Systems-Driven Identification and Experimental Validation of Actinomycetes-Derived Secondary Metabolites as Promising Antileishmanial Agents Targeting <i>Leishmania donovani</i> Methionyl-tRNA Synthetase

Visceral leishmaniasis (VL), caused mainly by Leishmania donovani in the Indian subcontinent, remains a major health concern due to the limited treatment options. This study aims to identify antileishmanial compounds from StreptomeDB 3.0, a database of Streptomycetes -derived secondary metabolites. High-throughput virtual screening (HTVS) of 6,524 compounds against 17 validated drug targets identified three multitargeting hits. Among them, oxytetracycline showed the highest potency against L. donovani promastigotes (IC 50 of 2.71 μM), comparable to the known antileishmanial drug miltefosine (IC 50 = 2.13 μM). The cytotoxicity evaluation on J774A.1 murine macrophage cells yielded a selectivity index of 32.65, underlining its safety profile. To elucidate its mode of action, we investigated the binding affinity of oxytetracycline with L. donovani methionyl-tRNA synthetase (LdMetRS) using Bio-Layer Interferometry (BLI), which suggested a binding affinity ( K d ) of 23.7 μM. Furthermore, a malachite green functional assay showed oxytetracycline inhibits LdMetRS, (IC 50 = 6.63 μM). These results confirm LdMetRS as one of the targets for oxytetracycline. This study highlights oxytetracycline as a promising repurposed lead for VL using an integrative in-silico screening, phenotypic screening, and biochemical target validation.