Contribution of the catalytic dyad of SARS-CoV-2 main protease to binding covalent and noncovalent inhibitors

The effect of mutations of the catalytic dyad residues of SARS-CoV-2 main protease (MPro^WT) on the thermodynamics of binding of covalent inhibitors comprising nitrile [nirmatrelvir (NMV), NBH2], aldehyde (GC373) and ketone (BBH1) warheads to MPro is examined together with room temperature X-ray crystallography. When lacking the nucleophilic C145, NMV binding is ∼400-fold weaker corresponding to 3.5 kcal/mol and 13.3 °C decreases in free energy (ΔG) and thermal stability (T_m), respectively, relative to MPro^WT. The H41A mutation results in a 20-fold increase in the dissociation constant (K_d), and 1.7 kcal/mol and 1.4 °C decreases in ΔG and T_m, respectively. Increasing the pH from 7.2 to 8.2 enhances NMV binding to MPro^H41A, whereas no significant change is observed in binding to MPro^WT. Structures of the four inhibitor complexes with MPro^1-304/C145A show that the active site geometries of the complexes are nearly identical to that of MPro^WT with the nucleophilic sulfur of C145 positioned to react with the nitrile or the carbonyl carbon. These results support a two-step mechanism for the formation of the covalent complex involving an initial non-covalent binding followed by a nucleophilic attack by the thiolate anion of C145 on the warhead carbon. Noncovalent inhibitor ensitrelvir (ESV) exhibits a binding affinity to MPro^WT that is similar to NMV but differs in its thermodynamic signature from NMV. The binding of ESV to MPro^C145A also results in a significant, but smaller, increase in K_d and decrease in ΔG and T_m, relative to NMV.

C145A/H41A mutations; SARS-CoV-2 main protease; inhibitor binding thermodynamics; main protease inhibitors; room-temperature X-ray crystallography; thermal stability.