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 111 ), through a Michael addition reaction. An inhibition mechanism can therefore be proposed, including four steps: (i) ligand entry into the protease pocket; (ii) Cys 111 deprotonation of the thiol group by a Brønsted-Lowry base; (iii) Cys 111 -S - addition to the ligand; and (iv) 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 111 , that is, prior to the Cys 111 deprotonation. For the second step, MD simulations revealed that His 272 would deprotonate Cys 111 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 111 deprotonation, whose equilibrium is largely shifted toward the neutral Cys 111 form. Although thermodynamically disfavored, if Cys 111 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 111 -S - negative charge redistribution over the bound ligand, proton transfer from the initially protonated His 272 is favored, finally leading to an irreversibly modified Cys 111 and a restored His 272 . These results elucidate the selectivity of Cys 111 to enable formation of a covalent bond, even if a weak proton acceptor is available, as His 272 .