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

Author(s)

Silva, Jose Rogerio A. ^[1] ; Cianni, Lorenzo ^[2] ; Araujo, Deborah ^[2] ; Jatai Batista, Pedro Henrique ^[2] ; de Vita, Daniela ^[2] ; Rosini, Fabiana ^[2] ; Leitao, Andrei ^[2] ; Lameira, Jeronimo ^[1] ; Montanari, Carlos A. ^[2]

Affiliation

^[1] Univ Fed Para, Lab Planejamento & Desenvolvimento Farmacos, Inst Ciencias Exatas & Nat, BR-66075110 Belem, Para - Brazil

^[2] Univ Sao Paulo, Inst Chem Sao Carlos, Med Chem Grp, BR-23566590 Sao Carlos, SP - Brazil

• Document type: Journal article
• Source: JOURNAL OF CHEMICAL INFORMATION AND MODELING; v. 60, n. 3, p. 1666-1677, MAR 23 2020.
• Web of Science Citations: 0

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.G 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. (AU)

• FAPESP's process: 13/18009-4 - Molecular design, synthesis and trypanocidal activity of cruzain reversible covalent inhibitors
• Grantee: Carlos Alberto Montanari
• Support Opportunities: Research Projects - Thematic Grants
• FAPESP's process: 18/21749-3 - Computational study of cruzain enzyme by reversible covalent inhibitors
• Grantee: Carlos Alberto Montanari
• Support Opportunities: Research Grants - Visiting Researcher Grant - Brazil