Impact of Citral and Phloretin, Alone and in Combination, on Major Virulence Traits of Streptococcus pyogenes.

Streptococcus pyogenes is well documented as a multi-virulent and exclusively human pathogen. The LuxS-based signaling in these bacteria has a crucial role in causing several infections through pathways that are pathogenic. This study evaluated the individual and synergistic effects of citral and phloretin against S. pyogenes in relation to major virulence traits. The in vitro synergy of citral and phloretin was evaluated by the checkerboard method. The fractional inhibitory concentration (FIC) values were calculated to determine the interactions between the inhibitors. The bacteria's virulence properties were tested in the presence of the molecules, individually as well as in combination. Molecules' cytotoxicity was tested using human tonsil epithelial cells. The synergistic effects of the molecules on the expression of biofilm and quorum sensing genes were tested using quantitative real-time polymerase chain reaction (qRT-PCR). The molecules were also tested for their impact on LuxS protein by molecular docking, modeling, and free-energy calculations. When the two molecules were assessed in combination (synergistic effect, FIC Index of 0.5), a stronger growth inhibitory activity was exhibited than the individual molecules. The cell surface hydrophobicity, as well as genes involved in quorum sensing and biofilm formation, showed greater suppression when the molecules were tested in combination. The in silico findings also suggest the inhibitory potential of the two molecules against LuxS protein. The binding orientation and the binding affinity of citral and phloretin well support the notion that there is a synergistic effect of citral and phloretin. The data reveal the combination of citral and phloretin as a potent antibacterial agent to combat the virulence of S. pyogenes.

Virtual screening of natural anti-filarial compounds against glutathione-S-transferase of Brugia malayi and Wuchereria bancrofti.

Glutathione-S-transferase also referred as GST is one of the major detoxification enzymes in parasitic helminths. The crucial role played by GST in various chronic infections has been well reported. The dependence of nematodes on detoxification enzymes to maintain their survival within the host established the crucial role of GST in filariasis and other related diseases. Hence, this well-established role of GST in filariasis along with its greater nonhomology with its human counterpart makes it an important therapeutic drug target. Here in this study, we have tried to explore the inhibitory potential of some of the well-reported natural ant-filarial compounds against the GST from Wuchereria bancrofti (W.bancrofti) and Brugia malayi (B.malayi). In silico virtual screening, approach was used to screen the selected natural compounds against GST from W.bancrofti and B.malayi. On the basis of our results, here we are reporting some of the natural compounds which were found to be very effective against GSTs. Along with we have also revealed the characteristic of the active site of BmGST and WbGST and the role of important active site residues involve in the binding of natural compounds within the active site of GSTs. This information will oped doors for using natural compounds as anti-filarial therapy and will also be helpful for future drug discovery.

The effect of Benzothiazolone-2 on the expression of Metallothionein-3 in modulating Alzheimer's disease.

INTRODUCTION: Metallothioneins (MTs) are a class of ubiquitously occurring low-molecular-weight cysteine- and metal-rich proteins containing sulfur-based metal clusters. MT-3 exhibits neuro-inhibitory activity. The possibility to enhance the expression of MT-3 or protect it from degradation is an attractive therapeutic target, because low levels of MT-3 were found in brains of Alzheimer's disease (AD) patients. OBJECTIVES: The primary objective of this study was to test an enhancement of MT-3 cellular concentration after MT-3 binding treatment, which could prevent MT-3 degradation. METHODS: MTT assay, flow-cytometry, fluorescence microscopy, quantitative real-time polymerase chain reaction, and immunodetection of MT3 were used for analysis of effect of STOCK1N-26544, STOCK1N-26929, and STOCK1N-72593 on immortalized human microglia-SV40 cell line. RESULTS: All three tested compounds enhanced concentration of MT-3 protein in cells and surprisingly also mRNA concentration. IC50 values of tested molecules exceeded about ten times the concentration that was needed for induction of MT-3 expression. The tested compound Benzothiazolone-2 enhanced apoptosis and necrosis, but it was not of severe effect. About 80% of cells were still viable. There was no serious ROS-generation and no severe decrease in mitochondria numbers or stress induced endoplasmic reticulum changes after test treatments. The selected compound showed stable hydrophobic and electrostatic interaction during MT-3 ligand interaction. CONCLUSION: Benzothiazolone-2 compounds significantly enhanced MT-3 protein and mRNA levels. The compounds can be looked upon as one of the probable lead compounds for future drug designing experiments in the treatment of Alzheimer's disease.

Magnetic Nanoparticles: Properties, Synthesis and Biomedical Applications.

Importance of magnetic nanoparticles in daily life including biomedical applications in near future cannot be overlooked. This review focuses on the properties of magnetic nanoparticles (MNPs), various approaches for their synthesis, and their biomedical applications. First part of this review focuses on the classes, physical properties, and characteristics of MNPs. The second part sheds light on strategies developed for the synthesis of MNPs, with special attention given to biological, physical, and chemical approaches as well as recent modifications in the preparation of monodispersed samples. Furthermore, this review deals with the biomedical applications of MNPs, which includes applications in targeted drug delivery, diagnostics, gene therapy, hyperthermia and advantages in the field of medicine.

Implication of Caspase-3 as a Common Therapeutic Target for Multineurodegenerative Disorders and Its Inhibition Using Nonpeptidyl Natural Compounds.

Caspase-3 has been identified as a key mediator of neuronal apoptosis. The present study identifies caspase-3 as a common player involved in the regulation of multineurodegenerative disorders, namely, Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), and amyotrophic lateral sclerosis (ALS). The protein interaction network prepared using STRING database provides a strong evidence of caspase-3 interactions with the metabolic cascade of the said multineurodegenerative disorders, thus characterizing it as a potential therapeutic target for multiple neurodegenerative disorders. In silico molecular docking of selected nonpeptidyl natural compounds against caspase-3 exposed potent leads against this common therapeutic target. Rosmarinic acid and curcumin proved to be the most promising ligands (leads) mimicking the inhibitory action of peptidyl inhibitors with the highest Gold fitness scores 57.38 and 53.51, respectively. These results were in close agreement with the fitness score predicted using X-score, a consensus based scoring function to calculate the binding affinity. Nonpeptidyl inhibitors of caspase-3 identified in the present study expeditiously mimic the inhibitory action of the previously identified peptidyl inhibitors. Since, nonpeptidyl inhibitors are preferred drug candidates, hence, discovery of natural compounds as nonpeptidyl inhibitors is a significant transition towards feasible drug development for neurodegenerative disorders.

Virtual screening, ADMET profiling, molecular docking and dynamics approaches to search for potent selective natural molecules based inhibitors against metallothionein-III to study Alzheimer's disease.

MOTIVATION: Metallothionein-III (MT-III) displays neuro-inhibitory activity and is involved in the repair of neuronal damage. An altered expression level of MT-III suggests that it could be a mitigating factor in Alzheimer's disease (AD) neuronal dysfunction. Currently there are limited marketed drugs available against MT-III. The inhibitors are mostly pseudo-peptide based with limited ADMET. In our present study, available database InterBioScreen (natural compounds) was screened out for MT-III. Pharmacodynamics and pharmacokinetic studies were performed. Molecular docking and simulations of top hit molecules were performed to study complex stability. RESULTS: Study reveals potent selective molecules that interact and form hydrogen bonds with amino acids Ser-6 and Lys-22 are common to established melatonin inhibitors for MT-III. These include DMHMIO, MCA B and s27533 derivatives. The ADMET profiling was better with comparable interaction energy values. It includes properties like blood brain barrier, hepatotoxicity, druggability, mutagenicity and carcinogenicity. Molecular dynamics studies were performed to validate our findings.

Improved efficiency and stability of secnidazole - An ideal delivery system.

Secnidazole (α,2-Dimethyl-5-nitro-1H-imidazole-1-ethanol) is a highly effective drug against a variety of G(+)/G(-) bacteria but with significant side effects because it is being used in very high concentration. In this study, gold nanoparticles (GNPS) were selected as a vehicle to deliver secnidazole drug at the specific site with more accuracy which made the drug highly effective at substantially low concentrations. The as-synthesized GNPs were capped with Human Serum Albumin (HSA) and subsequently bioconjugated with secnidazole because HSA provides the stability and improves the solubility of the bioconjugated drug, secnidazole. The quantification of covalently bioconjugated secnidazole with HSA encapsulated on enzymatically synthesized GNPs was done with RP-HPLC having SPD-20 A UV/VIS detector by using the C-18 column. The bioconjugation of GNPs with secnidazole was confirmed by Transmission Electron Microscopy (TEM) and Dynamic Light Scattering (DLS). The bioconjugated GNPs were characterized by UV-VIS spectroscopy, TEM, Scanning Electron Microscopy (SEM) and DLS. Zeta potential confirmed the stability and uniform distribution of particles in the emulsion of GNPs. The separation of bioconjugated GNPs, unused GNPs and unused drug was done by gel filtration chromatography. The minimal inhibitory concentration of secnidazole-conjugated gold nanoparticles (Au-HSA-Snd) against Klebsiella pneumonia (NCIM No. 2957) and Bacillus cereus (NCIM No. 2156) got improved by 12.2 times and 14.11 times, respectively, in comparison to pure secnidazole. Precisely, the MIC of Au-HSA-Snd against K. pneumonia (NCIM No. 2957) and B. cereus (NCIM No. 2156) were found to be 0.35 and 0.43 µg/ml, respectively whereas MIC of the pure secnidazole drug against the same bacteria were found to be 4.3 and 6.07 µg/ml, respectively.

Insight into the effect of inhibitor resistant S130G mutant on physico-chemical properties of SHV type beta-lactamase: a molecular dynamics study.

Bacterial resistance is a serious threat to human health. The production of ß-lactamase, which inactivates ß-lactams is most common cause of resistance to the ß-lactam antibiotics. The Class A enzymes are most frequently encountered among the four ß-lactamases in the clinic isolates. Mutations in class A ß-lactamases play a crucial role in substrate and inhibitor specificity. SHV and TEM type are known to be most common class A ß-lactamases. In the present study, we have analyzed the effect of inhibitor resistant S130G point mutation of SHV type Class-A ß-lactamase using molecular dynamics and other in silico approaches. Our study involved the use of different in silico methods to investigate the affect of S130G point mutation on the major physico-chemical properties of SHV type class A ß-lactamase. We have used molecular dynamics approach to compare the dynamic behaviour of native and S130G mutant form of SHV ß-lactamase by analyzing different properties like root mean square deviation (RMSD), H-bond, Radius of gyration (Rg) and RMS fluctuation of mutation. The results clearly suggest notable loss in the stability of S130G mutant that may further lead to decrease in substrate specificity of SHV. Molecular docking further indicates that S130G mutation decreases the binding affinity of all the three inhibitors in clinical practice.

A neuroinformatics study describing molecular interaction of Cisplatin with acetylcholinesterase: a plausible cause for anticancer drug induced neurotoxicity.

Several chemotherapeutic drugs are known to cause significant clinical neurotoxicity, which can result in the early cessation of treatment. To identify and develop more effective means of neuroprotection it is important to understand the toxicity of these drugs at the molecular and cellular levels. This study describes molecular interactions between human brain acetylcholinesterase (AChE) and the well-known anti-neoplastic drug, Cisplatin. Docking between Cisplatin and AChE was performed using 'GOLD 5.0' and accessible surface area of protein before and after ligand binding was calculated by NACCESS version 2.1.1. Hydrophobic interactions and hydrogen bonds both play an equally important role in the correct positioning of Cisplatin within the 'acyl pocket' as well as 'catalytic site' of AChE to permit docking. Gold fitness score of 'Cisplatin- acyl domain of AChE' interaction and 'Cisplatin-CAS domain of AChE' interaction were 38.78 and 39.91, respectively and free binding energy was found to be -5.82 Kcal/mol and -5.79 Kcal/mol, respectively. During 'Cisplatin-CAS site of AChE enzyme' interaction, it was found that out of the three amino acids constituting the catalytic triad (S203, H447 and E334), two amino acid residues namely S203 and H447 interact with Cisplatin by hydrogen bonding and hydrophobic interaction, respectively. The values for 'accessible surface area' for the amino acid residues H447 and S203 were found to be reduced by 14.398 Å(2) and 3.894 Å(2), respectively after interaction with Cisplatin. Hence, Cisplatin might act as a potent inhibitor of AChE. Scope still remains in the determination of the three-dimensional structure of AChE-Cisplatin complex by X-ray crystallography to validate the described data. Moreover, such information may aid in the design of versatile AChE-inhibitors, and is expected to aid in safe clinical use of Cisplatin.

Inhibitory effect of metformin and pyridoxamine in the formation of early, intermediate and advanced glycation end-products.

BACKGROUND: Non-enzymatic glycation is the addition of free carbonyl group of reducing sugar to the free amino groups of proteins, resulting in the formation of a Schiff base and an Amadori product. Dihydroxyacetone (DHA) is one of the carbonyl species which reacts rapidly with the free amino groups of proteins to form advanced glycation end products (AGEs). The highly reactive dihydroxyacetone phosphate is a derivative of dihydroxyacetone (DHA), and a product of glycolysis, having potential glycating effects to form AGEs. The formation of AGEs results in the generation of free radicals which play an important role in the pathophysiology of aging and diabetic complications. While the formation of DHA-AGEs has been demonstrated previously, no extensive studies have been performed to assess the inhibition of AGE inhibitors at all the three stages of glycation (early, intermediate and late) using metformin (MF) and pyridoxamine (PM) as a novel inhibitor. METHODOLOGY/PRINCIPAL FINDINGS: In this study we report glycation of human serum albumin (HSA) & its characterization by various spectroscopic techniques. Furthermore, inhibition of glycation products at all the stages of glycation was also studied. Spectroscopic analysis suggests structural perturbations in the HSA as a result of modification which might be due to generation of free radicals and formation of AGEs. CONCLUSION: The inhibition in the formation of glycation reaction reveals that Pyridoxamine is a better antiglycating agent than Metformin at all stages of the glycation (early, intermediate and late stages).

Design, synthesis, evaluation and thermodynamics of 1-substituted pyridylimidazo[1,5-a]pyridine derivatives as cysteine protease inhibitors.

Targeting papain family cysteine proteases is one of the novel strategies in the development of chemotherapy for a number of diseases. Novel cysteine protease inhibitors derived from 1-pyridylimidazo[1,5-a]pyridine representing pharmacologically important class of compounds are being reported here for the first time. The derivatives were initially designed and screened in silico by molecular docking studies against papain to explore the possible mode of action. The molecular interaction between the compounds and cysteine protease (papain) was found to be very similar to the interactions observed with the respective epoxide inhibitor (E-64c) of papain. Subsequently, compounds were synthesized to validate their efficacy in wet lab experiments. When characterized kinetically, these compounds show their Ki and IC50 values in the range of 13.75 to 99.30 µM and 13.40 to 96.50 µM, respectively. The thermodynamics studies suggest their binding with papain hydrophobically and entropically driven. These inhibitors also inhibit the growth of clinically important different types of Gram positive and Gram negative bacteria having MIC50 values in the range of 0.6-1.4 µg/ml. Based on Lipinski's rule of Five, we also propose these compounds as potent antibacterial prodrugs. The most active antibacterial compound was found to be 1-(2-pyridyl)-3-(2-hydroxyphenyl)imidazo[1,5-a]pyridine (3a).

Antioxidant and α -amylase inhibitory property of phyllanthus virgatus L.: an in vitro and molecular interaction study.

The present study on Phyllanthus virgatus, known traditionally for its remedial potential, for the first time provides descriptions of the antioxidant and inhibition of α -amylase enzyme activity first by in vitro analyses, followed by a confirmatory in silico study to create a stronger biochemical rationale. Our results illustrated that P. virgatus methanol extract exhibited strong antioxidant and oxidative DNA damage protective activity than other extracts, which was well correlated with its total phenolic content. In addition, P. virgatus methanol extract strongly inhibited the α -amylase activity (IC50 33.20 ± 0.556 µ g/mL), in a noncompetitive manner, than acarbose (IC50 76.88 ± 0.277 µ g/mL), which showed competitive inhibition. Moreover, this extract stimulated the glucose uptake activity in 3T3-L1 cells and also showed a good correlation between antioxidant and α -amylase activities. The molecular docking studies of the major bioactive compounds (9,12-octadecadienoic acid, asarone, 11-octadecenoic acid, and acrylic acid) revealed via GC-MS analysis from this extract mechanistically suggested that the inhibitory property may be due to the synergistic effect of these bioactive compounds. These results provide substantial basis for the future use of P. virgatus methanol extract and its bioactive compound in in vivo system for the treatment and management of diabetes as well as in the related condition of oxidative stress.

Predicted binding of certain antifilarial compounds with glutathione-S-transferase of human Filariids.

UNLABELLED: : Glutathione-S-transferase is a major phase-II detoxification enzyme in parasitic helminthes. Previous research highlights the importance of GSTs in the establishment of chronic infections in cytotoxic microenvironments. Filarial nematodes depend on these detoxification enzymes for their survival in the host. GST plays an important role in filariasis and other diseases. GST from W.bancrofti and B.malayi are very much different from human GST. This structural difference makes GST potential chemotherapeutic targets for antifilarial treatment. In this study we have checked the efficacy of some well known antifilarial compounds against GST from B.malayi and W.bancrofti. The structure of BmGST was modeled using modeller9v10 and was submitted to PMDB. Molecular docking study reveals arbindazole to be the most potent compounds against GST from both the filarial parasites. Role of some residues playing important role in the binding of compounds within the active site of GST has also been revealed in the present study. The BmGST and WbGST structural information and docking studies could aid in screening new antifilarials or selective inhibitors for chemotherapy against filariasis. ABBREVIATIONS: GST - Glutathione-S-transferase, Bm - Brugia malayi, Wb - Wuchereria bancrofti.

Screening of inhibitors for S130G inhibitor resistant mutants of TEM type beta-lactamase.

Bacteria are remarkably adaptable organisms that acquire an almost limitless competence to survive under unpleasant conditions. The drastic emergence of antibiotic resistance among ß-Lactamases is the most serious threat to hospitals and nosocomial settings. ß-lactam inhibitors came into existence in order to overcome the problem of antibibiotic resistance in bacteria. The emergence of inhibitor resistant mutants has raised the alarms. In this study we have used structured based virtual screening approach and have screened out some inhibitors against S130G TEM mutant. All the compounds were tested in presence and absence of conserved active site water molecules. These compounds were found be showing much higher efficacy than known ß-lactamase inhibitors. Amino acids G130, S70, N132, G130, Y105 and V216 were found crucial for the interaction of inhibitors within the active site.

Molecular docking analysis of new generation cephalosporins interactions with recently known SHV-variants.

Extended-spectrum-ß-lactamases (ESBLs), constitutes the growing class of betalactamses, these are enzymes produced by bacteria which impart resistance against advanced-generation-cephalosporins. SHV enzymes are among the most prevalent ESBLs. The mode of molecular interactions of recent SHV-variants to advanced generation cephalosporins has not been reported yet. This is the first time we are reporting the insilico study of these recent variants with new generation cephaosporins. Homology models for SHV-105, SHV-95, SHV-89, SHV-61 and SHV-48 were generated using MODELLER9v3. New generation Cephalosporins were selected to target the active site amino acid residues of these modeled SHV enzymes for predicting comparative efficacies of these inhibitors against the said enzymes on the basis of interaction energies of docking. The docked complexes were analyzed by using DISCOVERY STUDIO 2.5. In this study A237, S70, K234, R275, N132, R244 and S130 were found crucial to the correct positioning of drugs within the binding site of SHV enzymes in 11, 6, 6, 6, 5, 5 and 5 instances, respectively. On the basis of interaction energy and Ki calculations cefatoxime emerged as the most efficient among the other advanced cephalosporins against all the studied SHV variants, excluding SHV-48 where ceftazidime was found to be most effective drug. Furthermore, this study identified amino acid residues crucial to 'SHV-Cephalosporins' interactions and this information will be useful in designing effective and versatile drug candidates.