Dynamic Detection of the E3-PROTAC-Target Protein Ternary Complex In Vitro and In Vivo via Bimolecular Fluorescence...

Proteolysis-targeting chimeras (PROTACs) have played an important role in the development of protein-targeted degradation drugs. However, effective tools are urgently required for the further development and validation of PROTACs. We developed a high-potency reporter (AKT-PROTAC-Reporter; APR) for PROTACs that specifically targets AKT. The APR successfully detected the status and levels of the AKT-PROTAC-CRBN ternary complex in vivo and in vitro. The APR is based on a bimolecular fluorescence complementation system, where EGFP and luciferase were used as reporter signals for in vitro and in vivo experiments, respectively, with remarkable success. The absence of E3 ligase ubiquitin recruitment activity in the APR can significantly improve the reporting performance of the APR; however, this results in difficulties in the detection of the degradation efficiency of PROTAC target proteins. Our results show that the APR can sensitively, quickly, and effectively detect the presence of terpolymers. Furthermore, the APR can determine the specificity and degradation efficiency of the PROTAC via a fluorescence signal or bioluminescence signal intensity and can efficiently screen PROTACs for a certain target protein.