Abstact

The COVID-19 pandemic spurred the rapid development of nirmatrelvir, a main protease (Mpro) inhibitor now widely prescribed as part of Paxlovid (nirmatrelvir plus ritonavir). However, increasing use has raised concerns about drug resistance. Resistance selection studies have identified multiple Mpro mutations, with E166V emerging as a particularly resistant variant. Sequencing data from COVID-19 patients confirms E166V as a clinically relevant mutation, and importantly, this substitution also confers cross-resistance to several next-generation Mpro inhibitors under development. In response, this study reports the rational design of inhibitors active against nirmatrelvir-resistant E166V/A mutants. The lead candidate, Jun13698, shows potent inhibition of both wild-type Mpro and the E166V/A mutants. Structural studies and molecular dynamics simulations reveal that Jun13698 forms stable complexes with wild-type and mutant proteases, consistent with its potent enzymatic and antiviral activity. Together, these findings position Jun13698 as a promising next-generation Mpro inhibitor capable of overcoming clinically relevant nirmatrelvir resistance.