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Comprehensive fitness landscape of SARS-CoV-2 Mpro reveals insights into viral resistance mechanisms

Julia M. Flynn, Neha Samant, Gily Schneider-Nachum, David T. Barkan, Nese Kurt Yilmaz, Celia A. Schiffer, Stephanie A. Moquin, Dustin Dovala, Daniel N.A. Bolon
doi: https://doi.org/10.1101/2022.01.26.477860
Julia M. Flynn
1Department of Biochemistry and Molecular Biotechnology, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA
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  • For correspondence: Julia.Flynn@umassmed.edu Dan.Bolon@umassmed.edu
Neha Samant
1Department of Biochemistry and Molecular Biotechnology, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA
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Gily Schneider-Nachum
1Department of Biochemistry and Molecular Biotechnology, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA
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David T. Barkan
2Novartis Institutes for Biomedical Research, Emeryville, CA 94608, USA
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Nese Kurt Yilmaz
1Department of Biochemistry and Molecular Biotechnology, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA
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Celia A. Schiffer
1Department of Biochemistry and Molecular Biotechnology, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA
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Stephanie A. Moquin
2Novartis Institutes for Biomedical Research, Emeryville, CA 94608, USA
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Dustin Dovala
2Novartis Institutes for Biomedical Research, Emeryville, CA 94608, USA
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Daniel N.A. Bolon
1Department of Biochemistry and Molecular Biotechnology, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA
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  • For correspondence: Julia.Flynn@umassmed.edu Dan.Bolon@umassmed.edu
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Abstract

With the continual evolution of new strains of SARS-CoV-2 that are more virulent, transmissible, and able to evade current vaccines, there is an urgent need for effective anti-viral drugs. 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.

Competing Interest Statement

DTB, SAM, and DD are employees of Novartis Institutes for Biomedical Research

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC 4.0 International license.
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Posted January 30, 2022.
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Comprehensive fitness landscape of SARS-CoV-2 Mpro reveals insights into viral resistance mechanisms
Julia M. Flynn, Neha Samant, Gily Schneider-Nachum, David T. Barkan, Nese Kurt Yilmaz, Celia A. Schiffer, Stephanie A. Moquin, Dustin Dovala, Daniel N.A. Bolon
bioRxiv 2022.01.26.477860; doi: https://doi.org/10.1101/2022.01.26.477860
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Comprehensive fitness landscape of SARS-CoV-2 Mpro reveals insights into viral resistance mechanisms
Julia M. Flynn, Neha Samant, Gily Schneider-Nachum, David T. Barkan, Nese Kurt Yilmaz, Celia A. Schiffer, Stephanie A. Moquin, Dustin Dovala, Daniel N.A. Bolon
bioRxiv 2022.01.26.477860; doi: https://doi.org/10.1101/2022.01.26.477860

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