Finally, we note that the present work has some caveats that needs pointing out. We only explored different protonation states of the catalytic His41 in our simulations due to its critical role in the molecular mechanism. But in principle, all histidine protonation states should have been explored and considered. Nevertheless, we find that binding free energy estimates of the inhibitors to the M pro target are impacted when different protonation/tautomeric states are chosen for the active site His41 residue. We caution selection of appropriate residue protonation and tautomeric states (in particular, for His) for ionizable residues when performing binding free energy studies of the M pro target. Methods that can account for the dynamic variation in titratable residue and ligand protonation states at different solution pH will be valuable in this regard, enabling complex dynamic transitions in protein-ligand binding events to be modelled more accurately.