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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Improving Cell‐Free Protein Synthesis through Genome Engineering of Escherichia coli Lacking Release Factor 1

Site‐specific incorporation of non‐standard amino acids (NSAAs) into proteins opens the way to novel biological insights and applications in biotechnology. Here, we describe the development of a high yielding cell‐free protein synthesis (CFPS) platform for NSAA incorporation from crude extracts of genomically recoded Escherichia coli lacking release factor 1. We used genome engineering to construct synthetic organisms that, upon cell lysis, lead to improved extract performance. We targeted five potential negative effectors to be disabled: the nuclease genes rna, rnb, csdA, mazF, and endA. Using our most productive extract from strain MCJ.559 (csdA^? endA^?), we synthesized 550±40 μg mL^?1 of modified superfolder green fluorescent protein containing p‐acetyl‐L ‐phenylalanine. This yield was increased to ～1300 μg mL^?1 when using a semicontinuous method. Our work has implications for using whole genome editing for CFPS strain development, expanding the chemistry of biological systems, and cell‐free synthetic biology.     

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     

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     

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.     

Novel Aeruginosin‐865 from Nostoc sp. as a Potent Anti‐inflammatory Agent

Aeruginosin‐865 (Aer‐865), isolated from terrestrial cyanobacterium Nostoc sp. Luke?ová 30/93, is the first aeruginosin‐type peptide containing both a fatty acid and a carbohydrate moiety, and is the first aeruginosin to be found in the genus Nostoc. Mass spectrometry, chemical and spectroscopic analysis as well as one‐ and two‐dimensional NMR and chiral HPLC analysis of Marfey derivatives were applied to determine the peptidic sequence: D ‐Hpla, D ‐Leu, 5‐OH‐Choi, Agma, with hexanoic and mannopyranosyl uronic acid moieties linked to Choi. We used an AlphaLISA assay to measure the levels of proinflammatory mediators IL‐8 and ICAM‐1 in hTNF‐α‐stimulated HLMVECs. Aer‐865 showed significant reduction of both: with EC₅₀ values of (3.5±1.5) μg mL^?1 ((4.0±1.7) μM ) and (50.0±13.4) μg mL^?1 ((57.8±15.5) μM ), respectively. Confocal laser scanning microscopy revealed that the anti‐inflammatory effect of Aer‐865 was directly associated with inhibition of NF‐κB translocation to the nucleus. Moreover, Aer‐865 did not show any cytotoxic effect.     

Synthesis,characterization, and antifungal activity of nystatin—gum arabic conjugates

Nystatin, a polyene tetraene antibiotic widely used in the treatment of mycoses, was coupled with oxidized polysaccharide gum Arabic, by forming Schiff base structures between amine groups of antibiotics and aldehyde groups of modified carbohydrate. Imine conjugates synthesized in this way were reduced with sodium borohydride to secondary amines. Two imine and two amine conjugates were obtained with different nystatin content. The conjugates were characterized by UV–Vis, FTIR, ¹H NMR spectroscopy, and thermogravimetric analysis. Solubility in water, unlike nystatin, and significant activity against Candida albicans and Aspergillus niger with minimum inhibitory concentrations in range of 3.125–6.25 μg mL^?1 and 6.25–25 μg mL^?1, respectively, indicate that the chemical integrity and the biological function of these compounds were retained. A comparison of stability of the conjugates in the dry form, solution and under different pH values showed that the conjugates exhibited better stability than pure drug. © 2012 Wiley Periodicals, Inc. J. Appl. Polym. Sci., 2013     

Molecularly Imprinted Polymer for Solid-Phase Extraction of Phenolic Acids from Salicornia herbacea L.

This paper reports the application of a molecularly imprinted polymer (MIP) for the extraction and separation of phenolic acids from Salicornia herbacea L. (S. herbacea) using a solid phase extraction (SPE) system. The template of the MIP for phenolic acid extraction and separation was selected experimentally as one of seven templates. The obtained material with Benzoic acid (BA) as a template had the best extraction capacity of phenolic acids from S. herbacea. Water and methanol/HCl were selected as a washing solvent and elution solvent, respectively. The good linearity was obtained for the three targets, ranging from 10 to 500 μg mL^?1 (R² > 0.999), with relative standard deviations ≤ 5.0%. Overall, the proposed method using MIPs is expected to have a wide range of applications.     

Sequential optimization of culture medium composition for extracellular lipase production by Bacillus sphaericus using statistical methods

A sequential optimization strategy with the aid of statistical design of experiments was used to enhance the lipase (triacylglycerol acylhydrolases, EC 3.1.1.3) production by Bacillus sphaericus in submerged cultivation. A Plackett–Burman experimental design was used to evaluate the twelve medium components. Various vegetable oil inducers were tested for lipase production in the second step and the third step was to identify the optimal values of the significant medium components with sesame oil as the inducer using response surface methodology. A predictive model of the combined effects of the independent variables using response surface methodology and an artificial neural network was proposed. Unstructured kinetic models, a logistic model and a Luedeking–Piret model, were used to describe the cell mass and lipase production respectively. The significant variables affecting lipase production were found to be glucose, olive oil, peptone, NaCl and MnSO₄.H₂O. Sesame oil was found to be the best inducer for lipase production by Bacillus sphaericus. The maximum lipase activity of 4.45 U mL^?1, which was 1.5 times the maximum activity obtained in the Plackett–Burman experimental trials, was obtained at the optimum combination of medium constituents containing 12.695 g L^?1 glucose, 13.161 mL L^?1 sesame oil, 9.947 g L^?1 peptone, 3.25 g L^?1 NaCl, 0.5917 g L^?1 MnSO₄.H₂O and other insignificant components at the fixed level. The statistical design of experiments offers an efficient methodology to identify the significant variables and to optimize the factors with a minimum number of experiments for lipase production by Bacillus sphaericus. Copyright © 2007 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.     

Antibacterial Activities of Five Cationic Gemini Surfactants with Ethylene Glycol Bisacetyl Spacers

A series of cationic gemini surfactants containing two dimethylalkylammonium chains linked by ethylene glycol bisacetyl spacers were synthesized [G_m‐AnA‐m, G = gemini surfactant, m = 12 (–C₁₂H₂₅), 14 (–C₁₄H₂₉), or 16 (–C₁₆H₃₃), A = acetyl, and n = 2, 3, or 4 is the number of ethylene glycol units in the spacers]. Because of the inductive effect of the oxygen atom in the spacer, acylation can take place using chloroacetyl chloride instead of bromoacetyl bromide which helps to limit the use of environmentally harmful reagents. Critical micelle concentrations were determined using conductivity measurements. The antibacterial activities of the surfactants against Gram‐positive bacterium Staphylococcus aureus and Gram‐negative bacterium Escherichia coli were evaluated from the minimum inhibitory concentration (MIC), minimum bacterial concentration, a time–kill study, and the inhibitory zone. Increasing the length of the spacer did not result in an obvious change of antibacterial activity. However, increasing the length of the alkyl chain apparently increased the antibacterial activity against S. aureus but decreased the antibacterial activity against E. coli. The G_{12‐A2A‐12} surfactant had the lowest CMC of 1.26 mmol L^?1 and exhibited the best antibacterial activity with a MIC of 32 μg mL^?1 toward S. aureus and 64 μg mL^?1 toward E. coli in the presence of 10⁵ CFU of bacteria. This work indicated that these cationic gemini surfactants have potential applications as antibacterial agents and emulsifiers.     

Novel 1,4‐Substituted‐1,2,3‐Triazoles as Antitubercular Agents

Tuberculosis (TB) remains a pressing unmet medical need, particularly with the emergence of multidrug‐resistant and extensively drug‐resistant tuberculosis. Here, a series of 1,4‐substituted‐1,2,3‐triazoles have been synthesized and evaluated as potential antitubercular agents. These compounds were assembled via click chemistry in high crude purity and in moderate to high yield. Of the compounds tested, 12 compounds showed promising antitubercular activity with six possessing minimum inhibitory concentration (MIC) values <10 μg mL^?1, and total selectivity for Mycobacterium tuberculosis (Mtb) growth inhibition. A second set of 21 compounds bearing variations on ring C were synthesized and evaluated. This second library gave an additional six compounds displaying MIC values ≤10 μg mL^?1 and total selectivity for Mtb growth inhibition. These compounds serve as an excellent starting point for further development of antitubercular therapies.