Abstract

An assay platform that robustly and sensitively quantifies the kinetics of endogenous protein turnover is crucial for discovery of disease-relevant therapeutic agents. This need is particularly relevant for a new class oftherapeutics known as protein degraders, such as PROTACs that target specific disease-relevant proteins for degradation by the cellular ubiquitin-proteasome system. Using CRISPR technology and the well-established Enzyme Fragment Complementation system, we introduced a small β-galactosidase fragment into the BRD4 and c-Myc loci in physiologically relevant disease cell models. The homogeneous format and high sensitivity of the EFC assay allows for direct and rapid quantitation of drug-induced changes in endogenous BRD4 and c-Myc protein levels. We tested a panel of PROTACs targeting BRD4 in this system and observed differential kinetics for BRD4 and c-Myc degradation with individual PROTACs that were consistent with previous reports using cellproliferation assays. This suggests that discovery of new molecular entities that modulate the endogenous levels of these proteins is feasible using this assay format. We are currently expanding this cell-based platform, SPRINTer™ protein turnover biosensor assays, to additional protein targets and disease cell models where sensitive detection of endogenous protein modulation is critical.