Evolution of the SARS ‐ CoV ‐2 Main Protease Under Fitness Constraints Imposed by Folding Stability and Activity

ABSTRACT The severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) continues to evolve and diversify, leading to the emergence of immune‐evasive variants and underscoring the need for complementary antiviral strategies targeting conserved viral machinery. In this context, the main protease (Mpro) represents a potential therapeutic target because of its central role in viral replication and its low similarity to human proteases. However, the evolutionary consequences of Mpro variation on substrate recognition and potential resistance remain insufficiently explored. Through the analysis of multiple SARS‐CoV‐2 Mpro variants, we show that, despite ongoing viral diversification, Mpro evolution has remained remarkably constrained since the emergence of the Omicron‐associated P132H lineage in 2022, with limited amino‐acid substitutions in the active‐site and dimerization‐interface regions. Across the most prevalent SARS‐CoV‐2 variants, overall substrate recognition by Mpro was largely preserved, although specific variant‐substrate pairs exhibited some heterogeneity in per‐residue interactions and hydrogen‐bond patterns. Ensemble dynamics revealed conserved large‐scale motions alongside modest substrate‐dependent shifts in secondary motions in certain Mpro variants. Overall, SARS‐CoV‐2 Mpro evolution is strongly constrained by structural and functional requirements, supporting the suitability of this protein as an effective antiviral target. The findings further suggest that incorporating viral evolutionary dynamics may help inform the design of therapeutic strategies with improved long‐term robustness.