Prakash Saudagar

Miltefosine-unresponsive Leishmania donovani has a greater ability than miltefosine-responsive L. donovani to resist reactive oxygen species

Resistance of Leishmania parasites to miltefosine, which is only available oral drug, is a great concern. We have analyzed global gene expression profiles of miltefosine‐unresponsive and miltefosine‐responsive Leishmania donovani in order to understand the various metabolic processes involved in miltefosine drug resistance. The microarray data clearly indicated a role of oxidative metabolism in miltefosine resistance. Furthermore, fluorescence microscopy experiments suggested that miltefosine‐unresponsive L. donovani resists the accumulation of reactive oxygen species and subsequent mitochondrial membrane damage leading to apoptotic death. In contrast, in miltefosine‐responsive L. donovani, the accumulation of reactive oxygen species causes apoptotic death. Overall, this study provides fundamental insights into miltefosine resistance in L. donovani.

Molecular mechanism underlying antileishmanial effect of oxabicyclo[3.3.1]nonanones: Inhibition of key redox enzymes of the pathogen

We report oxabicyclo[3.3.1]nonanones as inhibitors of key redox enzymes, trypanothione synthetase (TryS), and trypanothione reductase (TryR) of Leishmania. Further, detailed cellular effects of 4-(4,4,8-Trimethyl-7-oxo-3-oxabicyclo[3.3.1]non-2-yl)-benzoic acid methyl ester, a oxabicyclo[3.3.1]nonanones, on the parasite were investigated. As these compounds inhibit key redox enzymes (TryR amd TryS), treatment of these compounds resulted in increased reactive oxygen species (ROS), mitochondrial membrane damage, activation of caspase like proteases, and DNA damage that finally leads to apoptosis. Although the compound has modest IC50 value against parasite (4.9±0.4 μM), they identify a novel chemical space to design and develop drugs based on these compounds against the Leishmania parasite. This is first report of oxabicyclo[3.3.1]nonanones as antileishmanial.

Cloning, expression, characterization and inhibition studies on trypanothione synthetase, a drug target enzyme, from Leishmania donovani.

Abstract Trypanothione synthetase, a validated drug target, synthesizes trypanothione from glutathione and spermidine. Here we report the gene cloning, expression, characterization and inhibition studies of trypanothione synthetase from Leishmania donovani (LdTryS). The purified recombinant LdTryS enzyme obeyed Michaelis-Menten kinetics. High substrate inhibition was observed with glutathione (Km=33.24 μm, kcat=1.3 s-1, Ki=866 μm). The enzyme shows simple hyperbolic kinetics with fixed glutathione concentration and with other substrates limiting Km values for Mg. ATP and spermidine of 14.2 μm and 139.6 μm, respectively. LdTryS was also screened for inhibitors. Tomatine, conessine, uvaol and betulin were identified as inhibitors of the enzyme and were tested for leishmanicidal activity. Finally, the effect of LdTryS inhibitors on redox homeostasis of the parasite gives a broader picture of their action against leishmaniasis.

Evaluation of a diospyrin derivative as antileishmanial agent and potential modulator of ornithine decarboxylase of Leishmania donovani

World health organization has called for academic research and development of new chemotherapeutic strategies to overcome the emerging resistance and side effects exhibited by the drugs currently used against leishmaniasis. Diospyrin, a bis-naphthoquinone isolated from Diospyros montana Roxb., and its semi-synthetic derivatives, were reported for inhibitory activity against protozoan parasites including Leishmania. Presently, we have investigated the antileishmanial effect of a di-epoxide derivative of diospyrin (D17), both in vitro and in vivo. Further, the safety profile of D17 was established by testing its toxicity against normal macrophage cells (IC₅₀∼20.7 μM), and also against normal BALB/c mice in vivo. The compound showed enhanced activity (IC₅₀∼7.2 μM) as compared to diospyrin (IC₅₀∼12.6 μM) against Leishmania donovani promastigotes. Again, D17 was tested on L. donovani BHU1216 isolated from a sodium stibogluconate-unresponsive patient, and exhibited selective inhibition of the intracellular amastigotes (IC₅₀∼0.18 μM). Also, treatment of infected BALB/c mice with D17 at 2mg/kg/day reduced the hepatic parasite load by about 38%. Subsequently, computational docking studies were undertaken on selected enzymes of trypanothione metabolism, viz. trypanothione reductase (TryR) and ornithine decarboxylase (ODC), followed by the enzyme kinetics, where D17 demonstrated non-competitive inhibition of the L. donovani ODC, but could not inhibit TryR.

Screening natural products database for identification of potential antileishmanial chemotherapeutic agents

Leishmaniasis is a parasitic infection caused by unicellular protozoan organism belonging to the family Trypanosomatidae. Among various forms of the disease, visceral leishmaniasis is the most lethal and caused by Leishmania infantum or Leishmania donovani. The redox metabolism of parasite requires a key enzyme, trypanothione reductase which is a validated drug target. In the past decade, it was observed that these protozoan parasites had developed resistance against many of available drugs. Importantly in the case of visceral leishmaniasis drug resistance is very high in the Indian subcontinent, a major endemic region of Leishmania donovani infection. In search for new drugs, we aim to identify potential natural product inhibitors of trypanothione reductase which can be further developed as anti-leishmanial drug. We have performed in silico virtual screening of a natural product data set of 800 diverse chemical entities. Leishmania infantum trypanothione reductase crystal structure (PDB ID: 2JK6) was used in the virtual screening process, docking studies to identify potential lead compounds. The compounds were sorted based upon their binding energy and the top 50 ranked protein-inhibitor complexes were clustered using AuPosSOM to ligand foot print the interactions. We report a few alkaloids and sterols for the first time, which could be potential trypanothione reductase inhibitors. The footprinting of protein-inhibitor interactions into clusters has also provided clues on various possible orientations that inhibitors can attain at the active site of Trypanothione reductase. Moreover, biological significance of the interactions has also been discussed.

Carbon nanotube based betulin formulation shows better efficacy against Leishmania parasite

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 cytotoxicities 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 Leishmania 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.

Discovery of novel anti-leishmanial agents targeting LdLip3 lipase

Leishmaniasis is a neglected tropical disease, caused by several species of Leishmania. Being an opportunistic lipid-scavenging pathogen, Leishmania relies extensively on lipid metabolism especially for host-pathogen interaction, utilizing host lipids for energy and virulence. The rational approach is to target lipid metabolism of the pathogen focusing lipid-catabolizing lipases. The LdLip3 lipase is considered as drug target as it is constitutively expressed in both promastigote and amastigote forms. Since the LdLip3 structure is not known, we modeled its three-dimensional structure to implement structure-based drug discovery approach. Similarity-based virtual screening was carried out to identify potential inhibitors utilizing NCI diversity set on ZINC database including natural products. Implementing computational and experimental approaches, four anti-leishmanial agents were discovered. The screened molecules ZINC01821375, ZINC04008765, ZINC06117316 and ZINC12653571 had anti-leishmanial activity with IC50 (% viable promastigotes vs. concentration) of 5.2±1.8μM, 13.1±2.6μM, 9.4±2.6μM and 17.3±3.1μM, respectively. The molecules showed negligible toxicity toward mouse macrophages. Based on the contact footprinting analysis, new molecules were designed with better predicted free energy of binding than discovered anti-leishmanial agents. Further validation for the therapeutic utility of discovered molecules can be carried out by the research community to combat leishmaniasis.

Episomal expression of human glutathione reductase (HuGR) in Leishmania sheds light on evolutionary pressure for unique redox metabolism pathway: Impaired stress tolerance ability of Leishmania donovani

Trypanothione based redox metabolism is unique to the Trypanosomatida family. Despite extensive studies on redox metabolism of Leishmania parasites, a prominent question of why Leishmania adopt this unique redox pathway remains elusive. We have episomally expressed human glutathione reductase (HuGR) in Leishmania donovani (LdGR+) and investigated its effect. LdGR+ strain has slower growth compared to the wild type (Ld) indicating decreased survival ability of the strain. Further, LdGR+ strain showed enhanced accumulation of intracellular reactive oxygen species (ROS) and more sensitivity to the anti-leishmanial drug, Miltefosine, inferring increased stress level. In contrast, the expression analyses of genes specific to redox metabolism were increased significantly in LdGR+ strain compared to wild type. Lower infectivity index of the LdGR+ strain substantiated the above findings and indicated that the expression of HuGR reduces the stress tolerance ability of the parasite. From molecular docking studies with HuGR, it was observed that oxidized trypanothione (TS2) binds much better than oxidized glutathione (GS2). These results also give us hints that the parasite is losing infectivity potential due to an overall increase in intracellular stress caused with the expression of HuGR, showcasing a possible role of evolutionary pressure on the Leishmania parasites posed by HuGR.