Dynamic Detection\nof the E3-PROTAC-Target Protein\nTernary Complex <i>In\nVitro</i> and <i>In Vivo</i> <i>via</i> Bimolecular\nFluorescence Complementation

Proteolysis-targeting chimeras (PROTACs) have played\nan important\nrole in the development of protein-targeted degradation drugs. However,\neffective tools are urgently required for the further development\nand validation of PROTACs. We developed a high-potency reporter (AKT-PROTAC-Reporter;\nAPR) for PROTACs that specifically targets AKT. The APR successfully\ndetected the status and levels of the AKT-PROTAC-CRBN ternary complex <i>in vivo</i> and <i>in vitro</i>. The APR is based\non a bimolecular fluorescence complementation system, where EGFP and\nluciferase were used as reporter signals for <i>in vitro</i> and <i>in vivo</i> experiments, respectively, with remarkable\nsuccess. The absence of E3 ligase ubiquitin recruitment activity in\nthe APR can significantly improve the reporting performance of the\nAPR; however, this results in difficulties in the detection of the\ndegradation efficiency of PROTAC target proteins. Our results show\nthat the APR can sensitively, quickly, and effectively detect the\npresence of terpolymers. Furthermore, the APR can determine the specificity\nand degradation efficiency of the PROTAC <i>via</i> a fluorescence\nsignal or bioluminescence signal intensity and can efficiently screen\nPROTACs for a certain target protein.