Dynamic monitoring of STAT3 activation in live cells using a novel STAT3 Phospho-BRET sensor.

Phosphorylation (pY705) mediated homodimerization is a rate-limiting step controlling STAT3 key oncogenic functions making it an attractive target for drug discovery. Hence, this study reports development of a sensitive and versatile STAT3 Phospho-BRET biosensor platform technology to monitor activation dynamics of STAT3 signalling directly from live cells. Categorically, we first demonstrate that NanoLuc donor and TurboFP635 acceptor serves as an excellent BRET system over other tested fluorophores like mOrange and TagRFP, both for live cells as well as in vivo optical imaging of protein-protein interactions. Based on initial multi-parametric optimizations, our Phospho-BRET sensor developed by fusing STAT3 with NanoLuc and TurboFP at the C-terminus, successfully captured the activation kinetics of STAT3 in response to different ligands (e.g. IL6 & EGF) and across multiple cancer cell types either with or without the endogenous STAT3 pool. Perturbation in EGF-mediated STAT3 BRET activation signal upon blocking with EGFR neutralizing antibody further confirms the specificity of the sensor to judge ligand-receptor pathway dependent STAT3 activation. Finally, we determine the high-throughput compatibility of the developed biosensor by testing a few known/unknown STAT3 inhibitors in a 96- and 384-well plate format. The results from this screen revealed that drug molecules such as curcumin and niclosamide are more efficient inhibitors over known molecule like Stattic. Thus, the STAT3 Phospho-BRET sensor is a first of its kind live cell platform technology developed for its use to study STAT3 pathway dynamics and screen potential drug molecules in vivo. AJNMMI