{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,14]],"date-time":"2026-08-14T15:56:31Z","timestamp":1786722991577,"version":"build-2736575974"},"reference-count":49,"publisher":"Oxford University Press (OUP)","issue":"5","license":[{"start":{"date-parts":[[2025,6,3]],"date-time":"2025-06-03T00:00:00Z","timestamp":1748908800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100000024","name":"Canadian Institutes of Health Research","doi-asserted-by":"publisher","id":[{"id":"10.13039\/501100000024","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000060","name":"National Institute of Allergy and Infectious Diseases","doi-asserted-by":"publisher","award":["1R44AI165188-01A1"],"award-info":[{"award-number":["1R44AI165188-01A1"]}],"id":[{"id":"10.13039\/100000060","id-type":"DOI","asserted-by":"publisher"}]},{"name":"Vancouver General Hospital Foundation"},{"DOI":"10.13039\/501100000196","name":"Canada Foundation for Innovation","doi-asserted-by":"publisher","id":[{"id":"10.13039\/501100000196","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100000038","name":"Natural Sciences and Engineering Research Council","doi-asserted-by":"crossref","id":[{"id":"10.13039\/501100000038","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/100008968","name":"National Research Council","doi-asserted-by":"publisher","id":[{"id":"10.13039\/100008968","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100021824","name":"Government of Saskatchewan","doi-asserted-by":"publisher","id":[{"id":"10.13039\/100021824","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100008920","name":"University of Saskatchewan","doi-asserted-by":"publisher","id":[{"id":"10.13039\/100008920","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2025,11,14]]},"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 severe acute respiratory syndrome coronavirus 2 (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\u2013treated 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 1528 SARS-CoV-2\u2013positive patients from 2 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 2 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>","DOI":"10.1093\/infdis\/jiaf294","type":"journal-article","created":{"date-parts":[[2025,6,2]],"date-time":"2025-06-02T07:22:51Z","timestamp":1748848971000},"page":"e789-e798","source":"Crossref","is-referenced-by-count":8,"title":["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"],"prefix":"10.1093","volume":"232","author":[{"given":"Natalie M","family":"Deschenes","sequence":"first","affiliation":[{"name":"Biological Sciences Platform, Sunnybrook Research Institute , Toronto, Ontario ,","place":["Canada"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4337-7903","authenticated-orcid":false,"given":"Jimena","family":"P\u00e9rez-Vargas","sequence":"additional","affiliation":[{"name":"Department of Microbiology and Immunology, Life Sciences Institute, University of British Columbia , Vancouver, British Columbia ,","place":["Canada"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zoe","family":"Zhong","sequence":"additional","affiliation":[{"name":"Sinai Health System, Mount Sinai Hospital , Toronto, Ontario ,","place":["Canada"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3430-0912","authenticated-orcid":false,"given":"Merrilee","family":"Thomas","sequence":"additional","affiliation":[{"name":"Montana Molecular , Bozeman, Montana ,","place":["USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Calem","family":"Kenward","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Biology and Centre for Blood Research, University of British Columbia , Vancouver, British Columbia ,","place":["Canada"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wesley A","family":"Mosimann","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Biology and Centre for Blood Research, University of British Columbia , Vancouver, British Columbia ,","place":["Canada"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Liam J","family":"Worrall","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Biology and Centre for Blood Research, University of British Columbia , Vancouver, British Columbia ,","place":["Canada"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nicholas","family":"Waglechner","sequence":"additional","affiliation":[{"name":"Shared Hospital Laboratory , Toronto, Ontario ,","place":["Canada"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Angel XinLiu","family":"Li","sequence":"additional","affiliation":[{"name":"Sinai Health System, Mount Sinai Hospital , Toronto, Ontario ,","place":["Canada"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1203-9514","authenticated-orcid":false,"given":"Finlay","family":"Maguire","sequence":"additional","affiliation":[{"name":"Shared Hospital Laboratory , Toronto, Ontario ,","place":["Canada"]},{"name":"Faculty of Computer Science, Dalhousie University , Halifax, Nova Scotia ,","place":["Canada"]},{"name":"Department of Community Health and Epidemiology, Dalhousie University , Halifax, Nova Scotia ,","place":["Canada"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Patryk","family":"Aftanas","sequence":"additional","affiliation":[{"name":"Shared Hospital Laboratory , Toronto, Ontario ,","place":["Canada"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7380-2192","authenticated-orcid":false,"given":"Jason R","family":"Smith","sequence":"additional","affiliation":[{"name":"Department of Chemistry, Simon Fraser University , Burnaby, British Columbia ,","place":["Canada"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0009-2640-033X","authenticated-orcid":false,"given":"Jared","family":"Lim","sequence":"additional","affiliation":[{"name":"Department of Molecular Biology and Biochemistry, Simon Fraser University , Burnaby, British Columbia ,","place":["Canada"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Robert N","family":"Young","sequence":"additional","affiliation":[{"name":"Department of Chemistry, Simon Fraser University , Burnaby, British Columbia ,","place":["Canada"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Artem","family":"Cherkasov","sequence":"additional","affiliation":[{"name":"Vancouver Prostate Centre, University of British Columbia , Vancouver, British Columbia ,","place":["Canada"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lubna","family":"Farooqi","sequence":"additional","affiliation":[{"name":"Sinai Health System, Mount Sinai Hospital , Toronto, Ontario ,","place":["Canada"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Adnan","family":"Moinuddin","sequence":"additional","affiliation":[{"name":"Sinai Health System, Mount Sinai Hospital , Toronto, Ontario ,","place":["Canada"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0006-6354-0292","authenticated-orcid":false,"given":"Lina","family":"Siddiqi","sequence":"additional","affiliation":[{"name":"Sinai Health System, Mount Sinai Hospital , Toronto, Ontario ,","place":["Canada"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Imaan","family":"Malik","sequence":"additional","affiliation":[{"name":"Sinai Health System, Mount Sinai Hospital , Toronto, Ontario ,","place":["Canada"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0009-8827-0905","authenticated-orcid":false,"given":"Maxime","family":"Lefebvre","sequence":"additional","affiliation":[{"name":"Sinai Health System, Mount Sinai Hospital , Toronto, Ontario 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,","place":["Canada"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Allison","family":"McGeer","sequence":"additional","affiliation":[{"name":"Sinai Health System, Mount Sinai Hospital , Toronto, Ontario ,","place":["Canada"]},{"name":"Faculty of Computer Science, Dalhousie University , Halifax, Nova Scotia ,","place":["Canada"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Robert A","family":"Kozak","sequence":"additional","affiliation":[{"name":"Biological Sciences Platform, Sunnybrook Research Institute , Toronto, Ontario ,","place":["Canada"]},{"name":"Shared Hospital Laboratory , Toronto, Ontario ,","place":["Canada"]},{"name":"Department of Laboratory Medicine and Pathology, University of Toronto , Toronto, Ontario 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