Structural basis of ligand selectivity in FAD/NAD(P)H-dependent dehydrogenases: insights from trypanothione reductase and type II NADH dehydrogenase

FAD/NAD(P)H-dependent dehydrogenases form a structurally conserved family of redox enzymes that participate in essential metabolic processes across parasites and higher organisms. Among them, trypanothione reductase (TR) is a key component of the redox metabolism of Leishmania species and represents an attractive target for antileishmanial drug development. However, because several flavoproteins share similar folds and cofactor-binding architectures, the selectivity of TR inhibitors remains a critical issue during early drug discovery. To explore this aspect, we investigated the structural landscape of FAD/NAD(P)H-dependent dehydrogenases in Leishmania infantum by integrating sequence analysis, structural modeling, docking simulations, and in vitro biochemical validation. Reciprocal sequence searches revealed eleven parasite flavoproteins structurally related to TR, including a dihydrolipoamide dehydrogenase (DLD)-like protein, a type II NADH dehydrogenase (NDH2)-like protein, and a dienoyl-CoA reductase (deCoAR)-like protein. Comparative docking analyses across parasite and mammalian homologues allowed us to examine potential cross-reactivity patterns among these enzymes. Enzymatic assays performed on recombinant Leishmania infantum TR (LiTR) and Caldalkalibacillus thermarum NDH2 (CtNDH2) confirmed that selected ligands can exert enzymedependent effects. In particular, auranofin, and to a lesser extent nitrofurazone, inhibited LiTR, whereas a terpyridine–Pt-derived compound strongly inhibited LiTR while stimulating CtNDH2 activity. Overall, the results illustrate how structurally related flavoproteins may accommodate common ligands while responding with distinct catalytic outcomes. The integrated computational and biochemical workflow presented here provides a practical framework for assessing ligand selectivity within the FAD/NAD(P)H-dependent dehydrogenase family and may support the development of selective modulators targeting parasite redox metabolism.