The lignan niranthin poisons Leishmania donovani topoisomerase IB and favours a Th1 immune response in mice

Abstract

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.

Disuccinyl Betulin Triggers Metacaspase-Dependent Endonuclease G-Mediated Cell Death in Unicellular Protozoan Parasite Leishmania donovani

“…Topoisomerases are DNA manipulators that relieve the torsional strain in DNA that is built up during vital cellular processes. The heterodimeric topoisomerase IB of Leishmania has been established as an attractive therapeutic target (10). In higher eukaryotes, socalled DNA sensors recognize inhibitor trapped topoI-DNA cleavable complex and activate Bax to subtly permeabilize the mitochondrial outer membrane.…”

A Novel Spirooxindole Derivative Inhibits the Growth of Leishmania donovani Parasites both In Vitro and In Vivo by Targeting Type IB Topoisomerase

“…The reactions were rapidly quenched using stop solution and kept on ice. The gels were stained with ethidium bromide (EtBr) (0.5 g/ml), and the amount of supercoiled monomer DNA band fluorescence was quantified by integration using Gel Doc 2000 under UV illumination (Bio-Rad Quantity One software) as described previously (18). Initial velocities (nanomolar DNA base pairs relaxed per minute) were calculated using the following equation: initial velocity ϭ Plasmid cleavage assay.…”

“…Initial velocities (nanomolar DNA base pairs relaxed per minute) were calculated using the following equation: initial velocity ϭ Plasmid cleavage assay. Cleavage assay was carried out as described previously (18). Briefly, 50 fmol of pHOT1 supercoiled DNA (containing the topoisomerase I cleavage site) and 100 fmol of reconstituted LdTopIB were incubated in a standard reaction mixture (50 l) containing 50 mM Tris-HCl (pH 7.5), 100 mM KCl, 10 mM MgCl 2 , 0.5 mM DTT, 0.5 mM EDTA, and 30 g/ml BSA in the presence of various concentrations of compound 4c at 37°C for 30 min.…”

“…Macrophages were isolated from BALB/c mice (female, 4 to 6 weeks old) at 36 to 48 h after injection (intraperitoneal) with 2% (wt/vol) hydrolyzed starch by peritoneal lavage with ice-cold phosphate-buffered saline. In vitro infection with promastigotes of the AG83 wild-type strain, laboratory-grown SAG-resistant (Sb r ) strain GE1, the laboratory-grown miltefosine-resistant (MIL r ) strain, and the camptothecin-resistant (CPT r ) strain were carried out as described previously (18). Compound 4c was added at different concentrations (0.25, 0.5, 0.75, 1, 1.25, 1.5, 1.75, and 2 M) to infected macrophages and left for another 24 h. Cells were then fixed in methanol and stained with 2% Giemsa stain.…”

“…This unique bisubunit topoisomerase IB of the kinetoplastid parasites is a very attractive chemotherapeutic target because of its difference in structure from human topoisomerase I (13). Several L. donovani topoisomerase IB (LdTopIB) poisons which can stabilize the DNA-LdTopIB cleavable complex and kill Leishmania parasite have been reported in literature, viz., camptothecin (14), diospyrin (15), 3,3=-diindolyl methane (DIM) (16), baicalein, luteolin, and quercetin (17), niranthin (18), lyoniside and saracoside (19), etc. Catalytic inhibitors of LdTopIB are not well studied, and only a few, such as dihydrobetulinic acid (DHBA) (20) and betulin and dihydrobetulin derivatives (21), have been reported.…”