Fragment-Based Discovery of BRD4-BD2 Selective Binders: Biophysical Validation and Structural Insights for Domain-Selective Inhibitor Design.

BRD4, a member of the bromodomain and extra-terminal (BET) family of epigenetic readers, plays a central role in transcriptional regulation and is implicated in a wide spectrum of diseases. Although BRD4 inhibitors targeting both bromodomains have advanced into clinical studies, their development and therapeutic use are limited by dose-limiting toxicities associated with nonselective BET inhibition. These limitations underscore the need for domain-selective targeting strategies. In this study, we performed fragment-based screening of a 200-compound diversity library against BRD4-BD2, identifying three chemically distinct hits: CCBT-T-226 (methyl 2-(2-(3,5-dimethylisoxazol-4-yl)-acetamido)-5-propylthiazole-4-carboxylate), CCBT-F-70 (2-(benzylamino)-pyrimidin-4-(3H)-one), and CCBT-F-182 (3-(8-methyl-3,8a-dihydroimidazo-[1,2-a]-pyridin-2-yl)-aniline). These fragments were validated as binders using TSA, ITC, STD-NMR, and 1H-15N HSQC NMR. Among them, CCBT-T-226 and CCBT-F-182 exhibited measurable BD2-over-BD1 selectivity, with CCBT-F-182 showing a pronounced domain preference, and CCBT-T-226 demonstrating approximately 7-fold selectivity for BD2 over BD1. X-ray crystallography of the BRD4-BD2-CCBT-F-70 complex, together with cofolding models for CCBT-T-226 and CCBT-F-182, provides a structural framework for the rational, structure-guided development of potent and selective BRD4-BD2 inhibitors.