A SARS-CoV-2 Mpro mutation conferring ensitrelvir resistance paradoxically increases nirmatrelvir susceptibility

Seong Cheol Min, Jin-Ju Seo, Ju Hwan Jeong, Beom Kyu Kim, Ji-Hyun Park, Ju Ryeong Lee, Dong Gyu Lee, Gi Chan Lee, Se Hee An, Yun Hee Baek, Young Ki Choi, Hyunah Choo, Hyo Yong Park, Gyeongmin Kim, Byungsun Jeon, Sang Chul Shin () and Min-Suk Song ()
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Seong Cheol Min: Chungbuk National University College of Medicine and Medical Research Institute, Department of Microbiology
Jin-Ju Seo: Korea Institute of Science and Technology (KIST), Technological Convergence Center
Ju Hwan Jeong: Chungbuk National University College of Medicine and Medical Research Institute, Department of Microbiology
Beom Kyu Kim: Chungbuk National University College of Medicine and Medical Research Institute, Department of Microbiology
Ji-Hyun Park: Chungbuk National University College of Medicine and Medical Research Institute, Department of Microbiology
Ju Ryeong Lee: Chungbuk National University College of Medicine and Medical Research Institute, Department of Microbiology
Dong Gyu Lee: Chungbuk National University College of Medicine and Medical Research Institute, Department of Microbiology
Gi Chan Lee: Chungbuk National University College of Medicine and Medical Research Institute, Department of Microbiology
Se Hee An: Chungbuk National University College of Medicine and Medical Research Institute, Department of Microbiology
Yun Hee Baek: Chungbuk National University College of Medicine and Medical Research Institute, Department of Microbiology
Young Ki Choi: Institute for Basic Science (IBS), Center for Study of Emerging and Re-emerging Viruses, Korea Virus Research Institute
Hyunah Choo: Korea Institute of Science and Technology (KIST), Medicinal Materials Research Center, Biomedical Research Division
Hyo Yong Park: Korea Institute of Science and Technology (KIST), Medicinal Materials Research Center, Biomedical Research Division
Gyeongmin Kim: Korea Institute of Science and Technology (KIST), Medicinal Materials Research Center, Biomedical Research Division
Byungsun Jeon: Korea Institute of Science and Technology (KIST), Medicinal Materials Research Center, Biomedical Research Division
Sang Chul Shin: Korea Institute of Science and Technology (KIST), Technological Convergence Center
Min-Suk Song: Chungbuk National University College of Medicine and Medical Research Institute, Department of Microbiology

Nature Communications, 2025, vol. 16, issue 1, 1-15

Abstract: Abstract 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 (Mpro), their distinct susceptibility profiles may allow alternative therapeutic approaches. Here, we identify a deletion mutation at glycine 23 (Δ23G) in Mpro 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 Mpro-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 Mpro inhibitors, supporting potential sequential or alternative use of nirmatrelvir and ensitrelvir in patients requiring prolonged antiviral treatment.

Date: 2025
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DOI: 10.1038/s41467-025-65767-z

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