Dihydrobetulinic Acid Induces Apoptosis in Leishmania donovani by Targeting DNA Topoisomerase I and II: Implications in Antileishmanial Therapy

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 IC 50 of 2.6 and 4.1 M 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.

Toxicity of betulin derivatives and in vitro effect on promastigotes and amastigotes of Leishmania infantum and L. donovani

“…Some compounds did not show any noticeable toxicity with this method (2, 7, 16, 17 and 20), and no apparent structure-activity relationship was observed between toxicity for the J774 cells and the compound subgroup (that is, derivatives having C-3 ketone carbonyl: compounds 10 and 11 were toxic, but L-aspartyl amide of betulinic acid 12 was non-toxic). However, all betulin derivatives having both hydroxyl groups acetylated (6,16,17,18) were non-toxic to J774 cells. It is noticeable that even betulin 1 exhibited certain toxicity (18.4 ± 0.4% inhibition).…”

“…In the subgroup of heterocyclic betulin derivatives (16)(17)(18)(19)(20), clearer structure-activity relationship trend could be observed. Compounds with sterically less bulky R3 and R4 groups showed better activity, when compared with more hindered derivatives.…”

“…The concentration of dimethyl sulfoxide in the working solution was no greater than 0.5% in any experiment. A standard concentration of 50 mM was used in the first screenings after Chowdhury et al 18 Approximate IC 50 of compound 16 was estimated with a range of concentrations (0, 10, 25 and 50 mM).…”

“…On the other hand, 28-O-cinnamoylbetulin 2 was less active against amastigotes. From the subgroup of heterocyclic betulin derivatives (16)(17)(18)(19)(20), compounds 16, …”

“…14,15 It has been suggested that betulinic acid has anticancer, anti-HIV and antimicrobial properties. 16,17 Some related compounds have shown antileishmanial activity against L. donovani (promastigotes and amastigotes) 18 and L. amazonensis. 19 More recently, studies have been carried out on the effect of a high number of betulinic derivatives on axenic amastigotes and amastigotes of L. donovani in the THP-1 cell line.…”