Mitochondria are the principal site for the generation of cellular ATP by oxidative phosphorylation. F0F1-ATP synthase, a complex V of the electron transport chain, is an important constituent of mitochondria-dependent signaling pathways involved in apoptosis. In the present study, we have shown for the first time that 3,3Ј-diindolylmethane (DIM), a DNA topoisomerase I poison, inhibits mitochondrial F0F1-ATP synthase of Leishmania donovani and induces programmed cell death (PCD), which is a novel insight into the mechanism in protozoan parasites. DIM-induced inhibition of F0F1-ATP synthase activity causes depletion of mitochondrial ATP levels and significant stimulation of mitochondrial reactive oxygen species (ROS) production, followed by depolarization of mitochondrial membrane potential (⌬⌿ m ). Because ⌬⌿ m is the driving force for mitochondrial ATP synthesis, loss of ⌬⌿ m results in depletion of cellular ATP level. The loss of ⌬⌿ m causes the cellular ROS generation and in turn leads to the oxidative DNA lesions followed by DNA fragmentation. In contrast, loss of ⌬⌿ m leads to release of cytochrome c into the cytosol and subsequently activates the caspase-like proteases, which lead to oligonucleosomal DNA cleavage. We have also shown that mitochondrial DNA-depleted cells are insensitive to DIM to induce PCD. Therefore, mitochondria are necessary for cytotoxicity of DIM in kinetoplastid parasites. Taken together, our study indicates for the first time that DIM-induced mitochondrial dysfunction by inhibition of F0F1-ATP synthase activity leads to PCD in Leishmania spp. parasites, which could be exploited to develop newer potential therapeutic targets.Apoptosis, a form of programmed cell death (PCD), is a genetically regulated active physiological process of cell suicide that causes cell deletion without inflammation, scarring, or release of cellular contents. Mitochondria of living cells play a pivotal role in controlling life and death (Green and Reed, 1998). Mitochondria are an important cellular source for the generation of reactive oxygen species (ROS) inside the cells (Halliwell and Gutteridge, 1990). Maintenance of proper mitochondrial transmembrane potential (⌬⌿ m ) is essential for the survival of the cell because it derives the synthesis of ATP and maintains oxidative phosphorylation (Gottlieb, 2001).3,3Ј-Diindolylmethane (DIM) is a major acid condensation product of indole-3-carbinol, a natural compound found in vegetables of the genus Brassica. It has an anticarcinogenic effect and inhibits the growth of human cancer cells. DIM-

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Niranthin, a lignan isolated from the aerial parts of the plant Phyllanthus amarus, exhibits a wide spectrum of pharmacological activities. In the present study, we have shown for the first time that niranthin is a potent anti-leishmanial agent. The compound induces topoisomerase I-mediated DNA–protein adduct formation inside Leishmania cells and triggers apoptosis by activation of cellular nucleases. We also show that niranthin inhibits the relaxation activity of heterodimeric type IB topoisomerase of L. donovani and acts as a non-competitive inhibitor interacting with both subunits of the enzyme. Niranthin interacts with DNA–protein binary complexes and thus stabilizes the ‘cleavable complex’ formation and subsequently inhibits the religation of cleaved strand. The compound inhibits the proliferation of Leishmania amastigotes in infected cultured murine macrophages with limited cytotoxicity to the host cells and is effective against antimony-resistant Leishmania parasites by modulating upregulated P-glycoprotein on host macrophages. Importantly, besides its in vitro efficacy, niranthin treatment leads to a switch from a Th2- to a Th1-type immune response in infected BALB/c mice. The immune response causes production of nitric oxide, which results in almost complete clearance of the liver and splenic parasite burden after intraperitoneal or intramuscular administration of the drug. These findings can be exploited to develop niranthin as a new drug candidate against drug-resistant leishmaniasis.

DIM (3,3'-di-indolylmethane), an abundant dietary component of cruciferous vegetables, exhibits a wide spectrum of pharmacological properties. In the present study, we show that DIM is a potent inhibitor of Leishmania donovani topoisomerase I with an IC50 of 1.2 microM. Equilibrium dialysis shows that DIM binds strongly to the free enzyme with a binding constant of 9.73x10(-9) M. The binding affinity of DIM to the small subunit is 8.6-fold more than that of the large subunit of unusual LdTOP1LS (bi-subunit L. donovani topoisomerase I). DIM stabilizes topoisomerase I-DNA cleavage complexes in vitro and also in vivo. Like CPT (camptothecin), DIM inhibits the religation step when the drug was added to preformed topoisomerase I-DNA binary complex. Hence, DIM is similar to CPT with respect to its ability to form the topoisomerase I-mediated 'cleavable complexes' in vitro and in vivo. But unlike CPT, DIM interacts with both free enzyme and substrate DNA. Therefore DIM is a non-competitive class I inhibitor of topoisomerase I. DIM also inhibits the relaxation activity of the CPT-resistant mutant enzyme LdTOP1Delta39LS (N-terminal deletion of amino acids 1-39 of LdTOP1LS). The IC50 values of DIM in simultaneous and enzyme pre-incubation relaxation assays were 3.6 and 2.9 muM respectively, which are higher than that of wild-type topoisomerase I (LdTOP1LS), indicating that the affinity of DIM to LdTOP1Delta39LS is less than that for LdTOP1LS. This is the first report on DIM as an L. donovani topoisomerase I poison. Our study illuminates a new mode of action of enzyme inhibition by DIM that might be exploited for rational drug design in human leishmaniasis.