Oxygenation Of Arachidonic Acid Research Articles

Characterisation of (R)-2-(2-Fluorobiphenyl-4-yl)-N-(3-Methylpyridin-2-yl)Propanamide as a Dual Fatty Acid Amide Hydrolase: Cyclooxygenase Inhibitor.

BackgroundIncreased endocannabinoid tonus by dual-action fatty acid amide hydrolase (FAAH) and substrate selective cyclooxygenase (COX-2) inhibitors is a promising approach for pain-relief. One such compound with this profile is 2-(2-fluorobiphenyl-4-yl)-N-(3-methylpyridin-2-yl)propanamide (Flu-AM1). These activities are shown by Flu-AM1 racemate, but it is not known whether its two single enantiomers behave differently, as is the case towards COX-2 for the parent flurbiprofen enantiomers. Further, the effects of the compound upon COX-2-derived lipids in intact cells are not known.Methodology/Principal FindingsCOX inhibition was determined using an oxygraphic method with arachidonic acid and 2-arachidonoylglycerol (2-AG) as substrates. FAAH was assayed in mouse brain homogenates using anandamide (AEA) as substrate. Lipidomic analysis was conducted in unstimulated and lipopolysaccharide + interferon γ- stimulated RAW 264.7 macrophage cells. Both enantiomers inhibited COX-2 in a substrate-selective and time-dependent manner, with IC50 values in the absence of a preincubation phase of: (R)-Flu-AM1, COX-1 (arachidonic acid) 6 μM; COX-2 (arachidonic acid) 20 μM; COX-2 (2-AG) 1 μM; (S)-Flu-AM1, COX-1 (arachidonic acid) 3 μM; COX-2 (arachidonic acid) 10 μM; COX-2 (2-AG) 0.7 μM. The compounds showed no enantiomeric selectivity in their FAAH inhibitory properties. (R)-Flu-AM1 (10 μM) greatly inhibited the production of prostaglandin D2 and E2 in both unstimulated and lipopolysaccharide + interferon γ- stimulated RAW 264.7 macrophage cells. Levels of 2-AG were not affected either by (R)-Flu-AM1 or by 10 μM flurbiprofen, either alone or in combination with the FAAH inhibitor URB597 (1 μM).Conclusions/SignificanceBoth enantiomers of Flu-AM1 are more potent inhibitors of 2-AG compared to arachidonic acid oxygenation by COX-2. Inhibition of COX in lipopolysaccharide + interferon γ- stimulated RAW 264.7 cells is insufficient to affect 2-AG levels despite the large induction of COX-2 produced by this treatment.

Competition and allostery govern substrate selectivity of cyclooxygenase-2

Cyclooxygenase-2 (COX-2) oxygenates arachidonic acid (AA) and its ester analog, 2-arachidonoylglycerol (2-AG), to prostaglandins (PGs) and prostaglandin glyceryl esters (PG-Gs), respectively. Although the efficiency of oxygenation of these substrates by COX-2 in vitro is similar, cellular biosynthesis of PGs far exceeds that of PG-Gs. Evidence that the COX enzymes are functional heterodimers suggests that competitive interaction of AA and 2-AG at the allosteric site of COX-2 might result in differential regulation of the oxygenation of the two substrates when both are present. Modulation of AA levels in RAW264.7 macrophages uncovered an inverse correlation between cellular AA levels and PG-G biosynthesis. In vitro kinetic analysis using purified protein demonstrated that the inhibition of 2-AG oxygenation by high concentrations of AA far exceeded the inhibition of AA oxygenation by high concentrations of 2-AG. An unbiased systems-based mechanistic model of the kinetic data revealed that binding of AA or 2-AG at the allosteric site of COX-2 results in a decreased catalytic efficiency of the enzyme toward 2-AG, whereas 2-AG binding at the allosteric site increases COX-2's efficiency toward AA. The results suggest that substrates interact with COX-2 via multiple potential complexes involving binding to both the catalytic and allosteric sites. Competition between AA and 2-AG for these sites, combined with differential allosteric modulation, gives rise to a complex interplay between the substrates, leading to preferential oxygenation of AA.

Is prostaglandin E2 (PGE2) involved in the thermogenic response to environmental cooling in healthy humans?

Prostaglandin E2 (PGE2) is an eicosanoid derived from cyclooxygenase, an enzyme responsible for the cyclisation and oxygenation of arachidonic acid. In response to bacterial infection, PGE2 binds to EP3 receptors on a population of GABAergic neurons in the pre-optic area. Activation of the EP3 receptor decreases the intracellular cyclic adenosine monophosphate (cAMP) concentrations of these neurons, and the resulting dis-inhibition activates spinal motor outputs responsible for shivering thermogenesis, tachycardia, and brown adipose tissue activation. These involuntary responses increase core body temperature to varying degrees depending on the magnitude of infection; an immune response which is crucial for the survival of the host. However, evidence in animal and human models, primarily through the use of cyclooxygenase inhibitors (which block the production of PGE2), suggests that PGE2 may also be an important molecule for the defence of core temperature against body cooling and cold stress (in the absence of fever). In this paper, evidence within human and animal models is discussed which supports the hypothesis that the eicosanoid PGE2 has a role in maintaining human core temperature during environmental cooling. Given that over-the-counter PGE2 inhibiting drugs [i.e. Non-Steroidal Anti Inflammatory Drugs (NSAIDS)] are frequently used worldwide, it is possible that the use of such medication during environmental cooling could impair one’s ability to thermoregulate. Support for such findings could have major implications in the pathology of hypothermia, thus, we suggest that future researchers investigate this specific hypothesis in vivo, using healthy human models. Suggestions for the implementation of such experiments are provided in the present work.

Versatility of tandem mass spectrometry for focused analysis of oxylipids

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) in the multiple reaction monitoring (MRM) scan mode has been the primary MS method applied for the target identification of specific and minor oxylipids in complex matrices, such as eicosanoids and docosanoids, which are potent lipid mediators derived from polyunsaturated fatty acid oxygenation. However, the high specificity of MRM can limit the detection of species with m/z MRM transitions not covered by the method. In addition to MRM, tandem-quadrupole mass analyzers enable other experiments to be conducted, by fragmenting ions via collision-induced dissociation process (CID). This paper presents the potential of tandem mass spectrometry for the focused analysis of oxylipids. We have successfully developed an LC-MS/MS method for the identification of precursor ions of m/z 115, a diagnostic product ion of 5-hydroxy- and 5-epoxy-fatty acids. As a proof of concept, the developed method was used to discover several oxylipids oxidized at C5 derived from arachidonic acid (C20 : 4) oxygenation in a hypothalamus rat extract that were not identified using the target MRM methodology. The proposed focused MS/MS-based approach in a tandem mass analyzer has proven to be a powerful strategy to accelerate the identification of oxylipids with structural similarities and assist the field of lipidomic research.

13-Methylarachidonic Acid Is a Positive Allosteric Modulator of Endocannabinoid Oxygenation by Cyclooxygenase

Cyclooxygenase-2 (COX-2) oxygenates arachidonic acid (AA) and the endocannabinoids 2-arachidonoylglycerol (2-AG) and arachidonylethanolamide to prostaglandins, prostaglandin glyceryl esters, and prostaglandin ethanolamides, respectively. A structural homodimer, COX-2 acts as a conformational heterodimer with a catalytic and an allosteric monomer. Prior studies have demonstrated substrate-selective negative allosteric regulation of 2-AG oxygenation. Here we describe AM-8138 (13(S)-methylarachidonic acid), a substrate-selective allosteric potentiator that augments 2-AG oxygenation by up to 3.5-fold with no effect on AA oxygenation. In the crystal structure of an AM-8138·COX-2 complex, AM-8138 adopts a conformation similar to the unproductive conformation of AA in the substrate binding site. Kinetic analysis suggests that binding of AM-8138 to the allosteric monomer of COX-2 increases 2-AG oxygenation by increasing kcat and preventing inhibitory binding of 2-AG. AM-8138 restored the activity of COX-2 mutants that exhibited very poor 2-AG oxygenating activity and increased the activity of COX-1 toward 2-AG. Competition of AM-8138 for the allosteric site prevented the inhibition of COX-2-dependent 2-AG oxygenation by substrate-selective inhibitors and blocked the inhibition of AA or 2-AG oxygenation by nonselective time-dependent inhibitors. AM-8138 selectively enhanced 2-AG oxygenation in intact RAW264.7 macrophage-like cells. Thus, AM-8138 is an important new tool compound for the exploration of allosteric modulation of COX enzymes and their role in endocannabinoid metabolism.

Development and evaluation of ST-1829 based on 5-benzylidene-2-phenylthiazolones as promising agent for anti-leukotriene therapy.

Different inflammatory diseases and allergic reactions are mediated by leukotrienes, which arise from the oxygenation of arachidonic acid catalyzed by 5-lipoxygenase (5-LO). One promising approach for an effective anti-leukotriene therapy is the inhibition of this key enzyme. This study presents the synthesis and development of a potent and direct 5-LO inhibitor based on the well characterized 5-benzylidene-2-phenylthiazolone C06, whose further pharmacological investigation was precluded due to its low solubility. Through optimization of C06, evaluation of structure-activity relationships including profound assessment of the thiazolone core and consideration of the solubility, the 5-benzyl-2-phenyl-4-hydroxythiazoles represented by 46 (ST-1829, 5-(4-chlorobenzyl)-2-p-tolylthiazol-4-ol) were developed. Compound 46 showed an improved 5-LO inhibitory activity in cell-based (IC50 values 0.14 μM) and cell-free assays (IC50 values 0.03 μM) as well as a prominent enhanced solubility. Furthermore, it kept its promising inhibitory potency in the presence of blood serum, excluding excessive binding to serum proteins. These facts combined with the non-cytotoxic profile mark a major step towards an effective anti-inflammatory therapy.

12-lipoxygenase promotes obesity-induced oxidative stress in pancreatic islets.

High-fat diets lead to obesity, inflammation, and dysglycemia. 12-Lipoxygenase (12-LO) is activated by high-fat diets and catalyzes the oxygenation of cellular arachidonic acid to form proinflammatory intermediates. We hypothesized that 12-LO in the pancreatic islet is sufficient to cause dysglycemia in the setting of high-fat feeding. To test this, we generated pancreas-specific 12-LO knockout mice and studied their metabolic and molecular adaptations to high-fat diets. Whereas knockout mice and control littermates displayed identical weight gain, body fat distribution, and macrophage infiltration into fat, knockout mice exhibited greater adaptive islet hyperplasia, improved insulin secretion, and complete protection from dysglycemia. At the molecular level, 12-LO deletion resulted in increases in islet antioxidant enzymes Sod1 and Gpx1 in response to high-fat feeding. The absence or inhibition of 12-LO led to increases in nuclear Nrf2, a transcription factor responsible for activation of genes encoding antioxidant enzymes. Our data reveal a novel pathway in which islet 12-LO suppresses antioxidant enzymes and prevents the adaptive islet responses in the setting of high-fat diets.

Inhibition of endocannabinoid metabolism by the metabolites of ibuprofen and flurbiprofen.

BackgroundIn addition to their effects upon prostaglandin synthesis, the non-steroidal anti-inflammatory drugs ibuprofen and flurbiprofen inhibit the metabolism of the endocannabinoids 2-arachidonoylglycerol (2-AG) and anandamide (AEA) by cyclooxygenase-2 (COX-2) and fatty acid amide hydrolase (FAAH), respectively. Here, we investigated whether these effects upon endocannabinoid metabolism are shared by the main metabolites of ibuprofen and flurbiprofen.Methodology/Principal FindingsCOX activities were measured via changes in oxygen consumption due to oxygenation of arachidonic acid (for COX-1) and arachidonic acid and 2-AG (for COX-2). FAAH activity was quantified by measuring hydrolysis of tritium labelled AEA in rat brain homogenates. The ability of ibuprofen and flurbiprofen to inhibit COX-2-catalysed oxygenation of 2-AG at lower concentrations than the oxygenation of arachidonic acid was seen with 4′-hydroxyflurbiprofen and possibly also 3′-hydroxyibuprofen, albeit at lower potencies than the parent compounds. All ibuprofen and flurbiprofen metabolites retained the ability to inhibit FAAH in a pH-dependent manner, although the potency was lower than seen with the parent compounds.Conclusions/SignificanceIt is concluded that the primary metabolites of ibuprofen and flurbiprofen retain some of the properties of the parent compound with respect to inhibition of endocannabinoid metabolism. However, these effects are unlikely to contribute to the actions of the parent compounds in vivo.

Regio- and stereospecificity in the oxygenation of arachidonic acid catalyzed by Leu597 mutants of rabbit 15-lipoxygenase: a QM/MM study.

We combined quantum mechanics/molecular mechanics calculations with molecular dynamics simulations to study the addition of O2 to the pentadienyl radical of arachidonic acid (AA) catalyzed by the Leu597Val and Leu597Ala mutants of rabbit 15-lipoxygenase (15-rLO). In the Leu597Val mutant, the addition of O2 to C15 of AA is the predominant path, although it reduces the C15/C11 product ratio by almost ten times with respect to the wildtype enzyme. The S stereochemistry is kept. Mutation to Ala causes just the opposite effect: regiospecificity favoring addition to C15 is somewhat sharper than that in the wildtype, but the stereochemistry is R. This is because the extra space created by the mutation to Ala is big enough for AA to move so that it can adopt an alternative binding mode, and this opens new feasible paths for the attack of O2 . So, we showed that the Leu597Ala mutant of 15r-LO works as an aspirin-acetylated cyclooxygenase-2, which makes 15-(R)- hydroperoxyeicosatetraenoic acid.

Cyclooxygenase-2 catalysis and inhibition in lipid bilayer nanodiscs

Cyclooxygenases (COX-1 and COX-2) oxygenate arachidonic acid (AA) to generate prostaglandins. The enzymes associate with one leaflet of the membrane bilayer. We utilized nanodisc technology to investigate the function of human (hu) COX-2 and murine (mu) COX-2 in a lipid bilayer environment. huCOX-2 and muCOX-2 were incorporated into nanodiscs composed of POPC, POPS, DOPC, or DOPS phospholipids. Size-exclusion chromatography and negative stain electron microscopy confirm that a single COX-2 homodimer is incorporated into the nanodisc scaffold. Nanodisc-reconstituted COX-2 exhibited similar kinetic profiles for the oxygenation of AA, eicosapentaenoic acid, and 1-arachidonoyl glycerol compared to those derived using detergent solubilized enzyme. Moreover, changing the phospholipid composition of the nanodisc did not alter the ability of COX-2 to oxygenate AA or to be inhibited by various nonselective NSAIDs or celecoxib. The cyclooxygenase activity of nanodisc-reconstituted COX-2 was reduced by aspirin acetylation and potentiated by the nonsubstrate fatty acid palmitic acid to the same extent as detergent solubilized enzyme, independent of phospholipid composition. The stabilization and maintenance of activity afforded by the incorporation of the enzyme into nanodiscs generates a native-like lipid bilayer environment to pursue studies of COX utilizing solution-based techniques that are otherwise not tractable in the presence of detergents.

Pre-existent Asymmetry in the Human Cyclooxygenase-2 Sequence Homodimer

Prostaglandin endoperoxide H synthase-2 (PGHS-2), also known as cyclooxygenase-2 (COX-2), is a sequence homodimer. However, the enzyme exhibits half-site heme and inhibitor binding and functions as a conformational heterodimer having a catalytic subunit (Ecat) with heme bound and an allosteric subunit (Eallo) lacking heme. Some recombinant heterodimers composed of a COX-deficient mutant subunit and a native subunit (i.e. Mutant/Native PGHS-2) have COX activities similar to native PGHS-2. This suggests that the presence of heme plus substrate leads to the subunits becoming lodged in a semi-stable Eallo-mutant/Ecat-Native∼heme form during catalysis. We examined this concept using human PGHS-2 dimers composed of combinations of Y385F, R120Q, R120A, and S530A mutant or native subunits. With some heterodimers (e.g. Y385F/Native PGHS-2), heme binds with significantly higher affinity to the native subunit. This correlates with near native COX activity for the heterodimer. With other heterodimers (e.g. S530A/Native PGHS-2), heme binds with similar affinities to both subunits, and the COX activity approximates that expected for an enzyme in which each monomer contributes equally to the net COX activity. With or without heme, aspirin acetylates one-half of the subunits of the native PGHS-2 dimer, the Ecat subunits. Subunits having an S530A mutation are refractory to acetylation. Curiously, aspirin acetylates only one-quarter of the monomers of S530A/Native PGHS-2 with or without heme. This implies that there are comparable amounts of two noninterchangeable species of apoenzymes, Eallo-S530A/Ecat-Native and Eallo-Native/Ecat-S530A. These results suggest that native PGHS-2 assumes a reasonably stable, asymmetric Eallo/Ecat form during its folding and processing.

Quantification of selected endogenous hydroxy-oxylipins from tropical marine macroalgae.

The present study investigated the contents of hydroxy-oxylipins hydroxyoctadecadienoic acids (HODEs), hydroxyoctadecatrienoic acids (HOTrEs), and hydroxyeicosatetraenoic acids (HETEs) in 40 macroalgae belonging to the Chlorophyceae, Rhodophyceae and, Phaeophyceae. The hydroxy-oxylipin content was low and ranged from 0.14 ± 0.012ng/g (Codium dwarkense) to 8,161.9 ± 253ng/g (Chaetomorpha linum) among the Chlorophyceae, 345.4 ± 56.8ng/g (Scytosiphon lomentaria) to 2,574.5 ± 155.5ng/g (Stoechospermum marginatum) among the Phaeophyceae, and 19.4 ± 2.2ng/g (Laurencia cruciata) to 1,753.1 ± 268.2ng/g in Gracilaria corticata v. folifera) among the Rhodophyceae on fresh weight basis (p ≤ 0.01). The concentrations of C18-oxylipins were greater than C20-oxylipins in all the investigated macroalgae, except forUlva linza, Codium sursum, Dictyopteris deliculata, S. marginatum, Sargassum tenerrimum, Gracilaria spp. (except G. textorii), Rhodymenia sonderi, and Odonthalia veravalensis.The macroalgal species rich in HODEs, HOTrEs, and HETEs were segregated using principal component analysis. The red macroalgae showed the highest contents of HETEs, followed by brown and green macroalgae in consistent with their PUFA profiles. The relative contents of isomeric forms of oxylipins displayed the species-specific positional selectivity of lipoxygenase (LOX) enzyme in macroalgae. All the species exhibited 13-LOX specificity for linoleic acid analogous of higher plants, while 21 out of 40 species showed 9-LOX selectivity for the oxygenation of α-linolenic acid. No trend was observed for the oxygenation of arachidonic acid in macroalgae, except for in the Halymeniales, Ceramiales (except L. cruciata), and Corallinales. This study infers that LOX products, octadecanoids and eicosanoids, described in macroalgal taxa were similar to those of higher plants and mammals, respectively, and thus can be utilized as an alternative source of chemically synthesized oxylipin analogues in therapeutics, cosmetics, and nutritional oil supplements.

Molecular basis for the catalytic inactivity of a naturally occurring near-null variant of human ALOX15

Mammalian lipoxygenases belong to a family of lipid-peroxidizing enzymes, which have been implicated in cardiovascular, hyperproliferative and neurodegenerative diseases. Here we report that a naturally occurring mutation in the hALOX15 gene leads to expression of a catalytically near-null enzyme variant (hGly422Glu). The inactivity may be related to severe misfolding of the enzyme protein, which was concluded from CD-spectra as well as from thermal and chemical stability assays. In silico mutagenesis experiments suggest that most mutations at hGly422 have the potential to induce sterical clash, which might be considered a reason for protein misfolding. hGly422 is conserved among ALOX5, ALOX12 and ALOX15 isoforms and corresponding hALOX12 and hALOX5 mutants also exhibited a reduced catalytic activity. Interestingly, in the hALOX5 Gly429Glu mutants the reaction specificity of arachidonic acid oxygenation was shifted from 5S- to 8S- and 12R-H(p)ETE formation. Taken together, our data indicate that the conserved glycine is of functional importance for these enzyme variants and most mutants at this position lose catalytic activity.

Rational Design of Novel Pyridinol-Fused Ring Acetaminophen Analogues

Acetaminophen (ApAP) is an electron donor capable of reducing radicals generated by redox cycling of hemeproteins. It acts on the prostaglandin H synthases (cyclooxygenases; COXs) to reduce the protoporphyrin radical cation in the peroxidase site of the enzyme, thus preventing the intra-molecular electron transfer that generates the Tyr385 radical required for abstraction of a hydrogen from arachidonic acid to initiate prostaglandin synthesis. Unrelated to this pharmacological action, metabolism of ApAP by CYPs yields an iminoquinone electrophile that is responsible for the hepatotoxicity, which results from high doses of the drug. We synthesized novel heterocyclic phenols predicted to be electron donors. Two of these inhibited the oxygenation of arachidonic acid by PGHS-1 and myoglobin and also were shown to be more metabolically stable and exhibited less direct cytotoxicity than acetaminophen. They are leading candidates for studies to determine whether they are free of the metabolism-based hepatotoxicity produced by acetaminophen.

In Vivo and In Vitro Antıneoplastic Actions of Caffeic Acid Phenethyl Ester (CAPE): Therapeutic Perspectives

Cancer prevention and treatment strategies have attracted increasing interest. Caffeic acid phenethyl ester (CAPE), an active component of propolis extract, specifically inhibits NF-κB at μM concentrations and shows ability to stop 5-lipoxygenase-catalyzed oxygenation of linoleic acid and arachidonic acid. Previous studies have demonstrated that CAPE exhibits antioxidant, antiinflammatory, antiproliferative, cytostatic, antiviral, antibacterial, antifungal, and, most improtantly, antineoplastic properties. The primary goal of the present review is to summarize and critically evaluate the current knowledge regarding the anticancer effect of CAPE in different cancer types.

Deciphering the Interaction of FLAP and 5LO

Leukotrienes are pro-inflammatory lipid mediators involved in chronic inflammatory diseases like asthma and atherosclerosis. Different enzymes and proteins are involved in the leukotriene biosynthesis pathway that stems from oxygenation of arachidonic acid (AA) by 5LO (5-lipoxygenase). FLAP (5-lipoxygenase activating protein) is an integral membrane protein, belonging to the MAPEG (Membrane Associated Proteins in Eicosanoid and Glutathione Metabolism) family. Till now, there is no prominent evidence for any biochemical or enzymatic activity for FLAP, except involvement as an “activator” in leukotriene biosynthesis. The hypothesis is that, on increase in intracellular calcium concentration, 5LO moves from the cytosol to the nuclear membrane and localizes near FLAP. The AA is then transferred from the nuclear membrane to 5LO by homo-trimeric FLAP. Then 5LO converts the AA to leukotriene A4. This interaction between FLAP and 5LO is ambiguous due to intricate mechanisms which occur at the interface of the nuclear membrane and also between an enzyme and an integral membrane protein with no documented function. To understand the function and involvement of FLAP in leukotriene biosynthesis in vitro, we employ soluble phospholipid bilayers called “Nanodiscs” which mimic a membrane environment. The nanodisc will act as a stable platform for structural and functional characterization of the interaction between 5LO and FLAP. After initial studies of the Ca2+dependent recruitment of 5LO to empty nanodiscs as well as the reconstitution of FLAP into nanodiscs, the entire complex 5LO-FLAP-nanodisc can be targeted. We employ biochemical assays to characterize the interactions and transmission electron microscopy (single particle analysis) to create a 3D model of the functional complex of 5LO and FLAP. Here, we portray our outcomes from the above mentioned subprojects encompassed in understanding the interaction of 5LO and FLAP.

Method Development and Validation for Ultra-High Pressure Liquid Chromatography/Tandem Mass Spectrometry Determination of Multiple Prostanoids in Biological Samples

Following oxygenation of arachidonic acid by cyclooxygenase to form prostaglandin H2 (PGH2), a variety of prostanoids can be generated with diverse physiologic effects on pain, inflammation, allergy, cardiovascular system, cancer, etc. To facilitate the quantitative analysis of prostanoids in human serum of cell culture, an ultra-high pressure LC (UHPLC)/MS/MS method was developed and validated for the measurement of six eicosanoids belonging to the cyclooxygenase pathway: PGE2, PGD2, 8-iso-PGF2alpha, PGF2alpha, 6-keto-PGF1alpha, and thromboxane B2 (TXB2). Selectivity, matrix effects, calibration model, precision, and accuracy (intraday and interday), lower limit of quantitation (LLOQ), recovery, stability, and sample dilution were evaluated. Fast UHPLC separation was carried out in only 0.5 min with isocratic elution, and each prostanoid was measured using negative electrospray ionization MS with collision-induced dissociation and selected reaction monitoring. UHPLC/MS/MS provided high throughput with peak widths of approximately 3 s and an LLOQ of 0.020 ng/mL for PGE2, 0.027 ng/mL for PGD2, 0.152 ng/mL for 8-iso-PGF2alpha, 0.179 ng/mL for PGF2alpha and 6-keto-PGF1alpha, and 0.013 ng/mL for TXB2.

Biosynthesis, isolation, and NMR analysis of leukotriene A epoxides: substrate chirality as a determinant of the cis or trans epoxide configuration

Leukotriene (LT)A₄ and closely related allylic epoxides are pivotal intermediates in lipoxygenase (LOX) pathways to bioactive lipid mediators that include the leukotrienes, lipoxins, eoxins, resolvins, and protectins. Although the structure and stereochemistry of the 5-LOX product LTA₄ is established through comparison to synthetic standards, this is the exception, and none of these highly unstable epoxides has been analyzed in detail from enzymatic synthesis. Understanding of the mechanistic basis of the cis or trans epoxide configuration is also limited. To address these issues, we developed methods involving biphasic reaction conditions for the LOX-catalyzed synthesis of LTA epoxides in quantities sufficient for NMR analysis. As proof of concept, human 15-LOX-1 was shown to convert 15S-hydroperoxy-eicosatetraenoic acid (15S-HPETE) to the LTA analog 14S,15S-trans-epoxy-eicosa-5Z,8Z,10E,12E-tetraenoate, confirming the proposed structure of eoxin A₄. Using this methodology we then showed that recombinant Arabidopsis AtLOX1, an arachidonate 5-LOX, converts 5S-HPETE to the trans epoxide LTA₄ and converts 5R-HPETE to the cis epoxide 5-epi-LTA₄, establishing substrate chirality as a determinant of the cis or trans epoxide configuration. The results are reconciled with a mechanism based on a dual role of the LOX nonheme iron in LTA epoxide biosynthesis, providing a rational basis for understanding the stereochemistry of LTA epoxide intermediates in LOX-catalyzed transformations.

Prostaglandin H Synthase-2-catalyzed Oxygenation of 2-Arachidonoylglycerol Is More Sensitive to Peroxide Tone than Oxygenation of Arachidonic Acid

The endocannabinoid, 2-arachidonoylglycerol (2-AG), is a selective substrate for the inducible isoform of prostaglandin H synthase (PGHS), PGHS-2. Its turnover leads to the formation of glyceryl esters of prostaglandins (PG-Gs), a subset of which elicits agonism at unique, as yet unidentified, receptors. The k(cat)/K(m) values for oxygenation of arachidonic acid (AA) and 2-AG by PGHS-2 are very similar, but the sensitivities of the two substrates to peroxide-dependent activation have not been compared. 15-Hydroperoxy derivatives of AA and 2-AG were found to be comparable in their ability to serve as substrates for the peroxidase activities of PGHS-2, PGHS-1, and glutathione peroxidase (GPx). They also were comparable in the activation of AA oxygenation by cyanide-inhibited PGHS-2. However, oxygenation of 2-AG was significantly suppressed relative to AA by the presence of GPx and GSH. Furthermore, 2-AG oxygenation by peroxidase-deficient H388YmPGHS-2 was much less efficient than AA oxygenation. Wild-type rates of 2-AG oxygenation were restored by treatment of H388YmPGHS-2 with hydroperoxide derivatives of AA or 2-AG. RNAi silencing of phospholipid hydroperoxide-specific GPx (GPx4) in NIH/3T3 cells led to increases in cellular peroxidation and in the levels of the isoprostane product, 8-epi-PGF(2α). GPx4 silencing led to 2-4-fold increases in PG-G formation but no change in PG formation. Thus, cellular peroxide tone may be an important determinant of the extent of endocannabinoid oxygenation by PGHS-2.

Substrate-Selective Inhibition of Cyclooxygenase-2: Development and Evaluation of Achiral Profen Probes

Cyclooxygenase-2 (COX-2) oxygenates arachidonic acid and the endocannabinoids 2-arachidonoylglycerol (2-AG) and arachidonoylethanolamide (AEA). We recently reported that (R)-profens selectively inhibit endocannabinoid oxygenation but not arachidonic acid oxygenation. In this work, we synthesized achiral derivatives of five profen scaffolds and evaluated them for substrate-selective inhibition using in vitro and cellular assays. The size of the substituents dictated the inhibitory strength of the analogs, with smaller substituents enabling greater potency but less selectivity. Inhibitors based on the flurbiprofen scaffold possessed the greatest potency and selectivity, with desmethylflurbiprofen (3a) exhibiting an IC50 of 0.11 μM for inhibition of 2-AG oxygenation. The crystal structure of desmethylflurbiprofen complexed to mCOX-2 demonstrated a similar binding mode to other profens. Desmethylflurbiprofen exhibited a half-life in mice comparable to that of ibuprofen. The data presented suggest that achiral profens can act as lead molecules toward in vivo probes of substrate-selective COX-2 inhibition.