Tanuj Sharma

Recombinant NAD-dependent SIR-2 Protein of Leishmania donovani: Immunobiochemical Characterization as a Potential Vaccine against Visceral Leishmaniasis

Background The development of a vaccine conferring long-lasting immunity remains a challenge against visceral leishmaniasis (VL). Immunoproteomic characterization of Leishmania donovani proteins led to the identification of a novel protein NAD+-dependent Silent Information regulatory-2 (SIR2 family or sirtuin) protein (LdSir2RP) as one of the potent immunostimulatory proteins. Proteins of the SIR2 family are characterized by a conserved catalytic domain that exerts unique NAD-dependent deacetylase activity. In the present study, an immunobiochemical characterization of LdSir2RP and further evaluation of its immunogenicity and prophylactic potential was done to assess for its possible involvement as a vaccine candidate against leishmaniasis. Methodology/Principal Findings LdSir2RP was successfully cloned, expressed and purified. The gene was present as a monomeric protein of ~45 kDa and further established by the crosslinking experiment. rLdSir2RP shown cytosolic localization in L. donovani and demonstrating NAD+-dependent deacetylase activity. Bioinformatic analysis also confirmed that LdSir2RP protein has NAD binding domain. The rLdSir2RP was further assessed for its cellular response by lymphoproliferative assay and cytokine ELISA in cured Leishmania patients and hamsters (Mesocricetus auratus) in comparison to soluble Leishmania antigen and it was observed to stimulate the production of IFN-γ, IL-12 and TNF-α significantly but not the IL-4 and IL-10. The naïve hamsters when vaccinated with rLdSir2RP alongwith BCG resisted the L. donovani challenge to the tune of ~75% and generated strong IL-12 and IFN-γ mediated Th1 type immune response thereof. The efficacy was further supported by remarkable increase in IgG2 antibody level which is indicative of Th1 type of protective response. Further, with a possible implication in vaccine design against VL, identification of potential T-cell epitopes of rLdSir2RP was done using computational approach. Conclusion/Significance The immunobiochemical characterization strongly suggest the potential of rLdSir2RP as vaccine candidate against VL and supports the concept of its being effective T-cell stimulatory antigen.

Dehydroascorbate-derivatized chitosan particles for targeting antimalarial agents to infected erythrocytes

The mammalian glucose transporter GLUT-1 and Plasmodium falciparum hexose transporter PfHT1 are overexpressed on human RBC infected with the parasite (iRBC), presumably for enhanced glucose uptake. Dehydroascorbic acid (DHA) competes out glucose in GLUT-1 binding. We prepared particles containing chloroquine phosphate using novel derivatives of chitosan (CSN). CSN was either pre-derivatized with DHA (PRE) or particles made of CSN were derivatized by surface-grafting DHA (POST). The optimized formulations were analyzed for size (170-200nm) drug content (about 40%) entrapment efficiency (50-57%), in vitro drug release (80% in 72h, Higuchis model), hemolysis on exposure to whole blood or RBC at 5% hematocrit, cytotoxicity towards cultured HEK 293T (kidney) and HepG2 (hepatic) cells, targeting iRBC and in vitro efficacy against P. falciparum. PRE particles were superior to POST CSN particles in terms of uptake and extent of preferential targeting to iRBCs than RBCs. Unlike starch particles reported earlier, dextrose did not competitively inhibit uptake of DHA-derivatized CSN particles. Both formulations significantly induced parasite inhibition at 1nM while free drug showed comparable activity at 100nM. Both PRE and POST particles were superior to free drug in efficacy. Targeting with high efficiency promises dose reduction and possibility of overcoming efflux-based drug resistance.

Ammonium trichloro [1,2-ethanediolato- O , O ′]-tellurate cures experimental visceral leishmaniasis by redox modulation of Leishmania donovani trypanothione reductase and inhibiting host integrin linked PI3K/Akt pathway

In an endeavor to search for affordable and safer therapeutics against debilitating visceral leishmaniasis, we examined antileishmanial potential of ammonium trichloro [1,2-ethanediolato-O,O′]-tellurate (AS101); a tellurium based non toxic immunomodulator. AS101 showed significant in vitro efficacy against both Leishmania donovani promastigotes and amastigotes at sub-micromolar concentrations. AS101 could also completely eliminate organ parasite load from L. donovani infected Balb/c mice along with significant efficacy against infected hamsters (˃93% inhibition). Analyzing mechanistic details revealed that the double edged AS101 could directly induce apoptosis in promastigotes along with indirectly activating host by reversing T-cell anergy to protective Th1 mode, increased ROS generation and anti-leishmanial IgG production. AS101 could inhibit IL-10/STAT3 pathway in L. donovani infected macrophages via blocking α4β7 integrin dependent PI3K/Akt signaling and activate host MAPKs and NF-κB for Th1 response. In silico docking and biochemical assays revealed AS101’s affinity to form thiol bond with cysteine residues of trypanothione reductase in Leishmania promastigotes leading to its inactivation and inducing ROS-mediated apoptosis of the parasite via increased Ca2+ level, loss of ATP and mitochondrial membrane potential along with metacaspase activation. Our findings provide the first evidence for the mechanism of action of AS101 with excellent safety profile and suggest its promising therapeutic potential against experimental visceral leishmaniasis.

Benzofuran-dihydropyridine hybrids: A new class of potential bone anabolic agents

A series of novel benzofuran-dihydropyridine hybrids were designed by molecular hybridization approach and evaluated for bone anabolic activities. Among the screened library, ethyl 4-(7-(sec-butyl)-2-(4-methylbenzoyl)benzofuran-5-yl)-2-methyl-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate (compound 21) significantly enhanced the ALP production and mineralized nodule formation, which are primary requisites in the process of in vitro osteogenesis. Oral administration of compound 21 at 10 mg.kg-1 day-1 for two weeks led to restoration of trabecular bone microarchitecture in drill hole fracture model by significantly increasing BV/TV and Tb.N. Furthermore, histological and molecular studies showed compound 21 triggering the new bone regeneration in a drill hole defect site by increasing BMP expression. Furthermore, molecular modeling studies were performed to gain insight into the binding approach, which revealed that both benzofuran and dihydropyridine moieties are essential to show similar binding interactions to fit into the active site of BMP2 receptor, an important target of the osteogenic agents. Our results suggest that compound 21 stimulates BMP2 synthesis in osteoblast cells that promotes new bone formation (∼40%) at the fracture site which helps in shorten the healing period.

Involvement of PKA/DARPP-32/PP1α and β- arrestin/Akt/GSK-3β Signaling in Cadmium-Induced DA-D2 Receptor-Mediated Motor Dysfunctions: Protective Role of Quercetin

Given increasing risk of cadmium-induced neurotoxicity, the study was conducted to delineate the molecular mechanisms associated with cadmium-induced motor dysfunctions and identify targets that govern dopaminergic signaling in the brain involving in vivo, in vitro, and in silico approaches. Selective decrease in dopamine (DA)-D2 receptors on cadmium exposure was evident which affected the post-synaptic PKA/DARPP-32/PP1α and β-arrestin/Akt/GSK-3β signaling concurrently in rat corpus striatum and PC12 cells. Pharmacological inhibition of PKA and Akt in vitro demonstrates that both pathways are independently modulated by DA-D2 receptors and associated with cadmium-induced motor deficits. Ultrastructural changes in the corpus striatum demonstrated neuronal degeneration and loss of synapse on cadmium exposure. Further, molecular docking provided interesting evidence that decrease in DA-D2 receptors may be due to direct binding of cadmium at the competitive site of dopamine on DA-D2 receptors. Treatment with quercetin resulted in the alleviation of cadmium-induced behavioral and neurochemical alterations. This is the first report demonstrating that cadmium-induced motor deficits are associated with alteration in postsynaptic dopaminergic signaling due to a decrease in DA-D2 receptors in the corpus striatum. The results further demonstrate that quercetin has the potential to alleviate cadmium-induced dopaminergic dysfunctions.

In silico identification and design of potent peptide inhibitors against PDZ-3 domain of Post Synaptic Density Protein (PSD-95)

Unique intrinsic properties of peptides like low toxicity, high biological activity, and specificity make them attractive therapeutic agents. PDZ-binding peptide inhibitors have been demonstrated for curing of Alzheimer, Parkinson, Dementia, and other central nervous system ailments. In this article, we report the successful use of an integrated computational protocol to analyze the structural basis of how peptides bind to the shallow groove of the third PDZ domain (PDZ-3) from the postsynaptic density (PSD-95) protein. This protocol employs careful and precise computational techniques for design of new strategy for predicting novel and potent peptides against PDZ protein. We attempted to generate a pharmacophore model using crystal structure of peptide inhibitor bound to the PDZ-3. A highly specific and sensitive generated pharmacophore model was used for screening virtual database generated using different combination of amino acid substitutions as well as decoy peptide database for its sensitivity and specificity. Identified hit peptides were further analyzed by docking studies, and their stability analyzed using solvated molecular dynamics. Quantum Mechanics/Molecular Mechanics (QM/MM) interaction energy and GMX-PBSA scoring schemes were used for ranking of stable peptides. Computational approach applied here generated encouraging results for identifying peptides against PDZ interaction model. The workflow can be further exercised as a virtual screening technique for reducing the search space for candidate target peptides against PDZ domains.

Phosphorylation of Wat1, human Lst8 homolog is critical for the regulation of TORC2 –Gad8 dependent pathway in fission yeast Schizosacchromyces pombe

Mammalian Lst8 interacts with the kinase domain of mTOR and stabilizes its interaction with Raptor regulating cell growth through the mTOR-S6K1 signalling pathway. Fission yeast Wat1, an ortholog of mammalian Lst8 is also an essential component of TOR complex 1 (TORC1) and TOR Complex 2 (TORC2) that control protein kinases essential for metabolic pathways. Here, we show that in response to osmotic stress, the Wat1 protein undergoes hyper-phosphorylation at S116 position. Wat1 interacts with the C-terminal region of Tor1 that also contain kinase domain. Co-immunoprecipitation and molecular modelling studies suggest that Wat1-Tor1 interaction is stabilized by FATC domain of Tor1 protein present at the C-terminal region. We have also demonstrated a physical interaction of Wat1 with Gad8, an AGC family protein kinase that is dependent on phosphorylation of Wat1 at S116 residue. Wat1 phosphorylation is required for the maintenance of vacuolar integrity and sexual differentiation. Collectively, our study reveals Wat1 phosphorylation regulates Gad8 function in a manner dependent on Tor1 interaction.