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/ 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.
Prostate cancer (PC) is the most common cancer and second leading cause of cancer death among men in western countries. Initial response rates to the standard androgen ablation therapy (AATs) are typically good, however, PC inevitably transforms and progresses to a castration resistant (CRPC) state that in the metastatic setting is incurable. No effective chemotherapy regimen has emerged for metastatic CRPC and current AATs are limited by toxicities including; muscle atrophy, osteoporosis, hot flashes, sexual dysfunction, fatigue, anemia, depression, cognitive dysfunction, and treatment-induced bone loss. Since 2010, the FDA has approved six new drugs for metastatic CRPC, and many more are in development. However, the new drugs largely represent extensions of existing chemotherapies, AR antagonists or androgen synthesis inhibitors. The newly approved drugs share the same toxicology liabilities as their precursors and only improve survival by 3-5 months, because the PC cells inevitably develop resistance and patients die from metastatic CRPC. We will screen a novel AR-TIF2 protein-protein interaction biosensor (PPIB) assay that recapitulates the ligand- induced translocation of AR into the nucleus and the recruitment interactions with the TIF2 coactivator. Small molecule probes that prevent the formation of AR-TIF2 protein-protein interactions or disrupt existing AR-TIF2 complexes, will be utilized to explore the role of these interactions in the development and progression of CRPC. The AR-TIF2 PPIB assay can identify compounds that bind either AR or TIF2 and are not limited to a specific method of AR activation. By disrupting AR-TIF2 interactions we hope to identify novel compounds to block AR transactivation with therapeutic potential to inhibit the development of resistance and recurrence of CRPC.
| Hua, Yun; Camarco, Daniel P; Strock, Christopher J et al. (2018) High Content Positional Biosensor Assay to Screen for Compounds that Prevent or Disrupt Androgen Receptor and Transcription Intermediary Factor 2 Protein-Protein Interactions. Methods Mol Biol 1683:211-227 |
| Fancher, Ashley T; Hua, Yun; Camarco, Daniel P et al. (2016) Reconfiguring the AR-TIF2 Protein-Protein Interaction HCS Assay in Prostate Cancer Cells and Characterizing the Hits from a LOPAC Screen. Assay Drug Dev Technol 14:453-477 |
| Hua, Yun; Shun, Tong Ying; Strock, Christopher J et al. (2014) High-content positional biosensor screening assay for compounds to prevent or disrupt androgen receptor and transcriptional intermediary factor 2 protein-protein interactions. Assay Drug Dev Technol 12:395-418 |
