KR20010042373A - Methods and compounds for modulating nuclear receptor activity - Google Patents

Abstract

The present invention relates to methods and agonists / antagonists for modulating nuclear receptor activity and nuclear receptor ligand binding. The present invention relates to a method for identifying residues comprising a ligand binding domain for a nuclear receptor of interest. The invention also includes methods for identifying agonists and / or antagonists that bind to the ligand binding domain of nuclear receptors, in particular estrogen receptors. The present invention is exemplified by the identification and manipulation of ligand binding domains of estrogen receptors and compounds that bind to these sites. The method of the present invention can be applied to other nuclear receptors including TR, GR and PR.

Description

METHODS AND COMPOUNDS FOR MODULATING NUCLEAR RECEPTOR ACTIVITY

SEQUENCE LISTING

〈110〉 Shiau, Andrew

Kushner, Peter J

Agard, David A

Greene, Geoffrey L

〈120〉 Methods and Compounds for Modulating Nuclear Receptor

Activity

<130> UCAL 256 / 01WO

〈140〉

141

〈150〉 60 / 079,956

〈151〉 1998-03-30

〈150〉 60 / 113,146

(151) 1998-12-16

〈150〉 60 / 113,014

(151) 1998-12-16

〈160〉 26

<170> Patent In Ver. 2.0

〈210〉 1

<211> 5

<212> PRT

〈213〉 Homo sapiens

〈220〉

〈221〉 BINDING

222 (1) .. (5)

〈223〉 residues 2 and 3 can be any amino acid

<400> 1

Leu Xaa Xaa Leu Leu

1 5

〈210〉 2

<211> 5

<212> PRT

〈213〉 Homo sapiens

〈220〉

〈221〉 BINDING

222 (1) .. (5)

〈223〉 residues 2 and 3 can be any amino acid

<400> 2

Leu Xaa Xaa Met Leu

1 5

〈210〉 3

<211> 5

<212> PRT

〈213〉 Homo sapiens

<400> 3

Leu Leu Gln Met Leu

1 5

〈210〉 4

<211> 13

<212> PRT

〈213〉 Homo sapiens

<400> 4

Lys His Lys Ile Leu His Arg Leu Leu Gln Asp Ser Ser

1 5 10

〈210〉 5

<211> 33

<212> PRT

〈213〉 Homo sapiens

<400> 5

Thr Pro Ala Ile Thr Arg Val Val Asp Phe Ala Lys Lys Leu Pro Met

1 5 10 15

Phe Cys Glu Leu Pro Cys Glu Asp Gln Ile Ile Leu Leu Lys Gly Cys

20 25 30

Cys

〈210〉 6

<211> 12

<212> PRT

〈213〉 Homo sapiens

<400> 6

Leu Phe Pro Pro Leu Phe Leu Glu Val Phe Glu Asp

1 5 10

〈210〉 7

<211> 33

<212> PRT

〈213〉 Homo sapiens

<400> 7

Thr Pro Ala Ile Thr Arg Val Val Asp Phe Ala Lys Lys Leu Pro Met

1 5 10 15

Phe Ser Glu Leu Pro Cys Glu Asp Gln Ile Ile Leu Leu Lys Cys Cys

20 25 30

Cys

〈210〉 8

<211> 12

<212> PRT

〈213〉 Homo sapiens

<400> 8

Leu Phe Pro Pro Leu Phe Leu Glu Val Phe Glu Asp

1 5 10

〈210〉 9

<211> 33

<212> PRT

〈213〉 Homo sapiens

〈400〉 9

Thr Lys Cys Ile Ile Lys Ile Val Glu Phe Ala Lys Arg Leu Pro Gly

1 5 10 15

Phe Thr Gly Leu Ser Ile Ala Asp Gln Ile Thr Leu Leu Lys Ala Ala

20 25 30

Cys

<210> 10

<211> 12

<212> PRT

〈213〉 Homo sapiens

<400> 10

Pro Met Pro Pro Leu Ile Arg Glu Met Leu Glu Asn

1 5 10

<210> 11

<211> 33

<212> PRT

〈213〉 Homo sapiens

<400> 11

Asp Lys Gln Leu Phe Thr Leu Val Glu Trp Ala Lys Arg Ile Pro His

1 5 10 15

Phe Ser Glu Leu Pro Leu Asp Asp Gln Val Ile Leu Leu Arg Ala Gly

20 25 30

Trp

<210> 12

<211> 12

<212> PRT

〈213〉 Homo sapiens

<400> 12

Pro Ile Asp Thr Phe Leu Met Glu Met Leu Glu Ala

1 5 10

〈210〉 13

<211> 33

<212> PRT

〈213〉 Homo sapiens

<400> 13

Val Glu Ala Val Gln Glu Ile Thr Glu Tyr Ala Lys Asn Ile Pro Gly

1 5 10 15

Phe Ile Asn Leu Asp Leu Asn Asp Gln Val Thr Leu Leu Lys Tyr Gly

20 25 30

Val

〈210〉 14

<211> 12

<212> PRT

〈213〉 Homo sapiens

<400> 14

Ser Leu His Pro Leu Leu Gln Glu Ile Tyr Lys Asp

1 5 10

<210> 15

<211> 33

<212> PRT

〈213〉 Homo sapiens

<400> 15

Ser Tyr Ser Ile Gln Lys Val Ile Gly Phe Ala Lys Met Ile Pro Gly

1 5 10 15

Phe Arg Asp Leu Thr Ser Glu Asp Gln Ile Val Leu Leu Lys Ser Ser

20 25 30

Ala

<210> 16

<211> 12

<212> PRT

〈213〉 Homo sapiens

<400> 16

Lys Leu Thr Pro Leu Val Leu Glu Val Phe Gly Asn

1 5 10

〈210〉 17

<211> 33

<212> PRT

〈213〉 Homo sapiens

〈400〉 17

Asp Arg Glu Leu Val His Met Ile Asn Trp Ala Lys Arg Val Pro Gly

1 5 10 15

Phe Val Asp Leu Thr Leu His Asp Gln Val His Leu Leu Glu Cys Ala

20 25 30

Trp

〈210〉 18

<211> 12

<212> PRT

〈213〉 Homo sapiens

〈400〉 18

Pro Leu Tyr Asp Leu Leu Leu Glu Met Leu Asp Ala

1 5 10

〈210〉 19

<211> 33

<212> PRT

〈213〉 Homo sapiens

〈400〉 19

Gly Arg Gln Val Ile Ala Ala Val Lys Trp Ala Lys Ala Ile Pro Gly

1 5 10 15

Phe Arg Asn Leu His Leu Asp Asp Gln Met Thr Leu Leu Gln Tyr Ser

20 25 30

Trp

<210> 20

<211> 12

<212> PRT

〈213〉 Homo sapiens

<400> 20

Glu Phe Pro Glu Met Leu Ala Glu Ile Ile Thr Asn

1 5 10

〈210〉 21

<211> 33

<212> PRT

〈213〉 Homo sapiens

<400> 21

Glu Arg Gln Leu Leu Ser Val Val Lys Trp Ser Lys Ser Leu Pro Gly

1 5 10 15

Phe Arg Asn Leu His Ile Asp Asp Gln Ile Thr Leu Ile Gln Tyr Ser

20 25 30

Trp

<210> 22

<211> 12

<212> PRT

〈213〉 Homo sapiens

<400> 22

Glu Phe Pro Glu Met Met Ser Glu Val Ile Ala Ala

1 5 10

<210> 23

<211> 33

<212> PRT

〈213〉 Homo sapiens

<400> 23

Gly Lys Gln Met Ile Gln Val Val Lys Trp Ala Lys Val Leu Pro Gly

1 5 10 15

Phe Lys Asn Leu Pro Leu Glu Asp Gln Ile Thr Leu Ile Gln Tyr Ser

20 25 30

Trp

<210> 24

<211> 12

<212> PRT

〈213〉 Homo sapiens

<400> 24

Glu Phe Pro Ala Met Leu Val Glu Ile Ile Ser Asp

1 5 10

<210> 25

<211> 33

<212> PRT

〈213〉 Homo sapiens

<400> 25

Glu Arg Gln Leu Val His Val Val Lys Trp Ala Lys Ala Leu Pro Gly

1 5 10 15

Phe Arg Asn Leu His Val Asp Asp Gln Met Ala Val Ile Gln Tyr Ser

20 25 30

Trp

〈210〉 26

<211> 12

<212> PRT

〈213〉 Homo sapiens

<400> 26

Asp Phe Pro Glu Met Met Ala Glu Ile Ile Ser Val

1 5 10

Cells contain receptors that can bind to various molecules, including proteins, hormones and / or agents, to eliminate biological responses. Nuclear receptors represent a superfamily of proteins that are hormone / ligand activated transcription factors that enhance or inhibit transcription by cell type dependent methods, ligand dependent methods, and promoter dependent methods. Conventional nuclear receptors, estrogen receptor α (ERα), are key factors that regulate the differentiation and maintenance of nerves, bones, cardiovascular, and replicating tissue (Korach, Science 266: 1524-1527 (1994); Smith, et al. , New Engl. J. Med. 331: 1056-1061 (1994)). Nuclear receptor families also include receptors for glucocorticoids, androgens, mineral corticoids, progestins, thyroid hormones, vitamin D, peroxysome proliferators and icosanoids. A subset of the nuclear receptor family is steroidal receptors, which include estrogens, glucocorticoids and progestin receptors.

Overall sequence conservation on nuclear receptors depends on the type of receptor family; However, sequence conservation rates between functional regions of modules or between modules are high. For example, the nuclear receptor can be bound into a functional module comprising an N-terminal transcriptional activation domain, a central DNA binding domain (DBD), and a C-terminal ligand binding domain (LBD). The LBD module of the nuclear receptor represents a hormone / ligand dependent molecular switch and recognizes many compounds that differ in size, shape, and chemical properties. Thus, estrogen hormones exhibit their physiological effects by binding to estrogen receptors (BEATRO, et al., Cell 83 (6): 851-857 (1995): Tsai. Et al., Annu. Rev. Biochem. 63 451-86 (1994). The binding of the hormone to the LBD of the nuclear receptor also changes its ability to regulate DNA transcription, although the following changes are transcription dependent.

All ERα ligands bind exclusively with C-terminal LBD. Some of these ligands include endogenous estrogens, 17-estradiol (E ₂ ), and synthetic nonsteroidal estrogens, diethylstilbestrol (DES) and act as pure agents, such as, for example, ICI-164,384. Others act as pure antagonists. Synthetic ligands For example, ligands such as tamoxifen and raloxifene (RAL) belong to proliferation-based molecules known as selective estrogen receptor modulators (SERMs), which act as antagonists in certain tissues and promoter contexts (Grese, et al. , Proc. Natl. Acad. Sci. USA 94: 14105-10 (1997). The excellent tissue specific propensity of tamoxifen has been demonstrated in recently reported breast cancer prevention trials (Samigel, J. Natl. Cancer Inst, 90: 647-8) (1998), where breast cancer has been treated with tamoxifen for 6 years Groups of women at high risk of developing endometrial cancer showed an increased incidence of endometrial cancer, but a reduction in the incidence of certain fractures and a dramatic reduction in breast cancer by 45%. The design of new SERMs and the optimization of the source of exposure require an understanding of the effects of different ligand chemistries and structures on ERα transcriptional activity.

Nuclear receptors also bind to proteins such as chaperone complexes, cosuppressants, or coactivators that are involved in receptor function. In particular, ligand dependent activation of transcription by nuclear receptors is regulated by interaction with other active agents. Receptor agonists promote coactivator binding and antagonist block coactivator binding. Hormonal binding by nuclear receptors may reduce or increase binding affinity for these proteins and may affect or regulate multiple actions on the transcription of nuclear receptors.

Transcriptional activity by ERα is regulated by two or more separate active functions (AFs) located in different domains of the protein, with AF-1 at the N-terminus and AF-2 at the LBD. These AFs can act independently or in concert depending on the cell type and promoter context. The AF-1 is regulated by the MAP kinase pathway (Kato, et al., Science 270: 1491-1494 (1995)) and is generally believed to be activated by ligand dependent methods, whereas AF-2 activity (" Transcriptional activity ") is reactive to ligand binding (Kumar, et al., Cell 51 (6): 941-951 (1987)). Binding of agents causes transcriptional activity, whereas binding of antagonists is not (Berry, et al., EMBO J. 9: 2811-8 (1990)). In addition, coactivators modulate transcriptional activity. The structure and functional properties of the site to which the coactivator binds have only recently been discovered. US Provisional Application No. 60 / 079,956, filed March 30, 1998, filed by Aprili et al., Incorporated herein by reference.

Recent studies suggest that ligands regulate transcriptional activity by directly affecting the structure of LBD. Human retinoid X receptor α LBD without ligand binding (Bourguet, et al., Nature 375: 377-82 (1995)) and human retinoic acid receptor γ (RARγ) (Renaud, et al., Nature 378: 681-689 (1995) ) And Wurtz, et al., Nat. Struct. Biol. 3: 87-94 (1996)), thyroid hormone receptor α (TRα) (Wagner, et al., Nature 378: 690-697 (1995)), Progesterone receptor (Williams, et al., Nature 393: 392-395 (1998)) and ERα (Brzozowski, et al., Nature 389: 753-758 (1977); Tanenbaum, et al., Proc. Natl. Acad. Comparison of ligand-bound LBDs of Sci. USA 95: 5998-6003 (1998) suggests that structural changes induced by agents involving the rearrangement of helix 12, the C-terminal helix of LBD, are essential for transcriptional activity. Since any point mutation in Helix 3, 5, and 12 eliminates transcriptional activity but does not affect ligand or DNA binding, this region of LBD is produced in the presence of an agent, linking a general transcriptional mechanism with a receptor. The state was expected to form part of the recognition surface for the molecule (Danielian, et al., EMBO J. 11: 1025-33 (1992); Feng, et al., Science 280: 1747-9 (1998); Henttu , et al., Mol. Cell. Biol. 17: 1832-9 (1997); Wrenn et al., Biol. Chem. 268: 24089-24098 (1993)). The structure of LBD complexed with E ₂ and RAL is that each of these ligands forms a different conformation of helix 12, even though both ligands bind to the same site in the center of the LBD (Brozozowski, et al., Supra). Suggested induction. Helix 12 in the E ₂ -LBD complex packs for helix 3, 5/6, and 11 in the observed configuration for the corresponding helix in the NR LBD structure of the other agent bond, whereas in the RAL-LBD complex Helix 12 binds into hydrophobic grooves consisting of residues from Helix 3 and 5. This selective orientation of Helix 12 partially incorporates residues necessary for transcriptional activity into the grooves, suggesting that RAL and other possible antagonists block transcriptional activity by damaging the topography of the coactivator binding site surface.

Biochemical and genetic approaches are described in SRC-1 / N-CoA1 (Onate, et al., Science 270: 1354- 1357 (1995)), GRIP1 / TIF2 / SRC-2 (Hong, et al., Proc. Natl. Acad. Sci. USA 93 (10): 4948-4952 (1996) and Vogel, et al., EMBO J. 15: 3667-3675 (1996)), p / CIP / RAC3 / ACTR / AIBI / SRC-3 ( Anzick, et al., Science 277: 965-968 (1997), Chen, et al., Cell 90 (3): 569-80 (1997), Li et al., Proc. Natl. Acad. Sci. USA 94 : 8479-84 (1997) and Torchia et al., Nature 387: 677-684 (1997) and CBP / p300 (Hanstein, et al., Proc. Natl. Acad. Sci. USA 93: 11540-11545 (1996) The identification of several proteins associated with ERα and ligand-dependent methods has resulted in the identification of ERα and several other NRs (Glass et al., Curr. Opin. Cell Biol. 9: 222-32, supra). (1997): It is classified as a transcriptional adjuvant because it enhances ligand dependent transcriptional activity by Torchia, et al .. The cleaved SRC-1 gene (Xu, et al., Science 279: 1922-1925 (1998)) Partial horde in mouse Observation of resistance is strong evidence that co-activators are required for the action of NR in the body, with the proposed role in AF-2 leading to transcriptional activity, SRC-1 is not only a histone acetylase activity and agonist binding receptor. But also has the ability to interact with other co-activators and several conventional transcription factors (Kamei, et al., Cell 85 (3): 403-14 (1996); Onate, et al .; Spencer, et al. , Nature 389: 194-8 (1977); Takeshita, et al., Endocrinology 137: 3594-7 (1996)). SRC-1 and GRIP1 also bind to agonist binding LBDs of both human TRβ and ERα using putative coactivator binding sites (Feng, et al., Supra).

Members of the p160 family of coactivators, for example SRC-1, GRIP1 / TIF2 / SRC-2, and p / CIP / RAC3 / ACTR / AIB1 / SRC-3 and other coactivators, are short signature sequence motifs known as NR boxes. Recognize agonist binding NR LBDs via LXXLL (SEQ ID NO: 1), where L is leucine and X is any amino acid (Ding, et al., Mol. Endoocrinol. 12: 302). -313 (1998); Heery, et al., Nature 387: 733-736 (1997); Le Douarin, et al., EMBO J. 15: 6701-15 (1996); Torchia, et al.). The study of mutations indicates that the affinity of the coactivator for LBDs is determined primarily by, but not limited to, these NR boxes (DING, et al., Supra). Each of the p160 coactivators contains several NR boxes. It has been found that the NR boxes in SRC-1, GRIP1, and TIF recognize different NRs with different affinity (Ding, et al .; Kalkhoven, et al., EMBO J. 17: 232-43 (1998). Vogel, et al., EMBO J. 17: 507-19 (1998), the reason for these binding affinity is unknown.

Darimont, et al., "Structure and specificity of nuclear receptor-coactivator interactions" Genes Dev. 12: 3343-3356 (1998), provides a structural study of the complex between TRβ and GRIP1 NR Box II peptides, and TRβ and Describe biochemical studies of GRIP1 in combination with GR.

Nuclear receptors are of great medical importance. They are involved in breast cancer, prostate cancer, arrhythmia diseases, infertility, osteoporosis, hyperthyroidism, hypercholesterolemia, obesity, and other diseases. For example, compounds that modulate ERα transcriptional activity are currently used in the treatment of osteoporosis, cardiovascular disease, and breast cancer (Gradishar, et al., J. Clin. Oncol. 15: 840-52 (1997) and Jordan. J. Natl. Cancer Inst. 90: 967-71 (1998).

There is a need for further identification and characterization of key residues in the ligand binding domains of nuclear receptors, and molecules affected by defects in these binding sites. Understanding these interactions provides the basis for iterative drug design, synthesis, and selection. It is also advantageous to design methods and compositions to reduce the time required to find compounds targeting these binding sites, and to administer the compositions to organs to control physiological processes regulated by nuclear receptors, particularly estrogen receptors. Will be.

The present invention relates to improved methods and compounds for the regulation of nuclear activity. In particular, the present invention relates to methods and compounds for the modulation of estrogen receptor activity.

1 is a stereogram of the electron density of the complex, Figure 1a is a stereogram of the electron density of the DES-ERα LBD-GRIP1 NR Box II peptide complex, Figure 1b is a stereogram of the electron density of the OHT-ERα LBD complex It is also. 1 is generated by BOBSCRIPT (Esnouf, J. Mol. Graph. Model. 15, 132-4. 112-3 (1997)), and Raster3D (Merritt, et al., Acta Crystallogr. D 50: 869-873 (1994)) is black and white graphics.

2 is generated by BOBSCRIPT as described above and created using Raster3D. 2A shows the overall structure of the DES-ERα LBD-GRIP1 NR Box II peptide complex in two orthogonal degrees. FIG. 2B shows the overall structure of the OHT-ERα LBD complex in two orthogonal views similar to the diagram for the agent complex of FIG. 2A.

3A and 3B are generated by BOBSCRIPT and created using Raster3D. 3C and 3D were generated using GRASP (Nicholls, GRASP Manual (New York: Columbia University, 1992)). FIG. 3A is an enlarged view of a coactivator, NR Box II peptide / LBD interface, associated with LBD. FIG. LBD regions that do not interact with the peptide were removed for clarity. Helix 3, 4, and 5 are labeled H3, H4, and H5, respectively. The side chains of the receptor residues interacting with the peptide are shown except Lys362 (blue) and Glu542 (red), and the side chains were colored according to the type of atom (green for carbon and sulfur atoms, red for oxygen atoms, and Nitrogen atom was blue). Helix 12 is fuchsia. Peptides that are gold are presented as Cα worms: only the side chains of Ile 689 and the three motifs leucine (Leu 690, Leu 693, and Leu 694) are shown (FIG. 3C). 3B shows Helix 12 / LBD as an enlarged view of OHT-LBD showing Helix 12 bound to a portion of the coactivator binding site. Only the side chains of residues interacting with helix 12 are shown (side chains of H373 are omitted for clarity). Except for Lys362 (blue) and Glu380 (red), the side chains varied in color depending on the atom as specified in FIG. 3A. Residues 530-551 represented the Cα worm; Residues 536-544 are magenta. Side chains of Leu 536, Tyr 537, Leu 540, Met 543, and Leu544 are shown. FIG. 3C shows a molecular surface showing the electrostatic surface of ERα LBD molecules bound to NR box II in the positive (blue) and negative (red) regions, calculated by GRASP. The coactivator peptide is shown in FIG. 3A, which is equivalent to FIG. 3A. The side chains of Leu690 and Leu694 are combined with hydrophobic grooves, and the side chains of Ile689 and Leu693 lie at the ends of the grooves. FIG. 3D shows the electrostatic surface of ERα LBD complexed with OHT and shows the positive (blue) and negative (red) regions as in FIG. 3C. Residues 530-511 are shown as in FIG. 3B, which is equivalent to that of FIG. 3B. The side chains of Leu540 and Leu544 are incorporated into the hydrophobic grooves, while the side chains of Met543 lie along the ends of the grooves.

FIG. 4 is generated using LIGPLOT (Wallace, et al., Protein Eng, 8: 127-34 (1995)) and depicts the interaction of LBD with DES (FIG. 4A) and OHT interaction with ligand binding pockets. Schematic diagram (FIG. 4B). Residues that interact with the ligand are shown at approximate actual positions. Residues that form van der Waals bonds with ligands are represented by labeled arcs with radial spokes facing the ligand atoms with which the surface interacts. These residues hydrogenated to the ligand are represented by spheres and rods. Hydrogen bonds are indicated by dashed blue lines and the spacing of each bond is indicated. Ligand rings and independent ligand atoms were labeled.

FIG. 5 shows a comparison of Helix 12 with NR Box II peptides generated using BOBSC0RIPT and generated using Raster3D as above, from the OHT complex. 5A and 5B are three-dimensional views. The structures of the OHT-LBD complex and the DES-LBD-NR Box II peptide complex were overlapped using the Cα coordinates of residues 306-526 of LBD. Helix 12 from the DES-LBD-adjuvant peptide complex is omitted for clarity. The residue 536-551 (helix 12 = residue 536-544) complex from OHT-LBD is magenta and the peptide is shown in gold. Hydrogen bonds between the ε-amino group of Lys362 and the backbone carbonyls of residues 543 and 544 of helix 12 are shown in magenta dotted lines. The hydrogen bond between the ε-amino group of Lys362 and the backbone carbonyl of residues 693 and 696 of the coactivator peptide is indicated by the dotted orange line. The following abbreviations are used in Helix 12: L540 = Leu540, M543 = Met543, and L544 = Leu544. The following abbreviations are used in the peptides: L690 = Leu690, L693 = Leu693 and L694 = Leu694.

6A and 6D are generated using BOBSC0RIPT and described using Raster3D as described above. 6B and 6C are shown using MidasPlus (Huang, et al., J. Mol. Graph. 9: 230-6,242 (1991)). FIG. 6A shows another LBD by depicting a DES complex (with no adjuvant peptide), an OHT complex, and an E ₂ complex as a ribbon, as described, for example, in Tanenbaum et al., Supra. Agents and antagonists that promote conformation are shown. The hormone is shown in space-filled state. In each complex, helix 12 is magenta and the main chains of residues 339-341, 421-423, and 527-530 are red. Helix 3, 8, and 11 (H3, 8, and 11, respectively) were labeled in the DES complex. 6B shows DES bound in a ligand binding cavity. The cross section of the space filled model of LBD coupled with DES (green) shows that the ligand is fully incorporated into the ligand binding cavity. The DES A 'ring (A'), Phe404 (404), Met421 (421), and Phe425 (425) were labeled. The carbon atoms in the side chain of Met421 are magenta and the sulfur atoms are yellow. 6C is a cross-sectional view of a space filled model of OHT (red) bound within a ligand binding pocket. This figure is equivalent to FIG. 6B. The B ring (B), Phe404 (404), Met421 (421), and Phe425 (425) of the OHT were labeled. The side chain of Met421 is as shown in FIG. 6B. The B ring configuration allows Met421 to have a different composition than that taken in the DES complex (compare FIG. 6B). 6D provides a comparison of ligand binding pockets that bind DES (green) and OHT (red). The structures of the OHT complex and the DES complex were overlapped as shown in FIG. 5. The A ring of both ligands is a point outside the ground, and the B ring of OHT and the A 'ring of DES are points in the ground. The LBD combined with the OHT is blue, and the LBD coupled with DES is pale blue gray. The side chains of several residues where the composition of these two complexes differed significantly: Met342 (342), Met343 (343), Phe404 (404), Met421 (421), Ile424 (424), Phe425 (425), His 524 (524), Leu525 (525), Met528 (528). The sulfur atom of Met421 is shown in yellow in both structures.

7 illustrates a model of antagonist action. Agonist (white triangle) binding stabilizes the formation of LBD that promotes coactivator (yellow) binding. Residues 527-530 (red) are part of helix 11 (blue) and the length of the loop between the helicals prevents helix 12 (magenta) from binding to the stactic region of the surface involved in transcriptional activity. Antagonist (white cross) side chains prevent helix 12 from being located on ligand binding pockets. Residues 527-530 (red) have an extended conformation as a result of structural interference induced by antagonists in or around the ligand binding pocket. The length of the loop between helix 11 and helix 12 causes helix 12 to bind to the electrostatic region of the surface and inhibits coactivator recognition.

FIG. 8 shows an arrangement of amino acid sequences (single letter designated amino acid sequences) containing residues that form coactivator binding sites of several nuclear receptors: human and recombinant thyroid hormones (hTRβ and rTRα) (SEQ ID NO: 5 And 6 and SEQ ID NOs: 7 and 8), retinoids (hPARγ and hRXRα) (SEQ ID NOs: 9 and 10 and SEQ ID NOs: 11 and 12) peroxysomes (hPPARγ) (SEQ ID NOs: 13 and 14), vitamin D ( hVDR) (SEQ ID NOs: 15 and 16), Estrogen (hERα) (SEQ ID NOs: 17 and 18), Glucocorticoid (hGR) (SEQ ID NOs: 19 and 20), Progestin (hPR) (SEQ ID NOs: 21 and 22) Mineral corticoids (hMR) (SEQ ID NOs: 23 and 24), and androgens (hAR) (SEQ ID NOs: 25 and 26). The boxes represent residues of alpha helix (H3, H4, H5, H6, and H12); Lowercase letters "h" and "q" represent hydrophobic and polar residues, respectively. The numbers at the top of the table, 280, 281, and the like correspond to hTRβ residues.

Detailed description of the invention

The present invention provides methods and compositions for modulating nuclear receptor activity, in particular steroid receptor activity, and more particularly estrogen receptor activity. The compound is a nuclear receptor agonist or antagonist that binds to a ligand binding domain. There is also provided a compound that binds to LBD. The compound is natural or synthetic. Preferred compounds are small organic molecules, peptides and peptidomimetics (eg, cyclic peptides, peptide analogs, or constrained peptides).

In particular, certain residues in important ERα LBDs have been identified: Met343, Leu346, Ala350, Glu353, Leu384, Leu387, Leu391, Arg394, Phe404, Met421, Leu428, Gly521, His524, Leu525, and Met528 (see FIG. 4A). Some of these have been found to affect the position of helix 12 directly or indirectly: Met 343, Met421, His524, Leu525 and Met528. For example, the interaction with these specific residues that occur when DES binds to the receptor stabilizes the formation of LBSD which promotes binding of the coactivator. Modifications to ligands that enhance binding or interaction with these residues provide improved receptor activity. Similarly, modifications of ligands that adversely affect binding or interaction with these residues are provided for improved antagonists.

It is also believed that ERα Tyr537 residues serve to stabilize unliganded receptors, and helix 12 freely interacts with coactivator binding sites. The ER has hTRβ, rTRα, hRARγ, hGR, hPR, hMR, and hAR all have proline residues, hRXRα has aspartic acid residues, hPPARγ has histidine residues, and hVDR corresponds to threonine residues with the Tyr537 residue of hERα. It is very unique in that it has a tyrosine in the position as it has a position. Thus, selective agents and antagonists may allow the design of estrogens to interact with Tyr537.

Conventional antagonists such as OHT or RAL have side chains that directly block the placement of helix 12 by being present in the agent complex (DES or E ₂ ). In addition, ligand-induced perturbation is required to allow helix 12 to reach coactivator helix binding sites, thereby blocking coactivator binding and inhibiting transcriptional activity. It has been found that this disturbance and interference with one or more LBD residues relaxes the receptor, disassembles the receptor's secondary structure and consequently unwinds helix 11. The unwinded helix 11 increases the length of the loop between helix 11 and 12 and directs helix 12 away from the ligand binding pocket to the coactivator binding site, where helix 12 mimics the action of the coactivator By occupying the recognition site it inhibits the coactivator recognition (Fig. 7). Modifications to ligands that interfere with binding or interaction with one or more amino acid points indicated result in relaxation of the receptor and affect the secondary structure of the receptor leading to unwinding of helix 12. Compounds according to these modified ligands will act as antagonists.

Accordingly, one aspect of the invention is a method of identifying a compound that modulates (eg, increases or decreases) nuclear activity, wherein the nuclear receptor ligand binding of interest using an estrogen α receptor ligand binding domain or an atomic structure model of a portion thereof. Modeling a test compound that spatially matches the domain; Assays characterized by binding of the test compound to the ligand binding domain, eg, screening the test compound in a biological assay; And identifying test compounds that modulate nuclear receptor activity, wherein the atomic structure model is a human estrogen receptor α Met343, Leu346, Ala350, Glu353, Leu384, Leu387, Leu391, Arg394, Phe404, Met421, Leu428, Gly521, And atomic coordinates of amino acid residues corresponding to residues of His524, Leu525 and Met528, preferably Met343, Met421 His524, Leu525 and Met528. In a preferred embodiment, the nuclear receptor is ER. The test compound may be an agent and nucleic acid receptor activity is modulated by the binding of the coactivator or compound mimicking the coactivator with the binding site of the coactivation as defined below. On the other hand, the test compound may be an antagonist and the activity of the nuclear receptor is measured by blocking the binding of the coactivator to the unwinding and / or coactivator binding site of helix 12. The screening is preferably high throughput screening in vitro. As discussed below, appropriate test compounds can be designed or obtained from a library of compounds, including, but not limited to, small organic molecules, peptides, and peptidomimetics. The method may also include providing an estrogen receptor α ligand binding domain or portion thereof to the computerized modeling system prior to the modeling step.

As used herein, the term “part of it” is intended to mean an atomic coordinate corresponding to a sufficient number of residues or atoms thereof of an LBD capable of interacting with a compound capable of binding to a site of the LBD. This includes acceptor residues having atoms within 4.5 kPa of the binding compound or fragment thereof. Thus, for example, the atomic coordinates provided in the modeling system may include a nuclear receptor LBD, a portion of the LBD such as an atom or a subset of atoms corresponding to the LBD useful for modeling and designing a compound that binds to the LBD.

The atomic coordinates of the compound that matches the ligand binding domain can also be used in modeling to identify the compound or fragment that binds to the site. "Modeling" is intended for qualitative and quantitative analysis of molecular structure / function based on atomic structure information and receptor ligand agonist / antagonist interaction models. This includes conventional water-based molecular dynamics and energy minimization models, interactive computer graphics models, modified molecular mechanism models, spacing geometry, and structure-based constraint models. Preferably, modeling is performed using a computer and can be further optimized using known methods. "Spatially coincident" means that the three-dimensional structure of the compound is geometrically received by the cavity or pocket of the nuclear receptor LBD.

Targeting the corresponding amino acid to other nuclear receptors is expected to have the same effect. Thus, one embodiment of the present invention is directed to a method of designing an antagonist that binds to the LBD of a nuclear receptor but does not interact with one or more residues in the LBD, wherein the residues are human ERα residues Met343, Leu346, Ala350, Corresponding to Glu353, Leu384, Leu387, Leu391, Arg394, Phe404, Met421, Leu428, Gly521, His524, Leu525 and Met528, preferably Met343, Met421 His524, Leu525 and Met528 (ie, identical or equivalent). Similarly, another embodiment of the present invention is directed to a method of designing an agent that binds to LBD of a nuclear receptor and has increased interaction with one or more residues in the LBD, wherein the residues are human ERα residues Met343, Leu346, Ala350, Corresponding to Glu353, Leu384, Leu387, Leu391, Arg394, Phe404, Met421, Leu428, Gly521, His524, Leu525 and Met528, preferably Met343, Met421 His524, Leu525 and Met528. One example of increased interaction is an agent that has a greater binding affinity for one or more of these residues compared to an endogenous ligand. For other members of the nuclear receptor family, these corresponding points are shown in Table 1, which shows human estrogen receptor (hERα): recombinant thyroid hormone (rTRα), retinoids (hPARγ and hRXRα), glucocorticoid (hGR), An array of amino acid sequences (single letter designation) comprising residues from the ligand binding domains of several nuclear receptors corresponding to designated points on progestin (hPR), mineralocorticoids (hMR), and androgens (hAR). Table 1 below is merely illustrative and does not limit the invention. Thus, other nuclear receptors such as other estrogen receptors, thyroid receptors, androgen receptors, peroxysome receptors, and vitamin D receptors may also be used in the methods of the present invention.

Table 1

As used herein, "adjuvant binding site" means a structural fragment or fragment of a nuclear receptor polypeptide chain that is folded to provide the appropriate geometry and amino acid residues for the binding aid. It is the physical arrangement of protein atoms in the three-dimensional space that forms the coactivator binding site pocket or cavity. As described by Aprili et al., The residues forming the coactivator binding site on the nuclear receptor are C-terminal helix 3 (Ile280, Thr281, Val283, Val284, Ala287, and Lys288), and Helix 4 (Phe293), Helix 5 (Gln301, Ile302, Leu305, Lys306), Helix 6 (Cys309), and Helix 12 (Pro453, Leu454, Glu457, Val458, and Phe459) (Ie, identical or equivalent) amino acid residues. The coactivator binding site is highly conserved in the nuclear receptor superfamily. Thus, the site corresponds to a surprisingly small cluster of residues on the surface of the LBD that form a significant hydrophobic cleft. The hydrophobic moieties include C-terminal helix 3 (Ile280, Val283, Val284, and Ala287), and helix 4 (Phe293), helix 5 (Ile302 and Leu305), helix 6 (Cys309), and helix 12 (Leu454, Val458, and Formed by the hydrophobic moiety corresponding to Phe459). The hydrophobic portions of these coactivator binding sites are also highly conserved in the nuclear receptor superfamily.

Based on the above examples herein, it was found that the residues forming the coactivator binding sites on the estrogen receptor correspond to the points described for the human TR. Thus, the residues forming the coactivator binding site on ERα are C-terminal helix 3 (Leu354, Val355, Met357, Ile358, Ala361, and Lys 362), and helix 4 (Phe367), helix, as shown in FIG. Human ERα residues of 5 (Gln375, Val376, Leu379, Glu380), Helix 6 (Trp383), and Helix 12 (Asp538, Leu539, Glu542, Met543, and Leu544). As in the general description of the nuclear receptor family, the site corresponds to residues on the surface of the LBD that form significant hydrophobic moieties, and C-terminal helix 3 (Leu354, Val355, Met357, Ile358, Ala361, and Lys 362). ), And hydrophobicity corresponding to human ERα residues of Helix 4 (Phe367), Helix 5 (Gln375, Val376, Leu379, Glu380), Helix 6 (Trp383), and Helix 12 (Asp538, Leu539, Glu542, Met543, and Leu544). Formed by residues. This corroborates the data presented by Aprilree et al. For the nuclear receptor family.

Structural analysis revealed the mechanism by which tamoxifen and other SERMs bind ligand binding domains, block coactivator binding, and thus transcriptional activity. Accordingly, ligand and coactivator binding is understood. Thus, the above coactivator binding moiety is useful for designing coactivator mimics with broad applicability to the methods of the present invention. Such "adjuvant pseudomaterials" are peptides or polypeptides that mimic a coactivator binding site recognition region on the surface of a coactivator so that the "coactivator pseudomaterial" acts as a competitive inhibitor of the coactivator to the coactivator binding site. Adjuvant pseudomaterials can be used to assay the agonist or antagonist properties of a test compound in that a measure of receptor activity and the agonist allow the adjuvant to bind to the adjuvant binding site, whereas the antagonist interferes with this binding. Can be. In addition, adjuvant pseudomaterials may have therapeutic utility when administered in combination with an agent compound of the present invention.

Another embodiment of the invention relates generally to a method for identifying a compound that modulates the binding of a ligand receptor and a nuclear receptor by binding to a ligand binding domain. The method comprises modeling a test compound that spatially matches a nuclear receptor ligand binding domain of interest using an atomic structure model of the estrogen α receptor ligand binding domain or portion thereof; Screening the test compound in an assay characterized by binding of the test compound to the binding domain; And identifying test compounds that modulate ligand binding to nuclear receptors, wherein the atomic structure model comprises human estrogen receptors α Met343, Leu346, Ala350, Glu353, Leu384, Leu387, Leu391, Arg394, Phe404, Met421, Leu428 , Atomic coordinates of amino acid residues corresponding to residues of Gly521, His524, Leu525 and Met528, preferably Met343, Met421 His524, Leu525 and Met528. In a preferred method, the nuclear receptor is ER, TR, GR, and PR. The screening is usually by high throughput screening in vitro. Appropriate test compounds can be designed or obtained from compound libraries, including, but not limited to, small organic molecules, peptides, and peptidomimetics. The test compound may be an agonist or antagonist of ligand binding.

The invention also includes compositions and methods for identifying key residues in ligand binding domains of nuclear receptors. The method involves examining the surface of the nuclear receptor of interest to identify residues that regulate ligand and / or coactivator binding. Such residues may be identified by homology to key residues on the LBD described herein. Preferred methods are human ERα residues of Met343, Leu346, Ala350, Glu353, Leu384, Leu387, Leu391, Arg394, Phe404, Met421, Leu428, Gly521, His524, Leu525 and Met528, preferably Met343, Met421, His524, Leu525 and Met528 Is an arrangement with residues of any nuclear receptor that corresponds to (ie, is equivalent to). Overlaying or superpositioning of the nuclear receptor LBD, or a three-dimensional model of the residue or a portion thereof comprising the corresponding residue may also be used for this purpose. For example, three-dimensional structures of TR, GR, PR, and LBDs can be used for this purpose. For example, nuclear receptors that can be identified by homology arrangements are proteins that are structurally associated with normal nuclear receptors or nuclear receptors found in humans, natural mutations of nuclear receptors found in humans, and organs of animals other than animals and mammals. For example, normal or mutant receptors found in plagues or infectious organs, or viruses.

Arraying and / or modeling can also be used as a guide for the placement of mutations on the LBD surface to characterize the site in the context of the cell. Selected receptors are mutated as in the case of antagonists, in order to preserve the global receptor structure and solubility in the case of an agonist, or to break up such a structure, and to allow the helix 12 to unwind. Mutants can be tested for relative changes in ligand binding and intensity of binding interactions. Ligand dependent coactivator interaction assays can also be tested for this purpose as described herein.

In particular, the present invention relates to a structural combination of LBD of the estrogen receptor, a binding of two chemically related compounds, an agonist, diethylstelbestrol (DES), and a selective antagonist 4-hydroxytamoxifen (OHT), which is an active metabolite of tamoxifen. And functional effects. As described in the Examples, studies of mutations and binding coupled with assays of atomic models derived from cocrystals revealed that GRIP1 NR Box II peptide sequences (SEQ ID NO: 4) bound to coactivator binding sites and agents, DES (ie , a peptide molecule comprising a peptide derived from the NR box II region of the p160 co-activator GRIP1) is shown in the structure of a co-crystallized human estrogen receptor ligand binding domain (ERα LBD). Also shown is the ERα LBD structure co-crystallized with the antagonist OHT. The example provides a 2.03 'spacing crystal structure of hERα LBD coupled with DES, a coactivator, and a 1.9' x-ray crystal structure of hERα LBD coupled with OHT. In other words, the crystals are diffracted at 2.03 ms or 1.9 ms intervals, respectively.

In another aspect of the invention, compounds of interest, namely agonists and antagonists of ligand binding, have been found by methods of identifying agonists or antagonists of ligand binding to nuclear receptors. The method comprises providing atomic coordinates of ERα LBD or a portion thereof to a computerized modeling system, modeling a compound that spatially matches the LBD, and binding the LBD of the nuclear receptor in an assay for nuclear receptor activity. Identifying compounds that increase or decrease the activity of nuclear receptors. Preferably, the atomic coordinates are human estrogen receptors α Met343, Leu346, Ala350, Glu353, Leu384, Leu387, Leu391, Arg394, Phe404, Met421, Leu428, Gly521, His524, Leu525 and Met528, preferably Met343, Met421 His524, Amino acid residues corresponding to the residues of Leu525 and Met528. Of particular interest are compounds that are in spatial and preferential matching with a ligand binding domain. "Spatially and preferentially" is a compound having a three-dimensional structure and conformation for selectively interacting with nuclear receptor LBD. Compounds that spatially and preferentially match the LBD interact with amino acid residues that form a hydrophobic portion of the site. The invention also includes methods for identifying compounds that can selectively modulate the activity of nuclear receptors. The method uses the atomic structure model of the nuclear receptor to model a test compound that spatially and preferentially matches the LBD of the nuclear receptor of interest, wherein the nuclear receptor activity is characterized by preferential binding of the LBD of the nuclear receptor to the test compound. Screening test compounds for assays, and identifying test compounds that selectively modulate nuclear receptor activity. Such receptor specific compounds are selected to make use of the difference between LBDs of the first type of nuclear receptor versus the second type of nuclear receptor.

The invention can also be applied to the preparation of novel compounds that distinguish nuclear receptor isoforms. It can be used to prepare tissue specific or function specific compounds. For example, members of the GR subfamily typically have one receptor encoded by one gene, with exceptions. For example, there are PR isoforms, A and B, translated from the same mRNA by selective initiation from different AUG codons. There are two GR forms, one of which does not bind to the ligand. The method is particularly applicable to ER subfamilies which generally have several receptors encoded by two or more (ER: α, β) genes or have selective RNA splicing.

Said receptor specific compound of the invention preferably interacts with the conformally bound moiety of LBD conserved in the nuclear receptor of the first type compared to the second type of nuclear receptor. "Conformatively bound" means a three-dimensional structure of a chemical or portion thereof that has a specific rotation around the bond fixed by various local geometries and physico-chemical bonds. Features of the conformally bound structure of the LBD include residues with inherent flexible conformations fixed by various geometric and physico-chemical bonds, for example, local backbones, local side chains, and topological bonds. This type of bond is used to limit receptor-adjuvant recognition and the placement of atoms involved in binding.

The present invention also provides a computerized method using a three-dimensional model of nuclear receptors based on nuclear receptor determination. In general, the computerized design method of nuclear receptor ligands involves determining which amino acids or amino acids of nuclear receptor LBD interact with the chemical component (one or more) of the ligand, three-dimensional of the crystallized protein comprising nuclear receptor LBD with binding ligand. The model is used to determine and select one or more modifications of a chemical component that produces a secondary chemical component with a structure that increases or decreases the interaction of the interactive amino acid with the chemical component. In the present invention, the structure of hERα having DES / peptide and the crystal structure of hERα having OHT are represented by the amino acid residues hERα Met343, Leu346, Ala350, Glu353, Leu384, Leu387, Leu391, Arg394, Phe404, Met421, Leu428, Gly521, And corresponding residues of His524, Leu525, and Met528, preferably Met343, Met421 His524, Leu525, and Met528.

Thus, one embodiment of the present invention is a computerized method of designing nuclear receptor ligands wherein human receptor estrogens α Met343, Leu346, Ala350, Glu353, Leu384, Leu387, Leu391, Arg394, Phe404, Met421, Leu428, Gly521, One or more amino acid residues of the nuclear receptor LBD corresponding to residues of His524, Leu525, and Met528, preferably Met343, Met421 His524, Leu525, and Met528, interact with one or more first chemical components of the ligand, Selecting one or more chemical modifications of the first chemical component to produce a second chemical component having a structure that increases or decreases the interaction of the amino acid with the second chemical component as compared to the interaction between the components. It includes.

The computerized method may include measuring the change in interaction between the interacting amino acid and the ligand after chemical modification of the first chemical component. The chemical modification is compared to the interaction with amino acids interacting with the first chemical component, such as hydrogen bond interactions, charge interactions, hydrophobic interactions, van der Waals interactions between amino acids interacting with the second chemical component. Or enhance or reduce bipolar interaction.

Chemical modifications often increase or decrease the interaction of atoms of LBD amino acids with atoms of LBD ligands. Stereotic interference will be a common means of changing the interaction of LBD binding cavities with activation domains. Chemical modifications are preferably induced at the C-H, C-, and C-OH points of the ligand, where the carbon moiety is the structural moiety of the ligand that remains the same after the modification ends. In the case of C-H, C may have 1, 2, or 3 hydrogen atoms, but usually only one hydrogen atom is substituted. The H or OH is removed after modification and substituted with the desired components.

Such chemical modifications preferably preferentially hERα Met343, Leu346, Ala350, Glu353, Leu384, Leu387, Leu391, Arg394, Phe404, Met421, Leu428, Gly521, His524, Leu525, and Met528, preferably Met343, Met421 His524, Addition of a substituent which places said substituent on any one of the free carbons of the A 'ring of DES to conflict with or bind to one or more residues corresponding to Leu525 and Met528. Typical substituents include, but are not limited to, hydrophobic groups, alkyl groups such as ethyl, propyl, isopropyl, and the like, and aromatic groups such as benzyl groups.

Indeed, one skilled in the art can design an agent / antagonist based on a known ligand for a nuclear receptor of interest as a chemical backbone. Known ligands can be modified as described above. For example, 17β-estradiol is an endogenous ligand for hERα. In the case of estradiol, the points of interest are C6α, C7α, C12α, C15α, and C17α. Modifications of one or more of the free carbons on the 17β-estradiol backbone may include one or more of Met343, Leu346, Ala350, Glu353, Leu384, Leu387, Leu391, Arg394, Phe404, Met421, Leu428, Gly521, His524, Leu525, and Met528, preferably Will interact with Met343, Met421 His524, Leu525, and Met528 residues.

The chemical backbone of other known ligands can be used in a similar manner. For example, other known agents include diethylstilbestrol (synthesis), magestrol (synthesis), mesohexestrol (synthesis), comethrol (clover), Δ ⁹ -THC (canabis), op-DDT (Insecticides), giarlenone (fungi), and ketones (insecticides). Known estrogen receptor antagonists include the ICI family of modified steroids such as ICI 164,384, and EM800. Known SERM's include tamoxifen, raproxyfen, and GW5638.

In addition, known agents can be computerized or placed into ligand binding pockets via manual docking. The arrangement for substitution is then selected based on the predicted binding orientation of these compounds. In addition, hybrid molecules may be created having side chains that prevent helix 12 from occupying an agent binding point. Novel SERMs can be produced by modifying two other effects: substitution of helix 12 and release of the secondary structure.

Conventional efforts to prepare antagonists include attaching large size substituents to the agent, usually through trial and error, and the design of the medicaments described herein can be very important for the development of new potent antagonists. Provides an alternative strategy.

For modeling, docking algorithms and computer programs can be used to identify compounds that match the ligand binding domain. For example, a docking program can be used to predict how molecules of interest can interact with nuclear receptor LBD. Fragment-based docking can also be used to construct de novo molecules in LBD by substituting chemical fragments to supplement the site, in order to optimize intramolecular interactions. The technique can be used to optimize the geometry of binding interactions. This design approach allows for the identification of LBD structures, and thus molecular recognition principles can be used in the design of compounds complementary to these sites. Compounds that match LBD serve as a repetitive design, synthesis, and starting point for them, where the new compounds are selected for desirable properties including affinity, efficiency, and selectivity. For example, the compounds may be subjected to additional modifications such as substitution and / or addition of R-group substituents of the central structure identified for a particular class of binding compounds, modeling and / or activity screening if necessary, The following additional test steps can be carried out.

Computerized small molecule databases can be used to screen chemical entities or compounds capable of binding in whole or in part to the nuclear receptor ligand binding domain of interest. In such screening, the qualities of the chemicals or compounds that closely match the binding sites are determined by shape complementarity (Des Jalais et al., J. Med. Chem. 31: 722-729 (1988)), (Meng, et al., J. Comp. Chem. 13: 505-524 (1992)). The molecular database includes any visual or physical database such as electronic and physical compound library databases, and is preferably used for the development of compounds that modulate coactivator binding.

Compounds can be intelligently designed by using available structural and functional information by understanding of quantitative structural activity, and this understanding can be extended to new compound libraries, particularly libraries with chemical diversity of specific groups of one or more central structures. By using a focused library and incorporating arbitrary structural data into an iterative design process. For example, those skilled in the art will be able to use any of several methods for screening their ability to associate chemicals or fragments. This method may begin with, for example, visual observation of the LBD on a computer screen. The selected fragments and chemicals can then be placed into all or part of the site. Docking can be performed using software such as Quanta and Sybyl, followed by energy minimization and molecular dynamics using standard molecular dynamics force fields such as CHARMM and AMBER.

Specialized computer programs can assist in the selection of chemical fragments or entire compounds. This includes: GRID (Goodford. J. Med. Chem. 28: 849-857 (1985), available from Oxford University, Oxford UK); Ranker. Et. Al., "Proteins: Structure, Function and Genetics" 11: 29-34 (1991), available from Molecular Simulations, Burlington, Mass .; AUTODOCK (Goodsell, et. Al., "Proteins: Structure, Function and Genetics" 8: 195-202 (1990), available from Scripps Research Institute, La Jolla, CA); And DOCK (Kuntz, et al., J. Mol. Biol. 161: 269-288 (1982), available from the University of California, San Francisco, CA).

Additional commercially available computer databases for small molecular compounds include the Cambridge Structural Database and Fine Chemical Database (Rusinko, Chem. Des. Auto. News 8: 44-47 (1993)).

Once suitable chemicals or fragments are selected, they can be incorporated into one compound. The coalescence is visually examined for the interrelationship of the fragments on a three-dimensional image presented on a computer screen in relation to the structural coordinates of the nuclear receptor.

In linking independent chemicals or fragments, those skilled in the art can use: CAVEAT (Bartlett, et al., "CAVEAT: A Program to Facilitate the Structure-Derived Design of Biologically Active Molecules", in Molecular Recognition in Chemical and Biological Problems, Special Pub., Royal Chem. Soc. 78: 182-196 (1989), available from the University of California, Berkeley, CA; 3D database systems, eg, MACCS-3D (MDL Informational Systems, San Leandro, CA, reviewed in Martin, J. Med. Chem. 35: 2145-2154 (1992)); And HOOK (available from molecular simulations, Burlington, MA).

Further, in order to construct the compound step by step, a compound which binds the ligand binding domain of interest, the chemical or fragment described above, is known to bind to an unfilled LBD or any site, in whole or in de novo format. Can be designed using LBDs comprising, for example, known ligands. Such methods include: LUDI (Bohm, J. Comp. Aid. Molec. Design 6: 61-78 (1992), available from Biosm Technologyes, San Diego, CA); LEGEND (Nishbata, et al., Tetrahedron 47: 8985 (1991), available from Molecular Simulations, Burlinton, Mass.); And Leapfrog (available from Tripos Associations, St. Louis, MO).

Other molecular modeling techniques can be used in accordance with the present invention. See, eg, Cohen et al., J. Med. Chem. 33: 883-894 (1990) and Navia, et al., Current Opinions in Structural Biology 2: 202-210 (1991). . For example, if the structure of the test compound is known, the test compound model can be overlaid with the structural model of the present invention. Various methods and techniques for carrying out this process are known in the art, any of which may be used. See, eg, Farmer, "Drug Design" 10: 119-143, Ariens, ed., Academic Press, New York (1980); US Pat. No. 5,331,573; US Pat. No. 5,500,807; Verlinde, Structure 2: 577 -587 (1994) and Kuntz, et al., Science 257: 1078-1082 (1992). The model building techniques and computer evaluation systems described herein are not limited to the present invention.

Using this computer modeling system, many compounds can be investigated quickly and easily, avoiding costly and prolonged biochemical testing. In addition, the need for actual synthesis for many compounds has been substantially reduced and / or effectively eliminated.

Compounds identified through modeling can be screened in assays as are well known in the art. Such assays include biological assays and are characterized by the binding of a compound with a ligand binding domain of interest for ligand binding activity. Screening can be performed in cell culture, for example in vitro and / or in vivo. Preferably, biological screening measures activity-based response models, binding assays (which measure how well the compounds bind to receptors), and bacterial, yeast, and animal cell lines (which measure the effects of compounds in cells). Focus on). Such assays can be automated for high dose-high throughput screening, where a large number of compounds can be tested for the identification of compounds with the desired activity.

For example, an in vitro binding assay is performed where the compound is tested for its ability to block the binding of ligand, fragment, fusion or peptide thereof with the ligand binding domain of interest. In cell and tissue culture assays, the compound may be a cell adjuvant such as the p160 family of adjuvant proteins such as SRC-1, AIB1, RAC3, p / CIP, and GRIP1 and its homologues TIF2 and NcoA-2, and Tests for the ability to block the function of coactivators exhibiting receptor and / or isoform specific binding activity are performed. In a preferred embodiment, the compounds of the present invention bind with ligand binding compounds with much greater affinity than intrinsic ligands. Tissue profiling and appropriate animal models can also be used to select compounds. Other cell types or tissues can be used for this biological assay. Suitable assays for such screening are described in the literature (Shibata, et al., Recent Prog. Horm. Res. 52: 141-164 (1997); Tagami, et al., Mol. Cell. Biol. 17 (5). ): 2642-2648 (1997); Zhu, et al., J. Biol. Chem. 272 (14): 9048-9054 (1997); Lin. Et al., Mol. Cell. Biol. 17 (10): 6131-6138 (1997); Kakizawa, et al., J. Biol. Chem. 272 (38): 23799-23804 (1997); and Chang, et al., Proc. Natl. Acad. Sci. USA 94 (17 ): 900-9045 (1997)), which are incorporated herein by reference.

The selected compound may be characterized as an agent and / or an antagonist. The compound may also include compounds which exhibit novel properties depending on the various mixing of agonist and antagonist activity, depending on the effect of selective ligand binding within the context of other activities of nuclear receptors, which are neither hormone dependent or hormone dependent. These compounds are mediated by proteins rather than coactivators and interact with receptors at locations rather than at coactivator binding sites. The compounds may also be identical, similar to those induced in the receptor by stabilizing or destabilizing the hormone binding conformation of the receptor through binding to a receptor location that is conformally sensitive to hormone binding, or by binding of the hormone. Direct induction of, or other conformational changes results in an allosteric effect on the receptor.

Of particular interest is the use of such compounds in a method of modulating nuclear receptor activity in a mammal by administering to a mammal in need thereof an amount of a compound conforming to the ligand binding domain of the nuclear receptor of interest spatially and preferentially. to be. "Modulation" means increasing or decreasing the activity of nuclear receptors. For example, preliminary clinical compounds can be tested to determine efficiency, adsorption, pharmacokinetics and toxicity in appropriate mammalian models, followed by standard methods known in the art. Compounds showing the desired properties can then be clinically tested for use in the treatment of various nuclear receptor related diseases. This includes postmenopausal signs and cancer resulting from ER related diseases such as loss of estrogen production, and osteoporosis and cardiovascular disease caused by conventional estrogen replacement therapy. Others include GR-related diseases including type II diabetes and infectious diseases such as arthritis.

Although for many nuclear receptors, the goal is to find new synthetic or antagonists, it is important to understand the value of nuclear compounds, especially estrogen receptors, of developing compounds with desirable agonist and antagonist effects on target tissues. It is important. Such compounds may be found and / or designed according to the methods listed herein and may be screened for tissue specificity according to methods known in the art. For example, there is a great need for improvements in current therapies and the development of new agents that act like estrogen receptors in cardiovascular and brain tissues, and new antagonists that act on estrogens in uterine and breast tissues. Ideally, there may be compounds with one or more of these properties, ie compounds that act as agonists in one tissue, while acting as antagonists in other tissues. Such tissue-specific antagonists of SERMs, such as OHT and RAL, have various factors (Grainger, et al., Nature Medicine 2 (4): 381-385 (1996); Grease, et al., Alc Jordan, J. Natl. Cancer Inst. 90: 967-71 (1998)), the dissolution of the mechanism of these ligands requires an understanding of how they act on LBD and how they regulate their interaction with other cellular factors. The present invention has surprisingly shown that ligand mediated structures perturb inside and around ligand binding pockets and contribute not only to the side chain effects but also to receptor antagonism. Thus, it provides a novel strategy for the design of SERMs that is improved by adjusting the balance between these two effects.

Based on this knowledge, human estrogen receptors α Met343, Leu346, Ala350, Glu353, Leu384, Leu387, Leu391, Arg394, Phe404, Met421, Leu428, Gly521, His524, Leu525, and Met528, preferably Met343, Met421 His524, Leu525 and Of particular interest is the design of therapeutic compounds that modify one or more amino acid residues corresponding to residues of Met528. Accordingly, one aspect of the invention is a method of modulating nuclear receptor activity by administering a sufficient amount of ligand to be spatially and preferentially matched with a ligand binding domain of a nuclear receptor of interest to a mammal in need thereof. Are designed by computerized methods, wherein hERα Met343, Leu346, Ala350, Glu353, Leu384, Leu387, Leu391, Arg394, Phe404, Met421, Leu428, Gly521, His524, Leu525, and Met528, preferably Met343, Met421 His524 The amino acid residues of at least one nuclear receptor LBD corresponding to Leu525 and Met528 interact with the first chemical component of the ligand. This method produces a second chemical component having a structure that increases or decreases the interaction between the interactive amino acid and the second chemical component as compared to the interaction of the interactive amino acid with the first chemical component. Selecting one or more variations of the first chemical component.

The compound designed by the above method is an agonist or antagonist, and the method of modulating nuclear receptor activity may include the administration of an agent alone, an antagonist alone, or an agent combined with a compound that mimics an adjuvant by binding to an adjuvant or adjuvant binding site. Administering.

The co-activator may be a known co-activator. Such co-activators can be designed in a computerized manner, wherein hERα helix 3 residues Leu354, Val355, Met357, Ile358, Ala361, and Lys 362, and helix 4 residues Phe367, helix 5 residues Gln375 Val376, Leu379, and Glu380, One or more amino acid residues of the nuclear receptor coactivator corresponding to helix 6 residue Trp383, and helix 12 residue Asp538, Leu539, Glu542, Met543, and Leu544 interact with one or more first chemical components of the coactivator analog. The method compares the interaction of the interactive amino acid with the first chemical component to produce a second chemical component having a structure that increases or decreases the interaction between the interactive amino acid and the second chemical component, Selecting one or more variations of the first chemical component.

The use of agents in combination with adjuvant or adjuvant pseudomaterials also provides unique means for delivering therapeutic agents with novel tissue specific effects. For example, the coactivator pseudomaterial may be designed to bind to a site associated with transcriptional activity only if helix 12 is at its agent binding site. If such coactivator pseudomaterials are specific for the site of a particular receptor, it may be possible to selectively inhibit the receptor only in the presence of an agent. This can lead to new, tissue specific antagonists based on the level of the endogenous agent. Agents designed by the present invention can be used in assays to determine the specificity of a coactivator pseudomaterial. In addition, the effective level in a given tissue can be controlled by known agents or antagonists designed according to the methods of the present invention. The crystal structure of the LBD / DES / GRIP1 peptide complex described herein precisely defines the binding site required to be targeted.

As described in the Examples, ER LBDs comprise a peptide molecule comprising a GRIP1 NR Box II peptide sequence (SEQ ID NO: 4) bound to a coactivator binding site and DES having a co-crystal structure purified at intervals of 2.03 μs; Cocrystallized together and co-crystallized with OHT having a purified co-crystal structure at 1.9 μs intervals. Accordingly, the present invention is also a method for providing a crystal made from a nuclear receptor ligand binding domain having a molecule bound to a ligand binding domain and a ligand bound to a ligand binding domain. Preferably, the co-crystal structure is refined at intervals greater than 3.6 mm 3, ie less than 3.6 mm 3. Even more preferably, the co-crystal is refined to greater than 3.4 kV, 3.2 kV, 3.0 kV, 2.8 kV, 2.6 kV, 2.4 kV, 2.2 kV and even more preferably at intervals greater than 2.03 kV. The present invention further provides cocrystals prepared from nuclear receptor ligand binding domains having ligands bound to ligand binding domains. Preferably, the co-crystal structure is purified at intervals greater than 3.6 mm 3, ie has a gap less than 3.6 mm 3. Even more preferably, the cocrystal is refined to greater than 3.4 kV, 3.2 kV, 3.0 kV, 2.8 kV, 2.6 kV, 2.4 kV, 2.2 kV and even more preferably at intervals greater than 1.9 kV.

Crystals are generally prepared from purified nuclear receptor LBDs expressed by cell cultures such as, for example, E. coli. E. coli is often the preferred expression system. The thyroid receptor has been successfully expressed in E. coli (Ref. Apriletti et al.). However, it has long been believed that human heat shock proteins are required for successful recombinant expression of the estrogen receptor. Thus, it was not expected that the estrogen receptor could be expressed as an active protein in E. coli.

Preferably, different crystals (cocrystals) for the same nuclear receptor are prepared separately using different coactivator family proteins such as protein fragments, fusions or small peptides. The coactivator family protein preferably contains an NR box sequence, which is required to bind the coactivator binding site, or a derivative of the NR box sequence. Other molecules such as coactivators or small organs that bind to hormone binding sites can be used for cocrystallization. Heavy element substituents may be used in the LBD and / or cocrystallization molecules.

After the determination of the three-dimensional structure of the co-crystal, the structural information can be used in a computerized method to design synthetic compounds for nuclear receptors, and the structural activity relationship is used in general tests using the assays herein and known. Can be further measured.

As provided in Appendix 1 and 2, nuclear receptor LBD crystallizes the structural coordinates of said receptor or part thereof in one or more crystalline forms, so it is particularly useful to reveal the structure of said other crystalline forms of nuclear receptor. It can also be used to identify the structure of a mutant or cocomplex with sufficient homology.

Annex 1 to the DES-ERα LBD-GRIP1 NR Box II peptide complex and Appendix 2 to the OHT-ERα LBD complex have been deposited with the Brookhaven National Laboratory Protein Data Bank and have a deposit number (Brookhaven Protein). Data Bank Accession Numbers 2erd and 2ert). The method can provide a more precise structure for unknown crystals more quickly and effectively than attempting to measure information such as, for example, ab initio.

Atomic coordinate data collected from the crystals of the present invention can be accumulated. In a preferred embodiment, it may be provided in the form of a device decrypted data storage medium. The medium may comprise information for the construction and / or manipulation of the ligand binding domain or portion thereof. For example, device detoxifying media include hERα Met343, Leu346, Ala350, Glu353, Leu384, Leu387, Leu391, Arg394, Phe404, Met421, Leu428, Gly521, His524, Leu525, and Met528, preferably Met343, Met421 His524, Leu525, And structural coordinates of the amino acids corresponding to Met528, or homologues of molecules or molecular complexes comprising the sites. The homologue includes an LBD having a root mean square deviation of 1.5 ms or less from the backbone atom of the amino acid. One preferred molecule and complex represents a compound bound to LBD.

Device detox data storage media can be used in the design of interactive agents and in the study of molecular substitution. For example, the data storage material may be encoded into a first set of device readable data that may be combined with a second set of device readable data. In the case of molecular substitution, the first set of data may comprise Fourier conversions of at least a portion of the structural coordinates of the nuclear receptor or portion thereof, and the second set of data may comprise the molecules or molecular complexes of interest. X-ray diffraction pattern. Some or all of the structural coordinates corresponding to the second set of data may be measured using the programmed device with instructions for using the first and second sets of data.

Proteins for the determination and analysis described herein can be prepared using known and expression and purification techniques described herein. For example, expression of high levels of nuclear receptor LBD can be obtained in a suitable expression host such as E. coli. For example, expression of LBD in E. coli includes ERα LBD and other nuclear receptors including GR and PR. Since these nuclear receptors are generally more difficult to express in bacteria except for the expression of ER, yeast and other eukaryotic expression systems can be used with nuclear receptors that bind heat shock proteins. Representative nuclear receptors or their ligand binding domains have been cloned and sequenced: human ER (Seielstad, et al., Molecular Endocrinology 9 (6): 647-658 (1995), incorporated herein by reference), human GR , Human PR. LBD for each of these receptors was identified.

Coactivator proteins are members of the p160 family of coactivator proteins that have already been cloned and / or expressed, for example SRC-1, AIB1, RAC3, p / CIP, and GRIP1 and homologs thereof. And NcoA-2. Preferred methods of expression of coactivator proteins include fragments having transcriptional activity, as described in Hong, et al., Mol. Cell. Biol. 17: 2735-44 (1997) and Proc. Natl. Acad. Sci. Expression using the "Song and Field" method (also referred to as "yeast 2-hybrid") on USA 93 (10): 4948-52 (1996).

The protein may be expressed alone as a fragment of a mature or full-legth sequence or by fusion with a heterogous sequence. For example, ERα can be expressed without any portion of the DBD or amino terminal domain. If additional structural information is needed with amino acids adjacent to the LBD, a portion of the DBD or amino terminus may be included. In general, the ERα LBD used for the crystal is less than 320 amino acids in length. Preferably, the ERα LBD is at least 220 amino acids in length and most preferably at least 250 amino acids in length. For example, the LBD used for crystallization may comprise 297 to 554 amino acid spanning of ERα.

Typically, the LBDs can be measured using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), mass spectrometry (MS), and hydrophobic high performance liquid chromatography (HPLC). The LBD purified for the crystallization should be at least 97.5% pure, preferably at least 99.05 and even more preferably at least 00.5% pure.

Initially, purification of non-ligand bound receptors can be obtained by known methods such as hydrophobic interaction chromatography (HPLC), and heparin affinity chromatography. To achieve higher purity for improved determination of nuclear receptors, in particular estrogen receptors, the receptors are separated using a column that separates the receptors by charge, e.g., an ion exchange or hydrophobic interaction column, and then The eluted receptor can be ligand-shift purified using columns that bind ligands, in particular agents. The ligand induces charge within the surface charge of the receptor such that the ligand bound receptor is fused at a different position than the receptor to which the ligand is not bound. Generally, ligands of saturation concentration are used, and proteins are preincubated before passing the ligand through the column. The structural studies described herein dictate the general applicability of the method for obtaining very high purity nuclear receptors for crystallization.

Purification can also be completed, for example, using a purification handle or “tag” with histidine amino acids engineered to be located at the terminal, eg, the N, terminus of the protein, and subsequently using a nickel or cobalt column ( Reference: Janknect, Proc. Natl. Acad. Sci. USA, 88: 8972-8976 (1991). Generally purified LBDs, such as ERα LBD, are in equilibrium at ligand saturation, under temperatures that preserve the integrity of the protein. Although the receptor is more stable in the 2-20 ° C. range, ligand equilibrium is established at 2 to 37 ° C. In order to improve the stability and quality of the crystals, controlled temperature control is desirable. In general, temperatures of 4-25 ° C. are used, and it is often desirable to test crystallization above this temperature range. The crystals are then gas diffused and bombarded with x-rays according to standard methods to obtain an x-ray diffraction pattern. Ligands of varying concentrations containing a sequence that binds to the binding site of the nuclear receptor of interest for cocrystallization with a peptide that binds to the ligand binding domain alone or to a coactivator binding site and a ligand that binds to the conjugated ligand binding domain. And peptides can be used among the appropriate compounds selected for microcrystallization testing, and further crystallization. Ligands and peptides can be assayed for binding to ligand binding domains and coactivator binding sites of the nuclear receptor of interest by a variety of methods. For the crystallization test using ERα LBD, the hanging drop vapor diffusion method is preferred. pH, solvent, solute component and concentration, and temperature can be adjusted, for example, as described in the Examples. In the hanging drop method, seeding of drops prepared with microcrystals of the composite can be used to obtain appropriate crystals for x-ray diffraction analysis. Collection of structural information can be measured by molecular substitution using the structure of ERα LBD as measured herein. The structure can be purified according to known standard methods.

The present invention provides many uses and advantages. For example, the methods and advantages described herein are useful for the identification of peptides, peptidomimetics, or small natural or synthetic organic molecules that modulate nuclear receptor activity. The compounds are useful for the treatment of nuclear receptor related diseases. The methods and compositions of the present invention are useful for characterizing the structure / function of natural and synthetic ligands.

In the following, a basic understanding of the examples is provided along with the results obtained and / or the conclusions reached.

As noted above, many coactivators recognize coactivators bound via nuclear receptor LBDs via the sequence LXXLL (SEQ ID NO: 1), where L is leucine and X is any amino acid (the NR box above). The structure of the DES-hERαLBDS-GRIP1 peptide complex sequence suggested that the LXXLL motif (SEQ ID NO: 1) forms the center of a short bipolar α helix recognized by a highly complementary hydrophobic groove on the receptor surface. Consistent with the conclusions in the study of other mutations and structures (B rozozowski, et al., And Feng, et al., Supra), residues from this helix 3, 4, 5, and 12, and between helix 3 and 4 The peptide binding groove formed by the turn is regarded as the surface of the ERα, ie, coactivator binding site, involved in transcriptional activity. In addition, studies of complexes between TRβ and GRIP1 NR Box II peptides, and studies of GRIP1 bound to TRβ and GR (Darimont, et al., Supra), and studies of the general properties of PPARγ / SRC-1 peptide complexes (above) , Nolte, et al.) Are similar to the studies for the ERα / GRIP1 NR Box II peptide complex described herein, suggesting that NR box recognition mechanisms are conserved across the nuclear receptor family.

Of the 11 AF-2 residues whose side chains interact with the coactivator helix (FIG. 3A), only four (Lys 362, Leu 379, Gln 375, and Gln 542) are highly conserved across the nuclear receptor family (above) Wurtz et al.). The side chains of Lys 362 and Leu 379 are buried even in the absence of GRIP1 binding, and appear to form integral parts of the surface structure involved in transcriptional activity. In contrast, the Gln 375 and Gln 542 side chains are generally solvents exposed in the absence of a coactivator and form both nonpolar contact and only receptor mediated interactions with the coactivator helix. The two capping interaction moieties are located opposite the coactivator binding groove to not only stabilize the backbone conformation of the coactivator, but also act as a molecular caliper; The 15 mm 3 spacing between Lys 362 and Glu542 was suitable for distinguishing the short 2 rotational co-activator α helix, the axis length of ˜11 mm 3 (FIG. Similar receptor mediated capping interactions were also observed in the complex between TRβLBD and the NR box II peptide (Darimont, et al., Supra). Mutations in either of these two residues severely impair coactivator binding by TRβ as well as ERα (Ref .: Apriletti, et al. And Feng, et al. And Henttu, et al.). Therefore, the formation of helix capping interactions may be a general feature of coactivator recognition by nuclear receptors.

The side chains of the six hydrophobic AF-2 residues (Ile 358, Leu 372, Val 376, Leu 379, Leu 539, and Met 543) show a high degree of van der Waals binding produced by the receptor with the hydrophobic side of the coactivator helix. Form the majority of the cooperative network of (FIGS. 3A and 3C). Mutations in Ile358, Val376, and Leu539 abolish GRIP1 binding (Feng, et al., Supra). Typically, although these residues are poorly conserved across nuclear receptors than Lys 362 or Glu542, their hydrophobic properties are conserved except Leu 372. Since different NR LBDs adopt the same overall fold (Moras, et al., Curr. Opin. CELL Biol. 10: 384-91 (1998)), this indicates that the hydrophobic portion of the surface of other nuclear receptor coactivator binding sites is distinctly organized. it means. In support of this hypothesis, the NR box II peptide used for crystallization inhibited binding to ERα, TRβ, and glucocorticoid receptor (GR) with very different efficiencies of GRIP1 (Ding, et al., Supra).

The hydrophobic side of the NR box helix is formed by the side chains of three motifs leucine and isoleucine (Ile 689) preceding the motif. The functional significance of leucine preserved during receptor binding has been demonstrated in many in vitro and in vivo studies. In contrast, the characterization of the role of residues preceding this motif in receptor binding was poor. Both biochemical and structural data associate Ile 689 as a determinant of major receptor conjugates. In this crystal, only the side chains of motif leucine and Ile 689 bind extensively with LBD in both non-crystalline symmetry related peptides. Mutation of Ile 689 to alanine reduces the peptide's ability to inhibit binding of GRIP1 to ERα by -30-fold in competitive assays (data not shown). The side chain of Ile 689 is located in a somewhat chemically distinct environment; This residue forms a van der Waals bond with the Asp 538 side chain, the side chain of Leu 539, and the aliphatic portion of the γ-carboxylate of Glu 542 (FIGS. 1A and 3A). Proximity of the hydrophobic side chain of Ile 689 with the negatively charged portion of Glu 542 increases the electrostatic potential of the carboxylate side chain and stabilizes the interaction with the N-terminus of the coactivator helix. Despite the apparently decisive role in receptor recognition, the conservation of identity of residues immediately preceding known NR boxes was poor. This sequence variability affects not only the packing interaction with ERα but also the chemical environment and very important orientation of Glu 542. This will alternately translate into a variety of affinity for the receptor. Indeed, three NR boxes from GRIP1, each containing a different residue preceded by the LXXLL motif (SEQ ID NO: 1), have different affinity for ERα (see Ding, et al .; Vogel, et al. EMBO J. 17: 507-19 (1998).

The NR box of TIF2, the human homologue of GRIP1, was found to be partially functionally overlapping despite individual differences in receptor binding affinity. All three NR boxes of TIF2 must be mutated to completely eliminate the interaction with ERα (Ding, et al .; Vogel, et al. EMBO J. 17: 507-19 (1998), supra). The data herein suggest that a single NR box peptide is sufficient to form a robust complex with a single ERαLBD. However, the p160 coactivator occupies multiple NR boxes. A possible explanation for the presence of multiple NR boxes is that they provide coactivators with broad specificity. Although receptor binding relies on the complex formation of multiple van der Waals interactions, many nuclear receptors appear to have different coactivator binding site surfaces. Other amino acids at positions immediately preceding the LXXLL motif (SEQ ID NO: 1) allow certain adaptability to these distinct surfaces; There may be no NR boxes that may have utility to bind all nuclear receptors. Thus, multiple NR boxes can provide the co-activator with the various interfaces needed to recognize many targets.

ERα transcriptional activity is blocked by antagonists such as OHT and RAL. The most surprising feature of the OHT and RAL ligand bound ERαLBDs structure is that helix 12 binds to the electrostatic region of the coactivator recognition groove (FIG. 3B and Brzozowski, et al.). Comparison of these two structures with the structure of the coactivator / LBD complex shows that within the agent complex, the region of Helix 12 with the NR box-like sequence (LXXML vs. LXXLL) (SEQ ID NO: 2 vs. SEQ ID NO: 1) It acts as an intramolecular pseudomaterial of the coactivator helix (FIG. 5 and Brzozowski, et al.).

In line with other proposals (Brzozowski, et al. And Darmont, et al.), The placement of Helix 12 directly affects the structure and function of the surface involved in transcriptional activity in two pathways. First, because the helix 12 residue forms an integral portion of the surface of the coactivator binding site, the surface is incomplete when helix 12 is an antagonist bound conformation. In particular, Leu 539, Glu 542, and Met 543 are incorrectly oriented for coactivator recognition. Second, residues from the electrostatic region of the surface bind to helix 12 and interfere with interaction with the coactivator (FIGS. 3A and 3B).

Sequence similarity to the LXXLL motif (SEQ ID NO: 1) of Helix 12 of ERαLBD appears to be unique in nuclear receptors. Although generally hydrophobic, the identity of the residues in this region of Helix 12 in other nuclear receptors is not as closely as the sequence of the NR box as in ERα (Wurtz, et al., Supra). However, intramolecular inhibitors with suboptimal recognition sequences can compete for coactivator binding when given at high local concentrations. However, if an endogenous inhibitor with a suboptimal recognition sequence is given at a very high local concentration, it may compete for the binding of the active agent.

Binding of OHT to ERα promotes Helix 12 formation, which inhibits binding of coactivators. OHT does not interact directly with any Helix 12 residues (FIG. 4B). In addition, the structure of LBD in the region of the coactivator binding site groove that interacts with helix 12 in the OHT complex is the same as in the DES and E ₂ complexes (FIGS. 3A, 3B and 5).

Numerous studies have demonstrated the importance of OHT side chains in receptor antagonism (Jordan, et al. And Robertson, et al., J. Med. Chem. 25: 167-71 (1982)). Comparison of the OHT and DES complex structures suggest that the binding mode of the OHT side chain excludes the agent induced formation of Helix 12. The OHT side chain protrudes out of the ligand binding pocket between helix 3 to 11 (FIGS. 2B, 6B, and 6C). As a result, the placement of Helix 12 across the ligand binding pocket, as in the agent binding configuration, incorporates the charged dimethylamino group in the amount of the OHT side chain in the hydrophobic cavity, and is steric between the dimethylamino group region of the side chain and the side chain of Leu540. Cause a conflict.

In terms of action, OHT is not simply a “side chain agonist.” OHT binding promotes the formation of LBD, which is distinct from that stabilized by DES or E ₂ binding. Imposes another restriction on

Helix 3, 8, and 11 in the DES and E ₂ complexes are 1 to 2 turns longer than they are in the OHT complex (Brzozowski, et al., Supra). Helix 11 terminates at Cys 530 in the DES and E ₂ complexes and at Tyr 526 in the OHT complex. Helix 12 starts at Leu 536 in the OHT complex. In the antagonist complex, it appears that Leu 536 forms a cooperative network of nonpolar bonds and hydrogen bonds with Glu 380 and Tyr 537, which stabilize the N-terminus of helix 12 (FIG. 1B). Thus, if helix 12 binds to the electrostatic region of the coactivator binding site in the presence of an agent, loop linking helixes 11 and 12 will have to span ˜17Å spanning five residues. Although theoretically possible, such a configuration is very tight and difficult to exist. In contrast, the long loop-linked helixes 11 and 12 in the OHT complex allow helix 12 to extend to the electrostatic region of the coactivator binding groove.

In DES and E ₂ complexes, helix 12 and loop linked helix 11 and 12 pack for helix 3 and 11, whereas in the OHT complex they do not pack (FIGS. 2A and 2B and Brzozowski, et al). The structure of the recently described E ₂ -LBD complex indicates that longer helixes in the DES and E ₂ complexes do not depend on the directed interaction of helix 12 in the agent-binding composition (Tanenbaum, et al. al). In this structure, the crystal packing composite causes helix 12 to bind with the symmetry-related molecule. It is clear that helix 12 is not located across the ligand binding pocket in this structure. Nevertheless, helix 3, 8 and 11 are longer than when they are in the OHT complex (FIG. 6A). Therefore, the long helix of the agonist complex occurs independently of the placement of helix 12 across the ligand binding pocket and instead is a direct result of the agonist binding.

The difference in secondary structure between the agent complex and the OHT complex results from a distinct arrangement of packing interactions induced by other ligands. A cooperative network of van der Waals bonds and βhairpins between many hydrophobic residues from helix 3, 7, 8, and 11 generated around DES or E ₂ appears to stabilize longer helix than in the agent complex (FIG. 4a and 6d). The placement of the OHT B ring allows many of the ligand binding pocket residues surrounding it to adopt a composition that is significantly different from those adopted in the DES or E ₂ structures. As a result, most of the packing interactions between residues present in the DES and E ₂ structures are lost or altered in the OHT structure (FIG. 6D). This structural modification causes the backbone of residues 339-341, 421-423, and 527-530 (which form part of the helix 3, 8, and 11 in the agonist structure, respectively) to adopt an extended conformation in the OHT structure ( 6a-d).

Thus, binding of OHT has two distinct effects, each contributing to the antagonism of the batch of helix 12 (FIG. 7). Helix 12 is prevented from locating across the ligand binding pocket by the OHT side chain. In addition, the selective packing arrangement of ligand binding pocket residues around OHT stabilizes the conformation of LBD allowing Helix 12 to reach the coactivator binding site and the electrostatic region of the pseudomaterial binding coactivator.

The mechanism does not appear to be specific for OHT. As in the case of OHT, the side chains of RAL stericly interfere with the agonist binding conformation of helix 12 (Brzozowski, et al., Supra). Helix 11 also appears to terminate at Met 528 in the RAL complex. This may be due to torsion in the binding pocket near His 542 by RAL binding (Brzozowski, et al., Supra).

The results are supported by the examples provided below to illustrate various aspects of the invention. The following examples do not limit the scope of the invention.

Example

Example 1

Experiment method

Protein Expression and Purification

BL21 (transformed with a modified pET-23d-ERG vector containing a Met-Asp-Pro sequence fused to residue 297 via 554 of human ERα-LBD 297-554fmf hERα (provided by Paul Sigler of Yale University) DE3) overexpressed in pLysS cells (Seielstad, et al., Mol. Endocrinol. 9: 647-658 (1995)). Purified bacterial lysate was adjusted to 3 M in urea and 0.7 M in NaCL, and then applied to an estradiol-sepharose 10 ml column (Greene, et al., Proc. Natl. Acad. Sci. USA 77: 5115). 5119 (1980); Landel, et al., Mol. Endocrinol. 8: 1407-1419 (1994); Landel, et al., J. Steroid Biochem. Molec. Biol. 63: 59-73 (1997).

To carboxymethylate the solvent-accessible cysteine, the bound hERαLBD was treated with 5 mM iodiacetic acid at pH 8.1, 10 mM Tris, 250 mM NaSCN (Hegy, et al., Steroids 61: 367-373 (1996)). Proteins were eluted using 3 × 10 ⁻⁵ M ligands (DES or OHT) in 30-100 ml of pH8.2, 50 mM Tris, 1 mM EDTA, 1 mM DTT and 250 mM NaSCN. The yield of hERαLBD was typically close to 100% (Seielstad, et al., Biochemistry 34: 12605-12615 (1995)). Affinity-purified material was concentrated by ultrafiltration and replaced with 20 mM Tris, 1 mM EDTA, 4 mM DTT at pH 8.1. The protein was combined with a Resource Q column (pharmacia) and then eluted under a linear gradient of 25-350 mM NaCl in 20 mM Tris, 1 mM DTT, pH 8.1. hERαLBD ligand complex was eluted at 150-200 mM NaCl. Fractions were concentrated by ultrafiltration and analyzed by SDS-PAGE, PAGE, and electrospray ionization mass spectrometry.

GST-Pulldown Analysis

Fusion between glutathione-S-transferase (GST) and amino acids 282-595 of hERα was prepared by subcloning EcoRI fragments from pSG5ERα-LBD (Lopez et al.) To pGEX-3X (pharmacia). Ile 358-> Arg, Lys 362-> Ala, and Leu 539-> Arg mutations were introduced into the GST-LBD construct using the Quick Change Kit (Stratagne) according to manufacturer's instructions. Subcloning the Val 376-> Arg, and Glu 54-> Lys mutations to the Bsm1 / HinIII fragment of pSG5-ER-HEGO to where the mutation was already introduced (Tora, et al., EMBO J, 8: 1981-6 (1989)) to produce within the GST-LBD construct. All constructs were identified by automated sequencing (University of Chicago Cancer Research Center DNA Sequencing Facility).

Wild-type and mutant GST-LBDs were expressed in BL21 (DE3) cells. Total ligand binding activity was determined by a controlled porous glass bead assay (Greene, et al., Mol. Endocrinol. 2: 714-726 (1988)) and the protein level was measured with a monoclonal antibody against hERα (H222). Monitor by blotting. Purified extracts containing GST-LBDs were added to a single buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 2 mM EDTA, 1 mM DTT, 0.5% NP-40 and protease inhibitor cocktail), or 1 μM of DES or OHT. Incubate at 4 ° C. in buffer for 1 hour. Extracted samples containing 30 pmol of GST-LBD were incubated with 10 μl glutathione-sepharose-4B beads (Falmacia) at 4 ° C. for 1 hour. Beads were washed five times with 20 mM HEPES, pH 7.4, 400 mM NaCl, 0.05% NP-40. ³⁵ S-labeled GRIP1 was synthesized by in vitro transcription and translation using the TNT Coupled Reticulocyte Lysate System (Promega) according to manufacturer's instructions and using pSG5-GRIP1 (presented by Michal Stallcup of the University of Southern California) as a template. . Immobilized GST-LBDs were incubated with 2.5 μl of a crude transcription reaction mixture diluted in 300 μl of Tris-buffered saline (TBS) for 2.5 h. After washing five times in TBS containing 0.05% NP-40, the protein was eluted by boiling the beads in sample buffer for 10 minutes. Binding ³⁵ S-GRIP1 was quantified by fluorography according to SDS-PAGE.

Crystallization and Data Collection

Crystals of the DES-hERα LBD-GRIP1 NR Box II peptide complex were obtained by rinsing drop vapor diffusion. Prior to crystallization, the DES-hERα LBD (residue 297-554) complex was incubated for 7-16 hours with a 2-4 fold molar excess of GRIP1 NR Box II peptide (SEQ ID NO: 4). 2 μL of a sample of this solution is mixed with an equal volume of storage buffer consisting of 25-27% (w / v) PEG 4000, 90 mM Tris (pH 8.75-9.0) and 180 mM Na acetate, and added to a well containing 800 μL of storage buffer. Suspended. After 4-7 days at 19-21 ° C., rod-shaped crystals were obtained. Coactivator complex crystals lie in space group P2 ₁ with cell specifications a = 54.09, b = 82.22, c = 58.04, and β = 111.34. Two molecules each of the DES-LBD and the coactivator peptide formed an asymmetric unit. A 200 μm × 40 μm × 40 μm crystals containing 25% (w / v) PEG 4000, 10% (w / v) ethylene glycol, 100 mM Tris (pH 8.5), 200 mM Na acetate, and 10 μm peptide Transfer to cryosolvent solution and freeze in nitrogen environment at -170 ° C in a rayon loop. Data from the crystals were measured at 170 ° C. in a Stanford Synchrotron Rsdiation Laboratory (SSRL) using a 300 mm MAR image plate at a wavelength of 1.08 GHz and beamline 7-1.

Crystals of the hERα LBD complex against OHT were obtained by rinsing drop vapor diffusion. Equivalent volume of a solution comprising a 3.9 mg / mL protein ligand complex and a stock solution containing 9% (w / v) PEG 8000, 6% (w / v) ethylene glycol, 50 mM HEPES (pH 6.7) and 200 mM NaCl Quotas (2 μL) were mixed and suspended for 800 μL of stock solution. Hexagonal plate crystals were obtained after 4-7 days at 21-23 ° C. The size and quality of the crystals were all improved by micro seeding technology. The decision belongs to space group P6 ₅ 22 with cell variables a = b = 58.24 ms and c = 277.47 ms. The asymmetric unit consists of a single LBD monomer; The dimer axis lies along two folds of crystallography. Single incubation (400 μm × 250 μm × 40 μm) is simply incubated in a cold protective solution consisting of 10% (w / v) PEG 8000, 25% (w / v) ethylene glycol 50 mM HEPES (pH 7.0), and 200 mM NaCl It was then frozen in liquid N ₂ suspended in a rayon loop. Diffraction data were measured at −170 ° C. using 345 mm MAR image plates at SSRL at wavelengths of beamlines 9-1 and 0.98 Hz.

Both images of both data sets were processed with DENZO and scaled with SCALEPACK (Otwinowski, et al, Methods Enzymol. 276: 307-326 (1997)) using a default value -3 cutoff.

Structure Determination and Purification

Early efforts to structure the DES-LBD-GRIP1 NR Box II peptide complex used a low interval (3.1 ms) data set (data not shown). Autorotation search was performed using POLARRFN (“CCP4 suite: Program for Protein Crystallography”, Acta Crystallogr. D 50: 760-763 (1994)), indicating the presence of an amorphous two minute chromosome (dyad). Two asymmetric LBDs were placed by molecular rearrangement in AMoRe (CCP4, 1994) using some polyalanine models of human RARγLBD (Renaud, et al.) As a search probe. If a model is given at this point, the model is inaccurate (rmsd 17 ms between the model and the last model based on the Cα position), incomplete (only 45% of the total scattered material in the asymmetric unit), and active density correction Instructions were followed. Repeated DM (CCp4, 1994) 2-fold NCS averaging interspersed with models in MOLOC (Muller, et al., Bull. Soc. Chim. Belg. 97: 655-667 (1988)), and models of LBD alone Model purification in REFMAC (Murshudov, et al., Acta Crystallor. D 53: 240-225 (1997)) was used to quickly build the system. For this process, MAMA (Kleywert, et al., "Halloween ... mask and bones.In form First Map to Final Model", Bailey, et al., Eds, Warington, England, SERC Daresbury Laboratory, 1994) All mask manipulations were used and PHASES (Furey, et al., PA33 Am. Cryst. Assoc. Mtg. Abstr. 18:73 (1990)) and CCP4 chute (CCP4) were used for the generation and weight calculation of the structural factors. .

However, although the DES-LBD-GRIP1 NR Box II peptide complex model is ˜90% of the scattering material in the asymmetric unit, purification is hindered by severe model bias. Next, the OHT complex data set was collected. Using monomers of the preliminary DES-LBD model as search probes, molecular rearrangements in AMoRe were used to search for LBD positions in these crystal forms in both P6 ₁ 22 and P6 ₅ 22. The translation search within P6 ₁ 22 yielded the correct result (R = 55.3%, CC = 38.2%). To reduce model bias, DMMULTI (CCP4, 1994) was used to calculate the averaged density from DES complex cells into OHT complex cells. The model was first purified in REFMAC and subsequently simulated annealing, batch and B-factor purification instructions using the maximum similarity target at X-PLOR (Bruger, X-PlLOR Version 3.843, New Havenm Connecticut: Yale University, 1996). (Adams et ai., Proc. Natl. Acad. Sci. USA 94: 5018-23 (1997)). Anisotropic scaling and bulk solvent correction were performed and the B-factor was isotropically purified. Except for the R _free set (8% random sampling of the data set), all data between 41 and 1.9 ms (σ cutoff absence) were included. The final model constitutes residues 306-551, ligands and 79 waters. According to PROCHEK (CCP4), 91.6% of all residues in the model were in the central region of the Ramachandran plot and there were no residues in the disallowed region.

Highly spaced data sets of the DES-LBD-GRIP NR Box II peptide complex were available when R _free of the OHT-LBD model was ˜31%. Both monomers in the asymmetric unit of the DES complex crystals were rearranged using AMoRe and a fully purified OHT-LBD model (with helix 12 and the loop between helix 11 and 12 removed) as a search model. The deletion portion of the model was built and the rest of the model was corrected using the 2-fold averaging maps generated in MOLOC and DM. First, purification was performed using REFMAC using solid NCS bondage. In the next step, the model was purified without NCS confinement using annealing, batch and B-factor purification instructions simulated in X-PlLOR, and maximum similarity targets. All B-factors were purified anisotropic and isotropic and bulk solvent calibration was performed. The R _free set contained 65 random sampling of all data. During purification, all data from 27 to 2.03 ms (no sigma cutoff) were used. The final model is residues 305-549 of monomer A, residues 305-461 and 470-554 of monomer B, residues 687-697 of peptide A, residues 686-696 of peptide B, two ligand molecules, 147 waters, two carboxyl groups And chlorine ions. According to PROCHEK, 93.7% of all residues in the model were in the central region of the Ramachandran plot and there was nothing in the unacceptable region.

FIG. 1A provides a 2Fo-Fc electron density map calculated at 1.03 ms intervals and created at 1.0σ, illustrating the interaction of GRIP1 NR box II with LBD. The GRIP1 NR Box II peptide (SEQ ID NO: 4) was removed prior to map calculation. ILe 689 from the peptide and two of the three receptor residues with which it interacts (Glu 542 and Leu 539) were labeled. Asp 538 has been removed for clarity. The hydrogen bonds between the γ-carboxylates of Glu 542 and the amides of residues 689 and 690 of the peptide are shown as dotted orange bonds. FIG. 1B provides a 2Fo-Fc electron density map calculated at 1.90 ms intervals and created at 1.0σ, showing the N-terminal region of helix 12. FIG. The dotted orange bond depicts a water-mediated hydrogen bond network of His377, an imidazole ring of γ-carboxylate with an amide of Tyr 537. The labeled three residues (Glu 380, Leu 536, and Tyr 537) bind to one another via van der Waals bonds and / or hydrogen bonds. Interestingly, mutations in each of these three residues greatly increased the transcriptional activity of the ligand-bound ERα LBD (Eng, et al., Mol. Cell. Biol. 17: 4644-4653 (1997): Lazennec, et. al., Mol.Endrcrinol. 11: 1375-86 (1997)); White, et al., EMBO J. 16: 1427-35 (1997))

Example 2

Structural measurement

GRIP1, a mouse p160 co-activator, interacts in vivo and in vitro with ERαLBD bound to an agent (Ding et al., Supra) but binds an antagonist with LBD (Norris, et al., J. Biol. Chem. 273 6679-88 (1998) does not. Mutation studies of GRIP1 and the human homologue TIF2 showed that NR box II (residues 690-694) of the three NR boxes from GRIP1 bound ERαLBD (Ding, et al., And Voegel. Et al.) Suggest to take.

The comparative assay is a 13 residues GRIP1 NR Box II peptide, NH ₂ -KHHILHRLLQDSS-CO ₂ H (SEQ ID NO: 4), synthesized by standard solid-phase synthesis corresponding to residues 686-698 of GRIP1 (Ding, et al., Supra). It is pointed out that it specifically binds to agonist binding ERαLBD (IC ₅₀ <0.4 μM; Kushner. Unpublished) and other agent binding NR LBDs (Ding, et al. And Darimont, et al.).

A migration shift assay of electrophoresis was used to demonstrate that GRIP1 NR Box II peptide (SEQ ID NO: 4) binds ERαLBD in the presence of agonist DES but not in the presence of antagonist OHT. Eight microgram samples of purified hERα-LBD combined with either DES or OHT are incubated in the absence of GRIP1 NR box II peptide (SEQ ID NO: 4), ie in buffer alone, or in 2 or 10 fold molar excesses. Cultured in the presence of GRIP1 NR Box II peptide. The binding reaction was carried out on ice for 45 minutes in 10 μl of buffer containing 20 mM Tris, pH8.1, 1 mM DTT, and 200 mM NaCl, followed by 6% PAGE. Gels were stained with GELCODE blue staining reagent (Pierce)

In the presence of the NR box II peptide, the migration of the DES-LBD complex was delayed. The addition of peptide did not have any effect on the mobility of the OHT-LBD complex. Thus, the peptide fragment of GRIP1 has the ligand dependent receptor binding activity of the full length protein. This observation suggests that GRIP1 NR Box II peptide is an effective model for studying the interaction between GRIP1 and ERαLBD.

To structurally characterize the interaction of the GRIP1 NR Box II peptide with ERαLBD, recombinant human ERαLBD (residues 297-554) was determined by binding to DES and the GRIP1 NR Box II peptide. In addition, ERαLBD bound to OHT was crystallized to determine the mechanism by which the antagonist blocked the coactivator / ERα interaction. X-ray diffraction data were measured from the crystals and the structure was determined by a combination of molecular rearrangement (using a modified version of the coordinates of the human retinoic acid receptor γ (RAAγ) LBD) and aggressive density adjustment.

The structure of the DES-ERα LBD-GRIP1 NR Box II peptide complex, as shown in FIGS. 1A and 2, was used to determine the 19.9% crystallographic R-factor (R _free = 25.0) using data for the 2.03 ′ interval. %). The structure of the OHT-ERα LBD complex was purified to 23.0% (R _free = 26.2%) crystallographic R-factor using data for 1.9 μs intervals, as shown in FIG. 1B and Table 2.

TABLE 2

Summary of Crystallographic Statistics

Ligand Data collection DES OHT Space group P2 ₁ P6 ₅ 22 interval 2.03 1.90 Observation 104189 269253 Eigenvalue 30265 23064 completeness(%) 98.4 99.1 R _sym (%) ^a 7.8 7.0 Average I / σI 9.8 16.1 Miniaturization Number of atoms other than hydrogen 4180 2070 R _cryst (%) ^b / R _free (%) 19.9 / 25.0 23.0 / 26.1 Combined r.m.s. Deviation (Å) 0.006 0.006 Angle r.m.s. Deviation) 1.05 1.05 Mean B factor (Å ² ) 34.0 40.4 ^a R _sym = ∑ _i │I _i- <I _i 〉 │∑I _i (where 〈I _i 〉 is the mean intensity for symmetrical equivalents) ^b R _cryst = ∑│F _O -F _C │ / ∑│F _O │

Example 3

Total Structure of DES-LBD-GRIP1 NR Box II Peptide Complex

The asymmetric unit of the DES-LBD-GRIP1 NR Box II peptide complex crystal contained the same non-crystalline dimer of LBDs bound with both E ₂ and RAL (Brozozowski, et al and Tanenbaum, et al). In addition to the flexible loop between Helix 2 and 3, and Helix 9 and 10, the two LBDSs of the dimer adopt a similar structure (rmsd 0.47 μs based on the Cα position). The structure of LBD combined with each DES was very similar to that of LBD combined with E ₂ ; Each monomer is a wedge shaped molecule consisting of three layers of 11-12 helixes and a single beta hairpin. The hydrophobic side of helix 12 in each LBD is packed for helix 3, 5/6, and 11 covered with ligand binding pockets. One GRIP1 NR Box II peptide binds to each LBD in a hydrophobic fragment consisting of a rotation between residues 3 and 4 of Helix 3, 4, 5, and 12 (FIGS. 2A and 3A). The density for both peptides in the asymmetric unique is continuous and unclear (FIG. 1A). Residues 687, 697 from the GRIP1 NR Box II peptide named Peptide A and residues 686 and 696 from the GRIP1 NR Box II peptide designated Peptide B were modeled; The remaining residues were irregular. When each peptide is placed in a different environment in the crystal, each peptide of residues Ile 689 to Gln 695 forms a two-turned amphoteric α helix (FIGS. 2A and 3A). During flanking of this region of the secondary structure usually, the peptide adopts a dissimilar random coil form.

The overall configuration of the DES-ERα-GRIP1NR box II peptide complex and the OHR-ERαLBD complex is shown in FIGS. 2 and 2A. Co-activator peptides and LBDs are shown as ribbons. The peptide is gold and helix 12 (residues 538-546) is magenta. Helix 3, 4 and 5 (denoted H3, H4, and H5, respectively) are shown in blue. DES in green suggests space charging. In FIG. 2B, LBD is represented by a ribbon. As in FIG. 2A, helix 12 (residues 536-544) indicated magenta, helix 3, 4, and 5 in blue. OHT shown in red suggests space filling.

Example 4

NR Box II Peptide-LBD Interface

GRIP1 NR Box II peptide binding to ERαLBD incorporated 1000Å ² of mainly hydrophobic surface region from each molecule. The GRIP1 NR box II peptide binding sites include residues Leu354, Val358, Ala361 and Lys362 from Helix 3; Leu372 from rotation between helix 3 and 4; Gln375, Val376, Leu379, and Glu380 from Helix 5; And a shallow groove consisting of Asp538, Leu539, Glu542, and Met543 from Helix 12 (FIG. 3 a). The bottom and sides of the groove are completely nonpolar, but the distal end of the groove is charged (FIG. 3C).

LBD interacts primarily in hydrophobicity of the GRIP1 NR box II peptide α helix formed by the side chain of Ile689 and the three LXXLL motifs (SEQ ID NO: 1), leucine (Leu690, Leu693, and Leu694). The side chains of Leu690 are deeply incorporated in the grooves, Ile358, Val376. Together with the side chains of Leu379, Glu380, and Met543 (FIGS. 3A and 3C) form van der Waals bonds. The side chains of Leu694 are similarly isolated in the grooves and form van der Waals bonds with the side chains of Ile358, Lys362, Leu372, Gln375, Val376 and Leu379 (FIGS. 3A and 3C). In contrast, both side chains of Ile689 and the second GRIP1 NR box leucine, Leu693 lie on the rim of the groove (FIGS. 3A and 3C). The side chain of Ile689 is placed in a shallow depression formed by the side chains of Asp538, Leu539, Glu542. The side chain of Leu693 forms a nonpolar bond with the side chains of Ile358 and Leu359.

Charged polar side chains that form the hydrophilic side of the peptide helix protrude from the receptor and interact strongly with the solvent or form a symmetric bond (FIG. 1A). With the exception of Lys688 of peptide B, the side chains of the polar and charged residues outside the helical region of the peptide in the asymmetric unit neither form hydrogen bonds bound to the LBD monomer nor create salt bridges. The ε-amino group of Lys688 of peptide B binds to the side chain carboxylate of Glu380 of monomer B. The interaction appears to be a crystal synthesizer; The backbone atoms of the three N-terminal residues of peptide B are substituted from monomer B and interact extensively with symmetric-related LBD.

In addition to interacting with the hydrophobic side of the peptide helix, the LBD stabilizes the backbone of the NR box peptide by forming a capping interaction with both ends of the peptide helix. Glu542 and Lys362 are located at opposite ends of the peptide binding moiety (FIG. 3A). The side chains of Glu542 and Lys362 form van der Waals bonds with the main and side chains at the N- and C-terminal rotations of the respective peptide helix. This interaction places the stabilizing charge of the γ-carboxylate of Gly542 and the ε-amino group of Lys362 near the GRIP1 NR box II peptide helix end (FIG. 3C). The side chain carboxylate (γ-carboxylate) of Glu542 hydrogen bonds with the residue amide of the N-terminal rotation of peptide helix (residues 688 and 689 of peptide A; residues 689 and 690 of peptide B) (FIG. 1A). Similarly, the ε-amino group of Lys362 hydrogen bonds with the carbonyl of the residue of the C-terminal rotation of the peptide helix (residue 693 of peptide A; residues 693 and 694 of peptide B) (FIG. 5).

To test the importance of the GRIP1 NR Box II peptide / LBD interface observed during the determination, a series of positional mutations were introduced into ERαLBD. These mutations simultaneously disturb the structural integrity and formation of capping interactions (Lys362-> Ala and Glu542-> Lys) at the same time with the nonpolarity of the bottom of the structural integrity and binding grooves (Ile358-> Arg, Val376-> Arg and Leu539-> Arg). Designed to prevent. Fusion of glutathione-S-transferase (GST) to wild-type and mutant LBDs assayed the binding of ³⁵ S-labeled GRIP1 in the absence of ligand or in the presence of DES or OHT. ³⁵ S-labeled GRIP1 was incubated with either immobilized GST, immobilized wild type GST-hERαLBD, or without ligand, or immobilized mutant GST-LBD in the presence of DES or OHT. The bound GRIP1 was quantified after SDS-PAGE. I358R, mutant LBD containing Ile-> Arg substitution at residue 358; K362A, mutant LBD containing Lys-> Ala substitution at residue 362; V376R, mutant LBD containing Val-> Arg substitution at residue 376; L539R, mutant LBD containing Leu-> Arg substitution at residue 539; E542K, mutant LBD containing Glu-> Lys substitution at residue 542.

In the absence of ligand or in the presence of OHT, fusion to wild type protein with all mutant LBD did not suggest detectable binding with GRIP1. The Ile358-> Arg, Val376-> Arg and Leu539-> Arg mutations are incapable of interacting with co-activators in the presence of agents that confirm the importance of the packing interactions observed in the crystals. Cleavage of either the N-terminal or C-terminal capping interaction also included GRIP1 binding in the presence of the agent. Only wild type GST-LBD was able to recognize the coactivator in the presence of DES.

Example 5

Agent Awareness

Despite different shape and volume and large molecules, DES (273Å ³⁾ were accommodated in the same binding pocket that recognizes the E ₂ (252Å ^3). In its receptor complex, DES was wrapped in a narrow half of LBD in a strong hydrophobic cavity consisting of residues from S1 / S2 hairpins (FIGS. 2A and 4A) as well as helix 3, 6, 7, 8, 11, and 12. The interaction between ERα and DES was similar to that of E ₂ . One of the DES phenolic rings was placed in the same position adjacent to helix 3 and 6 as the E ₂ A ring. Like the aromatic ring of E ₂ , the DES A ring (FIG. 4A) is a Phe404 having phenolic hydroxyl forming hydrogen bonds to the γ-carboxylate of Glu353, guanidinium of Arg394 and structurally conserved water molecules, Connected by the side chains of Ala350, Leu387 and Leu391. The A 'ring of DES (FIG. 4A) binds near helix 7, 8, and 11 with the locations of the E ₂ C and D rings. The ring forms a van der Waals bond with Gly521 and Leu525 as well as Met343, Leu346 and Met421 as the D ring of E ₂ (FIG. 4A). Even located at 1.7 kPa from the D ring hydroxyl, the DES A 'ring phenolic hydroxyl is still capable of hydrogen bonding to the imidazole ring of His524 (FIG. 4A).

DES also forms a bond with LBD, but E ₂ does not. There is an empty cavity adjacent to the α side of the B ring of E ₂ and the β side of the C ring (Brzozowski, et al., Tanenbaum, et al., Supra). The ethyl group of DES projecting perpendicularly from the plane of the phenolic ring fits into the space. The results of further nonpolar binding of the Ala350, Leu384, Phe404, and LeU428 with the side chain (FIG. 4A) account for the high affinity of DES for the receptor (Kuiper, et al., Endocrinology 138: 863-70 (1991)). . Except for Met421 and Met528, both of which only bind E ₂ , the ER can use the same moiety to form both the observed hydrogen bonds and van der Waals bonds with both agents (FIG. 4A, Brzozowski, et. al., and Tanenbaum, et al.). The excellent adaptability is probably the result of the relatively large molecular volume and pronounced structural plasticity of the binding pockets (˜500 mm ³ in both complexes). In particular, at the end of the DES A 'ring / steroid D ring of the binding pocket, Met343, Met421, His524 and Met528 adopt different packing forms in response to their respective ligands (not shown). This flexibility is necessary to allow the receptor to recognize endogenous estrogens such as estrone and estriol that are structurally different from E ₂ in the D ring.

Example 6

Structure of the OHT-LBD Complex

OHT binding induced a milky LBD conformation that differed in secondary and tertiary structures from those induced by DES binding. In the DES complex, the backbones of residues 339-341, 421-423, and 527-530 formed portions of helix 3, 8, and 11, respectively. In contrast, the region adopted extended conformation in the OHT complex (FIGS. 2A and 6A). In addition, the composition and orientation of helix 12 were different in the two structures. Helix 12 in the OHT complex constitutes residues 536-544, while helix 12 in the DES complex constitutes residues 538-546. In particular, rather than covering the ligand binding pockets as in the DES complex, helix 12 in the OHT complex is an adjuvant formed by residues from helix 3, 4 and 5 and rotationally bound helix 3 and 4 (FIGS. 2A, 2B and 3B). Occupies a portion of the activator binding groove. The alternative Helix 12 morphology appears to be similar to that observed in the RAL complex (Brozozoski, et al.).

Example 7

Helix 12-LBD Shared Area

Except for the orientation of helix 12, the structure of the peptide binding grooves was almost identical in the DES and OHT complexes (FIGS. 3A and 3B). The region outside the groove of helix 12 is referred to herein as the "electrostatic region" at the NR box junction. Helix 12 in the OHT complex and NR box peptide helix in the DES complex interact with the electrostatic region of the coactivator recognition groove in a significantly similar manner.

Helix 12 mimics the hydrophobic interaction of the NR box peptide with the electrostatic region of the groove with a stretch of residues similar to the NR box (LLEML instead of LXXLL) (SEQ ID NO: 1 instead of SEQ ID NO: 1) (residues 540 to 544). . The side chains of Leu 540 and Met 543 lie generally in the same position as the side chains of the first and second motifs leucine (Leu 690 and Leu 693) in the peptide complex (FIG. 5). Leu 540 was inserted into the grooves and formed van der Waals bonds with Leu 354, Val 376, and Glu 380 (FIGS. 3B and 3D). Met 543 lies along the edge of the groove and forms a van der Waals bond with the side chains of Leu 354, Val 355, and Ile 358 (FIGS. 3B and 3D). The side chain position of the Leu 544 almost exactly overlapped the position of the Leu694 (FIG. 5), the third NR box leucine. Deep in the grooves, Leu 544 side chains formed van der Waals bonds with the side chains of Ile358, Lys362, Leu372, Gln 375, Val 376, and Leu 379 (FIGS. 3B and 3D).

Helix 12 in the OHT complex is also stabilized by N- and C-terminal capping interactions. Lys 362 interacted with the C-terminal rotation of helix 12 as it interacted with the equivalent rotation of the helix peptides (FIGS. 3A and 3B). Lys 362 side chains were packed for C-terminal rotation of helix 12 with ε-amino group hydrogen bonds to the carbonyls of residues 543 and 544 (FIG. 5). The capping interaction given in the N-terminal rotational co-activator helix is formed by the helix 12 residue (Glu 542), and the N-terminal rotation of helix 12 in the antagonist complex is such that it interacts with another residue, Glu380 (Figures 3B and 3D) did. The Glu 380 γ-carboxylate forms a van der Waals bond with Tyr 537 and interacts with the amide of Tyr 537 through a series of water-mediated hydrogen bonds (FIG. 1B).

In addition to forming the "NR boxes" interconnections, helix 12 also forms van der Waals bonds with regions outside the LBD of the coactivator recognition groove. The side chains of Leu536 form van der Waals bonds with Glu 380 and Trp 383, and the side chains of Tyr 537 form van der Waals bonds with His 373, Val 376, and Glu380 (FIG. Lb, 3b and 3d). The binding results in helix 12 in the OHT complex containing more solvent proximity surface area (˜1200 Hz) than the NR box peptide in the DES complex.

Example 8

OHT recognition

OHT was combined into the same pocket recognizing DES, E ₂ and RAL. The orientation of the OHT in the binding pocket appears to be indicated by the two structural features of the ligand, the placement of the phenolic A ring and the large side chains (FIGS. 4B and 6C). The A ring of OHT binds to approximately the same position near helix 3 and 6 with phenolic hydroxyl hydrogen bonds to the side chains of Glu 353 and Arg 394 (FIG. 4b) and structurally conserved water like the A ring of DES. Like the huge side chains of the RAL, the side chains of the OHT vented the binding pockets between Helix 3 and 11 (FIGS. 2B and 4B). The OHT C ring (FIG. 4B) formed van der Waals bonds with the side chains of Met 343, Leu 346, Thr 347, Ala 350, Trp 383, Leu 384, Leu 387, and Leu 525. The arrangement in the flowable dimethylaminoethyl region of the side chain was stabilized by van der Waals bonds with Thr 347, Ala 350 and Trp 383, with a 3.8 kPa spacing between the dimethylamino group of the side chain and the β-carboxylate of Asp 351 It was stabilized by laying salt crosslinks. In the context of the robust triphenylethylene framework of OHT, the position of the A ring and side chains requires that the ethylene group of the OHT be placed in a direction that is nearly orthogonal to the position of the ethylene group of the DES (FIGS. 4A, 4B and 6D). As a result, the B ring of OHT is incorporated into the binding pocket deeper than the A 'ring of DES (FIGS. 6B and 6C).

OHT B ring It is clear that this position is unacceptable for the same mechanism that allows it to adapt to the other structural features of the DES A 'ring / E ₂ D ring ends DES and E ₂ of the binding pocket. Instead, residues that bind to the B ring (Met 343, Leu 346, Met421, Ile424, Gly% @ !, His 524 and Leu 525), which interact mostly with the A 'ring of DES, have been identified in the DES structure (FIG. 6D). Adopt a different configuration from the one adopted. Indeed, the location of the B ring substantially prevents the side chain of one residue Met 421 from adopting the same configuration as employed in the DES structure (FIGS. 6B and 6C). As a result of the B ring-induced side chain formation, no van der Waals bonds between many residues present in the DES complex were present in the OHT complex. For example, Met 421 packs for His 524 from Helix 11 and Met 343 from Helix 3 in the agonist complex, while Met is bound to any of these residues in the antagonist complex (FIG. 6D) by the OHT B ring location. Prevents mutual coupling with one.

The structural effect of the placement of the B ring is not limited to residues that bind to the B ring; the shape of the residue allows alternate residues across the binding pocket to alternately employ selective formation. For example, the conformation adopted by Met 421 in the OHT complex prevents the Phe 404 and Phe 425 side chains from occupying the positions occupied in the DES complex (FIGS. 6B and 6C). As a result, no bond was formed with the A ring of DES or E ₂ (FIG. 6C). Indeed, Phe 404 binds to the ethyl group of OHT (FIGS. 6C and 6D). Selective conformation of the side chains of the two residues, which are directly bound to and indirectly affected by the B ring, allows the main chain across the binding pocket to also adopt another configuration (FIG. 6D).

Identification and characterization of key residues in the ligand binding domain of ERα and expansion of these information to other nuclear receptors showed that these residues are common for all nuclear receptors identified in the data. As such, the examples presented herein can be applied to the design and selection of compounds that modulate the binding of compounds to nuclear receptors for all members of the nuclear receptor family, with information obtained in the structure and function of the ERα ligand binding domain. Proved.

All publications and patents mentioned in the above specification are herein incorporated by reference.

The present invention described above is, of course, capable of many changes and modifications without departing from the spirit and scope of the appended claims.

Summary of the Invention

The present invention relates to further identification and manipulation of ligand binding domains (LBD), which allows the design of compounds that bind to LBD and modulate the activity of nuclear receptors, in particular estrogen receptors. Such compounds include agents and antagonists that modulate nuclear activity and may be receptor-, cell-, and / or tissue specific. In particular, the compounds affect coactivator-coactivator reaction site interactions to modulate nuclear receptor activity.

The present invention also encompasses protein cocrystals of nuclear receptors having peptides bound to LBD-binding agents and co-activator binding sites, and methods for their preparation. Similarly, the present invention also includes protein cocrystallization of nuclear receptors with antagonists bound to LBD and methods for their preparation. The cocrystal provides a method for obtaining atomic modeling of specific amino acids and their atoms to form LBD, and information about coactivator binding sites that interact with molecules that bind to their sites. The cocrystal also provides modeling information for the ligand: nuclear receptor, and coactivator: nuclear receptor interaction, and the structure of the ligand bound thereto.

The invention further provides methods for the identification and design of molecules that regulate ligand binding to nuclear receptors using atomic models of nuclear receptors. The method can be used to model a test compound that specifically fits within a nuclear receptor LBD using an atomic structure model comprising a nuclear receptor LBD or a protein thereof, for example, screening of a test compound by a biological assay, test compound and nuclear receptor Characterization by binding to LBD, and identification of test compounds that modulate ligand binding to receptors.

The invention also includes compositions and methods for identifying key residues in LBDs of nuclear receptors. The method involves examining the surface of the nuclear receptor of interest to identify residues that regulate the binding of ligands and / or coactivators. Such residues may be identified by homology search for key residues in the LBD of human ERα described herein. Overlaying and superpositioning using LBDs of nuclear receptors, and / or stereoscopic models of their proteins can also be used for this purpose. In addition, 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 within the context of the cell.

Also provided are methods for modulating the activity of nuclear receptors. The method may be in vivo or in vitro. The method comprises administering a sufficient amount of a compound that binds to a ligand binding domain in vitro or in vivo and is active as an agent or antagonist. Preferred compounds are those that bind sites with greater affinity than ligands found in the cells of interest.

The present invention further includes methods for identifying agonists or antagonists of ligands that bind nuclear receptors. The method comprises providing atomic coordinates comprising a nuclear receptor binding domain or a protein thereof to a computerized modeling system; Modeling a compound that spatially fits into the ligand binding domain; Assays for Nuclear Receptor Activity For example, biological assays include identifying compounds that increase or decrease the activity of nuclear receptors through ligand binding domains.

Also provided is a device readable data storage medium with information on the ligand binding domain or portion thereof. The medium comprises a data storage material encoded with device decipherable data, and when using a programmed device with instructions for using the data, the data is a stereoscopic graphic of a molecule or molecular complex for a nuclear receptor ligand binding domain. Can show reproduction

Also provided are methods for identifying compounds that selectively modulate the activity of one type of nuclear receptor compound compared to other nuclear receptors. The method comprises modeling a test compound that spatially and preferentially fits into the nuclear receptor binding domain of interest using an atomic structure model of nuclear receptor LBD, wherein the compound interacts with one or more residues of the LBD unique within the context of the site. Selecting, and identifying, for example, biologically assaying, compounds that selectively bind LBD to other nuclear receptors. Such unique features included in receptor selective ligands can be identified by isoforms of other nuclear receptors or of the same type of receptor.

The invention relates to peptides, peptidomimetics, and other compounds identified by the methods of the invention, such as small organic molecules, in particular nuclear receptor related diseases, in particular steroid receptor related diseases, and even more specifically estrogen receptor related It can be used for the selection and characterization of novel inducing compounds useful for the treatment of diseases.

Claims (71)

Model of the atomic structure of the estrogen receptor α ligand binding domain or part thereof Modeling a test compound that spatially conforms into the nuclear receptor ligand binding domain of interest using Dell; Screening the test compound with an assay characterized by the binding of the test compound with the ligand binding domain; And A method of identifying a compound that modulates nuclear receptor activity, the method comprising identifying a test compound that modulates nuclear receptor activity, the method comprising: The atomic structure model comprises atomic coordinates of amino acid residues corresponding to residues of human estrogen receptor α Met343, Leu346, Ala350, Glu353, Leu384, Leu387, Leu391, Arg394, Phe404, Met421, Leu428, Gly521, His524, Leu525 and Met528. Way. The method of claim 1, wherein the amino acid residues correspond to residues Met343, Met421, His 524, Leu525, and Met528. The method of claim 1 wherein the test compound is an agent and the nuclear receptor activity is measured by binding of the coactivator to the coactivator binding site. The method of claim 1, wherein the test compound is an antagonist and nuclear receptor activity is measured by annealing of helix 12. The method of claim 1, wherein the test compound is an antagonist and the nuclear receptor activity is measured by blocking the coactivator bonds. The method of claim 1, wherein the screening is in vitro screening. 7. The method of claim 6, wherein the screening is high throughput screening. The method of claim 1, wherein the test compound is derived from a library of compounds. The method of claim 1 wherein the test compound is a small organic molecule, peptide, or peptidomimetic. The method of claim 1, further comprising providing atomic coordinates of the estrogen receptor α ligand binding domain or portion thereof to the computerized modeling system prior to the modeling step. 2. The nuclear receptor of claim 1, wherein the nuclear receptor is selected from the group consisting of estrogen receptor, thyroid receptor, retinoid receptor, glucocorticoid receptor, progestin receptor, mineralocorticoid receptor, androgen receptor, peroxysome receptor, and vitamin D receptor. How to. 12. The method of claim 11, wherein the nuclear receptor is an estrogen receptor. 13. The method of claim 12, wherein the estrogen receptor is estrogen receptor α. Modeling a test compound that spatially conforms into the nuclear receptor ligand binding domain of interest using an atomic structure model of the estrogen receptor α ligand binding domain or portion thereof; Screening the test compound in an assay characterized by binding of the test compound to the binding domain; And A method of identifying a compound that modulates binding of a ligand to a nuclear receptor, the method comprising identifying a test compound that modulates binding of the ligand to a nuclear receptor, the method comprising: The atomic structure model comprises atomic coordinates of amino acid residues corresponding to residues of human estrogen receptor α Met343, Leu346, Ala350, Glu353, Leu384, Leu387, Leu391, Arg394, Phe404, Met421, Leu428, Gly521, His524, Leu525 and Met528. Way. The method of claim 14, wherein the amino acid residues correspond to residues Met343, Met421, His 524, Leu525, and Met528. 15. The nuclear receptor of claim 14, wherein the nuclear receptor is selected from the group consisting of estrogen receptor, thyroid receptor, retinoid receptor, glucocorticoid receptor, progestin receptor, mineralocorticoid receptor, androgen receptor, peroxysome receptor, and vitamin D receptor. How to. The method of claim 16, wherein the nuclear receptor is an estrogen receptor. 18. The method of claim 17, wherein the estrogen receptor is estrogen receptor α. The method of claim 14, wherein the screening is in vitro screening. 20. The method of claim 19, wherein the screening is high throughput screening. The method of claim 14, wherein the test compound is derived from a library of compounds. The method of claim 14, wherein the test compound is an agonist or antagonist of ligand binding. The method of claim 14, wherein the test compound is a small organic molecule, peptide, or peptidomimetic. Providing atomic coordinates of the estrogen receptor α ligand binding domain or portion thereof to the computerized modeling system; Modeling a compound that spatially fits into the ligand binding domain; And Assays for nuclear receptor activity include identifying a compound that binds to the ligand binding domain of the nuclear receptor to increase or decrease the activity of the nuclear receptor, wherein the agonist and antagonist of ligand binding are identified, Identifies agonists or antagonists of ligand binding to nuclear receptors whose atomic coordinates are the human estrogen receptor α Met343, Leu346, Ala350, Glu353, Leu384, Leu387, Leu391, Arg394, Phe404, Met421, Leu428, Gly521, His524, Leu525 and Met528 How to. The method of claim 24, wherein the amino acid residues correspond to residues Met343, Met421, His 524, Leu525, and Met528. 25. The nuclear receptor of claim 24, wherein the nuclear receptor is selected from the group consisting of estrogen receptor, thyroid receptor, retinoid receptor, glucocorticoid receptor, progestin receptor, mineralocorticoid receptor, androgen receptor, peroxysome receptor, and vitamin D receptor. How to. 27. The method of claim 26, wherein the nuclear receptor is an estrogen receptor. The method of claim 27, wherein the estrogen receptor is estrogen receptor α. A method of modulating nuclear receptor activity in a mammal by administering to an animal in need of modulation of nuclear receptor activity a mammal that is spatially and preferentially incorporating into the ligand binding domain of the nuclear receptor of interest a compound wherein the compound is a human estrogen receptor α Met343. , Leu346, Ala350, Glu353, Leu384, Leu387, Leu391, Arg394, Phe404, Met421, Leu428, Gly521, His524, Leu525, and a compound designed to modify one or more amino acid residues corresponding to the residues of Metu. The method of claim 29, wherein the one or more amino acid residues correspond to residues Met343, Met421, His 524, Leu525, and Met528. 30. The nuclear receptor of claim 29, wherein the nuclear receptor is selected from the group consisting of estrogen receptor, thyroid receptor, retinoid receptor, glucocorticoid receptor, progestin receptor, mineralocorticoid receptor, androgen receptor, peroxysome receptor, and vitamin D receptor. How to. 32. The method of claim 31, wherein the nuclear receptor is an estrogen receptor. 33. The method of claim 32, wherein the estrogen receptor is estrogen receptor α. A device readable data storage medium comprising a data storage material encoded with device readable data, wherein the human estrogen receptor α Met343, Leu346, Ala350, Glu353, Leu384, Three-dimensional graphics of molecular complexes of compounds bound to nuclear receptor ligand binding domains or their homologues comprising structural coordinates of amino acids corresponding to Leu387, Leu391, Arg394, Phe404, Met421, Leu428, Gly521, His524, Leu525 and Met528 Media that can be displayed. 35. The medium of claim 34, wherein the amino acid residues correspond to residues Met343, Met421, His 524, Leu525, and Met528. 35. The nuclear receptor of claim 34, wherein the nuclear receptor is selected from the group consisting of estrogen receptor, thyroid receptor, retinoid receptor, glucocorticoid receptor, progestin receptor, mineralocorticoid receptor, androgen receptor, peroxysome receptor, and vitamin D receptor. Media to say. 37. The medium of claim 36, wherein the nuclear receptor is an estrogen receptor. 38. The medium of claim 37, wherein the estrogen receptor is estrogen receptor α. 35. The molecular complex of claim 34, wherein the molecular complex is defined as a set of structural coordinates as shown in Appendix 1 and Appendix 2, or as a homologue of a molecular complex having a mean square root deviation from a skeletal atom of amino acids of less than 1.5 kHz. media. At least a portion of the structural coordinates corresponding to the second set of device readable data, when combined with the second set of device readable data, using a device programmed with instructions for use of the first and second sets of data. A device readable data storage medium comprising a data storage material encoded with a first set of device readable data, wherein The first set of data comprises a Fourier transform of some or more structural coordinates selected from the group consisting of the coordinates described in Appendix 1 or Appendix 2; And the second set of data comprises an X-ray diffraction pattern of the molecule or molecular complex. 41. The nuclear receptor of claim 40, wherein the nuclear receptor is selected from the group consisting of estrogen receptor, thyroid receptor, retinoid receptor, glucocorticoid receptor, progestin receptor, mineralocorticoid receptor, androgen receptor, peroxysome receptor, and vitamin D receptor. Media made. 42. The medium of claim 41 wherein the nuclear receptor is an estrogen receptor. The medium of claim 42 wherein the estrogen receptor is estrogen receptor α. A nuclear receptor cocrystal diffracted at intervals of 2.03 μs or more comprising an agent bound to a ligand binding domain and a molecule bound to a coactivator binding site of a nuclear receptor. The cocrystal of claim 44, wherein the nuclear receptor is an estrogen receptor α. 46. The cocrystal of claim 45, which is a human estrogen receptor α. 48. The cocrystal of claim 46, wherein the molecule is a peptide. 48. The cocrystal of claim 47, wherein the peptide comprises an NR box amino acid sequence or derivative thereof. A nuclear receptor cocrystal diffracted at intervals of 1.9 mm 3 or more, comprising an antagonist bound to the ligand binding domain of the nuclear receptor. 50. The cocrystal of claim 49, wherein the nuclear receptor is estrogen receptor α. 51. The co-crystal of claim 50 which is a human estrogen receptor α. Corresponds to the residues of human receptor estrogen α Met343, Leu346, Ala350, Glu353, Leu384, Leu387, Leu391, Arg394, Phe404, Met421, Leu428, Gly521, His524, Leu525, and Met528, preferably Met343, Met421 His524, Leu525 and Met528 A computerized method of designing a nuclear receptor ligand wherein one or more amino acid residues of the nuclear receptor LBD interact with one or more first chemical components of the ligand, In order to produce a second chemical having a structure that increases or decreases the interaction between interacting amino acids and the second chemical, as compared to the interaction between interacting amino acids and the first chemical. Selecting one or more chemical modifications of the chemical component. The method of claim 52, wherein the one or more amino acid residues correspond to residues Met343, Met421, His 524, Leu525, and Met528. 53. The method of claim 52, further comprising measuring a change in interaction between the interacting amino acid and the ligand after chemical modification of the first chemical component. 53. The method of claim 52, wherein the chemical modification is hydrogen bond interaction, charge interaction, hydrophobic interaction, van der Waals between the interacting amino acid and the second chemical component in comparison to the interacting amino acid and the first chemical component. Interaction, or bipolar interaction. 53. The method of claim 52, wherein the chemical modification is hydrogen bond interaction, charge interaction, hydrophobic interaction, van der Waals between the interacting amino acid and the second chemical component in comparison to the interacting amino acid and the first chemical component. Reducing interactions, or bipolar interactions. 53. The nuclear receptor of claim 52, wherein the nuclear receptor is selected from the group consisting of estrogen receptor, thyroid receptor, retinoid receptor, glucocorticoid receptor, progestin receptor, mineralocorticoid receptor, androgen receptor, peroxysome receptor, and vitamin D receptor. How to. 58. The method of claim 57, wherein the nuclear receptor is an estrogen receptor. The method of claim 52, wherein the estrogen receptor is estrogen receptor α. 60. The method of claim 59, wherein the ligand is an agent. 61. The method of claim 60, wherein the ligand is from the group consisting of 17β-estradiol, diethylstilbestrol, magestrol, mesohexestrol, comethrol, Δ ⁹ -THC, op-DDT, giralenone, and kepon. A method characterized by being white. 62. The method of claim 61, wherein the ligand is 17β-estradiol and the first chemical is free carbon of the A 'ring disposed at a position selected from the group consisting of C6α, C7α, C12α, C15α, and C17α. 60. The method of claim 59, wherein the ligand is an antagonist. 64. The method of claim 63, wherein the ligand is selected from the group consisting of ICI 164,384 and EM800. 60. The method of claim 59, wherein the ligand is a selective estrogen receptor modulator. 66. The method of claim 65, wherein the ligand is selected from the group consisting of tamoxifen, raloxifene, and GW5638. A method of modulating nuclear receptor activity by administering to a mammal in need thereof a sufficient amount of ligand that is spatially and preferentially in agreement with the ligand binding domain of the nuclear receptor of interest, The ligands are designed by computerized methods and the human estrogen receptors α Met343, Leu346, Ala350, Glu353, Leu384, Leu387, Leu391, Arg394, Phe404, Met421, Leu428, Gly521, His524, Leu525, and Met528, preferably Met343 Amino acid residues of at least one nuclear receptor ligand binding domain corresponding to Met421 His524, Leu525 and Met528, interact with the first chemical component of the ligand, To generate a second chemical component having a structure that increases or decreases the interaction between the interacting amino acid and the second chemical component as compared to the interaction of the interacting amino acid with the first chemical component Selecting one or more variations of the component. 67. The method of claim 67, wherein the amino acid residues correspond to residues Met343, Met421, His 524, Leu525, and Met528. 68. The method of claim 67, wherein the ligand is an antagonist. 68. The method of claim 67, wherein the ligand is an agent. 71. The method of claim 70, further comprising administering a coagent active agent designed in a computerized manner, Human Estrogen Receptor α Helix 3 residue Leu354, Val355, Met357, Ile358, Ala361 and Lys 362, Helix 4 residue Phe367, Helix 5 residue Gln375 Val376, Leu379 and Glu380, Helix 6 residue Trp383, and Helix 12 residue Asp538, Leu539, Glu542, One or more amino acid residues of the nuclear receptor coactivator binding site corresponding to Met543 and Leu544 interact with one or more first chemical components of the coactivator pseudo-material, compared to the interaction between the interacting amino acids and the first chemical component. Selecting one or more chemical modifications of the first chemical component to produce a second chemical component having a structure that increases or decreases the interaction between the interacting amino acid and the second chemical component. How to.