A yeast-based system to study SARS-CoV-2 M pro structure and to identify nirmatrelvir resistant mutations.
A yeast-based system to study SARS-CoV-2 M pro structure and to identify nirmatrelvir resistant mutations.
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基于酵母的系统，用于研究 SARS-CoV-2 M 原结构并识别 nirmatrelvir 耐药突变。
DOI：
10.1101/2022.08.06.503039
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发表时间：
2022
期刊：
影响因子：
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通讯作者：
Choy,JohnS
中科院分区：
文献类型：
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作者：
Ou,Jin;Lewandowski,EricM;Hu,Yanmei;Lipinski,AustinA;Morgan,RyanT;Jacobs,LianMC;Zhang,Xiujun;Bikowitz,MelissaJ;Langlais,Paul;Tan,Haozhou;Wang,Jun;Chen,Yu;Choy,JohnS
SARS-CoV-2 主要蛋白酶 (Mpro) 是主要的治疗靶点。 Mproinhibitor，nirmatrelvir，是 Paxlovid 的抗病毒成分，Paxlovid 是一种治疗 COVID-19 的口服药物。随着 Mproinhibitor 使用的增加，耐药突变可能会出现。我们建立了一个非致病性系统，其中酵母生长可作为 Mproactivity 的近似值，从而能够快速识别酶活性和药物敏感性改变的突变体。众所周知，E166 残基是耐药性的潜在热点，酵母分析确定了赋予尼马曲韦强耐药性的替代和其他损害活性的替代。另一方面，Omicron 变体携带的 N142A 和 P132H 突变对药物反应和活性几乎没有影响。标准酶测定证实了酵母结果。反过来，我们解析了 MproE166R 和 MproE166N 的结构，从而深入了解精氨酸如何驱动耐药性，而天冬酰胺如何导致活性降低。这里介绍的工作将有助于表征 Mpro 的新型耐药变体，随着 Mpro 抗病毒药物的更广泛使用，可能会出现这种变体。
The SARS-CoV-2 main protease (Mpro) is a major therapeutic target. The Mproinhibitor, nirmatrelvir, is the antiviral component of Paxlovid, an orally available treatment for COVID-19. As Mproinhibitor use increases, drug resistant mutations will likely emerge. We have established a non-pathogenic system, in which yeast growth serves as an approximation for Mproactivity, enabling rapid identification of mutants with altered enzymatic activity and drug sensitivity. The E166 residue is known to be a potential hot spot for drug resistance and yeast assays identified substitutions which conferred strong nirmatrelvir resistance and others that compromised activity. On the other hand, N142A and the P132H mutation, carried by the Omicron variant, caused little to no change in drug response and activity. Standard enzymatic assays confirmed the yeast results. In turn, we solved the structures of MproE166R, and MproE166N, providing insights into how arginine may drive drug resistance while asparagine leads to reduced activity. The work presented here will help characterize novel resistant variants of Mprothat may arise as Mproantivirals become more widely used.