Leishmaniasis, caused by around 20 species of Leishmania, affects millions of people in endemic areas, particularly in tropical and subtropical regions. However, current treatments face challenges due to significant side effects and increasing drug resistance. This study aims to develop new anti-leishmanial therapies by focusing on the threonine biosynthesis pathway, due to its uniqueness in parasites. This pathway allows the parasite to synthesize threonine, an essential amino acid, while humans must obtain it through their diet. Therefore, targeting this pathway presents a promising opportunity for selective drug action. Homoserine kinase (HSK), a rate-limiting enzyme in the threonine biosynthesis pathway, was identified as a promising therapeutic target due to its crucial role in converting L-homoserine to ortho-phospho homoserine, a precursor to threonine. Using a multifaceted approach—including in silico virtual screening, in vitro biochemical assays, and parasite culture techniques—we screened the Maybridge chemical library and discovered two HSK inhibitors, RH00038 and S02587, with strong potential for further development. Both inhibitors demonstrated significant efficacy, inducing mortality in L. donovani parasites at both the amastigote (intracellular) and promastigote (extracellular) stages, effectively targeting multiple life stages vital to the parasite's life cycle. Of these, S02587 showed particularly high selectivity, effectively killing the parasite while preserving host macrophage cell viability at the same dose, and minimizing risks of side effects on human cells. This selective toxicity underscores S02587's promise as a therapeutic candidate for leishmaniasis with fewer adverse effects. The findings establish HSK as a viable drug target for L. donovani and validate S02587 as a potent and selective inhibitor worth further exploration. By focusing on essential, parasite-specific metabolic pathways, this study advances a strategic approach for safer, more effective anti-leishmanial therapies, potentially leading to new treatment options with improved safety profiles.
