Comprehensive fitness landscape of SARS-CoV-2 M(pro) reveals insights into viral resistance mechanisms.
Comprehensive fitness landscape of SARS-CoV-2 M(pro) reveals insights into viral resistance mechanisms.
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DOI：
10.7554/elife.77433
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发表时间：
2022-06-20
期刊：
影响因子：
7.7
通讯作者：
Bolon, Daniel N. A.
中科院分区：
文献类型：
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作者：
Flynn, Julia M.;Samant, Neha;Schneider-Nachum, Gily;Barkan, David T.;Yilmaz, Nese Kurt;Schiffer, Celia A.;Moquin, Stephanie A.;Dovala, Dustin;Bolon, Daniel N. A.
随着严重急性呼吸综合征冠状病毒-2 （SARS-CoV-2）新毒株的不断进化，这些毒株的毒性、传染性更强，并且能够逃避现有疫苗，迫切需要有效的抗病毒药物。SARS-CoV-2主蛋白酶（Mpro）因其在病毒生命周期中保守且不可或缺的作用而成为药物设计的主要靶点。靶向Mpro的药物看起来很有希望，但会引发耐药性的选择压力。为了了解Mpro的耐药潜力，我们对蛋白酶进行了全面的突变扫描，分析了所有可能的单氨基酸变化的功能。我们开发了三种单独的高通量测定酵母中Mpro功能的方法，基于Mpro变异体在特定切割位点的切割能力或其表达对酵母的毒性。我们使用深度测序来量化每个筛选中每个变体的功能影响。这三种筛选的蛋白质适合度景观具有很强的相关性，表明它们捕获了对Mpro功能至关重要的生物物理特性。适应度景观揭示了表面上一个对突变极其敏感的非活性位点位置，使其成为抑制剂的有利靶点。此外，我们发现了一个关键氨基酸网络，它在物理上连接了Mpro二聚体的两个活性位点。在我们的筛选中，Mpro的临床变异主要具有功能，这表明Mpro在人群中受到强大的选择压力。我们的研究结果提供了突变的预测，这些突变将很容易被Mpro进化所利用，并可能导致耐药性。这种完整的Mpro突变指南可用于设计具有降低病毒进化抗性潜力的抑制剂。
With the continual evolution of new strains of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) that are more virulent, transmissible, and able to evade current vaccines, there is an urgent need for effective anti-viral drugs. The SARS-CoV-2 main protease (Mpro) is a leading target for drug design due to its conserved and indispensable role in the viral life cycle. Drugs targeting Mpro appear promising but will elicit selection pressure for resistance. To understand resistance potential in Mpro, we performed a comprehensive mutational scan of the protease that analyzed the function of all possible single amino acid changes. We developed three separate high throughput assays of Mpro function in yeast, based on either the ability of Mpro variants to cleave at a defined cut-site or on the toxicity of their expression to yeast. We used deep sequencing to quantify the functional effects of each variant in each screen. The protein fitness landscapes from all three screens were strongly correlated, indicating that they captured the biophysical properties critical to Mpro function. The fitness landscapes revealed a non-active site location on the surface that is extremely sensitive to mutation, making it a favorable location to target with inhibitors. In addition, we found a network of critical amino acids that physically bridge the two active sites of the Mpro dimer. The clinical variants of Mpro were predominantly functional in our screens, indicating that Mpro is under strong selection pressure in the human population. Our results provide predictions of mutations that will be readily accessible to Mpro evolution and that are likely to contribute to drug resistance. This complete mutational guide of Mpro can be used in the design of inhibitors with reduced potential of evolving viral resistance.