Multiscale Simulations of SARS-CoV-2 3CL Protease Inhibition with Aldehyde Derivatives. Role of Protein and Inhibitor Conformational Changes in the Reaction Mechanism

Abstract

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.

The Se–S Bond Formation in the Covalent Inhibition Mechanism of SARS-CoV-2 Main Protease by Ebselen-like Inhibitors: A Computational Study

“…The analysis of the calculated RMSD for the residue pair Cys145-His41 in both the Nδ and Nε forms (shown in Figure S3) allows us to observe that the spatial orientation of the dyad remains constant during the simulation time in the case of His41-Nδ, while a dissimilar trend assumes the RMSD value of His41-Nε. The major fluctuations observed in RMSD for His41-Nε could suggest different conformations of the Cys145 side chain also previously found in the ortholog protease of SARS-CoV [46,58].…”

“…Moreover, stimulated by the most recent literature regarding the inhibition mechanism of M pro enzyme [43,[45][46][47] we took into account the protonation state of the catalytic histidine (His41) [43,48,49]. Therefore, we have also performed computational simulations by using molecular docking and classical molecular dynamics (cMD) devoted to accurately model the structure and dynamics of M pro in both the Nδ and Nε protonated states of His41 to better evaluate their eventual influence on the covalent inhibition process by EBS.…”

“…They play a central role in the enzyme's activation [40,41] and also in deiodinase biomimetics [42]. So far the SARS-CoV-2 M pro inhibition mechanism has been studied by using Michael acceptor and peptidomimetic inhibitors at the QM-MM level of theory [43][44][45][46][47]. So, the mechanistic understanding of the Se-S covalent bond formation promoted by inhibitors, such as EBS and EBS-OH in the present study, could contribute to enlarge the molecular inhibition mechanism of SARS-CoV-2 M pro and stimulate the design of other similar drugs.…”

“…So, the mechanistic understanding of the Se-S covalent bond formation promoted by inhibitors, such as EBS and EBS-OH in the present study, could contribute to enlarge the molecular inhibition mechanism of SARS-CoV-2 M pro and stimulate the design of other similar drugs. Moreover, stimulated by the most recent literature regarding the inhibition mechanism of M pro enzyme [43,[45][46][47] we took into account the protonation state of the catalytic histidine (His41) [43,48,49]. Therefore, we have also performed computational simulations by using molecular docking and classical molecular dynamics (cMD) devoted to accurately model the structure and dynamics of M pro in both the Nδ and Nε protonated states of His41 to better evaluate their eventual influence on the covalent inhibition process by EBS.…”

SARS-CoV-2 Mproinhibition by a zinc ion: structural features and hints for drug design

“…6,7 Several inhibitors of SARS-CoV and SARS-CoV-2 M pro have been designed that interfere with the formation of the acyl-enzyme complex due to the promotion of a covalent bond between the thiol group of the catalytic cysteine and the inihibitor. 3,4,8 A similar approach has been exploited using zinc-coordinating inhibitors, 9,10 where the zinc ion plays a role in stabilizing the protein-inhibitor complex through a tetrahedral coordination shared by the active site cysteine and histidine with two donor atoms of the inhibitor. The solved experimental structures [9][10][11] however leave open interesting questions concerning the zinc binding ability of the protease in the cellular environment, even in the absence of a coordinating ligand, and the possible role of zinc ions in modulating its biological activity.…”