title: High-content positional biosensor screening assay for compounds to prevent or disrupt androgen receptor and transcriptional intermediary factor 2 protein-protein interactions.
abstract: The androgen receptor-transcriptional intermediary factor 2 (AR-TIF2) positional protein-protein interaction (PPI) biosensor assay described herein combines physiologically relevant cell-based assays with the specificity of binding assays by incorporating structural information of AR and TIF2 functional domains along with intracellular targeting sequences and fluorescent reporters. Expression of the AR-red fluorescent protein (RFP) "prey" and TIF2-green fluorescent protein (GFP) "bait" components of the biosensor was directed by recombinant adenovirus constructs that expressed the ligand binding and activation function 2 surface domains of AR fused to RFP with nuclear localization and nuclear export sequences, and three α-helical LXXLL motifs from TIF2 fused to GFP and an HIV Rev nucleolar targeting sequence. In unstimulated cells, AR-RFP was localized predominantly to the cytoplasm and TIF2-GFP was localized to nucleoli. Dihydrotestosterone (DHT) treatment induced AR-RFP translocation into the nucleus where the PPIs between AR and TIF2 resulted in the colocalization of both biosensors within the nucleolus. We adapted the translocation enhanced image analysis module to quantify the colocalization of the AR-RFP and TIF2-GFP biosensors in images acquired on the ImageXpress platform. DHT induced a concentration-dependent AR-TIF2 colocalization and produced a characteristic condensed punctate AR-RFP PPI nucleolar distribution pattern. The heat-shock protein 90 inhibitor 17-N-allylamino-17-demethoxygeldanamycin (17-AAG) and antiandrogens flutamide and bicalutamide inhibited DHT-induced AR-TIF2 PPI formation with 50% inhibition concentrations (IC50s) of 88.5±12.5 nM, 7.6±2.4 μM, and 1.6±0.4 μM, respectively. Images of the AR-RFP distribution phenotype allowed us to distinguish between 17-AAG and flutamide, which prevented AR translocation, and bicalutamide, which blocked AR-TIF2 PPIs. We screened the Library of Pharmacologically Active Compounds (LOPAC) set for compounds that inhibited AR-TIF2 PPI formation or disrupted preexisting complexes. Eleven modulators of steroid family nuclear receptors (NRs) and 6 non-NR ligands inhibited AR-TIF2 PPI formation, and 10 disrupted preexisting complexes. The hits appear to be either AR antagonists or nonspecific inhibitors of NR activation and trafficking. Given that the LOPAC set represents such a small and restricted biological and chemical diversity, it is anticipated that screening a much larger and more diverse compound library will be required to find AR-TIF2 PPI inhibitors/disruptors. The AR-TIF2 protein-protein interaction biosensor (PPIB) approach offers significant promise for identifying molecules with potential to modulate AR transcriptional activity in a cell-specific manner that is distinct from the existing antiandrogen drugs that target AR binding or production. Small molecules that disrupt AR signaling at the level of AR-TIF2 PPIs may also overcome the development of resistance and progression to castration-resistant prostate cancer.
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.forename: Tong Ying
authors.initials: TY
authors.lastname: Shun
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Strock
authors.forename: Paul A
authors.initials: PA
authors.lastname: Johnston
mesh_terms: D059002:Androgen Receptor Antagonists / Q000494:pharmacology; D045744:Cell Line, Tumor; D055808:Drug Discovery / Q000379:methods*; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D008297:Male; D057054:Molecular Imaging / Q000379:methods; D051958:Nuclear Receptor Coactivator 2 / Q000187:drug effects* / Q000378:metabolism; D011471:Prostatic Neoplasms / Q000188:drug therapy; D054730:Protein Interaction Domains and Motifs / Q000187:drug effects*; D011944:Receptors, Androgen / Q000187:drug effects* / Q000378:metabolism
chemical_list: D059002:Androgen Receptor Antagonists; D051958:Nuclear Receptor Coactivator 2; D011944:Receptors, Androgen