Thermodynamic Drivers of Covalent Cruzain Inhibition Beyond Enthalpy-Entropy Compensation

Cysteine proteases are pivotal enzymes involved in diverse physiological and pathological processes, with cruzain and human cathepsin L emerging as key therapeutic targets. We report the design, synthesis, and characterization of a series of dipeptidyl inhibitors incorporating engineered modifications at the P1, P2, and P3 positions. Among these, Neq1276, featuring an indole substituent at P2, displayed the highest affinity for hCatL (pKi = 9.9) while maintaining activity against Cz (pKi = 7.4), whereas Neq1277 showed enhanced affinity for Cz (ΔpKi = +1.0) with preserved selectivity. Thermodynamic analyses by isothermal titration calorimetry uncovered a rare synergistic additivity of enthalpic and entropic contributions in Neq1153, Neq1260, and Neq1265, deviating from the widely observed entropy–enthalpy compensation. This phenomenon provides a unique framework for rational ligand design by enabling simultaneous ΔH and ΔS gains in binding. Selectivity patterns highlighted the key role of P2 substituents, particularly the indole group, in fine-tuning recognition. Retrospective database analysis further identified Neq1276 as a novel chemical entity, underscoring its potential as a promising lead compound for future development.