A novel coronavirus SARS-CoV-2, also called novel coronavirus 2019 (nCoV-19), 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 6.96% as of May 4, 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 M^pro inhibitors. A complex crystal structure of SARS-CoV-2 M^pro with GC-376, determined at 2.15 Å resolution with three monomers 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.

中文翻译：

---

Boceprevir，GC-376和钙蛋白酶抑制剂II，XII通过靶向病毒主要蛋白酶来抑制SARS-CoV-2病毒复制。

新型冠状病毒SARS-CoV-2也称为新型冠状病毒2019（nCoV-19），于2019年12月左右开始在人类中传播，如今已成为全球大流行病。由SARS-CoV-2病毒引起的疾病被称为COVID-19，它具有高度传染性，截至2020年5月4日的总死亡率为6.96％。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的酶测定法，鉴定出包括boceprevir，GC-376和钙蛋白酶抑制剂II和XII在内的几种抑制剂具有有效的活性，其单位数至亚微摩尔IC ₅₀酶法测定中的值。使用酶动力学研究，热移结合测定和天然质谱进一步表征命中的作用机理。值得注意的是，四种化合物（boceprevir，GC-376，钙蛋白酶抑制剂II和XII）在细胞培养物中抑制SARS-CoV-2病毒复制，其EC ₅₀值为0.49至3.37μM。值得注意的是，boceprevir，钙蛋白酶抑制剂II和XII代表了不同于已知的M ^pro抑制剂的新型化学型。SARS-CoV-2 M ^pro的复杂晶体结构GC-376的质谱以2.15Å的分辨率测定，每个不对称单元含3个单体，揭示了两种独特的结合构型，阐明了分子亲和力以及M ^pro与底物和抑制剂结合的蛋白质构象柔性。总体而言，本文鉴定的化合物为SARS-CoV-2治疗剂的进一步开发提供了有希望的起点。