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Functional and structural characterization of treatment-emergent nirmatrelvir resistance mutations at low frequencies in the main protease (Mpro) reveals a unique evolutionary route for SARS-CoV-2 to gain resistance

Deschenes et al., The Journal of Infectious Diseases, doi:10.1093/infdis/jiaf294, Jun 2025
https://c19early.org/deschenes.html
In Vitro and In Silico study showing that novel SARS-CoV-2 main protease (Mpro) mutations confer resistance to paxlovid. Authors identified new Mpro clinical variants, including D48D/L58F/P132H and D48D/L67V/K90R/P132H, in patients who remained SARS-CoV-2 positive after paxlovid treatment. Experiments in Caco-2 cells revealed these mutations, distant from the Mpro active site, led to significant nirmatrelvir and C5a resistance while retaining the enzyme's activity.
Deschenes et al., 3 Jun 2025, Canada, peer-reviewed, 25 authors, study period February 2022 - July 2023. Contact: rkozak@shn.ca, fjean@mail.ubc.ca.
In Vitro studies are an important part of preclinical research, however results may be very different in vivo.
Abstract: The Journal of Infectious Diseases MAJOR ARTICLE SC RI PT A N U Natalie M. Deschenes1* , Jimena Pérez-Vargas2* , Zoe Zhong3 , Merrilee Thomas4 , Calem Kenward5 , Wesley A. Mosimann5 , Liam J. Worrall5 , Nicholas Waglechner6 , Angel XinLiu Li3 , Finlay Maguire6,7,8 , Patryk Aftanas6 , Jason R. Smith9 , Jared Lim11 , Robert N. Young9 , Artem Cherkasov10 , Lubna Farooqi3 , Adnan Moinuddin3 , Lina Siddiqi3 , Imaan Malik3 , Maxime Lefebvre3 , Mark Paetzel11 , Natalie C.J. Strynadka5 , François Jean2*# , Allison McGeer3,7* , Robert A. Kozak1,6,12*# TE D M 1) Biological Sciences Platform, Sunnybrook Research Institute, Toronto, ON, Canada; 2) Department of Microbiology and Immunology, Life Sciences Institute, University of British Columbia, Vancouver, Canada; 3) Sinai Health System, Mount Sinai Hospital, Toronto, ON Canada; 4) Montana Molecular, Bozeman, Montana, United States; 5)Department of Biochemistry and Molecular Biology and Centre for Blood Research, University of British Columbia, Vancouver, BC, Canada; 6) Shared Hospital Laboratory, Toronto, ON, Canada; 7) CC EP —————————————————————————————————————————— *Authors contributed equally to this study # Corresponding author: Dr Robert Kozak, rkozak@shn.ca; Sunnybrook Health Sciences Centre, Microbiology Laboratory HB-01, 2075 Bayview Ave, Toronto, Ontario, Canada, M4N 3M4, 416-4806100 Alternate corresponding author : Dr. François Jean, fjean@mail.ubc.ca; University of British Columbia, 3558 - 2350 Health Sciences Mall, Life Sciences Centre, Vancouver, British Columbia, Canada, V6T 1Z3, 604-822-0256 A © The Author(s) 2025. Published by Oxford University Press on behalf of Infectious Diseases Society of America. This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs licence (https://creativecommons.org/licenses/by-nc-nd/4.0/), which permits non-commercial reproduction and distribution of the work, in any medium, provided the original work is not altered or transformed in any way, and that the work is properly cited. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com. 1 DOI: 10.1093/infdis/jiaf294 Functional and structural characterization of treatmentemergent nirmatrelvir resistance mutations at low frequencies in the main protease (M pro) reveals a unique evolutionary route for SARS-cov-2 to gain resistance Background: The main protease (Mpro ) is one of the most attractive targets for antiviral drug discovery against SARS-CoV-2. Mutations in Mpro have been linked to resistance against nirmatrelvir-ritonavir (NIR-RIT), an important therapy for SARS-CoV-2 infection. This study aimed to identify low-frequency antiviral resistance mutations in Mpro from NIR-RIT-treated patients and to analyze the enzymatic properties, inhibitor susceptibility, and structural features of new Mpro clinical variants. M A N U Methods: We screened 1,528 SARS-CoV-2-positive patients from two hospitals and identified 17 who remained positive after treatment. Whole genome sequencing of nasopharyngeal specimens was conducted to identify Mpro clinical variants. The impact..
DOI record: { "DOI": "10.1093/infdis/jiaf294", "ISSN": [ "0022-1899", "1537-6613" ], "URL": "http://dx.doi.org/10.1093/infdis/jiaf294", "abstract": "<jats:title>Abstract</jats:title>\n <jats:sec>\n <jats:title>Background</jats:title>\n <jats:p>The main protease (Mpro) is one of the most attractive targets for antiviral drug discovery against SARS-CoV-2. Mutations in Mpro have been linked to resistance against nirmatrelvir-ritonavir (NIR-RIT), an important therapy for SARS-CoV-2 infection. This study aimed to identify low-frequency antiviral resistance mutations in Mpro from NIR-RIT-treated patients and to analyze the enzymatic properties, inhibitor susceptibility, and structural features of new Mpro clinical variants.</jats:p>\n </jats:sec>\n <jats:sec>\n <jats:title>Methods</jats:title>\n <jats:p>We screened 1,528 SARS-CoV-2-positive patients from two hospitals and identified 17 who remained positive after treatment. Whole genome sequencing of nasopharyngeal specimens was conducted to identify Mpro clinical variants. The impact of these mutations on Mpro activity and inhibitor susceptibility was investigated using a fluorescent enzymatic biosensor in human cells, along with in vitro thermal stability and structure-based analyses of the Mpro mutants and Mpro-NIR complexes.</jats:p>\n </jats:sec>\n <jats:sec>\n <jats:title>Results</jats:title>\n <jats:p>The analysis identified two novel Mpro clinical variants: D48D/L58F/P132H (variant 1) and D48D/L67V/K90R/P132H (variant 2). Our data show that the selected clinical mutations are localized in the Mpro N-terminal domain, are far from the catalytic site, and strongly impact NIR resistance without affecting Mpro activity. Structural analysis and thermal denaturation analyses revealed that these mutations may disrupt the substrate binding site's structure and dynamics, reducing protein stability and potentially impacting substrate binding or dimerization without compromising catalytic activity.</jats:p>\n </jats:sec>\n <jats:sec>\n <jats:title>Conclusions</jats:title>\n <jats:p>Our new Mpro clinical mutations that confer complete resistance to NIR were not identified during previous cell-culture-based studies. More research is needed to explore resistance mechanisms, providing insights into strategies that mitigate resistance and protect therapeutic efficacy.</jats:p>\n </jats:sec>", "author": [ { "affiliation": [ { "name": "Biological Sciences Platform, Sunnybrook Research Institute , Toronto, ON ,", "place": [ "Canada" ] } ], "family": "Deschenes", "given": "Natalie M", "sequence": "first" }, { "ORCID": "https://orcid.org/0000-0002-4337-7903", "affiliation": [ { "name": "Department of Microbiology and Immunology, Life Sciences Institute, University of British Columbia , Vancouver ,", "place": [ "Canada" ] } ], "authenticated-orcid": false, "family": "Pérez-Vargas", "given": "Jimena", "sequence": "additional" }, { "affiliation": [ { "name": "Sinai Health System, Mount Sinai Hospital , Toronto, ON  ", "place": [ "Canada" ] } ], "family": "Zhong", "given": "Zoe", "sequence": "additional" }, { "ORCID": "https://orcid.org/0000-0002-3430-0912", "affiliation": [ { "name": "Montana Molecular , Bozeman, Montana ,", "place": [ "United States" ] } ], "authenticated-orcid": false, "family": "Thomas", "given": "Merrilee", "sequence": "additional" }, { "affiliation": [ { "name": "Department of Biochemistry and Molecular Biology and Centre for Blood Research, University of British Columbia , Vancouver, BC ,", "place": [ "Canada" ] } ], "family": "Kenward", "given": "Calem", "sequence": "additional" }, { "affiliation": [ { "name": "Department of Biochemistry and Molecular Biology and Centre for Blood Research, University of British Columbia , Vancouver, BC ,", "place": [ "Canada" ] } ], "family": "Mosimann", "given": "Wesley A", "sequence": "additional" }, { "affiliation": [ { "name": "Department of Biochemistry and Molecular Biology and Centre for Blood Research, University of British Columbia , Vancouver, BC ,", "place": [ "Canada" ] } ], "family": "Worrall", "given": "Liam J", "sequence": "additional" }, { "affiliation": [ { "name": "Shared Hospital Laboratory , Toronto, ON ,", "place": [ "Canada" ] } ], "family": "Waglechner", "given": "Nicholas", "sequence": "additional" }, { "affiliation": [ { "name": "Sinai Health System, Mount Sinai Hospital , Toronto, ON  ", "place": [ "Canada" ] } ], "family": "Li", "given": "Angel XinLiu", "sequence": "additional" }, { "ORCID": "https://orcid.org/0000-0002-1203-9514", "affiliation": [ { "name": "Shared Hospital Laboratory , Toronto, ON ,", "place": [ "Canada" ] }, { "name": "Faculty of Computer Science, Dalhousie University , Halifax, NS ,", "place": [ "Canada" ] }, { "name": "Department of Community Health & Epidemiology, Dalhousie University , Halifax, NS ,", "place": [ "Canada" ] } ], "authenticated-orcid": false, "family": "Maguire", "given": "Finlay", "sequence": "additional" }, { "affiliation": [ { "name": "Shared Hospital Laboratory , Toronto, ON ,", "place": [ "Canada" ] } ], "family": "Aftanas", "given": "Patryk", "sequence": "additional" }, { "ORCID": "https://orcid.org/0000-0001-7380-2192", "affiliation": [ { "name": "Department of Chemistry, Simon Fraser University , Burnaby, BC ,", "place": [ "Canada" ] } ], "authenticated-orcid": false, "family": "Smith", "given": "Jason R", "sequence": "additional" }, { "ORCID": "https://orcid.org/0009-0009-2640-033X", "affiliation": [ { "name": "Department of Molecular Biology and Biochemistry, Simon Fraser University , Burnaby, British Columbia ,", "place": [ "Canada" ] } ], "authenticated-orcid": false, "family": "Lim", "given": "Jared", "sequence": "additional" }, { "affiliation": [ { "name": "Department of Chemistry, Simon Fraser University , Burnaby, BC ,", "place": [ "Canada" ] } ], "family": "Young", "given": "Robert N", "sequence": "additional" }, { "affiliation": [ { "name": "Vancouver Prostate Centre, University of British Columbia , Vancouver, BC ,", "place": [ "Canada" ] } ], "family": "Cherkasov", "given": "Artem", "sequence": "additional" }, { "affiliation": [ { "name": "Sinai Health System, Mount Sinai Hospital , Toronto, ON  ", "place": [ "Canada" ] } ], "family": "Farooqi", "given": "Lubna", "sequence": "additional" }, { "affiliation": [ { "name": "Sinai Health System, Mount Sinai Hospital , Toronto, ON  ", "place": [ "Canada" ] } ], "family": "Moinuddin", "given": "Adnan", "sequence": "additional" }, { "ORCID": "https://orcid.org/0009-0006-6354-0292", "affiliation": [ { "name": "Sinai Health System, Mount Sinai Hospital , Toronto, ON  ", "place": [ "Canada" ] } ], "authenticated-orcid": false, "family": "Siddiqi", "given": "Lina", "sequence": "additional" }, { "affiliation": [ { "name": "Sinai Health System, Mount Sinai Hospital , Toronto, ON  ", "place": [ "Canada" ] } ], "family": "Malik", "given": "Imaan", "sequence": "additional" }, { "ORCID": "https://orcid.org/0009-0009-8827-0905", "affiliation": [ { "name": "Sinai Health System, Mount Sinai Hospital , Toronto, ON  ", "place": [ "Canada" ] } ], "authenticated-orcid": false, "family": "Lefebvre", "given": "Maxime", "sequence": "additional" }, { "ORCID": "https://orcid.org/0000-0002-7408-5487", "affiliation": [ { "name": "Department of Molecular Biology and Biochemistry, Simon Fraser University , Burnaby, British Columbia ,", "place": [ "Canada" ] } ], "authenticated-orcid": false, "family": "Paetzel", "given": "Mark", "sequence": "additional" }, { "affiliation": [ { "name": "Department of Biochemistry and Molecular Biology and Centre for Blood Research, University of British Columbia , Vancouver, BC ,", "place": [ "Canada" ] } ], "family": "Strynadka", "given": "Natalie C J", "sequence": "additional" }, { "affiliation": [ { "name": "Department of Microbiology and Immunology, Life Sciences Institute, University of British Columbia , Vancouver ,", "place": [ "Canada" ] } ], "family": "Jean", "given": "François", "sequence": "additional" }, { "affiliation": [ { "name": "Sinai Health System, Mount Sinai Hospital , Toronto, ON  ", "place": [ "Canada" ] }, { "name": "Faculty of Computer Science, Dalhousie University , Halifax, NS 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