Development and Cellular Evaluation of Selective N-Terminal BET Bromodomain Inhibitors

Abstract

As regulators of transcription, proteins that interpret post-translational modifications to N-terminal histone tails are essential for maintaining cellular homeostasis. When dysregulated, these ‘reader’ proteins become drivers of disease. In the case of bromodomains, which recognize N-acetylated-lysine, developing domain selective inhibitors has been a significant challenge to medicinal chemists. However, recent development of inhibitors with domain-selectivity within the Bromodomain and Extra Terminal (BET) family of bromodomains suggest the tandem BET bromodomains, BD1 and BD2, play differential roles in regulating gene expression. We identified tri-substituted imidazole-based inhibitors that are > 50-fold selective for the N-terminal bromodomains of BET proteins (BET-BD1) and display an unusual mode of domain selectivity via the displacement of conserved structured waters. We subsequently described a structure-based design approach to eliminate off-target p38a kinase affinity, while improving BET selectivity and affinity for BD1 of the bromodomain-containing protein 4 (BRD4). Using these molecules, we observe differential transcriptional effects relative to pan-BD1 inhibitors. Whereas BRD4-BD1 inhibition can reduce the expression of inflammatory cytokines, a weak effect on MYC super-enhancer regulation is only observed at concentrations when both BD1 and BD2 bromodomains are inhibited. Using our structural insights, new chemical-biology strategies were developed to study the role of BRD4 functional modulation through selective bromodomain targeting. Existing strategies to selectively target BRD4 rely on the use of pan-BET inhibitors. In the case of selective BRD4 degraders, pan-BET inhibitors are optimized for BRD4:protein-ubiquitin ligase (E3) ternary complex formation. Here, we present and validate a strategy for selectively targeting BRD4 through bivalent inhibition of both bromodomains, as well as a strategy to degrade BRD4 through its N-terminal bromodomain. Based on our novel BRD4-BD1 selective inhibitors, our unoptimized degrader dBRD4-BD1 induces BRD4 degradation at a half-maximal degradation concentration (DC50) of 0.28 μM, and results in the upregulation of BRD2 and 3. The design of selectivity upfront enables the study of BRD4 biology in the absence of wider BET-inhibition and simplifies design of future BRD4-selective degraders as new E3 recruiting ligands are discovered. Together, these approaches highlight the value of chemical probes that selectively target the unique functions of epigenetic reader domains.