Madhu Chopra

Flow-Cytometric Analysis of Reactive Oxygen Species in Peripheral Blood Mononuclear Cells of Patients with Thyroid Dysfunction

Thyroid hormones are major regulators of energy metabolism and increased levels of the hormones (hyperthyroidism) results in an increase in the metabolic rate. Thyroid dysfunction causing alteration in hormone secretion leads to perturbations in the metabolic status. The hypermetabolic state may cause increased generation of reactive oxygen species (ROS), leading to oxidative stress in these patients. This study was carried out to verify our proposition by measuring the ROS in the terminally differentiated cells like the peripheral blood mononuclear cells of the patients.

Gallic acid loaded disulfide cross-linked biocompatible polymeric nanogels as controlled release system: synthesis, characterization, and antioxidant activity

In this article, a sustained release formulation of the antioxidant gallic acid (GA) is presented in the form of glutathione responsive disulfide cross-linked poly(ethylene glycol)-based nanogels synthesized via aqueous inverse miniemulsion using atom transfer radical polymerization. The particle size was found to be in the range from 227 ± 51.78 to 573.3 ± 207.2 nm at three drug loading levels achieved i.e. 6.6, 14.26, and 18.29 wt.% of the nanogels with loading efficiency in the range of 60–70%. The release profile of the GA studied at three drug loading levels suggested a controlled release and the nanogels were capable of scavenging radicals and retained the antioxidant activity. The GA-loaded nanogels were found to be biocompatible on human cervical cancer cell lines (HeLa). DCFH-DA (2,7-dichlorofluorescin diacetate) assay evidenced that the nanogels were capable of scavenging the reactive oxygen species in cellular environment.

Synthesis, characterization, and evaluation of radical scavenging ability of ellagic acid-loaded nanogels

Ellagic acid (EA), a potential antioxidant phytochemical has low aqueous solubility and bioavailability. In this paper, encapsulation of ellagic acid has been carried out into the biodegradable disulfide crosslinked poly (ethylene glycol) PEO-based nanogels synthesized via AGET (activator generated electron transfer) ATRP (atom transfer radical polymerization), and their radical scavenging ability was evaluated. The encapsulation of the EA was carried out at two drug loading percentages, that is, 10 and 20 wt.% of the nanogels. 1,1-Diphenyl-2-picryldrazyl (DPPH) assay was utilized in order to assess the radical scavenging ability of the ellagic acid-loaded nanogels. A drug-loading level of about 2.5 wt.% was achieved with encapsulation efficiency of about 25% at 10 wt.% of the EA w.r.t nanogels, which was found to increase to about 4.7 wt.% with decreased encapsulation efficiency of 23.5% as EA content was increased to 20wt.% of the nanogels. Ellagic acid loading was found to be accompanied with increase in the size of the nanogels from 144.6 ± 39.52nm for neat nanogels to 217.8 ± 105.5 and 633 ± 160.1 nm at 2.5 and 4.7 wt.% drug loading level. The nanogels were found to be capable of scavenging radicals and biocompatible on human cervical cancer cell lines (HeLa cells) at appropriate concentrations.

Autoacetylation of Purified Calreticulin Transacetylase Utilizing Acetoxycoumarin as the Acetyl Group Donor

Our earlier reports documented that calreticulin, a multifunctional Ca2+-binding protein in endoplasmic reticulum lumen, possessed protein acetyltransferase function termed Calreticulin Transacetylase (CRTAase). The autoacetylation of purified human placental CRTAase concomitant with the acetylation of receptor proteins by a model acetoxycoumarin, 7,8-Diacetoxy-4-methylcoumarin, was observed. Here, we have examined the autoacetylation property of CRTAase by immunoblotting and mass spectrometry. Ca2+ was found to inhibit CRTAase activity. The inhibition of both autoacetylation of CRTAase as well as acetylation of the receptor protein was apparent when Ca2+ was included in the reaction mixture as visualized by interaction with anti-acetyl lysine antibody. The acetylation of lysines residues: −48, −62, −64, −153, and −159 in N-domain and −206, −207, −209, and −238 in P-domain of CRTAase were located by high-performance liquid chromatography-electronspray ionization tandem mass spectrometry. Further, computer assisted protein structure modeling studies were undertaken to probe the effect of autoacetylation of CRTAase. Accordingly, the predicted CRTAase 3D model showed that all the loop regions of both N- and P-domain bear the acetylated lysines. Energy minimization of the acetylated residues revealed charge neutralization of lysines due to the N-ε-acetylation which may facilitate the interaction of CRTAase with the protein substrate and the subsequent transacetylase action.

2D-QSAR, Docking Studies, and In Silico ADMET Prediction of Polyphenolic Acetates as Substrates for Protein Acetyltransferase Function of Glutamine Synthetase of Mycobacterium tuberculosis

A novel transacetylase (TAase) function of glutamine synthetase (GS) in bacterial species such as Mycobacterium smegmatis and Mycobacterium tuberculosis H37Rv was established by us, termed as mycobacterial TAase (MTAase). Several polyphenolic acetates (PAs) were found to be substrates for MTAase by inhibiting certain receptor proteins such as glutathione S-transferase by way of acetylation. The present work describes the descriptor-based 2D-QSAR studies developed for a series of PA synthesized by us and evaluated for MTAase and antimycobacterial activity using stepwise multiple linear regression method with the kinetic constants and the minimum inhibitory constant (MIC) as the dependent variables, to address the fact that TAase activity was leading to the antimycobacterial activity. Further, blind docking methods using AutoDock were carried out to study the interaction of potent PA with the crystal structure of M. tuberculosis GS. PAs were predicted to bind M. tuberculosis GS on the protein surface away from the known active site of GS. Subsequent focussed/refined docking of potent PA with GS showed that the -amino group of Lys4 of GS formed a cation- interaction with the benzene ring of PA. Also, ADMET-related descriptors were calculated to predict the pharmacokinetic properties for the selection of the effective and bioavailable compounds.

Effect of varying chain length between P1 and P1′ position of tripeptidomimics on activity of angiotensin-converting enzyme inhibitors

One of the efficient modes of treatments of chronic hypertension and cardiovascular disorders has been to restrain the formation of angiotensin-II by inhibiting the action of angiotensin-converting enzyme (ACE) on angiotensin-I. The efforts continue towards achieving superior molecules or drugs with improved affinities, better bioavailability and thus to achieve long duration of action with minimum side effects. Previously, we reported a library of tripeptidomimics of Ornithyl-Proline (Orn-Pro) conjugated with various unnatural amino acids and carboxylic acid derived heterocyclics based on the SAR studies of existing ACE inhibitors. Their synthesis and screening for possible inhibitors of angiotensin-converting enzyme (ACE) revealed that increase in the backbone chain length by one carbon atom results in a sudden decrease in their activity. Therefore, in the present study heterocycles with different chain length were introduced to interact with the hydrophobic S(2) sub-site of ACE and screened for their in vitro ACE inhibition activity. Further, their binding interaction with C-domain of somatic ACE was also determined. Docking and consequent LUDI scores showed good correlation with binding of these molecules in the active site of ACE. Results suggest that heterocycles with C(3) chain length are more appropriate for the effective binding of the tripeptidomimics within the active site of ACE.

Synthesis of novel bifunctional Schiff-base ligands derived from condensation of 1-(p-nitrobenzyl)ethylenediamine and 2-(p-nitrobenzyl)-3-monooxo-1,4,7-triazaheptane with salicylaldehyde

Two potentially tetradentate (N2O2) and pentadentate (N3O3) bifunctional Schiff-base ligands, N,N′-bis(2-hydroxybenzyl)-1-(p-aminobenzyl)ethylenediamine (7) and N,N′-bis(2-hydroxybenzyl)-2-(p-aminobenzyl)-3-monooxo-1,4,7-triazaheptane (5′) have been prepared and characterized by various spectroscopic methods (IR, FAB-MS, NMR). They are derived from the condensation reactions of the C-functionalized diamines 1-(p-nitrobenzyl)ethylenediamine and 2-(p-nitrobenzyl)-3-monooxo-1,4,7-triazaheptane with 2.1 equiv. of salicylaldehyde. The first complexation trials with 99mTc are reported.

Ligand-based molecular modeling study on a chemically diverse series of cholecystokinin-B/gastrin receptor antagonists: generation of predictive model.

Pharmacophore hypotheses were developed for six structurally diverse series of cholecystokinin-B/gastrin receptor (CCK-BR) antagonists. A training set consisting of 33 compounds was carefully selected. The activity spread of the training set molecules was from 0.1 to 2100 nM. The most predictive pharmacophore model (hypothesis 1), consisting of four features, namely, two hydrogen bond donors, one hydrophobic aliphatic, and one hydrophobic aromatic feature, had a correlation (r) of 0.884 and a root-mean-square deviation of 1.1526, and the cost difference between null cost and fixed cost was 81.5 bits. The model was validated on a test set consisting of six different series of 27 structurally diverse compounds and performed well in classifying active and inactive molecules correctly. This validation approach provides confidence in the utility of the predictive pharmacophore model developed in this work as a 3D query tool in the virtual screening of drug-like molecules to retrieve new chemical entities as potent CCK-BR antagonists. The model can also be used to predict the biological activities of compounds prior to their costly and time-consuming synthesis.

PEG-coumarin based biocompatible self-assembled fluorescent nanoaggregates synthesized via click reactions and studies of aggregation behavior.

HYPOTHESIS Click chemistry has found wide application in drug discovery, bioconjugation reactions, polymer chemistry and synthesis of amphiphilic materials with pharmaceutical and biomedical applications. Triazole substitution via a click reaction alters photophysical properties of coumarin. Both coumarin and triazole moieties participate in π-π stacking interactions. Hence it should be possible to prepare fluorescent self-assembly systems by conjugation of coumarin to poly (ethylene glycol) (PEG) via click reactions exhibiting hydrophilic, hydrophobic and π-π stacking interactions. Moreover, the materials can be suitable platforms to assess fluorescence modulation effect of triazole substitution on coumarins. EXPERIMENTS PEG supported coumarin conjugates were synthesized and the fluorescence modulation effect of the formation of triazole on coumarin was assessed. Their aggregation properties were studied by surface tension measurements, dynamic light scattering (DLS), transmission electron microscopy (TEM), fluorescence and (1)H NMR spectroscopy. FINDINGS The conjugates were found to form nanoaggregates in the size range of 100-120 nm with a negative free energy of micellization (~-27 kJ mol(-1)) confirming aggregation and self-assembly. The Quantum yield of 4-methyl-7-propargylcoumarin (7P4MC) was enhanced after triazole formation with azide functionalized PEG (methoxy-PEG350 azide). The conjugates were found to exhibit π-π stacking interactions in addition to hydrophilic and hydrophobic interactions. They were found to be biocompatible with human pancreatic cancer cells.

Poly(ethylene glycol)-co-methacrylamide-co-acrylic acid based nanogels for delivery of doxorubicin

Abstract Polymeric nanogels have been widely explored for their potential application as delivery carriers for cancer therapeutics. The ability of nanogels to encapsulate therapeutics by simple diffusion mechanism and the ease of their fabrication to impart target specificity in addition to their ability to get internalized into target cells make them good candidates for drug delivery. The present study aims to investigate the applicability of poly(ethylene glycol)-co-methacrylamide-co-acrylic acid (PMA)-based nanogels as a viable option for the delivery of doxorubicin (DOX). The nanogels were synthesized by free radical polymerization in an inverse mini-emulsion and characterized by nuclear magnetic resonance spectroscopy (1H NMR), Fourier transform infrared spectroscopy, dynamic light scattering, transmission electron microscopy (TEM), X-ray diffraction and differential scanning calorimetry. DOX was physically incorporated into the nanogels (PMA-DOX) and the mechanism of its in vitro release was studied. TEM experiment revealed spherical morphology of nanogels and the hydrodynamic diameter of the neat nanogels was in the range of 160 ± 46.95 nm. The size of the nanogels increased from 235.1 ± 28.46 to 403.7 ± 89.89 nm with the increase in drug loading capacity from 4.68 ± 0.03 to 13.71 ± 0.01%. The sustained release of DOX was observed upto 80 h and the release rate decreased with increased loading capacity following anomalous release mechanism as indicated by the value of diffusion exponent (n = 0.64–0.75) obtained from Korsmeyer–Peppas equation. Further, cytotoxicity evaluation of PMA-DOX nanogels on HeLa cells resulted in relatively higher efficacy (IC50~5.88 μg/mL) as compared to free DOX (IC50~7.24 μg/mL) thus demonstrating that the preparation is potentially a promising drug delivery carrier.