Structural and virologic mechanism of the emergence of resistance to Mpro inhibitors in SARS-CoV-2

  • Proc Natl Acad Sci U S A. 2024 Sep 10;121(37):e2404175121. doi: 10.1073/pnas.2404175121.
Shin-Ichiro Hattori  1 ,  Haydar Bulut  2 ,  Hironori Hayashi  3 ,  Naoki Kishimoto  4 ,  Nobutoki Takamune  4 ,  Kazuya Hasegawa  5 ,  Yuri Furusawa  6  7 ,  Seiya Yamayoshi  6  7  8 ,  Kazutaka Murayama  9 ,  Hirokazu Tamamura  10 ,  Mi Li  11  12 ,  Alexander Wlodawer  11 ,  Yoshihiro Kawaoka  6  7  13 ,  Shogo Misumi  4 ,  Hiroaki Mitsuya  1  2  14
Affiliations
  • 1. Department of Refractory Viral Diseases, National Center for Global Health and Medicine Research Institute, Tokyo 162-8655, Japan.
  • 2. Experimental Retrovirology Section, HIV and AIDS Malignancy Branch, National Cancer Institute, NIH, Bethesda, MD 20892.
  • 3. Division of Infectious Diseases, International Research Institute of Disaster Science, Tohoku University, Miyagi 980-8575, Japan.
  • 4. Department of Environmental and Molecular Health Sciences, Faculty of Life Sciences, Kumamoto University, Kumamoto 862-0973, Japan.
  • 5. Structural Biology Division, Japan Synchrotron Radiation Research Institute, Hyogo 679-5198, Japan.
  • 6. Division of Virology, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan.
  • 7. The Research Center for Global Viral Diseases, National Center for Global Health and Medicine Research Institute, Tokyo 162-8655, Japan.
  • 8. International Research Center for Infectious Diseases, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan.
  • 9. Division of Biomedical Measurements and Diagnostics, Graduate School of Biomedical Engineering, Tohoku University, Miyagi 980-8575, Japan.
  • 10. Department of Medicinal Chemistry, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, Tokyo 101-0062, Japan.
  • 11. Center for Structural Biology, National Cancer Institute, Frederick, MD 21702.
  • 12. Basic Science Program, Frederick National Laboratory for Cancer Research, Frederick, MD 21702.
  • 13. Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, WI 53711.
  • 14. Department of Clinical Sciences, Kumamoto University Hospital, Kumamoto 860-8556, Japan.
PMID: 39236245 DOI: 10.1073/pnas.2404175121
Abstract

We generated SARS-CoV-2 variants resistant to three SARS-CoV-2 main protease (Mpro) inhibitors (nirmatrelvir, TKB245, and 5h), by propagating the ancestral SARS-CoV-2WK521WT in VeroE6TMPRSS2 cells with increasing concentrations of each inhibitor and examined their structural and virologic profiles. A predominant E166V-carrying variant (SARS-CoV-2WK521E166V), which emerged when passaged with nirmatrelvir and TKB245, proved to be resistant to the two inhibitors. A recombinant SARS-CoV-2E166V was resistant to nirmatrelvir and TKB245, but sensitive to 5h. X-ray structural study showed that the dimerization of Mpro was severely hindered by E166V substitution due to the disruption of the presumed dimerization-initiating Ser1'-Glu166 interactions. TKB245 stayed bound to MproE166V, whereas nirmatrelvir failed. Native mass spectrometry confirmed that nirmatrelvir and TKB245 promoted the dimerization of Mpro, and compromised the enzymatic activity; the Ki values of recombinant MproE166V for nirmatrelvir and TKB245 were 117±3 and 17.1±1.9 µM, respectively, indicating that TKB245 has a greater (by a factor of 6.8) binding affinity to MproE166V than nirmatrelvir. SARS-CoV-2WK521WT selected with 5h acquired A191T substitution in Mpro (SARS-CoV-2WK521A191T) and better replicated in the presence of 5h, than SARS-CoV-2WK521WT. However, no significant enzymatic or structural changes in MproA191T were observed. The replicability of SARS-CoV-2WK521E166V proved to be compromised compared to SARS-CoV-2WK521WT but predominated over SARS-CoV-2WK521WT in the presence of nirmatrelvir. The replicability of SARS-CoV-2WK521A191T surpassed that of SARS-CoV-2WK521WT 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's drug resistance acquisition and the development of resistance-repellant COVID-19 therapeutics.

Keywords
SARS-CoV-2; drug resistance; main protease.
Products