Assessment of the Cruzain Cysteine Protease Reversible and Irreversible Covalent Inhibition Mechanism

Abstract

Reversible and irreversible covalent ligands are advanced cysteine protease inhibitors in the drug development pipeline. K777 is an irreversible inhibitor of cruzain, a necessary enzyme for the survival of the Trypanosoma cruzi (T. cruzi) parasite, the causative agent of Chagas disease. Despite their importance, irreversible covalent inhibitors are still often avoided due to the risk of adverse effects. Herein, we replaced the K777 vinyl sulfone group with a nitrile moiety to obtain a reversible covalent inhibitor (Neq0682) of cysteine protease. Then, we used advanced experimental and computational techniques to explore details of the inhibition mechanism of cruzain by reversible and irreversible inhibitors. The isothermal titration calorimetry (ITC) analysis shows that inhibition of cruzain by an irreversible inhibitor is thermodynamically more favorable than by a reversible one. The hybrid Quantum Mechanics/Molecular Mechanics (QM/MM) and Molecular Dynamics (MD) simulations were used to explore the mechanism of the reaction inhibition of cruzain by K777 and Neq0682. The calculated free energy profiles show that the Cys25 nucleophilic attack and His162 proton transfer occur in a single step for a reversible inhibitor and two steps for an irreversible covalent inhibitor. The hybrid QM/MM calculated free energies for the inhibition reaction correspond to −26.7 and −5.9 kcal mol–1 for K777 and Neq0682 at the MP2/MM level, respectively. These results indicate that the ΔG of the reaction is very negative for the process involving K777, consequently, the covalent adduct cannot revert to a noncovalent protein–ligand complex, and its binding tends to be irreversible. Overall, the present study provides insights into a covalent inhibition mechanism of cysteine proteases.

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.…”

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 MD simulations were performed using the fDynamo library, 49 Once the covalent enzyme-inhibitor E-I complex was equilibrated, the Michael addition reaction and the proton transfer from the protonated H41 to the C atom of the inhibitor (see S7 in Supporting Information) show that the most stable protonation state of the C145/H41 dyad corresponds to that in which both residues are neutral, designated E:I. This result is in contrast with previous computational studies of proteolysis 21 and inhibition 25,29,51 and earlier suggestions for other SARS-CoV main proteases that inhibitor binding may favor formation of the ion pair, 52 but it is in agreement with our previous study of the proteolysis reaction of M pro . 22 From this initial state, the proton transfer from C145 to H41 to form the ionic dyad E (+/-) :I…”

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

“…The mechanism is also slightly different because a water molecule is used in the transfer of the proton from His41 to C of the inhibitor while, as commented above, our simulations show that the direct transfer can take place, in an almost barrierless process, as previously for other CP inhibition reactions by Michael acceptors. 28,50 We note that, all other things being equal, the lower barrier found here (not involving an intervening water molecule), indicates that the mechanism we find would dominate the experimentally observed kinetics. Analysis of the structures of the key states in the reaction (Fig.…”