Ankur Chaudhuri

One new azido bridged dinuclear copper(II) thiosemicarbazide complex: synthesis, DNA/protein binding, molecular docking study and cytotoxicity activity

One new azido bridged dinuclear copper(II) thiosemicarbazide complex, [Cu(L)2(N3)2] (1), has been synthesised from the Schiff base ligand derived from 2-acetyl pyridine and thiosemicarbazide. Complex 1 is characterised by elemental analysis, IR, UV-Vis, ESR spectroscopy, cyclic voltammetry and single crystal X-ray structure analysis. Crystallographic results show that the ligand (HL) is coordinated to the metal in a uninegative tridentate fashion. The DNA binding properties of copper(II) complex 1 are explored by employing UV-vis, fluorescence spectral methods, cyclic voltammetry and also viscosity measurements. In addition, the protein binding behaviour in vitro is studied by multispectroscopic techniques using both BSA and HSA. The cytotoxicity profile of the compound along with a cell line study has also been evaluated. Furthermore, the molecular docking technique has also been used to ascertain the mechanism of action of the complex towards DNA, BSA and HSA. Notably, the in vitro cytotoxicity of complex 1 towards two cell lines (AGS and A549 cancer cells) demonstrates that complex 1 has very good broad spectrum anticancer activity.

Successful Therapy of Murine Visceral Leishmaniasis with Astrakurkurone, a Triterpene Isolated from the Mushroom Astraeus hygrometricus, Involves the Induction of Protective Cell-Mediated Immunity and TLR9

ABSTRACT In our previous report, we showed that astrakurkurone, a triterpene isolated from the Indian mushroom Astraeus hygrometricus (Pers.) Morgan, induced reactive oxygen species, leading to apoptosis in Leishmania donovani promastigotes, and also was effective in inhibiting intracellular amastigotes at the 50% inhibitory concentration of 2.5 μg/ml. The aim of the present study is to characterize the associated immunomodulatory potentials and cellular activation provided by astrakurkurone, leading to effective antileishmanial activity in vitro and in vivo. Astrakurkurone-mediated antileishmanial activity was evaluated by real-time PCR and flow cytometry. The involvement of Toll-like receptor 9 (TLR9) was studied by in vitro assay in the presence of a TLR9 agonist and antagonist and by in silico modeling of a three-dimensional structure of the ectodomain of TLR9 and its interaction with astrakurkurone. Astrakurkurone caused a significant increase in TLR9 expression of L. donovani-infected macrophages along with the activation of proinflammatory responses. The involvement of TLR9 in astrakurkurone-mediated amastigote killing has been evidenced from the fact that a TLR9 agonist (CpG, ODN 1826) in combination with astrakurkurone enhanced the amastigote killing, while a TLR9 antagonist (bafilomycin A1) alone or in combination with astrakurkurone curbed the amastigote killing, which could be further justified by in silico evidence of docking between mouse TLR9 and astrakurkurone. Astrakurkurone was found to reduce the parasite burden in vivo by inducing protective cytokines, gamma interferon and interleukin 17. Moreover, astrakurkurone was nontoxic toward peripheral blood mononuclear cells of immunocompromised patients with visceral leishmaniasis. Astrakurkurone, a nontoxic antileishmanial, enhances the immune efficiency of host cells, leading to parasite clearance in vitro and in vivo.

Comparative analysis of binding sites of human meprins with hydroxamic acid derivative by molecular dynamics simulation study

Meprins are complex and highly glycosylated multi-domain enzymes that require post-translational modifications to reach full activity. Meprins are metalloproteases of the astacin family characterized by a conserved zinc-binding motif (HExxHxxGFxHExxRxDR). Human meprin-α and -β protease subunits are 55% identical at the amino acid level, however the substrate and peptide bond specificities vary markedly. Current work focuses on the critical amino acid residues in the non-primed subsites of human meprins-α and -β involved in inhibitor/ligand binding. To compare the molecular events underlying ligand affinity, homology modeling of the protease domain of humep-α and -β based on the astacin crystal structure followed by energy minimization and molecular dynamics simulation of fully solvated proteases with inhibitor Pro-Leu-Gly-hydroxamate in S subsites were performed. The solvent accessible surface area curve shows a decrease in solvent accessibility values at specific residues upon inhibitor binding. The potential energy, total energy, H-bond interactions, root mean square deviation and root mean square fluctuation plot reflect the subtle differences in the S subsite of the enzymes which interact with different residues at P site of the inhibitor.

PLVAP and GKN3 Are Two Critical Host Cell Receptors Which Facilitate Japanese Encephalitis Virus Entry Into Neurons

Japanese Encephalitis Virus (JEV), a globally important pathogen, belongs to the family Flaviviridae, is transmitted between vertebrate hosts by mosquitoes, principally by Culex tritaeniorhynchus. The E-glycoprotein of the virus mediates its attachment to the host cell receptors. In this study, we cloned and purified JEV E-glycoprotein in pET28a vector using E. coli BL21 (DE3) cells. A pull down assay was performed using plasma membrane fraction of BALB/c mouse brain and E-glycoprotein as a bait protein. 2-Dimensional Gel Electrophoresis based separation of the interacting proteins was analyzed by mass spectrometry. Among all the identified partners of E-glycoprotein, PLVAP (Plasmalemma vesicle associated protein) and GKN3 (Gastrokine3) showed significant up-regulation in both JEV infected mouse brain and neuro2a cells. In-silico studies also predicted significant interaction of these receptors with E-glycoprotein. Additionally, overexperssion and silencing of these receptors resulted in increase and reduction in viral load respectively, suggesting them as two critical cellular receptors governing JEV entry and propagation in neurons. In support, we observed significant expression of PLVAP but not GKN3 in post-mortem autopsied human brain tissue. Our results establish two novel receptor proteins in neurons in case of JEV infection, thus providing potential targets for antiviral research.

Cytotoxicity activity, in silico molecular docking, protein- and DNA-binding study of a new Ni(II) Schiff base complex

Abstract One new nickel(II) complex, [Ni(L)] (1), was synthesized from the Schiff base ligand derived from pyrrole-2-carboxaldehyde and 1,3-diaminopropane. Complex 1 was characterized by elemental analysis, IR, UV-Vis and ESI mass spectroscopy, cyclic voltammetry, and single-crystal X-ray structure analysis. Crystallographic results show that two Ni(II) monomeric moieties are present with similar structural features but with slightly different bond lengths and bond angles. The geometry around the Ni(II) center is distorted square planar. DNA-binding properties of complex 1 were well explored by employing UV-Vis and fluorescence spectral methods, cyclic voltammetry, and by viscosity measurements. Similarly the protein-binding study was studied by multispectroscopic techniques using both BSA and HSA. The cytotoxicity study of the compound has also been evaluated. Notably, the in vitro cytotoxicity of complex 1 on two cancer cell lines (AGS and A549) demonstrates that complex 1 has very good anticancer activity. MTT assay, cell-cycle analysis, and annexin-V assay have been performed to know the extent of effect of complex 1 as anticancer agent. Further, in silico molecular docking study revealed that the nickel(II) complex fits into the minor groove of duplex DNA by hydrophobic interaction with functional groups of B-DNA. Graphical Abstract

Exploring protein–protein intermolecular recognition between meprin-α and endogenous protease regulator cystatinC coupled with pharmacophore elucidation

Meprins are a group of zinc metalloproteases of the astacin family which play a pivotal role in several physiological and pathologocal diseases. The inhibition of the meprins by various inhibitors, macromolecular and small molecules, is crucial in the control of several diseases. Human cystatinC, an amyloidogenic protein, is reported to be an endogenous inhibitor of meprin-α. In this computational study, we elucidate a rational model for meprinα–cystatinC complex using protein–protein docking. The complex model as well as the unbound form was evaluated by molecular dynamics simulation. A simulation study revealed higher stability of the complex owing to the presence of several interactions. Virtual alanine mutagenesis helps in identifying the hotspots on both proteins. Based on the frequency of occurrence of hotspot amino acids, it was possible to enumerate the important amino acids primarily responsible for protein stability present at the amino-terminal end of cystatin. Finally, pharmacophore elucidation carried out based on the information obtained from a series of small molecular inhibitors against meprin-α can be utilized in future for rational drug design and therapy.

Example of two novel thiocyanato bridged copper (II) complexes derived from substituted thiosemicarbazone ligand: Structural elucidation, DNA/albumin binding, biological profile analysis and molecular docking study

Abstract Two novel copper (II) substituted thiosemicarbazone Schiff base complexes [Cu(L1)(µ-SCN)]n(NO3)2 (1) and [Cu2(µ-SCN)(SCN)(L2)2](NO3) (2) have been synthesized by condensing substituted thiosemicarbazides like 4-methyl-3-thiosemicarbazide or 4-ethyl-3-thiosemicarbazide with 2-acetylpyridine. Both the metal complexes 1 and 2 are characterized using different spectroscopic techniques like IR, UV-Vis, ESR spectroscopy followed by elemental analysis, cyclic voltammetric measurement and single crystal X-ray structure analysis. X-ray crystal structure analysis reveal that complex 1 is polymeric while complex 2 is dimeric in nature. The coordination geometry around Cu(II) are square pyramidal in which thiosemicarbazone Schiff base ligand coordinate to the central Cu(II) atom in tridentate fashion. The prominent interaction patterns of 1 and 2 with CT-DNA were examined by employing electronic absorption and emission spectral titrations, cyclic voltammetry and viscosity measurements. All the results show that CT-DNA binds with both copper (II) complexes 1 and 2. Furthermore, protein binding ability in vitro of complexes 1 and 2 with both BSA and HSA were carried out using multispectroscopic techniques and a static quenching pattern was observed in both cases. Molecular docking study was employed to ascertain the exact mechanism of action of 1 and 2 with DNA and protein molecules (BSA and HSA). In vitro cytotoxicity activity of complexes 1 and 2 toward AGS and A549 was evaluated using MTT assay which demonstrates that both complexes 1 and 2 have superior prospectus to act as anticancer agents. Communicated by Ramaswamy H. Sarma

Role of BMP1/Tolloid like Proteases in Bone Morphogenesis and Tissue Remodeling

BMP-1/ tolloid like proteases collectively referred as BTPs are members of the astacin family of metalloproteases belonging to the metzincin clan. Four members of BTP are identified in humans; BMP-1, mTLD, mTLL1 and mTLL2. The BTPs are involved in several pathophysiological diseases including bone morphogenesis, fibrosis, tissue remodeling and tumor progression. BTPs are important regulators to activate several growth factors and helps to release anti-angiogenic fragments from parent proteins. Three dimensional structure of BTPs reveal the presence of a highly unusual disulphide bridge present within the cysteine-rich loop region in the active site. The activity of BTPs is controlled mainly by activators. The only endogenous inhibitor of mammalian BTPs is α2-macroglobulin. Several small molecular inhibitors of BTPs have been reported. Further studies will help to explore the full spectrum of activities of BTPs which will help in recognition of BTPs as new targets for future therapies.

Metalloproteases and Human Diseases: The Astacin Family

Astacins are a group of zinc metalloproteases characterized by a signature sequence of 18 amino acid residues (HExxHxxGxxHxxxRxDR) and a conserved SXMHY domain containing the Met-turn characteristic of the metzincins. They are synthesized as inactive zymogens whereby they are activated after the post-translational removal of the amino-terminal pro-segments. Astacins are widely spread in bacteria and throughout animal kingdom. They are multi-domain, glycosylated complex endopeptidases which may be membrane-bound or secretory in nature. The astacins are known to play diverse role in bone morphogenesis, tissue differentiation, wound healing, digestion and in diseases like fibrosis, cancer, neurodegenerative and Alzheimer’s disease. The homologous protease domain containing about 200 residues and two conserved disulphide bridges present in most astacins is the common thread linking all the members of this family. Though similar in structure the astacins differ widely in their substrate specificity. In humans, six astacins have been reported. They are meprin-α and-β, bone morphogenetic protein-1 (BMP-1) with its major splice variant mammalian tolloid, mammalian tolloid-like enzymes and ovastacin. Among them meprins α and β and bone morphogenetic protein1 have been found to be expressed at elevated level in certain diseases and hence important druggable targets. Several small molecule inhibitors and macromolecular inhibitors are known to control the activity of the astacins thereby contributing to the control of several diseases.

Insights from Analysis of Binding Sites of Human Meprins: Screening of Inhibitors by Molecular Dynamics Simulation Study

Human meprin-α and-β are important regulators of angiogenesis, cancer, inflammation, fibrosis, and neurodegenerative diseases and hence important therapeutic targets. Meprins are the only astacin proteases that are expressed in membrane-bound and secreted form. The cleavage specificity of human meprins is similar in certain cases but differs markedly in others. The inhibitor selectivity of human meprins is controlled by the specific residues involved in binding at the active-site cleft of the proteases. Meprins are inhibited by various small molecular inhibitors as well as macromolecular endogenous inhibitors, making them good drug targets. In the current study, molecular dynamics simulation was performed for 10 ns on ten systems consisting of two apoenzymes of meprin -α/β and eight complexes of human meprin-α and -β complexed to four inhibitors with different metal binding moieties and comparable Ki values. These simulation studies helped to elucidate the molecular details of how several parameters influence protein-inhibitor binding affinity. Analysis of the interaction energies of the protein-inhibitor complexes revealed the diverse binding nature of this series of inhibitors. Several structural segments of human meprins exhibited certain conformational changes during the simulation time course. Among the inhibitors studied captopril had a different disposition in the meprin-bound complexes compared to the other three inhibitors, namely Pro- Leu-Gly-hydroxamate, galardin and EDTA. Comparison of the interaction energies for each system helped us to conclude that the hydroxamic acid-based inhibitors are the most potent inhibitors of meprins.