Substrate Arachidonic Acid Research Articles

Anti-Inflammatory, Anti-Oxidant, GC-MS Profiling and Molecular Docking Analyses of Non-Polar Extracts from Five Salsola Species

Genus Salsola (family Amaranthaceae) is one of the most prevailing genera in Saudi Arabia. Although several species were reported for their traditional uses, the majority of Salsola species still need to be phytochemically and biologically explored. The current study presents the GC-MS profiling as well as an in vitro investigation of the bioactivities of the n-hexane extracts from the five Salsola species: Salsola arabica, S. cyclophylla, S. imbricata, S. incanescens and S. villosa. Additionally, the compounds identified in the most active extracts were screened for their interaction with the active sites of cyclooxygenase enzyme isoforms (COX-1 and COX-2). GC-MS analysis of the n-hexane extracts from the five species resulted in the identification of 67 constituents. Oleic acid (75.57%), 1-octadecene (14.46%), cinnamaldehyde α-hexyl (57.15%), octacosyl heptafluorobutyrate (25.36%) and hexadecanoic acid methyl ester (26.15%) represent the major constituents in S. arabica, S. cyclophylla, S. imbricata, S. inscanescence and S. villosa, respectively. Results of bioactivity testing highlighted S. villosa as having the highest anti-oxidant activity (IC50 0.99 ± 0.05 mg/mL), which was closely followed by S. cyclophylla (IC50 1.36 ± 0.06 mg/mL) compared to the IC50 of 0.16 ± 0.01 mg/mL recorded by ascorbic acid. S. villosa was further noted for having the strongest COX-2 inhibitory activity (IC50 4.6 ± 0.13 µg/mL) among the tested extracts followed by S. arabica (IC50 13.1 ± 0.37 µg/mL) and S. cyclophylla (IC50 20.1 ± 0.57 µg/mL). On the other hand, S. imbricata extract displayed the most characteristic inhibition activity against COX-1 (IC50 10.2 ± 0.52 µg/mL), which was non-significant from the standard drug celecoxib. Based upon bioactivity results, the phytoconstituents identified in S. villosa and S. imbricata extracts were investigated for their capability to interact with the active sites of both cyclooxygenase enzyme isoforms adopting molecular docking. Results indicated the possibility to incorporate the compounds to active sites of the enzymes where some of them bind with their polar end into the cavity beyond Arg120 and their aliphatic chain oriented to the catalytically important Tyr385 similar to the natural substrate arachidonic acid, indicating that they could be promising candidates for the future development of selective COX inhibitors.

Highly potent and selective 5-lipoxygenase inhibition by new, simple heteroaryl-substituted catechols for treatment of inflammation

5-Lipoxygenase (LO) catalyzes the first steps in the formation of pro-inflammatory leukotrienes (LT) that are pivotal lipid mediators contributing to allergic reactions and inflammatory disorders. Based on its key role in LT biosynthesis, 5-LO is an attractive drug target, demanding for effective and selective inhibitors with efficacy in vivo, which however, are still rare. Encouraged by the recent identification of the catechol 4-(3,4-dihydroxyphenyl)dibenzofuran 1 as 5-LO inhibitor, simple structural modifications were made to yield even more effective and selective catechol derivatives. Within this new series, the two most potent compounds 3,4-dihydroxy-3′-phenoxybiphenyl (6b) and 2-(3,4-dihydroxyphenyl)benzo[b]thiophene (6d) potently inhibited human 5-LO in cell-free (IC506b and 6d = 20 nM) and cell-based assays (IC506b = 70 nM, 6d = 60 nM). Inhibition of 5-LO was reversible, unaffected by exogenously added substrate arachidonic acid, and not primarily mediated via radical scavenging and antioxidant activities. Functional 5-LO mutants expressed in HEK293 cells were still prone to inhibition by 6b and 6d, and docking simulations revealed distinct binding of the catechol moiety to 5-LO at an allosteric site. Analysis of 5-LO nuclear membrane translocation and intracellular Ca2+ mobilization revealed that these 5-LO-activating events are hardly affected by the catechols. Importantly, the high inhibitory potency of 6b and 6d was confirmed in human blood and in a murine zymosan-induced peritonitis model in vivo. Our results enclose these novel catechol derivatives as highly potent, novel type inhibitors of 5-LO with high selectivity and with marked effectiveness under pathophysiological conditions.

Effects of sub-chronic, in vivo administration of sigma-1 receptor ligands on platelet and aortic arachidonate cascade in streptozotocin-induced diabetic rats.

Diabetes mellitus is a chronic metabolic disorder which induces endothelial dysfunction and platelet activation. Eicosanoids produced from arachidonic acid regulate cellular and vascular functions. Sigma-1 receptors (S1R) are expressed in platelets and endothelial cells and S1R expression is protective in diabetes. Our aim was to examine the influence of sub-chronic, in vivo administered S1R ligands PRE-084, (S)-L1 (a new compound) and NE-100 on the ex vivo arachidonic acid metabolism of platelets and aorta in streptozotocin-induced diabetic rats. The serum level of the S1R ligands was detected by LC-MS/MS before the ex vivo analysis. Sigma-1 receptor and cyclooxygenase gene expression in platelets were determined by RT-qPCR. The eicosanoid synthesis was examined with a radiolabelled arachidonic acid substrate and ELISA. One month after the onset of STZ-induced diabetes, in vehicle-treated, diabetic rat platelet TxB2 and aortic 6-k-PGF1α production dropped. Sub-chronic in vivo treatment of STZ-induced diabetes in rats for one week with PRE-084 enhanced vasoconstrictor and platelet aggregator and reduced vasodilator and anti-aggregator cyclooxygenase product formation. (S)-L1 reduced the synthesis of vasodilator and anti-aggregator cyclooxygenase metabolites and promoted the recovery of physiological platelet function in diabetic rats. The S1R antagonist NE-100 produced no significant changes in platelet arachidonic acid metabolism. (S)-L1 decreased the synthesis of vasoconstrictor and platelet aggregator cyclooxygenase metabolites, whereas NE-100 increased the quantity of aortic vasodilator and anti-aggregator cyclooxygenase products and promoted the recovery of diabetic endothelial dysfunction in the aorta. The novel S1R ligand, (S)-L1 had similar effects on eicosanoid synthesis in platelets as the agonist PRE-084 and in aortas as the antagonist NE-100. S1R ligands regulate cellular functions and local blood circulation by influencing arachidonic acid metabolism. In diabetes mellitus, the cell-specific effects of S1R ligands have a compensatory role and aid in restoring physiological balance between the platelet and vessel.

Effects of sub-chronic, in vivo administration of sigma non-opioid intracellular receptor 1 ligands on platelet and aortic arachidonate cascade in rats

Platelets regulate cell-cell interactions and local circulation through eicosanoids from arachidonic acid. Sigma non-opioid intracellular receptor 1 (sigma-1 receptor) expressed in platelets and endothelial cells can regulate intracellular signalization. Our aim was to examine the influence of sub-chronic, in vivo-administered sigma-1 receptor ligands 2-morpholin-4-ylethyl 1-phenylcyclohexane-1-carboxylate (PRE-084); N-benzyl-2-[(1S)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinolin-1-yl]ethan-1-amine; dihydrochloride, a new compound ((S)-L1); and N-[2-[4-methoxy-3-(2-phenylethoxy)phenyl]ethyl]-N-propylpropan-1-amine (NE-100) on the ex vivo arachidonic acid metabolism of the platelets and aorta of male rats. The serum level of sigma-1 receptor ligands was determined by liquid chromatography-mass spectrometry. Sigma-1 receptor and cyclooxygenase gene expression in the platelets were determined by a reverse transcription-coupled quantitative polymerase chain reaction. The eicosanoid synthesis was examined using a radiolabeled arachidonic acid substrate and enzyme-linked immunosorbent assay. We confirmed the absorption of sigma-1 receptor ligands and confirmed that the ligands were not present during the ex vivo studies, so their acute effect could be excluded. We detected no changes in either sigma-1 receptor or cyclooxygenase mRNA levels in the platelets. Nevertheless, (S)-L1 and NE-100 increased the quantity of cyclooxygenases there. Both platelet and aortic eicosanoid synthesis was modified by the ligands, although in different ways. The effect of the new sigma-1 receptor ligand, (S)-L1, was similar to that of PRE-084 in most of the parameters studied but was found to be more potent. Our results suggest that sigma-1 receptor ligands may act at multiple points in arachidonic acid metabolism and play an important role in the control of the microcirculation by modulating the eicosanoid synthesis of the platelets and vessels.

Synthesis, Biological Evaluation, and Molecular Docking Study of 3-Amino and 3-Hydroxy-seco A Derivatives of α-Amyrin and 3-Epilupeol as Inhibitors of COX-2 Activity and NF-kB Activation.

In this study, a series of novel 3-seco-A derivatives of the natural triterpenes α-amyrin (1) and 3-epilupeol (2) were synthesized by a one-pot radical scission-oxidation procedure and evaluated in vitro and in vivo for their capacity to inhibit the inflammatory process. For the in vitro studies, the trans-4-hydroxy-l-proline methyl ester derivatives (1f and 2f) were consistently effective in inhibiting NO, IL-6, and TNF-α secretion, as well as inhibition of NF-κB activation, in RAW cells stimulated by LPS. The further in vivo anti-inflammatory study revealed that the trans-4-hydroxy-l-proline methyl ester derivatives (1f and 2f), together with 1g, were the most effective in inhibiting TPA-induced edema. Interestingly, the α-amyrin derivatives were the most potent inhibitors of COX-2, but inhibited COX-1 only to some extent. The hydroxyl derivative (1c) was selective for COX-2 inhibition (66.3 ± 1.1% at 17.5 μM) without affecting the COX-1 isoform and did not present toxicity. Molecular docking studies revealed that these compounds bind with their polar region in the cavity over Arg-120, and their lipophilic part is orientated to the HEM cofactor similarly to the natural substrate arachidonic acid in the catalytic site of COX-2. These results indicated that seco-A ursane derivatives could be considered promising candidates for the future development of selective NF-κB and COX-2 inhibitors.

Nanocluster-assisted protein-film voltammetry for direct electrochemical signal acquisition.

Acquisition of the direct electrochemical response of protein is the cornerstone for the development of the third generation of electrochemical biosensors. In this work, we developed a nanocluster-assisted protein-film voltammetry technique (NCA-PFV) which can achieve the acquisition of the electrochemical signal and maintain the activity without affecting of the protein's structure. With this strategy, a lipid bilayer membrane is used to immobilize the membrane protein so as to maintain its natural state. Copper nanoclusters with a size smaller than most proteins are then used to function at sub-protein scale and to mediate the electron hopping from the electroactive center of the electrode. As a model, the direct electrochemical signal of cyclooxygenase (COX) is successfully obtained, with a pair of well-defined redox peaks located at -0.39mV and -0.31mV, which characterize the heme center of the enzyme. Its catalytic activity towards the substrate arachidonic acid (AA) is also retained. The detection range for AA is 10-1000μM and the detection limit is 2.4μM. Electrochemical monitoring of the regulation of the catalytic activity by an inhibitor DuP-697 is also achieved. This work provides a powerful tool for the fabrication of enzyme-based electrochemical biosensors, and is also of great significance for promoting the development and application of next-generation electrochemical biosensors.

Microglia depletion diminishes key elements of the leukotriene pathway in the brain of Alzheimer\u2019s Disease mice

Leukotrienes (LTs) contribute to the neuropathology of chronic neurodegenerative disorders including Alzheimer’s Disease (AD), where they mediate neuroinflammation and neuronal cell-death. In consequence, blocking the action of Leukotrienes (LTs) ameliorates pathologies and improves cognitive function in animal models of neurodegeneration. Surprisingly, the source of Leukotrienes (LTs) in the brain is largely unknown. Here, we identified the Leukotriene (LT) synthesis rate-limiting enzyme 5-Lipoxygenase (5-Lox) primarily in neurons and to a lesser extent in a subpopulation of microglia in human Alzheimer´s Disease (AD) hippocampus brain sections and in brains of APP Swedish PS1 dE9 (APP-PS1) mice, a transgenic model for Alzheimer´s Disease (AD) pathology. The 5-Lipoxygenase (5-Lox) activating protein (FLAP), which anchors 5-Lipoxygenase (5-Lox) to the membrane and mediates the contact to the substrate arachidonic acid, was confined exclusively to microglia with the entire microglia population expressing 5-Lipoxygenase activating protein (FLAP). To define the contribution of microglia in the Leukotriene (LT) biosynthesis pathway, we ablated microglia using the colony stimulating factor 1 receptor (CSF1R) inhibitor PLX5622 in wildtype (WT) and APP-PS1 mice. Microglia ablation not only diminished the expression of FLAP and of the Leukotriene (LT) receptor Cysteinylleukotriene receptor 1 (CysLTR1), as expected based on their microglia cell type-specific expression, but also drastically reduced 5-Lipoxygenase (5-Lox) mRNA expression in the brain and its protein expression in neurons, in particular in wildtype (WT) mice. In conclusion i) microglia are key in Leukotriene (LT) biosynthesis, and ii) they regulate neuronal 5-Lipoxygenase (5-Lox) expression implying a yet unknown signaling mechanism between neurons and microglia.

Elucidating the Open and Active Conformation of Human 5‐Lipoxygenase

Human 5‐Lipoxygenase (5‐LOX) is an enzyme that responds to the call for inflammation by initiating the synthesis of leukotrienes involved in asthmatic and allergic reactions and acts to sustain inflammatory reactions. Acting at the nuclear membrane in leukocytes, 5‐LOX oxygenates its lipid substrate arachidonic acid to leukotriene A4. The structure of 5‐LOX was solved in a closed conformation with helix‐α2 shortened and two large hydrophobic residues from the helix corking the active site. Most other animal lipoxygenase structures were solved with an elongated helix‐α2 allowing for an open conformation of the active site. Our goal is to study the molecular determinants important for the transition from a closed to open structure. We used site‐directed mutagenesis of helix‐α2 residues to favor either a more opened or closed conformation. We also used inhibitors to probe the conformational flexibility of 5‐LOX by testing susceptibility to cleavage by limited proteolysis and changes in melting temperature by differential scanning fluorimetry. So far, we have observed that by mutating key residues to encourage a closed conformation, 5‐LOX was less susceptible to proteolysis and melted at a higher temperature. We also showed that by mutating the same residues to encourage an open conformation, 5‐LOX was more susceptible to proteolysis. When bound to a competitive inhibitor nordihydroguaiaretic acid (NDGA), 5‐LOX melts at significantly lower temperatures and appears more susceptible to proteolytic cleavage. However, mutations that stabilize the closed conformation reduce these NDGA‐induced effects. By enhancing the presence of a closed or open conformation by mutagenesis, our results continue to hint at the structural significance of the elongated helix‐α2 in 5‐LOX dynamics. Determination of a structure of 5‐LOX in an open conformation will enable understanding of substrate acquisition and inhibitor binding. An open conformation of 5‐LOX could aid in structure‐based drug design and would foster the development of new therapies for inflammatory disorders.

Cyclooxygenase activity in bradykinin-induced dermal extravasation. A study in mice and humans

BackgroundNon-allergic angioedema is largely driven by increased plasma levels of bradykinin and over-activation of bradykinin receptor type II (B2), but the specific downstream signalling pathways remain unclear. The aim of this study was to identify signal transduction events involved in bradykinin-induced dermal extravasation. MethodsQuantification of dermal extravasation was accomplished following intradermal (i.d.) injection of bradykinin or the B2 agonist labradimil in mice with endothelial NO-synthase (eNOS) deficiency and in C57BL/6J mice pre-treated with vehicle, NO-synthase or cyclooxygenase (COX) inhibitors. In the multicentre clinical study ABRASE, 38 healthy volunteers received i.d. bradykinin injections into the ventral forearm before and after oral treatment with the COX inhibitor ibuprofen (600 mg). The primary endpoint of ABRASE was the mean time to complete resolution of wheals (TTCR) and the secondary endpoint was the change of maximal wheal size. ResultsNeither NOS inhibitors nor eNOS deficiency altered bradykinin-induced extravasation. In striking contrast, the COX inhibitors ibuprofen, diclofenac, SC560 and celecoxib significantly diminished this extravasation when given before injection. As for diclofenac, a similar but significantly lower effect was observed when given after i.d. injection of bradykinin. Similar results were obtained when bradykinin was replaced by labradimil. In volunteers, ibuprofen significantly reduced TTCR (P < 0.001) and maximal wheal size (P = 0.0044). ConclusionThese data suggest that COX activity contributes to bradykinin-induced dermal extravasation in mice and humans. In addition, our findings may open new treatment options and point to a potential activity of drugs interfering with the release of the COX substrate arachidonic acid, e.g. glucocorticoids.

Modification of the lead molecule: Tryptophan and piperidine appended triazines reversing inflammation and hyeperalgesia in rats

The structural optimization of the molecules making them to fit into the active site pocket of COX-2 occupying the same space as covered by the natural substrate arachidonic acid helped in the emergence of compound 10 as an efficacious anti-inflammatory agent. Selective for COX-2 over COX-1, compound 10 exhibited IC50 0.02 µM for COX-2 and reversed acetic acid induced inflammation in rats by 73% when used at 10 mg kg−1 dose and the same dose of the compound also rescued the animals from inflammatory phase of formalin induced hyperalgesia. As evidenced by the results of molecular modeling studies supported by the nuclear Overhauser enhancement data, the appropriate geometry of the molecule in the active site pocket of COX-2 contributing to its H-bond/hydrophobic interactions with Ser530, Trp387 and Tyr385 seems responsible for the enzyme inhibitory activity of the compound.

Entamoeba histolytica Cyclooxygenase-Like Protein Regulates Cysteine Protease Expression and Virulence.

The intestinal protozoan parasite Entamoeba histolytica (Eh) causes amebiasis associated with severe diarrhea and/or liver abscess. Eh pathogenesis is multifactorial requiring both parasite virulent molecules and host-induced innate immune responses. Eh-induced host pro-inflammatory responses plays a critical role in disease pathogenesis by causing damage to tissues allowing parasites access to systemic sites. Eh cyclooxygenase (EhCox) derived prostaglandin E2 stimulates the chemokine IL-8 from mucosal epithelial cells that recruits neutrophils to the site of infection to exacerbate disease. At present, it is not known how EhCox is regulated or whether it affects the expression of other proteins in Eh. In this study, we found that gene silencing of EhCox (EhCoxgs) markedly increased endogenous cysteine protease (CP) protein expression and virulence without altering CP gene transcripts. Live virulent Eh pretreated with arachidonic acid substrate to enhance PGE2 production or aspirin to inhibit EhCox enzyme activity or addition of exogenous PGE2 to Eh had no effect on EhCP activity. Increased CP enzyme activity in EhCoxgs was stable and significantly enhanced erythrophagocytosis, cytopathic effects on colonic epithelial cells and elicited pro-inflammatory cytokines in mice colonic loops. Acute infection with EhCoxgs in colonic loops increased inflammation associated with high levels of myeloperoxidase activity. This study has identified EhCox protein as one of the important endogenous regulators of cysteine protease activity. Alterations of CP activity in response to Cox gene silencing may be a negative feedback mechanism in Eh to limit proteolytic activity during colonization that can inadvertently trigger inflammation in the gut.

Residual cyclooxygenase activity of aspirin-acetylated COX-2 forms 15 R-prostaglandins that inhibit platelet aggregation.

Aspirin (acetylsalicylic acid) inhibits prostaglandin (PG) synthesis by transfer of its acetyl group to a serine residue in the cyclooxygenase (COX) active site. Acetylation of Ser530 inhibits catalysis by preventing access of arachidonic acid substrate in the COX-1 isoenzyme. Acetylated COX-2, in contrast, gains a new catalytic activity and forms 15 R hydroxy-eicosatetraenoic acid (15 R-HETE) as alternate product. Here we show that acetylated COX-2 also retains COX activity, forming predominantly 15 R-configuration PGs (70 or 62% 15 R, respectively, determined using radiolabeled substrate or LC-MS analysis). Although the Km of arachidonic acid for acetylated COX-2 was ∼3-fold lower than for uninhibited COX-2, the catalytic efficiency for PG formation by the acetylated enzyme was reduced 10-fold due to a concomitant decrease in Vmax. Aspirin increased 15 R-PGD2 but not 15 R-PGE2 in isolated human leukocytes activated with LPS to induce COX-2. 15 R-PGD2 inhibited human platelet aggregation induced by the thromboxane receptor agonist U46,619, and this effect was abrogated by an antagonist of the DP1 prostanoid receptor. We conclude that acetylation of Ser530 in COX-2 not only triggers formation of 15 R-HETE but also allows oxygenation and cyclization of arachidonic acid to a 15 R-PG endoperoxide. 15 R-PGs are novel products of aspirin therapy via acetylation of COX-2 and may contribute to its antiplatelet and other pharmacologic effects.-Giménez-Bastida, J. A., Boeglin, W. E., Boutaud, O., Malkowski, M. G., Schneider, C. Residual cyclooxygenase activity of aspirin-acetylated COX-2 forms 15 R-prostaglandins that inhibit platelet aggregation.

Simulation Study of Interactions Between Two Bioactive Components from Zingiber cassumunar and 5-Lipoxygenase.

Compound D and DMPBD are compounds extracted from Plai or Zingiber cassumunar Roxb., which have antiasthmatic properties. Thai herbal pharmacopoeia have indicated that approximate 50% of Thai prescriptions for asthma contain Plai. However, the inhibition mechanisms of these compounds are not clearly known. In this study, molecular docking and molecular dynamics (MD) simulations have been used to simulate complex systems and analyze molecular interactions between these compounds and protein target, 5-lipoxygenase (5-LO) enzyme, which is an enzyme involved with asthma symptoms. From our MD simulations, Compound D and DMPBD molecules bind at the same binding site of its natural substrate (arachidonic acid) on 5-LO enzyme, which is similar to the binding of commercial asthma drug (Zileuton). Molecular mechanics generalized born surface area binding energy calculations of the 5-LO complex with Compound D and DMPBD are -26.83 and -29.15kcal/mol, respectively. This work indicated that Compound D and DMPBD are competitive inhibitors, which are able to bind at the same 5-LO substrate binding site. This reveals opportunities for using Compound D and DMPBD as novel antiasthmatic drugs.

Expression, Purification, and Biochemical Characterization of the Flavocytochrome P450 CYP505A30 from Myceliophthora thermophila.

The cytochrome P450/P450 reductase fusion enzyme CYP505A30 from the thermophilic fungus Myceliophthora thermophila and its heme (P450) domain were expressed in Escherichia coli and purified using affinity, ion exchange, and size exclusion chromatography. CYP505A30 binds straight chain fatty acids (from ∼C10 to C20), with highest affinity for tridecanoic acid (KD = 2.7 μM). Reduced nicotinamide adenine dinucleotide phosphate is the preferred reductant for CYP505A30 (KM = 3.1 μM compared to 330 μM for reduced nicotinamide adenine dinucleotide in cytochrome c reduction). Electron paramagnetic resonance confirmed cysteine thiolate coordination of heme iron in CYP505A30 and its heme domain. Redox potentiometry revealed an unusually positive midpoint potential for reduction of the flavin adenine dinucleotide and flavin mononucleotide cofactors (E0′ ∼ −118 mV), and a large increase in the CYP505A30 heme domain FeIII/FeII redox couple (ca. 230 mV) on binding arachidonic acid substrate. This switch brings the ferric heme iron potential into the same range as that of the reductase flavins. Multiangle laser light scattering analysis revealed CYP505A30’s ability to dimerize, whereas the heme domain is monomeric. These data suggest CYP505A30 may function catalytically as a dimer (as described for Bacillus megaterium P450 BM3), and that binding interactions between CYP505A30 heme domains are not required for dimer formation. CYP505A30 catalyzed hydroxylation of straight chain fatty acids at the ω-1 to ω-3 positions, with a strong preference for ω-1 over ω-3 hydroxylation in the oxidation of dodecanoic and tetradecanoic acids (88 vs 2% products and 63 vs 9% products, respectively). CYP505A30 has important structural and catalytic similarities to P450 BM3 but distinct regioselectivity of lipid substrate oxidation with potential biotechnological applications.

Aspirin as a COX inhibitor and anti-inflammatory drug in human skeletal muscle.

Although aspirin is one of the most common anti-inflammatory drugs in the world, the effect of aspirin on human skeletal muscle inflammation is almost completely unknown. This study examined the potential effects and related time course of an orally consumed aspirin dose on the inflammatory prostaglandin E2 (PGE2)/cyclooxygenase (COX) pathway in human skeletal muscle. Skeletal muscle biopsies were taken from the vastus lateralis of 10 healthy adults (5 male and 5 female, 25 ± 2 yr old) before (Pre) and 2, 4, and 24 h after (Post) a standard dose (975mg) of aspirin and partitioned for analysis of 1) in vivo PGE2 levels in resting skeletal muscle and 2) ex vivo skeletal muscle PGE2 production when stimulated with the COX substrate arachidonic acid (5 μM). PGE2 levels in vivo and PGE2 production ex vivo were generally unchanged at each time point after aspirin consumption. However, most individuals clearly showed suppression of PGE2, but at varying time points after aspirin consumption. When the maximum suppression after aspirin consumption was examined for each individual, independent of time, PGE2 levels in vivo (184 ± 17 and 104 ± 23pg/g wet wt at Pre and Post, respectively) and PGE2 production ex vivo (2.74 ± 0.17 and 2.09 ± 0.11pg·mg wet wt-1·min-1 at Pre and Post, respectively) were reduced ( P < 0.05) by 44% and 24%, respectively. These results provide evidence that orally consumed aspirin can inhibit the COX pathway and reduce the inflammatory mediator PGE2 in human skeletal muscle. Findings from this study highlight the need to expand our knowledge regarding the potential role for aspirin regulation of the deleterious influence of inflammation on skeletal muscle health in aging and exercising individuals. NEW & NOTEWORTHY This study demonstrated that orally consumed aspirin can target the prostaglandin/cyclooxygenase pathway in human skeletal muscle. This pathway has been shown to regulate skeletal muscle metabolism and inflammation in aging and exercising individuals. Given the prevalence of aspirin consumption, these findings may have implications for skeletal muscle health in a large segment of the population.

Membrane-dependent Activities of Human 15-LOX-2 and Its Murine Counterpart: IMPLICATIONS FOR MURINE MODELS OF ATHEROSCLEROSIS

The enzyme encoded by the ALOX15B gene has been linked to the development of atherosclerotic plaques in humans and in a mouse model of hypercholesterolemia. In vitro, these enzymes, which share 78% sequence identity, generate distinct products from their substrate arachidonic acid: the human enzyme, a 15-S-hydroperoxy product; and the murine enzyme, an 8-S-product. We probed the activities of these enzymes with nanodiscs as membrane mimics to determine whether they can access substrate esterified in a bilayer and characterized their activities at the membrane interface. We observed that both enzymes transform phospholipid-esterified arachidonic acid to a 15-S-product. Moreover, when expressed in transfected HEK cells, both enzymes result in significant increases in the amounts of 15-hydroxyderivatives of eicosanoids detected. In addition, we show that 15-LOX-2 is distributed at the plasma membrane when the HEK293 cells are stimulated by the addition Ca(2+) ionophore and that cellular localization is dependent upon the presence of a putative membrane insertion loop. We also report that sequence differences between the human and mouse enzymes in this loop appear to confer distinct mechanisms of enzyme-membrane interaction for the homologues.

Substrate-selective Inhibition of Cyclooxygeanse-2 by Fenamic Acid Derivatives Is Dependent on Peroxide Tone

Cyclooxygenase-2 (COX-2) catalyzes the oxygenation of arachidonic acid (AA) and endocannabinoid substrates, placing the enzyme at a unique junction between the eicosanoid and endocannabinoid signaling pathways. COX-2 is a sequence homodimer, but the enzyme displays half-of-site reactivity, such that only one monomer of the dimer is active at a given time. Certain rapid reversible, competitive nonsteroidal anti-inflammatory drugs (NSAIDs) have been shown to inhibit COX-2 in a substrate-selective manner, with the binding of inhibitor to a single monomer sufficient to inhibit the oxygenation of endocannabinoids but not arachidonic acid. The underlying mechanism responsible for substrate-selective inhibition has remained elusive. We utilized structural and biophysical methods to evaluate flufenamic acid, meclofenamic acid, mefenamic acid, and tolfenamic acid for their ability to act as substrate-selective inhibitors. Crystal structures of each drug in complex with human COX-2 revealed that the inhibitor binds within the cyclooxygenase channel in an inverted orientation, with the carboxylate group interacting with Tyr-385 and Ser-530 at the top of the channel. Tryptophan fluorescence quenching, continuous-wave electron spin resonance, and UV-visible spectroscopy demonstrate that flufenamic acid, mefenamic acid, and tolfenamic acid are substrate-selective inhibitors that bind rapidly to COX-2, quench tyrosyl radicals, and reduce higher oxidation states of the heme moiety. Substrate-selective inhibition was attenuated by the addition of the lipid peroxide 15-hydroperoxyeicosatertaenoic acid. Collectively, these studies implicate peroxide tone as an important mechanistic component of substrate-selective inhibition by flufenamic acid, mefenamic acid, and tolfenamic acid.

Understanding the Mechanism of the Hydrogen Abstraction from Arachidonic Acid Catalyzed by the Human Enzyme 15-Lipoxygenase-2. A Quantum Mechanics/Molecular Mechanics Free Energy Simulation.

Lipoxygenases (LOXs) are a family of enzymes involved in the biosynthesis of several lipid mediators. In the case of human 15-LOX, the 15-LOX-1 and 15-LOX-2 isoforms show slightly different reaction regiospecificity and substrate specificity, indicating that substrate binding and recognition may be different, a fact that could be related to their different biological role. Here, we have used long molecular dynamics simulations, QM(DFT)/MM potential energy and free energy calculations (using the newly developed DHAM method), to investigate the binding mode of the arachidonic acid (AA) substrate into 15-LOX-2 and the rate-limiting hydrogen-abstraction reaction 15-LOX-2 catalyzes. Our results strongly indicate that hydrogen abstraction from C13 in 15-LOX-2 is only consistent with the "tail-first" orientation of AA, with its carboxylate group interacting with Arg429, and that only the pro-S H13 hydrogen will be abstracted (being the pro-R H13 and H10 too far from the acceptor oxygen atom). At the B3LYP/6-31G(d) level the potential and free energy barriers for the pro-S H13 abstraction of AA by 15-LOX-2 are 18.0 and 18.6 kcal/mol, respectively. To analyze the kinetics of the hydrogen abstraction process, we determined a Markov model corresponding to the unbiased simulations along the state-discretized reaction coordinate. The calculated rates based on the second largest eigenvalue of the Markov matrices agree well with experimental measurements, and also provide the means to directly determine the pre-exponential factor for the reaction by comparing with the free energy barrier height. Our calculated pre-exponential factor is close to the value of kBT/h. On the other hand, our results suggest that the spin inversion of the complete system (including the O2 molecule) that is required to happen at some point along the full process to lead to the final hydroperoxide product, is likely to take place during the hydrogen transfer, which is a proton coupled electron transfer. Overall, a different binding mode from the one accepted for 15-LOX-1 is proposed, which provides a molecular basis for 15-LOX-2 exclusive 15-HPETE production in front of the double (although highly 15-) 12/15 regiospecificity of 15-LOX-1. Understanding how these different isoenzymes achieve their regiospecificity is expected to help in specific inhibitor design.

The Interaction Between Lipoxygenase-Catalyzed Oxidation and Autoxidation in Dry-Cured Bacon and a Model System.

A model system was conducted to characterize the interaction between lipid autoxidation and enzyme-catalyzed oxidation in dry-cured bacon. This involved the use of a hydroxyl radical (HO•) generating system and the extraction and purification of lipoxygenases (LOX) from pork belly. The results showed that LOX activity rapidly (P < 0.05) increased during the curing of dry-cured bacon. This may be because of the hydroxyl-radical-mediated oxidation from LOX-Fe(2+) to LOX-Fe(3+), which activates LOX. In addition, experiments of the model system also showed that LOX activity could be inhibited by increasing the substrate concentration, although substrate type and concentration had no effect on autoxidation. Moreover, LOX enzyme-catalyzed oxidation and autoxidation could act synergistically to promote lipid oxidation irrespective of the substrate (linoleic or arachidonic acid). These results provide useful information for regulating lipid oxidation during the production of dry-cured pork products.

Characterization of the interaction of human 5-lipoxygenase with its activating protein FLAP

Human 5-lipoxygenase (5-LO) is the key enzyme in the formation of leukotrienes (LTs), important mediators of inflammation. Cellular 5-LO activity is regulated in a complex manner, e.g. by calcium influx, the cellular redox status or 5-LO phosphorylation. Being a mobile enzyme, 5-LO migrates from the cytosol to the nuclear envelope where it is believed to interact with 5-lipoxygenase-activating protein (FLAP) and receives the substrate arachidonic acid (AA). 5-LO contains four cysteine residues located close to the AA entry site. In the present study, we show that in vitro glutathionylation of recombinant purified 5-LO wildtype (WT) as well as 5-LO 4C, a mutant where the four surface cysteines are replaced by serines (Cys159/300/416/418Ser), does not alter the product synthesis. However, in 5-LO/FLAP-transfected HeLa cells, treatment with the thiol-oxidizing agent diamide which promotes glutathionylation at surface Cys residues led to a decreased LT synthesis by 5-LO WT. In contrast to the WT enzyme, LT formation of the 4C mutant was stimulated by addition of diamide. Immunofluorescence studies in human monocytes and HEK293 cells, expressing 5-LO and FLAP, revealed that diamide prevented the translocation of 5-LO WT whereas it enhanced the translocation of the fourfold cysteine mutant. Therefore, we could demonstrate that the interface, involving the four cysteines 159, 300, 416 and 418, is important for the translocation to the nuclear membrane and the colocalization with FLAP.