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

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 SARSCoV-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.