title: Development and Implementation of a High-Throughput High-Content Screening Assay to Identify Inhibitors of Androgen Receptor Nuclear Localization in Castration-Resistant Prostate Cancer Cells.
abstract: Patients with castration-resistant prostate cancer (CRPC) can be treated with abiraterone, a potent inhibitor of androgen synthesis, or enzalutamide, a second-generation androgen receptor (AR) antagonist, both targeting AR signaling. However, most patients relapse after several months of therapy and a majority of patients with relapsed CRPC tumors express the AR target gene prostate-specific antigen (PSA), suggesting that AR signaling is reactivated and can be targeted again to inhibit the relapsed tumors. Novel small molecules capable of inhibiting AR function may lead to urgently needed therapies for patients resistant to abiraterone, enzalutamide, and/or other previously approved antiandrogen therapies. Here, we describe a high-throughput high-content screening (HCS) campaign to identify small-molecule inhibitors of AR nuclear localization in the C4-2 CRPC cell line stably transfected with GFP-AR-GFP (2GFP-AR). The implementation of this HCS assay to screen a National Institutes of Health library of 219,055 compounds led to the discovery of 3 small molecules capable of inhibiting AR nuclear localization and function in C4-2 cells, demonstrating the feasibility of using this cell-based phenotypic assay to identify small molecules targeting the subcellular localization of AR. Furthermore, the three hit compounds provide opportunities to develop novel AR drugs with potential for therapeutic intervention in CRPC patients who have relapsed after treatment with antiandrogens, such as abiraterone and/or enzalutamide.
authors.affiliation: 1 Department of Pharmaceutical Sciences, School of Pharmacy, University of Pittsburgh , Pittsburgh, Pennsylvania.|2 University of Pittsburgh Cancer Institute , Pittsburgh, Pennsylvania.
authors.forename: Paul A
authors.initials: PA
authors.lastname: Johnston
authors.affiliation: 3 Department of Urology, School of Medicine, University of Pittsburgh , Pittsburgh, Pennsylvania.
authors.forename: Minh M
authors.initials: MM
authors.lastname: Nguyen
authors.affiliation: 3 Department of Urology, School of Medicine, University of Pittsburgh , Pittsburgh, Pennsylvania.|4 Central Laboratory, College of Science, King Saud University , Riyadh, Saudi Arabia .
authors.forename: Javid A
authors.initials: JA
authors.lastname: Dar
authors.affiliation: 3 Department of Urology, School of Medicine, University of Pittsburgh , Pittsburgh, Pennsylvania.
authors.forename: Junkui
authors.initials: J
authors.lastname: Ai
authors.affiliation: 3 Department of Urology, School of Medicine, University of Pittsburgh , Pittsburgh, Pennsylvania.
authors.forename: Yujuan
authors.initials: Y
authors.lastname: Wang
authors.affiliation: 3 Department of Urology, School of Medicine, University of Pittsburgh , Pittsburgh, Pennsylvania.|5 Transcriptomics and Proteomics Lab, Centre for Plant Biotechnology, Sher-e-Kashmir University of Agricultural Sciences and Technology of Kashmir (SKUAST-K) , Shalimar, Srinagar, India .
authors.forename: Khalid Z
authors.initials: KZ
authors.lastname: Masoodi
authors.affiliation: 6 Pittsburgh Specialized Application Center, University of Pittsburgh Drug Discovery Institute , Pittsburgh, Pennsylvania.
authors.forename: Tongying
authors.initials: T
authors.lastname: Shun
authors.affiliation: 6 Pittsburgh Specialized Application Center, University of Pittsburgh Drug Discovery Institute , Pittsburgh, Pennsylvania.
authors.forename: Sunita
authors.initials: S
authors.lastname: Shinde
authors.affiliation: 1 Department of Pharmaceutical Sciences, School of Pharmacy, University of Pittsburgh , Pittsburgh, Pennsylvania.
authors.forename: Daniel P
authors.initials: DP
authors.lastname: Camarco
authors.affiliation: 1 Department of Pharmaceutical Sciences, School of Pharmacy, University of Pittsburgh , Pittsburgh, Pennsylvania.
authors.forename: Yun
authors.initials: Y
authors.lastname: Hua
authors.affiliation: 1 Department of Pharmaceutical Sciences, School of Pharmacy, University of Pittsburgh , Pittsburgh, Pennsylvania.|7 University of Pittsburgh Chemical Diversity Center , Pittsburgh, Pennsylvania.
authors.forename: Donna M
authors.initials: DM
authors.lastname: Huryn
authors.affiliation: 8 Department of Health Services and Health Administration, College of Nursing and Health Professions, University of Southern Indiana , Evansville, Indiana.
authors.forename: Gabriela Mustata
authors.initials: GM
authors.lastname: Wilson
authors.affiliation: 9 Departments of Pharmacology and Chemistry, University of Virginia , Charlottesville, Virginia.
authors.forename: John S
authors.initials: JS
authors.lastname: Lazo
authors.affiliation: 2 University of Pittsburgh Cancer Institute , Pittsburgh, Pennsylvania.|3 Department of Urology, School of Medicine, University of Pittsburgh , Pittsburgh, Pennsylvania.
authors.forename: Joel B
authors.initials: JB
authors.lastname: Nelson
authors.affiliation: 1 Department of Pharmaceutical Sciences, School of Pharmacy, University of Pittsburgh , Pittsburgh, Pennsylvania.|2 University of Pittsburgh Cancer Institute , Pittsburgh, Pennsylvania.|7 University of Pittsburgh Chemical Diversity Center , Pittsburgh, Pennsylvania.|10 Department of Chemistry, University of Pittsburgh , Pittsburgh, Pennsylvania.
authors.forename: Peter
authors.initials: P
authors.lastname: Wipf
authors.affiliation: 2 University of Pittsburgh Cancer Institute , Pittsburgh, Pennsylvania.|3 Department of Urology, School of Medicine, University of Pittsburgh , Pittsburgh, Pennsylvania.|11 Department of Pharmacology and Chemical Biology, University of Pittsburgh , Pittsburgh, Pennsylvania.
authors.forename: Zhou
authors.initials: Z
authors.lastname: Wang
mesh_terms: D021581:Active Transport, Cell Nucleus / Q000187:drug effects* / Q000502:physiology; D059002:Androgen Receptor Antagonists / Q000378:metabolism* / Q000494:pharmacology*; D016227:Benzoquinones / Q000378:metabolism / Q000494:pharmacology; D045744:Cell Line, Tumor; D004305:Dose-Response Relationship, Drug; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D047029:Lactams, Macrocyclic / Q000378:metabolism / Q000494:pharmacology; D008297:Male; D064129:Prostatic Neoplasms, Castration-Resistant / Q000378:metabolism*; D011944:Receptors, Androgen / Q000378:metabolism*
chemical_list: C410798:AR protein, human; D059002:Androgen Receptor Antagonists; D016227:Benzoquinones; D047029:Lactams, Macrocyclic; D011944:Receptors, Androgen; C112765:tanespimycin