Mechanistic Insights into Nitrile and Alkyne Covalent Inhibitors of the SARS-CoV-2 Main Protease

The treatment of SARS-CoV-2 can be accomplished by effective suppression of its 3CL protease (3CL^pro), also known as the main protease (M^pro) and nonstructural protein 5 (nsp5). Covalent inhibitors can irreversibly and selectively disable the protease, particularly when they are highly exothermic. Herein we investigated the distinct kinetic behaviors exhibited by two covalently linked SARS-CoV-2 inhibitors. One of these inhibitors features a nitrile reactive group, while the other has this group replaced by an alkyne group, a less reactive electrophile. Our investigations involve the assessment of the free energy surfaces of the key feasible mechanisms: that is, direct and water-assisted mechanisms involved in the rate-determining proton-transfer nucleophilic attack step through the utilization of both ab initio and empirical valence bond (EVB) simulations. The calculated free energy profiles show that substituting the nitrile group with alkyne increases the chemical barrier but leads to very exothermic reaction energy and is an irreversible process as opposed to nitrile, which is moderately exothermic and reversible. We also examine the time dependence of IC50 inhibition by applying an innovative kinetic simulation approach, which is particularly important in studies of covalent inhibitors with a very exothermic bonding step. Our computational approach provides a good agreement between the calculated and observed values of the time dependence results for the nitrile and alkyne inhibitors. Our approach, which is rather unique in combining calculations of the chemical barriers and the binding energy is likely to be very effective in studies of the effectiveness of other covalent inhibitors related cases.