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Structural Basis for Accommodation of Nonsteroidal Ligands in the Androgen Receptor

2005; Elsevier BV; Volume: 280; Issue: 45 Linguagem: Inglês

10.1074/jbc.m507464200

ISSN

1083-351X

Autores

Casey E. Bohl, Duane D. Miller, Jiyun Chen, Charles E. Bell, James T. Dalton,

Tópico(s)

Estrogen and related hormone effects

Resumo

The mechanism by which the androgen receptor (AR) distinguishes between agonist and antagonist ligands is poorly understood. AR antagonists are currently used to treat prostate cancer. However, mutations commonly develop in patients that convert these compounds to agonists. Recently, our laboratory discovered selective androgen receptor modulators, which structurally resemble the nonsteroidal AR antagonists bicalutamide and hydroxyflutamide but act as agonists for the androgen receptor in a tissue-selective manner. To investigate why subtle structural changes to both the ligand and the receptor (i.e. mutations) result in drastic changes in activity, we studied structure-activity relationships for nonsteroidal AR ligands through crystallography and site-directed mutagenesis, comparing bound conformations of R-bicalutamide, hydroxyflutamide, and two previously reported nonsteroidal androgens, S-1 and R-3. These studies provide the first crystallographic evidence of the mechanism by which nonsteroidal ligands interact with the wild type AR. We have shown that changes induced to the positions of Trp-741, Thr-877, and Met-895 allow for ligand accommodation within the AR binding pocket and that a water-mediated hydrogen bond to the backbone oxygen of Leu-873 and the ketone of hydroxyflutamide is present when bound to the T877A AR variant. Additionally, we demonstrated that R-bicalutamide stimulates transcriptional activation in AR harboring the M895T point mutation. As a whole, these studies provide critical new insight for receptor-based drug design of nonsteroidal AR agonists and antagonists. The mechanism by which the androgen receptor (AR) distinguishes between agonist and antagonist ligands is poorly understood. AR antagonists are currently used to treat prostate cancer. However, mutations commonly develop in patients that convert these compounds to agonists. Recently, our laboratory discovered selective androgen receptor modulators, which structurally resemble the nonsteroidal AR antagonists bicalutamide and hydroxyflutamide but act as agonists for the androgen receptor in a tissue-selective manner. To investigate why subtle structural changes to both the ligand and the receptor (i.e. mutations) result in drastic changes in activity, we studied structure-activity relationships for nonsteroidal AR ligands through crystallography and site-directed mutagenesis, comparing bound conformations of R-bicalutamide, hydroxyflutamide, and two previously reported nonsteroidal androgens, S-1 and R-3. These studies provide the first crystallographic evidence of the mechanism by which nonsteroidal ligands interact with the wild type AR. We have shown that changes induced to the positions of Trp-741, Thr-877, and Met-895 allow for ligand accommodation within the AR binding pocket and that a water-mediated hydrogen bond to the backbone oxygen of Leu-873 and the ketone of hydroxyflutamide is present when bound to the T877A AR variant. Additionally, we demonstrated that R-bicalutamide stimulates transcriptional activation in AR harboring the M895T point mutation. As a whole, these studies provide critical new insight for receptor-based drug design of nonsteroidal AR agonists and antagonists. The androgen receptor (AR) 3The abbreviations used are: ARandrogen receptorDHTdihydrotestosteroneHFhydroxyflutamideTwild typeLBDligand binding domainDTTdithiothreitolCHAPS3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid 3The abbreviations used are: ARandrogen receptorDHTdihydrotestosteroneHFhydroxyflutamideTwild typeLBDligand binding domainDTTdithiothreitolCHAPS3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid is a ligand-inducible nuclear hormone receptor involved in regulation of prostate growth, muscle and bone mass, and spermatogenesis in males. Endogenous ligands for the AR include the steroid, testosterone, and its more potent metabolite, dihydrotestosterone (DHT). Agonist compounds for the AR provide therapeutic potential in the treatment of osteoporosis, cachexia, contraception, and androgen deficiency (1Negro-Vilar A. J. Clin. Endocrinol. Metab. 1999; 84: 3459-3462Crossref PubMed Scopus (237) Google Scholar). Antagonist AR ligands are commonly used in the treatment of androgen-dependent prostate cancer. The clinically available nonsteroidal antiandrogens, flutamide and bicalutamide, are advantageous over steroidal drug treatments because of their oral bioavailability and lack of cross-reactivity with other steroid receptors. Mutations to the AR that cause resistance to antiandrogens by converting the compounds to agonists exist and represent a significant problem with currently prescribed prostate cancer drugs that target the AR (2Hara T. Miyazaki J. Araki H. Yamaoka M. Kanzaki N. Kusaka M. Miyamoto M. Cancer Res. 2003; 63: 149-153PubMed Google Scholar, 3Veldscholte J. Berrevoets C.A. Ris-Stalpers C. Kuiper G.G. Jenster G. Trapman J. Brinkmann A.O. Mulder E. J. Steroid Biochem. Mol. Biol. 1992; 41: 665-669Crossref PubMed Scopus (361) Google Scholar, 4Suzuki H. Akakura K. Komiya A. Aida S. Akimoto S. Shimazaki J. Prostate. 1996; 29: 153-158Crossref PubMed Scopus (179) Google Scholar). Different AR mutations cause agonism for hydroxyflutamide (HF, active form of flutamide) as compared with bicalutamide, suggesting that these ligands do not antagonize the AR by the same mechanism. Our recent report of the W741L-R-bicalutamide crystal structure (5Bohl C.E. Gao W. Miller D.D. Bell C.E. Dalton J.T. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 6201-6206Crossref PubMed Scopus (323) Google Scholar) demonstrated that R-bicalutamide induces the same overall fold of the AR ligand binding domain (LBD) observed in steroidal androgen-bound AR LBD crystal structures when bound to a mutant AR associated with resistance. Additionally, mutations to Thr-877 (4Suzuki H. Akakura K. Komiya A. Aida S. Akimoto S. Shimazaki J. Prostate. 1996; 29: 153-158Crossref PubMed Scopus (179) Google Scholar, 6Steketee K. Timmerman L. Ziel-van der Made A.C. Doesburg P. Brinkmann A.O. Trapman J. Int. J. Cancer. 2002; 100: 309-317Crossref PubMed Scopus (105) Google Scholar) have been shown to result in agonist activity for HF as well as steroidal AR antagonists (7Poujol N. Wurtz J.M. Tahiri B. Lumbroso S. Nicolas J.C. Moras D. Sultan C. J. Biol. Chem. 2000; 275: 24022-24031Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). Subtle differences in the AR-ligand interaction therefore are responsible for the activity of androgen antagonists. The recent discovery of nonsteroidal AR agonists by structural modification of bicalutamide and HF further reinforces this finding (8Dalton J.T. Mukherjee A. Zhu Z. Kirkovsky L. Miller D.D. Biochem. Biophys. Res. Commun. 1998; 244: 1-4Crossref PubMed Scopus (197) Google Scholar, 9Marhefka C.A. Gao W. Chung K. Kim J. He Y. Yin D. Bohl C. Dalton J.T. Miller D.D. J. Med. Chem. 2004; 47: 993-998Crossref PubMed Scopus (118) Google Scholar, 10Yin D. He Y. Perera M.A. Hong S.S. Marhefka C. Stourman N. Kirkovsky L. Miller D.D. Dalton J.T. Mol. Pharmacol. 2003; 63: 211-223Crossref PubMed Scopus (106) Google Scholar).To date, multiple docking solutions have been reported to explain how the extra bulk on bicalutamide as compared with steroidal androgens can be accommodated in the AR ligand binding pocket (7Poujol N. Wurtz J.M. Tahiri B. Lumbroso S. Nicolas J.C. Moras D. Sultan C. J. Biol. Chem. 2000; 275: 24022-24031Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 11Marhefka C.A. Moore II, B.M. Bishop T.C. Kirkovsky L. Mukherjee A. Dalton J.T. Miller D.D. J. Med. Chem. 2001; 44: 1729-1740Crossref PubMed Scopus (81) Google Scholar, 12Bohl C.E. Chang C. Mohler M.L. Chen J. Miller D.D. Swaan P.W. Dalton J.T. J. Med. Chem. 2004; 47: 3765-3776Crossref PubMed Scopus (66) Google Scholar, 13Soderholm A.A. Lehtovuori P.T. Nyronen T.H. J. Med. Chem. 2005; 48: 917-925Crossref PubMed Scopus (50) Google Scholar, 14Balog A. Salvati M.E. Shan W. Mathur A. Leith L.W. Wei D.D. Attar R.M. Geng J. Rizzo C.A. Wang C. Krystek S.R. Tokarski J.S. Hunt J.T. Gottardis M. Weinmann R. Bioorg. Med. Chem. Lett. 2004; 14: 6107-6111Crossref PubMed Scopus (32) Google Scholar). None of these models resembled the bound conformation of R-bicalutamide in the W741L mutant (5Bohl C.E. Gao W. Miller D.D. Bell C.E. Dalton J.T. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 6201-6206Crossref PubMed Scopus (323) Google Scholar). Thus, questions regarding how R-bicalutamide and structurally related selective androgen receptor modulators could be accommodated in the wild type (WT) AR remained unanswered. The presence of the Trp-741 side chain would seemingly preclude the B-ring of nonsteroidal selective androgen receptor modulators from binding in the same position occupied by the B-ring of R-bicalutamide in the W741L AR. Although our efforts to obtain crystal structures of the AR LBD complexed to pure antiandrogens have been unsuccessful due to tight association with the groEL chaperone protein, crystal structures of the WT AR bound to agonists that closely resemble bicalutamide and HF in structure were obtained. Herein we present the first evidence as to how nonsteroidal ligands are accommodated in the WT AR by inducing changes to the positions of residues in the binding pocket. We also report the crystal structure of HF bound to the well known resistance mutation, T877A. Lastly, we present evidence that M895T represents a novel resistance mutation for R-bicalutamide and may also be involved in bicalutamide withdrawal syndrome similar to mutations to Trp-741 (2Hara T. Miyazaki J. Araki H. Yamaoka M. Kanzaki N. Kusaka M. Miyamoto M. Cancer Res. 2003; 63: 149-153PubMed Google Scholar, 5Bohl C.E. Gao W. Miller D.D. Bell C.E. Dalton J.T. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 6201-6206Crossref PubMed Scopus (323) Google Scholar).MATERIALS AND METHODSCloning, Expression, and Purification—An AR-LBD-(663–919) was obtained by PCR amplification from a full-length human AR expression construct (pCMVhAR; generously provided by Dr. Donald J. Tindall, Mayo Clinic and Mayo Foundation, Rochester, MN) with primers containing flanking restriction sites and inserted into the pGEX6P-1 plasmid (Amersham Biosciences). Mutations were created in the pGEX6P1-AR-(663–919) and the pCMVhAR via the Stratagene QuikChange mutagenesis kit according to the manufacturer's instructions. AR LBD expression and purification were performed essentially as previously described (5Bohl C.E. Gao W. Miller D.D. Bell C.E. Dalton J.T. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 6201-6206Crossref PubMed Scopus (323) Google Scholar, 15Hur E. Pfaff S.J. Payne E.S. Gron H. Buehrer B.M. Fletterick R.J. PLoS Biol. 2004; 2: E274Crossref PubMed Scopus (179) Google Scholar, 16Matias P.M. Donner P. Coelho R. Thomaz M. Peixoto C. Macedo S. Otto N. Joschko S. Scholz P. Wegg A. Basler S. Schafer M. Egner U. Carrondo M.A. J. Biol. Chem. 2000; 275: 26164-26171Abstract Full Text Full Text PDF PubMed Scopus (498) Google Scholar). The AR LBD was expressed as a glutathione S-transferase (GST) fusion protein in Escherichia coli BL21 DE3 at 15 °C for 16 h by induction with 30 μm isopropyl-1-thio-β-d-galactopyranoside. Cells were lysed in a buffer containing 150 mm NaCl, 50 mm Tris, pH 8.0, 5 mm EDTA, 10% glycerol, 1 mg/ml lysozyme, 10 units/ml DNase I, 10 mm MgCl2, 10 mm DTT, 0.5% CHAPS, 100 μm ligand, and 100 μm phenylmethylsulfonyl fluoride by three cycles of freeze-thaw. The supernatant from ultracentrifugation was incubated for 1 h at 4°C with glutathione-Sepharose resin (Amersham Biosciences) and washed with 150 mm NaCl, 50 mm Tris, pH 8.0, 5 mm EDTA, 10% glycerol, 10 mm ATP, 10 μm ligand (S-1, R-3, or HF), 0.1% n-octyl-β-glucoside, and 1 mm DTT. The GST-LBD fusion protein was cleaved in a buffer containing 150 mm NaCl, 50 mm Tris, pH 7.0, 10% glycerol, 10 μm ligand, 0.1% n-octyl-β-glucoside, 1 mm DTT, and 5 units/mg protein PreScission protease (Amersham Biosciences) at 4 °C overnight, releasing the AR LBD from the glutathione-Sepharose resin. The supernatant was then diluted 3-fold in 10 mm Hepes, pH 7.2, 10% glycerol, 10 μm ligand, 0.1% n-octyl-β-glucoside, and 1 mm DTT and loaded onto an HP SP cation exchange column (Amersham Biosciences). Protein was eluted with a gradient of 50–500 mm NaCl in the same dilution buffer. The buffer was exchanged in a Millipore 10-kDa cutoff concentrator to a buffer containing 150 mm Li2SO4, 50 mm Hepes, pH 7.2, 10% glycerol, 100 μm ligand, 0.1% n-octyl-β-glucoside, and 10 mm DTT, and protein was concentrated to above 4 mg/ml.Crystallization, Data Collection, and Structure Determination—AR LBD crystals formed in 1–2 days using the hanging drop vapor diffusion method in 0.1 m Hepes, pH 7.5, and 0.5–0.8 m sodium citrate for the T877A-HF, T877A-S-1, and W741L-S-1 complexes. WT-S-1 crystals were obtained in 0.1 m Hepes, pH 7.5, and 0.6 m sodium citrate after 4 weeks. Nucleation sites from these WT-S-1 crystals were transferred with a cat whisker into freshly mixed protein and reservoir solution ranging from 0.3 to 0.6 m sodium citrate with 0.1 m Hepes, pH 7.5, to improve WT-S-1 crystal quality and size. Crystals formed in 1–2 days using this technique allowing for optimization. The WT-R-3 crystals were obtained by transferring microseeds with a cat whisker from T877A-HF crystals in 0.3-0.6 m sodium citrate with 0.1 m Hepes, pH 7.5. Prior to flash freezing in liquid nitrogen, AR LBD crystals were transferred to a solution consisting of 0.1 m Hepes, pH 7.5, 0.7 m sodium citrate, and 20% ethylene glycol. Diffraction data were collected using a Rigaku RU300 rotating anode generator and an R-axis IV++ image plate (Rigaku, The Woodlands, TX) and processed with Crystal Clear software (Molecular Structure Corporation, The Woodlands, TX). The W741L-R-bicalutamide structure (Protein Data Bank code 1Z95) was used as a starting structure for refinement using Crystallography and NMR System (CNS) (17Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16930) Google Scholar). After an initial round of refinement, electron density maps allowed for accurate fitting of the ligand. Model building and water molecules were added using the program O (18Jones T.A. Zou J.Y. Cowan Kjeldgaard S.W. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13004) Google Scholar), and further rounds of refinement were performed using rigid body, torsion angle simulated annealing, and individual temperature factor modules of CNS. Figures were prepared with PyMOL (The PyMOL Molecular Graphics System).Cotransfection Assay—CV-1 cells (green monkey kidney cells) were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 2 mm glutamine, and 1% streptomycin and penicillin to confluency in T175 tissue culture flasks. Cells were then transfected with 3 μg of the CMVhAR expression vector (WT or mutant), 30 μg of an androgen-dependent luciferase reporter construct (pMMTV-luc; generously provided by Dr. Ronald Evans at The Salk Institute, San Diego, CA), and 30 μg of a β-galactosidase expression construct (pSV-β-galactosidase; Promega, Madison, WI) via 30 μl of Plus reagent (Invitrogen) and 40 μl of Lipofectamine (Invitrogen) in serum-free Dulbecco's modified Eagle's medium. After 4 h, the medium was exchanged to Dulbecco's modified Eagle's medium supplemented with 0.2% fetal bovine serum and 2 mm glutamine. Cells were transferred 12 h later to 24-well tissue culture plates and after another 6 h were treated with 0.1–1000 nm, drug or no drug (control). After another 24 h, cells were washed twice with cold phosphate-buffered saline and harvested by incubation with 100 μl of passive lysis buffer (Promega) for 30 min. An aliquot (50 μl) of the lysate was then added to an opaque 96-well plate, and luciferase activity was monitored after automated injection of 50 μl of luciferase substrate (Promega) with a MicroLumatPlus LB96V luminometer (Berthold Technologies, Oak Ridge, TN) using the WinGlow software package. An aliquot (50 μl) of the lysate was also added to a clear 96-well plate along with 50 μl of β-galactosidase assay buffer. An absorbance measurement at 420 nm was taken following a 2-h incubation at 37 °C on a Dynex MRX plate reader. Luciferase activity was normalized with β-galactosidase activity to account for differences in cell number and/or loss in transfection efficiency. Transcriptional activation was interpreted as the -fold increase (relative luciferase units) over control (i.e. non-drug treated) wells.RESULTSAll crystal structures of AR LBD solved to date have been in an orthorhombic P212121 space group (5Bohl C.E. Gao W. Miller D.D. Bell C.E. Dalton J.T. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 6201-6206Crossref PubMed Scopus (323) Google Scholar, 15Hur E. Pfaff S.J. Payne E.S. Gron H. Buehrer B.M. Fletterick R.J. PLoS Biol. 2004; 2: E274Crossref PubMed Scopus (179) Google Scholar, 16Matias P.M. Donner P. Coelho R. Thomaz M. Peixoto C. Macedo S. Otto N. Joschko S. Scholz P. Wegg A. Basler S. Schafer M. Egner U. Carrondo M.A. J. Biol. Chem. 2000; 275: 26164-26171Abstract Full Text Full Text PDF PubMed Scopus (498) Google Scholar, 19He B. Gampe Jr., R.T. Kole A.J. Hnat A.T. Stanley T.B. An G. Stewart E.L. Kalman R.I. Minges J.T. Wilson E.M. Mol. Cell. 2004; 16: 425-438Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar, 20Sack J.S. Kish K.F. Wang C. Attar R.M. Kiefer S.E. An Y. Wu G.Y. Scheffler J.E. Salvati M.E. Krystek Jr., S.R. Weinmann R. Einspahr H.M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4904-4909Crossref PubMed Scopus (383) Google Scholar, 21Matias P.M. Carrondo M.A. Coelho R. Thomaz M. Zhao X.Y. Wegg A. Crusius K. Egner U. Donner P. J. Med. Chem. 2002; 45: 1439-1446Crossref PubMed Scopus (76) Google Scholar) with helix 12 in a similar conformation as other agonist-bound steroid receptor LBD structures (16Matias P.M. Donner P. Coelho R. Thomaz M. Peixoto C. Macedo S. Otto N. Joschko S. Scholz P. Wegg A. Basler S. Schafer M. Egner U. Carrondo M.A. J. Biol. Chem. 2000; 275: 26164-26171Abstract Full Text Full Text PDF PubMed Scopus (498) Google Scholar, 22Brzozowski A.M. Pike A.C. Dauter Z. Hubbard R.E. Bonn T. Engstrom O. Ohman L. Greene G.L. Gustafsson J.A. Carlquist M. Nature. 1997; 389: 753-758Crossref PubMed Scopus (2917) Google Scholar, 23Tanenbaum D.M. Wang Y. Williams S.P. Sigler P.B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5998-6003Crossref PubMed Scopus (589) Google Scholar, 24Kauppi B. Jakob C. Farnegardh M. Yang J. Ahola H. Alarcon M. Calles K. Engstrom O. Harlan J. Muchmore S. Ramqvist A.K. Thorell S. Ohman L. Greer J. Gustafsson J.A. Carlstedt-Duke J. Carlquist M. J. Biol. Chem. 2003; 278: 22748-22754Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar). Purification of AR LBD bound to agonist ligands is well documented and involves removal of contamination chaperones, dnak and groEL, through cation exchange chromatography (21Matias P.M. Carrondo M.A. Coelho R. Thomaz M. Zhao X.Y. Wegg A. Crusius K. Egner U. Donner P. J. Med. Chem. 2002; 45: 1439-1446Crossref PubMed Scopus (76) Google Scholar). The AR LBD is not retained by cation exchange chromatography in the presence of antagonist compounds and co-elutes with groEL after anion exchange chromatography, suggesting that antagonist-bound AR LBD is tightly associated with groEL, possibly as a result of partial receptor unfolding (25Ellis R.J. Hartl F.U. FASEB J. 1996; 10: 20-26Crossref PubMed Scopus (210) Google Scholar, 26Gragerov A. Nudler E. Komissarova N. Gaitanaris G.A. Gottesman M.E. Nikiforov V. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10341-10344Crossref PubMed Scopus (184) Google Scholar, 27Langer T. Lu C. Echols H. Flanagan J. Hayer M.K. Hartl F.U. Nature. 1992; 356: 683-689Crossref PubMed Scopus (781) Google Scholar, 28Martin J. Langer T. Boteva R. Schramel A. Horwich A.L. Hartl F.U. Nature. 1991; 352: 36-42Crossref PubMed Scopus (718) Google Scholar). Thus, mutations to the AR that confer resistance to antiandrogens offer a viable method to obtain crystallographic evidence for antagonist binding conformations to the AR and provide insight as to how they would alter the conformation in the WT AR to stabilize association with heat shock proteins (3Veldscholte J. Berrevoets C.A. Ris-Stalpers C. Kuiper G.G. Jenster G. Trapman J. Brinkmann A.O. Mulder E. J. Steroid Biochem. Mol. Biol. 1992; 41: 665-669Crossref PubMed Scopus (361) Google Scholar, 29Kallio P.J. Janne O.A. Palvimo J.J. Endocrinology. 1994; 134: 998-1001Crossref PubMed Scopus (46) Google Scholar). AR LBD bound to the partial agonists S-1 and R-3 demonstrated a fractional amount of cation exchange retention compared with DHT-bound LBD. The yields of purified S-1-associated AR LBD were, however, substantially improved in the T877A and W741L AR mutants. Additionally, crystal formation of the AR LBD-S-1 complex was also significantly facilitated in these mutants. WT-S-1 crystal formation took nearly one month as opposed to 1–2 days for the mutant complexes, likely because of slowed nucleation, and was circumvented by using microseeding. Furthermore, WT-R-3 crystals did not form in similar conditions or any of numerous crystal screens. Because WT-S-1 and T877A-HF complexes crystallized in the same space group with nearly the same unit cell dimensions and same overall protein fold (TABLE ONE), we predicted that the WT-R-3 crystals would be similar. Therefore, we obtained crystals of the WT-R-3 complex by transferring microseeds from T877A-HF crystals. Clear electron density for Thr-877 and the bromine atom insured that we had, in fact, obtained the WT-R-3 complex.TABLE ONECrystallographic data and refinement statistics Values for data in the last resolution shell are shown in parenthesis. R.m.s.d., root mean square deviation.ComplexWT-R-3T877A-HFWT-S-1T877A-S-1W741L-S-1Protein Data Bank code2AX92AX62AXA2AX72AX8Space groupP212121P212121P212121P212121P212121Unit cellA55.3854.7854.7354.8255.09B66.0165.9666.3966.3766.11C69.0669.6368.9469.0869.03Resolution range (Å)18.88–1.65 (1.71–1.65)18.03–1.50 (1.55–1.50)22.13–1.80 (1.86–1.80)30.64–1.90 (1.97–1.90)23.01–1.70 (1.76–1.70)Number of unique reflections31,12139,98722,97919,96227,002Average redundancy8.27 (6.57)6.04 (4.88)12.55 (8.84)13.48 (9.71)11.95 (7.04)% completeness100.0 (99.6)97.4 (85.2)96.0 (70.1)97.3 (80.8)95.0 (67.5)RmergeaRmerge = Σ|Ih – 〈I〉h|/ΣIh, where 〈I〉h is average intensity over symmetry equivalents0.095 (0.358)0.153 (0.480)0.092 (0.316)0.115 (0.530.079 (0.332)I/σ12.6 (4.9)17.5 (3.5)16.9 (6.9)11.5 (3.6)20.5 (5.1)RfactorbRfactor = Σ|Fobs – Fcalc|/ΣFobs. The free Rfactor is calculated from 10% of the reflections that are omitted from the refinement0.224 (0.321)0.246 (0.409)0.210 (0.276)0.206 (0.244)0.212 (0.279)Rfree0.248 (0.339)0.274 (0.455)0.250 (0.311)0.247 (0.284)0.251 (0.311)R.m.s.d. bonds (Å)0.0050.0060.0050.0050.006R.m.s.d. angles1.11.11.11.11.1Mean B value (Å2)17.620.220.323.620.1a Rmerge = Σ|Ih – 〈I〉h|/ΣIh, where 〈I〉h is average intensity over symmetry equivalentsb Rfactor = Σ|Fobs – Fcalc|/ΣFobs. The free Rfactor is calculated from 10% of the reflections that are omitted from the refinement Open table in a new tab Structure of HF-like Nonsteroidal Agonist in the WT AR—R-3 is an analog of HF that differs only by the addition of a bromine atom to a methyl group, which creates a stereo-selective compound (8Dalton J.T. Mukherjee A. Zhu Z. Kirkovsky L. Miller D.D. Biochem. Biophys. Res. Commun. 1998; 244: 1-4Crossref PubMed Scopus (197) Google Scholar, 10Yin D. He Y. Perera M.A. Hong S.S. Marhefka C. Stourman N. Kirkovsky L. Miller D.D. Dalton J.T. Mol. Pharmacol. 2003; 63: 211-223Crossref PubMed Scopus (106) Google Scholar) (Fig. 1). Addition of a bromine at this position results in a large increase in binding affinity to the AR (8Dalton J.T. Mukherjee A. Zhu Z. Kirkovsky L. Miller D.D. Biochem. Biophys. Res. Commun. 1998; 244: 1-4Crossref PubMed Scopus (197) Google Scholar). However, R-3 elicits agonist activity for the AR only at high concentrations (i.e. ≥100 nm) (Fig. 2a). The crystal structure of R-3 bound to the native AR LBD demonstrates a very similar bound conformation as R-bicalutamide in the W741L (5Bohl C.E. Gao W. Miller D.D. Bell C.E. Dalton J.T. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 6201-6206Crossref PubMed Scopus (323) Google Scholar). Arg-752 and Gln-711 hydrogen bond with the nitro group of R-3 directly and by way of a water molecule (Fig. 3, a and b), resembling the hydrogen bond pattern seen with the cyano group of R-bicalutamide. However, Gln-711 is likely too distant (3.7 Å) from the cyano in the W741L-R-bicalutamide complex for direct hydrogen bond formation. The amide nitrogen of R-3 is within hydrogen bond distance to the backbone oxygen of Leu-704, and the chiral hydroxyl group of R-3 is within distance for hydrogen bonds with Asn-705 Oδ1 and the backbone oxygen of Leu-704, identical to the interactions observed with R-bicalutamide in the W741L mutant AR. Thr-877 is also oriented as in the W741L-R-bicalutamide complex, which is rotated 180° compared with steroidal bound AR structures. The Trp-741 indole ring, which is absent in the W741L-R-bicalutamide complex, borders the region occupied by bromine along with Met-742 and Met-895 residues as shown in Fig. 3b. Bulk provided by the bromine atom in this region therefore appears to greatly enhance binding affinity as compared with HF. Furthermore, the W741L and M895T mutations, which decrease bulk around the location of the bromine atom of R-3, demonstrate a decrease in transcriptional activation with R-3 relative to the WT (Fig. 2, c and d). A gain of AR-mediated transcription with R-3, however, is observed in the T877A AR (Fig. 2b).FIGURE 2AR-mediated transcriptional activation by DHT, R-bicalutamide (R-bic), S-1, HF, and R-3 in the WT (a), T877A (b), W741L (c), and M895T (d) AR variants. Relative luciferase units (RLU) normalized with β-galactosidase (β-gal) activity. Inter-experiment variation was observed with RLU/β-galactosidase values due to transfection efficiency, but relative drug-induced transcriptional activation was found consistent and therefore more accurately represents changes in functional activity observed in AR mutations.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 3Stereo representations of AR interactions with R-3 and S-1. a, WT-R-3 complex fit into the 2Fo - Fc electron density maps contoured at the 2σ level (cyan) with R-3 shown in the Fo - Fc simulated annealing omit map contoured at the 4σ level (red). AR carbons, gray; R-3 carbons, gold; oxygen, red; nitrogen, blue; sulfur, orange; bromine, yellow. b, AR surface contacts with the R-3 binding pocket. Possible hydrogen bonds within 3.5 Å are represented by dashed lines. Notice that Gln-711 and Arg-752 hydrogen bond with the nitro group of R-3. Leu-704 and Asn-705 also form hydrogen bonds with R-3. Also notice the hydrophobic contacts from Trp-741, Met-742, and Met-895 with the R-3 bromine atom. Electron density for Trp-741 in the WT-R-3 structure cannot be visualized at the 2σ level, but electron density is more evident at lower σ levels. c, WT-S-1 complex fit into the 2Fo - Fc electron density maps contoured at the 2σ level (cyan) with S-1 (carbons in green) shown in the Fo - Fc simulated annealing omit map contoured at the 4σ level (red). d, AR surface contacts with the S-1 binding pocket. Similar hydrogen bonds to the AR are present in S-1 as with R-3. Notice the increased size of the S-1 binding pocket as compared with R-3 due to the S-1 B-ring. A water molecule hydrogen bonds to the His-874 side chain and backbone atoms of helices 4 and 5 within 3.0 Å of the B-ring fluorine atom, similar to that seen in the W741L-R-bicalutamide complex. Also notice the clear electron density for Trp-741 at the 2σ level as compared with the R-3-bound structure, likely because of decreased mobility of Trp-741 from binding of the S-1 B-ring.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Structure of Ether-linked Selective Androgen Receptor Modulator in WT AR—S-1 is an agonist for the AR that exhibits tissue-selective pharmacologic effects in vivo and therefore represents a novel class of AR ligands known as selective androgen receptor modulators (1Negro-Vilar A. J. Clin. Endocrinol. Metab. 1999; 84: 3459-3462Crossref PubMed Scopus (237) Google Scholar, 30Gao W. Kearbey J.D. Nair V.A. Chung K. Parlow A.F. Miller D.D. Dalton J.T. Endocrinology. 2004; 145: 5420-5428Crossref PubMed Scopus (97) Google Scholar). S-1 resembles R-3 structurally but contains an ether-linked phenyl ring with a para-fluoro substituent in place of the bromine atom (Fig. 1). S-1 also closely resembles R-bicalutamide in structure, differing only at the linkage group and A-ring para position (Fig. 1). It is therefore surprising that S-1 elicits significantly more agonist activity than these structural analogs in the WT AR (Fig. 2a). The crystal structure of the WT-S-1 complex (Fig. 3, c and d) demonstrates that S-1 superimposes identically to the A-ring and amide portion of R-3. At the chiral hydroxyl group, S-1 bends and orients the B-ring nearly parallel to helix 12. Displacement of the Trp-741 indole ring by the B-ring of S-1 opens up an additional cavity in the AR binding pocket (Fig.

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