We present a detailed theoretical analysis of the reaction mechanism of proteolysis catalyzed by the main protease of SARS-CoV-2. Using multiscale simulation methods, we have characterized the interactions established by a peptidic substrate in the active site, and then we have explored the free energy landscape associated with the acylation and deacylation steps of the proteolysis reaction, characterizing the transition states of the process. Our mechanistic proposals can explain most of the experimental observations made on the highly similar ortholog protease of SARS-CoV. We point to some key interactions that may facilitate the acylation process and thus can be crucial in the design of more specific and efficient inhibitors of the main protease activity. In particular, from our results, the P1′ residue can be a key factor to improve the thermodynamics and kinetics of the inhibition process.

Inhibition of SARS-CoV-2 3CL protease by a Michael acceptor is studied using classical and QM/MM simulations. Results point out to a transition state with a key water molecule stabilizing the catalytic dyad and assisting the protonation step.

We here investigate the mechanism of SARS-CoV-2 3CL protease inhibition by one of the most promising families of inhibitors, those containing an aldehyde group as a warhead. These compounds are covalent inhibitors that inactivate the protease, forming a stable hemithioacetal complex. Inhibitor 11a is a potent inhibitor that has been already tested in vitro and in animals. Using a combination of classical and QM/MM simulations, we determined the binding mode of the inhibitor into the active site and the preferred rotameric state of the catalytic histidine. In the noncovalent complex, the aldehyde group is accommodated into the oxyanion hole formed by the NH main-chain groups of residues 143 to 145. In this pose, P1–P3 groups of the inhibitor mimic the interactions established by the natural peptide substrate. The reaction is initiated with the formation of the catalytic dyad ion pair after a proton transfer from Cys145 to His41. From this activated state, covalent inhibition proceeds with the nucleophilic attack of the deprotonated Sγ atom of Cys145 to the aldehyde carbon atom and a water-mediated proton transfer from the Nε atom of His41 to the aldehyde oxygen atom. Our proposed reaction transition-state structure is validated by comparison with X-ray data of recently reported inhibitors, while the activation free energy obtained from our simulations agrees with the experimentally derived value, supporting the validity of our findings. Our study stresses the interplay between the conformational dynamics of the inhibitor and the protein with the inhibition mechanism and the importance of including conformational diversity for accurate predictions about the inhibition of the main protease of SARS-CoV-2. The conclusions derived from our work can also be used to rationalize the behavior of other recently proposed inhibitor compounds, including aldehydes and ketones with high inhibitory potency.

We present a detailed computational analysis of the binding mode and reactivity of the novel oral inhibitor PF-07321332 developed against SARS-CoV-2 3CL protease. Alchemical free energy calculations suggest that positions...

We present the results of classical and QM/MM simulations for the inhibition of SARS-CoV-2 3CL protease by ah ydroxymethylketone inhibitor,P F-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,t he P1' hydroxymethyl group also contributes to the binding free energy.C ovalent inhibition of the enzyme is triggered by the proton transfer from Cys145 to His41. This step is followed by the nucleophilic attackofthe Sg atom on the carbonyl carbon atom of the inhibitor and ap roton transfer from His41 to the carbonyl oxygen atom mediated by the P1' hydroxyl group. Computational simulations showt hat the addition of ac hloromethyl substituent to the P1' group may lower the activation free energy for covalent inhibition