Letters pubs.acs.org/acschemicalbiology
Development of 5N-Bicalutamide, a High-Affinity Reversible Covalent Antiandrogen Felipe de Jesus Cortez,† Phuong Nguyen,‡ Charles Truillet,§ Boxue Tian,† Kristopher M. Kuchenbecker,‡ Michael J. Evans,§ Paul Webb,∥ Matthew P. Jacobson,† Robert J. Fletterick,‡ and Pamela M. England*,†
Downloaded via KAOHSIUNG MEDICAL UNIV on July 26, 2018 at 12:30:30 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.
†
Department of Pharmaceutical Chemistry, University of California San Francisco, 600 16th Street, San Francisco, California 94158, United States ‡ Department of Biochemistry and Biophysics, University of California San Francisco, 600 16th Street, San Francisco, California 94158, United States § Department of Radiology, University of California San Francisco, 185 Berry Street, San Francisco, California 94107, United States ∥ California Institute of Regenerative Medicine Therapeutics Program, Lake Merritt Plaza, 1999 Harrison Street, Suite #1650, Oakland, California 94612, United States S Supporting Information *
ABSTRACT: Resistance to clinical antiandrogens has plagued the evolution of effective therapeutics for advanced prostate cancer. As with the first-line therapeutic bicalutamide (Casodex), resistance to newer antiandrogens (enzalutamide, ARN-509) develops quickly in patients, despite the fact that these drugs have ∼10-fold better affinity for the androgen receptor than bicalutamide. Improving affinity alone is often not sufficient to prevent resistance, and alternative strategies are needed to improve antiandrogen efficacy. Covalent and reversible covalent drugs are being used to thwart drug resistance in other contexts, and activated aryl nitriles are among the moieties being exploited for this purpose. We capitalized on the presence of an aryl nitrile in bicalutamide, and the existence of a native cysteine residue (Cys784) in the androgen receptor ligand binding pocket, to develop 5N-bicalutamide, a cysteine-reactive antiandrogen. 5N-bicalutamide exhibits a 150-fold improvement in Ki and 20-fold improvement in IC50 over the parent compound. We attribute the marked improvement in affinity and activity to the formation of a covalent adduct with Cys784, a residue that is not among the more than 160 androgen receptor point mutations associated with prostate cancer. Increasing the residence time of bound antiandrogen via formation of a covalent adduct may forestall the drug resistance seen with current clinical antiandrogens.
A
Antiandrogen resistance is attributed to several factors, including the upregulation of AR itself, which promotes signaling from low levels of residual hormone, and mutations in the AR ligand binding pocket, which cause antiandrogens to stimulate (rather than inhibit) receptor activity.7 Efforts to thwart this resistance with newer, higher affinity antiandrogens (e.g., enzalutamide, ARN-509) do extend median survival time, but acquired resistance nevertheless ensues in most patients.8 Alternative strategies to increase antiandrogen efficacy are needed to combat advanced prostate cancer. One approach to averting drug resistance is to slow the off-rate of bound drug.9,10 For example, acquired drug resistance in patients with nonsmall cell lung cancer was curbed when gefitinib, a non-covalent EGF receptor antagonist, was replaced with the covalent analog afatinib.11,12
ndrogen receptor (AR) is a hormone-activated transcription factor that plays a critical role in the development and normal functioning of the prostate.1 The initiation and progression of prostate cancer is also uniquely dependent on AR, with receptor activation driving tumor growth.2 Like other nuclear receptors, AR is comprised of three main functional domains: a variable N-terminal domain, a highly conserved DNA-binding domain (DBD), and a conserved ligand binding domain (LBD).3 Binding of endogenous hormones (testosterone, dihydrotestosterone) to the LBD induces a conformational change in the receptor that results in its translocation into the nucleus, interaction with DNA, and modulation of specific gene transcription (e.g., prostate specific antigen, PSA).4 Competitive antagonists of AR, so-called antiandrogens, are intended to inhibit these processes and are commonly employed in the treatment of advanced prostate cancer.5 Unfortunately, existing antiandrogen therapies are largely considered only palliative, as resistance to these drugs develops in nearly all patients.6 In fact, prostate cancer is projected to take the lives of over 26000 U.S. males in 2017. © 2017 American Chemical Society
Received: August 14, 2017 Accepted: October 5, 2017 Published: October 5, 2017 2934
DOI: 10.1021/acschembio.7b00702 ACS Chem. Biol. 2017, 12, 2934−2939
Letters
ACS Chemical Biology We set out to investigate this strategy for prostate cancer, by first developing a cysteine-reactive analog of the widely utilized clinical antiandrogen bicalutamide (1, Figure 1). In particular,
Figure 1. Structures of bicalutamide, 5N-bicalutamide, and the AR ligand binding pocket bound to bicalutamide. The structure of 1 bound to AR is derived from the crystal structure of the agonist DHT bound to wildtype AR (PDB: 1T79) and bicalutamide bound to a mutant AR (Trp741Leu; PDB: 1Z95), where it functions as an agonist. Helices 1−2 have been removed for clarity. Highlighted in the binding pocket are a native cysteine residue (Cys784) in close proximity to the aryl nitrile and a neighboring arginine residue (Arg752) poised to facilitate thioimidate formation. Note the steric clash between the aromatic ring of 1 and Trp741 that likely displaces bicalutamide toward the Cys and Arg residues.
we converted the aryl nitrile in bicalutamide into an activated aryl nitrile by replacing the methine (CH) unit ortho to the aryl nitrile with a nitrogen (N) atom, to produce the cysteinereactive analog 5N-bicalutamide (2, Figure 1). Thiols are known to form reversible covalent adducts with activated aryl nitriles,13,14 and the crystal structure of AR (Trp741Leu) bound to bicalutamide places the aryl nitrile in proximity to an endogenous cysteine (Cys784) within the AR ligand binding pocket (Figure 1).15 As described in detail below, we observed dramatic improvements in the reactivity, affinity, and efficacy of 5N-bicalutamide compared to bicalutamide. To initially assess the relative electrophilicity of bicalutamide (1) and 5N-bicalutamide (2), we used density functional theory (DFT) to calculate the free energy of thioimidate formation derived from the reaction of each aryl nitrile with methanethiol. These calculations predicted a 10-fold increase in the electrophilicity of 5N-bicaluatmide (−3.589 kcal/mol) relative to bicalutamide (−0.375 kcal/mol). Next, we directly evaluated the reactivity of 1 and 2 toward thiols by measuring the extent of covalent adduct formation with cysteine in a physiological buffer. Aryl nitriles are known to react with the f ree amino acid cysteine to form irreversible covalent adducts (thiazoline derivatives), which can be readily isolated and quantified.13 Consistent with theory, 5N-bicalutamide (2) is nearly completely converted to the thiazoline adduct within 4 h, whereas bicalutamide (1) remains unreactive to cysteine even after 24 h (Figure 2). Next, two cellular assays commonly employed to study antiandrogens were used to measure the affinity and efficacy of 1 and 2. In a standard competition binding assay,7 5Nbicalutamide (Ki = 0.15 nM) binds AR with ∼150-fold greater affinity than bicalutamide (Ki = 22.3 nM; Figure 3A). To evaluate the efficacy of these antiandrogens, we employed a transactivation assay in which hormone-induced activation of AR drives the transcription of the reporter luciferase.16 In particular, the AR LBD fused to the Gal4 DNA binding domain was expressed in HeLa cells, and AR-driven lucifierase expression was measured in the presence of hormone and increasing concentrations of each antiandrogen. In these assays, 5N-bicalutamide is 20-fold more potent than bicalutamide at
Figure 2. 5N-bicalutamide rapidly reacts with cysteine to form the covalent thiazoline adduct. LC-MS analysis of the reaction of 5Nbicalutamide with cysteine in phosphate buffered saline (pH 7.4, 37 °C) showing 29% and 99% conversion to the thiazoline adduct after 1 and 4 h, respectively. No adduct formation was observed for bicalutamide after 24 h under identical conditions.
inhibiting AR activation (Figure 3B). To investigate the importance of the nitrile in 1 versus 2, we prepared the corresponding control compounds 3 and 4, in which the nitrile has been replaced with a proton. Removing the nitrile in bicalutamide reduces the IC50 by only 4.5-fold, whereas removing it in 5N-bicalutamide reduces the IC50 88-fold (Figure 3B). To rule out the possibility that the improved activity of 5N-bicalutamide is rather a reflection of cell death, we measured the cytotoxicity of 5N-bicalutamide and found that it is negligible and indistinguishable from that of bicalutamide (Supporting Information Figure 1). Next, we sought to confirm that the activity of 5Nbicalutamide depends on reaction with Cys784 in the AR LBD. We reasoned that if adduct formation with Cys784 underlies the enhanced activity of 5N-bicalutamide, then mutating this cysteine residue should dramatically reduce ligand activity. Thus, we conservatively mutated Cys784 to six different amino acids and reassessed antiandrogen activity using the aforementioned luciferase reporter assay. Intriguingly, while each of the resulting mutant receptors is efficiently expressed in cells, none are functional, each failing to activate in response to agonist (Figure 4A). Western blot analyses show that the mutated receptors are not degraded in cells, suggesting that they are properly folded (Figure 4B). Notably, Cys784 is not among the ∼160 AR point mutations identified in prostate cancer, making this residue a good target for covalent antiandrogens.17 Next, we attempted to directly observe formation of the thioimidate adduct using a 15N-(nitrile)enriched sample of 5N-bicalutamide and NMR spectroscopy. Unfortunately, a combination of two insurmountable factors 2935
DOI: 10.1021/acschembio.7b00702 ACS Chem. Biol. 2017, 12, 2934−2939
Letters
ACS Chemical Biology
Figure 3. The affinity and efficacy of 5N-bicalutamide are markedly better than that of the parent compound bicalutamide. (A) The binding affinity of 5N-bicalutamide is two orders of magnitude greater than bicalutamide. Concentration-dependence curves from competition binding assays in LNCaP/AR cells expressing wildtype AR and treated with 3DHT in the presence of increasing concentrations of the cold competitors bicalulamide 1 (Ki = 22.3 ± 5.7 nM) or 5Nbicalutamide 2 (Ki = 0.15 ± 0.03 nM). (B) 5N-Bicalutamide is 20-fold more effective than bicalutamide at inhibiting AR activation. Concentration-dependence curves from transactivation assays in HeLa cells expressing AR following treatment with DHT (1 nM) and increasing concentrations of the antiandrogens 1−4. Bicalutamide 1 (IC50 = 0.31 ± 0.02 μM), 5N-bicalutamide 2 (IC50 = 0.015 ± 0.001 μM), bicalutamide(ΔCN) 3 (IC50 = 1.41 ± 0.18 μM), and 5Nbicalutamide(ΔCN) 4 (IC50 = 1.33 ± 0.22 μM). Data are representative of at least three independent experiments with error bars representing the SEM.
Figure 4. Cys784 is required for AR function, but not expression. (A) Quantification of AR activity, as a function of luciferase bioluminescence, carried out as described in the Methods. (B) Western blot showing relative expression levels of wildtype and Cys784Xaa mutant AR protein in HeLa cells. (C) Quantification of relative AR protein levels from Western blots.
receptor. Absent the nitrile, bicalutamide (1) and 5Nbicalutamide (2) have the same activity in cells (see Figure 3B, compounds 3 and 4), ruling out the possibility that the functional differences between the two antiandrogens are due to an electrostatic interaction between the receptor and the nitrogen atom added to 5N-bicalutamide. Consistent with this notion, the antiandrogens RD-162 and ARN-509 also only differ by the absence and presence, respectively, of a nitrogen atom ortho to the aryl nitrile, yet have similar IC50 values.20,21 Unlike the bicalutamides, these antiandrogens have highly rigidified structures that undoubtedly prevent reaction with Cys784 in the AR ligand binding pocket. Specifically, the thioxo-diazaspiro[3.4]octan-8-one bridge precludes the spatial relationship between the aryl (A and B) rings required to support both binding in the AR pocket and nucleophilic attack by the Cys784 side chain thiol. In fact, like 5N-bicalutamide, ARN-509 reacts rapidly with the free amino acid cysteine in solution, whereas RD-162 does not (data not shown). Previous reports suggest that the aryl nitrile in bicalutamide contributes to ligand affinity by forming a hydrogen bond with Arg752 in the ligand binding pocket, mimicking the interaction between the 3-keto functionality of the hormone DHT and the receptor.22 With this in mind, an alternative explanation for the improved activity of 5N-bicalutamide compared to bicaluta-
stymied these efforts. First, we observed rapid precipitation of AR from solution upon addition of the antiandrogen, and efforts to resolubilize the receptor while retaining the reversibly bound antagonist were unsuccessful under a variety of conditions. This observation is consistent with previous reports establishing that antiandrogens destabilize the androgen receptor, causing it to unfold.18 Second, whereas the reaction between aryl nitriles and the free amino acid cysteine yields an irreversible covalent adduct (see Figure 2), the reaction with thiols lacking a β-amine (e.g., cysteine side chain) yields a reversible thioimidate adduct that generally cannot be isolated under aqueous conditions.19 The marked improvements in affinity and activity observed with 5N-bicalutmaide are most consistent with formation of a reversible covalent adduct between the aryl nitrile and the 2936
DOI: 10.1021/acschembio.7b00702 ACS Chem. Biol. 2017, 12, 2934−2939
Letters
ACS Chemical Biology
Antiandrogen Reactivity Assays. The reaction of aryl nitriles 1 and 2 with cysteine was carried out as previously reported.13 Briefly, the nitrile (100 μM) and cysteine (1 mM) were combined in sodium phosphate buffer (200 mM, pH 7.4) and warmed to 37 °C. Aliquots of the reaction were analyzed at 1, 4, and 24 h by LC-MS to quantify the extent of thiazoline adduct formation. Ligand Binding (Ki) Assays. The binding affinity of bicalutamide and 5N-bicalutamide to full-length AR, relative to dihydrotestosterone (DHT), was determined using a competition assay in which increasing concentrations of cold competitor are added to cells preincubated with 3 H-DHT. LNCaP/AR cells (human prostate cancer cells engineered to express higher amounts of wild-type AR to mimic the clinical scenario7,20) were grown in RPMI media supplemented with 10% FBS. Cells, plated 1 day before use, were washed in PBS and mixed with 3HDHT and increasing amounts of cold competitor (1 pM to 1 μM) in standard buffer media (50 mM Tris HCl, 10 mM MgCl2, 0.1 mM EDTA, pH 7.4). After incubating at ambient temperature for 1.5 h, the cells were washed twice with ice cold Tris-buffered saline and then lysed with a solution of 1 M NaOH. Isolated cell samples were counted using a scintillation counter, with appropriate standards of total activity and blank controls, and the specific uptake of 3H-DHT determined. These data were plotted against the concentration of the cold competitor to give sigmoidal displacement curves. The binding curves were fit using a single binding site competition model, with the Prism statistical analysis software package. Ki values were calculated as averages across experiments and binding affinities reported as a percentage relative to the tight-binding ligand control for that receptor (DHT). Cellular Assays. The efficacy of antiandrogens 1−4 at inhibiting AR-mediated transcription was evaluated using a commonly employed luciferase reporter assay. Briefly, cells are transfected with (1) a plasmid containing the AR-LBD fused to the Gal4-DBD, (2) a plasmid that expresses luciferase under the control of the Gal4-DBD binding element, and (3) a plasmid expressing β-galactosidase under the control of the SV40 promoter (which is not recognized by the ARLBD or the Gal4-DBD) to control for transfection efficiency and cell number across assays. Activation of the AR-LBD−Gal4-DBD fusion protein with agonist (e.g., DHT) drives the expression of luciferase. The IC50 values are determined by measuring the amount of luciferase activity in the presence of DHT and varying concentrations of the antiandrogens. Hela cells were maintained in Dulbecco’s modified Eagle’s medium H-21 4.5 g L−1 of glucose, containing 10% steroid depleted fetal bovine serum and 50 units mL−1 of penicillin, at 37 °C. Cells were plated at a density of 1 × 105 cells per well (24-well plate) and then transfected with the AR expression plasmid (10 ng), a luciferase reporter plasmid (200 ng), and a β-galactosidase positive control plasmid (10 ng) in lipofectamine (0.5 μL, BioRad). After incubating overnight, cells were treated with DHT (1 nM) and varying concentrations of antiandrogen (0.1 nM-30 uM) and then incubated overnight. The next day, the cells were collected, the pellets were lysed in 100 mM Tris·HCl (100 μL, pH 7.5) containing 0.1% Triton X-100, and the luciferase and β-galactosidase activities measured using the Luciferase Assay System (Promega) and Galacto-Light Plusgalactosidase reporter gene assay system (Applied Biosystems) according to the manufacturer’s instructions. Cytotoxicity Assays. The cytotoxicity of antiandrogens 1−4 was evaluated in LNCaP/AR cells expressing full-length wildtype AR. Cells were plated at a density of 104 cells per well in 96-well plates, incubated overnight, and then treated with antiandrogens (3, 10, 30 μM) or DMSO (0.1%). After 24 h, the relative number of dead cells was determined using the CytoTox-Glo Cytotoxicity Assay (Promega) according to the manufacturer’s instructions. Western Blotting Assays. The effect of mutating Cys784 on the expression and activity of AR was evaluated using Western blotting and the aforementioned luciferase reporter assay, respectively. HeLa cells were plated in six-well plates and transfected with 100 μg of either the AR-LBD−Gal4-DBD or the control Gal4-DBD expression plasmid. After 24 h, plated cells were treated with DHT (1 nM). After 20 h, cells were chilled on ice, washed with cold PBS, incubated
mide is that the former establishes a much stronger hydrogen bond to Arg752. However, the calculated electron density (Mulliken charges) on the nitrile in bicalutamide (−0.265) is slightly more negative than in 5N-bicalutamide (−0.254), which is inconsistent with latter forming a stronger electrostatic interaction. We propose instead that Arg752 stabilizes formation of the reversible thioimidate adduct with 5Nbicalutamide, hydrogen bonding to the imido nitrogen. The cocrystal structure of bicalutamide bound to AR places the nucleophilic Cys784 thiol ∼10 Å from the electrophilic nitrile, seemingly too far away for a chemical reaction to occur between the two. However, this is most certainly not an accurate measure of the spatial relationship between Cys784 and the nitrile. First, in this and related antiandrogen AR structures,15,23 the receptor contains a resistance point mutation (Trp741Leu) that drastically alters the activity of bicalutamide, causing it to function as an agonist. Thus, bicalutamide most certainly binds somewhat differently within the wildtype AR pocket.24 Second, nuclear receptors are dynamic proteins, sampling disparate conformations in solution.25 In fact, covalent adducts are formed between reactive ligands and cysteine residues within the ligand binding pockets of other nuclear receptors across similarly long distances. For example, estrogen receptor forms a covalent adduct with tamoxifine aziridine,26 despite a distance of ∼7 Å between the nucleophilic cysteine and electrophilic aziridine (PDB: 3ERT). Similarly, glucocorticoid receptor forms a covalent adduct with dexamethasone 21-mesylate,27 despite a distance of ∼11 Å between the nucleophilic cysteine and electrophilic mesylate (PDB: 4UDC). In summary, 5N-bicalutamide was developed by introducing a single nitrogen atom into the clinical antiandrogen bicalutamide (Casodex). The resulting cysteine-reactive antiandrogen displays dramatically improved affinity and efficacy compared to the parent non-covalent drug, owing to the formation of a reversible covalent adduct with Cys784 in the AR ligand binding pocket. Functions of the androgen receptor are determined by its stability, recruitment of coregulators, and cellular localization. The potential value of 5N-bicalutamide and related molecules needs to be established by determining ligand effects on transcription regulation in appropriate cellular and animal contexts. Future studies will explore the impact of 5N̈ prostate cancer bicalutamide on the time to resistance in naive cells and the proliferation of prostate cancer cells that have developed resistance to existing clinical antiandrogens.
■
METHODS
Computational Chemistry. Density functional theory (DFT) calculations were performed to assess the electrophilicity of bicalutamide and 5N-bicalutamide, according to procedures described for related aryl nitriles.13 Briefly, the free energy of thioimidate formation, derived from the reaction of the aryl nitrile with methanethiol, was calculated by optimizing the geometry of the substrates and product in the gas phase using the B3LYP/6-311G(d,p) level of theory, followed by a single point calculation with the polarization continuum model (PCM) method in water. The latter was done to avoid overestimation of intramolecular H-bonds potentially formed within the thioimidate. The electrophilcity of the nitrile was then calculated as follows: electrophiliicty (kcal/mol) = E(adduct) − E(nitrile) − E(methanethiol). Synthetic Chemistry. The synthesis of bicalutamide analogs 2−4 is described in detail in the Supporting Information. The purity of compounds 1−4 used in bioassays was judged to be ≥98% by 1H NMR spectroscopy. 2937
DOI: 10.1021/acschembio.7b00702 ACS Chem. Biol. 2017, 12, 2934−2939
Letters
ACS Chemical Biology with lysis buffer for 10 min, scraped from the wells, and then centrifuged at 4 °C for 10 min. The protein concentration from the cell extracts was quantified using standard methods, and 50 μg of protein per well was loaded onto an SDS-PAGE gel. Proteins were transferred to a wet Immuno-Blot membrane at 400 mA for 2 h. The membrane was then incubated in 5% nonfat milk in TBS-T (20 mM Tris, 500 mM NaCl pH7.5, 0.1% Tween-20) for 15 min, washed twice with TBS-T at ambient temperature, and then incubated with the primary Gal4-DBD antibody (Santa Cruz Biotechnology, diluted 1:500 in TBS-T) overnight. After three 15 min washes with TBS-T, the membrane was incubated with secondary antimouse IgG (1:2000 dilution), followed by three 15 min washes with TBS-T. The membrane was then treated with the ECL Western Blotting Substrate (Promega) and exposed to film.
■
(7) Chen, C. D., Welsbie, D. S., Tran, C., Baek, S. H., Chen, R., Vessella, R., Rosenfeld, M. G., and Sawyers, C. L. (2004) Molecular determinants of resistance to antiandrogen therapy. Nat. Med. 10, 33− 39. (8) Shore, N. D., Chowdhury, S., Villers, A., Klotz, L., Siemens, D. R., Phung, D., van Os, S., Hasabou, N., Wang, F., Bhattacharya, S., and Heidenreich, A. (2016) Efficacy and safety of enzalutamide versus bicalutamide for patients with metastatic prostate cancer (TERRAIN): a randomised, double-blind, phase 2 study. Lancet Oncol. 17, 153−163. (9) Swinney, D. C. (2004) Biochemical mechanisms of drug action: what does it take for success? Nat. Rev. Drug Discovery 3, 801−808. (10) Singh, J., Petter, R. C., Baillie, T. A., and Whitty, A. (2011) The resurgence of covalent drugs. Nat. Rev. Drug Discovery 10, 307−317. (11) Kwak, E. L., Sordella, R., Bell, D. W., Godin-Heymann, N., Okimoto, R. A., Brannigan, B. W., Harris, P. L., Driscoll, D. R., Fidias, P., Lynch, T. J., Rabindran, S. K., McGinnis, J. P., Wissner, A., Sharma, S. V., Isselbacher, K. J., Settleman, J., and Haber, D. A. (2005) Irreversible inhibitors of the EGF receptor may circumvent acquired resistance to gefitinib. Proc. Natl. Acad. Sci. U. S. A. 102, 7665−7670. (12) Li, D., Ambrogio, L., Shimamura, T., Kubo, S., Takahashi, M., Chirieac, L. R., Padera, R. F., Shapiro, G. I., Baum, A., Himmelsbach, F., Rettig, W. J., Meyerson, M., Solca, F., Greulich, H., and Wong, K. K. (2008) BIBW2992, an irreversible EGFR/HER2 inhibitor highly effective in preclinical lung cancer models. Oncogene 27, 4702−4711. (13) Oballa, R. M., Truchon, J. F., Bayly, C. I., Chauret, N., Day, S., Crane, S., and Berthelette, C. (2007) A generally applicable method for assessing the electrophilicity and reactivity of diverse nitrilecontaining compounds. Bioorg. Med. Chem. Lett. 17, 998−1002. (14) Fleming, F. F., Yao, L., Ravikumar, P. C., Funk, L., and Shook, B. C. (2010) Nitrile-containing pharmaceuticals: efficacious roles of the nitrile pharmacophore. J. Med. Chem. 53, 7902−7917. (15) Bohl, C. E., Gao, W., Miller, D. D., Bell, C. E., and Dalton, J. T. (2005) Structural basis for antagonism and resistance of bicalutamide in prostate cancer. Proc. Natl. Acad. Sci. U. S. A. 102, 6201−6206. (16) Estebanez-Perpina, E., Moore, J. M., Mar, E., DelgadoRodrigues, E., Nguyen, P., Baxter, J. D., Buehrer, B. M., Webb, P., Fletterick, R. J., and Guy, R. K. (2005) The molecular mechanisms of coactivator utilization in ligand-dependent transactivation by the androgen receptor. J. Biol. Chem. 280, 8060−8068. (17) Gottlieb, B., Beitel, L. K., Nadarajah, A., Paliouras, M., and Trifiro, M. (2012) The androgen receptor gene mutations database: 2012 update. Hum. Mutat. 33, 887−894. (18) Waller, A. S., Sharrard, R. M., Berthon, P., and Maitland, N. J. (2000) Androgen receptor localisation and turnover in human prostate epithelium treated with the antiandrogen, casodex. J. Mol. Endocrinol. 24, 339−351. (19) Chaturvedi, R. K., Macmahon, A. E., and Schmir, G. L. (1967) Hydrolysis of Thioimidate Esters. Tetrahedral Intermediates and General Acid Catalysis. J. Am. Chem. Soc. 89, 6984−6993. (20) Tran, C., Ouk, S., Clegg, N. J., Chen, Y., Watson, P. A., Arora, V., Wongvipat, J., Smith-Jones, P. M., Yoo, D., Kwon, A., Wasielewska, T., Welsbie, D., Chen, C. D., Higano, C. S., Beer, T. M., Hung, D. T., Scher, H. I., Jung, M. E., and Sawyers, C. L. (2009) Development of a second-generation antiandrogen for treatment of advanced prostate cancer. Science 324, 787−790. (21) Clegg, N. J., Wongvipat, J., Joseph, J. D., Tran, C., Ouk, S., Dilhas, A., Chen, Y., Grillot, K., Bischoff, E. D., Cai, L., Aparicio, A., Dorow, S., Arora, V., Shao, G., Qian, J., Zhao, H., Yang, G., Cao, C., Sensintaffar, J., Wasielewska, T., Herbert, M. R., Bonnefous, C., Darimont, B., Scher, H. I., Smith-Jones, P., Klang, M., Smith, N. D., De Stanchina, E., Wu, N., Ouerfelli, O., Rix, P. J., Heyman, R. A., Jung, M. E., Sawyers, C. L., and Hager, J. H. (2012) ARN-509: a novel antiandrogen for prostate cancer treatment. Cancer Res. 72, 1494− 1503. (22) Bassetto, M., Ferla, S., Pertusati, F., Kandil, S., Westwell, A. D., Brancale, A., and McGuigan, C. (2016) Design and synthesis of novel bicalutamide and enzalutamide derivatives as antiproliferative agents for the treatment of prostate cancer. Eur. J. Med. Chem. 118, 230−243.
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acschembio.7b00702. Chemical synthesis and characterization of 5N-bicalutamide and analogs, and cytotoxicity data (PDF)
■
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Matthew P. Jacobson: 0000-0001-6262-655X Pamela M. England: 0000-0002-2308-1048 Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS P.M.E. thanks the California Institute for Quantitative Biosciences (QB3) and the UCSF Prostate Cancer T3 Grant (#A124472) for funding and Charles Ryan and Fred Schaufele for helpful discussions. M.J.E. was supported by the National Institutes of Health (R01CA17661), a DOD Idea Development Award (PC140107), and an ACS Research Scholar Grant (130635-RSG-17-005-01-CCE). F.d.J.C. was supported by an IRACDA postdoctoral fellowship (K12GM081266). C.T. was supported by a postdoctoral fellowship from the DOD Prostate Cancer Research Program (PC151060).
■
REFERENCES
(1) Bennett, N. C., Gardiner, R. A., Hooper, J. D., Johnson, D. W., and Gobe, G. C. (2010) Molecular cell biology of androgen receptor signalling. Int. J. Biochem. Cell Biol. 42, 813−827. (2) Lonergan, P. E., and Tindall, D. J. (2011) Androgen receptor signaling in prostate cancer development and progression. J. Carcinog. 10, 20. (3) Jenster, G., van der Korput, H. A., van Vroonhoven, C., van der Kwast, T. H., Trapman, J., and Brinkmann, A. O. (1991) Domains of the human androgen receptor involved in steroid binding, transcriptional activation, and subcellular localization. Mol. Endocrinol. 5, 1396− 1404. (4) Jenster, G. (1998) Coactivators and corepressors as mediators of nuclear receptor function: an update. Mol. Cell. Endocrinol. 143, 1−7. (5) Dalton, J. T., Miller, D. D., Rakox, I., Bohl, C. E., and Mohler, M. L. (2008) Selective androgen receptor modulators, analogs and derivatives thereof and uses thereof, W02008011072A2. (6) Watson, P. A., Arora, V. K., and Sawyers, C. L. (2015) Emerging mechanisms of resistance to androgen receptor inhibitors in prostate cancer. Nat. Rev. Cancer 15, 701−711. 2938
DOI: 10.1021/acschembio.7b00702 ACS Chem. Biol. 2017, 12, 2934−2939
Letters
ACS Chemical Biology (23) Bohl, C. E., Miller, D. D., Chen, J., Bell, C. E., and Dalton, J. T. (2005) Structural basis for accommodation of nonsteroidal ligands in the androgen receptor. J. Biol. Chem. 280, 37747−37754. (24) Liu, H., An, X., Li, S., Wang, Y., Li, J., and Liu, H. (2015) Interaction mechanism exploration of R-bicalutamide/S-1 with WT/ W741L AR using molecular dynamics simulations. Mol. BioSyst. 11, 3347−3354. (25) Nagy, L., and Schwabe, J. W. (2004) Mechanism of the nuclear receptor molecular switch. Trends Biochem. Sci. 29, 317−324. (26) Harlow, K. W., Smith, D. N., Katzenellenbogen, J. A., Greene, G. L., and Katzenellenbogen, B. S. (1989) Identification of cysteine 530 as the covalent attachment site of an affinity-labeling estrogen (ketononestrol aziridine) and antiestrogen (tamoxifen aziridine) in the human estrogen receptor. J. Biol. Chem. 264, 17476−17485. (27) Simons, S. S., Jr., Pumphrey, J. G., Rudikoff, S., and Eisen, H. J. (1987) Identification of cysteine 656 as the amino acid of hepatoma tissue culture cell glucocorticoid receptors that is covalently labeled by dexamethasone 21-mesylate. J. Biol. Chem. 262, 9676−9680.
2939
DOI: 10.1021/acschembio.7b00702 ACS Chem. Biol. 2017, 12, 2934−2939
