Structure-Based Design of Covalent SARS-CoV-2 Main Protease Inhibitors Targeting the Nirmatrelvir-Resistant E166 Mutants

The COVID-19 pandemic spurred the rapid development of nirmatrelvir, a main protease (M^pro) 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 M^pro 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 M^pro 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 M^pro 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 M^pro inhibitor capable of overcoming clinically relevant nirmatrelvir resistance.