Analysis of ibuzatrelvir's activity against SARS-CoV-2 circulating variants and in vitro resistance mutations.

The continued evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) underscores the ongoing need for effective antiviral therapies targeting this now-endemic virus. Ibuzatrelvir, a second-generation SARS-CoV-2 main protease (Mpro) inhibitor, has demonstrated potent antiviral activity without the need for a pharmacokinetic booster such as ritonavir. In the present study, we show that ibuzatrelvir maintains antiviral efficacy against all major SARS-CoV-2 variants circulating between 2020 and 2024 (alpha (α), beta (β), gamma (γ), lambda (λ), delta (δ), mu (μ) and omicron (ο)). Additionally, we characterized the in vitro resistance profile of ibuzatrelvir by selecting for mutations under drug pressure. This approach identified several resistance-associated amino acid changes in Mpro, including T21I, L232R, and S144A, as well as substitution combinations E166V+L232R and S144A+L232R+L253L/F. Among these, the E166V+L232R double substitution conferred the highest level of resistance to ibuzatrelvir. Surveillance studies have shown low prevalence of the E166V substitution in both GISAID datasets and in breakthrough cases from clinical trials. Cross-resistance testing revealed that this double substitution retained susceptibility to remdesivir and also conferred resistance to nirmatrelvir. Ibuzatrelvir and nirmatrelvir remained active against viruses containing the ensitrelvir resistance-associated Mpro substitution, M49L. The sustained efficacy of ibuzatrelvir against circulating variants, combined with the low prevalence of the E166V substitution, supports its continued evaluation in phase 3 studies.