Enhanced Target Binding by Leritrelvir Restores Dimerization of M ^pro Mutants and Mitigates Drug Resistance

The SARS-CoV-2 main protease (M ^pro ) has been a major target of antiviral drug development, leading to the development of inhibitors such as nirmatrelvir, the antiviral component of the COVID-19 drug Paxlovid. However, resistance-associated mutations that reduce the efficacy of current M ^pro inhibitors, particularly nirmatrelvir, have emerged. Here, we evaluated the inhibitory activity of leritrelvir (RAY1216), an M ^pro inhibitor approved in China for COVID-19 monotherapy, against a panel of M ^pro variants carrying mutations at 12 resistance-associated residues distributed across four catalytic subsites. Using integrated biochemical, biophysical, structural, and cellular analyses, we demonstrate that leritrelvir retains stronger inhibitory activity against most tested resistant mutants compared with nirmatrelvir. Most of the tested mutations promote M ^pro dimer dissociation, with E166V showing a particularly pronounced effect and markedly compromising nirmatrelvir binding. In contrast, thermal shift and size-exclusion chromatography assays demonstrate that leritrelvir binding restores dimerization of these M ^pro mutants. Sixteen high-resolution crystal structures reveal that leritrelvir binding re-instates key dimer-interface interactions disrupted by resistance mutations. Mini-replicon assays further confirm leritrelvir to possess enhanced cellular antiviral efficacy compared with nirmatrelvir. Our findings indicate that tighter leritrelvir binding enables more effective inhibition of dissociation-prone M ^pro mutants than nirmatrelvir, supporting its use as a more resilient antiviral agent for SARS-CoV-2 treatment.