HCS to Identify Inhibitors and Disruptors of AR-TIF2 Interactions

University Of Pittsburgh At Pittsburgh, Pittsburgh PA

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Abstract

? DESCRIPTION (provided by applicant): A significant body of clinical data implicates elevated TIF2 coactivator expression levels with prostate cancer recurrence after androgen ablation therapy. Over- expressed TIF2 leads to androgen receptor (AR) hypersensitivity and transactivation by lower affinity androgens or other steroids that may contribute to the recurrence of castration resistant prostate cancer (CRPC) after ablation therapy. Our intent is to identify small molecules that will disrupt the protein-protein interactions (PPIs) between TIF-2 and AR. Disruptors of AR- TIF2 PPIs will provide novel small molecule probes to investigate the role of these interactions in the development and progression to CRPC, and may lead to the development of novel therapeutics for prostate cancer. We will screen 143,000 compounds in a novel image-based biosensor assay developed to measure and quantify AR-TIF2 PPIs. The AR-TIF2 biosensors recapitulate the ligand-induced translocation of AR from the cytoplasm to the nucleus and the subsequent recruitment and interaction with the TIF2 coactivator. The biosensor assay will be screened in two formats; 1) to find compounds that block the induction of AR-TIF2 PPIs, and 2) to identify compounds that disrupt established AR-TIF2 complexes. We will confirm that actives identified in both of the screens conducted in U-2 OS cells are also concentration dependent inhibitors and/or disruptors of AR-TIF2 PPIs in the PC-3 prostate cancer cell background. Since the AR-TIF2 PPIB assay is dependent upon a ligand-induced translocation event, we will investigate non-specific effects on cargo trafficking in three established HCS counter screens; the p53-hDM2 PPIB assay, the DHT-induced AR nuclear localization assay, and the Dexamethasone-induced GR-GFP nuclear translocation assay. The counter screens will identify and eliminate compounds that interfere with the assay format, are non-specific PPI disruptors, inhibit NR ligand binding, disrupt dynein-mediated NR trafficking to the nucleus, or block importing a/ß mediated passage through the nuclear pore complex. We will characterize the confirmed hits in our established secondary and tertiary assays to identify their mechanism of action and demonstrate activity in prostate cancer growth models. By disrupting AR-TIF2 interactions we hope to identify novel compounds that block AR transactivation with therapeutic potential to block the development of resistance and the recurrence of CRPC.