Discovery of chalcone derivatives as antileishmanial agents: phenotypic screening, exploratory docking and enzymatic assays on trypanothione reductase and related flavoproteins.

Leishmaniasis is a neglected tropical disease caused by intracellular protozoa of the genus Leishmania. As current treatments of leishmaniasis are limited by toxicity, drug resistance, and difficult administration, novel therapies targeting parasite-specific pathways are needed. In this context, 25 flavonoid-related compounds, including 19 chalcone analogues, were evaluated for their antiparasitic activity against Leishmania. Among the tested compounds, chalcones 1, 2, 5, and 12 showed potent inhibitory effects on the growth of promastigotes and amastigotes of Leishmania infantum as well as other Leishmania species. These lead compounds were selectively toxic to the parasite, but not to human cells. Furthermore, chalcones 1 and 5 strongly suppressed cytokines (IL-1β, IL-18 and IL-10) release in Leishmania-infected macrophages. Given the structural similarity of the chalcone scaffold to flavin cofactors, we hypothesized that the lead chalcones may preferentially interfere with Leishmania flavoproteins. To test this, we computationally explored the cofactor-binding regions of four parasite flavoproteins to assess their affinity for chalcones 1, 2, 5, and 12. Among them, the FAD binding pocket of trypanothione reductase (TR), a Leishmania-specific flavoprotein absent in humans, showed the highest predicted binding affinity for the investigated chalcones. However, in vitro enzymatic assays revealed only modest and compound-specific inhibition of TR activity. In conclusion, we identified promising chalcone derivatives with potent in vitro activity against Leishmania and favourable selectivity. While TR represents a suitable target for in silico analyses, our findings suggest that disrupting parasite redox homeostasis rather than selective TR inhibition could contribute to the antileishmanial effects of these chalcones.