Structure-based Virtual Screening and Identification of a Novel Androgen Receptor Antagonist*

Background: The androgen receptor (AR) is the primary drug target for prostate cancer treatment. Results: We have identified a novel AR antagonist, the compound 6-(3,4-dihydro-1H-isoquinolin-2-yl)-N-(6-methylpyridin-2-yl)nicotinamide (DIMN) that inhibits the growth of AR-positive prostate cancer cells. Conclusion: DIMN has been identified as a new lead structure targeting the AR. Significance: This novel AR antagonist could be a useful therapeutic agent for prostate cancer treatment. Hormonal therapies, mainly combinations of anti-androgens and androgen deprivation, have been the mainstay treatment for advanced prostate cancer because the androgen-androgen receptor (AR) system plays a pivotal role in the development and progression of prostate cancers. However, the emergence of androgen resistance, largely due to inefficient anti-hormone action, limits the therapeutic usefulness of these therapies. Here, we report that 6-(3,4-dihydro-1H-isoquinolin-2-yl)-N-(6-methylpyridin-2-yl)nicotinamide (DIMN) acts as a novel anti-androgenic compound that may be effective in the treatment of both androgen-dependent and androgen-independent prostate cancers. Through AR structure-based virtual screening using the FlexX docking model, fifty-four compounds were selected and further screened for AR antagonism via cell-based tests. One compound, DIMN, showed an antagonistic effect specific to AR with comparable potency to that of the classical AR antagonists, hydroxyflutamide and bicalutamide. Consistent with their anti-androgenic activity, DIMN inhibited the growth of androgen-dependent LNCaP prostate cancer cells. Interestingly, the compound also suppressed the growth of androgen-independent C4–2 and CWR22rv prostate cancer cells, which express a functional AR, but did not suppress the growth of the AR-negative prostate cancer cells PPC-1, DU145, and R3327-AT3.1. Taken together, the results suggest that the synthetic compound DIMN is a novel anti-androgen and strong candidate for useful therapeutic agent against early stage to advanced prostate cancer.

[1]  S. Chattopadhyay,et al.  Antiandrogenic effects of bisphenol A and nonylphenol on the function of androgen receptor. , 2003, Toxicological sciences : an official journal of the Society of Toxicology.

[2]  V. Georget,et al.  Evidence that chlormadinone acetate exhibits antiandrogenic activity in androgen-dependent cell line , 2002, Molecular and Cellular Endocrinology.

[3]  J. Dalton,et al.  Pharmacokinetics and Pharmacodynamics of Nonsteroidal Androgen Receptor Ligands , 2006, Pharmaceutical Research.

[4]  S. Liao,et al.  Molecular action of androgen in the normal and neoplastic prostate. , 1999, Vitamins and hormones.

[5]  David Heber,et al.  Limitations of MTT and MTS-Based Assays for Measurement of Antiproliferative Activity of Green Tea Polyphenols , 2010, PloS one.

[6]  Hyun Joo Lee,et al.  The CCAAT Enhancer-Binding Protein-α Negatively Regulates the Transactivation of Androgen Receptor in Prostate Cancer Cells , 2006 .

[7]  Keesook Lee,et al.  Expression of MIS in the Testis Is Downregulated by Tumor Necrosis Factor Alpha through the Negative Regulation of SF-1 Transactivation by NF-κB , 2003, Molecular and Cellular Biology.

[8]  Zijie Sun,et al.  Phosphatidylinositol 3-Kinase/Akt Stimulates Androgen Pathway through GSK3β Inhibition and Nuclear β-Catenin Accumulation* , 2002, The Journal of Biological Chemistry.

[9]  Yanping Zhang,et al.  Targeting prostate cancer with conditionally replicative adenovirus using PSMA enhancer. , 2004, Molecular therapy : the journal of the American Society of Gene Therapy.

[10]  Tommi H. Nyrönen,et al.  Computationally Identified Novel Diphenyl- and Phenylpyridine Androgen Receptor Antagonist Structures , 2008, J. Chem. Inf. Model..

[11]  S. Yeh,et al.  From estrogen to androgen receptor: a new pathway for sex hormones in prostate. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[12]  Daniel Gioeli,et al.  Ras signaling in prostate cancer progression , 2004, Journal of cellular biochemistry.

[13]  R A Rifkind,et al.  Suberoylanilide hydroxamic acid, an inhibitor of histone deacetylase, suppresses the growth of prostate cancer cells in vitro and in vivo. , 2000, Cancer research.

[14]  J. Ferlay,et al.  Global Cancer Statistics, 2002 , 2005, CA: a cancer journal for clinicians.

[15]  H. Scher,et al.  Development of a Second-Generation Antiandrogen for Treatment of Advanced Prostate Cancer , 2009, Science.

[16]  Hideo Araki,et al.  Novel mutations of androgen receptor: a possible mechanism of bicalutamide withdrawal syndrome. , 2003, Cancer research.

[17]  T. Shiraishi,et al.  Biological properties of androgen receptor pure antagonist for treatment of castration‐resistant prostate cancer: Optimization from lead compound to CH5137291 , 2011, The Prostate.

[18]  J. Wong,et al.  The corepressors silencing mediator of retinoid and thyroid hormone receptor and nuclear receptor corepressor are involved in agonist- and antagonist-regulated transcription by androgen receptor. , 2006, Molecular endocrinology.

[19]  O. Ogawa,et al.  Antiandrogen bicalutamide promotes tumor growth in a novel androgen-dependent prostate cancer xenograft model derived from a bicalutamide-treated patient. , 2005, Cancer research.

[20]  S. Yeh,et al.  From transforming growth factor-β signaling to androgen action: Identification of Smad3 as an androgen receptor coregulator in prostate cancer cells , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[21]  L. Chung,et al.  LNCaP progression model of human prostate cancer: Androgen‐independence and osseous metastasis , 2000, The Prostate.

[22]  N. Dubrawsky.  Cancer statistics , 1989, CA: a cancer journal for clinicians.

[23]  C. Sawyers,et al.  Structure-activity relationship for thiohydantoin androgen receptor antagonists for castration-resistant prostate cancer (CRPC). , 2010, Journal of medicinal chemistry.

[24]  J. Nelson,et al.  HDAC6 regulates androgen receptor hypersensitivity and nuclear localization via modulating Hsp90 acetylation in castration-resistant prostate cancer. , 2009, Molecular endocrinology.

[25]  Duane D. Miller,et al.  Structural Basis for Accommodation of Nonsteroidal Ligands in the Androgen Receptor* , 2005, Journal of Biological Chemistry.

[26]  Keesook Lee,et al.  Reduced testicular steroidogenesis in tumor necrosis factor-α knockout mice , 2008, The Journal of Steroid Biochemistry and Molecular Biology.

[27]  C. de la Piedra,et al.  Comparative Effects of Bicalutamide (Casodex) versus Orchidectomy on Bone Mineral Density, Bone Remodelling, and Bone Biomechanics in Healthy Rats , 2005, Urologia Internationalis.

[28]  G. Bubley,et al.  Bicalutamide Functions as an Androgen Receptor Antagonist by Assembly of a Transcriptionally Inactive Receptor* , 2002, The Journal of Biological Chemistry.

[29]  R. Silverman,et al.  Resveratrol regulates the PTEN/AKT pathway through androgen receptor-dependent and -independent mechanisms in prostate cancer cell lines , 2010, Human molecular genetics.

[30]  R. Vessella,et al.  Molecular determinants of resistance to antiandrogen therapy , 2004, Nature Medicine.

[31]  P. Kantoff,et al.  Androgen receptor mutations in androgen-independent prostate cancer: Cancer and Leukemia Group B Study 9663. , 2003, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[32]  Duane D. Miller,et al.  Structural basis for antagonism and resistance of bicalutamide in prostate cancer , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[33]  H. Leung,et al.  Evaluation of an in vitro model of androgen ablation and identification of the androgen responsive proteome in LNCaP cells , 2007, Proteomics.

[34]  R. Vessella,et al.  Inactivation of the tumor suppressor PTEN/MMAC1 in advanced human prostate cancer through loss of expression. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[35]  H. Klocker,et al.  Targeting the androgen receptor in hormone-refractory prostate cancer--new concepts. , 2005, Future oncology.

[36]  M. Okada,et al.  N-Arylpiperazine-1-carboxamide derivatives: a novel series of orally active nonsteroidal androgen receptor antagonists. , 2005, Chemical & pharmaceutical bulletin.

[37]  M. Garabedian,et al.  GRIP1, a transcriptional coactivator for the AF-2 transactivation domain of steroid, thyroid, retinoid, and vitamin D receptors , 1997, Molecular and cellular biology.

[38]  S. Yeh,et al.  Akt suppresses androgen-induced apoptosis by phosphorylating and inhibiting androgen receptor , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[39]  J. Trapman,et al.  The androgen receptor in prostate cancer. , 1996, Pathology, research and practice.

[40]  J. A. Kemppainen,et al.  Intermolecular NH2-/Carboxyl-terminal Interactions in Androgen Receptor Dimerization Revealed by Mutations That Cause Androgen Insensitivity* , 1998, The Journal of Biological Chemistry.

[41]  V. Njar,et al.  Pregnenolone stimulates LNCaP prostate cancer cell growth via the mutated androgen receptor , 2000, The Journal of Steroid Biochemistry and Molecular Biology.

[42]  David E. Williams,et al.  Regression of castrate-recurrent prostate cancer by a small-molecule inhibitor of the amino-terminus domain of the androgen receptor. , 2010, Cancer cell.

[43]  M. Wirth,et al.  Antiandrogens in the treatment of prostate cancer. , 2007, European urology.

[44]  M. Rubin,et al.  Current thoughts on the role of the androgen receptor and prostate cancer progression. , 2005, Advances in anatomic pathology.

[45]  Artem Cherkasov,et al.  Inhibitors of androgen receptor activation function-2 (AF2) site identified through virtual screening. , 2011, Journal of medicinal chemistry.

[46]  E. Wilson,et al.  Distinguishing androgen receptor agonists and antagonists: distinct mechanisms of activation by medroxyprogesterone acetate and dihydrotestosterone. , 1999, Molecular endocrinology.

[47]  Hyun Joo Lee,et al.  Hepatocyte nuclear factor-3 alpha (HNF-3alpha) negatively regulates androgen receptor transactivation in prostate cancer cells. , 2008, Biochemical and biophysical research communications.

[48]  Chawnshang Chang,et al.  Androgen Receptor Coregulators in Prostate Cancer , 2004, Clinical Cancer Research.

[49]  K. Hamil,et al.  Dehydroepiandrosterone activates mutant androgen receptors expressed in the androgen-dependent human prostate cancer xenograft CWR22 and LNCaP cells. , 1997, Molecular endocrinology.

[50]  Biaoyang Lin,et al.  The program of androgen-responsive genes in neoplastic prostate epithelium , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[51]  Duane D. Miller,et al.  Preclinical Pharmacology of a Nonsteroidal Ligand for Androgen Receptor-Mediated Imaging of Prostate Cancer , 2006, Journal of Pharmacology and Experimental Therapeutics.

[52]  Ramesh Narayanan,et al.  Nonsteroidal selective androgen receptor modulators (SARMs): dissociating the anabolic and androgenic activities of the androgen receptor for therapeutic benefit. , 2009, Journal of medicinal chemistry.

[53]  J. Veldscholte,et al.  Anti-androgens and the mutated androgen receptor of LNCaP cells: differential effects on binding affinity, heat-shock protein interaction, and transcription activation. , 1992, Biochemistry.

[54]  Keesook Lee,et al.  Activating Signal Cointegrator 1 Is Highly Expressed in Murine Testicular Leydig Cells and Enhances the Ligand-Dependent Transactivation of Androgen Receptor1 , 2002, Biology of reproduction.

[55]  S. Yeh,et al.  From HER2/Neu signal cascade to androgen receptor and its coactivators: a novel pathway by induction of androgen target genes through MAP kinase in prostate cancer cells. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[56]  B. Fraile,et al.  MAP Kinases and Prostate Cancer , 2011, Journal of signal transduction.

[57]  E. Messing,et al.  Androgen deprivation therapy for prostate cancer: Current status and future prospects , 2004, The Prostate.

[58]  John Anderson,et al.  The role of antiandrogen monotherapy in the treatment of prostate cancer , 2003, BJU international.

[59]  G. Coetzee,et al.  Prostate specific antigen gene regulation by androgen receptor , 2004, Journal of cellular biochemistry.

[60]  J. Nicolas,et al.  Mechanism of antiandrogen action: key role of hsp90 in conformational change and transcriptional activity of the androgen receptor. , 2002, Biochemistry.

[61]  C. Heinlein,et al.  Androgen receptor (AR) coregulators: an overview. , 2002, Endocrine reviews.

[62]  Y. Wang,et al.  Regulation of androgen receptor transcriptional activity by rapamycin in prostate cancer cell proliferation and survival , 2008, Oncogene.

[63]  S. Kato,et al.  Stabilization of androgen receptor protein is induced by agonist, not by antagonists. , 2002, Biochemical and biophysical research communications.

[64]  Duane D. Miller,et al.  Crystal Structure of the T877A Human Androgen Receptor Ligand-binding Domain Complexed to Cyproterone Acetate Provides Insight for Ligand-induced Conformational Changes and Structure-based Drug Design* , 2007, Journal of Biological Chemistry.

[65]  Takashi Kamikubo,et al.  (+)-(2R,5S)-4-[4-cyano-3-(trifluoromethyl)phenyl]-2,5-dimethyl-N-[6-(trifluoromethyl)pyridin-3- yl]piperazine-1-carboxamide (YM580) as an orally potent and peripherally selective nonsteroidal androgen receptor antagonist. , 2006, Journal of medicinal chemistry.

[66]  T. H. van der Kwast,et al.  Domains of the human androgen receptor involved in steroid binding, transcriptional activation, and subcellular localization. , 1991, Molecular endocrinology.

[67]  D. Osguthorpe,et al.  Mechanism of androgen receptor antagonism by bicalutamide in the treatment of prostate cancer. , 2011, Biochemistry.

[68]  H. Scher,et al.  Hormone-refractory (D3) prostate cancer: refining the concept. , 1995, Urology.

[69]  E. Small,et al.  Selection for androgen receptor mutations in prostate cancers treated with androgen antagonist. , 1999, Cancer research.

[70]  K. Kish,et al.  Structure based approach to the design of bicyclic-1H-isoindole-1,3(2H)-dione based androgen receptor antagonists. , 2005, Bioorganic & medicinal chemistry letters.

[71]  J. Kreisberg,et al.  AKT regulates androgen receptor-dependent growth and PSA expression in prostate cancer. , 2008, Advances in experimental medicine and biology.

[72]  Martin R. Schneider,et al.  Switch from antagonist to agonist of the androgen receptor blocker bicalutamide is associated with prostate tumour progression in a new model system , 1999, British Journal of Cancer.

[73]  A. Jemal,et al.  Cancer Statistics, 2008 , 2008, CA: a cancer journal for clinicians.

[74]  H. Klocker,et al.  Mutant androgen receptor detected in an advanced-stage prostatic carcinoma is activated by adrenal androgens and progesterone. , 1993, Molecular endocrinology.

[75]  Clifford A. Meyer,et al.  Androgen Receptor Regulates a Distinct Transcription Program in Androgen-Independent Prostate Cancer , 2009, Cell.

[76]  K. Grigor,et al.  Androgen receptor gene amplification and protein expression in hormone refractory prostate cancer , 2003, British Journal of Cancer.

[77]  Z. Dong,et al.  A novel synthetic compound that interrupts androgen receptor signaling in human prostate cancer cells , 2007, Molecular Cancer Therapeutics.

[78]  Hyun Joo Lee,et al.  Modulation of the expression and transactivation of androgen receptor by the basic helix-loop-helix transcription factor Pod-1 through recruitment of histone deacetylase 1. , 2005, Molecular endocrinology.

[79]  B. Fraile,et al.  MAPKinases and Prostate Cancer , 2014 .

[80]  F. S. French,et al.  A mechanism for androgen receptor-mediated prostate cancer recurrence after androgen deprivation therapy. , 2001, Cancer research.

[81]  P. Kantoff,et al.  Antiandrogens in Prostate Cancer , 2004, Investigational New Drugs.

[82]  Desok Kim,et al.  Androgen receptor expression in androgen-independent prostate cancer is associated with increased expression of androgen-regulated genes. , 1998, Cancer research.

[83]  Keesook Lee,et al.  CR6-interacting factor 1 represses the transactivation of androgen receptor by direct interaction. , 2008, Molecular endocrinology.

[84]  M. A. Carrondo,et al.  Structural evidence for ligand specificity in the binding domain of the human androgen receptor. Implications for pathogenic gene mutations. , 2000, The Journal of biological chemistry.

[85]  G. Wilding,et al.  Promotion of agonist activity of antiandrogens by the androgen receptor coactivator, ARA70, in human prostate cancer DU145 cells. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[86]  C. Sawyers,et al.  Mechanistic Concepts in Androgen-dependence of Prostate Cancer , 2004, Cancer and Metastasis Reviews.

[87]  A. Jemal,et al.  Global cancer statistics , 2011, CA: a cancer journal for clinicians.

[88]  G. Jenster,et al.  A mutation in the ligand binding domain of the androgen receptor of human LNCaP cells affects steroid binding characteristics and response to anti-androgens. , 1990, Biochemical and biophysical research communications.

[89]  Jorma Isola,et al.  In vivo amplification of the androgen receptor gene and progression of human prostate cancer , 1995, Nature Genetics.

[90]  P. Poyet,et al.  Comparison of the antiandrogenic/androgenic activities of flutamide, cyproterone acetate and megestrol acetate , 1985, Molecular and Cellular Endocrinology.

[91]  H. Klocker,et al.  Androgen receptor activation in prostatic tumor cell lines by insulin-like growth factor-I, keratinocyte growth factor and epidermal growth factor. , 1995, European urology.

[92]  D. Troyer,et al.  Signal transduction pathways in androgen-dependent and -independent prostate cancer cell proliferation. , 2005, Endocrine-related cancer.

[93]  D. Feldman,et al.  The development of androgen-independent prostate cancer , 2001, Nature Reviews Cancer.

[94]  B. O’Malley,et al.  Sequence and Characterization of a Coactivator for the Steroid Hormone Receptor Superfamily , 1995, Science.

[95]  L. Morel,et al.  Androgen receptor expression is regulated by the phosphoinositide 3-kinase/Akt pathway in normal and tumoral epithelial cells. , 2002, The Biochemical journal.

[96]  Charis Eng,et al.  PTEN: One Gene, Many Syndromes , 2003, Human mutation.

[97]  M. Chen,et al.  Aberrant response in vitro of hormone‐responsive prostate cancer cells to antiandrogens , 1989, The Prostate.

[98]  K. Makino,et al.  HER-2/neu promotes androgen-independent survival and growth of prostate cancer cells through the Akt pathway. , 2000, Cancer research.

[99]  P. Meltzer,et al.  A Nuclear Factor, ASC-2, as a Cancer-amplified Transcriptional Coactivator Essential for Ligand-dependent Transactivation by Nuclear Receptors in Vivo * , 1999, The Journal of Biological Chemistry.