{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,7]],"date-time":"2026-10-07T02:17:57Z","timestamp":1791339477413,"version":"4.3.1"},"reference-count":39,"publisher":"National Academy of Sciences","issue":"37","license":[{"start":{"date-parts":[[2024,9,5]],"date-time":"2024-09-05T00:00:00Z","timestamp":1725494400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100009619","name":"Japan Agency for Medical Research and Development","doi-asserted-by":"publisher","award":["20fk0108257h0001"],"award-info":[{"award-number":["20fk0108257h0001"]}],"id":[{"id":"10.13039\/100009619","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100009619","name":"Japan Agency for Medical Research and Development","doi-asserted-by":"publisher","award":["20fk0108510h0001"],"award-info":[{"award-number":["20fk0108510h0001"]}],"id":[{"id":"10.13039\/100009619","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100012319","name":"National Center for Global Health and Medicine","doi-asserted-by":"publisher","award":["20A2001D"],"award-info":[{"award-number":["20A2001D"]}],"id":[{"id":"10.13039\/100012319","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100012319","name":"National Center for Global Health and Medicine","doi-asserted-by":"publisher","award":["22A2009D"],"award-info":[{"award-number":["22A2009D"]}],"id":[{"id":"10.13039\/100012319","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000054","name":"HHS | NIH | National Cancer Institute","doi-asserted-by":"publisher","award":["HHSN261200800001E"],"award-info":[{"award-number":["HHSN261200800001E"]}],"id":[{"id":"10.13039\/100000054","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["www.pnas.org"],"crossmark-restriction":true},"short-container-title":["Proc. Natl. Acad. Sci. U.S.A."],"published-print":{"date-parts":[[2024,9,10]]},"abstract":"\n We generated SARS-CoV-2 variants resistant to three SARS-CoV-2 main protease (M\n pro<\/jats:sup>\n ) inhibitors (nirmatrelvir, TKB245, and 5h), by propagating the ancestral SARS-CoV-2\n WK521<\/jats:sup>\n WT<\/jats:sub>\n in VeroE6\n TMPRSS2<\/jats:sup>\n cells with increasing concentrations of each inhibitor and examined their structural and virologic profiles. A predominant E166V-carrying variant (SARS-CoV-2\n WK521<\/jats:sup>\n E166V<\/jats:sub>\n ), which emerged when passaged with nirmatrelvir and TKB245, proved to be resistant to the two inhibitors. A recombinant SARS-CoV-2\n E166V<\/jats:sub>\n was resistant to nirmatrelvir and TKB245, but sensitive to 5h. X-ray structural study showed that the dimerization of M\n pro<\/jats:sup>\n was severely hindered by E166V substitution due to the disruption of the presumed dimerization-initiating Ser1\u2019-Glu166 interactions. TKB245 stayed bound to M\n pro<\/jats:sup>\n E166V<\/jats:sub>\n , whereas nirmatrelvir failed. Native mass spectrometry confirmed that nirmatrelvir and TKB245 promoted the dimerization of M\n pro<\/jats:sup>\n , and compromised the enzymatic activity; the Ki values of recombinant M\n pro<\/jats:sup>\n E166V<\/jats:sub>\n for nirmatrelvir and TKB245 were 117\u00b13 and 17.1\u00b11.9 \u00b5M, respectively, indicating that TKB245 has a greater (by a factor of 6.8) binding affinity to M\n pro<\/jats:sup>\n E166V<\/jats:sub>\n than nirmatrelvir. SARS-CoV-2\n WK521<\/jats:sup>\n WT<\/jats:sub>\n selected with 5h acquired A191T substitution in M\n pro<\/jats:sup>\n (SARS-CoV-2\n WK521<\/jats:sup>\n A191T<\/jats:sub>\n ) and better replicated in the presence of 5h, than SARS-CoV-2\n WK521<\/jats:sup>\n WT<\/jats:sub>\n . However, no significant enzymatic or structural changes in M\n pro<\/jats:sup>\n A191T<\/jats:sub>\n were observed. The replicability of SARS-CoV-2\n WK521<\/jats:sup>\n E166V<\/jats:sub>\n proved to be compromised compared to SARS-CoV-2\n WK521<\/jats:sup>\n WT<\/jats:sub>\n but predominated over SARS-CoV-2\n WK521<\/jats:sup>\n WT<\/jats:sub>\n in the presence of nirmatrelvir. The replicability of SARS-CoV-2\n WK521<\/jats:sup>\n A191T<\/jats:sub>\n surpassed that of SARS-CoV-2\n WK521<\/jats:sup>\n WT<\/jats:sub>\n in the absence of 5h, confirming that A191T confers enhanced viral fitness. The present data should shed light on the understanding of the mechanism of SARS-CoV-2\u2019s drug resistance acquisition and the development of resistance-repellant COVID-19 therapeutics.\n <\/jats:p>","DOI":"10.1073\/pnas.2404175121","type":"journal-article","created":{"date-parts":[[2024,9,5]],"date-time":"2024-09-05T14:32:13Z","timestamp":1725546733000},"update-policy":"https:\/\/doi.org\/10.1073\/pnas.cm10313","source":"Crossref","is-referenced-by-count":21,"title":["Structural and virologic mechanism of the emergence of resistance to M\n pro<\/sup>\n inhibitors in SARS-CoV-2"],"prefix":"10.1073","volume":"121","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-5637-3397","authenticated-orcid":false,"given":"Shin-ichiro","family":"Hattori","sequence":"first","affiliation":[{"name":"Department of Refractory Viral Diseases, National Center for Global Health and Medicine Research Institute, Tokyo 162-8655, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0262-6296","authenticated-orcid":false,"given":"Haydar","family":"Bulut","sequence":"additional","affiliation":[{"name":"Experimental Retrovirology Section, HIV and AIDS Malignancy Branch, National Cancer Institute, NIH, Bethesda, MD 20892"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5892-0040","authenticated-orcid":false,"given":"Hironori","family":"Hayashi","sequence":"additional","affiliation":[{"name":"Division of Infectious Diseases, International Research Institute of Disaster Science, Tohoku University, Miyagi 980-8575, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Naoki","family":"Kishimoto","sequence":"additional","affiliation":[{"name":"Department of Environmental and Molecular Health Sciences, Faculty of Life Sciences, Kumamoto University, Kumamoto 862-0973, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7173-240X","authenticated-orcid":false,"given":"Nobutoki","family":"Takamune","sequence":"additional","affiliation":[{"name":"Department of Environmental and Molecular Health Sciences, Faculty of Life Sciences, Kumamoto University, Kumamoto 862-0973, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0331-1624","authenticated-orcid":false,"given":"Kazuya","family":"Hasegawa","sequence":"additional","affiliation":[{"name":"Structural Biology Division, Japan Synchrotron Radiation Research Institute, Hyogo 679-5198, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuri","family":"Furusawa","sequence":"additional","affiliation":[{"name":"Division of Virology, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan"},{"name":"The Research Center for Global Viral Diseases, National Center for Global Health and Medicine Research Institute, Tokyo 162-8655, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Seiya","family":"Yamayoshi","sequence":"additional","affiliation":[{"name":"Division of Virology, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan"},{"name":"The Research Center for Global Viral Diseases, National Center for Global Health and Medicine Research Institute, Tokyo 162-8655, Japan"},{"name":"International Research Center for Infectious Diseases, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1749-4038","authenticated-orcid":false,"given":"Kazutaka","family":"Murayama","sequence":"additional","affiliation":[{"name":"Division of Biomedical Measurements and Diagnostics, Graduate School of Biomedical Engineering, Tohoku University, Miyagi 980-8575, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2788-2579","authenticated-orcid":false,"given":"Hirokazu","family":"Tamamura","sequence":"additional","affiliation":[{"name":"Department of Medicinal Chemistry, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, Tokyo 101-0062, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mi","family":"Li","sequence":"additional","affiliation":[{"name":"Center for Structural Biology, National Cancer Institute, Frederick, MD 21702"},{"name":"Basic Science Program, Frederick National Laboratory for Cancer Research, Frederick, MD 21702"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5510-9703","authenticated-orcid":false,"given":"Alexander","family":"Wlodawer","sequence":"additional","affiliation":[{"name":"Center for Structural Biology, National Cancer Institute, Frederick, MD 21702"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5061-8296","authenticated-orcid":false,"given":"Yoshihiro","family":"Kawaoka","sequence":"additional","affiliation":[{"name":"Division of Virology, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan"},{"name":"The Research Center for Global Viral Diseases, National Center for Global Health and Medicine Research Institute, Tokyo 162-8655, Japan"},{"name":"Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin\u2013Madison, Madison, WI 53711"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shogo","family":"Misumi","sequence":"additional","affiliation":[{"name":"Department of Environmental and Molecular Health Sciences, Faculty of Life Sciences, Kumamoto University, Kumamoto 862-0973, 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