Isoprenoid Biosynthesis Inhibitors Targeting Bacterial Cell Growth

We synthesized potential inhibitors of farnesyl diphosphate synthase (FPPS), undecaprenyl diphosphate synthase (UPPS), or undecaprenyl diphosphate phosphatase (UPPP), and tested them in bacterial cell growth and enzyme inhibition assays. The most active compounds were found to be bisphosphonates with electron‐withdrawing aryl‐alkyl side chains which inhibited the growth of Gram‐negative bacteria (Acinetobacter baumannii, Klebsiella pneumoniae, Escherichia coli, and Pseudomonas aeruginosa) at ～1–4 μg mL^?1 levels. They were found to be potent inhibitors of FPPS; cell growth was partially “rescued” by the addition of farnesol or overexpression of FPPS, and there was synergistic activity with known isoprenoid biosynthesis pathway inhibitors. Lipophilic hydroxyalkyl phosphonic acids inhibited UPPS and UPPP at micromolar levels; they were active (～2–6 μg mL^?1) against Gram‐positive but not Gram‐negative organisms, and again exhibited synergistic activity with cell wall biosynthesis inhibitors, but only indifferent effects with other inhibitors. The results are of interest because they describe novel inhibitors of FPPS, UPPS, and UPPP with cell growth inhibitory activities as low as ～1–2 μg mL^?1.     

Spectrophotometric Determination of Benzalkonium Bromide in Pharmaceutical Samples with Alizarin Green

A direct, extraction‐free spectrophotometric method was developed for the determination of benzalkonium bromide (BAK). The method is based on the formation of mixed dye–surfactant aggregates between alizarin green (AG) and BAK in alkaline medium by measuring the decrease in absorbance of AG at 460 and 700 nm. Beer's law was obeyed in the concentration range 3–40 μg mL^?1 with good precision and accuracy. The limits of detection were 0.4 μg mL^?1 at 460 nm and 0.3 μg mL^?1 at 700 nm, which reduced to 0.2 μg mL^?1 by combining the absorbance at the two wavelengths. The proposed method was successfully applied to the determination of BAK in disinfectant solution and eye drops. The analytical results of the real samples were in good agreement with those of an HPLC method.     

New Antimicrobial Hexapeptides: Synthesis,Antimicrobial Activities,Cytotoxicity, and Mechanistic Studies

Synthetic antimicrobial peptides have recently emerged as promising candidates against drug‐resistant pathogens. We identified a novel hexapeptide, Orn‐D ‐Trp‐D ‐Phe‐Ile‐D ‐Phe‐His(1‐Bzl)‐NH₂, which exhibits broad‐spectrum antifungal and antibacterial activity. A lead optimization was undertaken by conducting a full amino acid scan with various proteinogenic and non‐proteinogenic amino acids depending on the hydrophobic or positive‐charge character of residues at various positions along the sequence. The hexapeptide was also cyclized to study the correlation between the linear and cyclic structures and their respective antimicrobial activities. The synthesized peptides were found to be active against the fungus Candida albicans and Gram‐positive bacteria such as methicillin‐resistant Staphylococcus aureus and methicillin‐resistant Staphylococcus epidermidis, as well as the Gram‐negative bacterium Escherichia coli; MIC values for the most potent structures were in the range of 1–5 μg mL^?1 (IC₅₀ values in the range of 0.02–2 μg mL^?1). Most of the synthesized peptides showed no cytotoxic effects in an MTT assay up to the highest test concentration of 200 μg mL^?1. A tryptophan fluorescence quenching study was performed in the presence of negatively charged and zwitterionic model membranes, mimicking bacterial and mammalian membranes, respectively. The results of the fluorescence study demonstrate that the tested peptides are selective toward bacterial over mammalian cells; this is associated with a preferential interaction between the peptides and the negatively charged phospholipids of bacterial cells.     

Synthesis,characterization, and application of a new chelating resin functionalized with dithiooxamide

Chloromethylated polystyrene‐divinylbenzene has been functionalized with dithiooxamide. The resulting chelating resin (DTOA) has been characterized by elemental analyses, infrared spectroscopy, thermogravimetric analysis, and metal ion sorption capacities. It has been used for the preconcentration and separation of Cu(II), Zn(II), Cd(II), and Pb(II) prior to their determination by FAAS. Parameters such as the amount of the resin, effect of pH, equilibration rate, sorption and desorption of metal ions, and effect of diverse ions have been studied. The maximum sorption capacities found are 0.97, 0.12, 0.08, and 0.12 mmol g^?1 for Cu(II), Zn(II), Cd(II), and Pb(II) at pH 6.0, 5.5, 1.0, and 5.5, respectively. The preconcentration factors are 100, 100, 50, and 50 for Cu(II), Zn(II), Cd(II), and Pb(II), respectively. Recoveries of the metal ions were 96 ± 5, 97 ± 6, 96 ± 5, and 96 ± 5 at 95% confidence level, whereas the limits of detection are 2.0, 1.3, 2.5, and 25.0 μg L^?1 for Cu(II), Zn(II), Cd(II), and Pb(II), respectively. The calibration curves were linear up to 12 μg mL^?1 (R² = 1.000), 2 μg mL^?1 (R² = 0.998), 2 μg ml^?1 (R² = 1.000), and 5 μg mL^?1 (R² = 0.979) for Cu(II), Zn(II), Cd(II), and Pb(II), respectively. The reliability of the method has been tested by analyzing certified samples. © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 103: 2281–2285, 2007     

Synthesis and Anti-Listeria Properties of Odorless Hybrid Bio-Based n-Phenolic Vegetable Branched-Chain Fatty Acids

Consumers are becoming concerned about the impact of synthetic chemicals on human health and environments, and demanding natural compounds to reduce risk of antibiotic resistance of microorganisms. However, natural compounds are often less effective than synthetic antimicrobials. This challenge may be addressed with the development of bio-based antimicrobial agents. In this study, bio-based n-phenolic branched-chain fatty acids (n-phenolic-branched chain fatty acid [BCFA]) were synthesized from vegetable oil (soybean and safflower) fatty acids and four natural phenolics (phenol, thymol, carvacrol, and creosote), and tested against Listeria innocua. Results revealed that the newly synthesized products in crude form had minimum inhibitory concentrations (MIC) against L. innocua ranging from 3.6 to 116.4 μg mL⁻¹, with phenol-BCFA products having the lowest MIC (3.6 μg mL⁻¹) and minimum bactericidal concentration (MBC) (7.3 μg mL⁻¹). The precursors (unsaturated free fatty acids and phenolics) and noncovalently bound mixture of free fatty acids and phenolics had MIC above 232.7 μg mL⁻¹. After purification by molecular fractionation, n-phenolic-BCFA in the free fatty acid/monomer form were shown to be responsible for the anti-Listeria activity with MIC of 3.6–7.3 μg mL⁻¹ and MBC of 7.3–29.1 μg mL⁻¹. These promising results pave the road for further study of this new class of bio-based compounds, which may lead to their widespread use.     

Two Spectrophotometric Methods for the Assay of Benzalkonium Chloride in Bandage Samples

Two rapid, direct, extraction‐free spectrophotometric procedures were developed for the determination of benzalkonium chloride. The procedures were based on the formation of mixed dye‐surfactant aggregates between eosine Y and benzalkonium chloride (Method A), or between eosine B and benzalkonium chloride (Method B) in Clark‐Lubs medium by measuring the increase in absorbance at 556 and 550 nm, respectively. Beer's law was obeyed in the concentration range of 0.5–12 and 0.5–12 μg mL^?1 for benzalkonium chloride with good precision and accuracy, whose limits of detection were 0.1 μg mL^?1 at 556 nm for Method A and 0.2 μg mL^?1 at 550 nm for Method B, respectively. The proposed procedures were successfully applied to the assay of benzalkonium chloride in bandages. The analytical results of the real samples were in good agreement with those by HPLC.     

In silico Optimization of a Fragment‐Based Hit Yields Biologically Active,High‐Efficiency Inhibitors for Glutamate Racemase

A novel lead compound for inhibition of the antibacterial drug target, glutamate racemase (GR), was optimized for both ligand efficiency and lipophilic efficiency. A previously developed hybrid molecular dynamics–docking and scoring scheme, FERM‐SMD, was used to predict relative potencies of potential derivatives prior to chemical synthesis. This scheme was successful in distinguishing between high‐ and low‐affinity binders with minimal experimental structural information, saving time and resources in the process. In vitro potency was increased approximately fourfold against GR from the model organism, B. subtilis. Lead derivatives show two‐ to fourfold increased antimicrobial potency over the parent scaffold. In addition, specificity toward B. subtilis over E. coli and S. aureus depends on the substituent added to the parent scaffold. Finally, insight was gained into the capacity for these compounds to reach the target enzyme in vivo using a bacterial cell wall lysis assay. The outcome of this study is a novel small‐molecule inhibitor of GR with the following characteristics: K_i=2.5 μM , LE=0.45 kcal mol^?1 atom^?1, LiPE=6.0, MIC₅₀=260 μg mL^?1 against B. subtilis, EC_{50, lysis}=520 μg mL^?1 against B. subtilis.     

Salimyxins and Enhygrolides: Antibiotic,Sponge‐Related Metabolites from the Obligate Marine Myxobacterium Enhygromyxa salina

Unlike their terrestrial counterparts, marine myxobacteria are hardly investigated for their secondary metabolites. This study describes three new compounds ( 1 – 3 ), named salimyxins and enhygrolides, obtained from the obligate marine myxobacterium Enhygromyxa salina. These are the first natural products obtained from Enhygromyxa species. Their structures were elucidated by spectroscopic analysis, including NMR and CD spectroscopy. Enhygrolides are closely related to the nostoclides, which were initially isolated from a cyanobacterium of the genus Nostoc. The salimyxins, representing structurally most unusual degraded sterols, are close to identical to demethylincisterol from the sponge Homaxinella sp. Salimyxin B and enhygrolide A inhibit the growth of the Gram‐positive bacterium Arthrobacter cristallopoietes (MIC salimyxin B, 8 μg mL^?1; enhygrolide A, 4 μg mL^?1).     

A Consistent Dataset of Kinetic Solubilities for Early‐Phase Drug Discovery

Herein, we describe a new dataset of kinetic aqueous solubilities determined by nephelometry for 711 druglike compounds. The solubilities are reported in twelve classes ranging from <2 μg mL^?1 to >250 μg mL^?1. The measurements were designed to provide the appropriate data for applications in the early phases of drug discovery. Three class classification models (insoluble, moderately soluble, soluble) were built using the random forest algorithm and their performance for this dataset was analyzed.     

Synthetic Arabinomannan Heptasaccharide Glycolipids Inhibit Biofilm Growth and Augment Isoniazid Effects in Mycobacterium smegmatis

Biofilm formation, involving attachment to an adherent surface, is a critical survival strategy of mycobacterial colonies in hostile environmental conditions. Here we report the synthesis of heptasaccharide glycolipids based on mannopyranoside units anchored on to a branched arabinofuranoside core. Two types of glycolipids—2,3‐branched and 2,5‐branched—were synthesized and evaluated for their efficacies in inhibiting biofilm growth by the non‐pathogenic mycobacterium variant Mycobacterium smegmatis. Biofilm formation was inhibited at a minimum biofilm growth inhibition concentration (MBIC) of 100 μg mL^?1 in the case of the 2,5‐branched heptasaccharide glycolipid. Further, we were able to ascertain that a combination of the drug isoniazid with the branched heptasaccharide glycolipid (50 μg mL^?1) potentiates the drug, making it three times more effective, with an improved MBIC of 30 μg mL^?1. These studies establish that synthetic glycolipids not only act as inhibitors of biofilm growth, but also provide a synergistic effect when combined with significantly lowered concentrations of isoniazid to disrupt the biofilm structures of the mycobacteria.     

Studies on a new water-soluble sulfur-containing cellulose derivative

A new sulfur-containing cellulose derivative, carboxymethyl cellulose (CMC) xanthate (CMCX), has been prepared by the reaction of CMC with CS₂ in 10–15% NaOH solution for 1 h. Degree of esterification (DE) of the product, determined by UV, may reach 3.3% by mole (or about 1.1% by weight). The product was water soluble. The stability of CMCX has been studied by UV, DSC, and TGA, and it was found that CMCX was more stable in the solid state than in solution. The precipitation of gold ion in dilute solution (10 μg Au · mL^?1) by CMCX was very rapid, and the recovery efficiency of gold could reach 96% or higher. The optimum dosage was about 0.1 g CMCX for 10 mL 10 μg Au · mL^?1 solution. © 1994 John Wiley & Sons, Inc.     

Optimization of chitosanase production from Bacillus sp. RKY3 using statistical experimental designs

BACKGROUND: The culture medium and fermentation conditions for the production of constitutive chitosanase from a newly isolated Bacillus sp. RKY3 were optimized statistically. RESULTS: The variables significantly influencing both chitosanase production and cell growth were screened through the Plackett–Burman design, by which maltose, beef extract, MgSO₄, and incubation time were identified as the most significant variables. The optimum values of the selected variables and their mutual interactions were determined through the steepest ascent method and Box–Behnken experimental design. The results demonstrated that 62.30 U mL^?1 chitosanase activity was predicted with optimum conditions of maltose (30.18 g L^?1), beef extract (15.25 g L^?1), MgSO₄ (0.26 g L^?1), and incubation time (50.02 h). The predicted response was verified by the validation experiments, and the optimum conditions resulted in a maximum chitosanase activity of 63.53 ± 1.22 U mL^?1. CONCLUSION: The optimization of fermentation variables resulted in an approximately 11.3‐fold increase in chitosanase activity compared with that observed under unoptimized conditions (from 5.63 U mL^?1 to 63.53 U mL^?1). Copyright © 2009 Society of Chemical Industry     

The effect of amino acids and amino acid analogues on growth of an obligate methanotroph,Methylosinus trichosporium OB3b

The sensitivity of the obligate methanotroph, Methylosinus trichosporium OB3b to a number of amino acid analogues and amino acids has been examined. Sulphaguanidine (5 μg ml^?1) norleucine (100 μg ml^?1) m-fluorophenylalanine (50 μg ml^?1), p-fluorophenylalanine (50 μg ml^?1) and S2-aminoethyl-C-cysteine (1000 μg ml^?1) inhibited growth. Proline, threonine, methionine and lysine (2–4 μg ml^?1) also inhibited growth, but arginine and leucine at similar concentrations had no effect. Attempts were made to isolate amino acid analogue resistant mutants, which would be expected to overproduce amino acids. Two approaches were made, selection of spontaneous and induced mutants in plate culture, and selection of a spontaneous mutant during growth in continuous culture under selective conditions, but no mutants were obtained. Possible reasons for failure to obtain such mutants are discussed.     

Production and Quantitative Analysis of Trehalose Lipid Biosurfactants Using High‐Performance Liquid Chromatography

Trehalose lipids (THL) are glycolipid biosurfactants having a wide range of biomedical and environmental applications. Low yield, high purification cost, and the absence of a valid analytical method hinders their application. Hence, in the present study a simple, rapid, and reliable isocratic high‐performance liquid chromatography (LC) method was developed for the identification and quantification of trehalose lipid biosurfactants from Rhodococcus erythropolis. THL having a minimum surface tension of 24 mN m^?1 and a critical micellar concentration of 25 mg L^?1 were produced using hexadecane as a substrate. A standard was developed from the crude THL mixture using thin‐layer chromatography and column chromatography and its structure was confirmed using infrared spectroscopy, mass spectroscopy, and ¹H NMR. A high performance liquid chromatography (HPLC) method for quantitation was developed using a C18 column with water/acetonitrile (80:20) as the mobile phase at a 1 mL min^?1 flow rate and UV detection at 208 nm. This method was validated according to International Conference on Harmonization guidelines for linearity, precision, accuracy, robustness, LOD, and LOQ. This method was found to be linear over the range 10‐50 μg m L^?1 (r² =0.99801), precise, accurate, and robust. This method can detect minimum 3.2 μg mL^?1 and quantify minimum 9.2 μg mL^?1 of THL. Standards were developed from R. erythropolis, broth and purified standard trehalose 6,6′‐dimycolate from Mycobacterium bovis, having the same retention time of 2.0 min. The yield was calculated from the calibration curve and was found to be 25 g L^?1.     

Direct electrochemistry of double strand DNA on ionic liquid modified screen-printed graphite electrode

An ionic liquid modified screen-printed graphite electrode (SPE) was used for direct electrochemistry of herring sperm double strand DNA (dsDNA) by voltammetry. Due to the high conductivity of ionic liquid n-octylpyridinum hexafluorophosphate (OPPF), this electrode exhibited excellent electrochemical activity for the oxidation of dsDNA. Two irreversible oxidation peaks were obtained at the developed electrode, which corresponded to the oxidation of guanine and adenine residues present in the dsDNA. The basic electrochemical behavior of dsDNA at the OPPF modified SPE was carefully investigated. Combined with the differential pulse voltammetry, this electrode exhibited a good linear range from 20 μg mL⁻¹ to 120 μg mL⁻¹ with a detection limit of 5 μg mL⁻¹ for the direct determination of dsDNA. Furthermore, the OPPF modified electrode displayed high reproducibility and stability for the dsDNA determination.     

Structural Optimization of Berberine as a Synergist to Restore Antifungal Activity of Fluconazole against Drug‐Resistant Candida albicans

We have conducted systematic structural modification, deconstruction, and reconstruction of the berberine core with the aim of lowering its cytotoxicity, investigating its pharmacophore, and ultimately, seeking novel synergistic agents to restore the effectiveness of fluconazole against fluconazole‐resistant Candida albicans. A structure–activity relationship study of 95 analogues led us to identify the novel scaffold of N‐(2‐(benzo[d][1,3]dioxol‐5‐yl)ethyl)‐2‐(substituted phenyl)acetamides 7 a – l , which exhibited remarkable levels of in vitro synergistic antifungal activity. Compound 7 d (N‐(2‐(benzo[d][1,3]dioxol‐5‐yl)ethyl)‐2‐(2‐fluorophenyl)acetamide) significantly decreased the MIC₈₀ values of fluconazole from 128.0 μg mL^?1 to 0.5 μg mL^?1 against fluconazole‐resistant C. albicans and exhibited much lower levels of cytotoxicity than berberine toward human umbilical vein endothelial cells.     

Synthesis and Antimicrobial Activities of Oximes Derived from O-Benzylhydroxylamine as FabH Inhibitors

Forty‐three oxime derivatives were synthesized by allowing O‐benzylhydroxylamines to react with primary benzaldehydes or salicylaldehydes; these products were gauged as potential inhibitors of β‐ketoacyl‐(acyl‐carrier‐protein) synthase III (FabH). Among the 43 compounds, 38 are reported herein for the first time. These compounds were assayed for antimicrobial activities against Escherichia coli, Pseudomonas aeruginosa, Pseudomonas fluorescens, Bacillus subtilis, Staphylococcus aureus, and Enterococcus faecalis. Compounds with prominent antibacterial activities were tested for their E. coli FabH inhibitory activities. 3‐((2,4‐Dichlorobenzyloxyimino)methyl)benzaldehyde O‐2,4‐dichlorobenzyl oxime ( 44 ) showed the best antibacterial activity, with minimum inhibitory concentrations of 3.13–6.25 μg mL^?1 against the tested bacterial strains, exhibiting the best E. coli FabH inhibitory activity, with an IC₅₀ value of 1.7 mM . Docking simulations were performed to position compound 44 into the E. coli FabH active site in order to determine the most probable binding conformation.     

Design of a probe based on poly(glycidyl methacrylate‐co‐vinylferrocene)‐coated Pt electrode for electrochemical detection of PTEN gene in PCR amplified samples from prostate tissues

In this study, a new probe based on immobilization of amino linked oligonucleotide (NH₂‐linked DNA) on poly(glycidyl methacrylate‐co‐vinylferrocene)‐coated Pt electrode was fabricated for the electrochemical detection of PTEN gene from human prostate tissues. The experimental parameters such as DNA immobilization time, DNA concentration, and target concentration were optimized. The selectivity of the NH₂‐linked DNA probe was assessed with mismatch (MM) and noncomplementary (NC) sequences. The applicability of the NH₂‐linked DNA probe to the PCR amplified samples correspond to PTEN gene from prostate tissues was evaluated. The immobilization of DNA on the copolymer was confirmed by FTIR, AFM, CV and DPV analysis. The PCR products were also identified by using agarose gel electrophoresis. The prepared probe indicated a linear range (10–100 μg mL^?1) with a detection limit (4.7 μg mL^?1) and a good selectivity of the NH₂‐linked DNA probe toward target DNA sequence. © 2014 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014 , 131, 40638.     

Solvent extraction separation and spectrophotometric determination of ruthenium(III) with p-methylphenyl thiourea: sequential separation of ruthenium,osmium and iron

ABSTRACT

A selective and sensitive solvent extraction and spectrophotometric study of the ruthenium(III)–p-methylphenyl thiourea (PMPT) system is presented. The optimum conditions were determined by a critical study of acid concentration, reagent concentration, equilibration period, heating time and effect of solvent on the equilibrium. Ruthenium(III) forms 1:1 complex with PMPT in 20% ethanol and extracted into chloroform. Conformity to Beer’s law at 600 nm was observed up to 40 µg mL^–1 of ruthenium. Molar absorptivity and Sandell’s sensitivity were found to be 2.31 × 10³ L mol^?1cm^?1 and 0.044 μg cm^?2, respectively. The detection limits were 0.11 μg mL^?1 of ruthenium. The method is free from interferences from large number of cations and anions. Proposed method was successfully applied to the separation and determination of ruthenium from synthetic alloys, catalyst and water samples. Sequential separation and determination method for ruthenium(III), osmium(VIII) and iron(II) has been developed.     

C2 Arylated Benzo[b]thiophene Derivatives as Staphylococcus aureus NorA Efflux Pump Inhibitors

An innovative and straightforward synthesis of second‐generation 2‐arylbenzo[b]thiophenes as structural analogues of INF55 and the first generation of our laboratory‐made molecules was developed. The synthesis of C2‐arylated benzo[b]thiophene derivatives was achieved through a method involving direct arylation, followed by simple structural modifications. Among the 34 compounds tested, two of them were potent NorA pump inhibitors, which led to a 16‐fold decrease in the ciprofloxacin minimum inhibitory concentration (MIC) against the SA‐1199B strain at concentrations of 0.25 and 0.5 μg mL^?1 (1 and 1.5 μm , respectively). This is a promising result relative to that obtained for reserpine (MIC=20 μg mL^?1), a reference compound amongst NorA pump inhibitors. These molecules thus represent promising candidates to be used in combination with ciprofloxacin against fluoroquinolone‐resistant strains.