Characterization of the Cross-Resistance of SARS-CoV-2 Main Protease Inhibitors, Ibuzatrelvir, Ensitrelvir, and Nirmatrelvir

The emergence of resistance to SARS-CoV-2 main protease (Mpro) inhibitors such as nirmatrelvir poses a significant threat to the long-term effectiveness of COVID-19 antivirals. Ibuzatrelvir (PF-07817883) and ensitrelvir are next-generation Mpro inhibitors with enhanced metabolic stability, eliminating the need for coadministration with ritonavir, unlike nirmatrelvir. Ibuzatrelvir is currently in Phase 3 clinical trials in the United States, and ensitrelvir is approved in Japan. In this study, we assessed the cross-resistance of ibuzatrelvir, nirmatrelvir, and ensitrelvir against a panel of clinically relevant Mpro mutants using FRET-based enzymatic assays, thermal shift binding assays, and cell-based antiviral plaque assays. Our results reveal a cross-resistance pattern of ibuzatrelvir, nirmatrelvir, and ensitrelvir against Q192, S144, H172, and E166 mutants. Notably, the recombinant SARS-CoV-2 virus containing the Mpro L50F/E166A/L167F triple mutant is highly resistant to all three drugs in the antiviral plaque assay. These findings underscore the challenge posed by E166 mutations and highlight the need for resistance-resistant Mpro inhibitors as future therapeutics.