Nidhi Singh

Increased levels of thiols protect antimony unresponsive Leishmania donovani field isolates against reactive oxygen species generated by trivalent antimony

The current trend of antimony (Sb) unresponsiveness in the Indian subcontinent is a major impediment to effective chemotherapy of visceral leishmaniasis (VL). Although contributory mechanisms studied in laboratory-raised Sb-R parasites include an up-regulation of drug efflux pumps and increased thiols, their role in clinical isolates is not yet substantiated. Accordingly, our objectives were to study the contributory role of thiols in the generation of Sb unresponsiveness in clinical isolates. Promastigotes were isolated from VL patients who were either Sb responsive (n=2) or unresponsive (n=3). Levels of thiols as measured by HPLC and flow cytometry showed higher basal levels of thiols and a faster rate of thiol regeneration in Sb unresponsive strains as compared with sensitive strains. The effects of antimony on generation of reactive oxygen species (ROS) in normal and thiol-depleted conditions as also their H2O2 scavenging activity indicated that in unresponsive parasites, Sb-mediated ROS generation was curtailed, which could be reversed by depletion of thiols and was accompanied by a higher H2O2 scavenging activity. Higher levels of thiols in Sb-unresponsive field isolates from patients with VL protect parasites from Sb-mediated oxidative stress, thereby contributing to the antimony resistance phenotype.

Differential gene expression analysis in antimony-unresponsive Indian kala azar (visceral leishmaniasis) clinical isolates by DNA microarray.

In this study, cDNA microarray analysis of a closely related species, Leishmania major, was used as a screening tool to compare antimonial-resistant and susceptible clinical isolates of Leishmania donovani in order to to identify candidate genes on the basis of antimony resistance. Clinically confirmed resistant isolate 39 and sensitive isolate 2001 were used in this study. Many differentially regulated genes were identified whose expression levels differ in sodium antimony gluconate (SAG)-treated patients. Interestingly, genes on the array, showing changes in expression of over 2-fold revealed the identity of ABC transporters, which are known determinants of drug resistance in laboratory mutants. The functionality of the transporters was validated by flow cytometry which, being biologically informative, provides direct clues to gene function. The results suggest that isolate 39 could have developed resistance by an increased multidrug resistance protein (MRP)-like pump. This study provides preliminary clues to the role of a thiol-dependent efflux system in antimonial resistant clinical isolates of Leishmania donovani.

Plant defense activation and management of tomato root rot by a chitin-fortified Trichoderma/Hypocrea formulation

Tomato root rot caused by Rhizoctonia solani is a major soilborne disease resulting in significant yield loss. The culture filtrates of six isolates of Trichoderma/Hypocrea species were evaluated for in vitro production of hydrolytic enzymes. Results demonstrated that all the six isolates were able to produce chitinase, β-1, 3 glucanase and protease in the range of 76–235xa0μmol GlcNAc min-1 mg-1 protein, 31.90–37.72xa0nmol glucose min-1 mg-1 proteins and 63.05–86.22xa0μmolxa0min-1xa0mg-1 proteins, respectively. Trichoderma/Hypocrea-based formulation(s) were prepared with chitin (1% v:v) and CMC (0.5% w:v) for root rot management in a greenhouse. Root dip application with bioformulation(s) resulted in a significant reduction of the root rot index. In addition, bioformulations increased plant growth attributing traits significantly relative to untreated control. Accumulation of total phenols, peroxidase, polyphenoloxidase and phenylalanine ammonia lyase increased in chitin-supplemented Trichoderma/Hypocrea formulation-treated plants challenged with R. solani. The results suggest that chitin-fortified bioformulation(s) could be an effective system to control root rot of tomato in an eco-compatible manner.

Proteophosphoglycan is differentially expressed in sodium stibogluconate-sensitive and resistant Indian clinical isolates of Leishmania donovani.

Leishmania produce several types of mucin-like glycoproteins called proteophosphoglycans (PPGs) some of which are secreted while others are found on the surface of promastigotes and amastigotes. These proteins are thought to be important in the transmission, invasion and subsequent intracellular survival of parasites. The structure and function of PPGs are species and stage-specific in the case of L. major and L. mexicana, but no such information has hitherto been available for L. donovani. This study presents, for the first time, an initial characterization (localization) of PPG in sodium stibogluconate (SSG)-resistant and sensitive clinical isolates of L. donovani from Bihar (India) by confocal microscopy, flow cytometry and Western blotting using antibodies to L. major PPG. Confocal microscopy analysis revealed that both promastigotes and amastigotes possess PPG on their cell membrane and flagellar pocket membrane but its expression was variable in different isolates. The quantitative analysis by FACS and Western blotting showed that the expression and intensity of PPG bands was higher in SSG-resistant isolates. This study suggests the possibilities of involvement of PPG in drug-resistant mechanisms and of using PPG abundance as a marker for identifying drug-resistant clinical isolates in Indian kala azar.

[6]-Gingerol induces bone loss in ovary intact adult mice and augments osteoclast function via the transient receptor potential vanilloid 1 channel.

SCOPEn[6]-Gingerol, a major constituent of ginger, is considered to have several health beneficial effects. The effect of 6-gingerol on bone cells and skeleton of mice was investigated.nnnMETHODS AND RESULTSnThe effects of 6-gingerol on mouse bone marrow macrophages and osteoblasts were studied. 6-Gingerol-stimulated osteoclast differentiation of bone marrow macrophages but had no effect on osteoblasts. Capsazepine, an inhibitor of TRPV1 (transient receptor potential vanilloid 1) channel, attenuated the pro-osteoclastogenic effect of 6-gingerol or capsaicin (an agonist of TRPV1). Similar to capsaicin, 6-gingerol stimulated Ca(2) + influx in osteoclasts. The effect of daily feeding of 6-gingerol for 5 wk on the skeleton of adult female Balb/cByJ mice was investigated. Mice treated with capsaicin and ovariectomized (OVx) mice served as controls for osteopenia. 6-Gingerol caused increase in trabecular osteoclast number, microarchitectural erosion at all trabecular sites and loss of vertebral stiffness, and these effects were comparable to capsaicin or OVx group. Osteoclast-specific serum and gene markers of 6-gingerol-treated mice were higher than the OVx group. Bone formation was unaffected by 6-gingerol.nnnCONCLUSIONnDaily feeding of 6-gingerol to skeletally mature female mice caused trabecular osteopenia, and the mechanism appeared to be activation of osteoclast formation via the TRPV1 channel.

Sulfur Mobilization for Fe-S Cluster Assembly by the Essential SUF Pathway in the Plasmodium falciparum Apicoplast and Its Inhibition

ABSTRACT The plastid of the malaria parasite, the apicoplast, is essential for parasite survival. It houses several pathways of bacterial origin that are considered attractive sites for drug intervention. Among these is the sulfur mobilization (SUF) pathway of Fe-S cluster biogenesis. Although the SUF pathway is essential for apicoplast maintenance and parasite survival, there has been limited biochemical investigation of its components and inhibitors of Plasmodium SUFs have not been identified. We report the characterization of two proteins, Plasmodium falciparum SufS (PfSufS) and PfSufE, that mobilize sulfur in the first step of Fe-S cluster assembly and confirm their exclusive localization to the apicoplast. The cysteine desulfurase activity of PfSufS is greatly enhanced by PfSufE, and the PfSufS-PfSufE complex is detected in vivo. Structural modeling of the complex reveals proximal positioning of conserved cysteine residues of the two proteins that would allow sulfide transfer from the PLP (pyridoxal phosphate) cofactor-bound active site of PfSufS. Sulfide release from the l-cysteine substrate catalyzed by PfSufS is inhibited by the PLP inhibitor d-cycloserine, which forms an adduct with PfSufS-bound PLP. d-Cycloserine is also inimical to parasite growth, with a 50% inhibitory concentration close to that reported for Mycobacterium tuberculosis, against which the drug is in clinical use. Our results establish the function of two proteins that mediate sulfur mobilization, the first step in the apicoplast SUF pathway, and provide a rationale for drug design based on inactivation of the PLP cofactor of PfSufS.

Innovative Disulfide Esters of Dithiocarbamic Acid as Women-Controlled Contraceptive Microbicides: A Bioisosterism Approach.

In an ongoing effort to discover an effective, topical, dual‐function, non‐surfactant contraceptive vaginal microbicide, a novel series of 2,2′‐disulfanediylbis(3‐(substituted‐1‐yl)propane‐2,1‐diyl) disubstituted‐1‐carbodithioates were designed by using a bioisosterism approach. Thirty‐three compounds were synthesized, and interestingly, most demonstrated multiple activities: they were found to be spermicidal at a minimal effective concentration of 1–0.001u2009%, trichomonacidal against drug‐susceptible and resistant Trichomonas strains at minimal inhibitory concentration (MIC) ranges of 10.81–377.64 and 10.81–754.14u2005μM, respectively, and fungicidal at MIC 7.93–86.50u2005μM. These compounds were also found to be non‐cytotoxic to human cervical (HeLa) epithelial cells and vaginal microflora (Lactobacilli) in vitro. The most promising compound, 2,2′‐disulfanediylbis(3‐(pyrrolidin‐1‐yl)propane‐2,1‐diyl)dipyrrolidine‐1‐carbodithioate (5), exhibited spermicidal activity 15‐fold higher than that of the marketed spermicide Nonoxynol‐9 (N‐9) and also demonstrated microbicidal potency. To identify common structural features required for spermicidal activity, a 3D‐QSAR analysis was carried out, as well as in vivo efficacy studies and fluorescent labeling studies to determine the biological targets of compound 5.

[Fe–S] cluster assembly in the apicoplast and its indispensability in mosquito stages of the malaria parasite

The relict plastid (apicoplast) of the malaria parasite is the site for important biochemical pathways and is essential for parasite survival. The sulfur mobilization (SUF) pathway of iron–sulfur [Fe–S] cluster assembly in the apicoplast of Plasmodium spp. is of interest due to its absence in the human host suggesting the possibility of antimalarial intervention through apicoplast [Fe–S] biogenesis. We report biochemical characterization of components of the Plasmodium falciparum apicoplast SUF pathway after the first step of SUF. In vitro interaction experiments and in vivo cross‐linking showed that apicoplast‐encoded PfSufB and apicoplast‐targeted PfSufC and PfSufD formed a complex. The PfSufB‐C2‐D complex could function as a scaffold to assemble [4Fe–4S] clusters in vitro and activity of the PfSufC ATPase was enhanced by PfSufD. Two carrier proteins, the NifU‐like protein PfNfu and the A‐type carrier PfSufA are homodimers, the former mediating transfer of [4Fe–4S] from the scaffold to a model [4Fe–4S] target protein with higher efficiency. Conditional knockout of SufS, the enzyme catalyzing the first step of SUF, by selective excision in the mosquito stages of Plasmodium berghei severely impaired development of sporozoites in oocysts establishing essentiality of the SUF machinery in the vector. Our results delineate steps of the complete apicoplast SUF pathway and demonstrate its critical role in the parasite life cycle.

2-Methyl-4/5-nitroimidazole derivatives potentiated against sexually transmitted Trichomonas: Design, synthesis, biology and 3D-QSAR study

Trichomoniasis is the most prevalent, non-viral sexually transmitted diseases (STD) caused by amitochondriate protozoan Trichomonas vaginalis. Increased resistance of T.xa0vaginalis to the marketed drug Metronidazole necessitates the development of newer chemical entities. A library of sixty 2-methyl-4/5-nitroimidazole derivatives was synthesized via nucleophilic ring opening reaction of epoxide and the efficacies against drug-susceptible and -resistant Trichomonas vaginalis were evaluated. All the molecules except two were found to be active against both susceptible and resistant strains with MICs ranging 8.55-336.70xa0μM and 28.80-1445.08xa0μM, respectively. Most of the compounds were remarkably more effective than the standard Metronidazole. This study analyzes the inxa0vitro and inxa0vivo activities of the new 5-nitroimidazoles, which were found to be safe against human cervical HeLa cells with good selectivity index. The exploration of SAR by the synthesis of four different prototypes and 3D-QSAR study has shown the importance of prototype 1 over other prototypes.

Discovery of novel inhibitors for Leishmania nucleoside diphosphatase kinase (NDK) based on its structural and functional characterization

Nucleoside diphosphate kinases (NDKs) are ubiquitous enzymes that catalyze the transfer of the γ-phosphate moiety from an NTP donor to an NDP acceptor, crucial for maintaining the cellular level of nucleoside triphosphates (NTPs). The inability of trypanosomatids to synthesize purines de novo and their dependence on the salvage pathway makes NDK an attractive target to develop drugs for the diseases they cause. Here we report the discovery of novel inhibitors for Leishmania NDK based on the structural and functional characterization of purified recombinant NDK from Leishmania amazonensis. Recombinant LaNDK possesses auto-phosphorylation, phosphotransferase and kinase activities with Histidine 117 playing an essential role. LaNDK crystals were grown by hanging drop vapour diffusion method in a solution containing 18% PEG-MME 500, 100xa0mM Bis-Tris propane pH 6.0 and 50xa0mM MgCl2. It belongs to the hexagonal space group P6322 with unit cell parameters au2009=u2009bu2009=u2009115.18, cu2009=u200962.18xa0Å and αu2009=u2009βu2009=u200990°, γu2009=u2009120°. The structure solved by molecular replacement methods was refined to crystallographic R-factor and Rfree values of 22.54 and 26.52%, respectively. Molecular docking and dynamics simulation-based virtual screening identified putative binding compounds. Protein inhibition studies of selected hits identified five inhibitors effective at micromolar concentrations. One of the compounds showed ~45% inhibition of Leishmania promastigotes proliferation. Analysis of inhibitor-NDK complexes reveals the mode of their binding, facilitating design of new compounds for optimization of activities as drugs against leishmaniasis.