Lopinavir targets Leishmania topoisomerase I and its combination with Ritonavir exhibits enhanced antileishmanial efficacy in clinical isolates of Leishmania donovani.

DNA topoisomerase I of Leishmania donovani (LdTopILS) represents a specific enzymatic vulnerability with structural and catalytic features distinct from its human counterpart. Targeting this enzyme offers opportunities for selective therapeutic intervention. In this study, we demonstrate that the HIV-1 protease inhibitor lopinavir (LPV), particularly in combination with ritonavir (RTV), functions as a selective catalytic inhibitor of LdTopILS. Biochemical analyses reveal that LPV inhibits LdTopILS activity without stabilizing the topoisomerase-DNA cleavable complex, distinguishing its mechanism from classical topoisomerase poisons. Importantly, LPV exhibits marked selectivity over human topoisomerase I, highlighting its biochemical specificity. At the cellular level, LPV-RTV treatment induces an oxidative imbalance characterized by increased reactive oxygen species generation, depletion of intracellular glutathione, and enhanced lipid accumulation and lipid peroxidation. These biochemical alterations precede mitochondrial membrane depolarization and cytochrome c release, culminating in DNA fragmentation through a caspase-independent pathway. Importantly, mitochondrial DNA-depleted parasites fail to undergo LPV-RTV induced oxidative stress and mitochondrial dysfunction, establishing the indispensability of functional kinetoplast mitochondria for drug mediated cytotoxicity. Thus, the present study establishes LPV as a mechanistically defined catalytic inhibitor with selective biochemical activity against L. donovani and identify a topoisomerase I linked mitochondrial cell death pathway in kinetoplastids, supporting its potential as a targeted antileishmanial strategy.