Solution Phase Combinatorial Chemistry. Purine- and Pyrimidine-Based Libraries with Antibacterial Activity via Solution Phase Simultaneous Addition of Functionalities

Abstract

We have effectively used the SPSAF method with purine and pyrimidine based scaffolds to rapidly generate diverse libraries with antibacterial activity. Deconvolution via HPLC fractionation resulted in libraries with reduced complexity (2–10 compounds), a number of which retained good antibacterial activity.     

Mode of Action of the Antibacterial Compound Ar26 Produced by Agrobacterium vitis Strain E26 with Activity against A. tumefaciens, A. rhizogenes and A. vitis

The biological control bacterium Agrobacterium vitis strain E26 was previously shown to produce an antibacterial compound, Ar26. This compound was involved in the biocontrol process and inhibited grapevine crown gall-causing A. vitis strains in vitro by an unknown mechanism. This work was undertaken to determine the antibacterial properties and mode of action of Ar26. In a well agar plate diffusion assay against 29 tumorigenic isolates of Agrobacterium spp., Ar26 displayed broad inhibitory activity against 27. All of the 10 A. vitis , 8 of 9 A. tumefaciens and 9 of 10 A. rhizogenes strains were sensitive to this compound. Agrobacterium vitis strains were more sensitive to Ar26 than A. tumefaciens or A. rhizogenes strains, with larger inhibition zones and lower minimal inhibitory concentration (MIC). Ar26 exhibited a bactericidal effect against A. vitis. This compound did not cause bacterial cell lysis, as determined by morphological observation with an electronic microscope. Also, no leakage of cytoplasmic materials from cells of A. vitis occurred after treatment with Ar26 at concentrations equivalent to the MIC. However, an inhibition of the incorporation of radiolabelled precursors into DNA, RNA and protein was observed after treatment with Ar26. Results obtained suggest that Ar26 inhibited DNA, RNA and protein syntheses in tumorigenic A. vitis .     

Molecular Interaction of Novel Compound 2-Methylheptyl Isonicotinate Produced by Streptomyces sp. 201 with Dihydrodipicolinate Synthase (DHDPS) Enzyme of Mycobacterium tuberculosis for its Antibacterial Activity

Antibiotic resistance is a growing problem in multi-drug-resistant tuberculosis which is caused by Mycobacterium tuberculosis (MTB). Hence there is an urgent need for designing or developing a novel or potent anti-tubercular agent. The Lysine/DAP biosynthetic pathway is a promising target because of its role in cell wall and amino acid biosynthesis. In our study we performed a molecular docking analysis of a novel antibacterial isolated from Streptomyces sp. 201 at three different binding site of dihydrodipicolinate synthase (DHDPS) enzyme of MTB. The molecular docking studies suggest that the novel molecule shows favourable interaction at the three different binding sites as compared to five experimentally known inhibitors of DHDPS.     

Single major polypeptide of a calicivirus: characterization by polyacrylamide gel electrophoresis and stabilization of virions by cross-linking with dimethyl suberimidate.

A calicivirus, San Miguel sea lion virus serotype 4, isolate 15FT, externally labelled with 125I, was shown by gel electrophoresis to possess a single major polypeptide. The polypeptide migrated anomalously upon electrophoresis in two sodium dodecyl sulfate (SDS) systems: more slowly than bovine serum albumin in a continuous phosphate-buffered system and more rapidly than bovine serum albumin in a discontinuous system. Estimated molecular weights in the two systems were approximately 71,000 and 64,000, respectively. There was no clear evidence for a minor virion polypeptide. Treatment of purified San Miguel sea lion virions with dimethyl suberimidate, a cross-linking reagent, preserved virion integrity during long-term storage at 4 degrees C. Oligomeric species of the polypeptide were observed upon electrophoresis of products from cross-linked virions. Based upon a preferred polypeptide molecular weight estimate of 71,000 and distribution of oligomeric species, a calicivirion model with 120 monomeric protein units is proposed as an alternative to a 180-unit model.     

Conversion of NfsA, the Major Escherichia coli Nitroreductase, to a Flavin Reductase with an Activity Similar to That of Frp, a Flavin Reductase in Vibrio harveyi, by a Single Amino Acid Substitution

NfsA is the major oxygen-insensitive nitroreductase of Escherichia coli, similar in amino acid sequence to Frp, a flavin reductase of Vibrio harveyi. Here, we show that a single amino acid substitution at position 99, which may destroy three hydrogen bonds in the putative active center, transforms NfsA from a nitroreductase into a flavin reductase that is as active as the authentic Frp and a tartrazine reductase that is 30-fold more active than wild-type NfsA.     

Spatial Distribution of Ethylene Production by Individual Needles Along a Shoot ofPinus sylvestrisL.: Relationship with Peroxidase Activity

Spatial distribution of ethylene production by individual currentyear needles and the relationship with peroxidase activity wasstudied along a shoot of Scots pine (Pinus sylvestrisL.). Muchvariation in ethylene production between and within individualseedlings was found. There was a significant difference in meanintensity of ethylene production between orthostichies withinindividual seedlings. The capacity of individual needles toconvert 1-aminocyclopropane-1-carboxylic acid (ACC) to ethylenedid not correlate with the basal rate of ethylene production.Mean ethylene production intensity by orthostichies correlatedpositively with mean peroxidase activity. A positive correlationbetween the intensity of ethylene production by individual needlesand detachment-induced peroxidase activity was found. The datasuggests that the rate of ethylene production by individualneedles may be directly related to their peroxidase activityand at least in part to their antioxidative capacity.Copyright1998 Annals of Botany Company Pinus sylvestris,pine, ethylene production, spatial distribution, individual needles, peroxidase, seasonal changes.     

Adrenocortical Suppression by a Glucocorticoid: Effect of a Single i. m. Injection of Betamethasone Depot Versus Placebo given Prior to Orthopaedic Surgery in Dogs

The suppression of the endogenous serum Cortisol level by a single i. m. injection of 3 mg betamethasone depot (Celeston Chronodose∞) prior to orthopaedic surgical interventions on the forelimbs of 8 dogs was studied in a placebo–controlled cross–over trial. Compared to placebo the endogenous serum Cortisol level was significantly (p < 0.05) reduced for a period of 4 days, and then returned to the pre–operative range. During the 28–day observation period after each surgical intervention no impairment of wound healing, bleeding episodes or side–effects which could be attributed to the glucocorticoid, was seen.     

Effective Immunity to Dental Caries: Protection of Gnotobiotic Rats by Local,Immunization with a Ribosomal Preparation from Streptococcus mutans

A ribosomal preparation from Streptococcus mutans 6715 was characterized for its ability to induce an immune response in gnotobiotic rats which was protective against S. mutans-induced dental caries. Animals injected in the salivary gland region with the S. mutans ribosomal vaccine developed significantly higher (P < 0.01) salivary IgA and serum IgG antibody activities against whole S. mutans cells and ribosomal preparations than nonimmunized rats. Vaccinated animals had significantly lower (67%; P < 0.01) levels of S. mutans adherent to their molar surfaces than the control rats after infection with the homologous, cariogenic S. mutans. The immunized animals had significantly fewer (P < 0.01) carious lesions on their buccal, sulcal, and proximal molar surfaces than the nonimmunized rats following challenge with the virulent organism. Animals injected with the ribosomal preparation developed salivary IgA and serum IgG antibodies with specificities to various cell surface-associated antigens such as lipoteichoic acid and glucosyltransferase, suggesting that the observed protection may be due to antibodies against cell surface contaminants of the ribosomal vaccine. These results are the first demonstration that a ribosomal preparation from S. mutans protected rats from caries formation after challenge with the homologous, virulent S. mutans.     

Simultaneous determination of metformin and rosiglitazone in human plasma by liquid chromatography/tandem mass spectrometry with electrospray ionization: application to a pharmacokinetic study

A selective and sensitive high performance liquid chromatography-electrospray ionization-tandem mass spectrometry (ESI-MS/MS) method for simultaneous determination of metformin and rosiglitazone in human plasma using phenformin as internal standard (IS) has been first developed and validated. Plasma samples were precipitated by acetonitrile and the analytes were separated on a prepacked Phenomenex Luna 5u CN 100A (150 mm x 2.0 mm I.D.) column using a mobile phase comprised of methanol:30 mM ammonium acetate pH 5.0 (80:20, v/v) delivered at 0.2 ml/min. Detection was performed on a Finnigan TSQ triple-quadrupole tandem mass spectrometer in positive ion selected reaction monitoring (SRM) mode using electrospray ionization. The ion transitions monitored were m/z 130.27-->71.11 for metformin, m/z 358.14-->135.07 for rosiglitazone and m/z 206.20-->105.19 for the IS. The standard curves were linear (r(2)>0.99) over the concentration range of 5-3000 ng/ml for metformin and 1.5-500 ng/ml for rosiglitazone with acceptable accuracy and precision, respectively. The within- and between-batch precisions were less than 15% of the relative standard deviation. The limit of detection (LOD) of both metformin and rosiglitazone was 1 ng/ml. The method described is precise and sensitive and has been successfully applied to the study of pharmacokinetics of compound metformin and rosiglitazone capsules in 12 healthy Chinese volunteers.     

Two-dimensional crystallization of streptavidin by nonspecific binding to a surface film: study with a scanning electron microscope.

A two-dimensional (2D) crystal of streptavidin has been obtained by a nonspecific binding method. The protein molecules were bound and formed a dense packing on the film of poly(1-benzyl-L-histidine) spread at the surface of protein solution. The surface film was moderately heated to stimulate crystallization of bound streptavidin. A potential of this method for obtaining 2D crystals of soluble proteins is demonstrated. The present 2D crystal structure of streptavidin resembles that previously obtained by specific binding to biotinylated lipid. We show in addition that the 2D array of protein with usual size approximately 50 A can be imaged using a high resolution scanning electron microscope (HR-SEM) and subject to structural analysis at low resolution. Various limitations in HR-SEM degrade considerably the image quality. However, the usability of a bulk plate as specimen support would make HR-SEM a convenient tool, when such a substrate must be considered in application of protein arrays, and if an intrinsic low resolution is acceptable.     

Simultaneous determination of didanosine and its amino acid prodrug,valdidanosine by hydrophilic interaction chromatography coupled with electrospray ionization tandem mass spectrometry: Application to a pharmacokinetic study in rats

A rapid, sensitive and selective ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method with hydrophilic interaction chromatography has been developed and validated for the simultaneous determination of didanosine and valdidanosine (L-valine amino acid ester prodrug of didanosine) in rat plasma. Solid-phase extraction (SPE) column was employed to extract the analytes from rat plasma, with high extraction recovery (>85%) for both didanosine and valdidanosine. The analytes were then separated by hydrophilic interaction chromatography (HILIC column) and detected by a triple-quadrupole mass spectrometry equipped with an electrospray ionization (ESI) source. The method was linear over the concentration ranges of 2–20,000 ng/mL for didanosine and 4–300 ng/mL for valdidanosine. The lower limit of quantitation (LLOQ) of didanosine and valdidanosine was 2 and 4 ng/mL, respectively. The intra-day and inter-day relative standard deviation (RSD) were less than 15% and the relative errors (RE) were all within 15%. Finally, the validated UPLC–MS/MS method was successfully applied to the pharmacokinetic study after either didanosine or valdidanosine orally administrated to the Sprague–Dawley rats.     

Simultaneous analysis of insect repellent DEET, sunscreen oxybenzone and five relevant metabolites by reversed-phase HPLC with UV detection: application to an in vivo study in a piglet model

N,N-Diethyl-m-toluamide (DEET) and oxybenzone are two essential active ingredients in insect repellent and sunscreen preparations. We developed and validated a simple, sensitive, and selective HPLC assay to simultaneously measure DEET, oxybenzone and five primary metabolites of DEET and oxybenzone in biological samples including plasma, urine and skin strips. The compounds were separated on a reversed-phase C18 column using three-stage gradient steps with methanol and water. DEET and two relevant metabolites were detected at 254 nm, while oxybenzone and three relevant metabolites were detected at 289 nm. The limit of detection was 0.6 ng for DEET and 0.5 ng for oxybenzone, respectively. The developed method was further applied to analyze various biological samples from an in vivo animal study that evaluated concurrent use of commercially available insect repellent and sunscreen preparations.     

Immunodetection of 3-nitrotyrosine in the liver of zymosan-treated rats with a new monoclonal antibody: comparison to analysis by HPLC.

Zymosan-induced peritonitis is associated with an increased production of reactive nitrogen oxides that may contribute to the often-observed failure of multiple organ systems in this model of acute inflammation. Quantitative biochemical evidence is provided for a marked 13-fold increase in protein-bound 3-nitrotyrosine (NTyr), a biomarker of reactive nitrogen oxides, in liver tissue of zymosan-treated rats. In order to investigate the localization of NTyr in this affected tissue, a monoclonal antibody, designated 39B6, was raised against 3-(4-hydroxy-3-nitrophenylacetamido) propionic acid-bovine serum albumin conjugate and its performance characterized. 39B6 was judged by competition ELISA to be approximately 2 orders of magnitude more sensitive than a commercial anti-NTyr monoclonal antibody. Binding characteristics of 39B6 were similar, but not identical, to that of a commercial affinity-purified polyclonal antibody in ELISA and immunohistochemical analyses. Western blot experiments revealed high specificity of 39B6 against NTyr and increased immunoreactivity of specific proteins from liver tissue homogenates of zymosan-treated rats. Immunohistochemical analysis of liver sections indicated a marked zymosan-induced increase in immunofluorescent staining, which was particularly intense in or adjacent to nonparenchymal cells, but not in the parenchymal cells of this tissue. Quantitative analysis of fractions enriched in these cell populations corroborated the immunofluorescent data, although the relative amounts detected in response to zymosan treatment was greatly reduced compared to whole liver tissue. These results demonstrate the high specificity of the newly developed antibody and its usefulness in Western blot and immunohistochemical analysis for NTyr, confirm the presence of NTyr by complementary methods, and suggest the possible involvement of reactive nitrogen oxides in hepatic vascular dysfunction.