Novel betulin derivatives as antileishmanial agents with mode of action targeting type IB DNA topoisomerase

Molecular Pharmacology (Impact Factor: 4.12). 10/2011; 80(4):694.

ABSTRACT Toward developing antileishmanial agents with mode of action targeted to DNA topoisomerases of Leishmania donovani, we have synthesized a large number of derivatives of betulin. The compound, a natural triterpene isolated from the cork layer of Betula spp. plants exhibits several pharmacological properties. Three compounds (disuccinyl betulin, diglutaryl dihydrobetulin, and disuccinyl dihydrobetulin) inhibit growth of the parasite as well as relaxation activity of the enzyme type IB topoisomerase [Leishmania donovani topoisomerase I (LdTOP1LS)] of the parasite. Mechanistic studies suggest that these compounds interact with the enzyme in a reversible manner. The stoichiometry of these compounds binding to LdTOP1LS is 1:1 (mole/mole) with a dissociation constant on the order of ∼10(-6) M. Unlike CPT, these compounds do not stabilize the cleavage complex; rather, they abrogate the covalent complex formation. In processive mode of relaxation assay condition, these compounds slow down the strand rotation event, which ultimately affects the relaxation of supercoiled DNA. It is noteworthy that these compounds reduce the intracellular parasite burden in macrophages infected with wild-type L. donovani as well as with sodium antimony gluconate resistant parasite (GE1). Taken together, our data suggest that these betulin derivatives can be exploited as potential drug candidates against threatening drug resistant leishmaniasis.

  • [Show abstract] [Hide abstract]
    ABSTRACT: Although leishmanial infections of humans occur globally, the major health impact lies in developing nations, thus, leishmaniases remain "neglected" diseases for new drugs development. Multidrug resistance has been documented in most countries where leishmaniases is endemic. Betulin is a widely available and affordable natural product exerting leishmanicidal activity at micromolar concentration. In this study, the molecular mechanisms of death that contribute to the anti-leishmanial activity of betulin are investigated. In promastigotes, betulin stimulated reactive oxygen species generation at micromolar concentrations in Leishmania. Apoptosis was observed in betulin-treated promastigotes using flow cytometric analysis of treated cells stained with annexin V-FITC and propidium iodide. Furthermore, betulin treatment of promastigotes led to mitochondrial membrane damage, activation of caspase-like proteases, and DNA fragmentation in Leishmania donovani promastigotes. Betulin treatment of amastigotes cultured within macrophages, resulted in a reduced number of amastigotes, with no substantive cytotoxic damage to the host macrophage cells at leishmanicidal drug concentrations.
    The American journal of tropical medicine and hygiene 01/2014; · 2.53 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Leishmania, a protozoan parasite, causes a wide range of human diseases ranging from the localized self-healing cutaneous lesions to fatal visceral leishmaniasis. Toxicity of traditional first line drugs and emergence of drug-resistant strains have worsened the situation. DNA topoisomerase II in kinetoplastid protozoan parasites are of immense interest as drug target because they take part in replication of unusual kinetoplast DNA network. In this study, we have taken target-based therapeutic approaches to combat leishmaniasis. Two isobenzofuranone compounds, viz., (1) 3,5-bis(4-chlorophenyl)-7-hydroxyisobenzofuran-1(3H)-one (JVPH3) and (2) (4-bromo)-3′-hydroxy-5′-(4-bromophenyl)-benzophenone(JVPH4) were synthesized chemically and characterized by NMR and mass spectrometry analysis. Activity of type II DNA topoisomerase of leishmania (LdTOPII) was monitored by decatenation assay and plasmid cleavage assay. The antiparasitic activity of these compounds was checked in experimental BALB/c mice model of visceral leishmaniasis. Isobenzofuranone derivatives exhibited potent antileishmanial effect on both antimony (Sb) sensitive and resistant parasites. Treatment with isobenzofuranone derivatives on promastigotes caused induction of reactive oxygen species (ROS)-mediated apoptosis like cell death in leishmania. Both the compounds inhibited the decatenation activity of LdTOPII but have no effect on bi-subunit topoisomerase IB. Treatment of LdTOPII with isobenzofuranone derivatives did not stabilize cleavage complex formation both in vitro and in vivo. Moreover, treatment with isobenzofuranone derivatives on Leishmania donovani-infected mice resulted in clearance of parasites in liver and spleen by induction of Th1 cytokines. Taken together, our data suggest that these compounds can be exploited as potential antileishmanial agents targeted to DNA topoisomerase II of the parasite.
    Pharmacology Research & Perspectives. 12/2014; 2(6).
  • [Show abstract] [Hide abstract]
    ABSTRACT: We report a novel antileishmanial formulation of betulin (BET) attached to functionalized carbon nanotubes (f-CNTs). We conjugated betulin, a pentacyclic triterpenoid secondary metabolite, to carboxylic acid chains on f-CNTs to obtain BET attached functionalized carbon nanotubes (f-CNT-Bet). The drug release profile demonstrated a fairly slow release of BET. The in-vitro cytotoxicity of BET, f-CNT and f-CNT-BET on J774A.1 macrophage cell line were 211.05±7.14μg/ml; 24.67±3.11μg/ml and 72.63±6.14μg/ml, respectively. The IC50 of BET and f-CNT-BET against intracellular L. donovani amastigotes were 8.33±0.41μg/ml and 0.69±0.08μg/ml, respectively. The results demonstrate better antileishmanial efficiency of f-CNT-BET formulation than BET alone and with no significant cytotoxicity observed on host cells.
    Parasitology International 07/2014; · 2.11 Impact Factor