Carol A. Rouzer

Cyclooxygenases: structural and functional insights

Cyclooxygenase (COX; prostaglandin G/H synthase, EC 1.14.99.1) catalyzes the first two steps in the biosynthesis of prostaglandins (PGs). The two COX isoforms COX-1 and COX-2 are the targets of the widely used nonsteroidal anti-inflammatory drugs, indicating a role for these enzymes in pain, fever, inflammation, and tumorigenesis. The ubiquitous constitutive expression of COX-1 and inducible expression of COX-2 have led to the widely held belief that COX-1 produces homeostatic PGs, while PGs produced by COX-2 are primarily pathophysiological. However, recent discoveries call this paradigm into question and reveal as yet underappreciated functions for both enzymes. This review focuses on some of these new insights.

Endocannabinoid oxygenation by cyclooxygenases, lipoxygenases, and cytochromes P450: cross-talk between the eicosanoid and endocannabinoid signaling pathways.

The discovery of the cannabinoid receptors CB1 and CB2 in 19881,2 and 1990,(3) respectively, and of the endogenous cannabinoid ligands (endocannabinoids) arachidonoylethanolamide (AEA) and 2-arachidonoylglycerol (2-AG) in 1992(4) and 1995,(5) respectively, represented major strides in the understanding of cannabinoid physiology and pharmacology. The realization that both endocannabinoids are derivatives of arachidonic acid (AA) also revealed a potential interrelationship between the endocannabinoid and eicosanoid signaling systems that is just beginning to be unraveled. In this review, we explore what is known about the interplay between the two lipid signaling networks and discuss the challenges and opportunities offered by this new field of inquiry. 1.1. Eicosanoid Biosynthetic Pathways AA is an ω-6 tetraunsaturated fatty acid that is a component of mammalian cell membrane phospholipids, where it is predominantly esterified at the sn-2 position. AA’s role in eicosanoid signaling was first discovered in 1964, when Van Dorp et al. and Bergstrom et al. showed that incubation of the radiolabeled fatty acid with bull seminal vesicles led to the formation of prostaglandin E2 (PGE2).6,7 Since that time, we have come to appreciate that a wide range of stimuli (depending on cell type, tissue context, and physiologic state) can trigger the activation of cytosolic phospholipase A2 (cPLA2) and/or other phospholipases, leading to the release of free AA from phospholipid pools. The free fatty acid is then subject to oxidative metabolism by cyclooxygenase 1 and/or 2 (COX-1 and/or COX-2), leading to the formation of the endoperoxide PGH2. Tissue-specific metabolism of PGH2 by a group of PG synthases yields the biologically active PGs (PGE2, PGD2, PGF2α), prostacyclin (PGI2), and thromboxane A2 (TxA2) (Figure ​(Figure11a).8,9 Alternatively, free AA may be metabolized by one of a variety of lipoxygenases (LOXs) that catalyze regio- and stereospecific oxygenation, yielding hydro-peroxyeicosatetraenoic acids (HPETEs). These compounds are enzymatically or chemically reduced to the corresponding hydroxy-eicosatetraenoic acids (HETEs) or undergo further metabolism. Multiple lipoxygenations produce the lipoxins,(10) or in the case of 5-HPETE, epoxidation followed by hydrolysis or glutathione adduction yields the leukotrienes (LTs)9,11 (Figure ​(Figure1b).1b). Finally, free AA may be oxidized at each of its double bonds or at the ω-terminus by cytochromes P450, leading to the epoxyeicosatrienoic acids (EETs) or HETEs(12) (Figure ​(Figure1c).1c). Members of each of these classes of compounds possess a unique range of biological activities. Figure 1 (a) Cyclooxygenase pathway of AA metabolism. AA is converted to PGG2 at the cyclooxygenase active site of COX-1 or COX-2 and is then reduced to PGH2 at the peroxidase active site. PGH2 spontaneously decomposes to yield PGE2 or PGD2, but these compounds, ...

Differential Sensitivity and Mechanism of Inhibition of COX-2 Oxygenation of Arachidonic Acid and 2-Arachidonoylglycerol by Ibuprofen and Mefenamic Acid

Ibuprofen and mefenamic acid are weak, competitive inhibitors of cyclooxygenase-2 (COX-2) oxygenation of arachidonic acid (AA) but potent, noncompetitive inhibitors of 2-arachidonoylglycerol (2-AG) oxygenation. The slow, tight-binding inhibitor, indomethacin, is a potent inhibitor of 2-AG and AA oxygenation whereas the rapidly reversible inhibitor, 2′-des-methylindomethacin, is a potent inhibitor of 2-AG oxygenation but a poor inhibitor of AA oxygenation. These observations are consistent with a model in which inhibitors bind in one subunit of COX-2 and inhibit 2-AG binding in the other subunit of the homodimeric protein. In contrast, ibuprofen and mefenamate must bind in both subunits to inhibit AA binding.

Non-redundant Functions of Cyclooxygenases: Oxygenation of Endocannabinoids

The two cyclooxygenase (COX) enzymes catalyze the oxygenation of arachidonic acid to prostaglandin endoperoxides, which are the common intermediates in the biosynthesis of the bioactive lipids prostaglandins and thromboxane. COX-1 and COX-2 are ∼60% identical in amino acid sequence, exhibit highly homologous three-dimensional structures, and appear functionally similar at the biochemical level. Recent work has uncovered a subtle functional difference between the two enzymes, namely the ability of COX-2 to efficiently utilize neutral derivatives (esters and amides) of arachidonic acid as substrates. Foremost among these neutral substrates are the endocannabinoids 2-arachidonoylglycerol and arachidonoylethanolamide. This raises the possibility that COX-2 oxygenation plays a role in a novel signaling pathway dependent on agonist-induced release of endocannabinoids and their selective oxygenation by COX-2. Among the products of COX-2 oxygenation of endocannabinoids are glyceryl prostaglandins, some of which (e.g. glyceryl prostaglandin E2 and glyceryl prostaglandin I2) exhibit interesting biological activities in inflammatory, neurological, and vascular systems. These compounds are produced in intact cells stimulated with physiological agonists and have been isolated from in vivo sources. Important concepts relevant to the hypothesis of a COX-2-selective signaling pathway are presented.

Chemical stability of 2-arachidonylglycerol under biological conditions.

Recent evidence indicates that 2-arachidonylglycerol (2-AG) is a potent and specific ligand for the central and peripheral cannabinoid receptors. Therefore, the chemical stability of this molecule under biological conditions is of interest. A method for the isolation and detection of 2-AG using HPLC with evaporative light scattering detection is described. The method provides an extraction recovery from aqueous media of 78%, and a limit of detection of 60 ng on column. Incubation of 2-AG in culture medium or biological buffers indicated that it is stable to oxidation and ester hydrolysis for up to 6 h at 37 degrees C. However, gradual disappearance of the compound was noted due to adherence to glass and plastic surfaces. During incubation in RPMI culture medium, 2-AG rearranged to 1(3)-arachidonylglycerol (1(3)-AG) in a first order process with a half-life of 10 min in the absence of serum and 2.3 min in the presence of 10% fetal calf serum. Further studies indicated that the acyl migration reaction is base catalyzed (k(cat)=78,000/min M), and that the reaction is affected slightly by changes in buffer (Tris) concentration and not at all by changes in ionic strength. The results indicate that 2-AG is readily converted to 1(3)-AG under conditions commonly used to study receptor-ligand interactions, findings that have significant implications for the interpretation of relative ligand potency between the two isomers.

Targeted Cyclooxygenase Gene (Ptgs) Exchange Reveals Discriminant Isoform Functionality

The prostaglandin G/H synthase enzymes, commonly termed COX-1 and COX-2, differ markedly in their responses to regulatory stimuli and their tissue expression patterns. COX-1 is the dominant source of “housekeeping” prostaglandins, whereas COX-2 synthesizes prostaglandins of relevance to pain, inflammation, and mitogenesis. Despite these distinctions, the two enzymes are remarkably conserved, and their subcellular distributions overlap considerably. To address the functional interchangeability of the two isozymes, mice in which COX-1 is expressed under COX-2 regulatory elements were created by a gene targeting “knock-in” strategy. In macrophages from these mice, COX-1 was shown to be lipopolysaccharide-inducible in a manner analogous to COX-2 in wild-type macrophages. However, COX-1 failed to substitute effectively for COX-2 in lipopolysaccharide-induced prostaglandin E2 synthesis at low concentrations of substrate and in the metabolism of the endocannabinoid 2-arachidonylglycerol. The marked depression of the major urinary metabolite of prostacyclin in COX-2 null mice was only partially rescued by COX-1 knock-in, whereas the main urinary metabolite of prostaglandin E2 was rescued totally. Replacement with COX-1 partially rescued the impact of COX-2 deletion on reproductive function. The renal pathology consequent to COX-2 deletion was delayed but not prevented, whereas the corresponding peritonitis was unaltered. Insertion of COX-1 under the regulatory sequences that drive COX-2 expression indicated that COX-1 can substitute for some COX-2 actions and rescue only some of the consequences of gene disruption. Manipulation of COX-2 also revealed a preference for coupling with distinct downstream prostaglandin synthases in vivo. These mice will provide a valuable reagent with which to elucidate the distinct roles of the COX enzymes in mammalian biology.

Capture and release of alkyne-derivatized glycerophospholipids using cobalt chemistry

Alkyne modified phospholipids can be unambiguously identified and differentiated from native species in complex mixtures by formation of dicobalthexacarbonyl complexes. This reaction is specific for alkynes and is unaffected by other glycerophospholipid related moieties. Enrichment of cells with alkyne-derivatized fatty acids or glycerophospholipids followed by solid phase sequestration and release is a promising new method for unequivocally monitoring individual glycerophospholipids following incorporation and facilitates lipidomic analysis of substrates and products.

The 2'-Trifluoromethyl Analogue of Indomethacin Is a Potent and Selective COX-2 Inhibitor.

Indomethacin is a potent, time-dependent, nonselective inhibitor of the cyclooxygenase enzymes (COX-1 and COX-2). Deletion of the 2′-methyl group of indomethacin produces a weak, reversible COX inhibitor, leading us to explore functionality at that position. Here, we report that substitution of the 2′-methyl group of indomethacin with trifluoromethyl produces CF3–indomethacin, a tight-binding inhibitor with kinetic properties similar to those of indomethacin and unexpected COX-2 selectivity (IC50 mCOX-2 = 267 nM; IC50 oCOX-1 > 100 μM). Studies with site-directed mutants reveal that COX-2 selectivity results from insertion of the CF3 group into a small hydrophobic pocket formed by Ala-527, Val-349, Ser-530, and Leu-531 and projection of the methoxy group toward a side pocket bordered by Val-523. CF3–indomethacin inhibited COX-2 activity in human head and neck squamous cell carcinoma cells and exhibited in vivo anti-inflammatory activity in the carrageenan-induced rat paw edema model with similar potency to that of indomethacin.

Zymosan-induced glycerylprostaglandin and prostaglandin synthesis in resident peritoneal macrophages: roles of cyclo-oxygenase-1 and -2

COX [cyclo-oxygenase; PG (prostaglandin) G/H synthase] oxygenates AA (arachidonic acid) and 2-AG (2-arachidonylglycerol) to endoperoxides that are converted into PGs and PG-Gs (glycerylprostaglandins) respectively. In vitro, 2-AG is a selective substrate for COX-2, but in zymosan-stimulated peritoneal macrophages, PG-G synthesis is not sensitive to selective COX-2 inhibition. This suggests that COX-1 oxygenates 2-AG, so studies were carried out to identify enzymes involved in zymosan-dependent PG-G and PG synthesis. When macrophages from COX-1-/- or COX-2-/- mice were treated with zymosan, 20-25% and 10-15% of the PG and PG-G synthesis observed in wild-type cells respectively was COX-2 dependent. When exogenous AA and 2-AG were supplied to COX-2-/- macrophages, PG and PG-G synthesis was reduced as compared with wild-type cells. In contrast, when exogenous substrates were provided to COX-1-/- macrophages, PG-G but not PG synthesis was reduced. Product synthesis also was evaluated in macrophages from cPLA(2alpha) (cytosolic phospholipase A2alpha)-/- mice, in which zymosan-induced PG synthesis was markedly reduced, and PG-G synthesis was increased approx. 2-fold. These studies confirm that peritoneal macrophages synthesize PG-Gs in response to zymosan, but that this process is primarily COX-1-dependent, as is the synthesis of PGs. They also indicate that the 2-AG and AA used for PG-G and PG synthesis respectively are derived from independent pathways.

Oxicams, a Class of Nonsteroidal Anti-inflammatory Drugs and Beyond

Oxicams are a class of nonsteroidal anti‐inflammatory drugs (NSAIDs) structurally related to the enolic acid class of 4‐hydroxy‐1,2‐benzothiazine carboxamides. They are used clinically to treat both acute and chronic inflammation by inhibiting the activity of the two cyclooxygenase (COX) isoforms, COX‐1 and COX‐2. Oxicams are structurally distinct from all other NSAIDs, exhibiting a novel binding pose in the COX active site. The 4‐hydroxyl group on the thiazine ring partners with Ser‐530 via hydrogen bonding while two coordinated water molecules mediate a polar interaction between the oxicam and COX. The rotation of Leu‐531 in the complex opens a new pocket, which is not used for binding other NSAIDs to the enzyme. This structure provides the basis for understanding documented structure–activity relationships within the oxicam class. In addition, from the oxicam template, a series of potent microsomal prostaglandin E synthase‐1 (mPGES‐1) inhibitors represents a new direction for drug development. Here, we review the major route of oxicam synthesis and structure–activity for COX inhibition, as well as recent advances in oxicam‐mediated mPGES‐1 inhibition.