Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA, USA.
Huck Institutes of the Life Sciences, The Pennsylvania State University, University Park, PA, USA.
Commun Biol. 2022 Feb 25;5(1):169. doi: 10.1038/s42003-022-03090-9.
SARS-CoV-2 proteases Mpro and PLpro are promising targets for antiviral drug development. In this study, we present an antiviral screening strategy involving a novel in-cell protease assay, antiviral and biochemical activity assessments, as well as structural determinations for rapid identification of protease inhibitors with low cytotoxicity. We identified eight compounds with anti-SARS-CoV-2 activity from a library of 64 repurposed drugs and modeled at protease active sites by in silico docking. We demonstrate that Sitagliptin and Daclatasvir inhibit PLpro, and MG-101, Lycorine HCl, and Nelfinavir mesylate inhibit Mpro of SARS-CoV-2. The X-ray crystal structure of Mpro in complex with MG-101 shows a covalent bond formation between the inhibitor and the active site Cys145 residue indicating its mechanism of inhibition is by blocking the substrate binding at the active site. Thus, we provide methods for rapid and effective screening and development of inhibitors for blocking virus polyprotein processing as SARS-CoV-2 antivirals. Additionally, we show that the combined inhibition of Mpro and PLpro is more effective in inhibiting SARS-CoV-2 and the delta variant.
SARS-CoV-2 的蛋白酶 Mpro 和 PLpro 是抗病毒药物开发的有前途的靶标。在这项研究中，我们提出了一种抗病毒筛选策略，涉及新型细胞内蛋白酶测定法、抗病毒和生化活性评估以及结构测定，以快速鉴定具有低细胞毒性的蛋白酶抑制剂。我们从 64 种重新利用药物的库中鉴定出 8 种具有抗 SARS-CoV-2 活性的化合物，并通过计算机对接模拟在蛋白酶活性部位。我们证明西他列汀和达卡他韦抑制 PLpro，MG-101、盐酸石蒜碱和奈非那韦甲磺酸盐抑制 SARS-CoV-2 的 Mpro。与 MG-101 结合的 Mpro 的 X 射线晶体结构显示抑制剂与活性位点 Cys145 残基之间形成共价键，表明其抑制机制是通过阻断底物在活性位点的结合。因此，我们提供了快速有效筛选和开发抑制剂的方法，以阻断病毒多蛋白加工作为 SARS-CoV-2 抗病毒药物。此外，我们表明 Mpro 和 PLpro 的联合抑制在抑制 SARS-CoV-2 和 delta 变体方面更有效。
