Abstract: The Journal of Infectious Diseases
MAJOR ARTICLE
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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*#
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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)
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*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/),
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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.
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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:
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"DOI": "10.1093/infdis/jiaf294",
"ISSN": [
"0022-1899",
"1537-6613"
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"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>",
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