Inhibition Mechanism of SARS-CoV-2 Main Protease with Ketone-Based Inhibitors Unveiled by Multiscale Simulations: Insights for Improved Designs**

We present the results of classical and QM/MM simulations for the inhibition of SARS-CoV-2 3CL protease by a hydroxymethylketone inhibitor, PF-00835231. In the noncovalent complex the carbonyl oxygen atom of the warhead is placed in the oxyanion hole formed by residues 143 to 145, while P1-P3 groups are accommodated in the active site with interactions similar to those observed for the peptide substrate. According to alchemical free energy calculations, the P1 ' hydroxymethyl group also contributes to the binding free energy. Covalent inhibition of the enzyme is triggered by the proton transfer from Cys145 to His41. This step is followed by the nucleophilic attack of the S gamma atom on the carbonyl carbon atom of the inhibitor and a proton transfer from His41 to the carbonyl oxygen atom mediated by the P1 ' hydroxyl group. Computational simulations show that the addition of a chloromethyl substituent to the P1 ' group may lower the activation free energy for covalent inhibition

Automated summary

The study demonstrates the mechanism of inhibition of SARS-CoV-2 3CL protease by PF-00835231 involving noncovalent interactions as well as covalent inhibition through proton transfer from Cys145 to His41 and nucleophilic attack of the S gamma atom. The hydroxymethyl group in the P1' position contributes to the binding free energy, and the addition of a chloromethyl substituent may lower the activation free energy for covalent inhibition.