Oxygenation Of Arachidonic Acid Research Articles

Low back pain: justification of treatment standards (literature review)

Low back pain: justification of treatment standards (literature review)

Modulation of the 5-Lipoxygenase Pathway by Chalcogen-Containing Inhibitors of Leukotriene A4 Hydrolase.

The 5-lipoxygenase (5-LOX) pathway gives rise to bioactive inflammatory lipid mediators, such as leukotrienes (LTs). 5-LOX carries out the oxygenation of arachidonic acid to the 5-hydroperoxy derivative and then to the leukotriene A4 epoxide which is converted to a chemotactic leukotriene B4 (LTB4) by leukotriene A4 hydrolase (LTA4H). In addition, LTA4H possesses aminopeptidase activity to cleave the N-terminal proline of a pro-inflammatory tripeptide, prolyl-glycyl-proline (PGP). Based on the structural characteristics of LTA4H, it is possible to selectively inhibit the epoxide hydrolase activity while sparing the inactivating, peptidolytic, cleavage of PGP. In the current study, chalcogen-containing compounds, 4-(4-benzylphenyl) thiazol-2-amine (ARM1) and its selenazole (TTSe) and oxazole (TTO) derivatives were characterized regarding their inhibitory and binding properties. All three compounds selectively inhibit the epoxide hydrolase activity of LTA4H at low micromolar concentrations, while sparing the aminopeptidase activity. These inhibitors also block the 5-LOX activity in leukocytes and have distinct inhibition constants with recombinant 5-LOX. Furthermore, high-resolution structures of LTA4H with inhibitors were determined and potential binding sites to 5-LOX were proposed. In conclusion, we present chalcogen-containing inhibitors which differentially target essential steps in the biosynthetic route for LTB4 and can potentially be used as modulators of inflammatory response by the 5-LOX pathway.

The 5-lipoxygenase/cyclooxygenase-2 cross-over metabolite, hemiketal E2, enhances VEGFR2 activation and promotes angiogenesis

Consecutive oxygenation of arachidonic acid by 5-lipoxygenase and cyclooxygenase-2 yields the hemiketal eicosanoids, HKE2 and HKD2. Hemiketals stimulate angiogenesis by inducing endothelial cell tubulogenesis in culture; however, how this process is regulated has not been determined. Here, we identify vascular endothelial growth factor receptor 2 (VEGFR2) as a mediator of HKE2-induced angiogenesis invitro and invivo. We found that HKE2 treatment of human umbilical vein endothelial cells dose-dependently increased the phosphorylation of VEGFR2 and the downstream kinases ERK and Akt that mediated endothelial cell tubulogenesis. Invivo, HKE2 induced the growth of blood vessels into polyacetal sponges implanted in mice. HKE2-mediated effects invitro and invivo were blocked by the VEGFR2 inhibitor vatalanib, indicating that the pro-angiogenic effect of HKE2 was mediated by VEGFR2. HKE2 covalently bound and inhibited PTP1B, a protein tyrosine phosphatase that dephosphorylates VEGFR2, thereby providing a possible molecular mechanism for how HKE2 induced pro-angiogenic signaling. In summary, our studies indicate that biosynthetic cross-over of the 5-lipoxygenase and cyclooxygenase-2 pathways gives rise to a potent lipid autacoid that regulates endothelial cell function invitro and invivo. These findings suggest that common drugs targeting the arachidonic acid pathway could prove useful in antiangiogenic therapy.

N-Substituted 5-(1H-Indol-2-yl)-2-methoxyanilines Are Allosteric Inhibitors of the Linoleate Oxygenase Activity of Selected Mammalian ALOX15 Orthologs: Mechanism of Action.

Here, we describe the first systematic study on the mechanism of substrate-selective inhibition of mammalian ALOX15 orthologs. For this purpose, we prepared a series of N-substituted 5-(1H-indol-2-yl)anilines and found that (N-(5-(1H-indol-2-yl)-2-methoxyphenyl)sulfamoyl)carbamates and their monofluorinated analogues are potent and selective inhibitors of the linoleate oxygenase activity of rabbit and human ALOX15. Introduction of a 2-methoxyaniline moiety into the core pharmacophore plays a crucial role in substrate-selective inhibition of ALOX15-catalyzed oxygenation of linoleic acid at submicromolar concentrations without affecting arachidonic acid oxygenation. Steady-state kinetics, mutagenesis studies, and molecular dynamics (MD) simulations suggested an allosteric mechanism of action. Using a dimer model of ALOX15, our MD simulations suggest that the binding of the inhibitor at the active site of one monomer induces conformational alterations in the other monomer so that the formation of a productive enzyme-linoleic acid complex is energetically compromised.

Oxygenation of endocannabinoids by mammalian lipoxygenase isoforms

Endocannabinoids, such as anandamide (ANA) and 2-arachidonoylglycerol (2AG), are lipid-signaling molecules that can be oxidized by lipid-peroxidizing enzymes, and this oxidation alters the bioactivity of these lipid mediators. Here, under strictly comparable experimental conditions, we explored whether ANA and 2AG function as substrates for four human (ALOX15, ALOX15B, ALOX12, ALOX5) and three mice Alox isoforms (Alox15, Alox12, Alox5) and compared the rates of product formation with those of arachidonic acid oxygenation. Except for ALOX5, the two endocannabinoids were more efficiently oxygenated than arachidonic acid by human ALOX isoforms. Mice Alox15 oxygenated ANA more efficiently than arachidonic acid, but the other mice Alox isoforms exhibited reduced reaction rates for endocannabinoid conversion. Like its human ortholog, mice Alox5 did not oxygenate ANA, but the formation of 5-HETE-containing 2AG derivatives was observed for this enzyme. 1AG and 2AG were similarly effective substrates for human ALOX isoforms. Molecular docking studies, the pattern of oxygenation products, and site-directed mutagenesis experiments suggested a similar substrate alignment of arachidonic acid and endocannabinoids at the active site of ALOX15 orthologs. The product specificity of arachidonic acid oxygenation was conserved for endocannabinoid metabolization, and the triad concept describing the molecular basis for the reaction specificity of ALOX15 orthologs is applicable for endocannabinoid oxygenation. Taken together, these data indicate that, except for ALOX5 orthologs, endocannabinoids are suitable substrates for most mammalian ALOX isoforms.

Human lipoxygenase isoforms form complex patterns of double and triple oxygenated compounds from eicosapentaenoic acid

Lipoxygenases (ALOX) are lipid peroxidizing enzymes that catalyze the biosynthesis of pro- and anti-inflammatory lipid mediators and have been implicated in (patho-)physiological processes. In humans, six functional ALOX isoforms exist and their arachidonic acid oxygenation products have been characterized. Products include leukotrienes and lipoxins which are involved in the regulation of inflammation and resolution. Oxygenation of n3-polyunsaturated fatty acids gives rise to specialized pro-resolving mediators, e.g. resolvins. However, the catalytic activity of different ALOX isoforms can lead to a multitude of potentially bioactive products. Here, we characterized the patterns of oxygenation products formed by human recombinant ALOX5, ALOX15, ALOX15B and ALOX12 from eicosapentaenoic acid (EPA) and its 18-hydroxy derivative 18-HEPE with particular emphasis on double and triple oxygenation products. ALOX15 and ALOX5 formed a complex mixture of various double oxygenation products from EPA, which include 5,15-diHEPE and various 8,15-diHEPE isomers. Their biosynthetic mechanisms were explored using heavy oxygen isotopes (H218O, 18O2 gas) and three catalytic activities contributed to product formation: i) fatty acid oxygenase activity, ii) leukotriene synthase activity, iii) lipohydroperoxidase activity. For ALOX15B and ALOX12 more specific product patterns were identified, which was also the case when these enzymes reacted in concert with ALOX5. Several double oxygenated compounds were formed from 18-HEPE by ALOX5, ALOX15B and ALOX12 including previously identified resolvins (RvE2, RvE3), while formation of triple oxygenation products, e.g. 5,17,18-triHEPE, required ALOX5. Taken together our data show that EPA can be converted by human ALOX isoforms to a large number of secondary oxygenation products, which might exhibit bioactivity.

Harmaline Analogs as Substrate-Selective Cyclooxygenase-2 Inhibitors.

We report the design, synthesis, and evaluation of a series of harmaline analogs as selective inhibitors of 2-arachidonylglycerol (2-AG) oxygenation over arachidonic acid (AA) oxygenation by purified cyclooxygenase-2 (COX-2). A fused tricyclic harmaline analog containing a CH3O substituent at C-6 and a CH3 group at the C-1 position of 4,9-dihydro-3H-pyrido[3,4-b]indole (compound 3) was the best substrate-selective COX-2 inhibitor of those evaluated, exhibiting a 2AG-selective COX-2 inhibitory IC50 of 0.022 μM as compared to >1 μM for AA. The 2.66 Å resolution crystal complex of COX-2 with compound 3 revealed that this series of tricyclic indoles binds in the cyclooxygenase channel by flipping the side chain of L531 toward the dimer interface. This novel tricyclic indole series provides the foundation for the development of promising substrate-selective molecules capable of increasing endocannabinoid (EC) levels in the brain to offer new treatments for a variety of diseases, from pain and inflammation to stress and anxiety disorders.

Flipped regiospecificity in L434F mutant of 8-lipoxygenase.

Lipoxygenases are non-heme iron containing enzymes that catalyze oxygenation of poly-unsaturated fatty acids in different animal and plant species with extremely high regio- and stereospecificity. Nature employs 8-lipoxygenase to produce 8R-hydroperoxide from the oxygenation of arachidonic acid. A single-point L434F mutation of 8-lipoxygenase alters the regio- and stereospecificity of the final products, with a product ratio of 66 : 34 for 8R- and 12S-hydroperoxide, respectively. A molecular level explanation of this flipped regiospecificity is presented in this work on the basis of molecular dynamics simulations and transition network analysis of oxygen migration in the protein matrix. Phe434 is shown to exist in two conformations, the so-called open and closed conformations. In the closed conformation, the phenyl group of Phe434 shields the C8 site of the substrate, thereby preventing access of the oxygen molecule to this site, which leads to a quenching of the 8R-product. On the other hand, both closed and open conformations of Phe434 allow the oxygen molecule to approach the pro-S face of the C12 site of the substrate, which enhances the propensity of the 12S-hydroperoxide.

Prostaglandins as the Agents That Modulate the Course of Brain Disorders.

Neurologic and neuropsychiatric diseases are associated with great morbidity and mortality. Prostaglandins (PGs) are formed by sequential oxygenation of arachidonic acid in physiologic and pathologic conditions. For the production of PGs cyclooxygenase is a necessary enzyme that has two isoforms, that are named COX-1 and COX-2. COX-1 produces type 1 prostaglandins and on the other hand, COX-2 produces type 2 prostaglandins. Recent studies suggest PGs abnormalities are present in a variety of neurologic and psychiatric disorders. In a disease state, type 2 prostaglandins are mostly responsible and type 1 PGs are not so important in the disease state. In this review, the importance of prostaglandins especially type 2 in brain diseases has been discussed and their possible role in the initiation and outcome of brain diseases has been assessed. Overall the studies suggest prostaglandins are the agents that modulate the course of brain diseases in a positive or negative manner. Here in this review article, the various aspects of PGs in the disease state have discussed. It appears more studies must be done to understand the exact role of these agents in the pathophysiology of brain diseases. However, the suppression of prostaglandin production may confer the alleviation of some brain diseases.

Atopic Patients Show Increased Interleukin 4 Plasma Levels but the Degree of Elevation Is Not Sufficient to Upregulate Interleukin-4-Sensitive Genes

Background: Atopic diseases constitute a major health challenge for industrialized countries, and elevated levels of interleukin 4 (IL-4) frequently characterize these disorders. Previous in vitroanalyses have indicated that IL-4 strongly upregulates the expression of IL-4-sensitive genes in human monocytes. Objective: To explore whether similar expression alterations may contribute to the pathomechanisms of atopic diseases in vivo we carried out a small-scale case-control clinical study (n = 43), in which we quantified the plasma levels of IgE and IL-4 as well as the expression of selected IL-4-sensitive genes in blood leukocytes. Methods: 34 allergic patients suffering from allergic rhinitis (n = 11), atopic eczema (n = 11) and allergic asthma (n = 12) as well as 9 healthy control individuals were recruited. IgE and IL-4 plasma levels were determined by ELISA, and the expression of selected IL-4-sensitive gene products in blood leukocytes was quantified by qRT-PCR. In addition, the fatty acid oxygenase activity of isolated monocytes was measured by RP-HPLC analysis of the arachidonic acid oxygenation products (ex vivo activity assays). Results: We found that plasma levels of IgE and IL-4 were significantly elevated in atopic patients but the degree of elevation was not sufficient to upregulate the expression of the selected IL-4-sensitive genes in circulating leukocytes. Moreover, the arachidonic acid oxygenase activity of blood monocytes was not significantly altered in atopic patients. Conclusion: Our data suggest that the IL-4 plasma levels of atopic patients are not high enough to impact the expression of IL-4-sensitive genes.

Lysophospholipid acyltransferases and leukotriene biosynthesis: intersection of the Lands cycle and the arachidonate PI cycle

Leukotrienes (LTs) are autacoids derived from the precursor arachidonic acid (AA) via the action of five-lipoxygenase (5-LO). When inflammatory cells are activated, 5-LO translocates to the nuclear membrane to initiate oxygenation of AA released by cytosolic phospholipase A2 (cPLA2) into leukotriene A4 (LTA4). LTA4 can also be exported from an activated donor cell into an acceptor cell by the process of transcellular biosynthesis. When thimerosal is added to cells, the level of free AA increases by inhibition of lysophospholipid acyltransferases of the Lands pathway of phospholipid remodeling. Another arachidonate phospholipid cycle involves phosphatidylinositol (PI) in the plasma membrane that undoubtedly intersects with the Lands pathway of phospholipid remodeling. The highest abundance of PI occurs between the ER and the plasma membrane and is probably a result of the importance of the PI signaling cascade in cellular biochemistry. Because transport proteins mediate the rapid intracellular movement of phospholipids, largely as result of physical membrane contact, 5-LO-dependent production of LTA4 could be mediated by the disappearance of free AA from the nuclear membrane, transfer to the ER for Lands cycle reesterification into PI, and population of PI(18:0/20:4) for cell membrane signaling.

5 S,15 S-Dihydroperoxyeicosatetraenoic Acid (5,15-diHpETE) as a Lipoxin Intermediate: Reactivity and Kinetics with Human Leukocyte 5-Lipoxygenase, Platelet 12-Lipoxygenase, and Reticulocyte 15-Lipoxygenase-1.

The reaction of 5 S,15 S-dihydroperoxyeicosatetraenoic acid (5,15-diHpETE) with human 5-lipoxygenase (LOX), human platelet 12-LOX, and human reticulocyte 15-LOX-1 was investigated to determine the reactivity and relative rates of producing lipoxins (LXs). 5-LOX does not react with 5,15-diHpETE, although it can produce LXA4 when 15-HpETE is the substrate. In contrast, both 12-LOX and 15-LOX-1 react with 5,15-diHpETE, forming specifically LXB4. For 12-LOX and 5,15-diHpETE, the kinetic parameters are kcat = 0.17 s-1 and kcat/ KM = 0.011 μM-1 s-1 [106- and 1600-fold lower than those for 12-LOX oxygenation of arachidonic acid (AA), respectively]. On the other hand, for 15-LOX-1 the equivalent parameters are kcat = 4.6 s-1 and kcat/ KM = 0.21 μM-1 s-1 (3-fold higher and similar to those for 12-HpETE formation by 15-LOX-1 from AA, respectively). This contrasts with the complete lack of reaction of 15-LOX-2 with 5,15-diHpETE [Green, A. R., et al. (2016) Biochemistry 55, 2832-2840]. Our data indicate that 12-LOX is markedly inferior to 15-LOX-1 in catalyzing the production of LXB4 from 5,15-diHpETE. Platelet aggregation was inhibited by the addition of 5,15-diHpETE, with an IC50 of 1.3 μM; however, LXB4 did not significantly inhibit collagen-mediated platelet activation up to 10 μM. In summary, LXB4 is the primary product of 12-LOX and 15-LOX-1 catalysis, if 5,15-diHpETE is the substrate, with 15-LOX-1 being 20-fold more efficient than 12-LOX. LXA4 is the primary product with 5-LOX but only if 15-HpETE is the substrate. Approximately equal proportions of LXA4 and LXB4 are produced by 12-LOX but only if LTA4 is the substrate, as described previously [Sheppard, K. A., et al. (1992) Biochim. Biophys. Acta 1133, 223-234].

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.

Dual cyclooxygenase–fatty acid amide hydrolase inhibitor exploits novel binding interactions in the cyclooxygenase active site

The cyclooxygenases COX-1 and COX-2 oxygenate arachidonic acid (AA) to prostaglandin H2 (PGH2). COX-2 also oxygenates the endocannabinoids 2-arachidonoylglycerol (2-AG) and arachidonoylethanolamide (AEA) to the corresponding PGH2 analogs. Both enzymes are targets of nonsteroidal anti-inflammatory drugs (NSAIDs), but NSAID-mediated COX inhibition is associated with gastrointestinal toxicity. One potential strategy to counter this toxicity is to also inhibit fatty acid amide hydrolase (FAAH), which hydrolyzes bioactive fatty acid ethanolamides (FAEs) into fatty acids and ethanolamine. Here, we investigated the mechanism of COX inhibition by ARN2508, an NSAID that inhibits both COXs and FAAH with high potency, target selectivity, and decreased gastrointestinal toxicity in mouse models, presumably due to its ability to increase levels of FAEs. A 2.27-Å-resolution X-ray crystal structure of the COX-2·(S)-ARN2508 complex reveals that ARN2508 adopts a binding pose similar to that of its parent NSAID flurbiprofen. However, ARN2508's alkyl tail is inserted deep into the top channel, an active site region not exploited by any previously reported NSAID. As for flurbiprofen, ARN2508's potency is highly dependent on the configuration of the α-methyl group. Thus, (S)-ARN2508 is more potent than (R)-ARN2508 for inhibition of AA oxygenation by both COXs and 2-AG oxygenation by COX-2. Also, similarly to (R)-flurbiprofen, (R)-ARN2508 exhibits substrate selectivity for inhibition of 2-AG oxygenation. Site-directed mutagenesis confirms the importance of insertion of the alkyl tail into the top channel for (S)-ARN2508's potency and suggests a role for Ser-530 as a determinant of the inhibitor's slow rate of inhibition compared with that of (S)-flurbiprofen.

Structural Dynamics of 15-Lipoxygenase-2 via Hydrogen-Deuterium Exchange.

Eicosanoids are inflammatory signaling lipids that are biosynthesized in response to cellular injury or threat. They were originally thought to be pro-inflammatory molecules, but members of at least one subclass, the lipoxins, are able to resolve inflammation. One step in lipoxin synthesis is the oxygenation of arachidonic acid by 15-lipoxygenase (15-LOX). 15-LOX contains two domains: a Ca2+ binding PLAT domain and a catalytic domain. 15-LOX is a soluble cytosolic protein until binding of Ca2+ to the PLAT domain promotes translocation to the membrane surface. The role of 15-LOX structural dynamics in this translocation has remained unclear. We investigated the dynamics of 15-LOX isoform B (15-LOX-2) upon binding of Ca2+ and ligands, as well as upon membrane association using hydrogen–deuterium exchange mass spectrometry (HDX-MS). We used HDX-MS to probe the solvent accessibility and backbone flexibility of 15-LOX-2, revealing significant differences in deuterium incorporation between the PLAT and catalytic domains, with the PLAT domain demonstrating higher flexibility. Comparison of HDX for 15-LOX-2 in the presence and absence of Ca2+ indicates there are few differences in structural dynamics. Furthermore, our HDX results involving nanodisc-associated 15-LOX-2 suggest that significant structural and dynamic changes in 15-LOX-2 are not required for membrane association. Our results also show that a substrate lipid binding to the active site in the catalytic domain does induce changes in incorporation of deuterium into the PLAT domain. Overall, our results challenge the previous hypothesis that Ca2+ binding induces major structural changes in the PLAT domain and support the hypothesis that is interdomain communication in 15-LOX-2.

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.

Interactions of 2-O-arachidonylglycerol ether and ibuprofen with the allosteric and catalytic subunits of human COX-2

Prostaglandin (PG) endoperoxide H synthase (PGHS)-2, also known as cyclooxygenase (COX)-2, can convert arachidonic acid (AA) to PGH2 in the committed step of PG synthesis. PGHS-2 functions as a conformational heterodimer composed of an allosteric (Eallo) and a catalytic (Ecat) monomer. Here we investigated the interplay between human (hu)PGHS-2 and an alternative COX substrate, the endocannabinoid, 2-arachidonoylglycerol (2-AG), as well as a stable analog, 2-O-arachidonylglycerol ether (2-AG ether). We also compared the inhibition of huPGHS-2-mediated oxygenation of AA, 2-AG, and 2-AG ether by the well-known COX inhibitor, ibuprofen. When tested with huPGHS-2, 2-AG and 2-AG ether exhibit very similar kinetic parameters, responses to stimulation by FAs that are not COX substrates, and modes of inhibition by ibuprofen. The 2-AG ether binds Ecat more tightly than Eallo and, thus, can be used as a stable Ecat-specific substrate to examine certain Eallo-dependent responses. Ibuprofen binding to Eallo of huPGHS-2 completely blocks 2-AG or 2-AG ether oxygenation; however, inhibition by ibuprofen of huPGHS-2-mediated oxygenation of AA engages a combination of both allosteric and competitive mechanisms.

The Structural Basis of Cyclooxygenase‐2 Inhibition by Fenamates

Two cyclooxygenase (COX) isoforms, COX‐1 and COX‐2, catalyze the first two steps of prostaglandin biosynthesis from arachidonic acid (AA) and are the pharmacological targets of non‐steroidal anti‐inflammatory drugs (NSAIDs). Mefenamic acid is one of several NSAIDs that have shown selective inhibition of 2‐arachidonylglycerol (2‐AG) oxygenation by COX‐2 (Prusakiewicz J.J. et al, Biochemistry 2009). While the X‐ray crystal structures of most classes of NSAIDs bound to COX have been solved, the binding mode of fenamates is still unknown. Here we report the structural determination of murine COX‐2 structures complexed with several fenamates and biochemical investigation of the role of individual residues in their inhibition. The crystal structures for the first time revealed that the fenamates are bound in the cyclooxygenase channel in an inverted binding mode similar to those of propionic acid derivatives such as diclofenac and lumiracoxib. The side chains of Tyr‐385 and Ser‐530 form critical H‐bonds with the carboxylic group of the fenamates. The aniline ring substituents of mefenamic acid, tolfenamic acid and flufenamic acid exhibit a preference for the space beneath Leu‐531 as opposed to the side pocket off the active site channel. In contrast, when there is an additional chloride substituent, as in the case of meclofenamic acid, this extra substituent orients toward the side pocket. These observations provide a structural basis for the fact that meclofenamic acid is a slow, tight‐binding inhibitor of COX‐2 with a low IC50 (31 nM) whereas mefenamic acid, flufenamic acid, niflumic acid, and tolfenamic acid are weak, rapidly reversible inhibitors with IC50's greater than 10 μM for AA oxygenation. Strikingly, when 2‐AG is used as the substrate, these weak, rapidly reversible inhibitors become potent inhibitors with IC50's ranging from 20 to 50 nM. In contrast, meclofenamic acid exhibits the same potency for both substrates. Our results provide the first atomic level detail of the interaction between fenamates and COX‐2, structural insights of slow, irreversible and rapid reversible fenamates, and a biochemical and structural foundation for future design and development of substrate‐selective inhibitors of COX‐2.Support or Funding InformationThe work was supported by NIH CA089450 (to L. J. M.).

Different Fatty Acids Compete with Arachidonic Acid for Binding to the Allosteric or Catalytic Subunits of Cyclooxygenases to Regulate Prostanoid Synthesis

Prostaglandin endoperoxide H synthases (PGHSs), also called cyclooxygenases (COXs), convert arachidonic acid (AA) to PGH2. PGHS-1 and PGHS-2 are conformational heterodimers, each composed of an (Eallo) and a catalytic (Ecat) monomer. Previous studies suggested that the binding to Eallo of saturated or monounsaturated fatty acids (FAs) that are not COX substrates differentially regulate PGHS-1 versus PGHS-2. Here, we substantiate and expand this concept to include polyunsaturated FAs known to modulate COX activities. Non-substrate FAs like palmitic acid bind Eallo of PGHSs stimulating human (hu) PGHS-2 but inhibiting huPGHS-1. We find the maximal effects of non-substrate FAs on both huPGHSs occurring at the same physiologically relevant FA/AA ratio of ∼20. This inverse allosteric regulation likely underlies the ability of PGHS-2 to operate at low AA concentrations, when PGHS-1 is effectively latent. Unlike FAs tested previously, we observe that C-22 FAs, including ω-3 fish oil FAs, have higher affinities for Ecat than Eallo subunits of PGHSs. Curiously, C-20 ω-3 eicosapentaenoate preferentially binds Ecat of huPGHS-1 but Eallo of huPGHS-2. PGE2 production decreases 50% when fish oil consumption produces tissue EPA/AA ratios of ≥0.2. However, 50% inhibition of huPGHS-1 itself is only seen with ω-3 FA/AA ratios of ≥5.0. This suggests that fish oil-enriched diets disfavor AA oxygenation by altering the composition of the FA pool in which PGHS-1 functions. The distinctive binding specificities of PGHS subunits permit different combinations of non-esterified FAs, which can be manipulated dietarily, to regulate AA binding to Eallo and/or Ecat thereby controlling COX activities.

Eicosanoid Mediators in the Airway Inflammation of Asthmatic Patients: What is New?

Lipid mediators contribute to inflammation providing both pro-inflammatory signals and terminating the inflammatory process by activation of macrophages. Among the most significant biologically lipid mediators, these are produced by free-radical or enzymatic oxygenation of arachidonic acid named "eicosanoids". There were some novel eicosanoids identified within the last decade, and many of them are measurable in clinical samples by affordable chromatography-mass spectrometry equipment or sensitive immunoassays. In this review, we present some recent advances in understanding of the signaling by eicosanoid mediators during asthmatic airway inflammation. Eicosanoid profiling in the exhaled breath condensate, induced sputum, or their metabolites measurements in urine is complementary to the cellular phenotyping of asthmatic inflammation. Special attention is paid to aspirin-exacerbated respiratory disease, a phenotype of asthma manifested by the most profound changes in the profile of eicosanoids produced. A hallmark of this type of asthma with hypersensitivity to non-steroid anti-inflammatory drugs (NSAIDs) is to increase biosynthesis of cysteinyl leukotrienes on the systemic level. It depends on transcellular biosynthesis of leukotriene C4 by platelets that adhere to granulocytes releasing leukotriene A4. However, other abnormalities are also reported in this type of asthma as a resistance to anti-inflammatory activity of prostaglandin E2 or a robust eosinophil interferon-γ response resulting in cysteinyl leukotrienes production. A novel mechanism is also discussed in which an isoprostane structurally related to prostaglandin E2 is released into exhaled breath condensate during a provoked asthmatic attack. However, it is concluded that any single eicosanoid or even their complex profile can hardly provide a thorough explanation for the mechanism of asthmatic inflammation.