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 (M(pro)) 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 M(pro) 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 M(pro) 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 M(pro) 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 M(pro) inhibitors as future therapeutics.