In Situ Monitoring PROTAC Molecule-Induced Target Protein Degradation Process in Living Cells by Multi-channel Fluorescence Cross-Correlation Spectroscopy

Abstract Proteolysis-targeting chimeras (PROTACs) are a novel technique for degrading proteins of interest (POIs) by the cellular protein ubiquitination degradation pathway, and this technique has emerged as an innovative strategy for targeting undruggable proteins. Despite significant progress in PROTAC technology, a major challenge remains in the in situ acquisition of concentration-dependent equilibrium profiles and kinetic information on the interaction between POIs and PROTAC molecules and the POI degradation kinetics within living cells. Here, we proposed a novel method for in situ monitoring PROTAC-induced target protein degradation by combining multi-channel fluorescence cross-correlation spectroscopy (FCCS) and protein fusion technology. In this study, ABL1 was used as the target protein. We designed three plasmids encoding fluorescent fusion proteins: a fusion protein comprising SNAP, mCherry, and ABL1; a fusion protein comprising eGFP and E3 ligase VHL; and a fusion protein comprising Halo and ubiquitin. We developed a multi-channel FCCS system and in situ studied the concentration-dependent equilibrium profiles and kinetics of ternary complex formation among PROTACs, ABL1, and VHL; the ubiquitination of ABL1; and PROTAC-induced degradation of ABL1. Furthermore, we propose a new approach for evaluating the efficacy of PROTAC molecules on the basis of the degradation processes of the target protein. The results demonstrated that our method can in real time monitor the formation of the PROTAC-induced ternary complex, the ubiquitination process, and the target protein degradation in living cells; evaluate the efficacy of PROTAC drugs; and will become an efficient platform for studying the pharmacodynamics of PROTAC drugs and developing new PROTAC drugs.