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Journal of Radiation Research Mar 2020Wortmannin, a fungal metabolite, is a specific inhibitor of the phosphatidylinositol 3-kinase (PI3K) family, which includes double-stranded DNA dependent protein kinase...
Wortmannin, a fungal metabolite, is a specific inhibitor of the phosphatidylinositol 3-kinase (PI3K) family, which includes double-stranded DNA dependent protein kinase (DNA-PK) and ataxia telangiectasia mutated kinase (ATM). We investigated the effects of wortmannin on DNA damage in DNA-PK-deficient cells obtained from severe combined immunodeficient mice (SCID cells). Survival of wortmannin-treated cells decreased in a concentration-dependent manner. After treatment with 50 μM wortmannin, survival decreased to 60% of that of untreated cells. We observed that treatment with 20 and 50 μM wortmannin induced DNA damage equivalent to that by 0.37 and 0.69 Gy, respectively, of γ-ray radiation. The accumulation of DNA double-strand breaks (DSBs) in wortmannin-treated SCID cells was assessed using pulsed-field gel electrophoresis. The maximal accumulation was observed 4 h after treatment. Moreover, the presence of DSBs was confirmed by the ability of nuclear extracts from γ-ray-irradiated SCID cells to produce in vitro phosphorylation of histone H2AX. These results suggest that wortmannin induces cellular toxicity by accumulation of spontaneous DSBs through inhibition of ATM.
Topics: Animals; Cell Line; Cell Survival; DNA Breaks, Double-Stranded; Histones; Humans; Phosphatidylinositol 3-Kinase; Phosphorylation; Protein Kinase Inhibitors; Radiation Tolerance; Wortmannin
PubMed: 32052028
DOI: 10.1093/jrr/rrz102 -
Journal of Natural Products Mar 2019In the course of our studies of coprophilous fungi as sources of antifungal agents, a strain of an undescribed species in the genus Niesslia (TTI-0426) was isolated from...
In the course of our studies of coprophilous fungi as sources of antifungal agents, a strain of an undescribed species in the genus Niesslia (TTI-0426) was isolated from horse dung collected in Texas. An extract from fermentation cultures of this strain afforded two new antifungal wortmannin derivatives, wortmannins C and D (1 and 2), as well as four additional new related compounds, wortmannines B1-B4 (3-6), containing an unusual ring system. The structures of these metabolites were established mainly by analysis of HRESIMS and 2D NMR data. Relative configurations were assigned using NOESY data, and the structure assignments were supported by NMR comparison with similar compounds. Wortmannins C and D showed activity against Cryptococcus neoformans and Candida albicans in disk assays, but low MIC potency observed for 1 was suggested to be due in part to efflux processes on the basis of assay results for a Schizosaccharomyces pombe efflux mutant in comparison to wild-type.
Topics: Candida albicans; Cryptococcus neoformans; Fermentation; Hypocreales; Microbial Sensitivity Tests; Molecular Structure; Schizosaccharomyces; Spectrum Analysis; Wortmannin
PubMed: 30844268
DOI: 10.1021/acs.jnatprod.8b00923 -
Trends in Biochemical Sciences Aug 1995Wortmannin is a fungal metabolite that so far has been shown to act as a selective inhibitor of phosphoinositide 3-kinase. It can therefore be used to investigate the... (Review)
Review
Wortmannin is a fungal metabolite that so far has been shown to act as a selective inhibitor of phosphoinositide 3-kinase. It can therefore be used to investigate the convergence between two major cellular signalling systems: those involving G-protein-coupled receptors and those involving receptor tyrosine kinases. Importantly, wortmannin can enter intact cells, making whole-cell studies of the above signalling pathways possible.
Topics: Androstadienes; Molecular Probes; Phosphatidylinositol 3-Kinases; Phosphatidylinositols; Phosphotransferases (Alcohol Group Acceptor); Signal Transduction; Wortmannin
PubMed: 7667888
DOI: 10.1016/s0968-0004(00)89056-8 -
Journal of Cellular Physiology Jun 2020Modifications on shear stress-based mechanical forces are associated with pathophysiological susceptibility and their effect on endothelial cells (EC) needs to be better...
Modifications on shear stress-based mechanical forces are associated with pathophysiological susceptibility and their effect on endothelial cells (EC) needs to be better addressed looking for comprehending the cellular and molecular mechanisms. This prompted us to better evaluate the effects of shear stress in human primary venous EC obtained from the umbilical cord, using an in vitro model to mimic the laminar blood flow, reaching an intensity 1-4 Pa. First, our data shows there is a significant up-expression of phosphatidylinositol 3-kinase (PI3K) in shear-stressed cells culminating downstream with an up-phosphorylation of AKT and up-expression of MAPK-ERK, concomitant to a dynamic cytoskeleton rearrangement upon integrin subunits (α4 and ß 3) requirements. Importantly, the results show there is significant involvement of nitric oxide synthase (eNOS), nNOS, and vascular endothelial growth factors receptor 2 (VEGFR2) in shear-stressed EC, while cell cycle-related events seem to being changed. Additionally, although diminution of 5-hydroxymethylcytosine in shear-stressed EC, suggesting a global repression of genes transcription, the promoters of PI3K and eNOS genes were significantly hydroxymethylated corroborating with their respective transcriptional profiles. Finally, to better address, the pivotal role of PI3K in shear-stressed EC we have revisited these biological issues by wortmannin targeting PI3K signaling and the data shows a dependency of PI3K signaling in controlling the expression of VGFR1, VGFR2, VEGF, and eNOS, once these genes were significantly suppressed in the presence of the inhibitor, as well as transcripts from Ki67 and CDK2 genes. Finally, our data still shows a coupling between PI3K and the epigenetic landscape of shear-stressed cells, once wortmannin promotes a significant suppression of ten-11 translocation 1 (TET1), TET2, and TET3 genes, evidencing that PI3K signaling is a necessary upstream pathway to modulate TET-related genes. In this study we determined the major mechanotransduction pathway by which blood flow driven shear stress activates PI3K which plays a pivotal role on guaranteeing endothelial cell phenotype and vascular homeostasis, opening novel perspectives to understand the molecular basis of pathophysiological disorders related with the vascular system.
Topics: Angiogenesis Inducing Agents; DNA-Binding Proteins; Dioxygenases; Endothelial Cells; Humans; Mechanotransduction, Cellular; Mixed Function Oxygenases; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Nitric Oxide Synthase Type III; Phosphatidylinositol 3-Kinase; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Shear Strength; Signal Transduction; Stress, Mechanical; Vascular Endothelial Growth Factor Receptor-2; Wortmannin
PubMed: 31858593
DOI: 10.1002/jcp.29412 -
Advances in Protein Chemistry and... 2016Oncogenic mutations in phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA) are the most frequently reported in association with various... (Review)
Review
Oncogenic mutations in phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA) are the most frequently reported in association with various forms of cancer. Several studies have reported the significance of hotspot mutations in a catalytic subunit of PIK3CA in association with breast cancer. Mutations are frequently observed in the highly conserved region of the kinase domain (797-1068 amino acids) of PIK3CA are activating or gain-of-function mutations. Mutation in codon 1047 occurs in the C-terminal region of the kinase domain with histidine (H) replaced by arginine (R), lysine (L), and tyrosine (Y). Pathogenicity and protein stability predictors PhD-SNP, Align GVGD, HANSA, iStable, and MUpro classified H1047R as highly deleterious when compared to H1047L and H1047Y. To explore the inhibitory activity of Wortmannin toward PIK3CA, the three-dimensional structure of the mutant protein was determined using homology modeling followed by molecular docking and molecular dynamics analysis. Docking studies were performed for the three mutants and native with Wortmannin to measure the differences in their binding pattern. Comparative docking study revealed that H1047R-Wortmannin complex has a higher number of hydrogen bonds as well as the best binding affinity next to the native protein. Furthermore, 100 ns molecular dynamics simulation was initiated with the docked complexes to understand the various changes induced by the mutation. Though Wortmannin was found to nullify the effect of H1047R over the protein, further studies are required for designing a better compound. As SNPs are major genetic variations observed in disease condition, personalized medicine would provide enhanced drug therapy.
Topics: Androstadienes; Breast Neoplasms; Catalytic Domain; Class I Phosphatidylinositol 3-Kinases; Codon; Female; Humans; Molecular Docking Simulation; Molecular Dynamics Simulation; Mutation; Phosphatidylinositol 3-Kinases; Polymorphism, Single Nucleotide; Precision Medicine; Protein Binding; Wortmannin
PubMed: 26827608
DOI: 10.1016/bs.apcsb.2015.09.008 -
Journal of the American Chemical Society May 2017A concise and enantioselective total synthesis of the potent PI3K inhibitor (+)-wortmannin is described. A Pd-catalyzed cascade reaction was first developed to connect a...
A concise and enantioselective total synthesis of the potent PI3K inhibitor (+)-wortmannin is described. A Pd-catalyzed cascade reaction was first developed to connect a synthon derived from Hajos-Parrish ketone to a furan moiety. The subsequent Friedel-Crafts alkylation of the β-position of a furan ring to an epoxide was optimized to establish the C10 quaternary center. (+)-Wortmannin was eventually accomplished by transformations following a late-stage oxidation of the furan allylic position. Kinome profiling and in vitro enzymatic assays were performed on 17-β-hydroxy-wortmannin and an epoxide analogue.
Topics: Androstadienes; Catalysis; Molecular Structure; Palladium; Stereoisomerism; Wortmannin
PubMed: 28474885
DOI: 10.1021/jacs.7b02515 -
Journal of Huazhong University of... Aug 2009The inhibitory effect of wortmannin on leukemic cells and the possible mechanisms were examined. K562 cells were treated with wortmannin of various concentrations...
The inhibitory effect of wortmannin on leukemic cells and the possible mechanisms were examined. K562 cells were treated with wortmannin of various concentrations (3.125-100 nmol/L) for 0-72 h. MTT assay was used to evaluate the inhibitory effect of wortmannin on the growth of K562 cells. Cell apoptosis was detected by both Annexin-V FITC/PI double-labeled cytometry and transmission electron microscopy (TEM). The expression of p-Akt, T-p-Akt, NF-kappaBp65 and IKK-kappaB was determined by Western blotting and reverse transcription-polymerase chain reaction (RT-PCR). Our results showed that wortmannin obviously inhibited growth and induced apoptosis of K562 cells in vitro in a time- and dose-dependent manner. The IC(50) value of wortmannin for 24 h was 25+/-0.14 nmol/L. Moreover, wortmannin induced K562 cells apoptosis in a dose-dependent manner. TEM revealed typical morphological changes of apoptosis in wortmannin-treated K562 cells, such as chromatin condensation, karyopyknosis, karyorhexis and apoptotic bodies. Additionally, several important intracellular protein kinases such as p-Akt, NF-kappaBp65 and IKK-kappaB experienced degradation of various degrees in a dose-dependent manner both at protein level and transcription level when cultured with wortmannin, but the expression of total Akt showed no change. It is concluded that wortmannin can inhibit the proliferation and induce apoptosis of K562 leukemia cells possibly by down-regulating the survival signaling pathways (PI3K/Akt and NF-kappaB channels).
Topics: Androstadienes; Apoptosis; Down-Regulation; Gene Expression Regulation, Neoplastic; Humans; K562 Cells; Oncogene Protein v-akt; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Signal Transduction; Transcription Factor RelA; Wortmannin
PubMed: 19662361
DOI: 10.1007/s11596-009-0412-x -
Molecular Biology of the Cell Mar 1996Treatment with the phosphatidylinositol 3-kinase inhibitor wortmannin promotes approximately 30% decrease in the steady-state number of cell-surface transferrin...
Treatment with the phosphatidylinositol 3-kinase inhibitor wortmannin promotes approximately 30% decrease in the steady-state number of cell-surface transferrin receptors. This effect is rapid and dose dependent, with maximal down-regulation elicited with 30 min of treatment and with an IC50 approximately 25 nM wortmannin. Wortmannin-treated cells display an increased endocytic rate constant for transferrin internalization and decreased exocytic rate constants for transferrin recycling. In addition to these effects in vivo, wortmannin is a potent inhibitor (IC50 approximately 15 nM) of a cell-free assay that detects the delivery of endocytosed probes into a common compartment. Inhibition of the in vitro assay involves the inactivation of a membrane-associated factor that can be recruited onto the surface of vesicles from the cytosol. Its effects on the cell-free assay suggest that wortmannin inhibits receptor sorting and/or vesicle budding required for delivery of endocytosed material to "mixing" endosomes. This idea is consistent with morphological changes induced by wortmannin, which include the formation of enlarged transferrin-containing structures and the disruption of the perinuclear endosomal compartment. However, the differential effects of wortmannin, specifically increased transferrin receptor internalization and inhibition of receptor recycling, implicate a role for phosphatidylinositol 3-kinase activity in multiple sorting events in the transferrin receptor's membrane traffic pathway.
Topics: Androstadienes; Down-Regulation; Endocytosis; Endosomes; HeLa Cells; Humans; Phosphatidylinositol 3-Kinases; Phosphotransferases (Alcohol Group Acceptor); Receptors, Transferrin; Tumor Cells, Cultured; Wortmannin
PubMed: 8868465
DOI: 10.1091/mbc.7.3.355 -
Journal of Lipid Mediators and Cell... Sep 1996
Review
Topics: Androstadienes; Animals; Enzyme Inhibitors; Humans; Phosphatidylinositol 3-Kinases; Phosphotransferases (Alcohol Group Acceptor); Signal Transduction; Wortmannin
PubMed: 8906571
DOI: 10.1016/0929-7855(96)00534-2 -
The Journal of Biological Chemistry May 1996Phosphatidylinositol (PI) 3'-kinases are a family of lipid kinases implicated in the regulation of cell growth by oncogene products and tyrosine kinase growth factor...
Phosphatidylinositol (PI) 3'-kinases are a family of lipid kinases implicated in the regulation of cell growth by oncogene products and tyrosine kinase growth factor receptors. The catalytic subunit of the p85/p110 PI 3'-kinase is homologous to VPS-34, a phosphatidylinositol-specific lipid kinase involved in the sorting of newly synthesized hydrolases to the yeast vacuole. This suggests that PI 3'-kinases may play analogous roles in mammalian cells. We have measured a number of secretory and endocytic trafficking events in Chinese hamster ovary cells in the presence of wortmannin, a potent inhibitor of PI 3'-kinase. Wortmannin caused a 40-50% down-regulation of surface transferrin receptors, with a dose dependence identical to that required for maximal inhibition of the p85/p110 PI 3'-kinase in intact cells. The redistribution of transferrin receptors reflected a 60% increase in the internalization rate and a 35% decrease in the recycling rate. Experiments with fluorescent transferrin showed that entry of transferrin receptors into the recycling compartment and efflux of receptors out of the compartment were slowed by wortmannin. Wortmannin altered the morphology of the recycling compartment, which was more vesiculated than in untreated cells. Using Semliki Forest virus as a probe, we also found that delivery of the endocytosed virus to its lysosomal site of degradation was slowed by wortmannin, whereas endosomal acidification was unaffected. In contrast to these effects on endocytosis and recycling, wortmannin did not affect intracellular processing of newly synthesized viral spike proteins. Wortmannin did induce missorting of the lysosomal enzyme cathepsin D to the secretory pathway, but only at a dose 20-fold greater than that required to inhibit p85/p110 PI 3'-kinase activity or to redistribute transferrin receptors. Our data demonstrate the presence of wortmannin-sensitive enzymes at three distinct steps of the endocytic cycle in Chinese hamster ovary cells: internalization, transit from early endosomes to the recycling and degradative compartments, and transit from the recycling compartment back to the cell surface. The wortmannin-sensitive enzymes critical for endocytosis and recycling are distinct from those involved in sorting newly synthesized lysosomal enzymes.
Topics: Androstadienes; Animals; Biological Transport; CHO Cells; Cricetinae; Cricetulus; Endocytosis; Enzyme Inhibitors; Hydrogen-Ion Concentration; Lysosomes; Phosphatidylinositol 3-Kinases; Phosphotransferases (Alcohol Group Acceptor); Wortmannin
PubMed: 8631914
DOI: 10.1074/jbc.271.18.10953