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）是关键的表观遗传调控因子，可通过溴结构域（bromodomains, BDs）结合组蛋白与转录因子的乙酰化赖氨酸（acetylated lysine, AcLys）残基来调控基因转录。通过小分子溴结构域抑制剂破坏此类相互作用，是治疗癌症、自身免疫性疾病及心血管疾病等多种病症的极具前景的策略。共价抑制剂或可实现更持久的靶点抑制，进而优化体内药理学表现。本文报道了针对BRD4(BD1)的共价抑制剂设计方案：将环氧弹头连接至定向合适的非共价抑制剂，以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 mass spectrometry）与X射线晶体结构分析，我们证实此类抑制剂可与BRD4(BD1)中的Met149特异性形成共价键，而不会结合其他溴结构域；且在细胞水平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路径。