Numerous organic molecules are known to inhibit the main protease (M^Pro) of SARS-CoV-2, the pathogen of Coronavirus Disease 2019 (COVID-19). Guided by previous research on zinc-ligand inhibitors of M^Pro and zinc-dependent histone deacetylases (HDACs), we identified BRD4354 as a potent inhibitor of M^Pro. The in vitro protease activity assays show that BRD4354 displays time-dependent inhibition against M^Pro with an IC₅₀ (concentration that inhibits activity by 50%) of 0.72 ± 0.04 μM after 60 min of incubation. Inactivation follows a two-step process with an initial rapid binding step with a K_I of 1.9 ± 0.5 μM followed by a second slow inactivation step, k_inact,max of 0.040 ± 0.002 min^–1. Native mass spectrometry studies indicate that a covalent intermediate is formed where the ortho-quinone methide fragment of BRD4354 forms a covalent bond with the catalytic cysteine C145 of M^Pro. Based on these data, a Michael-addition reaction mechanism between M^Pro C145 and BRD4354 was proposed. These results suggest that both preclinical testing of BRD4354 and structure–activity relationship studies based on BRD4354 are warranted to develop more effective anti-COVID therapeutics.

中文翻译：

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BRD4354 是针对 SARS-CoV-2 主要蛋白酶的有效共价抑制剂

已知许多有机分子可抑制2019 年冠状病毒病 (COVID-19) 病原体 SARS-CoV-2 的主要蛋白酶 (M ^{Pro )。}在之前对 M ^{Pro锌配体抑制剂和锌依赖性组蛋白脱乙酰酶 (HDAC) 的研究的指导下，我们确定 BRD4354 是 M Pro}的有效抑制剂。体外蛋白酶活性测定表明，孵育 60 分钟后，BRD4354 对 M ^Pro表现出时间依赖性抑制， IC ₅₀（抑制活性 50% 的浓度）为 0.72 ± 0.04 μM。灭活过程分为两步，首先是K _I为 1.9 ± 0.5 μM 的快速结合步骤，然后是第二个缓慢灭活步骤，k _inact,max为 0.040 ± 0.002 min ^–1。天然质谱研究表明， BRD4354 的邻醌甲基化物片段与 M ^Pro的催化半胱氨酸 C145 形成共价键，形成共价中间体。基于这些数据，提出了M ^{Pro C145 和 BRD4354 之间的迈克尔加成反应机理。}这些结果表明，BRD4354 的临床前测试和基于 BRD4354 的结构-活性关系研究有必要开发更有效的抗 COVID 疗法。