9ARS

Crystal structure of SARS-CoV-2 main protease E166V mutant in complex with an inhibitor TKB-245

Summary for 9ARS

• Entry DOI: 10.2210/pdb9ars/pdb
• Descriptor: 3C-like proteinase nsp5, (1R,2S,5S)-N-{(1S,2S)-1-(4-fluoro-1,3-benzothiazol-2-yl)-1-hydroxy-3-[(3S)-2-oxopyrrolidin-3-yl]propan-2-yl}-6,6-dimethyl-3-[3-methyl-N-(trifluoroacetyl)-L-valyl]-3-azabicyclo[3.1.0]hexane-2-carboxamide (3 entities in total)
• Functional Keywords: sars-cov-2 main protease, 3c-like proteinase, viral protein, sars-cov-2, hydrolase-inhibitor complex, hydrolase/inhibitor
• Biological source: Severe acute respiratory syndrome coronavirus 2
• Total number of polymer chains: 2
• Total formula weight: 68902.53

Authors

Bulut, H.,Hattori, S.,Hayashi, H.,Hasegawa, K.,Li, M.,Wlodawer, A.,Misumi, S.,Tamamura, H.,Mitsuya, H. (deposition date: 2024-02-23, release date: 2024-04-24, Last modification date: 2024-11-13)

Primary citation

Hattori, S.I.,Bulut, H.,Hayashi, H.,Kishimoto, N.,Takamune, N.,Hasegawa, K.,Furusawa, Y.,Yamayoshi, S.,Murayama, K.,Tamamura, H.,Li, M.,Wlodawer, A.,Kawaoka, Y.,Misumi, S.,Mitsuya, H.
Structural and virologic mechanism of the emergence of resistance to M pro inhibitors in SARS-CoV-2.
Proc.Natl.Acad.Sci.USA, 121:e2404175121-e2404175121, 2024

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

PubMed: 39236245
DOI: 10.1073/pnas.2404175121

Experimental method

X-RAY DIFFRACTION (2.4 Å)