Atomistic-Level Description of the Covalent Inhibition of SARS-CoV-2 Papain-like Protease.

Inhibition of the papain-like protease (PLpro) of SARS-CoV-2 has been demonstrated to be a successful target to prevent the spreading of the coronavirus in the infected body. In this regard, covalent inhibitors, such as the recently proposed VIR251 ligand, can irreversibly inactivate PLpro by forming a covalent bond with a specific residue of the catalytic site (Cys), through a Michael addition reaction. An inhibition mechanism can therefore be proposed, including four steps: ligand entry into the protease pocket; Cys deprotonation of the thiol group by a Brønsted-Lowry base; Cys-S addition to the ligand; and proton transfer from the protonated base to the covalently bound ligand. Evaluating the energetics and PLpro conformational changes at each of these steps could aid the design of more efficient and selective covalent inhibitors. For this aim, we have studied by means of MD simulations and QM/MM calculations the whole mechanism. Regarding the first step, we show that the inhibitor entry in the PLpro pocket is thermodynamically favorable only when considering the neutral Cys, that is, prior to the Cys deprotonation. For the second step, MD simulations revealed that His would deprotonate Cys after overcoming an energy barrier of ca. 32 kcal/mol (at the QM/MM level), but implying a decrease of the inhibitor stability inside the protease pocket. This information points to a reversible Cys deprotonation, whose equilibrium is largely shifted toward the neutral Cys form. Although thermodynamically disfavored, if Cys is deprotonated in close proximity to the vinylic carbon of the ligand, then covalent binding takes place in an irreversible way (third step) to form the enolate intermediate. Finally, due to Cys-S negative charge redistribution over the bound ligand, proton transfer from the initially protonated His is favored, finally leading to an irreversibly modified Cys and a restored His. These results elucidate the selectivity of Cys to enable formation of a covalent bond, even if a weak proton acceptor is available, as His.