First Quantum Mechanics/Molecular Mechanics Studies of the Inhibition Mechanism of Cruzain by Peptidyl Halomethyl Ketones

Abstract

Cruzain is a primary cysteine protease expressed by the protozoan parasite Trypanosoma cruzi during Chagas disease infection, and thus, the development of inhibitors of this protein is a promising target for designing an effective therapy against the disease. In this paper, the mechanism of inhibition of cruzain by two different irreversible peptidyl halomethyl ketones (PHK) inhibitors has been studied by means of hybrid quantum mechanics/molecular mechanics−molecular dynamics (MD) simulations to obtain a complete representation of the possible free energy reaction paths. These have been traced on free energy surfaces in terms of the potential of mean force computed at AM1d/MM and DFT/MM levels of theory. An analysis of the possible reaction mechanisms of the inhibition process has been performed showing that the nucleophilic attack of an active site cysteine, Cys25, on a carbon atom of the inhibitor and the cleavage of the halogen−carbon bond take place in a single step. PClK appears to be much more favorable than PFK from a kinetic point of view. This result would be in agreement with experimental studies in other papain-like enzymes. A deeper analysis of the results suggests that the origin of the differences between PClK and PFK can be the different stabilizing interactions established between the inhibitors and the residues of the active site of the protein. Any attempt to explore the viability of the inhibition process through a stepwise mechanism involving the formation of a thiohemiketal intermediate and a three-membered sulfonium intermediate has been unsuccessful. Nevertheless, a mechanism through a protonated thiohemiketal, with participation of His159 as a proton donor, appears to be feasible despite showing higher free energy barriers. Our results suggest that PClK can be used as a starting point to develop a proper inhibitor of cruzain.

Revealing the molecular mechanisms of proteolysis of SARS-CoV-2 Mproby QM/MM computational methods

“…First, according to the present results, the most stable protonation state of the Cys145/His41 dyad in E:S (and in E:P) corresponds to that where both residues are neutral (Fig. 3), in contrast with previous computational studies of the proteolysis 5 and inhibition 15,16,18 of other CPs. The Cys145 À /His41 + ion pair is located in high energy regions of the FESs of the rst and last steps (Fig.…”

“…14 This is in good agreement with our previous studies of the catalysis and inhibition of different cysteine proteases carried out with this hybrid functional and available experimental data. 5,15,16 Results and discussion Analysis of the FESs (Fig. S3 and S4 †) shows that both the acylation and the deacylation reactions take place in a stepwise manner (Fig.…”

“…While this has been supported by X-ray diffraction studies, 19 its presence has been questioned by others, 20 including ourselves. 5 Moreover, previous attempts to explore the viability of the inhibition of CPs, such as cruzain by peptidyl halomethyl ketones, 15 reveal that a stepwise mechanism involving the formation of a THA intermediate was unsuccessful. Nevertheless, despite being a metastable intermediate as discussed above, it appears that the presence of a THA (E:I1) in the active site of SARS-CoV-2 M pro is favored by strong hydrogen bond interactions with an "oxyanion hole" formed by Gly143, Ser144 and Cys145 (Table S3 †).…”

Quantum Mechanics/Molecular Mechanics Studies of the Mechanism of Cysteine Proteases Inhibition by Dipeptidyl Nitroalkenes

“…The inhibition mechanism of CPs by vinyl sulfones and other α,β‐unsaturated electron‐withdrawing groups proceeds by a Michael addition with an attack on the β‐carbon atom by the thiolate of the active‐site cysteine residue followed by protonation of the α‐carbon atom to form the thioether derivative (see Scheme ) . With regard to the cruzain cysteine protease, only a few inhibition mechanisms have been studied using computational tools that include protein environment effects . The inhibition mechanisms of cruzain by peptidyl halomethyl ketones (PHKs) and the dipeptidyl‐2,3‐epoxyketone Cbz‐Phe‐Hph‐(S) have been studied in our laboratory through quantum mechanics/molecular mechanics (QM/MM) simulations.…”

Mechanism of Inhibition of SARS-CoV-2 Mpro by N3 Peptidyl Michael Acceptor Explained by QM/MM Simulations and Design of New Derivatives with Tunable Chemical Reactivity

“…QM/MM simulations provide a good tool to investigate the reactivity of covalent inhibitors within their protein targets. [23][24][25][26][27][28][29]…”