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ChemMedChem Apr 2017The 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...
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; Glutaminase; Helicobacter pylori; Hydrogen Bonding; Isoxazoles; Ligands; Trypanocidal Agents; Trypanosoma brucei brucei; gamma-Glutamyltransferase
PubMed: 28333400
DOI: 10.1002/cmdc.201700118 -
Chemical Science Dec 2014Acivicin is a natural product with diverse biological activities. Several decades ago its clinical application in cancer treatment was explored but failed due to...
Target discovery of acivicin in cancer cells elucidates its mechanism of growth inhibition†Electronic supplementary information (ESI) available: Synthesis, cloning, protein expression, purification and biochemical assays. See DOI: 10.1039/c4sc02339k.
Acivicin is a natural product with diverse biological activities. Several decades ago its clinical application in cancer treatment was explored but failed due to unacceptable toxicity. The causes behind the desired and undesired biological effects have never been elucidated and only limited information about acivicin-specific targets is available. In order to elucidate the target spectrum of acivicin in more detail we prepared functionalized derivatives and applied them for activity based proteomic profiling (ABPP) in intact cancer cells. Target deconvolution by quantitative mass spectrometry (MS) revealed a preference for specific aldehyde dehydrogenases. Further in depth target validation confirmed that acivicin inhibits ALDH4A1 activity by binding to the catalytic site. In accordance with this, downregulation of ALDH4A1 by siRNA resulted in a severe inhibition of cell growth and might thus provide an explanation for the cytotoxic effects of acivicin.
PubMed: 25580214
DOI: 10.1039/c4sc02339k -
Frontiers in Plant Science 2019The vegetative phase change in flowering plants is controlled by microRNA156 (miR156) under transcriptional regulation. However, the developmental signals upstream of...
UNLABELLED
The vegetative phase change in flowering plants is controlled by microRNA156 (miR156) under transcriptional regulation. However, the developmental signals upstream of miR156 are not well understood. The glutathione/glutathione disulfide (GSH/GSSG) ratios and GSH levels decline significantly during phase change, which is consistent with miR156 expression in apple ( Borkh.). Here, we found that the content of protein conjugated glutathione was remarkably higher in chloroplasts and nuclei of adult than juvenile phase apple hybrids. The decrease in miR156 expression was most relevant to the activities of serine acetyltransferase (SAT) and soluble γ-glutamyl transpeptidase (GGT), and the expressions of or . Transgenic apples over-expressing or miR156-mimetic (MIM156) did not alter expression or the soluble GGT activity. Inhibition of GGT activity with serine-borate complex or acivicin led to significant reduction in GSH content, the GSH/GSSG ratio, and the expressions of , , and miR156. Depletion of GSH with diethyl maleate without altering GGT activity caused a dramatic decrease in the expression of , , and miR156. Manipulating GGT activity and GSH homeostasis by transgenic over-expressing or RNAi increased or decreased and levels, respectively. These data provided novel evidence that participates in transcriptional level of transcription regulation of miR156 precursors during ontogenesis.
HIGHLIGHTS
- MdGGT1 affects thiol redox status and indirectly participates in the regulation of miR156 expression during vegetative phase change.
PubMed: 31417600
DOI: 10.3389/fpls.2019.00994 -
The Journal of General and Applied... Nov 2016
Topics: Antifungal Agents; Culture Media; Fusarium; Genes, Fungal; Isoxazoles; Mycelium; Trichothecenes
PubMed: 27600357
DOI: 10.2323/jgam.2016.04.002 -
Frontiers in Pharmacology 2014Asthma is characterized by airway inflammation. Inflammation is associated with oxidant stress. Airway epithelial cells are shielded from this stress by a thin layer of...
Asthma is characterized by airway inflammation. Inflammation is associated with oxidant stress. Airway epithelial cells are shielded from this stress by a thin layer of lung lining fluid (LLF) which contains an abundance of the antioxidant glutathione. LLF glutathione metabolism is regulated by γ-glutamyl transferase (GGT). Loss of LLF GGT activity in the mutant GGT(enu1) mouse causes an increase in baseline LLF glutathione content which is magnified in an IL-13 model of allergic airway inflammation and protective against asthma. Normal mice are susceptible to asthma in this model but can be protected with acivicin, a GGT inhibitor. GGT is a target to treat asthma but acivicin toxicity limits clinical use. GGsTop is a novel GGT inhibitor. GGsTop inhibits LLF GGT activity only when delivered through the airway. In the IL-13 model, mice treated with IL-13 and GGsTop exhibit a lung inflammatory response similar to that of mice treated with IL-13 alone. But mice treated with IL-13 and GGsTop show attenuation of methacholine-stimulated airway hyper-reactivity, inhibition of Muc5ac and Muc5b gene induction, decreased airway epithelial cell mucous accumulation and a fourfold increase in LLF glutathione content compared to mice treated with IL-13 alone. Mice treated with GGsTop alone are no different from that of mice treated with saline alone, and show no signs of toxicity. GGsTop could represent a valuable pharmacological tool to inhibit LLF GGT activity in pulmonary disease models. The associated increase in LLF glutathione can protect lung airway epithelial cells against oxidant injury associated with inflammation in asthma.
PubMed: 25132819
DOI: 10.3389/fphar.2014.00179 -
Thrombosis Journal 2016Besides maintaining intracellular glutathione stores, gamma-glutamyltransferase(GGT) generates reactive oxygen species and activates NFkB, a redox-sensitive...
BACKGROUND
Besides maintaining intracellular glutathione stores, gamma-glutamyltransferase(GGT) generates reactive oxygen species and activates NFkB, a redox-sensitive transcription factor key in the induction of Tissue Factor (TF) gene expression, the principal initiator of the clotting cascade. Thus, GGT might be involved in TF-mediated coagulation processes, an assumption untested insofar.
METHODS
Experiments were run with either equine, enzymatically active GGT or human recombinant (hr) GGT, a wheat germ-derived protein enzymatically inert because of missing post-translational glycosylation. TF Procoagulant Activity (PCA, one-stage clotting assay), TF antigen(ELISA) and TFmRNA(real-time PCR) were assessed in unpooled human peripheral blood mononuclear cell(PBMC) suspensions obtained from healthy donors through discontinuous Ficoll/Hystopaque density gradient.
RESULTS
Equine GGT increased PCA, an effect insensitive to GGT inhibition by acivicin suggesting mechanisms independent of its enzymatic activity, a possibility confirmed by the maintained stimulation in response to hrGGT, an enzymatically inactive molecule. Endotoxin(LPS) contamination of GGT preparations was excluded by heat inactivation studies and direct determination(LAL method) of LPS concentrations <0.1 ng/mL practically devoid of procoagulant effect. Inhibition by anti-GGT antibodies corroborated that conclusion. Upregulation by hrGGT of TF antigen and mRNA and its downregulation by BAY-11-7082, a NFkB inhibitor, and N-acetyl-L-cysteine, an antioxidant, was consistent with a NFkB-driven, redox-sensitive transcriptional site of action.
CONCLUSIONS
GGT upregulates TF expression independent of its enzymatic activity, a cytokine-like behaviour mediated by NFκB activation, a mechanism contributing to promote acute thrombotic events, a possibility in need, however, of further evaluation.
PubMed: 27822142
DOI: 10.1186/s12959-016-0119-8