  • Compounds identified through modeling can be screened in an assay characterized by binding of the compound to a coactivator binding site of interest for coactivator binding activity, such as a biologically based assay. Screening can be in vitro and/or in vivo. Preferred assays include cell-free competition assays and cell culture based assays. The biological screening preferably centers on activity-based response models, binding assays (which measure how well a compound binds to the receptor), and bacterial, yeast and animal cell lines (which measure the biological effect of a compound in a cell). The assays can be automated for high capacity - high throughput screening (HTS) in which large numbers of compounds can be tested to identify compounds with the desired activity.
  • 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 coactivator protein, fragment, fusion or peptide thereof, to a coactivator binding site of interest.
  • a coactivator protein, fragment, fusion or peptide thereof to a coactivator binding site of interest.
  • cell and tissue culture assays they may be performed to assess a compound's ability to block function of cellular coactivators, such as members of the pl60 family of coactivator proteins, such as SRC-1, AIB1, RAC3, p/CIP, and GRIPl and its homologues TIF 2 and NcoA-2, and those that exhibit receptor and or isoform-specific binding affinity.
  • compounds of the invention bind to a nuclear receptor coactivator binding site with greater affinity than the cellular coactivator proteins.
  • Tissue profiling and appropriate animal models also can be used to select compounds. Different cell types and tissues also can be used for these biological screening assays. Suitable assays for such screening are described herein and in Shibata et al. (Recent Prog. Horm. Res. 52:141-164 (1997)); Tagami et al. (Mol. Cell Biol (1997) 77(5):2642-2648); Zhu et al. (J. Biol Chem. (1997) 272(14):9048-9054); Lin et al. (Mol. Cell Biol. (1997) 77(10):6131-6138); Kakizawa et al. (J. Biol. Chem.
  • coactivators or binding fragments thereof can be expressed and/or assayed for binding as for GRIPl (Hong et al., MCB supra; and Hong et al., PNAS supra) and/or SRC-1 (Spencer et al, Nature (1997) 359:194-198; Onate et al., Science (1995) 270:1354-1357), incorporated by reference.
  • This assay uses a fluorescently labeled peptide (peptide 2 as shown in Figure 20) which represents the second NR box of GRIPl as a probe for coactivator binding. Binding is detected by monitoring changes in fluorescence polarization of the probe. Potential agonists or antagonists are evaluated by their ability to displace this probe. Using this assay, the peptides shown in Figure 20 were evaluated. The results of this study are shown in Figure 21. TG-17, (peptide number 9) was shown to be a strong antagonist of coactivator binding.
  • 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 coactivator 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 conf ormational 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 coactivator binding site 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.
  • TR-based disorders including cardiovascular disease, metabolic disorders, hyperthyroidism, glaucoma and skin disorders.
  • GR-based disorders include Type IJ diabetes and inflammatory conditions such as rheumatic diseases.
  • the invention also provides for cocrystals made from nuclear receptor ligand binding domains with a molecule bound to the coactivator binding site.
  • TR LBDs are co-crystallized with a peptide molecule comprising a coactivator NR-box 2 peptide sequence bound to the coactivator binding site, and the hormone/ligand T 3 .
  • Crystals are made from purified nuclear receptor LBDs that are usually expressed by a cell culture, such as 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.
  • the machine readable data for the coactivator binding site comprises structure coordinates of amino acids corresponding to human TR amino, acids selected from 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), or a homologue of the molecule or molecular complex comprising the site.
  • the homologues comprise a coactivator binding site that has a root mean square deviation from the backbone atoms of the amino acids of not more than 1.5 A.
  • a preferred molecule or complex represents a compound bound to the coactivator binding site.
  • Representative nuclear receptors or their ligand binding domains have been cloned and sequenced: human RAR- ⁇ , human RAR- ⁇ , human RXR- ⁇ , human RXR- ⁇ , human PPAR- ⁇ , human PPAR- ⁇ , human PPAR- ⁇ , human VDR, human ER (as described in Seielstad et al, Molecular Endocrinol, (1995) 9:647-658, incorporated herein by reference), human GR, human PR, human MR, and human AR. The LBD for each of these receptors has been identified.
  • TR can be expressed alone, as fragments of the mature or full- length sequence, or as fusions to heterologous sequences.
  • TR 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 300 amino acids in length.
  • the TR LBD will be at least 150 amino acids in length, more preferably at least 200 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 Met 122 to Val 410 of the rat TR- ⁇ or Glu 202 to Asp 461 of the human TR- ⁇ .
  • 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).
  • SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis
  • MS mass spectrometry
  • HPLC hydrophobic high performance liquid chromatography
  • the purified LBD for crystallization should be at least 97.5 % pure, preferably at least 99.0% pure, 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 when re- chromatographed on the same column, ligand-bound receptor is separated from unliganded receptor.
  • concentrations of ligand are used in the column and the protein can be preincubated with the ligand prior to passing it over the column.
  • LBD typically purified LBD, such as TR 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.
  • a preferred peptide contains a NR-box (SEQ ID NO: 1) LxxLL motif, and suitable flanking sequences necessary for binding and forming complex with coactivator binding site of the nuclear receptor of interest, such as a TR LBD.
  • the binding peptides are then tested in crystallization trials at various concentrations and ratios of concentrations with a nuclear receptor of interest, for example, as described herein and in the Examples.
  • 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 TR LBD determined herein or previously by Wagner et al., supra. 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 coactivator compounds.
  • Human TR ⁇ LBD (His6-E202-D461) was expressed and purified as described (Shiau et al., Gene (1996) 779(2):205-10). Briefly, the protein was expressed from pET (e.g., pET3 and pET28) in BL21DE3 at 14°C, induced at OD(600nm) 0.7 with ImM IPTG and incubation was extended for 24 hours. Cells were harvested and lysed in 50mM sodium-phosphate buffer (pH 8.0), 0.3M NaCl, 10% glycerol, 25mM ⁇ - merceptoethanol and O.lmM PMSF as described above.
  • pET e.g., pET3 and pET28
  • the lysate was cleared by ultracentrifugation (Ti45, 36000 rpm, lh, 4°C), loaded on a Talon column equilibrated in the sodium phosphate buffer described above, washed with 12mM imidazole and eluted with an imidazole gradient (12 - 300 mM).
  • TR ⁇ LBD containing fractions were loaded in 0.6M ammonium sulfate on a TSK-phenyl hydrophobic interaction column and eluted with a reverse ammonium sulfate gradient [0.6 - 0 M] in 50% glycerol and 10% acetonitrile.
  • Thirty-seven thyroid receptor mutants were created by synthesizing double- stranded oligonucleotides which encode the mutant sequence and which have ends allowing them to be ligated as a cassette using pairs of the Nsil, Pstl, Sstl, AlwNI, Apol, PflMI, BstXI, BseRI, BsmFI, PvuJJ, Nspl, Smal, Pmll, BglU and Bsml restriction sites of the hTR ⁇ l cDNA sequence, or the 3' plasmid polylinker Sail, or BamHI restriction sites.
  • the hTR ⁇ l sequences thus mutated were subcloned into the pCMX vector encoding the full- length 461 amino acid hTR ⁇ l sequence.
  • Some of the mutations of the hTR ⁇ l in the CMX vector and all three mutations of the hER ⁇ in the pSG5-ER-HEGO vector were created using Quick Change Site-Directed Mutagenesis Kits (Stratagene). The mutated sequences were verified by DNA sequencing using Sequenase Kits (Stratagene).
  • the human ER ⁇ -LBD 297-554 was overexpressed as described previously (Seielstad, et al., supra) in BL21(DE3)pLysS cells transformed with a modified pET-23d- ERG vector that contained the sequence Met- Asp-Pro fused to residues 297 to 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. Sci. USA (1980) 77:5115-5119; Landel, et al., Mol. Endocrinol. (1994) 5:1407-1419; Landel, et al., J. Steroid Biochem. Molec. Biol. (1997) 63:59-73).
  • the bound hER ⁇ -LBD was treated with 5 mM iodoacetic acid in 10 mM Tris, pH 8.1, 250 mM NaSCN (Hegy, et al., Steroids (1996) (57:367-373). Protein was eluted with 3 x 10-5 M ligand (either DES or OHT) in 30-100 ml of 50 mM Tris, 1 mM EDTA, 1 mM DTT and 250 mM NaSCN, pH 8.5. The yield of hER ⁇ -LBD was typically close to 100% (Seielstad, et al., Biochemistry (1995) 34:12605-12615).
  • the affinity-purified material was concentrated and exchanged into 20 mM Tris, 1 mM EDTA, 4 mM DTT, pH 8.1 by ultrafiltration.
  • the protein was bound to a Resource Q column (Pharmacia) and then eluted with a linear gradient of 25-350 mM NaCl in 20 mM Tris, pH 8.1, 1 mM DTT.
  • 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 glutathione-S -transferase
  • 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 JJe 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.
  • Val 376- >Arg and Glu 542->Lys mutations were created in the GST-LBD construct by subcloning the Bsml Hindi ⁇ fragments of derivatives of pSG5-ER-HEGO (Tora, et al., supra) into which these mutations had already been introduced. All constructs were verified by automated sequencing (University of Chicago Cancer Research Center DNA Sequencing Facility). F. Radiolabeled full-length receptors and coactivator proteins
  • Wild-type (WT) or mutant pCMV-hTR ⁇ l vector and the pSG5-GPJPl and pCMX-SRC-la vectors were used to produce radiolabeled full-length receptors and coactivator proteins using the TNT coupled Reticulocyte Lysate System (Promega) and [ 35 S]-Met (DuPont).
  • GST-GRIPl amino acids 721-1221
  • GST-GRIPl amino acids 563- 1121
  • GST-SRC-la amino acids 381-882
  • GST- hTR ⁇ l full-length, WT or mutants, WT provided by. C. Costa
  • the GST-hRXR ⁇ full-length provided by. C. Costa
  • fusion proteins were produced in E. coli strain HB101 as per the manufacturer's protocol (Pharmacia Biotech).
  • GRIPl 563-767 was cloned as a Bam Hl-Xho I fragment derived from pG ⁇ X-2TK GPJPl 563-1121 into the corresponding sites of pGEX-4Tl.
  • a His6-tag was added by inserting a Xho I-Nae I fragment of pET23a into Xho I-Bsa Al sites of this pGEX- 4T1 construct yielding pGEX GRIPl 563-767His6.
  • Mutants of GRIPl 563-767 were generated by PCR or single stranded mutagenesis using oligonucleotides carrying the mutations and a pSG5 GRIPl vector as template.” The mutations were confirmed by sequence analysis and integrated into pGEX GRIPl 563-767His6 as NgoMI - Xho I fragments. The GRIPl 563-767 His6 GST fusion protein was expressed in HB101 at 37°C. Protein expression was induced with ImM JPTG at an optical density (600 nm) of 0.7 and extended for 4 hours after induction.
  • the fragment was associated with a 70 kDa protein which was removed by a MonoQ run in 50mM TrisHCl pH7.5, 10% glycerol, ImM EDTA, ImM DTT, O.lmM PMSF and protease inhibitors.
  • GRIPl 563-767His6 eluted in the flow through and was concentrated by ultrafiltration. At this step the protein was more than 95% pure.
  • Binding experiments were performed by mixing glutathione beads containing 10 ⁇ g of GST fusion proteins (Coomassie Plus Protein Assay Reagent, Pierce) with 1-2 ⁇ l of the [ 35 S]-labeled wild-type or mutant hTR ⁇ l (25 fmoles, 4000 cpm of receptor), or coactivators in 150 ⁇ l of binding buffer (20 mM HEPES, 150 mM KC1, 25 mM MgCl 2 , 10% glycerol, 1 mM dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride, and protease inhibitors) containing 2 mg/ml BSA for 1.5 hrs in the presence or absence of 1 ⁇ M T 3 . Beads were washed 3 times with 1 ml of binding buffer and the bound proteins were separated using 10% SDS-PAGE and visualized by autoradiography. Binding was quantitated by phosphorimaging using ImageQuant (Molecular Dynamics).
  • TR and their derivatives were translated in the presence of [ S]methionine using the TNT Coupled Reticulocyte System (Promega). Separate translations were performed in the presence and absence of lO ⁇ M dexamethasone or l ⁇ M RU486 for GR and lO ⁇ M triiodothyronine for TR. Expression was quantified by phosphoimager analysis (BAS2000, Fuji).
  • Example 3 A In vitro binding of [ 35 S]-labeled full-length GRIPl, [ 35 S]-labeled full-length SRC-la, and [ 35 S]-labeled full- length hRXR ⁇ , to GST- hTR ⁇ l wild-type (WT) and mutants was performed. Mutants V284R, K288A, I302R, L454R, and E457K all bound to hRXR ⁇ with an affinity equivalent to wild type hTR. All of these mutants showed decreased ability to bind GRIPl and SRC- la, as expected from the results of Example 3 A. The same results were obtained when a GST-SRC1 construct including SRC-la amino acids 381-882 was tested for binding of [ 35 S]-Met-labeled full-length hTR ⁇ 1 WT and mutants (data not shown).
  • the wild-type and mutant GST-hER ⁇ 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. (1988) 2:714-726) and protein levels were monitored by western blotting with a monoclonal antibody to hER ⁇ (H222).
  • Extract samples containing the GST- hER ⁇ 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 GRJJPl 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 as the template. Immobilized GST- hER ⁇ 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 35 S-GRIP1 was quantitated by fluorography following SDS-PAGE.
  • potential modulators can be evaluated by pre-equilibrating the receptor, probe, and hormone for as set period of time, for example, one hour. Then a set of samples can be titrated with increasing concentrations of each modulator, and evaluated for displacement of the probe after a set period of time, for example, one hour. All experiments can be carried out in quadruplicate, with each iteration containing the positive and negative competition controls and ten dose points. The data can then be analyzed using Klotz plots. IC50 values are determined using nonlinear regression analysis. Further analysis of binding data can involve Scatchard or Hill plots to verify the inhibitory constant and presence of a single class or multiple classes of binding sites. The various parameters of the experiment can be varied depending upon the dissociation constant of the receptor coactivator pair or the identity of the receptor or coactivator.
  • GRIPl a mouse pl60 coactivator, recognizes the ER ⁇ LBD in a ligand- dependent manner.
  • the binding of agonists to the ER ⁇ LBD promotes recruitment of GRIPl, whereas binding of antagonists prevents this interaction (Norris, et al., J. Biol. Chem. (1998) 273:6679-88). While agonist-bound receptor will bind to all three of the NR boxes from GRIPl, ER ⁇ strongly prefers NR-box 2 (Ding, et al., Mol. Endocrinol. (1998) 72:302-13).
  • An electrophoretic mobility shift assay was used to directly assess the ability of the NR-box 2 peptide to bind the purified ER ⁇ LBD in the presence of either DES or OHT.
  • Eight microgram samples of purified Her ⁇ -LBD bound to either DES or OHT were incubated in the absence of the peptide, i.e., buffer alone, or in the presence of either a 2- fold or 10-fold molar excess of the GRIPl NR-box 2 peptide.
  • 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).
  • this peptide fragment of GRTPl possesses the ligand-dependent receptor binding activity characteristic of the full-length protein.
  • hTR ⁇ l expression vector Pcmx-TR (WT or mutant), and 0.5 ⁇ g transfection control vector, Pj3LacZ, which contains the SV40 promoter linked to the ⁇ -galactosidase gene, were used.
  • Other cells co-transfected with vector or receptor constructs can be used for same purpose.
  • Alternative cells expressing sufficient levels of an endogenous receptor(s), or cells selected that express a single reporter, can be used for transfection assays, including MCF-7 cells expressing ER (Webb et al., supra) , and GC cells expressing TR (Norman et al., J. Biol. Chem. (1989) 26 " 4: 12063-12073).
  • T 3 binding affinity constants (Kd) for in vitro -translated WT and mutant TRs were measured using [ 125 I] 3,5,3' triiodo-L-thyronine ([ 125 I]T 3 ) in gel filtration binding assays as described (Apriletti et al., Protein Expr. Purif. (1995) 6:363). Both the Kd and standard enor (S.E.) values were calculated using the Prism computer program (GraphPad Software, Inc.). Mutations are indicated by the single-letter amino acid abbreviations, with the native residue name, followed by the primary sequence position number, and then the mutated residue name.
  • the affinity of the WT TR is 81 + 12 Pm.
  • the relative affinity was determined by dividing the WT Kd by each mutant Kd.
  • the 37 mutants tested with their relative affinities are: E217R (123%), E227R (109%), K242E (92%), E267R (117%), H271R (123%), T277R (7%), T281R (145%), V284R (105%), D285A (89%), K288A (98%), C294K (94%), E295R (118%), C298A (87%), C298R (141%), E299A (171%), I302A (86%), I302R (99%), K306A (6%), K306E (6%), P384R (164%), A387R (107%), E390R (151%), E393R (146%), L400R (95%), H413R (109%), H416R (153%), M423R (156%), R429A (48%), S437R (170%), L440R (174%), V444R (89%), T
  • Example 7 TR Residues Involved In Ligand-Dependent Transcription Activation In Context Of A
  • Residues involved in ligand-mediated transcription activation were identified by testing the TR mutants of Example 8 in HeLa cells.
  • T 3 increased reporter gene activity 5-fold in cells expressing either WT TR or mutated TRs showing normal GRIPl binding (representative mutants are shown in Figure 1.
  • TR mutants with diminished or absent GRIPl binding show a diminished or absent response to T 3 which correlates with the GRIPl binding defect.
  • Overexpression of GRTPl increases activation by the WT TR and rescues activation by TR mutants roughly in proportion to the severity of the defect of GRTPl binding and activation (Figure 2).
  • the ER mutants demonstrated a normal hormone-dependent ability to activate a vitellogenin-LUC hybrid reporter gene, GL45, which responds to the ER amino-terminal activation function (Berry et al., EMBO J (1990) 9:2811-2818) (data not shown).
  • GL45 vitellogenin-LUC hybrid reporter gene
  • Binding assays show that GRTPl NR-boxes 1, 2 and 3, interact differentially with hTR ⁇ LBD (Figure 7).
  • a GST-fusion of the GRTPl (563-767) fragment strongly binds TR (Kd or EC50 ) in a ligand depend fashion.
  • Replacement of the hydrophobic residues of NR-box 3 with alanine does not reduce binding of TR significantly, whereas similar replacement of NR-box 2 results in loss of TR binding of about 50%.
  • GR also was found to bind GRTPl (563-767) in a ligand-dependent manner ( Figure 8). However, in contrast to TR, extension of GRTPl (563-767) to residue 1121 increases binding to GR about 3 -fold suggesting an additional binding site on GRTPl for GR. Binding of the larger fragment remains ligand-dependent; no interaction can be observed in the presence of the GR partial antagonist RU486. These results are in agreement with in vivo 2-hybrid GR GRTPl interaction studies. In the presence of ligand no difference was detected in the binding of GRTPl by full length GR or a deletion mutant of GR that lacks the N-terminal activation domain AF-1.
  • NR-box 2 or 3 Peptides of NR-box 2 or 3 compete GRTPl (563-767) containing functional NR-boxes 2 and 3 or a mutant of this fragment that contains only a functional NR-box 2 with comparable affinity.
  • TR can bind both NR-box 2 and 3, in a GRTPl coactivator peptide fragment containing both boxes, TR preferentially binds NR-box 2.
  • the same types of assays for TR competition are performed to assess coactivator peptide binding affinity for GR.
  • the peptide concentrations are normalized relative to TR for obtaining comparable dose response curves.
  • NR-box 3 peptides (residues 8-24 of SEQ ID NO: 7) KENALLRYLLDKDDTKD and (residues 5-24 of SEQ JD NO: 7) PKKKENALLRYLLDKDDTKD).
  • a chimeric peptide containing the NR-box 3 motif in the context of the NR-box 2 flanking sequences (SEQ TD NO: 31; TSLKEKHKLLRYLLQDSS) binds like a NR-box 2 peptide.
  • the surface generated by the three conserved leucines (L690, L693, L694) of the NR-box 2 peptide (residues 12-24 of SEQ ID NO: 6) 686-KHKILHRLLQDSS-698 is highly complementary to the coreesponding binding site in the hTR ⁇ LBD ( Figures 16 and 17). Comparison of this binding site to other nuclear receptors shows that it contains a structural motif that is unique, highly conserved and present in all known structures of nuclear receptor LBDs (Wurtz et al., Nat Struct Biol.
  • complementarity of the interacting hydrophobic surfaces identified here seem to be a common feature of these interactions, cross-reactions between different motifs are possible. For instance, VP16, p53, and p65 (FxxFL) are able to functionally interact with TAFIT31, or p53 and E2F1-DP1 (FxxLL) both interact with MDM2. These interactions are sensitive to mutations in the Fxxxh motif. Therefore it appears that either complementarity of the hydrophobic surfaces is not an absolute requirement or that induced fitting of the interacting surfaces is possible.
  • GRTPl mutants changing NR-box 2 ( SEQ JD NO: 1; LxxLL) to VP16 ( SEQ ID NO: 4; FxxAL) or p53 (SEQ JD NO: 3; FxxLW) like binding sites also failed to bind TAFTI31 or MDM2 demonstrating that the presence of the correct binding site is not sufficient to create binding (data not shown).
  • peptides containing the VP16 or p53 binding sites are not able to compete the interaction of GRTPl with TR, even in very high concentration, but do compete the interaction with GR (data not shown).
  • the affinity of this interaction is weak, but comparable to affinity of a peptide of NR-box 2 that, in the context of a GRTPl mutant lacking NR-box 3, binds GR in vivo (Ding et al., supra). This binding is only about ten times less than a peptide containing NR-box 3, GR's primary binding site.
  • GR binds GRTPl (563-767) with about one-fifth the affinity than a comparable amount of TR.
  • the high concentration of NR-box 3 peptide required to compete the interaction of GR with GRTPl (563-767) may rather reflect a weak affinity of GR for the peptide rather than a particular strong interaction of GR with GRTPl (563-767).
  • the complex between the hTR ⁇ LBD and the GRTPl NR-box 2 peptide 686-KHKTLHRLLQDSS-698 was prepared by mixing (equal) volumes of a solution of 9mg ml hTR ⁇ LBD in 20Mm HEPES Ph 7.4 with a solution of 14 Mm GRTPl in 0.4Mm ammonium acetate Ph 4.72, and incubating the mixture on ice for 1 hour.
  • Translation function (t a , t b , ...) ⁇ h (
  • E 0 represents the normalized observed structure factor amplitudes
  • E c represents the normalized structure factors for the search model in a triclinic unit cell with dimensions identical to that of the crystal.
  • the reported peak height represents the value of the function for the translation (t a , t b ) of the NCS monomers, divided by the rms value of the translation function density.
  • R free is calculated the same as R factor, except only for 10% of the reflections that were set aside for cross validation and not used in refinement.
  • Crystals of a DES-Her ⁇ LBD-GRTPl NR-box 2 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 GRTPl NR- box 2 peptide 686-KHKTLHRLLQDSS-698 (residues 12-24 of SEQ JD NO: 6) for 7-16 hr.
  • 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 0.98 A.
  • the diffraction images were processed with DENZO and scaled with SCALEPACK (Otwinowski, et al., supra) using the default -3 ⁇ cutoff.
  • Both 2FoFc and FoFc electron density maps showed interpretable density, related by the NCS operator, near H12 of both molecules of the hTR ⁇ LBD.
  • the electron density could be modeled as a short ⁇ -helix, and the observed side chain density was used to tentatively assign the sequence and direction to the chain.
  • the refined model consists of residues of the hTR ⁇ LBD, and peptide residues of the GRTPl NR-box 2 peptide 686-KHKTLHRLLQDSS-698 (residues 12-24 of SEQ JD NO: 6).
  • Atomic coordinates of the hTR ⁇ LBD:GRP1 site 2 peptide complex are attached as Appendix 1.
  • refinement was carried out with REFMAC using tight NCS restraints.
  • the model was refined without NCS restraints using the simulated annealing, minimization 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 R free set contained a random sample of 6.5% of all data. In refinement, all data between 27 and 2.03 A (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, 164 waters, two carboxymethyl groups and a chloride ion.
  • 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.
  • Tie 689 from the peptide interacts with three receptor residues (Asp 538, Glu 542 and Leu 539).
  • the ⁇ -carboxylate of Glu 542 forms hydrogen bonds to the amides of residues 689 and 690 of the peptide.
  • a water-mediated hydrogen bond network is formed between the imidazole ring of His 377, the ⁇ -carboxylate of Glu 380, and the amide of Tyr 537.
  • Three 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 transcription activity of unliganded ER ⁇ LBD (Eng, et al., Mol. Cell. Biol.
  • the OHT complex data set was then collected.
  • molecular replacement in AmoRe was used to search for the location of LBD in this crystal form in both P6 t 22 and P6 5 22.
  • DMMULTI CCP4, 1994 was then used to project averaged density from the DES complex cell into the OHT complex cell.
  • MOLOC a model of the Her ⁇ LBD was built into the resulting density.
  • the final model consisted of residues 306-551, the ligand and 78 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.
  • Atomic coordinates of OHT-Her ⁇ LBD complex are attached as Appendix 3.
  • the asymetric unit (asu) of the crystal contains two monomers of the hTR ⁇ LBD and two molecules of the GRTPl NR-box 2 peptide 686-KHKTLHRLLQDSS-698 (residues 12-24 of SEQ JD NO: 6), which observes the NCS relation of the two TR monomers ( Figure 12).
  • the structure of the hTR ⁇ LBD which closely resembles that of the Rtr ⁇ LBD (Wagner et al., supra), consists of twelve alpha-helices and two ⁇ -strands organized in three layers, resembling an alpha-helical sandwich.
  • the only significant difference between the hTR ⁇ LBD and the Rtr ⁇ LBD is disorder in the loop between helices HI and H3.
  • the GRTPl NR-box 2 peptide forms an amphipathic ⁇ -helix of about 3 turns, preceded by 2 residues and followed by 3 residues in extended coil conformation.
  • the relation of the two monomers of the hTR ⁇ LBD is primarily translational, and does not resemble the homodimer structures reported for the Hrxr, or the Her (Bourguet et al., supra; Brzozowski et al., supra). Furthermore, the interface between the two monomers does not involve residues necessary for formation of the physiological TR dimer. Instead, one of the cocrystal peptides appears to bridge the interaction between the two monomers.
  • the hydrophobic face of the alpha-helix of the cocrystal peptide contacts monomer 1 of the hTR ⁇ LBD at H3, H5, and HI 2, while the hydrophilic face contacts monomer 2 at the hairpin turn preceding strand S3.
  • the second cocrystal peptide also contacts monomer 2 at H3, H5, and H12, and the two cocrystal peptides observe the same NCS relation as TR LBD monomers.
  • the common interface between both cocrystal peptides and the hTR ⁇ LBD buries the hydrophobic residues that define the cocrystal peptide (SEQ JD NO: 1) LxxLL sequence motif, residues Tle689, Leu690, Leu693, and Leu694; against the surface of the receptor LBD ( Figures 16 and 17).
  • the presence of the second peptide in the crystal, duplicating the interactions of the hydrophobic residues suggests those interactions are specific and drive the interaction of the peptide with the hTR ⁇ LBD, while the hydrophilic interactions provide a fortuitous crystal contact and account for the dependence of crystallization on the presence and concentration of the peptide.
  • the GRTPl NR-box 2 peptide used in the crystallization is 13 amino acids long (residues 12-24 of SEQ JD NO: 6; 686-K ⁇ KTLHRLLQDSS-698).
  • NR-box 2 peptide in monomer 1 (peptide 1), 12 amino acids are ordered in the crystal.
  • Residues K688 - Q694 form an amphipathic helix, with residues K686-H687 and D695-S698 on either end in extended coil conformations.
  • residues K688 - Q694 again form an amphipathic helix, but the ends of the peptide are disordered.
  • Stable helix formation may thus be induced by the interaction of the hydrophobic amino acids with the receptor LBD as it has been proposed in other proteimprotein interactions, such as p53:MDM2 (Kussie et al., Science (1996) 274:948-953), VP16:TAF31 (Uesugi et al., Science (1996) 277:1310-1313), and CREB:KIX-CBP (Radhakrishnan et al., Cell (1997) 97:741-752).
  • the hTR ⁇ LBD of the cocrystal contributes residues from three helices, H3, H5, and H12 to the interface, which pack against one another to create a hydrophobic cleft.
  • the residues lining the cleft are 1280, T281, V283, V284, A287, and K288 from H3; Q301, 1302, L305, and K306 from H5; and L454, E457, V458, and F459 from H12.
  • a cysteine residue (C309) from H6 appears to provide a partial surface that is buried deep within the bottom of the cleft.
  • the GRTPl NR-box 2 peptide 686-KHKILHRLLQDSS-698 (residues 12-24 of SEQ JD NO: 6) binds at the junction of H3 and H12.
  • Leu690 of the bound peptide inserts into a shallow but defined depression at the base of the cleft, making van der Waals contact with L454 and V458 of H12, while peptide residue Tle689 packs against L454 of H12 outside the edge of the cleft; L454, then, interdigitates between the two residues.
  • One further turn C-terminal along the alpha-helix, L693 and L694 of the bound peptide pack into complementary pockets within the hydrophobic cleft.
  • Peptide residue L693 forms van der Waals contact with V284 of H3, while peptide residue L694, bound more deeply in the cleft, makes contact with F298 and L305 of H4 and H5.
  • the hydrophobic interactions of the GRTPl NR-box 2 peptide with the hTR ⁇ LBD are observed for both cocrystal peptides 1 and 2 in their respective monomers of the crystal dimer complex, suggesting that the interactions are specific to the peptide, and not induced by crystallization.
  • the asymmetric unit of the DES-Her ⁇ LBD-NR-box 2 peptide 686- KHKTLHRLLQDSS-698 (residues 12-24 of SEQ JD NO: 6) complex crystals contains the same noncrystallographic dimer of LBDs that has been observed in the previously determined structures of the LBD bound to both E and RAL (Brzozowski, et al., supra and Tanenbaum, et al., supra). Beyond the flexible loops between helices 2 and 3 and helices 9 and 10, the two LBDs of the dimer adopt similar structures (r.m.s.d. 0.47 A 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.
  • the hydrophobic face of helix 12 is packed against helices 3,.5/6 and 11 covering the ligand binding pocket.
  • One NR-box 2 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.
  • the density for both peptides in the asymmetric unit is continuous and unambiguous.
  • Residues 687 to 697 from peptide A and residues 686 to 696 from peptide B have been modeled; the remaining residues are disordered. Given that each peptide lies within a different environment within the crystal, it is striking that from residues He 689 to Gin 695 each peptide forms a two turn, amphipathic ⁇ helix. Flanking this region of common secondary structure, the peptides adopt dissimilar random coil conformations.
  • the binding of OHT induces a conformation of the Her ⁇ 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 11 respectively.
  • these regions adopt an extended conformation in the OHT complex.
  • the composition and orientation of helix 12 are different in the two structures.
  • Helix 12 in the DES complex consists of residues 538 to 546 whereas helix 12 in the OHT complex consists of residues 536 to 544.
  • helix 12 in the OHT complex occupies the part of the coactivator binding groove formed by residues from helices 3, 4, and 5, and the turn connecting helices 3 and 4.
  • This alternative conformation of helix 12 appears to be similar to that observed in the RAL complex (Brzozowski, et al., supra).
  • nuclear receptors exemplified by TR, GR and ER
  • TR nuclear receptor
  • GR nuclear receptor
  • ER nuclear receptors
  • Identification and characterization of this coupling surface and the coactivator binding site of nuclear receptors offers a new target for the design and selection of compounds that modulate binding of coactivator to nuclear receptors.
  • Residues forming the coactivator binding site were found to cluster within a surprisingly small area with well-defined borders (see, e.g., Figures 5, 14, and 15). As is shown in above Examples, mutated residues nearby this area do not affect coactivator binding or transcriptional activation. Additionally, the coactivator binding assays and structural analyses demonstrated that NR-box containing proteins and peptides bind to this site. These results also showed that the GRTPl coactivator protein binds to the site through a highly (SEQ JD NO: 1) LxxLL.
  • residues which block GRTPl and SRC-1 coactivator binding when mutated are residues V284, K288, 1302, K306, L454, and V458. Mutated residues likely to undergo a conformational change upon hormone binding included Leu454 and Glu457.
  • the site identified by mutational, binding assays and crystallography conesponds to a surprisingly small cluster of residues on the surface of the LBD that define a prominent hydrophobic cleft formed by hydrophobic residues conesponding to human TR residues of C-terminal helix 3 (Tle280, Val283, Val284, and Ala287), helix 4 (Phe293), helix 5 (Lle302 and Leu305), helix 6 (Cys309), and helix 12 (Leu454, Val458 and Phe459).
  • residues forming the site are amino acids conesponding to human TR residues of C-terminal helix 3 OQe280, Thr281, Val283, Val284, Ala287, and Lys288), helix 4 (Phe293), helix 5 (Gln301, Tle302, Leu305, Lys306), helix 6 (Cys309), and helix 12 (Pro453, Leu454, Glu457, Val458 and Phe459).
  • the coactivator binding site is highly conserved among the nuclear receptor super family (Figure 19).
  • the coactivator binding site of TR contains charged and hydrophobic residues at its periphery, but only hydrophobic residues at its center (see, e.g., Figures 5 and 18).
  • the hydrophobic cleft at the center of the site may play a significant role in driving the coactivator binding reaction.
  • the site is comprised of two parts ( Figure 18), right). Residues contained in helices 3, 5 and 6 ( Figure 18, yellow residues) likely form a constitutive part, since their positions are identical in all nuclear receptor structures reported, including the liganded, activated states of the TR, RAR, and ER, the unliganded RXR, and the inhibitor-liganded ER.
  • the residues of helix 12 ( Figure 18, red residues) are differently positioned in the active and inactive states reported.
  • the coactivator binding site for the nuclear receptors is likely to be formed in response to an active hormone by positioning helix 12 against a scaffold formed by helices 3-6. Because the coactivator binding site is so small, it is easy to understand how even slight changes in the position of helix 12, which may, for example, be induced by an antagonist ligand, could impair coactivator binding, and thus receptor activation.
  • the NR-box 2 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.
  • the ER ⁇ LBD interacts primarily with the hydrophobic face of the NR-box 2 peptide 686-KHKTLHRLLQDSS-698 (residues 12-24 of SEQ JD NO: 6) ⁇ helix formed by the side chains of Tie 689 and the three (SEQ JD NO: 1) LxxLL motif 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.
  • 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.
  • the side chains of both De 689 and the second NR box leucine, Leu 693 rest against the rim of the groove.
  • the side chain of Tie 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 Tie 358 and Leu 539.
  • the ⁇ -amino group of Lys 688 of peptide B hydrogen bonds to the side chain carboxylate of Glu 380 of monomer B. This interaction is presumably a crystal artifact; the main chain atoms of the N-terminal three residues of peptide B are displaced from monomer B and interact extensively with a symmetry-related ER ⁇ LBD.
  • the ER ⁇ LBD stabilizes the main chain conformation of the NR box peptide by forming capping interactions with both ends of the peptide helix.
  • Glu 542 and Lys 362 are positioned at opposite ends of the peptide binding site.
  • the side chains of Glu 542 and Lys 362 form van der Waals contacts with main chain and side chain atoms at the N- and C- terminal turns of the peptide helix respectively. These interactions position the stabilizing charges of the ⁇ -carboxylate of Glu 542 and ⁇ -amino group of Lys 362 near the ends of the NR box peptide helix.
  • the side chain carboxylate of Glu 542 hydrogen bonds to the amides of the residues of N-terminal turn of the peptide helix (residues 688 and 689 of peptide A; residues 689 and 690 of peptide B).
  • the ⁇ -amino group of Lys 362 hydrogen bonds to the carbonyls of the residues of the C-terminal turn of the peptide helix (residue 693 of peptide A; residues 693 and 694 of peptide B).
  • the structure of the peptide binding groove of the ER ⁇ LBD is almost identical in the DES and OHT complexes.
  • 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 ((residues 6-10 of SEQ JD NO: 43) LLEML instead of (SEQ JD NO: 1) LxxLL).
  • the side chains of Leu 540 and Met 543 lie in approximately the same locations as those of the first and second motif leucines (Leu 690 and Leu 693) in the peptide complex.
  • Leu 540 is inserted into the groove and makes van der Waals contacts with Leu 354, Val 376 and Glu 380.
  • 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.
  • the side chain position of Leu 544 almost exactly overlaps that of the third NR box leucine, Leu 694. Deep within the groove, the Leu 544 side chain makes van der Waals contacts with the side chains of De 358, Lys 362, Leu 372, Gin 375, Val 376 and Leu 379.
  • Helix 12 in the OHT complex is also stabilized by N- and C-terminal capping interactions.
  • Lys 362 interacts with the C-terminal turn of helix 12 much as it does with the equivalent turn of the peptide helix.
  • 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.
  • the capping interaction at the N-terminal turn coactivator helix is formed by a helix 12 residue (Glu 542)
  • the N-terminal turn of helix 12 in the antagonist complex is forced to interact with another residue, Glu 380.
  • the 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.
  • TG-17 (9) was the only constrained peptide to exhibit significantly increased competitive ability relative to the linear probe.
  • the rest of the materials ranged from not significantly better than linear peptide 1 to significantly worse than 1.
  • TG-17 functions as a competitive inhibitor of GRTPl binding. Its synthesis was adapted to the format needed for the production of combinatorial libraries. The coupling reactions, deprotections, lactam formation, and cleavage reaction were optimized so that the material can be produced in good yield (approximately 60%) and purity (>85%) after cleavage and purification by precipitation in the block.
  • the parallel synthesis of ten samples of TG-17 carried out on the 50 ⁇ M scale in ten wells of a Robbins 96 well parallel synthesis apparatus. A set of peptides can be produced in less than a week. Analysis of these samples after cleavage and purification by precipitation revealed the same degree of purity as with other manual syntheses (>85%).
  • the resulting inhibitors were tested in the fluorescence assay, they revealed a high degree of homogeneity in response, showing less than twofold variation in IC 50 .
  • the effects of peptide length were also evaluated.
  • the constrained peptide can be shortened to a total length of 9 residues without any loss of binding affinity.
  • shortening the peptide can result in a substantial increase in the side reaction of dimerization during the macrolactam formation and also significantly lowers the efficiency of purification by precipitation.
  • the length of the peptide is as shown in TG- 17, which gives good purity peptide without requiring additional purification after precipitation.
  • the geometric information from the crystal structure of the GRTPl peptide bound to hTR ⁇ l was used to design chemical scaffolds that present pendant substituents to the three pockets normally occupied by the leucine side chains of the LxxLL triad.
  • CAVEAT was used to search databases of three-dimensional molecules using queries formed from geometric descriptors defined by vectors from the alpha and beta carbons of leucines 690, 693 and 694 of GRTPl. Searches were performed over databases obtained from Molecular Design Ltd. (MDL), the Available Chemical Directory (ACD), Pergamon's Comprehensive Medicinal Chemistry (CMC), and the MDL Drug Data Report (MDDR).
  • DOCK 4.0.1 was used to remove potential scaffolds that matched the vector queries but which had Van der Waals clashes with the receptors. This procedure generated several potential scaffolds of which two were chosen for further optimization (Figure 23).
  • the dibenzoazocine scaffold fits somewhat differently in the pocket with the scaffold itself binding into the hydrophobic cleft. In all three positions, the docking results favored a hydrocarbon linker with no apparent advantage being offered by heteroatoms or more complex linkages. In the Rl and R3 positions, the favored groups were aromatic rings, which could take advantage of some deep nanow pockets. All of the conesponding molecules that were visually inspected have the Rl substituent bound into the SI pocket of the protein. The pocket appears to be deeper than the R2 and R3 pockets. The R2 pocket benefited little from increased substitution with a simple ethyl group consistently providing the best average results.
  • the differences among binding pockets on different nuclear receptors were evaluated to determine how these differences could be utilized by synthetic inhibitors to provide specificity.
  • the structure of the nuclear receptor*coactivator complexes were aligned in a common frame of reference by performing an RMS fit of the alpha and beta carbons of the leucine residues of the LxxLL motif of the peptide coactivator.
  • Initial visual inspection of molecular surfaces, with potentials projected onto the surfaces, seemed to indicate that the receptor sites from hTR ⁇ l and PPAR ⁇ are quite similar to each other while that of hER ⁇ was quite distinct.
  • differences in the SI pocket may afford selective binding of ligands to these sites.
  • the SI pockets of hTR ⁇ l and PPAR ⁇ are distinctly different, with the PPAR ⁇ pocket being the larger of the two.
  • Computational docking studies have shown that hTR ⁇ l prefers planar rings such as benzene in this pocket, and that larger saturated rings such as cyclohexane do not score well.
  • Visual inspection of the S2 pocket of PPAR ⁇ suggests that rings the size of cyclohexane may preferentially bind there.
  • libraries of organic molecules with larger substituents at the PI position may bind PPAR ⁇ selectively over hTR ⁇ l.
  • the S 1 pocket of hER ⁇ is larger, shallower, and less distinct than the conesponding pockets of hTR ⁇ l and PPAR ⁇ .
  • the charge distribution on the surface of each of the proteins in the binding pocket ranges from neutral to electronegative.
  • the least apparent charge is located on the surface of PPAR ⁇ , while the greatest is located on hER ⁇ , suggesting polar groups and electrostatic interactions as potentially useful for differentiating between these pockets.
  • the S2 pocket is similar with respect to size, shape and charge for all three proteins.
  • the S2 pocket of hTR ⁇ l is longer than that of both PPAR ⁇ and hER ⁇ and may afford some advantage for selectivity.
  • the hER ⁇ protein appears to possess a secondary pocket between SI and S2 which suggests that larger, bifurcated substituents might be used at the R2 position of scaffolds to obtain specificity for the hER ⁇ receptor site.
  • the S2 pocket appears to possess a partial positive charge in all three protein-binding sites.
  • the S3 pocket of PPAR ⁇ is relatively indistinct with respect to shape when compared to conesponding pockets of the other two binding sites.
  • the hER ⁇ binding site contains a ridge between S3 and SI pockets that is not present in hTR ⁇ l.
  • the hER ⁇ S3 pocket is also slightly larger and deeper than the S3 pocket of hTR ⁇ l. Again, the potentials projected onto the surfaces of the S3 pockets appear to be quite similar. While the differences between the sites are sometimes subtle, compounds may be developed that bind selectively to each of these sites.
  • ATOM 8 CA PRO A 212 50.809 23. 379 40. 166 l.oo- 38. 35 6
  • ATOM 180 CA GLN A 235 20.562 24.363 ⁇ 25.629 1.00 76.32 6
  • CTl 4-. 4-. .t. ⁇ 1 ⁇ J CTl 4-. CTl ⁇ -> ⁇ -> ⁇ -J ⁇ j cn c ⁇ n NJ I- 1 !£> CTl c ⁇ -j ⁇ -» O -J cn cn ⁇ 1 45. c ⁇ C ⁇ 4 ⁇ .
  • ATOM 324 CA GLN A 252 16.541 50.930 15.207 1.00 74.10 6
  • G G G G G G C C G O O O 'U TJ O r O O T5 > M ( W ( N) I fO M ui ui ui n ui ui ui ui ui ui
  • CD NJ Cn J UJ t- 1 M -J 4 > en 00 0 00 4-. 0 cn t-> c ⁇ UJ 4s. ⁇ b ⁇ i H cn cn -J UJ c ⁇ ⁇ - j o c P 4 - > cn ⁇ ⁇ u J ⁇ uJ UJ ⁇ - > co o ⁇ 4 ⁇ 1 ⁇ i co ⁇ o c ⁇ cn cn cn -J ⁇ ⁇ OT ⁇ , o ⁇ , ⁇ ⁇ o c cn c c ⁇ ⁇ o ⁇ ⁇ c ⁇ o ⁇ c ⁇ ⁇ ⁇
  • ATOM 183 CE2 PHE A 286 27.307 29.233 39.387 1.00 38.08 6

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Abstract

Cette invention se rapporte à des procédés et à des composés agonistes/antagonistes servant à moduler la fixation de coactivateurs à des récepteurs nucléaires. Cette invention concerne un procédé servant à identifier des résidus comprenant un site de liaison de coactivateurs pour un récepteur nucléaire examiné. Cette invention concerne également un procédé servant à identifier des agonistes et/ou des antagonistes qui se fixent à un site de fixation de coactivateurs d'un récepteur nucléaire examiné. Des agonistes et des antagonistes de fixation de coactivateurs à des récepteurs nucléaires sont également présentés. Cette invention fournit des exemples d'identification et de manipulation du site de fixation de coactivateurs aux récepteurs thyroïdiens (TR), et des composés qui se fixent à ces sites. Ces procédés peuvent s'appliquer à d'autres récepteurs nucléaires, tels que RAR, RXR, PPAR, VDR, ER, GR, PR, MR et AR.
EP01950770A 2000-06-30 2001-06-29 Procedes et composes pour moduler la fixation de coactivateurs a des recepteurs nucleaires Withdrawn EP1297175A4 (fr)

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US20040005636A1 (en) * 2002-04-15 2004-01-08 Guy Rodney Kiplin Method for obtaining the binding affinities of a peptide library to a protein
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WO1999050658A2 (fr) * 1998-03-30 1999-10-07 The Regents Of The University Of California Methodes et composes de modulation d'activite de recepteurs nucleaires
WO1999060014A2 (fr) * 1998-03-30 1999-11-25 The Regents Of The University Of California Methodes et composes de modulation de la liaison d'un coactivateur a des recepteurs nucleaires

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GB9416571D0 (en) * 1994-08-16 1994-10-12 Battelle Memorial Institute Novel alkylamino derivatives as sigma 2 selective ligands
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WO1999027365A1 (fr) * 1997-11-21 1999-06-03 Tularik Inc. Cribles pour medicaments recepteurs de l'hormone nucleaire
WO1999050658A2 (fr) * 1998-03-30 1999-10-07 The Regents Of The University Of California Methodes et composes de modulation d'activite de recepteurs nucleaires
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CHANG C Y ET AL: "Dissection of the LXXLL nuclear receptor-coactivator interaction motif using combinatorial peptide libraries: discovery of peptide antagonists of estrogen receptors alpha and beta.", MOLECULAR AND CELLULAR BIOLOGY. DEC 1999, vol. 19, no. 12, December 1999 (1999-12-01), pages 8226 - 8239, XP001120691, ISSN: 0270-7306 *
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