Development of an N-Terminal BRD4 Bromodomain-Targeted Degrader.

Targeted protein degradation is a powerful induced-proximity tool to control cellular protein concentrations using small molecules. However, the design of selective degraders remains empirical. Among bromodomain and extra-terminal (BET) family proteins, BRD4 is the primary therapeutic target over family members BRD2/3/T. Existing strategies for selective BRD4 degradation use pan-BET inhibitors optimized for BRD4:E3 ubiquitin ligase (E3) ternary complex formation, but these result in residual inhibition of undegraded BET-bromodomains by the pan-BET ligand, obscuring BRD4-degradation phenotypes. Using our selective inhibitor of the first BRD4 bromodomain, iBRD4-BD1 (IC50 = 12 nM, 23- to 6200-fold intra-BET selectivity), we developed dBRD4-BD1 to selectively degrade BRD4 (DC50 = 280 nM). Notably, dBRD4-BD1 upregulates BRD2/3, a result not observed with degraders using pan-BET ligands. Designing BRD4 selectivity up front enables analysis of BRD4 biology without wider BET-inhibition and simplifies designing BRD4-selective heterobifunctional molecules, such as degraders with new E3 recruiting ligands or for additional probes beyond degraders.

A Structure-based Design Approach for Generating High Affinity BRD4 D1-Selective Chemical Probes.

Chemical probes for epigenetic proteins are essential tools for dissecting the molecular mechanisms for gene regulation and therapeutic development. The bromodomain and extra-terminal (BET) proteins are master transcriptional regulators. Despite promising therapeutic targets, selective small molecule inhibitors for a single bromodomain remain an unmet goal due to their high sequence similarity. Here, we address this challenge via a structure-activity relationship study using 1,4,5-trisubstituted imidazoles against the BRD4 N-terminal bromodomain (D1). Leading compounds 26 and 30 have 15 and 18 nM affinity against BRD4 D1 and over 500-fold selectivity against BRD2 D1 and BRD4 D2 via ITC. Broader BET selectivity was confirmed by fluorescence anisotropy, thermal shift, and CETSA. Despite BRD4 engagement, BRD4 D1 inhibition was unable to reduce c-Myc expression at low concentration in multiple myeloma cells. Conversely, for inflammation, IL-8 and chemokine downregulation were observed. These results provide new design rules for selective inhibitors of an individual BET bromodomain.