Structural and virologic mechanism of the emergence of resistance to M <sup>pro</sup> inhibitors in SARS-CoV-2-Reference-Cited by-同舟云学术

Structural and virologic mechanism of the emergence of resistance to M ^pro inhibitors in SARS-CoV-2

Published:2024-09-05 Issue:37 Volume:121 Page:
ISSN:0027-8424
Container-title:Proceedings of the National Academy of Sciences
language:en
Short-container-title:Proc. Natl. Acad. Sci. U.S.A.

Author:

Hattori Shin-ichiro¹^ORCID,Bulut Haydar²^ORCID,Hayashi Hironori³^ORCID,Kishimoto Naoki⁴,Takamune Nobutoki⁴^ORCID,Hasegawa Kazuya⁵^ORCID,Furusawa Yuri⁶⁷,Yamayoshi Seiya⁶⁷⁸,Murayama Kazutaka⁹^ORCID,Tamamura Hirokazu¹⁰^ORCID,Li Mi¹¹¹²,Wlodawer Alexander¹¹^ORCID,Kawaoka Yoshihiro⁶⁷¹³^ORCID,Misumi Shogo⁴,Mitsuya Hiroaki¹²¹⁴

Affiliation:

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

Abstract

We generated SARS-CoV-2 variants resistant to three SARS-CoV-2 main protease (M pro ) inhibitors (nirmatrelvir, TKB245, and 5h), by propagating the ancestral SARS-CoV-2 WK521 WT in VeroE6 TMPRSS2 cells with increasing concentrations of each inhibitor and examined their structural and virologic profiles. A predominant E166V-carrying variant (SARS-CoV-2 WK521 E166V ), which emerged when passaged with nirmatrelvir and TKB245, proved to be resistant to the two inhibitors. A recombinant SARS-CoV-2 E166V was resistant to nirmatrelvir and TKB245, but sensitive to 5h. X-ray structural study showed that the dimerization of M pro was severely hindered by E166V substitution due to the disruption of the presumed dimerization-initiating Ser1’-Glu166 interactions. TKB245 stayed bound to M pro E166V , whereas nirmatrelvir failed. Native mass spectrometry confirmed that nirmatrelvir and TKB245 promoted the dimerization of M pro , and compromised the enzymatic activity; the Ki values of recombinant M pro E166V 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 M pro E166V than nirmatrelvir. SARS-CoV-2 WK521 WT selected with 5h acquired A191T substitution in M pro (SARS-CoV-2 WK521 A191T ) and better replicated in the presence of 5h, than SARS-CoV-2 WK521 WT . However, no significant enzymatic or structural changes in M pro A191T were observed. The replicability of SARS-CoV-2 WK521 E166V proved to be compromised compared to SARS-CoV-2 WK521 WT but predominated over SARS-CoV-2 WK521 WT in the presence of nirmatrelvir. The replicability of SARS-CoV-2 WK521 A191T surpassed that of SARS-CoV-2 WK521 WT 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.

Funder

Japan Agency for Medical Research and Development

National Center for Global Health and Medicine

HHS | NIH | National Cancer Institute

Publisher

Proceedings of the National Academy of Sciences

Link

https://pnas.org/doi/pdf/10.1073/pnas.2404175121

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