Sushma Rathaur

Antifilarial Activity of 1,3-Diarylpropen-1-One: Effect on Glutathione-S-Transferase, a Phase II Detoxification Enzyme

Chalcone derivatives were evaluated for their antifilarial activity on Setaria cervi using glutathione-S-transferase (GST) as a drug target. The compounds 1-(4-benzotriazol-1-yl-phenyl)-3-(4-methoxyphenyl)prop-2-en-1-one (5), and 3-(4-methoxyphenyl)-1-(4-pyrrolidin-1-yl-phenyl) prop-2-en-1-one (7) showed a significant suppression (P < 0.01) in GST activity of adult female parasite extract at 3 microM concentration in vitro. However, GST activity was detected along with depletion in GSH level. Except Compounds 1 and 2, all exhibited a significant effect on the motility and viability of adult parasites. Compounds 3-(4-chlorophenyl)-1-(4-piperidin-1-yl-phenyl)prop-2-en-1-one (3), 1-(4-benzotriazol-1-yl-phenyl)-3-(4-methoxyphenyl)prop-2-en-1-one (5), and 3-(4-methoxyphenyl)-1-(4-pyrrolidin-1-yl-phenyl) prop-2-en-1-one (7) exhibited major irreversible effects on viability and resulted in parasite death and also inhibited the GST activity by 84-100% in vitro. We report for the first time the antifilarial activity of chalcones on GST of adult parasites. This study also strengthens our previous findings where GST is reported as a potential drug target for antifilarials.

Expression of rd29A::AtDREB1A/CBF3 in tomato alleviates drought-induced oxidative stress by regulating key enzymatic and non-enzymatic antioxidants

Transgenic tomato lines (cv. Kashi Vishesh) over-expressing AtDREB1A/CBF3 driven by stress-inducible rd29A promoter showed significantly higher activities of key antioxidant enzymes when exposed to water-deficit for 7, 14, and 21 days. Transgenic tomato plants exposed to water-deficit recorded lower levels of hydrogen peroxide and superoxide anion formation compared to the non-transgenic plants, suggesting alleviation of reactive oxygen species (ROS). A significant increase in activities of enzymes superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), glutathione reductase (GR), dehydroascorbate reductase (DHAR), and monodehydroascorbate reductase (MDHAR) was observed in response to the different durations of water-deficit conditions. In contrast, enzyme guaiacol peroxidase (POD) activity was lower in the transgenic lines and showed a negative correlation with ROS, ascorbic acid (AsA), and glutathione levels. The concentrations of AsA, glutathione and their reduced forms were higher in the transgenic plants and increased with ROS levels. These results indicate that AtDREB1A transgenic tomato lines are better adapted to water-deficit as they showed lower drought-induced oxidative stress due to activation of the antioxidant response.

Filarial glutathione-S-transferase: A potential vaccine candidate against lymphatic filariasis

Present report enumerates the vaccine potential of a glutathione-S-transferase purified from Setaria cervi against lymphatic filariasis. In jirds (Meriones unguiculatus) vaccination trial, a very significant 82.75% (p<0.005) reduction in adult parasite burden was observed in ScGST immunized group after 90 days post Brugia malayi L3 challenge. An inverse correlation between the antibody level and worm burden was found in ScGST immunized group (Persons correlation r=0.943, p<0.05). No recoveries of worms were obtained in heart and lungs of vaccinated group. The Antibodies reactive to ScGST appeared within four weeks of first dose and were able to neutralize the GST activity up to 86%. In an earlier study we have shown vaccine potential of ScGST against B. malayi by ADCC. Evaluation of cytokine profile in T-cells isolated from BALB/c mice immunized with ScGST were also showed predominance of Th2 response which further maintained the humoral immunity generated by ScGST administration in mice. The overall observations prompted us to envisage ScGST as a potential vaccine candidate against lymphatic filariasis.

Structural modeling and simulation studies of Brugia malayi glutathione-S-transferase with compounds exhibiting antifilarial activity: implications in drug targeting and designing.

Since glutathione-S-transferase (GST) mediated xenobiotic detoxification is a crucial mechanism in nematodes survival, we aimed to conduct an in silico analysis of filarial GST in order to predict the possible interactions for antifilarials. Present report depicts the homology modeling approach applied in the construction of molecular structure of Brugia malayi GST (BmGST) followed by its docking simulation with available antifilarials such as diethylcarbamazine, albendazole, Butylated Hydroxyanisole (BHA) and substituted chalcones. A very low root mean square deviation (0.82A) from template structure and stereochemical quality of constructed BmGST model proposed it as a significant framework for further analysis. In docking studies antifilarials and chalcones exhibited demarcation in their binding affinity and modes. Amongst all the compounds studied, albendazole and methyl-substituted chalcone showed the lowest binding energy and occupied binding pocket near to substrate binding site of GST. The side chain of these compounds interplayed as a potential interaction site which targeted mainly hydrophilic residues of the BmGST. The structural information and binding site mapping of BmGST for different antifilarials obtained from this study could aid in screening and designing new antifilarials or selective inhibitors for chemotherapy against filariasis.

Combination of DEC plus aspirin induced mitochondrial mediated apoptosis in filarial parasite Setaria cervi

Diethylcarbamazine (DEC) is the main drug used against lymphatic filariasis but it is only microfilaricidal. Hence there is an urgent need for adulticidal drug. Aspirin is known nonsteroidal anti-inflammatory drugs which can inhibit prostaglandin H synthase and also induces apoptosis. Studies presented in this paper demonstrated that exposure of worms to the combination of DEC plus aspirin (DEC + A) at 100 microM concentration irreversibly paralyzed adult worms as well as microfilariae within 2 h. Some of the apoptosis markers viz; DNA fragmentation with accompanying ladder formation, upregulation of Bax expression and decrease in Bcl-2 have suggested that the parasite may be killed due to mitochondrial mediated apoptosis. The levels of several apoptosis regulating proteins and enzymes have also shown to be altered. DEC + A treated worms showed significant decrease in prostaglandin H synthase activity (PGHS) and increase in the level of nitric oxide (NO) and cysteine proteases while glutathione (GSH) and peroxidase level was found to be decreased. NO is known inducer of mitochondrial mediated apoptosis and acts by increasing the permeability of mitochondrial membrane through Bax and allowing cytochrome c to release in cytosol, inducing caspases leading to apoptosis. The DEC + A concentration used in this study is much lower than recommended dose so its intake is safe. Here we report for the first time that combination of DEC and aspirin is more effective and could be used as an adulticidal for control of human filarial infections.

Designing, synthesis of selective and high-affinity chalcone-benzothiazole hybrids as Brugia malayi thymidylate kinase inhibitors: In vitro validation and docking studies

In our continuing search for safe and efficacious antifilarials, a series of novel chalcone-benzothiazole hybrids have been synthesized and evaluated for their Brugia malayi thymidylate kinase (BmTMK) enzyme inhibition activity. Their selectivity towards BmTMK was studied and compared to the human TMK (HsTMK) by an in silico method. Out of seventeen derivatives, compounds 34 and 42 showed higher interactions with the BmTMK active site. MolDock docking model revealed the interactions of these two derivatives and the results corroborated well with their in vitro antifilarial activities. Our studies suggest that these hybrids are selective towards the BmTMK enzyme and may serve as potential therapeutic agents against filariasis.

Brevundimonas diminuta mediated alleviation of arsenic toxicity and plant growth promotion in Oryza sativa L.

Arsenic (As), a toxic metalloid adversely affects plant growth in polluted areas. In the present study, we investigated the possibility of improving phytostablization of arsenic through application of new isolated strain Brevundimonas diminuta (NBRI012) in rice plant [Oryza sativa (L.) Var. Sarju 52] at two different concentrations [10ppm (low toxic) and 50ppm (high toxic)] of As. The plant growth promoting traits of bacterial strains revealed the inherent ability of siderophores, phosphate solubilisation, indole acetic acid (IAA), 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase production which may be associated with increased biomass, chlorophyll and MDA content of rice and thereby promoting plant growth. The study also revealed the As accumulation property of NBRI012 strain which could play an important role in As removal from contaminated soil. Furthermore, NBRI012 inoculation significantly restored the hampered root epidermal and cortical cell growth of rice plant and root hair elimination. Altogether our study highlights the multifarious role of B. diminuta in mediating stress tolerance and modulating translocation of As in edible part of rice plant.

Secretory acetylcholinesterase of Setaria cervi microfilariae and its antigenic cross‐reactivity with Wuchereria bancrofti

Setaria cervi, a bovine filarial parasite, secretes acetylcholinesterase during in vitro cultivation. A significant amount of enzyme activity was detected both in culture media and somatic extracts of different developmental stages of the parasite. The microfilarial stage showed a higher level of AChE activity than adult worms, with females being considerably more active than males. The secretory enzyme from microfilariae preferentially utilized acetylthiocholine iodide as substrate and showed two electrophoretically distinct isoforms in native PAGE. Secretory enzyme was purified from the excretory/secretory products of microfilariae using edrophonium chloride linked to epoxy‐activated sepharose. Analysis of purified acetylcholinesterase by SDS‐PAGE revealed the existence of two proteins of 75kD and 45kD under non‐reducing conditions. These secretory enzymes are antigenic and cross‐reactive with Wuchereria bancrofti‐infected asymptomatic microfilaraemic human sera when tested by enzyme linked immunosorbent assay and immunoblotting. The secretory AChE(s) from S. cervi microfilariae may be utilized for diagnosis of early filarial infections.

Effect of diethylcarbamazine, butylated hydroxy anisole and methyl substituted chalcone on filarial parasite Setaria cervi: Proteomic and biochemical approaches

For survival, parasite exerts several lines of defense of which drug neutralization is one of the major phenomena. Lack of phase I cytochrome P450 in some of the nematode render them depend on the phase II detoxification system involving GST as a major detoxifying enzymes. In present study, the antifilarial DEC, phenolic compound BHA and methyl chalcone have been evaluated for proteomic and biochemical studies in Setaria cervi. BHA and methyl chalcone showed cytotoxic effect leading to irreversible inhibition in motility and viability of parasites. These drugs showed marked alteration in proteomic profile of S. cervi at 100 μM concentration with 10.82, 8.52 and 6.75% downregulated (<0.5) and 7.64, 31.78 and 24.32% upregulated (>1.5) in DEC, BHA and methyl chalcone treatment respectively. Significant depletion in GSH level with increase in NO production was observed. Amongst these compounds, methyl chalcone demonstrated significant inhibitory effect (p<0.05) on GST, PGHS and PTP activity leading to loss of metabolic homeostasis and parasite death. The cytotoxic response and altered expression profile of major enzymes under drug exposure suggested the oxidative stress induced apoptosis as a major cause of parasite killing which was further supported by DNA fragmentation in BHA and methyl chalcone.

Brugia malayi and Wuchereria bancrofti: gene comparison and recombinant expression of π-class related glutathione S-transferases

The nucleotide sequences for Brugia malayi and Wuchereria bancrofti GST have been submitted to EMBL, GenBank, and DDBJ Nucleotide Sequence Databases under Accession Nos. Y12788 and AY195867.