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

Reversible and irreversible covalent ligands are advanced cysteine protease inhibitors in the drug development pipeline. is an irreversible inhibitor of cruzain, a necessary enzyme for the survival of the () 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 vinyl sulfone group with a nitrile moiety to obtain a reversible covalent inhibitor () 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 and . 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 for and at the MP2/MM level, respectively. These results indicate that the Δ of the reaction is very negative for the process involving , 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.