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一个基于酵母的系统,用于研究SARS-CoV-2 Mpro结构,并识别耐尼马特里尔维尔抗性突变

Jin Ou¹, Eric M Lewandowski², Yanmei Hu³

¹Department of Biology, School of Arts and Sciences, The Catholic University of America, Washington, Washington DC, United States of America.

|August 31, 2023

概括

耐药SARS-CoV-2变种对COVID-19治疗构成威胁. 酵母测定主要蛋白酶 (Mpro) 中快速识别的突变,使其对尼尔马特雷尔维尔产生耐药性,有助于描述潜在的抗病毒耐药性.

科学领域:

病毒学病毒学
药物发现药物发现药物发现
生物化学生物化学

背景情况:

SARS-CoV-2 主蛋白酶 (Mpro) 是尼尔马特里尔维尔 (Paxlovid) 等抗病毒疗法的关键标.
在Mpro中出现耐药突变是对持续的COVID-19治疗疗效的重大关注.
需要一个强大的系统来快速选具有药物敏感性和酶活性变化的Mpro变体.

研究的目的:

开发和验证一种基于酵母的系统,用于识别具有改变尼尔马特里尔维尔敏感性的SARS-CoV-2 Mpro突变.
描述特定的Mpro突变,包括在关注变异中发现的Mpro突变,对药物反应和蛋白酶活性的影响.
为Mpro药物耐药性和活性降低的机制提供结构性见解.

主要方法:

利用非致病性酵母生长系统作为SARS-CoV-2 Mpro活性和药物敏感性的代理.
选了Mpro突变,表现出改变的酶活性和对Mpro抑制剂耐药性的Mpro突变.
使用标准生化酶定量测试证实了这些发现.
确定了关键Mpro突变 (E166R,E166N) 的晶体结构,以阐明抗性机制.

主要成果:

酵母测定系统成功识别了Mpro突变,使其具有显著的耐尼马特里尔维尔耐药性 (例如,E166R) 和减少的蛋白酶活性 (例如,E166N).
在Omicron变异中存在的N142A和P132H突变对尼尔马特里尔维尔活性和Mpro功能的影响很小.
生物化学分析验证了从酵母查中获得的结果.
结构分析揭示了E166残留物的特定替代如何影响药物结合和蛋白酶功能.

结论:

开发的酵母系统提供了一个快速有效的平台,用于识别具有潜在耐药性的SARS-CoV-2 Mpro变体.
了解E166R和E166N等突变的影响对于预测和打击抗病毒耐药性至关重要.
结构数据为Mpro-nirmatrelvir相互作用和耐药性途径提供了有价值的机制性见解.
这项研究将有助于持续对新出现的Mpro变体进行表征,因为针对Mpro的抗病毒药物得到了更广泛的临床应用.

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