The fungal alkaloid Okaramine-B activates an L-glutamate-gated chloride channel from Ixodes scapularis, a tick vector of Lyme disease.

A novel L-glutamate-gated anion channel (IscaGluCl1) has been cloned from the black-legged tick, Ixodes scapularis, which transmits multiple pathogens including the agents of Lyme disease and human granulocytic anaplasmosis. When mRNA encoding IscaGluCl1 was expressed in Xenopus laevis oocytes, we detected robust 50-400 nA currents in response to 100 µM L-glutamate. Responses to L-glutamate were concentration-dependent (pEC50 3.64 ±â€¯0.11). Ibotenate was a partial agonist on IscaGluCl1. We detected no response to 100 µM aspartate, quisqualate, kainate, AMPA or NMDA. Ivermectin at 1 µM activated IscaGluCl1, whereas picrotoxinin (pIC50 6.20 ±â€¯0.04) and the phenylpyrazole fipronil (pIC50 6.90 ±â€¯0.04) showed concentration-dependent block of the L-glutamate response. The indole alkaloid okaramine B, isolated from fermentation products of Penicillium simplicissimum (strain AK40) grown on okara pulp, activated IscaGluCl1 in a concentration-dependent manner (pEC50 5.43 ±â€¯0.43) and may serve as a candidate lead compound for the development of new acaricides.

Mechanistic Basis for ATP-Dependent Inhibition of Glutamine Synthetase by Tabtoxinine-ß-lactam.

Tabtoxinine-ß-lactam (TßL), also known as wildfire toxin, is a time- and ATP-dependent inhibitor of glutamine synthetase produced by plant pathogenic strains of Pseudomonas syringae. Here we demonstrate that recombinant glutamine synthetase from Escherichia coli phosphorylates the C3-hydroxyl group of the TßL 3-(S)-hydroxy-ß-lactam (3-HßL) warhead. Phosphorylation of TßL generates a stable, noncovalent enzyme-ADP-inhibitor complex that resembles the glutamine synthetase tetrahedral transition state. The TßL ß-lactam ring remains intact during enzyme inhibition, making TßL mechanistically distinct from traditional ß-lactam antibiotics such as penicillin. Our findings could enable the design of new 3-HßL transition state inhibitors targeting enzymes in the ATP-dependent carboxylate-amine ligase superfamily with broad therapeutic potential in many disease areas.

Use of FDA approved therapeutics with hNTCP metabolic inhibitory properties to impair the HDV lifecycle.

Worldwide there are approximately 240million individuals chronically infected with the hepatitis B virus (HBV), including 15-20million coinfected with the hepatitis delta virus (HDV). Treatments available today are not fully efficient and often associated to important side effects and development of drug resistance. Targeting the HBV/HDV entry step using preS1-specific lipopeptides appears as a promising strategy to block viral entry for both HBV and HDV (Gripon et al., 2005; Petersen et al., 2008). Recently, the human Sodium Taurocholate Cotransporting Polypeptide (hNTCP) has been identified as a functional, preS1-specific receptor for HBV and HDV. This groundbreaking discovery has opened a very promising avenue for the treatment of chronic HBV and HDV infections. Here we investigated the ability of FDA approved therapeutics with documented inhibitory effect on hNTCP cellular function to impair viral entry using a HDV in vitro infection model based on a hNTCP-expressing Huh7 cell line. We demonstrate the potential of three FDA approved molecules, irbesartan, ezetimibe, and ritonavir, to alter HDV infection in vitro.

Determination of the thrombin inhibitor AZD0837 and its metabolites in human bile using mixed mode solid phase extraction and LC-MS/MS.

A method for the determination of AZD0837 and its two metabolites AR-H069927 and AR-H067637 in human bile was developed and validated. All three analytes and their stable isotope-labeled internal standards were isolated from bile using solid phase extraction on a mixed mode reversed phase/anion exchange column. Elution was done at high ionic strength with 0.125 M ammoniumacetate in 50% methanol. The extraction recoveries were >75%. Due to the high concentration of AR-H067637 a portion of the extract was diluted before injection on to the LC column, while undiluted extract was directly injected for the analysis of AZD0837 and AR-H069927. Chromatographic separation of all three analytes was achieved in a single system utilizing a C18 column based on fused core particle technology at high flow rate. The two metabolites were eluted when a gradient from 30 to 57% methanol was applied while the more hydrophobic pro-drug, AZD0837, eluted during a steeper second gradient from 57 to 80% methanol with the ammonium acetate concentration and acetic acid concentration kept constant at 3.8 mmol/L and 0.1%, respectively. The total cycle time was 3.2 min. Detection was performed using positive electrospray ionization tandem mass spectrometry. The linearity range was 0.02-20 µmol/L for AZD0837 and AR-H069927, and 1-1000 µmol/L for AR-H067637. The repeatability and the overall precision were less than 15% (RSD) and the accuracy was within the interval 93-100%.

Isolation and structure elucidation of major alkaline degradant of ezetimibe.

This work presents the isolation and characterization of the alkaline degradant of Ezetimibe. Ezetimibe, a selective inhibitor of intestinal cholesterol absorption, was subjected to alkaline degradation. Ezetimibe was reacted with 0.1M methanolic sodium hydroxide solution for 10min at 80°C to yield alkaline degradant to an extent of 90% of initial amount of the drug taken. This degradant was detected by high performance liquid chromatography (HPLC) at relative retention time (RRT) of 1.48 with respect to Ezetimibe. HPLC method involved an isocratic elution on a Waters Symmetry C(8) 150mm×4.6mm, 5µm column using ammonium acetate buffer (pH 4.5, 50mM) - acetonitrile (50:50, v/v) as the mobile phase at a flow rate of 1.0mL/min and UV detection at 242nm. The degradant was isolated by preparative HPLC. Purity of the isolated solid was found to be more than 99%. Structure of alkaline degradant was confirmed by LC-MS, (1)H and (13)C NMR and IR spectroscopy. On the basis of spectral data, the structure of the degradant was confirmed as 5-(4-fluorophenyl)-2-[(4-fluorophenyl amino)-(4-hydroxyphenyl)methyl]-pent-4-enoic acid. The route for the formation of this degradant is also proposed. Determining the structures of degradation products arouse during stress testing can be useful for preclinical discovery efforts.

Purification and characterization of 3,3-dihydroxyazetidine from culture medium of Bacillus mesentericus and B. subtilis.

A method is described for the purification of 3,3-dihydroxyazetidine (DHA), which accelerates the growth of Bifidobacterium spp., from the culture medium of Bacillus mesentericus (BM). Purification involved adsorption to an ion-exchange resin; it required less time and gave a higher yield than a previously reported method. Monitoring the inhibition of E. coli NIHJ JC-2, we searched for other strains that produced 3,3-dihydroxyazetidine. We found that not only B. mesentericus TO-A but also B. subtilis IFO13719 produced the compound of interest.

A new factor from Bacillus mesentericus which promotes the growth of Bifidobacterium.

It was reported previously that supernatants of cultures of Bacillus mesentericus TO-A promote the growth of Bifidobacterium species. In this study, a new growth-promoting factor, BM-1, was purified from the supernatant of such a culture and its chemical structure was determined. BM-1 was identified as 3,3-dihydroxyazetidine, and it promoted the growth of several strains of Bifidobacterium.

Antimetabolites produced by microorganisms. XII (S)-alanyl-3-[alpha-(S)-chloro-3-(S)-hydroxy 2-oxo-3-azetidinylmethyl]-(S)-alanine, a new beta-lactam containing natural product.

(S)-Alanyl-3-[alpha-(S)-chloro-3-(S)-hydroxy-2-oxo-3-azetidinylmethyl]-(S)-alanine was isolated from a fermentation broth of an unidentified Streptomyces species 372 A. The structure was determined by single crystal X-ray diffraction analysis. The substance inhibits the growth of several strains of gram-positive and gram-negative bacteria in a chemically defined medium but growth inhibition is relieved by addition of L-glutamine to the medium.