Bakkenolides and Caffeoylquinic Acids from the Aerial Portion of Petasites japonicus and Their Bacterial Neuraminidase Inhibition Ability.

Petasites japonicus have been used since a long time in folk medicine to treat diseases including plague, pestilential fever, allergy, and inflammation in East Asia and European countries. Bioactive compounds that may prevent and treat infectious diseases are identified based on their ability to inhibit bacterial neuraminidase (NA). We aimed to isolate and identify bioactive compounds from leaves and stems of P. japonicas (PJA) and elucidate their mechanisms of NA inhibition. Key bioactive compounds of PJA responsible for NA inhibition were isolated using column chromatography, their chemical structures revealed using 1 H NMR, 13 C NMR, DEPT, and HMBC, and identified to be bakkenolide B (1), bakkenolide D (2), 1,5-di-O-caffeoylquinic acid (3), and 5-O-caffeoylquinic acid (4). Of these, 3 exhibited the most potent NA inhibitory activity (IC50 = 2.3 ± 0.4 µM). Enzyme kinetic studies revealed that 3 and 4 were competitive inhibitors, whereas 2 exhibited non-competitive inhibition. Furthermore, a molecular docking simulation revealed the binding affinity of these compounds to NA and their mechanism of inhibition. Negative-binding energies indicated high proximity of these compounds to the active site and allosteric sites of NA. Therefore, PJA has the potential to be further developed as an antibacterial agent for use against diseases associated with NA.

Detection of Enzyme Inhibitors in Crude Natural Extracts Using Droplet-Based Microfluidics Coupled to HPLC.

Natural product screening for new bioactive compounds can greatly benefit from low reagents consumption and high throughput capacity of droplet-based microfluidic systems. However, the creation of large droplet libraries in which each droplet carries a different compound is a challenging task. A possible solution is to use an HPLC coupled to a droplet generating microfluidic device to sequentially encapsulate the eluting compounds. In this work we demonstrate the feasibility of carrying out enzyme inhibiting assays inside nanoliter droplets with the different components of a natural crude extract after being separated by a coupled HPLC column. In the droplet formation zone, the eluted components are mixed with an enzyme and a fluorogenic substrate that permits to follow the enzymatic reaction in the presence of each chromatographic peak and identify those inhibiting the enzyme activity. Using a fractal shape channel design and automated image analysis, we were able to identify inhibitors of Clostridium perfringens neuraminidase present in a root extract of the Pelargonium sidoides plant. This work demonstrates the feasibility of bioprofiling a natural crude extract after being separated in HPLC using microfluidic droplets online and represents an advance in the miniaturization of natural products screening.

Neuraminidase inhibition of Dietary chlorogenic acids and derivatives - potential antivirals from dietary sources.

Plants rich in chlorogenic acids (CGAs), caffeic acids and their derivatives have been found to exert antiviral effects against influenza virus neuroaminidase. In this study several dietary naturally occurring chlorogenic acids, phenolic acids and derivatives were screened for their inhibitory activity against neuroaminidases (NAs) from C. perfringens, H5N1 and recombinant H5N1 (N-His)-Tag using a fluorometric assay. There was no significant difference in inhibition between the different NA enzymes. The enzyme inhibition results indicated that chlorogenic acids and selected derivatives, exhibited high activities against NAs. It seems that the catechol group from caffeic acid was important for the activity. Dietary CGA therefore show promise as potential antiviral agents. However, caffeoyl quinic acids show low bioavailibility and are intensly metabolized by the gut micro flora, only low nM concentrations are observed in plasma and urine, therefore a systemic antiviral effect of these compounds is unlikely. Nevertheless, gut floral metabolites with a catechol moiety or structurally related dietary phenolics with a catechol moiety might serve as interesting compounds for future investigations.

Screening-based discovery of drug-like O-GlcNAcase inhibitor scaffolds.

O-GlcNAcylation is an essential posttranslational modification in metazoa. Modulation of O-GlcNAc levels with small molecule inhibitors of O-GlcNAc hydrolase (OGA) is a useful strategy to probe the role of this modification in a range of cellular processes. Here we report the discovery of novel, low molecular weight and drug-like O-GlcNAcase inhibitor scaffolds by high-throughput screening. Kinetic and X-ray crystallographic analyses of the binding modes with human/bacterial O-GlcNAcases identify some of these as competitive inhibitors. Comparative kinetic experiments with the mechanistically related human lysosomal hexosaminidases reveal that three of the inhibitor scaffolds show selectivity towards human OGA. These scaffolds provide attractive starting points for the development of non-carbohydrate, drug-like OGA inhibitors.

Evaluation of acid phosphatase as a confirmation test for Clostridium perfringens isolated from water.

AIMS: To evaluate testing for acid phosphatase as an alternative method for the confirmation of Clostridium perfringens isolated from water. METHODS AND RESULTS: Sixty-two reference strains of Clostridium were tested for their ability to produce acid phosphatase, as well as reduction of sulfite on tryptose sulfite cycloserine agar (TSC) and production of fluorescence in TSC supplemented with 4-methylumbelliferylphosphate (MUP). Additionally 155 environmental presumptive C. perfringens isolates from TSC incubated at 44 degrees C were identified and tested for acid phosphatase production and by the conventional MNLG (testing for motility, nitrate reduction, lactose fermentation and gelatin liquefaction) confirmation procedure. Twenty-seven strains from 15 species of Clostridium-reduced sulfite to some extent on TSC incubated at 44 degrees C, with a significant number of species being able to grow well at this temperature, indicating that a confirmation step is needed for the enumeration of C. perfringens on this medium. All 10 strains of C. perfringens tested, together with one strain each of Clostridium baratii and Clostridium rectum produced acid phosphatase. These also produced fluorescence on MUP supplemented TSC, as did 13 strains of acid phosphatase negative, sulfite-reducing clostridia, representing nine species. Of the environmental isolates, 114 were identified as C. perfringens of which 108 (94.7%) were confirmed by the acid phosphatase test compared with 104 (91.2%) by the MNLG tests. CONCLUSIONS: Testing for acid phosphatase production is at least as reliable, and much simpler to perform, than the current standard confirmation MNLG procedure. Incorporation of MUP into TSC does not reliably improve the identification of presumptive C. perfringens. SIGNIFICANCE AND IMPACT OF THE STUDY: Application of testing for acid phosphatase as a confirmation test for C. perfringens would substantially simplify the analysis for this bacterium from water samples, and reduce the analysis time to confirmed counts.

One-bead-one-inhibitor-one-substrate screening of neuraminidase activity.

Given the eminent threat of a 21st century flu pandemic, the search for novel antiviral compounds is an increasingly important area of research. Recent developments in antiviral research have established the viability of targeting viral neuraminidase (NA), an enzyme that cleaves sialic acid from the cell-surface-mediating passage of the virus in the respiratory tract. N-acetyl neuraminic acid (NeuAc) is the substrate for NA, and analogues of this core structure have been commercialized as antiviral therapeutics. Recent studies have established that this system is well suited for combinatorial approaches to drug discovery. An important step in the process is to develop solid-phase screening technologies. The feasibility of performing competitive solid-phase NA assays is reported herein. Initially, a fluorogenic NeuAc substrate was immobilized on solid support, and the ability of three NAs (Clostridium perfringens, Salmonella typhimurium, and Vibrio cholerae) to cleave the substrate was shown to be analogous to solution-phase assays. The solid support was then bifunctionalized with the fluorogenic NeuAc substrate and one of two known inhibitors (DANA and Zanamivir). The ability of NA to cleave NeuAc from the solid support when simultaneously presented with an inhibitor was shown to be enzyme dependent. As expected, simultaneous presentation of NeuAc and DANA, a nonspecific inhibitor, led to diminished activity for all three enzymes tested. In contrast, dual presentation of NeuAc and the selective inhibitor Zanamivir only showed significant activity against Vibrio cholerae.

Alterations in DNA gyrase and topoisomerase IV in resistant mutants of Clostridium perfringens found after in vitro treatment with fluoroquinolones.

To compare mutations in the DNA gyrase (gyrA and gyrB) and topoisomerase IV (parC and parE) genes of Clostridium perfringens, which are associated with in vitro exposure to fluoroquinolones, resistant mutants were selected from eight strains by serial passage in the presence of increasing concentrations of norfloxacin, ciprofloxacin, gatifloxacin, or trovafloxacin. The nucleotide sequences of the entire gyrA, gyrB, parC, and parE genes of 42 mutants were determined. DNA gyrase was the primary target for each fluoroquinolone, and topoisomerase IV was the secondary target. Most mutations appeared in the quinolone resistance-determining regions of gyrA (resulting in changes of Asp-87 to Tyr or Gly-81 to Cys) and parC (resulting in changes of Asp-93 or Asp-88 to Tyr or Ser-89 to Ile); only two mutations were found in gyrB, and only two mutations were found in parE. More mutants with multiple gyrA and parC mutations were produced with gatifloxacin than with the other fluoroquinolones tested. Allelic diversity was observed among the resistant mutants, for which the drug MICs increased 2- to 256-fold. Both the structures of the drugs and their concentrations influenced the selection of mutants.

Dietary antibiotic growth promoters enhance the bioavailability of alpha-tocopheryl acetate in broilers by altering lipid absorption.

The influence of intestinal microbial bile salt deconjugation on absorption of fatty acids and alpha- and gamma-tocopherol was investigated in a trial with Ross 208 broilers. Birds (n = 1600) were assigned to 4 dietary treatments: no supplementation or supplementation of antibiotics (salinomycin, 40 mg/kg feed and avilamycin, 10 mg/kg feed), and inclusion of either animal fat (10 g/100 g feed) or soybean oil (10 g/100 g feed) in the diet. At d 7, 14, 21, and 35 of age, the intestinal number of the bile salt hydrolase-active bacteria Clostridium perfringens, the concentration of conjugated and unconjugated bile salts, the ileal absorption of fatty acids and tocopherols, and the blood plasma concentrations of tocopherols were measured. All variables were significantly influenced by bird age. C. perfringens counts were lower and bile salt concentrations were greater in birds fed soybean oil. The supplementation of antibiotics reduced the numbers of C. perfringens in the small intestine and reduced the concentration of unconjugated bile salts. The ileal absorption of fatty acids and alpha-tocopherol, as well as the plasma concentration of alpha-tocopherol, was greater in birds fed antibiotics. The absorption and plasma concentration of gamma-tocopherol were not influenced by antibiotics. Unlike gamma-tocopherol, which is present solely as the free alcohol, the major proportion of dietary alpha-tocopherol is present as alpha-tocopheryl acetate, which requires a bile salt-dependent enzymatic hydrolysis before absorption. In conclusion, proper digestion of lipid-soluble compounds is highly dependent on an adequate concentration of bile salts in the small intestine to provide proper lipid emulsification and activation of lipolytic enzymes.

Synthesis and evaluation as sialidase inhibitors of xylo-configured cyclohexenephosphonates carrying glycerol side-chain mimics.

Based on a strategy previously reported by us, we have synthesized D-xylo configured cyclohexenephosphonates designed to mimic the transition state of the sialidase reaction. The double bond orientation corresponds to the benchmark inhibitor Neu5Ac2en and we could selectively introduce hydroxyalkyl substituents in order to simulate the glycerol side-chain of neuraminic acid. The inhibitory activity of a set of compounds towards bacterial sialidases was tested and interesting differences in activity were found.

Mechanism of chemical manipulation of the heat resistance of Clostridium perfringens spores.

The mechanism of chemical manipulation of the heat resistance of Clostridium perfringens type A spores was studied. Spores were converted to various ionic forms to base-exchange technique and these spores were heated at 95 degrees C. Of the four ionic forms, i.e. Ca2+, Na+, H+ and native, only hydrogen spores appeared to have been rapidly inactivated at this temperature, when survivors were enumerated on the ordinary plating medium. However, the recovery of the survivors was improved when the plating medium was supplemented with lysozyme, and more dramatically when the heated spores were pretreated with alkali followed by plating in the medium containing lysozyme. In contrast to crucial damage to germination, in particular to spore lytic enzyme, no appreciable amount of DPA was released from the heat-damaged H-spores. These results suggest that a germination system is involved in the thermal inactivation of the ionic forms of spores, and that exchangeable cation load plays a role in protection from thermal damage of the germination system within the spore. An enhancement of thermal stability of spore lytic enzyme in the presence of a high concentration of NaCl was consistent with the hypothesis.

Interactions between aerolysin, erythrocytes, and erythrocyte membranes.

Aerolysin, a hemolytic and lethal exotoxin of Aeromonas hydrophila, was analyzed for amino acids. Assuming 8 histidine residues/mol, the purified toxic protein has, by summation, a molecular weight of 49,000, a value in agreement with earlier estimates by other methods. Erythrocytes from different animal species differ greatly in sensitivity to aerolysin's lytic action. There is some correlation between sensitivity and phosphatidyl choline content. Erythrocyte membranes of different species bind the toxin, and the efficiency of binding is a function of sensitivity to lysis. Binding is temperature independent, is not dependent upon membrane sialic acid, and is decreased by prior treatment with phospholipase C and proteases. Preparations of aerolysin convert substantial amounts of membrane phosphorus to water-soluble form; the conversion is concentration and temperature dependent. Most of the conversion is attributable to contaminating phospholipase(s) that is separable from the toxin. Aerolysin purified by electrophoresis in polyacrylamide gel retains some phospholipase activity, and this activity may or may not be a contaminant.

Fusion of intact human erythrocytes and erythrocyte ghosts.

Sendai virus is able to induce the fusion of human erythrocytes. Bivalent cations or ATP are not essential for polyerythrocyte formation. High fusion indices were obtained when Sendai virus was added to cells incubated in the presence of both EDTA and iodoacetic acid. Human erythrocyte ghosts prepared by gradual hemolysis still retain the potential to undergo virus-induced fusion. Fusion of human red blood cells without the addition of viruses was obtained by incubation of erythrocytes at pH 10.5 in the presence of Ca(++) (40 mM) or by addition of phospholipase C Clostridium perfringens preparations to cells previously agglutinated or polylysine.

Inhibition of enzymes by human salivary immunoglobulin A.

Human whole saliva inhibited bacterial neuraminidases and the inhibition was found to reside in the salivary IgA fraction. Further, salivary immunoglobulin (Ig)A inhibited various bacterial enzymes and toxins: neuraminidases from Streptococcus mitis, Streptococcus sanguis, and Clostridium perfringens, hyaluronidase and chondroitin sulfatase from oral bacteria, diphtheria toxin, and streptolysin O. The inhibitory activity of salivary IgA did not correlate with that of serum on the basis of minimum inhibitory dose. A small amount of salivary IgA was required to inhibit oral bacterial neuraminidases, whereas a large amount was required to inhibit other bacterial neuraminidase. Therefore, it is concluded that the absence of neuraminidase activity of oral bacteria in whole saliva may be due to specific inhibition by salivary IgA.