Exploring Possible Drug-Resistant Variants of SARS-CoV-2 Main Protease (M^pro) with Noncovalent Preclinical Candidate, Mpro61

SARS-CoV-2 M^pro inhibitors, such as nirmatrelvir, have proven efficacy in clinical use. Nirmatrelvir was developed in a target-based approach against wild-type M^pro, with the anticipation that prolonged usage may cause enrichment of drug-resistant mutations and persistence of COVID infections. Although globally prevalent drug-resistant mutations have not yet been observed, individual cases have recently been reported among patients following treatment with Paxlovid-a formulation of nirmatrelvir. Mutations E166V and E166A have been detected in these drug-resistant clinical isolates, consistent with predictions from <i>in vitro</i> viral passage experiments and therefore necessitate ongoing drug development. In this study, we selected seven M^pro variants (T21I, L50F, E166V, A173V, T190I, E166V/L50F, and A173V/L50F), which have been repeatedly found in viral passage experiments. We investigated their kinetic and structural properties, as well as resistance level to M^pro inhibitors: nirmatrelvir, GC376-a similar peptidomimetic for feline COVID infections, and our in-house-developed nonpeptidomimetic inhibitor Mpro61. Mpro61 maintains potency against the single variants (except for E166V) and the A173/L50F double variant, with <i>K</i> _i values similar to those of the wild type. In contrast, while nirmatrelvir and GC376 were still effective against the A173V/L50F double variant, their <i>K</i> _i values significantly increased up to 10-fold. None of the inhibitors appeared to be potent against E166V-containing variants. Our structural analysis revealed a significant movement of Ser1 residue in all E166V-containing variants in the presence or absence of an inhibitor. The new orientation of the Ser1 suggested potential strategies for medicinal chemistry modifications of Mpro61 to enhance hydrogen-bonding interactions between these variants and Mpro61 derivatives. These studies provide critical insights into guiding the future design of additional Mpro61 derivatives that would potentially inhibit variants with the pan-drug-resistant E166V mutation.