基于细胞内生物发光共振能量转移(BRET)的检测发现 Ceritinib 和 CA-074 是 SARS-CoV-2 类木瓜蛋白酶抑制剂。

IF 5.6 2区 医学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY
Mei Li, Zhu-Chun Bei, Yongtian Yuan, Baogang Wang, Dongna Zhang, Likun Xu, Liangliang Zhao, Qin Xu, Yabin Song
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引用次数: 0

摘要

木瓜蛋白酶(Papain-like protease,PLpro)是一种极具吸引力的抗冠状病毒靶标。然而,现有 PLpro 检测方法的局限性和有效活性化合物的稀缺性阻碍了 PLpro 抑制剂的开发。我们开发了一种基于 BRET 的新型细胞内 PLpro 检测方法,并用它来评估和发现 SARS-CoV-2 PLpro 抑制剂。所开发的检测方法在检测细胞内 PLpro 活性降低方面表现出了极高的灵敏度,同时在抑制剂评估和高通量筛选方面也具有极高的可靠性和性能。利用这种检测方法,三种蛋白酶抑制剂被鉴定为新型 PLpro 抑制剂,它们在结构上与以前已知的抑制剂不同。随后进行的酶测定和基于分子对接的配体-蛋白质相互作用分析表明,塞瑞替尼可直接抑制PLpro,与PLpro中的底物结合口袋显示出高度的几何互补性,而CA-074甲酯则会发生细胞内水解,暴露出一个游离的羧羟基,该羧羟基对于与BL2槽中的G266发生氢键作用至关重要,从而导致PLpro受到抑制。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
In-cell bioluminescence resonance energy transfer (BRET)-based assay uncovers ceritinib and CA-074 as SARS-CoV-2 papain-like protease inhibitors.

Papain-like protease (PLpro) is an attractive anti-coronavirus target. The development of PLpro inhibitors, however, is hampered by the limitations of the existing PLpro assay and the scarcity of validated active compounds. We developed a novel in-cell PLpro assay based on BRET and used it to evaluate and discover SARS-CoV-2 PLpro inhibitors. The developed assay demonstrated remarkable sensitivity for detecting the reduction of intracellular PLpro activity while presenting high reliability and performance for inhibitor evaluation and high-throughput screening. Using this assay, three protease inhibitors were identified as novel PLpro inhibitors that are structurally disparate from those previously known. Subsequent enzymatic assays and ligand-protein interaction analysis based on molecular docking revealed that ceritinib directly inhibited PLpro, showing high geometric complementarity with the substrate-binding pocket in PLpro, whereas CA-074 methyl ester underwent intracellular hydrolysis, exposing a free carboxyhydroxyl group essential for hydrogen bonding with G266 in the BL2 groove, resulting in PLpro inhibition.

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来源期刊
CiteScore
10.30
自引率
10.70%
发文量
195
审稿时长
4-8 weeks
期刊介绍: Journal of Enzyme Inhibition and Medicinal Chemistry publishes open access research on enzyme inhibitors, inhibitory processes, and agonist/antagonist receptor interactions in the development of medicinal and anti-cancer agents. Journal of Enzyme Inhibition and Medicinal Chemistry aims to provide an international and interdisciplinary platform for the latest findings in enzyme inhibition research. The journal’s focus includes current developments in: Enzymology; Cell biology; Chemical biology; Microbiology; Physiology; Pharmacology leading to drug design; Molecular recognition processes; Distribution and metabolism of biologically active compounds.
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