Selectivity Profiling of Bromodomain PROTACs Using Chemical Inducers of Proximity DNA-Encoded Library Screening.

Chemical Inducers of Proximity DNA-Encoded Library (CIP-DEL) screening enables high-throughput discovery of compounds that induce protein-protein interactions, including Proteolysis-Targeting Chimeras (PROTACs). Simultaneous screening of protein paralogs with CIP-DEL allows profiling of compound selectivity and efficient identification of paralog-selective degraders, but such an application has not been reported. Here, we optimized CIP-DEL screening conditions and conducted a von Hippel-Lindau (VHL)-biased CIP-DEL screen with two million DNA-barcoded PROTAC compounds on eight closely related Bromodomain and Extra Terminal domain (BET) bromodomains: BRD2 BD1, BRD2 BD2, BRD3 BD1, BRD3 BD2, BRD4 BD1, BRD4 BD2, BRDT BD1, and BRDT BD2. We observed a marked tendency of compounds to bind the first bromodomain (BD1) preferentially over the second bromodomain (BD2), which contrasts with the predominantly BD2-selective inhibitors reported in the literature. Specifically, our screening approach yielded compound , which demonstrated promising BRD2 BD1 selectivity in both sequencing data of DEL screening output and in vitro assays. Additionally, normalized relative enrichment selectivity from sequencing data rather than unnormalized absolute enrichment selectivity correlated more closely with experimentally validated selectivity. Overall, we highlight the value of CIP-DEL in profiling PROTAC selectivity, which should be applicable to other protein families with high sequence homologies, where selective degrader discovery remains challenging.