Abstract
Leishmaniasis is the second-most dreaded parasitic disease in the modern world, behind malaria. The lack of effective vaccines demand improved chemotherapy along with the development of lead compounds and newer targets. We report here that the pentacyclic triterpenoid, dihydrobetulinic acid (DHBA), is a novel lead compound for antileishmanial therapy. It acts by targeting DNA topoisomerases. DNA topoisomerase I and II activity was studied using relaxation and decatenation assays. Mechanistic studies were based on the decreased mobility of enzyme-bound DNA compared with free DNA and the differential mobility of nicked and supercoiled monomers in 1% agarose gel. Pulsed field gradient gel electrophoresis, confocal microscopy, and transmission electron microscopy were performed to assess cytotoxicity of the compound and ultrastructural damage of the parasite. Apoptosis was studied by the isolation of DNA from DHBA-treated parasites and subsequent electrophoresis in 1% agarose gel. DHBA inhibits growth of Leishmania donovani promastigotes and amastigotes with an IC50 of 2.6 and 4.1 microM respectively. The compound is a dual inhibitor of DNA topoisomerases that fails to induce DNA cleavage and acts by preventing the formation of enzyme-DNA binary complex, ultimately inducing apoptosis. Treatment of infected golden hamsters with the compound markedly reduces (> 92%) parasitic burden, both in spleen and liver. Interestingly, the 17-decarboxylated analogue, dihydrolupeol, does not inhibit DNA topoisomerase I and II, has no effect on parasitic growth, and also fails to induce apoptosis. DHBA is a potent antileishmanial agent that induces apoptosis by primarily targeting DNA topoisomerases. Therefore it is a strong candidate for use in designing new antileishmanial drugs.
Highlights
Kala-azar, or visceral leishmaniasis, is the most fatal form of leishmaniasis, affecting millions around the world
We describe the antileishmanial activity of dihydrobetulinic acid (DHBA) (Figure 1A) on both L. donovani promastigotes and amastigotes
DHBA Inhibits DNA Topoisomerases I and II of L. donovani DNA relaxation and Kinetoplast DNA (kDNA) decatenation assays were performed with 2 units of topoisomerases I and II with different concentrations of DHBA
Summary
Kala-azar, or visceral leishmaniasis, is the most fatal form of leishmaniasis, affecting millions around the world. Pilot study has demonstrated itraconazole to be a well-tolerated alternative treatment of mucocutaneous leishmaniasis [3] whereas the leaf extract of Kalanchoe pinnata was reported to reduce lesion size in cutaneous leishmaniasis [4]. Various compounds, such as atovaquone [5], WR6026 [6], licochalcone A [7], ilmfosine [8], and formycin B [9] have been reported to inhibit Leishmania donovani infection, the causative agent of visceral leishmaniasis. The only effective new oral compound to date is alkyl phospholipid miltefosine, which is effective against even antimony unresponsive cases [12] and presently is under phase III clinical trials [13]
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