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

• 成果类型: Article
• 署名作者: Hattori, Shin-ichiro; Bulut, Haydar; Hayashi, Hironori; Kishimoto, Naoki; Takamune, Nobutoki; Hasegawa, Kazuya; Furusawa, Yuri; Yamayoshi, Seiya; Murayama, Kazutaka; Tamamura, Hirokazu; Li, Mi; Wlodawer, Alexander; Kawaoka, Yoshihiro; Misumi, Shogo; Mitsuya, Hiroaki
• 署名单位: Japan Institute for Health Security (JIHS); National Center for Global Health & Medicine - Japan; National Institutes of Health (NIH) - USA; NIH National Cancer Institute (NCI); Tohoku University; Kumamoto University; Japan Synchrotron Radiation Research Institute; University of Tokyo; Japan Institute for Health Security (JIHS); National Center for Global Health & Medicine - Japan; University of Tokyo; Tohoku University; Institute of Science Tokyo; Tokyo Medical & Dental University (TMDU); National Institutes of Health (NIH) - USA; NIH National Cancer Institute (NCI); National Institutes of Health (NIH) - USA; NIH National Cancer Institute (NCI); Frederick National Laboratory for Cancer Research; University of Wisconsin System; University of Wisconsin Madison; Kumamoto University
• 刊物名称: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• ISSN/ISSBN: 0027-9741
• DOI: 10.1073/pnas.2404175121
• 发表日期: 2024-09-10
• 关键词: hiv-1; virus

摘要

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- 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 WK521E166V ), 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 proE166V , 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 proE166V for nirmatrelvir and TKB245 were 117 +/- 3 and 17.1 +/- 1.9 mu M, respectively, indicating that TKB245 has a greater (by a factor of 6.8) binding affinity to M proE166V than nirmatrelvir. SARS- CoV- 2 WK521 WT selected with 5h acquired A191T substitution in M pro (SARS- CoV- 2 WK521A191T ) and better replicated in the presence of 5h, than SARS- CoV- 2 WK521WT . However, no significant enzymatic or structural changes in M proA191T were observed. The replicability of SARS- CoV- 2 WK521E166V proved to be compromised compared to SARS-CoV-2WK521 WT but predominated over SARS-CoV-2WK521 WT in the presence of nirmatrelvir. The replicabilityof SARS- CoV- 2 WK521A191T surpassed that of SARS- CoV- 2 WK521WT 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.