title: 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 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.
authors.affiliation: Division of Molecular Parasitology, Indian Institute of Chemical Biology, 4 Raja S.C. Mullick Road, Kolkata 700032, India.
authors.forename: Arnab Roy
authors.initials: AR
authors.lastname: Chowdhury
authors.forename: Suparna
authors.initials: S
authors.lastname: Mandal
authors.forename: Anindya
authors.initials: A
authors.lastname: Goswami
authors.forename: Monidipa
authors.initials: M
authors.lastname: Ghosh
authors.forename: Labanya
authors.initials: L
authors.lastname: Mandal
authors.forename: Debabani
authors.initials: D
authors.lastname: Chakraborty
authors.forename: Agneyo
authors.initials: A
authors.lastname: Ganguly
authors.forename: Gayatri
authors.initials: G
authors.lastname: Tripathi
authors.forename: Sibabrata
authors.initials: S
authors.lastname: Mukhopadhyay
authors.forename: Santu
authors.initials: S
authors.lastname: Bandyopadhyay
authors.forename: Hemanta K
authors.initials: HK
authors.lastname: Majumder
mesh_terms: D000818:Animals; D000981:Antiprotozoal Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D006224:Cricetinae; D004249:DNA Damage / Q000187:drug effects; D004264:DNA Topoisomerases, Type I / Q000378:metabolism; D004250:DNA Topoisomerases, Type II / Q000378:metabolism; D018105:DNA, Kinetoplast / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007893:Leishmania donovani / Q000201:enzymology*; D007898:Leishmaniasis, Visceral / Q000188:drug therapy*; D008647:Mesocricetus; D018613:Microscopy, Confocal; D059004:Topoisomerase I Inhibitors; D059005:Topoisomerase II Inhibitors; D014315:Triterpenes / Q000494:pharmacology*; D000094062:Betulinic Acid
chemical_list: D000981:Antiprotozoal Agents; D004264:DNA Topoisomerases, Type I; D004250:DNA Topoisomerases, Type II; D018105:DNA, Kinetoplast; D004791:Enzyme Inhibitors; D059004:Topoisomerase I Inhibitors; D059005:Topoisomerase II Inhibitors; D014315:Triterpenes; C098641:dihydrobetulinic acid; D000094062:Betulinic Acid