Cumulative evidence indicates that elevated TIF2 coactivator expression levels are associated with prostate cancer (CaP) recurrence after androgen ablation therapy (AAT). Overexpressed TIF2 leads to androgen receptor (AR) hypersensitivity and transactivation by lower affinity adrenal androgens or other steroids that may contribute to the recurrence of castration resistant (CR) CaP after AAT. Disruptors of AR-TIF2 coactivator interactions will provide small molecule probes to investigate the roles of these interactions in the progression to CR CaP, and may lead to development of new therapeutics for CaP. We are proposing to develop a novel high content image-based biosensor assay to measure and quantify the protein-protein interactions (PPIs) between AR and TIF2, and to screen for probe compounds that prevent the formation of AR-TIF2 PPIs and/or disrupt AR-TIF2 complexes. The AR-TIF2 PPI biosensor (PPIB) assay exploits features of protein targeting to organelles, AR and TIF2 functional domains, and fluorescent reporters to generate positional biosensors that measure and quantify the interactions between these partners in cell, and the subsequent interaction with TIF2. The biosensor can be screened in several formats to identify: 1) AR-agonists, 2) compounds that block induction of AR-TIF2 PPIs, and 3) compounds that disrupt established AR-TIF2 complexes. We will screen formats #2 and #3 because numerous assay formats exist to screen for AR agonists. We propose to complete the development of the AR-TIF2 PPIB HCS assay and generate recombinant adenovirus (rAV) banks to conduct an MLPCN HCS campaign against e 300,000 compounds. We will further optimize the assay in prostate cancer cell lines (PC-3, DU-45, LNCaP, LAPC4, C4-2, and CWR-R1) and select the most suitable cell model to represent CR-CaP for the HCS. We will then adapt and automate the assay to screen for molecules that prevent or disrupt AR-TIF2 PPIs, and validate its performance in pilot screens. We propose to integrate counter screens and secondary or tertiary assays to characterize and determine the mechanism of action of AR-TIF2 PPI hits. Our plan would then be to submit the fully optimized and validated AR-TIF2 PPIB HCS assay to the MLPCN for screening. By targeting a late stage in AR signaling, namely, disruption of AR-TIF2 interactions, we hope to identify novel compounds that inhibit AR transactivation with therapeutic potential to block the development of AAT-resistance and the recurrence of CR-CaP.
Despite initial responses to androgen ablation therapy,(AAT), prostate cancer (CaP) progresses to the castration resistant (CR) state. Although CaP is the second leading cause of cancer death among men, no chemotherapy regimen has emerged as the standard and current AATs are limited by toxicities and progression to CR. We are proposing to develop a novel protein-protein interaction biosensor (PPIB) assay that recapitulates the activation of AR and its interactions with the TIF2 coactivator, and to use this assay to identify small molecules that disrupt AR-TIF2 complexes with therapeutic potential to block progression to CR CaP.
|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|