Beyond the Active Site: First‐In‐Class Leishmania Infantum Trypanothione Reductase Dimerization Disruptors as Antileishmanial Leads

Leishmania infantum trypanothione reductase (Li-TryR) is an essential enzyme for parasite redox homeostasis and a validated target for antileishmanial therapy. Classical inhibitors acting at the active site have shown limited success due to poor selectivity, suboptimal pharmacological properties, and lack of in-cell efficacy. This work presents a comprehensive strategy to develop first-in-class Li-TryR dimerization disruptors, targeting the protein-protein interface required for enzymatic activity. Through molecular modeling, mutagenesis, and structural analysis, a key α-helical hotspot (around residue E436) was identified as a druggable region. Interface-derived peptides, helix-stabilized stapled analogs, α/β-foldamers, and cell-penetrating conjugates demonstrated that dimer disruption effectively inhibits LiTryR and can translate into antiparasitic activity. Building on these insights, several families of small-molecule proteomimetics, including imidazole-phenyl-thiazoles, triazole analogs, and triazolium salts, were developed, achieving low-micromolar to submicromolar inhibition and significant activity in parasite cultures and intracellular models. Overall, this work establishes dimer-interface targeting as a novel therapeutic paradigm for leishmaniasis and provides multiple optimized chemical scaffolds for future drug development.