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Opposing roles of LTB4 and PGE2 in regulating the inflammasome-dependent scorpion venom-induced mortality.
Zoccal KF, Sorgi CA, Hori JI, Paula-Silva FW, Arantes EC, Serezani CH, Zamboni DS, Faccioli LH
(2016) Nat Commun 7: 10760
MeSH Terms: Animals, Arachidonate 5-Lipoxygenase, Blotting, Western, Carrier Proteins, Celecoxib, Cyclic AMP, Cyclic AMP-Dependent Protein Kinases, Cyclooxygenase Inhibitors, Dinoprostone, In Vitro Techniques, Indoles, Indomethacin, Inflammasomes, Interleukin-1beta, Leukotriene B4, Lipoxygenase Inhibitors, Macrophages, Macrophages, Peritoneal, Mice, Mice, Knockout, NF-kappa B, NLR Family, Pyrin Domain-Containing 3 Protein, Phosphoproteins, Prostaglandin Antagonists, Receptors, Prostaglandin E, EP2 Subtype, Receptors, Prostaglandin E, EP4 Subtype, Reverse Transcriptase Polymerase Chain Reaction, Scorpion Stings, Scorpion Venoms, Scorpions, Xanthones
Show Abstract · Added May 4, 2017
Tityus serrulatus sting causes thousands of deaths annually worldwide. T. serrulatus-envenomed victims exhibit local or systemic reaction that culminates in pulmonary oedema, potentially leading to death. However, the molecular mechanisms underlying T. serrulatus venom (TsV) activity remain unknown. Here we show that TsV triggers NLRP3 inflammasome activation via K(+) efflux. Mechanistically, TsV triggers lung-resident cells to release PGE2, which induces IL-1β production via E prostanoid receptor 2/4-cAMP-PKA-NFκB-dependent mechanisms. IL-1β/IL-1R actions account for oedema and neutrophil recruitment to the lungs, leading to TsV-induced mortality. Inflammasome activation triggers LTB4 production and further PGE2 via IL-1β/IL-1R signalling. Activation of LTB4-BLT1/2 pathway decreases cAMP generation, controlling TsV-induced inflammation. Exogenous administration confirms LTB4 anti-inflammatory activity and abrogates TsV-induced mortality. These results suggest that the balance between LTB4 and PGE2 determines the amount of IL-1β inflammasome-dependent release and the outcome of envenomation. We suggest COX1/2 inhibition as an effective therapeutic intervention for scorpion envenomation.
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31 MeSH Terms
Inhibitors of the 5-lipoxygenase arachidonic acid pathway induce ATP release and ATP-dependent organic cation transport in macrophages.
da Silva-Souza HA, Lira MN, Costa-Junior HM, da Cruz CM, Vasconcellos JS, Mendes AN, Pimenta-Reis G, Alvarez CL, Faccioli LH, Serezani CH, Schachter J, Persechini PM
(2014) Biochim Biophys Acta 1838: 1967-77
MeSH Terms: Adenosine Triphosphate, Animals, Arachidonate 5-Lipoxygenase, Calcium, Cations, Ion Transport, Lipoxygenase Inhibitors, Macrophages, Mice, Mice, Inbred C57BL, Phospholipases A2, Receptors, Purinergic P2X7
Show Abstract · Added May 4, 2017
We have previously described that arachidonic acid (AA)-5-lipoxygenase (5-LO) metabolism inhibitors such as NDGA and MK886, inhibit cell death by apoptosis, but not by necrosis, induced by extracellular ATP (ATPe) binding to P2X7 receptors in macrophages. ATPe binding to P2X7 also induces large cationic and anionic organic molecules uptake in these cells, a process that involves at least two distinct transport mechanisms: one for cations and another for anions. Here we show that inhibitors of the AA-5-LO pathway do not inhibit P2X7 receptors, as judged by the maintenance of the ATPe-induced uptake of fluorescent anionic dyes. In addition, we describe two new transport phenomena induced by these inhibitors in macrophages: a cation-selective uptake of fluorescent dyes and the release of ATP. The cation uptake requires secreted ATPe, but, differently from the P2X7/ATPe-induced phenomena, it is also present in macrophages derived from mice deficient in the P2X7 gene. Inhibitors of phospholipase A2 and of the AA-cyclooxygenase pathway did not induce the cation uptake. The uptake of non-organic cations was investigated by measuring the free intracellular Ca(2+) concentration ([Ca(2+)]i) by Fura-2 fluorescence. NDGA, but not MK886, induced an increase in [Ca(2+)]i. Chelating Ca(2+) ions in the extracellular medium suppressed the intracellular Ca(2+) signal without interfering in the uptake of cationic dyes. We conclude that inhibitors of the AA-5-LO pathway do not block P2X7 receptors, trigger the release of ATP, and induce an ATP-dependent uptake of organic cations by a Ca(2+)- and P2X7-independent transport mechanism in macrophages.
Copyright © 2014 Elsevier B.V. All rights reserved.
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12 MeSH Terms
Effect of zileuton and celecoxib on urinary LTE4 and PGE-M levels in smokers.
Mohebati A, Milne GL, Zhou XK, Duffield-Lillico AJ, Boyle JO, Knutson A, Bosworth BP, Kingsley PJ, Marnett LJ, Brown PH, Akpa EG, Szabo E, Dannenberg AJ
(2013) Cancer Prev Res (Phila) 6: 646-55
MeSH Terms: Adult, Arachidonate 5-Lipoxygenase, Biomarkers, Celecoxib, Chromatography, Liquid, Cyclooxygenase 2, Cyclooxygenase 2 Inhibitors, Female, Humans, Hydroxyurea, Leukotriene E4, Lipoxygenase Inhibitors, Male, Maximum Tolerated Dose, Prognosis, Prostaglandins, Pyrazoles, Smoking, Sulfonamides, Tandem Mass Spectrometry
Show Abstract · Added March 7, 2014
COX-2 and 5-lipoxygenase (5-LO) use arachidonic acid for the synthesis of eicosanoids that have been implicated in carcinogenesis and cardiovascular disease. The ability of celecoxib, a selective COX-2 inhibitor, to redirect arachidonic acid into the 5-LO pathway can potentially reduce its efficacy as a chemopreventive agent and increase the risk of cardiovascular complications. Levels of urinary prostaglandin E metabolite (PGE-M) and leukotriene E4 (LTE4), biomarkers of the COX and 5-LO pathways, are elevated in smokers. Here, we investigated the effects of zileuton, a 5-LO inhibitor, versus zileuton and celecoxib for 6 ± 1 days on urinary PGE-M and LTE4 levels in smokers. Treatment with zileuton led to an 18% decrease in PGE-M levels (P = 0.03); the combination of zileuton and celecoxib led to a 62% reduction in PGE-M levels (P < 0.001). Levels of LTE4 decreased by 61% in subjects treated with zileuton alone (P < 0.001) and were unaffected by the addition of celecoxib. Although zileuton use was associated with a small overall decrease in PGE-M levels, increased PGE-M levels were found in a subset (19 of 52) of subjects. Notably, the addition of celecoxib to the 5-LO inhibitor protected against the increase in urinary PGE-M levels (P = 0.03). In conclusion, zileuton was an effective inhibitor of 5-LO activity resulting in marked suppression of urinary LTE4 levels and possible redirection of arachidonic acid into the COX-2 pathway in a subset of subjects. Combining celecoxib and zileuton was associated with inhibition of both the COX-2 and 5-LO pathways manifested as reduced levels of urinary PGE-M and LTE4.
©2013 AACR.
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20 MeSH Terms
Leukotriene biosynthesis inhibitor MK886 impedes DNA polymerase activity.
Ketkar A, Zafar MK, Maddukuri L, Yamanaka K, Banerjee S, Egli M, Choi JY, Lloyd RS, Eoff RL
(2013) Chem Res Toxicol 26: 221-32
MeSH Terms: Binding Sites, DNA-Directed DNA Polymerase, Humans, Indoles, Kinetics, Leukotrienes, Lipoxygenase Inhibitors, Molecular Docking Simulation, Nucleic Acid Synthesis Inhibitors
Show Abstract · Added May 27, 2014
Specialized DNA polymerases participate in replication stress responses and in DNA repair pathways that function as barriers against cellular senescence and genomic instability. These events can be co-opted by tumor cells as a mechanism to survive chemotherapeutic and ionizing radiation treatments and as such, represent potential targets for adjuvant therapies. Previously, a high-throughput screen of ∼16,000 compounds identified several first generation proof-of-principle inhibitors of human DNA polymerase kappa (hpol κ). The indole-derived inhibitor of 5-lipoxygenase activating protein (FLAP), MK886, was one of the most potent inhibitors of hpol κ discovered in that screen. However, the specificity and mechanism of inhibition remained largely undefined. In the current study, the specificity of MK886 against human Y-family DNA polymerases and a model B-family DNA polymerase was investigated. MK886 was found to inhibit the activity of all DNA polymerases tested with similar IC(50) values, the exception being a 6- to 8-fold increase in the potency of inhibition against human DNA polymerase iota (hpol ι), a highly error-prone enzyme that uses Hoogsteen base-pairing modes during catalysis. The specificity against hpol ι was partially abrogated by inclusion of the recently annotated 25 a.a. N-terminal extension. On the basis of Michaelis-Menten kinetic analyses and DNA binding assays, the mechanism of inhibition by MK886 appears to be mixed. In silico docking studies were used to produce a series of models for MK886 binding to Y-family members. The docking results indicate that two binding pockets are conserved between Y-family polymerases, while a third pocket near the thumb domain appears to be unique to hpol ι. Overall, these results provide insight into the general mechanism of DNA polymerase inhibition by MK886.
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9 MeSH Terms
Crystal structure of 12-lipoxygenase catalytic-domain-inhibitor complex identifies a substrate-binding channel for catalysis.
Xu S, Mueser TC, Marnett LJ, Funk MO
(2012) Structure 20: 1490-7
MeSH Terms: Animals, Arachidonate 12-Lipoxygenase, Biocatalysis, Catalytic Domain, Coordination Complexes, Crystallography, X-Ray, Hydrogen Bonding, Hydrophobic and Hydrophilic Interactions, Iron, Lipoxygenase Inhibitors, Models, Molecular, Oxygen, Phenylpropionates, Protein Binding, Protein Structure, Secondary, Sus scrofa
Show Abstract · Added June 1, 2014
Lipoxygenases are critical enzymes in the biosynthesis of families of bioactive lipids including compounds with important roles in the initiation and resolution of inflammation and in associated diseases such as diabetes, cardiovascular disease, and cancer. Crystals diffracting to high resolution (1.9 Å) were obtained for a complex between the catalytic domain of leukocyte 12-lipoxygenase and the isoform-specific inhibitor, 4-(2-oxapentadeca-4-yne)phenylpropanoic acid (OPP). In the three-dimensional structure of the complex, the inhibitor occupied a new U-shaped channel open at one end to the surface of the protein and extending past the redox-active iron site that is essential for catalysis. In models, the channel accommodated arachidonic acid, defining the binding site for the substrate of the catalyzed reaction. There was a void adjacent to the OPP binding site connecting to the surface of the enzyme and providing a plausible access channel for the other substrate, oxygen.
Copyright © 2012 Elsevier Ltd. All rights reserved.
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16 MeSH Terms
Protein kinase C regulation of 12-lipoxygenase-mediated human platelet activation.
Yeung J, Apopa PL, Vesci J, Kenyon V, Rai G, Jadhav A, Simeonov A, Holman TR, Maloney DJ, Boutaud O, Holinstat M
(2012) Mol Pharmacol 81: 420-30
MeSH Terms: Arachidonate 12-Lipoxygenase, Blood Platelets, Blotting, Western, Calcium, Enzyme Activation, Flow Cytometry, Gas Chromatography-Mass Spectrometry, Humans, Lipoxygenase Inhibitors, Platelet Activation, Platelet Glycoprotein GPIIb-IIIa Complex, Protein Kinase C, Protein Kinase Inhibitors
Show Abstract · Added March 27, 2014
Platelet activation is important in the regulation of hemostasis and thrombosis. Uncontrolled activation of platelets may lead to arterial thrombosis, which is a major cause of myocardial infarction and stroke. After activation, metabolism of arachidonic acid (AA) by 12-lipoxygenase (12-LOX) may play a significant role in regulating the degree and stability of platelet activation because inhibition of 12-LOX significantly attenuates platelet aggregation in response to various agonists. Protein kinase C (PKC) activation is also known to be an important regulator of platelet activity. Using a newly developed selective inhibitor for 12-LOX and a pan-PKC inhibitor, we investigated the role of PKC in 12-LOX-mediated regulation of agonist signaling in the platelet. To determine the role of PKC within the 12-LOX pathway, a number of biochemical endpoints were measured, including platelet aggregation, calcium mobilization, and integrin activation. Inhibition of 12-LOX or PKC resulted in inhibition of dense granule secretion and attenuation of both aggregation and αIIbβ(3) activation. However, activation of PKC downstream of 12-LOX inhibition rescued agonist-induced aggregation and integrin activation. Furthermore, inhibition of 12-LOX had no effect on PKC-mediated aggregation, indicating that 12-LOX is upstream of PKC. These studies support an essential role for PKC downstream of 12-LOX activation in human platelets and suggest 12-LOX as a possible target for antiplatelet therapy.
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13 MeSH Terms
Pyridine and pyrimidine analogs of acetaminophen as inhibitors of lipid peroxidation and cyclooxygenase and lipoxygenase catalysis.
Nam TG, Nara SJ, Zagol-Ikapitte I, Cooper T, Valgimigli L, Oates JA, Porter NA, Boutaud O, Pratt DA
(2009) Org Biomol Chem 7: 5103-12
MeSH Terms: Acetaminophen, Antioxidants, Cell Line, Tumor, Cyclooxygenase Inhibitors, Humans, Kinetics, Lipid Peroxidation, Lipoxygenase, Lipoxygenase Inhibitors, Prostaglandin-Endoperoxide Synthases, Prostaglandins, Pyridines, Pyrimidines, Soybeans, Structure-Activity Relationship
Show Abstract · Added November 19, 2013
Herein we report an investigation of the efficacy of pyridine and pyrimidine analogs of acetaminophen (ApAP) as peroxyl radical-trapping antioxidants and inhibitors of enzyme-catalyzed lipid peroxidation by cyclooxygenases (COX) and lipoxygenases (LOX). In inhibited autoxidations we find that ApAP, the common analgesic and antipyretic agent, is a very good antioxidant with a rate constant for reaction with peroxyl radicals (k(inh) = 5 x 10(5) M(-1) s(-1)) that is higher than many widely-used phenolic antioxidants, such as the ubiquitous butylated hydroxytoluene (BHT). This reactivity is reduced substantially upon incorporation of nitrogen into the phenolic ring, owing to an increase in the O-H bond dissociation enthalpy of pyridinols and pyrimidinols with respect to phenols. Incorporation of nitrogen into the phenolic ring of ApAP was also found to decrease its efficacy as an inhibitor of prostaglandin biosynthesis by ovine COX-1 (oCOX-1). This is explained on the basis of an increase in its oxidation potential and its reduced reactivity as a reducing co-substrate of the peroxidase protoporphyrin. In contrast, the efficacy of ApAP as an inhibitor of lipid hydroperoxide biosynthesis by soybean LOX-1 (sLOX-1) increased upon incorporation of nitrogen into the ring, suggesting a different mechanism of inhibition dependent on the acidity of the phenolic O-H which may involve chelation of the catalytic non-heme iron atom. The greater stability of the 3-pyridinols and 5-pyrimidinols to air oxidation as compared to phenols allowed us to evaluate some electron-rich pyridinols and pyrimidinols as inhibitors of oCOX-1 and sLOX-1. While the pyridinols had the best combination of activities as antioxidants and inhibitors of oCOX-1 and sLOX-1, they were found to be more toxic than ApAP in preliminary assays in human hepatocellular carcinoma (HepG2) cell culture. The pyrimidinols, however, were up to 17-fold more reactive to peroxyl radicals and up to 25-fold better inhibitors of prostaglandin biosynthesis than ApAP, with similar cytotoxicities to HepG2 cells at high levels of exposure.
1 Communities
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15 MeSH Terms
Kinetics of inhibition of leukocyte 12-lipoxygenase by the isoform-specific inhibitor 4-(2-oxapentadeca-4-yne)phenylpropanoic acid.
Moody JS, Marnett LJ
(2002) Biochemistry 41: 10297-303
MeSH Terms: 5,8,11,14-Eicosatetraynoic Acid, Animals, Arachidonate 12-Lipoxygenase, Binding Sites, Binding, Competitive, Enzyme Inhibitors, Isoenzymes, Kinetics, Leukocytes, Linoleic Acids, Lipid Peroxides, Lipoxygenase Inhibitors, Oxidation-Reduction, Oxygen, Phenylpropionates, Swine
Show Abstract · Added March 5, 2014
Lipoxygenases (LOXs) are a ubiquitous family of enzymes that catalyze the dioxygenation of polyunsaturated fatty acids. Their role in a diverse range of biological processes has prompted the development of a large number of lipoxygenase inhibitors of possible therapeutic and probative value. The isoform-selective inhibitor 4-(2-oxapentadeca-4-yne)phenylpropanoic acid (OPP) was previously shown to inhibit leukocyte-type 12-LOX by a novel mechanism in which it binds to both the ferrous and ferric forms of the enzyme. The current study provides a detailed kinetic model of this inhibition. Nonlinear regression analysis of OPP's inhibition of arachidonic acid dioxygenation indicated mixed inhibition toward the ferric form of 12-LOX with apparent K(I) values in the low micromolar range: 2.0 +/- 0.2 microM for the free enzyme and 4.5 +/- 0.7 microM for the substrate-bound form of the enzyme. Rapid kinetic techniques allowed OPP's inhibition of the activation of the enzyme from the ferrous to the ferric form to be investigated. Titration of ferrous 12-LOX with OPP indicated that it bound to the ferrous form with an apparent K(I) value of 70 +/- 20 nM, suggesting a significantly higher affinity for the ferrous form than for the ferric form of the enzyme. Investigation of the LOX inhibitors nordihydroguaiaretic acid, N-(4-chlorophenyl)-N-hydroxy-N'-(3-chlorophenyl)urea, BWA137C, and eicosatetraynoic acid revealed that eicosatetraynoic acid also inhibited the activation of 12-LOX. These results demonstrate that LOX inhibitors are capable of binding to multiple forms of LOXs with high affinity and suggest that inhibition of enzyme activation may be an unrecognized mechanism of inhibition of additional LOX inhibitors.
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16 MeSH Terms
Mechanism of inhibition of porcine leukocyte 12-lipoxygenase by the isoform-specific inhibitor 4-(2-oxapentadeca-4-yne)phenylpropanoic acid.
Richards KM, Moody JS, Marnett LJ
(1999) Biochemistry 38: 16529-38
MeSH Terms: Animals, Arachidonate 12-Lipoxygenase, Chromatography, High Pressure Liquid, Ferric Compounds, Ferrous Compounds, Isoenzymes, Leukocytes, Linoleic Acids, Lipid Peroxides, Lipoxygenase Inhibitors, Oxidation-Reduction, Peroxidases, Phenylpropionates, Recombinant Proteins, Swine
Show Abstract · Added March 5, 2014
The mechanism of inhibition of porcine leukocyte 12-lipoxygenase by 4-(2-oxapentadeca-4-yne)phenylpropanoic acid (OPP) was investigated. This compound is selective for the leukocyte form of the 12-lipoxygenase and inhibits the purified recombinant enzyme with an IC(50) value of approximately 2 microM. OPP induced a concentration-dependent lag phase in the oxygenation of arachidonic acid and decreased the maximal rate of reaction. Addition of the fatty acid hydroperoxide 13(S)-hydroperoxyoctadecadienoic acid (13-HPODE) to the reaction greatly reduced the OPP-induced lag. Lineweaver-Burk analysis of the effect of OPP on 12-lipoxygenase kinetics with arachidonic acid indicated that it was a mixed-type inhibitor. OPP was not metabolized by 12-lipoxygenase as evidenced by its quantitative recovery from incubations with stoichiometric amounts of enzyme and 13-HPODE or arachidonic acid. OPP inhibited the pseudoperoxidase activity of the enzyme with 13-HPODE and the reducing agent, BWA137C. Lineweaver-Burk analysis of the effect of OPP on pseudoperoxidase kinetics suggested that OPP was competitive with 13-HPODE. Single-turnover experiments indicated that OPP inhibited the reduction of 13-HPODE by a stoichiometric amount of ferrous 12-lipoxygenase. Addition of 13-HPODE shortened the OPP-induced lag phase but did not affect the maximal rate of enzyme activity. In addition, OPP had no effect on total product formation in either the presence or the absence of 5 microM 13-HPODE when the reaction was allowed to go to completion. All of these observations are consistent with a model for inhibition of 12-lipoxygenase activity in which OPP slows the oxidation of the inactive ferrous enzyme to the active ferric enzyme and competes with arachidonic acid for the ferric enzyme.
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15 MeSH Terms
Leukocyte 12-lipoxygenase: expression, purification, and investigation of the role of methionine residues in turnover-dependent inactivation and 5,8,11,14-eicosatetraynoic acid inhibition.
Richards KM, Marnett LJ
(1997) Biochemistry 36: 6692-9
MeSH Terms: Amino Acid Sequence, Ammonium Sulfate, Animals, Arachidonate 12-Lipoxygenase, Catalase, Chemical Precipitation, Chromatography, Ion Exchange, DNA, Complementary, Dithiothreitol, Escherichia coli, Gene Expression, Humans, Leukocytes, Lipoxygenase Inhibitors, Methionine, Molecular Sequence Data, Mutagenesis, Site-Directed, Recombinant Proteins, Sequence Alignment, Structure-Activity Relationship, Swine
Show Abstract · Added March 5, 2014
Porcine leukocyte 12-lipoxygenase cDNA was cloned into the expression vectors pSE280, pSE380, and pSE420. pSE380 yielded the highest level of 12-lipoxygenase activity when these vectors were tested for expression in Escherichia coli Top10 cells. Optimal expression of the protein from this vector occurred in cells cultured at 30 degrees C and harvested 18 h following induction of expression by 0.5 mM isopropyl thiogalactoside (IPTG). The enzyme was purified from the 100000 g supernatant to 98% homogeneity by a combination of ammonium sulfate precipitation, anion exchange chromatography, and chromatofocusing. Addition of dithiothreitol and catalase to buffers at various steps in the purification protocol enabled the isolation of enzyme having a specific activity of 12 micromol min(-1) mg(-1). The recovery of purified protein from this expression system was 56%, resulting in a 109-fold purification. On the basis of amino acid sequence comparisons between mammalian 15- and 12-lipoxygenases, three methionine residues in the porcine leukocyte 12-lipoxygenase (M338L, M367V, and M562L) were targeted for mutation to assess their potential role in turnover-dependent inactivation and inhibition by 5,8,11,14-eicosatetraynoic acid (ETYA). The mutants were expressed and purified by the same procedure used for the wild-type enzyme. These amino acid changes did not significantly alter enzyme catalysis as judged by the kinetic constants Km and k(cat)/Km, nor did they affect the rate of turnover-dependent inactivation or inhibition by ETYA. The results indicate that these methionine residues do not play a pivotal role in catalysis, autoinactivation, or sensitivity to inhibition by acetylenic compounds.
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21 MeSH Terms