tetano
Editor, Senior Moderator
Proc Natl Acad Sci U S A
. 2024 Sep 10;121(37):e2404175121.
doi: 10.1073/pnas.2404175121. Epub 2024 Sep 5. Structural and virologic mechanism of the emergence of resistance to M[SUP]pro[/SUP] inhibitors in SARS-CoV-2
Shin-Ichiro Hattori[SUP] 1 [/SUP], Haydar Bulut[SUP] 2 [/SUP], Hironori Hayashi[SUP] 3 [/SUP], Naoki Kishimoto[SUP] 4 [/SUP], Nobutoki Takamune[SUP] 4 [/SUP], Kazuya Hasegawa[SUP] 5 [/SUP], Yuri Furusawa[SUP] 6 7 [/SUP], Seiya Yamayoshi[SUP] 6 7 8 [/SUP], Kazutaka Murayama[SUP] 9 [/SUP], Hirokazu Tamamura[SUP] 10 [/SUP], Mi Li[SUP] 11 12 [/SUP], Alexander Wlodawer[SUP] 11 [/SUP], Yoshihiro Kawaoka[SUP] 6 7 13 [/SUP], Shogo Misumi[SUP] 4 [/SUP], Hiroaki Mitsuya[SUP] 1 2 14 [/SUP]
Affiliations
We generated SARS-CoV-2 variants resistant to three SARS-CoV-2 main protease (M[SUP]pro[/SUP]) inhibitors (nirmatrelvir, TKB245, and 5h), by propagating the ancestral SARS-CoV-2[SUP]WK521[/SUP][SUB]WT[/SUB] in VeroE6[SUP]TMPRSS2[/SUP] cells with increasing concentrations of each inhibitor and examined their structural and virologic profiles. A predominant E166V-carrying variant (SARS-CoV-2[SUP]WK521[/SUP][SUB]E166V[/SUB]), which emerged when passaged with nirmatrelvir and TKB245, proved to be resistant to the two inhibitors. A recombinant SARS-CoV-2[SUB]E166V[/SUB] was resistant to nirmatrelvir and TKB245, but sensitive to 5h. X-ray structural study showed that the dimerization of M[SUP]pro[/SUP] was severely hindered by E166V substitution due to the disruption of the presumed dimerization-initiating Ser1'-Glu166 interactions. TKB245 stayed bound to M[SUP]pro[/SUP][SUB]E166V[/SUB], whereas nirmatrelvir failed. Native mass spectrometry confirmed that nirmatrelvir and TKB245 promoted the dimerization of M[SUP]pro[/SUP], and compromised the enzymatic activity; the Ki values of recombinant M[SUP]pro[/SUP][SUB]E166V[/SUB] 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[SUP]pro[/SUP][SUB]E166V[/SUB] than nirmatrelvir. SARS-CoV-2[SUP]WK521[/SUP][SUB]WT[/SUB] selected with 5h acquired A191T substitution in M[SUP]pro[/SUP] (SARS-CoV-2[SUP]WK521[/SUP][SUB]A191T[/SUB]) and better replicated in the presence of 5h, than SARS-CoV-2[SUP]WK521[/SUP][SUB]WT[/SUB]. However, no significant enzymatic or structural changes in M[SUP]pro[/SUP][SUB]A191T[/SUB] were observed. The replicability of SARS-CoV-2[SUP]WK521[/SUP][SUB]E166V[/SUB] proved to be compromised compared to SARS-CoV-2[SUP]WK521[/SUP][SUB]WT[/SUB] but predominated over SARS-CoV-2[SUP]WK521[/SUP][SUB]WT[/SUB] in the presence of nirmatrelvir. The replicability of SARS-CoV-2[SUP]WK521[/SUP][SUB]A191T[/SUB] surpassed that of SARS-CoV-2[SUP]WK521[/SUP][SUB]WT[/SUB] 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.
. 2024 Sep 10;121(37):e2404175121.
doi: 10.1073/pnas.2404175121. Epub 2024 Sep 5. Structural and virologic mechanism of the emergence of resistance to M[SUP]pro[/SUP] inhibitors in SARS-CoV-2
Shin-Ichiro Hattori[SUP] 1 [/SUP], Haydar Bulut[SUP] 2 [/SUP], Hironori Hayashi[SUP] 3 [/SUP], Naoki Kishimoto[SUP] 4 [/SUP], Nobutoki Takamune[SUP] 4 [/SUP], Kazuya Hasegawa[SUP] 5 [/SUP], Yuri Furusawa[SUP] 6 7 [/SUP], Seiya Yamayoshi[SUP] 6 7 8 [/SUP], Kazutaka Murayama[SUP] 9 [/SUP], Hirokazu Tamamura[SUP] 10 [/SUP], Mi Li[SUP] 11 12 [/SUP], Alexander Wlodawer[SUP] 11 [/SUP], Yoshihiro Kawaoka[SUP] 6 7 13 [/SUP], Shogo Misumi[SUP] 4 [/SUP], Hiroaki Mitsuya[SUP] 1 2 14 [/SUP]
Affiliations
- PMID: 39236245
- DOI: 10.1073/pnas.2404175121
We generated SARS-CoV-2 variants resistant to three SARS-CoV-2 main protease (M[SUP]pro[/SUP]) inhibitors (nirmatrelvir, TKB245, and 5h), by propagating the ancestral SARS-CoV-2[SUP]WK521[/SUP][SUB]WT[/SUB] in VeroE6[SUP]TMPRSS2[/SUP] cells with increasing concentrations of each inhibitor and examined their structural and virologic profiles. A predominant E166V-carrying variant (SARS-CoV-2[SUP]WK521[/SUP][SUB]E166V[/SUB]), which emerged when passaged with nirmatrelvir and TKB245, proved to be resistant to the two inhibitors. A recombinant SARS-CoV-2[SUB]E166V[/SUB] was resistant to nirmatrelvir and TKB245, but sensitive to 5h. X-ray structural study showed that the dimerization of M[SUP]pro[/SUP] was severely hindered by E166V substitution due to the disruption of the presumed dimerization-initiating Ser1'-Glu166 interactions. TKB245 stayed bound to M[SUP]pro[/SUP][SUB]E166V[/SUB], whereas nirmatrelvir failed. Native mass spectrometry confirmed that nirmatrelvir and TKB245 promoted the dimerization of M[SUP]pro[/SUP], and compromised the enzymatic activity; the Ki values of recombinant M[SUP]pro[/SUP][SUB]E166V[/SUB] 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[SUP]pro[/SUP][SUB]E166V[/SUB] than nirmatrelvir. SARS-CoV-2[SUP]WK521[/SUP][SUB]WT[/SUB] selected with 5h acquired A191T substitution in M[SUP]pro[/SUP] (SARS-CoV-2[SUP]WK521[/SUP][SUB]A191T[/SUB]) and better replicated in the presence of 5h, than SARS-CoV-2[SUP]WK521[/SUP][SUB]WT[/SUB]. However, no significant enzymatic or structural changes in M[SUP]pro[/SUP][SUB]A191T[/SUB] were observed. The replicability of SARS-CoV-2[SUP]WK521[/SUP][SUB]E166V[/SUB] proved to be compromised compared to SARS-CoV-2[SUP]WK521[/SUP][SUB]WT[/SUB] but predominated over SARS-CoV-2[SUP]WK521[/SUP][SUB]WT[/SUB] in the presence of nirmatrelvir. The replicability of SARS-CoV-2[SUP]WK521[/SUP][SUB]A191T[/SUB] surpassed that of SARS-CoV-2[SUP]WK521[/SUP][SUB]WT[/SUB] 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.