Integrated Target-Based Screening, Characterization and Computational Profiling of a Natural Alkaloid Library Identifies Tryptanthrin as an Antileishmanial Hit Targeting Leishmania donovani Trypanothione Reductase

Abstract Leishmaniasis requires safer and more effective therapies, and the trypanothione-dependent redox metabolism of trypanosomatids, particularly trypanothione reductase (TryR), represents an attractive druggable target. An alkaloid library comprising 432 compounds was screened to identify inhibitors of Leishmania donovani TryR (LdTryR). Six compounds displayed both LdTryR inhibitory activity and antileishmanial effects against intramacrophage amastigotes: (−)-alkannin (EC50: 8.98 ± 1.96 μM; Ki: 2.83 ± 1.99 μM), chelerythrine (EC50: 1.61 ± 0.05 μM; Ki: 27.80 ± 9.43 μM), dehydronuciferine (EC50: > 50 μM; Ki: 15.77 ± 8.34 μM), ethoxysanguinarine (EC50: 0.84 ± 0.03 μM; Ki: 21.22 ± 7.32 μM), sanguinarine chloride (EC50: 0.85 ± 0.03 μM; Ki: 28.57 ± 9.62 μM) and tryptanthrin (EC50: 1.91 ± 0.21 μM; Ki: 16.80 ± 10.61 μM). Kinetic analyses revealed competitive inhibition patterns for benzophenanthridines, whereas (−)-alkannin, tryptanthrin, and dehydronuciferine exhibited uncompetitive inhibition behavior. Cytotoxicity analysis revealed that tryptanthrin was the only compound that fulfilled the hit criteria commonly applied in visceral leishmaniasis. Benzophenanthridine alkaloids induced rapid reactive oxygen species (ROS) accumulation, whereas (−)-alkannin produced delayed oxidative stress. Compounds inducing the highest parasite ROS levels also stimulated nitrite accumulation in macrophages. Molecular docking and molecular dynamics simulations indicated that the six compounds engaged a common region of the trypanothione-binding site, although binding was weak and transient and did not rank with their measured antileishmanial potency. (−)-Alkannin was a notable exception, reproducibly forming a ternary complex with the bound substrate, in line with its uncompetitive inhibition. Overall, these findings support LdTryR as a relevant antileishmanial target.