TLR7/8 agonist HYBRID2 as a dual-action compound against Leishmania donovani: antiparasitic and immunomodulatory effects

Abstract Background Visceral leishmaniasis (VL) remains a fatal parasitic disease with limited treatment options and growing drug resistance. To address this challenge, we synthesized and characterized HYBRID2, an imidazoquinoline-based TLR7/8 agonist and explored its untapped potential against Leishmania through in vitro and in silico studies. Results HYBRID2 effectively eliminated both promastigote and intracellular amastigote forms of Leishmania donovani (Promastigote IC 50 : 6.50 ± 2.20 µM; Amastigote IC 50 : 7.56 ± 1.58 µM), while exhibiting moderate cytotoxicity (Raw macrophages CC 50 : 42.30 ± 3.88 µM; HeLa CC 50 : 36.66 ± 4.37 µM). The mechanistic investigation confirmed that HYBRID2 induced a marked arrest of the parasite cell cycle in the sub-G 0 /G 1 population (84.64% vs. 7.23% in untreated; p < 0.0001), significantly inhibited parasite growth, and robustly enhanced Reactive Oxygen Species (ROS) (607.66 mean fluorescence intensity; p < 0.0001) and Nitric Oxide (NO) production (22.53 ± 0.73 µM; p < 0.0001), indicating a strong immunomodulatory role associated through its dual TLR7/8 agonist activity. Interestingly, the in silico studies and ADMET analyses of HYBRID2 suggested strong multi-target interactions, with the strongest affinity observed for anti-leishmanial drug target trypanothione reductase, alongside favorable pharmacokinetic properties. Conclusions This study highlights an innovative and disease-relevant application of a dual TLR7/8 agonist with a distinct immunopharmacological profile, reinforced by strong in silico target engagement, underscoring its potential for host-directed therapeutic development against visceral leishmaniasis. Graphical abstract