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

  1. Author:
    Hattori, Shin-Ichiro [ORCID]
    Bulut, Haydar [ORCID]
    Hayashi, Hironori [ORCID]
    Kishimoto, Naoki
    Takamune, Nobutoki [ORCID]
    Hasegawa, Kazuya [ORCID]
    Furusawa, Yuri
    Yamayoshi, Seiya
    Murayama, Kazutaka [ORCID]
    Tamamura, Hirokazu [ORCID]
    Li,Mi
    Wlodawer, Alexander [ORCID]
    Kawaoka, Yoshihiro [ORCID]
    Misumi, Shogo
    Mitsuya, Hiroaki

  2. Author Address

    Department of Refractory Viral Diseases, National Center for Global Health and Medicine Research Institute, Tokyo 162-8655, Japan., Experimental Retrovirology Section, HIV and AIDS Malignancy Branch, National Cancer Institute, NIH, Bethesda, MD 20892., Division of Infectious Diseases, International Research Institute of Disaster Science, Tohoku University, Miyagi 980-8575, Japan., Department of Environmental and Molecular Health Sciences, Faculty of Life Sciences, Kumamoto University, Kumamoto 862-0973, Japan., Structural Biology Division, Japan Synchrotron Radiation Research Institute, Hyogo 679-5198, Japan., Division of Virology, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan., The Research Center for Global Viral Diseases, National Center for Global Health and Medicine Research Institute, Tokyo 162-8655, Japan., International Research Center for Infectious Diseases, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan., Division of Biomedical Measurements and Diagnostics, Graduate School of Biomedical Engineering, Tohoku University, Miyagi 980-8575, Japan., Department of Medicinal Chemistry, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, Tokyo 101-0062, Japan., Center for Structural Biology, National Cancer Institute, Frederick, MD 21702., Basic Science Program, Frederick National Laboratory for Cancer Research, Frederick, MD 21702., Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, WI 53711., Department of Clinical Sciences, Kumamoto University Hospital, Kumamoto 860-8556, Japan.,
    1. Year: 2024
    2. Date: Sep 10
    3. Epub Date: 2024 09 05
  2. Journal: Proceedings of the National Academy of Sciences of the United States of America
    1. 121
    2. 37
    3. Pages: e2404175121
  4. Type of Article: Article
  5. Article Number: e2404175121
  1. 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 39;-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 117 177;3 and 17.1 177;1.9 181;M, 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 39;s drug resistance acquisition and the development of resistance-repellant COVID-19 therapeutics.

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External Sources

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  2. DOI: 10.1073/pnas.2404175121
  3. PMID: 39236245

Library Notes

  1. Fiscal Year: FY2024-2025
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