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Naturally occurring mutations of SARS-CoV-2 main protease confer drug resistance to nirmatrelvir

Yanmei Hu, Eric M. Lewandowski, Haozhou Tan, Ryan T. Morgan, Xiujun Zhang, Lian M. C. Jacobs, Shane G. Butler, Maura V. Mongora, John Choy, Yu Chen, View ORCID ProfileJun Wang
doi: https://doi.org/10.1101/2022.06.28.497978
Yanmei Hu
1Department of Medicinal Chemistry, Ernest Mario School of Pharmacy, Rutgers, the State University of New Jersey, NJ, 08854, United States
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Eric M. Lewandowski
2Department of Molecular Medicine, Morsani College of Medicine, University of South Florida, Tampa, FL, 33612, United States
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Haozhou Tan
1Department of Medicinal Chemistry, Ernest Mario School of Pharmacy, Rutgers, the State University of New Jersey, NJ, 08854, United States
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Ryan T. Morgan
2Department of Molecular Medicine, Morsani College of Medicine, University of South Florida, Tampa, FL, 33612, United States
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Xiujun Zhang
2Department of Molecular Medicine, Morsani College of Medicine, University of South Florida, Tampa, FL, 33612, United States
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Lian M. C. Jacobs
2Department of Molecular Medicine, Morsani College of Medicine, University of South Florida, Tampa, FL, 33612, United States
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Shane G. Butler
2Department of Molecular Medicine, Morsani College of Medicine, University of South Florida, Tampa, FL, 33612, United States
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Maura V. Mongora
2Department of Molecular Medicine, Morsani College of Medicine, University of South Florida, Tampa, FL, 33612, United States
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John Choy
3Department Biology, School of Arts and Sciences, the Catholic University of America, Washington DC, 20064, United States
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Yu Chen
2Department of Molecular Medicine, Morsani College of Medicine, University of South Florida, Tampa, FL, 33612, United States
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  • For correspondence: junwang@pharmacy.rutgers.edu ychen1@usf.edu
Jun Wang
1Department of Medicinal Chemistry, Ernest Mario School of Pharmacy, Rutgers, the State University of New Jersey, NJ, 08854, United States
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  • ORCID record for Jun Wang
  • For correspondence: junwang@pharmacy.rutgers.edu ychen1@usf.edu
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ABSTRACT

The SARS-CoV-2 main protease (Mpro) is a cysteine protease and a validated antiviral drug target. Paxlovid is an FDA-approved oral COVID-19 antiviral that contains the Mpro inhibitor nirmatrelvir and the metabolic booster ritonavir. The emergence of SARS-CoV-2 variants mutations in the Mpro raised the alarm of potential drug resistance. In this study, we aim to discover Mpro drug resistant mutants from naturally observed polymorphisms. Through analyzing the SARS-CoV-2 sequences deposited in Global initiative on Sharing Avian influenza Data (GISAID) database, we identified 66 prevalent Mpro mutations located at the nirmatrelvir binding site. The Mpro mutant proteins were expressed and characterized for enzymatic activity and nirmatrelvir inhibition. While the majority of the Mpro mutants had reduced enzymatic activity (kcat/Km >10-fold decrease), 11 mutants including S144M/F/A/G/Y, M165T, E166Q, H172Q/F, and Q192T/S/V showed comparable enzymatic activity as the wild-type (kcat/Km <10-fold change) and resistance to nirmatrelvir (Ki > 10-fold increase). We further demonstrate that the enzymatic activity and inhibitor resistance of these single mutations can be enhanced by additional substitutions in a double mutant. X-ray crystal structures were determined for six of the single mutants with and/or without GC-376/nirmatrelvir. The structures illustrate how mutations can reduce ligand binding by impacting the conformational stability of the active site. Overall, our study identified several drug resistant hot spots that warrant close monitoring for possible clinical evidence of Paxlovid resistance.

Competing Interest Statement

The authors have declared no competing interest.

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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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Naturally occurring mutations of SARS-CoV-2 main protease confer drug resistance to nirmatrelvir
Yanmei Hu, Eric M. Lewandowski, Haozhou Tan, Ryan T. Morgan, Xiujun Zhang, Lian M. C. Jacobs, Shane G. Butler, Maura V. Mongora, John Choy, Yu Chen, Jun Wang
bioRxiv 2022.06.28.497978; doi: https://doi.org/10.1101/2022.06.28.497978
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Naturally occurring mutations of SARS-CoV-2 main protease confer drug resistance to nirmatrelvir
Yanmei Hu, Eric M. Lewandowski, Haozhou Tan, Ryan T. Morgan, Xiujun Zhang, Lian M. C. Jacobs, Shane G. Butler, Maura V. Mongora, John Choy, Yu Chen, Jun Wang
bioRxiv 2022.06.28.497978; doi: https://doi.org/10.1101/2022.06.28.497978

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