HCS Assay to Identify Disruptors of AR-TIF2 Protein-Protein Interactions

Johnston, Paul

Abstract

There is a growing body of evidence that high TIF2 coactivator expression levels are associated with prostate cancer (CaP) recurrence after androgen ablation therapy (AAT). Over expressed TIF2 may lead to androgen receptor (AR) hypersensitivity and transactivation by lower affinity adrenal androgens or other steroids that could contribute to the growth of recurrent castration resistant (CR) CaP after AAT. Small molecule disruptors of the AR-TIF2 coactivator interaction will provide probes to investigate the role of this interaction in the development and progression to CR CaP, and may lead to the development of novel therapeutics for CaP. We are proposing to develop a novel high content image-based biosensor HCS assay to measure and quantify the protein-protein interactions (PPIs) between the AR and TIF2, and to screen for disruptors of these interactions. The AR-TIF2 protein-protein interaction biosensor (PPIB) assay exploits features of protein targeting to organelles, AR and TIF2 functional domains, and fluorescent reporters to generate positional biosensors to measure and quantify the interactions between these protein partners in cells. The AR-TIF2 PPIB proposed here recapitulates the ligand-induced translocation of AR from the cytoplasm to the nucleus and the subsequent recruitment and interaction with the TIF2 coactivator. The AR-TIF2 PPIB assay can be screened in two formats;to screen for compounds that block the DHT-induced formation of AR-TIF2 PPIs cells will be pre-exposed to compounds prior to DHT treatment, and to identify compounds capable of disrupting established AR-TIF2 PPI complexes cells will be pre-exposed to DHT prior to compound treatment. We recently described the development and characterization of p53-hDM2 PPIB assay utilizing this same technology that we successfully used to screen 220,000 compounds from the NIH library and to identify a novel chemotype series that disrupts p53-hDM2 PPIs in cells. We propose to optimize the AR-TIF2 biosensor HCS assay, adapt it to screen compound libraries for molecules that disrupt the AR-TIF2 PPIs, and to validate its performance by screening the LOPAC and NIH Clinical Collection compound libraries. After completing the development and validation of the AR-TIF2 PPIB HCS assay, our plan would be to submit the assay through the fast track mechanism into the MLPCN for screening.

Public Health Relevance

Prostate cancer (CaP) remains incurable in the metastatic setting and despite the initial response to androgen ablation therapy (AATs) the disease transforms and progresses to the castration resistant (CR) state and is the most common cancer and second leading cause of cancer death among men in western countries,. To date however, no chemotherapy regimen has emerged as the standard therapy for metastatic CR CaP and current AATs are limited by toxicities including;muscle atrophy, osteoporosis, hot flashes, sexual dysfunction, fatigue, anemia, depression and cognitive dysfunction. We are proposing to develop and validate a novel AR-TIF2 protein-protein interaction biosensor assay that recapitulates the ligand-induced translocation of AR into the nucleus and the recruitment interactions with the TIF2 coactivator, and to use this assay to identify small molecule probes that disrupt AR-TIF2 protein-protein interactions and to use these probes to explore the role of such interactions in the development and progression of CR CaP.