A new coronavirus SARS-CoV-2, also called novel coronavirus 2019 (2019-nCoV), started to circulate among humans around December 2019, and it is now widespread as a global pandemic. The disease caused by SARS-CoV-2 virus is called COVID-19, which is highly contagious and has an overall mortality rate of 6.35% as of May 26, 2020. There is no vaccine or antiviral available for SARS-CoV-2. In this study, we report our discovery of inhibitors targeting the SARS-CoV-2 main protease (M^pro). Using the FRET-based enzymatic assay, several inhibitors including boceprevir, GC-376, and calpain inhibitors II, and XII were identified to have potent activity with single-digit to submicromolar IC₅₀ values in the enzymatic assay. The mechanism of action of the hits was further characterized using enzyme kinetic studies, thermal shift binding assays, and native mass spectrometry. Significantly, four compounds (boceprevir, GC-376, calpain inhibitors II and XII) inhibit SARS-CoV-2 viral replication in cell culture with EC₅₀ values ranging from 0.49 to 3.37 µM. Notably, boceprevir, calpain inhibitors II and XII represent novel chemotypes that are distinct from known substrate-based peptidomimetic M^pro inhibitors. A complex crystal structure of SARS-CoV-2 M^pro with GC-376, determined at 2.15 Å resolution with three protomers per asymmetric unit, revealed two unique binding configurations, shedding light on the molecular interactions and protein conformational flexibility underlying substrate and inhibitor binding by M^pro. Overall, the compounds identified herein provide promising starting points for the further development of SARS-CoV-2 therapeutics.

一种新型冠状病毒 SARS-CoV-2，也称为新型冠状病毒 2019 (2019-nCoV)，于 2019 年 12 月左右开始在人类中传播，目前已成为全球大流行病。由 SARS-CoV-2 病毒引起的疾病称为 COVID-19，具有高度传染性，截至 2020 年 5 月 26 日，总体死亡率为 6.35%。目前还没有针对 SARS-CoV-2 的疫苗或抗病毒药物。在这项研究中，我们报告了针对 SARS-CoV-2 主要蛋白酶 (M ^pro ) 的抑制剂的发现。使用基于 FRET 的酶测定，包括 boceprevir、GC-376 和钙蛋白酶抑制剂 II 和 XII 在内的几种抑制剂被鉴定为具有有效活性，在酶测定中IC ₅₀值为个位数至亚微摩尔级。使用酶动力学研究、热位移结合测定和天然质谱进一步表征了命中的作用机制。值得注意的是，四种化合物（boceprevir、GC-376、钙蛋白酶抑制剂 II 和 XII）可抑制细胞培养物中的 SARS-CoV-2 病毒复制，EC 50 值范围为 0.49 至 3.37 _µM。^{值得注意的是，波普瑞韦、钙蛋白酶抑制剂 II 和 XII 代表了与已知的基于底物的拟肽 M pro}抑制剂不同的新型化学型。以 2.15 Å 分辨率测定每个不对称单元三个原聚体的 SARS-CoV-2 M pro 与 GC-376 的复杂晶体结构，揭示了两种独特的结合构型，揭示了底物和抑制剂结合背后的分子相互作用和蛋白质构^象灵活性由 M^亲。总体而言，本文鉴定的化合物为 SARS-CoV-2 疗法的进一步开发提供了有希望的起点。