Design and Characterization of Novel Covalent Bromodomain and Extra-Terminal Domain (BET) Inhibitors Targeting a Methionine

BET proteins are key epigenetic regulators
that regulate transcription
through binding to acetylated lysine (AcLys) residues of histones
and transcription factors through bromodomains (BDs). The disruption
of this interaction with small molecule bromodomain inhibitors is
a promising approach to treat various diseases including cancer, autoimmune
and cardiovascular diseases. Covalent inhibitors can potentially offer
a more durable target inhibition leading to improved in vivo pharmacology.
Here we describe the design of covalent inhibitors of BRD4­(BD1) that
target a methionine in the binding pocket by attaching an epoxide
warhead to a suitably oriented noncovalent inhibitor. Using thermal
denaturation, MALDI-TOF mass spectrometry, and an X-ray crystal structure,
we demonstrate that these inhibitors selectively form a covalent bond
with Met149 in BRD4­(BD1) but not other bromodomains and provide durable
transcriptional and antiproliferative activity in cell based assays.
Covalent targeting of methionine offers a novel approach to drug discovery
for BET proteins and other targets.

BET蛋白（BET proteins）是一类关键的表观遗传调控因子，它们通过溴结构域（bromodomains, BDs）结合组蛋白与转录因子的乙酰化赖氨酸（acetylated lysine, AcLys）残基，从而调控基因转录。利用小分子溴结构域抑制剂阻断此类蛋白与靶点的相互作用，是治疗癌症、自身免疫性疾病及心血管疾病等多种疾病的极具潜力的策略。共价抑制剂有望实现更持久的靶点抑制，进而优化体内药理学特性。本研究报道了BRD4(BD1)共价抑制剂的设计方案：通过将环氧弹头连接至定向合理的非共价抑制剂，靶向结合口袋内的甲硫氨酸残基。借助热变性实验、基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）以及X射线晶体结构分析，我们证实此类抑制剂可特异性与BRD4(BD1)中的Met149形成共价键，而不会与其他溴结构域发生反应，且在细胞实验中展现出持久的转录调控与抗增殖活性。针对甲硫氨酸的共价靶向策略，为BET蛋白及其他靶点的药物研发提供了全新的思路。