pH-dependent mechanical stability of SARS-CoV-2 Mpro-inhibitor complexes revealed by steered molecular dynamics

Understanding how environmental conditions alter the mechanical stability of drug-target interactions is essential for developing robust antiviral therapeutics. Here, we investigate the pH-dependent mechanical response of the SARS-CoV-2 main protease (Mpro) when bound to two herbal inhibitors (Gallocatechin gallate and Glabridin) and two synthetic compounds (Remdesivir and Teniposide) using steered molecular dynamics (SMD) simulations. Protonation states reflecting acidic endosomal conditions were assigned using PROPKA, and mechanical resilience was quantified through rupture forces and pulling work during forced unbinding. The results reveal two distinct pH-dependent behaviors: acidic environments strengthen the mechanical stability of Mpro-Remdesivir and Mpro-Glabridin complexes, whereas Gallocatechin gallate and Teniposide exhibit significantly reduced rupture forces and pulling work. Among the tested compounds, Gallocatechin gallate displays the highest mechanical stability at neutral pH, whereas Remdesivir demonstrates superior resistance to forced unbinding under acidic conditions. Mechanistic analysis reveals that protonation of catalytic residues His41 and Glu166 reshapes hydrogen-bond networks and electrostatic interactions, explaining the ligand-specific responses. These findings demonstrate that environmental pH can invert inhibitor performance rankings, with rupture forces spanning ∼120-850 pN across ligands and exhibiting pronounced pH-dependent shifts, including a decrease for Gallocatechin gallate (∼800 to ∼600 pN) and a substantial increase for Glabridin (∼120 to ∼450 pN), highlighting the importance of incorporating mechanical metrics and pH variability into antiviral therapeutic development for physiologically variable microenvironments.