Abstract 86: Live-cell profiling of inhibitors targeting the N/C interaction within the androgen receptor

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Androgen receptor (AR) is an important target for drug therapies combating prostate cancer. However, various acquired mutations within the AR sequence often render resistance to these treatments. Since intramolecular interactions between the N- and C-terminus of AR are implicated in AR activation, it is essential to understand how AR inhibitors alter the N/C interaction within the AR, both for the wt receptor and for its mutant forms. In this study we applied our recently developed Fluorescent-Two Hybrid (F2H) assay for live-cell analysis of AR N/C interactions. F2H enables real-time visualization and quantitative analysis of interactions between GFP- and RFP- tagged proteins in live mammalian cells. The F2H principle employs a tethering strategy: a GFP-tagged protein is enriched at the protein interaction platform of engineered F2H-BHK cells and serves as bait, whereas an RFP-tagged protein serves as a prey. Here, AR-LBD (wt or mut) was fused to GFP, AR-TAD was fused to RFP, both constructs were co-transfected into the F2H cells, treated with modulatory compounds and the LBD/TAD interactions were evaluated by fluorescence microscopy. With F2H we analyzed the effects of DHT +/- flutamide, abiraterone, bicalutamide and enzalutamide in live cells. As expected, we observed striking differences in the effects of these treatments on the interactions between the AR-TAD and wt or mut AR-LBD (wt vs W741L, F876L, T877A, F876L-T877A). Further, we compared the effects of the selected compounds on AR-TAD and AR-LBD (wt vs mut) interactions in dose-response F2H assays. We also compared kinetics of these inhibitors with real-time imaging in F2H cells. We further validated our F2H findings with cell-lysate-ELISA on GFP-multiTrap plates. The acquired results correlate well with the published proliferation/reporter-assay data and clarify the role of the intramolecular AR N/C interactions in downstream effects. Taken together, our results demonstrate that the fully reversible cellular F2H assay enables side-by-side profiling of new inhibitors and activators of protein-protein interactions with respect to their intracellular activity, cell penetration and kinetics. Citation Format: Larisa Yurlova, Andrea Buchfellner, Jacqueline Gregor, Tina Romer, Ian Hickson. Live-cell profiling of inhibitors targeting the N/C interaction within the androgen receptor. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 86. doi:10.1158/1538-7445.AM2015-86