glutaminase and acivicin

The natural product acivicin inhibits the glutaminase activity of cytidine triphosphate (CTP) synthetase and is a potent lead compound for drug discovery in the area of neglected tropical diseases, specifically trypanosomaisis. A 2.1-Å-resolution crystal structure of the acivicin adduct with the glutaminase domain from Trypanosoma brucei CTP synthetase has been deposited in the RCSB Protein Data Bank (PDB) and provides a template for structure-based approaches to design new inhibitors. However, our assessment of that data identified deficiencies in the model. We now report an improved and corrected inhibitor structure with changes to the chirality at one position, the orientation and covalent structure of the isoxazoline moiety, and the location of a chloride ion in an oxyanion binding site that is exploited during catalysis. The model is now in agreement with established chemical principles and allows an accurate description of molecular recognition of the ligand and the mode of binding in a potentially valuable drug target.

Topics: Bacillus subtilis; Carbon-Nitrogen Ligases; Catalytic Domain; gamma-Glutamyltransferase; Glutaminase; Helicobacter pylori; Hydrogen Bonding; Isoxazoles; Ligands; Trypanocidal Agents; Trypanosoma brucei brucei

Tumor cells intensely utilize glutamine as the major source of respiratory fuel. Glutamine-analogue acivicin inhibits tumor growth and tumor-induced angiogenesis in Ehrlich ascites carcinoma. In the present study, antitumor properties of acivicin in combination with glutaminase enzyme is reported. Acivicin along with E. coli glutaminase synergistically reduced in vitro proliferation and matrigel invasion of human MCF-7 and OAW-42 cells. Effects of single and combined treatments with acivicin and glutaminase on angiogenic factors were also analyzed in these cell lines. Co-administration of the treatment agents inhibits the release of VEGF and MMP-9 by cells in culture supernatant significantly than single agent treatments. The result suggests that combination of acivicin with glutaminase may provide a better therapeutic option than either of them given separately for treating human breast and ovarian cancer. However, further studies are required to be conducted in vivo for its confirmation.

Topics: Antimetabolites, Antineoplastic; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Collagen; Drug Combinations; Female; Glutaminase; Glutamine; Humans; In Vitro Techniques; Isoxazoles; Laminin; Matrix Metalloproteinase 9; Neoplasm Invasiveness; Ovarian Neoplasms; Proteoglycans; Tetrazolium Salts; Thiazoles; Vascular Endothelial Growth Factor A

Vitamin B6 is an essential nutrient in the human diet. It can act as a co-enzyme for numerous metabolic enzymes and has recently been shown to be a potent antioxidant. Plants and microorganisms have the ability to make the compound. Yet, studies of vitamin B6 biosynthesis have been mainly restricted to Escherichia coli, where the vitamin is synthesized from 1-deoxy-d -xylulose 5-phosphate and 4-phosphohydroxy-l-threonine. Recently, a novel pathway for its synthesis has been discovered, involving two genes (PDX1 and PDX2) neither of which is homologous to any of those participating in the E. coli pathway. In Bacillus subtilis, YaaD and YaaE represent the PDX1 and PDX2 homolog, respectively. The two proteins form a complex that functions as a glutamine amidotransferase, with YaaE as the glutaminase domain and YaaD as the acceptor and pyridoxal 5'-phosphate (PLP) synthesis domain. In this report we corroborate a recent report on the identification of the substrates of YaaD and provide unequivocal proof of the identity of the reaction product. We show that both the glutaminase and synthase reactions are dependent on the respective protein partner. The synthase reaction can also utilize an external ammonium source but, in contrast to other glutamine amidotransferases, is dependent on YaaE under certain conditions. Furthermore, we report on the detailed characterization of the inhibition of the glutaminase domain, and thus PLP synthesis, by the glutamine analog acivicin. Employing pull-out assays and native-PAGE, we provide evidence for the dissociation of the bi-enzyme complex under these conditions. The results are discussed in light of the nature of the interaction of the two components of the enzyme complex.

Topics: Antioxidants; Bacillus subtilis; Binding Sites; Carbon-Nitrogen Lyases; Chromatography; Chromatography, Gel; Chromatography, High Pressure Liquid; Cloning, Molecular; Cysteine; DNA; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Escherichia coli Proteins; Glutaminase; Glutamine; Immunochemistry; Isoxazoles; Kinetics; Mass Spectrometry; Models, Chemical; Organophosphates; Oxidoreductases; Pentosephosphates; Protein Structure, Tertiary; Pyridoxal Phosphate; Quaternary Ammonium Compounds; Spectrometry, Mass, Electrospray Ionization; Spectrophotometry; Threonine; Time Factors; Ultraviolet Rays; Vitamin B 6

The role of extracellular glutamate flux in regulating intracellular glutaminase activity was assessed in confluent monolayers of proximal tubule-like LLC-PK1-F+ cells grown on porous supports. Glutamate is a well-known inhibitor of phosphate-dependent glutaminase (PDG). We hypothesized that, by restricting the flux of glutamate from the extracellular media, cellular level would fall, effecting deinhibition of the cellular glutaminase activity. To test this, cellular glutamate uptake and extracellular production were inhibited for 18 h by the addition of D-aspartate (10 mM) or acivicin (0.7 mM) to both apical and basal media. Inhibiting glutamate flux depressed cellular glutamate content 43 and 41%, respectively. Intracellular relative glutaminase activity, monitored as the breakdown of 14C-radiolabeled glutamine to glutamate, measured over 60 s in the presence of D-aspartate or acivicin showed a 2- to 2.5-fold increase with the fall in cellular glutamate. Interestingly, enhanced glutamine uptake after PDG deinhibition was predominantly expressed on the basal surface. Indeed, measuring glutamine utilization after gamma-glutamyltranspeptidase inhibition over the entire 18-h time course revealed inhibition at the apical surface but relative enhancement of uptake at the basal surface. The increased intracellular glutaminase pathway was also reflected in increased alanine production measured over the 18-h time course, despite the reduction in overall glutamine utilization. These results point to a major role for extracellular glutamate fluxes in regulating cellular glutamine metabolism and suggest that the intracellular pathway may be suppressed under these conditions.

Topics: Animals; Aspartic Acid; Cell Line; Extracellular Space; gamma-Glutamyltransferase; Glutamic Acid; Glutaminase; Glutamine; Intracellular Fluid; Isoxazoles; Kidney Tubules, Proximal; Kinetics; LLC-PK1 Cells; Models, Biological; Swine

The metabolism of glutamine was studied in erythrocytes infected with Plasmodium falciparum, comparatively to normal cells, in presence or not of DON (6-diazo-5-oxo-L-norleucine) or acivicin, two glutamine antagonists which have been shown to inhibit the growth of P. falciparum in vitro. Extracellular glutamine was partially converted into glutamate using two routes corresponding to gamma-glutamyl transpeptidase (GGT) and glutaminase activities. In cells infected with mature trophozoites, the observed enhancement of the glutamine influx and of the glutamate formation was consistent with the enhancement of GGT and glutaminase activities.

Topics: Animals; Antimetabolites; Cells, Cultured; Diazooxonorleucine; Erythrocytes; gamma-Glutamyltransferase; Glutaminase; Glutamine; Humans; Isoxazoles; Plasmodium falciparum

Topics: Antimetabolites; Azo Compounds; Cell Cycle; Cell Line; Colonic Neoplasms; Diazooxonorleucine; Drug Evaluation, Preclinical; Glutaminase; Glutamine; Humans; Isoxazoles; Lung Neoplasms; Neoplasms, Experimental; Oxazoles

Topics: Ammonia; Animals; gamma-Glutamyltransferase; Glutaminase; Glutamine; Glycine; Isoxazoles; Kidney; Kinetics; Male; Oxazoles; Phosphates; Rats