The clinical durability of SARS-CoV-2 main protease (Mpro) inhibitors depends on their resilience to emerging resistance mutations. Recent genomic surveillance and functional reports have highlighted substitutions at positions 49, 165, and 301, raising questions about the robustness of the noncovalent inhibitor WU-04 in variant backgrounds. Here, we combined μs-scale, triplicate molecular dynamics simulations with end-state binding free energy estimates and a network-rewiring inference (NRI) framework that maps long-range dynamical communication across the full protease dimer. We evaluated wild type (WT), single mutants M49K, M165V, S301P, and selected double mutants (M49K its substitution rewires inter-domain coupling that can compensate for local disruptions at residue 49. Together, these results identify structural hotspots and network pathways that may inform the design of next-generation Mpro inhibitors with improved mutation tolerance—specifically by strengthening interactions that do not rely solely on the mutable S2 pocket and by engaging conserved backbone features near the 165–166 region.
Liu et al. (2026) studied this question.