The SARS-CoV-2 main protease (M^pro) is the drug target of Pfizer’s oral drug Paxlovid. The emergence of SARS-CoV-2 variants with mutations in M^pro raised the alarm of potential drug resistance. In this study, we identified 100 naturally occurring M^pro mutations located at the nirmatrelvir binding site, among which 20 mutants, including S144M/F/A/G/Y, M165T, E166G, H172Q/F, and Q192T/S/L/A/I/P/H/V/W/C/F, showed comparable enzymatic activity to the wild-type (k_cat/K_m <10-fold change) and resistance to nirmatrelvir (K_i >10-fold increase). X-ray crystal structures were determined for seven representative mutants with and/or without GC-376/nirmatrelvir. Viral growth assay showed that M^pro mutants with reduced enzymatic activity led to attenuated viral replication. Overall, our study identified several drug resistant hot spots that warrant close monitoring for possible clinical evidence of Paxlovid resistance.

中文翻译：

---

SARS-CoV-2 主要蛋白酶的自然发生突变赋予尼马瑞韦耐药性

SARS-CoV-2主蛋白酶（M ^pro）是辉瑞公司口服药物Paxlovid的药物靶点。^{带有 M pro}突变的 SARS-CoV-2 变体的出现引发了潜在耐药性的警报。在这项研究中，我们鉴定了 100 个天然存在的 M ^pro突变，位于 nirmatrelvir 结合位点，其中 20 个突变体，包括 S144M/F/A/G/Y、M165T、E166G、H172Q/F 和 Q192T/S/L/ A/I/P/H/V/W/C/F，显示出与野生型相当的酶活性（k _cat /K _m <10 倍变化）和对 nirmatrelvir 的抗性（K _i >10 倍增加） . 确定了具有和/或不具有 GC-376/nirmatrelvir 的七个代表性突变体的 X 射线晶体结构。病毒生长试验表明，M酶活性降低的^{前突变体导致病毒复制减弱。}总体而言，我们的研究确定了几个耐药热点，需要密切监测 Paxlovid 耐药的可能临床证据。