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 decrease 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.