Synthesis of benzofuran based 1,3,5-substituted pyrazole derivatives: As a new class of potent antioxidants and antimicrobials-A novel accost to amend biocompatibility

In search for a new antioxidant and antimicrobial agent with improved potency, we synthesized a series of benzofuran based 1,3,5-substituted pyrazole analogues (5a-l) in five step reaction. Initially, o-alkyl derivative of salicyaldehyde readily furnish corresponding 2-acetyl benzofuran 2 in good yield, on treatment with 1,8-diaza bicyclo5.4.0]undec-7-ene (DBU) in the presence of molecular sieves. Further, aldol condensation with vanillin, Claisen-Schmidt condensation reaction with hydrazine hydrate followed by coupling of substituted anilines afforded target compounds. The structures of newly synthesized compounds were confirmed by IR, H-1 NMR, C-13 NMR, mass, elemental analysis and further screened for their antioxidant and antimicrobial activities. Among the tested compounds 5d and 5f exhibited good antioxidant property with 50% inhibitory concentration higher than that of reference while compounds 5h and 5l exhibited good antimicrobial activity at concentration 1.0 and 0.5 mg/mL compared with standard, streptomycin and fluconazole respectively. (C) 2012 Elsevier Ltd. All rights reserved.

Differential modulation of intracellular survival of cytosolic and vacuolar pathogens by curcumin

Curcumin, a principal component of turmeric, acts as an immunomodulator regulating the host defenses in response to a diseased condition. The role of curcumin in controlling certain infectious diseases is highly controversial. It is known to alleviate symptoms of Helicobacter pylori infection and exacerbate that of Leishmania infection. We have evaluated the role of curcumin in modulating the fate of various intracellular bacterial pathogens. We show that pretreatment of macrophages with curcumin attenuates the infections caused by Shigella flexneri (clinical isolates) and Listeria monocytogenes and aggravates those caused by Salmonella enterica serovar Typhi CT18 (a clinical isolate), Salmonella enterica serovar Typhimurium, Staphylococcus aureus, and Yersinia enterocolitica. Thus, the antimicrobial nature of curcumin is not a general phenomenon. It modulated the intracellular survival of cytosolic (S. flexneri and L. monocytogenes) and vacuolar (Salmonella spp., Y. enterocolitica, and S. aureus) bacteria in distinct ways. Through colocalization experiments, we demonstrated that curcumin prevented the active phagosomal escape of cytosolic pathogens and enhanced the active inhibition of lysosomal fusion by vacuolar pathogens. A chloroquine resistance assay confirmed that curcumin retarded the escape of the cytosolic pathogens, thus reducing their inter- and intracellular spread. We have demonstrated that the membrane-stabilizing activity of curcumin is crucial for its differential effect on the virulence of the bacteria.

Biocides - resistance, cross-resistance mechanisms and assessment

Importance of the field: Antibiotic resistance in bacterial pathogens has increased worldwide leading to treatment failures. Concerns have been raised about the use of biocides as a contributing factor to the risk of antimicrobial resistance (AMR) development. In vitro studies demonstrating increase in resistance have often been cited as evidence for increased risks. It is therefore important to understand the mechanisms of resistance employed by bacteria toward biocides used in consumer products and their potential to impart cross-resistance to therapeutic antibiotics. Areas covered: In this review, the mechanisms of resistance and cross-resistance reported in the literature toward biocides commonly used in consumer products are summarized. The physiological and molecular techniques used in describing and examining these mechanisms are reviewed and application of these techniques for systematic assessment of biocides for their potential to develop resistance and/or cross-resistance is discussed. Expert opinion: The guidelines in the usage of biocides in household or industrial purpose should be monitored and regulated to avoid the emergence of any MDR strains. The genetic and molecular methods to monitor the resistance development to biocides should be developed and included in preclinical and clinical studies.

Laser receptive polyelectrolyte thin films doped with biosynthesized silver nanoparticles for antibacterial coatings and drug delivery applications

We report a simple method to fabricate multifunctional polyelectrolyte thin films to load and deliver the therapeutic drugs. The multilayer thin films were assembled by the electrostatic adsorption of poly (allylamine hydrochloride) (PAH) and dextran sulfate (DS). The silver nanoparticles (Ag NPs) biosynthesized from novel Hybanthus enneaspermus leaf extract as the reducing agent were successfully incorporated into the film. The biosynthesized Ag NPs showed excellent antimicrobial activity against the range of enteropathogens, which could be significantly enhanced when used with commercial antibiotics. The assembled silver nano composite multilayer films showed rupture and deformation when they are exposed to laser. The Ag NPs act as an energy absorption center, locally heat up the film and rupture it under laser treatment. The antibacterial drug, moxifloxacin hydrochloride (MH) was successfully loaded into the multilayer films. The total amount of MH release observed was about 63% which increased to 85% when subjected to laser light exposure. Thus, the polyelectrolyte thin film reported in our study has significant potential in the field of remote activated drug delivery, antibacterial coatings and wound dressings. (C) 2013 Elsevier B.V. All rights reserved.

Polyelectrolyte/silver nanocomposite multilayer films as multifunctional thin film platforms for remote activated protein and drug delivery

We demonstrate a nanoparticle loading protocol to develop a transparent, multifunctional polyelectrolyte multilayer film for externally activated drug and protein delivery. The composite film was designed by alternate adsorption of poly(allylamine hydrochloride) (PAH) and dextran sulfate (DS) on a glass substrate followed by nanoparticle synthesis through a polyol reduction method. The films showed a uniform distribution of spherical silver nanoparticles with an average diameter of 50 +/- 20 nm, which increased to 80 +/- 20 nm when the AgNO3 concentration was increased from 25 to 50 mM. The porous and supramolecular structure of the polyelectrolyte multilayer film was used to immobilize ciprofloxacin hydrochloride (CH) and bovine serum albumin (BSA) within the polymeric network of the film. When exposed to external triggers such as ultrasonication and laser light the loaded films were ruptured and released the loaded BSA and CH. The release of CH is faster than that of BSA due to a higher diffusion rate. Circular dichroism measurements confirmed that there was no significant change in the conformation of released BSA in comparison with native BSA. The fabricated films showed significant antibacterial activity against the bacterial pathogen Staphylococcus aureus. Applications envisioned for such drug-loaded films include drug and vaccine delivery through the transdermal route, antimicrobial or anti-inflammatory coatings on implants and drug-releasing coatings for stents. (C) 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Chitosan-dextran sulphate nanocapsule drug delivery system as an effective therapeutic against intraphagosomal pathogen Salmonella

Objectives: The ability to target conventional drugs efficiently inside cells to kill intraphagosomal bacteria has been a major hurdle in treatment of infective diseases. We aimed to develop an efficient drug delivery system for combating infection caused by Salmonella, a well-known intracellular and intraphagosomal pathogen. Chitosan dextran sulphate (CD) nanocapsules were assessed for their efficiency in delivering drugs against Salmonella. Methods: The CD nanocapsules were prepared using the layer-by-layer method and loaded with ciprofloxacin or ceftriaxone. Antibiotic-loaded nanocapsules were analysed in vitro for their ability to enter epithelial and macrophage cells to kill Salmonella. In vivo pharmacokinetics and organ distribution studies were performed to check the efficiency of the delivery system. The in vivo antibacterial activity of free antibiotic and antibiotic loaded into nanocapsules was tested in a murine salmonellosis model. Results: In vitro and in vivo experiments showed that this delivery system can be used effectively to clear Salmonella infection, CD nanocapsules were successfully employed for efficient targeting and killing of the intracellular pathogen at a dosage significantly lower than that of the free antibiotic. The increased retention time of ciprofloxacin in the blood and organs when it was delivered by CD nanocapsules compared with the conventional routes of administration may be the reason underlying the requirement for a reduced dosage and frequency of antibiotic administration. Conclusions: CD nanocapsules can be used as an efficient drug delivery system to treat intraphagosomal pathogens, especially Salmonella infection, This delivery system might be used effectively for other vacuolar pathogens including Mycobacteria, Brucella and Legionella.

In vitro and in silico biological studies of novel thiazolo3,2-a]pyrimidine-6-carboxylate derivatives

In the present study, we report the synthesis, characterization of new series of thiazolo3,2-a]pyrimidine-6-carboxylate derivatives 3a-f and 4a-f. The newly synthesized compounds were screened for in vitro antimicrobial and antiviral activities. The probable mode of action of these active compounds was determined through in silico docking study by docking the receptor methionyl-tRNA synthetase and human inosine-5'-monophosphate dehydrogenase (IMPDH) for antibacterial and antiviral activities, respectively. Among the compounds, 4c exhibited excellent in vitro antimicrobial activity against all tested strains with binding and docking energies -35.6 and -12.4 kcal/mol, respectively. The antiviral studies were carried out for the selected compounds in which 4a exhibited 73.69 and 54.42 % of inhibition of buffalopox and camelpox viruses, respectively. Furthermore, compound 4a showed minimum docking and binding energy along with the maximum hydrogen/hydrophobic interaction with IMPDH. The study contributes towards identification and screening of potential antimicrobial and antiviral agent's against the pathogens.

Molecular Basis for the Differential Quinolone Susceptibility of Mycobacterial DNA Gyrase

DNA gyrase is a type II topoisomerase that catalyzes the introduction of negative supercoils in the genomes of eubacteria. Fluoroquinolones (FQs), successful as drugs clinically, target the enzyme to trap the gyrase-DNA complex, leading to the accumulation of double-strand breaks in the genome. Mycobacteria are less susceptible to commonly used FQs. However, an 8-methoxy-substituted FQ, moxifloxacin (MFX), is a potent antimycobacterial, and a higher susceptibility of mycobacterial gyrase to MFX has been demonstrated. Although several models explain the mechanism of FQ action and gyrase-DNA-FQ interaction, the basis for the differential susceptibility of mycobacterial gyrase to various FQs is not understood. We have addressed the basis of the differential susceptibility of the gyrase and revisited the mode of action of FQs. We demonstrate that FQs bind both Escherichia coli and Mycobacterium tuberculosis gyrases in the absence of DNA and that the addition of DNA enhances the drug binding. The FQs bind primarily to the GyrA subunit of mycobacterial gyrase, while in E. coli holoenzyme is the target. The binding of MFX to GyrA of M. tuberculosis correlates with its effectiveness as a better inhibitor of the enzyme and its efficacy in cell killing.

Amine-functionalized multiwall carbon nanotubes impart osteoinductive and bactericidal properties in poly(epsilon-caprolactone) composites

Poly(epsilon-caprolactone) (PCL) is an aliphatic polyester widely used for biomedical applications but lacks the mechanical properties desired for many load-bearing orthopedic applications. The objective of this study was to prepare and characterize PCL composites incorporating multiwall carbon nanotubes (MWNTs) with different surface functional groups. PCL composites were prepared by melt-mixing with three different types of MWNTs: pristine (pMWNT), amine functionalized (aMWNT), and carboxyl functionalized (cMWNT). Melt rheology and scanning electron microscopy indicated good dispersion of the nanotubes in the matrix. Tensile strength and elastic modulus of the polymer was significantly increased by the incorporation of MWNTs and further enhanced by favorable interactions between PCL and aMWNTs. Thermal analysis revealed that MWNTs act as heterogeneous nucleation sites for crystallization of PCL and increase polymer crystallinity. Incorporation of functionalized MWNTs increased the surface water wettability of PCL. Osteoblast proliferation and differentiation was significantly enhanced on functionalized composites. aMWNT composites also exhibited the best bactericidal response. This study demonstrates that surface functionalization of MWNTs profoundly influences the properties of PCL and amine-functionalization offers the optimal combination of mechanical properties, osteogenesis and antimicrobial response. These results have important implications for designing nanocomposites for use in orthopedics.

Suramin is a potent and selective inhibitor of Mycobacterium tuberculosis RecA protein and the SOS response: RecA as a potential target for antibacterial drug discovery

In eubacteria, RecA is essential for recombinational DNA repair and for stalled replication forks to resume DNA synthesis. Recent work has implicated a role for RecA in the development of antibiotic resistance in pathogenic bacteria. Consequently, our goal is to identify and characterize small-molecule inhibitors that target RecA both in vitro and in vivo. We employed ATPase, DNA strand exchange and LexA cleavage assays to elucidate the inhibitory effects of suramin on Mycobacterium tuberculosis RecA. To gain insights into the mechanism of suramin action, we directly visualized the structure of RecA nucleoprotein filaments by atomic force microscopy. To determine the specificity of suramin action in vivo, we investigated its effect on the SOS response by pull-down and western blot assays as well as for its antibacterial activity. We show that suramin is a potent inhibitor of DNA strand exchange and ATPase activities of bacterial RecA proteins with IC50 values in the low micromolar range. Additional evidence shows that suramin inhibits RecA-catalysed proteolytic cleavage of the LexA repressor. The mechanism underlying such inhibitory actions of suramin involves its ability to disassemble RecA-single-stranded DNA filaments. Notably, suramin abolished ciprofloxacin-induced recA gene expression and the SOS response and augmented the bactericidal action of ciprofloxacin. Our findings suggest a strategy to chemically disrupt the vital processes controlled by RecA and hence the promise of small molecules for use against drug-susceptible as well as drug-resistant strains of M. tuberculosis for better infection control and the development of new therapies.

Characterisation and bioactivity of oosporein produced by endophytic fungus Cochliobolus kusanoi isolated from Nerium oleander L.

Bioactive compounds comprising secondary metabolites produced by endophytic fungi have wide applications in pharmacology and agriculture. Isolation, characterisation and evaluation of biological activities of secondary metabolites were carried out from Cochliobolus kusanoi an endophytic fungus of Nerium oleander L. The fungus was identified based on 18S rDNA sequence analysis. There are no reports available on the compounds of C. kusanoi hence, antimicrobial metabolite produced by this fungus was extracted and purified by fractionation using hexane, diethyl ether, dichloromethane, ethyl acetate and methanol. Out of all the solvent fractions, the methanol fraction exhibited better antimicrobial activity which was further purified and characterised as oosporein. Oosporein from C. kusanoi exhibited broad spectrum in vitro antimicrobial, antioxidant and cytotoxic activities. The characterisation and antioxidant activity of oosporein from C. kusanoi are reported for the first time.

Interactome analyses of Salmonella pathogenicity islands reveal SicA indispensable for virulence

Background: Serovars of Salmonella enterica, namely Typhi and Typhimurium, reportedly, are the bacterial pathogens causing systemic infections like gastroenteritis and typhoid fever. To elucidate the role and importance in such infection, the proteins of the Type III secretion system of Salmonella pathogenicity islands and two component signal transduction systems, have been mainly focused. However, the most indispensable of these virulent ones and their hierarchical role has not yet been studied extensively. Results: We have adopted a theoretical approach to build an interactome comprising the proteins from the Salmonella pathogeneicity islands (SPI) and two component signal transduction systems. This interactome was then analyzed by using network parameters like centrality and k-core measures. An initial step to capture the fingerprint of the core network resulted in a set of proteins which are involved in the process of invasion and colonization, thereby becoming more important in the process of infection. These proteins pertained to the Inv, Org, Prg, Sip, Spa, Ssa and Sse operons along with chaperone protein SicA. Amongst them, SicA was figured out to be the most indispensable protein from different network parametric analyses. Subsequently, the gene expression levels of all these theoretically identified important proteins were confirmed by microarray data analysis. Finally, we have proposed a hierarchy of the proteins involved in the total infection process. This theoretical approach is the first of its kind to figure out potential virulence determinants encoded by SPI for therapeutic targets for enteric infection. Conclusions: A set of responsible virulent proteins was identified and the expression level of their genes was validated by using independent, published microarray data. The result was a targeted set of proteins that could serve as sensitive predictors and form the foundation for a series of trials in the wet-lab setting. Understanding these regulatory and virulent proteins would provide insight into conditions which are encountered by this intracellular enteric pathogen during the course of infection. This would further contribute in identifying novel targets for antimicrobial agents. (C) 2014 Elsevier Ltd. All rights reserved.

Targeting Mycobacterium tuberculosis Topoisomerase I by Small-Molecule Inhibitors

We describe inhibition of Mycobacterium tuberculosis topoisomerase I (MttopoI), an essential mycobacterial enzyme, by two related compounds, imipramine and norclomipramine, of which imipramine is clinically used as an antidepressant. These molecules showed growth inhibition of both Mycobacterium smegmatis and Mycobacterium tuberculosis cells. The mechanism of action of these two molecules was investigated by analyzing the individual steps of the topoisomerase I (topoI) reaction cycle. The compounds stimulated cleavage, thereby perturbing the cleavage-religation equilibrium. Consequently, these molecules inhibited the growth of the cells overexpressing topoI at a low MIC. Docking of the molecules on the MttopoI model suggested that they bind near the metal binding site of the enzyme. The DNA relaxation activity of the metal binding mutants harboring mutations in the DxDxE motif was differentially affected by the molecules, suggesting that the metal coordinating residues contribute to the interaction of the enzyme with the drug. Taken together, the results highlight the potential of these small molecules, which poison the Mycobacterium tuberculosis and Mycobacterium smegmatis topoisomerase I, as leads for the development of improved molecules to combat mycobacterial infections. Moreover, targeting metal coordination in topoisomerases might be a general strategy to develop new lead molecules.

Tetrahydrofuran amino acid-containing gramicidin S analogues with improved biological profiles

Gramicidin S (GS) is a cyclic cationic antimicrobial peptide (CAP) with a wide spectrum of antibiotic activities whose usage has been limited to topical applications owing to its cytotoxic side effects. We have synthesized tetrahydrofuran amino acid (Taa)-containing GS analogues, and we have carried out conformational analysis and explored their structure activity relationships by evaluating their antitubercular, antibacterial and cytotoxic properties. Two of these analogues showed impressive as well as selective activity against Mycobacterium tuberculosis (MTB) without toxicity towards mammalian Vero cells or human RBCs, and are promising as potential leads.

Cytotoxicity of Ultrasmall Gold Nanoparticles on Planktonic and Biofilm Encapsulated Gram-Positive Staphylococci

The emergence of multidrug resistant bacteria, especially biofilm-associated Staphylococci, urgently requires novel antimicrobial agents. The antibacterial activity of ultrasmall gold nanoparticles (AuNPs) is tested against two gram positive: S. aureus and S. epidermidis and two gram negative: Escherichia coli and Pseudomonas aeruginosa strains. Ultrasmall AuNPs with core diameters of 0.8 and 1.4 nm and a triphenylphosphine-monosulfonate shell (Au0.8MS and Au1.4MS) both have minimum inhibitory concentration (MIC) and minimum bactericidal concentration of 25 x 10(-6)m Au]. Disc agar diffusion test demonstrates greater bactericidal activity of the Au0.8MS nanoparticles over Au1.4MS. In contrast, thiol-stabilized AuNPs with a diameter of 1.9 nm (AuroVist) cause no significant toxicity in any of the bacterial strains. Ultrasmall AuNPs cause a near 5 log bacterial growth reduction in the first 5 h of exposure, and incomplete recovery after 21 h. Bacteria show marked membrane blebbing and lysis in biofilm-associated bacteria treated with ultrasmall AuNP. Importantly, a twofold MIC dosage of Au0.8MS and Au1.4MS each cause around 80%-90% reduction in the viability of Staphylococci enveloped in biofilms. Altogether, this study demonstrates potential therapeutic activity of ultrasmall AuNPs as an effective treatment option against staphylococcal infections.