Structural insights into the nirmatrelvir-resistant SARS-CoV-2 M L50F/E166A/L167F triple mutant-inhibitor-complex reveal strategies for next generation coronaviral inhibitor design.

Drug-resistance is an eminent threat in antiviral therapy, and is currently a concern in nirmatrelvir-based therapy of SARS-CoV-2. Nirmatrelvir (antiviral component in Paxlovid) binds covalently to the active site cysteine of the main protease of SARS-CoV-2 (M), thereby blocking enzyme activity and halting viral replication. passage experiments mimicking a multi-dosage nirmatrelvir treatment regime, identified M variants with mutations in the active site and near the C-terminal dimerization interface with variable levels of nirmatrelvir resistance. One such variant harbors a triple mutation in M, L50F/E166A/L167F, that displays decreased potency for nirmatrelvir (IC ∼ 850-1600 nM) and ibuzatrelvir while viral replication remained similar to that of the wildtype (WT) virus. We here confirm a previously developed short peptide aldehyde bisulfite compound 4 as potent inhibitor for SARS-CoV-2 M L50F/E166A/L167F and related variants. A co-crystal structure reveals tight inhibitor binding that is stabilized by a network of hydrogen bonds formed by the mutated residues A166 and F167. This study provides the groundwork for optimized M inhibitors against potential emerging variants of SARS-CoV-2, as well as strategies for broad-spectrum inhibitor design against variants of M.