A SARS-CoV-2 Mpro mutation conferring ensitrelvir resistance paradoxically increases nirmatrelvir susceptibility
SARS-CoV-2 variants resistant to current antivirals remain a significant threat, particularly in high-risk patients. Although nirmatrelvir and ensitrelvir both target the viral 3CL protease (M<sup>pro</sup>), their distinct susceptibility profiles may allow alternative therapeutic approaches. Here, we identify a deletion mutation at glycine 23 (Δ23G) in M<sup>pro</sup> that conferred high-level resistance to ensitrelvir ( ~ 35-fold) while paradoxically increasing susceptibility to nirmatrelvir ( ~ 8-fold). This opposite susceptibility pattern is confirmed both in vitro and in a male hamster infection model. Recombinant viruses carrying M<sup>pro</sup>-Δ23G exhibit impaired replication, pathogenicity, and transmissibility compared to wild-type, though the co-occurring mutation T45I partially restore viral fitness. Structural analyses reveal critical conformational changes in the catalytic loop (Ile136-Val148) and β-hairpin loop (Cys22-Thr26), directly influencing inhibitor binding selectivity. These results highlight differential resistance profiles of M<sup>pro</sup> inhibitors, supporting potential sequential or alternative use of nirmatrelvir and ensitrelvir in patients requiring prolonged antiviral treatment.
Authors
- Gyeongmin Kim (ORCID: https://orcid.org/0000-0002-2851-0374)
- Seong Cheol Min (ORCID: https://orcid.org/0000-0001-8262-990X)
- Hyunah Choo (ORCID: https://orcid.org/0000-0002-3545-2678)
- Beom Kyu Kim (ORCID: https://orcid.org/0000-0003-4344-3488)
- Yun Hee Baek (ORCID: https://orcid.org/0000-0002-8453-4880)
- Young Ki Choi (ORCID: https://orcid.org/0000-0002-0872-0147)
- Sang Chul Shin (ORCID: https://orcid.org/0000-0003-0156-680X)
- Ju Hwan Jeong (ORCID: https://orcid.org/0000-0001-7226-1507)
- Shinki An (ORCID: https://orcid.org/0000-0002-9822-7961)
- Min‐Suk Song (ORCID: https://orcid.org/0000-0001-6073-0783)
- Byungsun Jeon (ORCID: https://orcid.org/0000-0003-4189-6774)
- Juhyun Lee (ORCID: https://orcid.org/0000-0001-6777-4447)
- Ji Hyun Park (ORCID: https://orcid.org/0000-0002-1534-0070)
- Dong Gyu Lee (ORCID: https://orcid.org/0000-0001-9071-5648)
- H. PARK (ORCID: https://orcid.org/0009-0004-5050-0326)
- Jin-Ju Seo
- Gi Chan Lee
Institutions
- Ewha Womans University (KR)
- Chungbuk National University (KR)
- Cheongju University (KR)
- Institute for Basic Science (KR)
- Bio-Medical Science (South Korea) (KR)
- Chungbuk National University Hospital (KR)
- Korea Institute of Science and Technology (KR)
- Korea Research Institute of Bioscience and Biotechnology (KR)
- Korea Institute of Science & Technology Information (KR)
- Korea University of Science and Technology (KR)
Publication Details
- Journal
- Nature Communications
- Published
- 2025-11-22
- DOI
- https://doi.org/10.1038/s41467-025-65767-z
- Primary Topic
- SARS-CoV-2 and COVID-19 Research
- Type
- article
- Field-Weighted Citation Impact
- 0.00
Funders
- Korea Disease Control and Prevention Agency
- National Research Foundation
- Korea Health Industry Development Institute
- National Research Foundation of Korea
- Advanced Research Projects Agency
- Korea National Institute of Health