Assessment of the Binding Affinity of Covalent Inhibitors of Cruzain and Cathepsin L Using Relative Binding Free Energy

Cysteine proteases are recognized as relevant therapeutic targets in both parasitic diseases and cancer due to their essential biological functions. Among these enzymes, cruzain from Trypanosoma cruzi is an important molecular target for the treatment of Chagas disease. Likewise, human cathepsin L (hCatL) is a potential target for anticancer drug development. Both enzymes share conserved catalytic features, particularly the cysteine–histidine catalytic dyad, which enables interaction with nitrile‐based reversible covalent inhibitors. Here, six nitrile‐derived inhibitors ( Neq1171.1, Neq1172.1, Neq1181.1, Neq1182.1, Neq1268, and Neq1276 ) had their cruzain and hCatL inhibition constants, K i , determined. Besides, we have used molecular dynamics simulation and alchemical free energy calculations for evaluating the overall relative binding free energies of these inhibitors. The calculated free energy differences closely reproduced experimental trends, confirming the reliability of the computational protocol. These findings demonstrate that thermodynamic integration is an effective strategy for predicting relative binding affinities and provides valuable molecular‐level insights for the rational optimization of cysteine protease inhibitors with potential applications in Chagas disease and cancer therapy.