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Comprehensive studies of prostaglandin (PG) synthesis in murine resident peritoneal macrophages (RPM) responding to bacterial lipopolysaccharide (LPS) revealed that the primary PGs produced by RPM were prostacyclin and PGE(2). Detectable increases in net PG formation occurred within the first hour, and maximal PG formation had occurred by 6-10 h after LPS addition. Free arachidonic acid levels rose and peaked at 1-2 h after LPS addition and then returned to baseline. Cyclooxygenase-2 (COX-2) and microsomal PGE synthase levels markedly increased upon exposure of RPM to LPS, with the most rapid increases in protein expression occurring 2-6 h after addition of the stimulus. RPM constitutively expressed high levels of COX-1. Studies using isoform-selective inhibitors and RPM from mice bearing targeted deletions of ptgs-1 and ptgs-2 demonstrated that COX-1 contributes significantly to PG synthesis in RPM, especially during the initial 1-2 h after LPS addition. Selective inhibition of either COX isoform resulted in increased secretion of tumor necrosis factor-alpha (TNF-alpha); however, this effect was much greater with the COX-1 than with the COX-2 inhibitor. These results demonstrate autocrine regulation of TNF-alpha secretion by endogenous PGs synthesized primarily by COX-1 in RPM and suggest that COX-1 may play a significant role in the regulation of the early response to endotoxemia.

The metabolism of 2-[1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl]-N-phenethyl-acetamide (indomethacin phenethylamide, LM-4108), a highly selective cyclooxygenase-2 inhibitor, was studied in rat, mouse, and human liver microsomes. The primary site of oxidation in all species examined was on the methylene carbons of the phenethyl side chain to form the 1'- and 2'-hydroxy and 2'-oxo metabolites as determined by electrospray ionization liquid chromatography-tandem mass spectrometry. Half-lives for the disappearance of 10 microM LM-4108 in rat, human, and mouse liver microsomes (0.15 pmol P450/ml) were 11 min, 21 min, and 51 min, respectively. Indomethacin formation was not observed in incubations with rat, mouse, or human liver microsomes. Both the 2'-hydroxy-LM-4108 and 2'-oxo-LM-4108 metabolites were synthesized and found to be equipotent to the parent compound with regard to COX-2 inhibitory potency and selectivity [2'-hydroxy-LM-4108: IC(50)(COX-2) = 0.06 microM, IC(50)(COX-1) >66 microM; 2'-oxo-LM-4108: IC(50)(COX-2) = 0.05 microM, IC(50)(COX-1) >66 microM]. The formation of the metabolites was strongly inhibited by specific CYP3A4 inhibitors ketoconazole and troleandomycin but not by other isoform-selective inhibitors. These findings were confirmed by demonstrating that cloned, expressed CYP3A4 catalyzed side chain oxidation. O-Demethylation was a minor oxidative pathway in contrast to the metabolism of indomethacin and was catalyzed by CYP2D6. Upon intravenous administration of LM-4108 to Sprague-Dawley rats, oxidative metabolism on the phenethyl side chain constituted the rate-limiting steps in its clearance. The active metabolites, 2'-oxo- and 2'-hydroxy-LM-4108, as well as 1'-hydroxy-LM-4108, were all observed in rat plasma and thus may contribute to COX-2 inhibition in vivo. The glucuronides of 2'hydroxy-LM-4108 and O-desmethyl-2'-hydroxy-LM-4108 were also identified in rat bile.

The last decade has witnessed a rapid expansion in our understanding of the mammalian endogenous cannabinoid system. In just a few short years since the discovery of endogenous lipids that serve as cannabinoids in vivo, these molecules have been shown to participate in a broad array of physiological and pathological processes. Consequently, attention has been directed at defining the proteins responsible for endocannabinoid synthesis, transport, and metabolism. Recently, multiple fatty acid oxygenases including, most notably, cyclooxygenase-2 (COX-2), have been implicated in endocannabinoid metabolism. This review will highlight connections between COX-2 and the endogenous cannabinoid system. The available biochemical evidence supporting a role for COX-2 in endocannabinoid metabolism will be presented. Finally, the potential biological consequences of COX-2-mediated endocannabinoid oxygenation will be discussed.

A variety of drugs inhibit the conversion of arachidonic acid to prostaglandin G2 by the cyclooxygenase (COX) activity of prostaglandin endoperoxide synthases. Several modes of inhibitor binding in the COX active site have been described including ion pairing of carboxylic acid containing inhibitors with Arg-120 of COX-1 and COX-2 and insertion of arylsulfonamides and sulfones into the COX-2 side pocket. Recent crystallographic evidence suggests that Tyr-385 and Ser-530 chelate polar or negatively charged groups in arachidonic acid and aspirin. We tested the generality of this binding mode by analyzing the action of a series of COX inhibitors against site-directed mutants of COX-2 bearing changes in Arg-120, Tyr-355, Tyr-348, and Ser-530. Interestingly, diclofenac inhibition was unaffected by the mutation of Arg-120 to alanine but was dramatically attenuated by the S530A mutation. Determination of the crystal structure of a complex of diclofenac with murine COX-2 demonstrates that diclofenac binds to COX-2 in an inverted conformation with its carboxylate group hydrogen-bonded to Tyr-385 and Ser-530. This finding represents the first experimental demonstration that the carboxylate group of an acidic non-steroidal anti-inflammatory drug can bind to a COX enzyme in an orientation that precludes the formation of a salt bridge with Arg-120. Mutagenesis experiments suggest Ser-530 is also important in time-dependent inhibition by nimesulide and piroxicam.

The endocannabinoid arachidonylethanolamide (AEA, anandamide) is an endogenous ligand for the cannabinoid receptors and has been shown to be oxygenated by cyclooxygenase-2 (COX-2). We examined the structural requirements for COX-mediated, AEA oxygenation using a number of substrate analogues and site-directed mutants of COX-2. Fourteen AEA analogues were synthesized and tested as COX substrates. These studies identified the hydroxyl moiety of AEA as a critical determinant in the ability of COX enzymes to effect robust endocannabinoid oxygenation. In addition, these studies suggest that subtle structural modifications of AEA analogues near the ethanolamide moiety can result in pronounced changes in their ability to serve as COX-2 substrates. Site-directed mutagenesis studies have permitted the development of a model of AEA binding within the COX-2 active site. As with arachidonic acid, the omega-terminus of AEA binds in a hydrophobic alcove near the top of the COX-2 active site. The polar ethanolamide moiety of AEA, like the carboxylate of arachidonate, interacts with Arg-120 at the bottom of the COX-2 active site. Mutation of Tyr-385 prevents AEA oxygenation, suggesting that, as in the case of other COX substrates, AEA metabolism is initiated by Tyr-385-mediated hydrogen abstraction. Thus, AEA binds within the COX-2 active site in a conformation roughly similar to that of arachidonic acid. However, important differences have been identified that account for the isoform selectivity of AEA oxygenation. Importantly, the COX-2 side pocket and Arg-513 in particular are critical determinants of the ability of COX-2 to efficiently generate prostaglandin H(2) ethanolamide. The reduced efficiency of COX-1-mediated, AEA oxygenation can thus be explained by the absence of an arginine residue at position 513 in this isoform. Mutational analysis of Leu-531, an amino acid located directly across from the COX-2 side pocket, suggests that AEA is shifted away from this hydrophobic residue and toward Arg-513 relative to arachidonic acid. Coupled with earlier observations with the endocannabinoid 2-arachidonylglycerol, these results indicate that one possible function of the highly conserved COX-2 active site side pocket is to promote endocannabinoid oxygenation.

At antipyretic and analgesic doses, salicylate has no antiplatelet or anti-inflammatory effects, unlike typical inhibitors of the prostaglandin H synthases (PGHSs). We demonstrated that salicylate inhibits PGHS-1 and -2 with a potency inversely related to ambient hydroperoxide concentrations. Salicylate inhibition of PGHS-1 was prevented by 12-hydroperoxyeicosatetraenoic acid (12-HPETE). Increasing the production of prostaglandin G2 (the peroxide product of PGHS-cyclooxygenase activity) by elevating the concentration of either enzyme or substrate reversed inhibition. Using analogs of benzoic acid differing only at the hydroxyl position, we revealed the importance of this moiety to salicylate's inhibitory mechanism. Unlike typical phenolic inhibitors, e.g., acetaminophen, salicylate was ineffective as a reducing cosubstrate for PGHS-peroxidase activity, implicating the cyclooxygenase site as its putative target. PGHS-cyclooxygenase activity depends upon the oxidation of an active site tyrosine by electron transfer to the oxidized ferriprotoporphyrin of the peroxidase. The PGHS-1 apoenzyme reconstituted with manganese protoporphyrin instead of iron protoporphyrin has very little peroxidase activity. 12-HPETE does not prevent the inhibition of Mn-PGHS-1 by salicylate, indicating that reversal of salicylate inhibition by hydroperoxides depends upon electron transfer between the cyclooxygenase and peroxidase active sites. These results are consistent with an inhibitory action of salicylate at the PGHS-cyclooxygenase site that is dependent on the PGHS-peroxidase activity.

Selective inhibition of cyclooxygenase-2 (COX-2) leads to relief of pain and inflammation with reduced gastrointestinal side effects relative to nonsteroidal anti-inflammatory drugs. 2-Acetoxyphenylhept-2-ynyl sulfide (APHS) is a selective COX-2 inhibitor that covalently modifies the protein by acetylating Ser-530. We utilized site-directed mutants in the COX-2 active site to probe the molecular determinants of APHS acetylation of COX-2. Incorporation of acetyl groups into Ser-530 was monitored by HPLC and mass spectrometry. Mutations that introduce steric bulk into a channel at the top of the active site (e.g., G533A, G533V) lead to a significant reduction in APHS acetylation. Reduction in acetylation is also observed by mutation of the active-site tyrosine (Tyr-385) to phenylalanine. Mutations in the side-pocket region, into which diarylheterocycle inhibitors insert, do not affect the ability of APHS to acetylate COX-2. Surprisingly, mutation of Arg-120, which is located on the floor of the active site, strongly reduces acetylation. Based on these results, we propose that the heptynyl side chain of APHS inserts into the top channel and acetylates Ser-530 with the assistance of hydrogen bonding from Tyr-385. Arg-120 is proposed to fix the conformation of the active site to one that favors acetylation.

Nonsteroidal anti-inflammatory drugs prevent hyperalgesia and inflammation by inhibiting the cyclooxygenase-2 (COX-2) catalyzed oxygenation of arachidonic acid to prostaglandin (PG) H(2). The lipoamino acid N-arachidonylglycine (NAGly) has also been shown to suppress tonic inflammatory pain and is naturally present at significant levels in many of the same mammalian tissues that express COX-2. Here, we report that COX-2 selectively metabolizes NAGly to PGH(2) glycine (PGH(2)-Gly) and hydroxyeicosatetraenoic glycine (HETE-Gly). Site-directed mutagenesis experiments identify the side pocket residues of COX-2, especially Arg-513, as critical determinants of the COX-2 selectivity towards NAGly. This is the first report of a charged arachidonyl derivative that is a selective substrate for COX-2. These results suggest a possible role for COX-2 in the regulation of NAGly levels and the formation of a novel class of eicosanoids from NAGly metabolism.

Recent studies of the mechanism and selectivity of inhibition of cyclooxygenase enzymes are reviewed. The structural determinants of inhibition by the non-selective inhibitor, aspirin, and COX-2-selective diarylheterocycles are considered. Kinetic investigations indicate that the time-dependence of binding and inhibition of COX-1 and COX-2 by diarylheterocycles is more complex than originally postulated. The selectivity of inhibition is not determined by differences in the rates of association of the inhibitors with the two enzymes but rather by differences in the rates of dissociation. New strategies for the development of COX-2-selective inhibitors are highlighted.

Conversion of carboxylate-containing nonsteroidal antiinflammatory drugs, such as indomethacin, to esters or amides provides potent and selective inhibitors of cyclooxygenase-2 (COX-2) [Kalgutkar et al. (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 925-930]. Synthesis of cinnamyl- or coumarinyl-substituted ethanolamide derivatives of indomethacin produced fluorescent probes of inhibitor interaction with COX-2 and COX-1. Binding of either derivative to apoCOX-2 or apoCOX-1 resulted in a rapid, reversible enhancement of fluorescence. Following this rapid phase, a slow additional increase in fluorescence was observed with apoCOX-2 but not with apoCOX-1. The slow, COX-2-specific increase in fluorescence was prevented or reversed by addition of the nonfluorescent COX inhibitor (S)-flurbiprofen. Detailed kinetic studies of the interaction of the coumarinyl derivative with COX-2 showed that the observed changes in fluorescence could be described by two sequential equilibria, the first of which is rapid, reversible, and bimolecular in the forward direction. The second equilibrium is slower, reversible, and unimolecular in both directions. The forward rate constant for the slow equilibrium determined by fluorescence enhancement [(3.1 +/- 0.6) x 10(-3) s(-1)] corresponded closely to the forward rate constant for inhibition of COX-2 activity [(6.8 +/- 2.3) x 10(-3) s(-1)], suggesting that the slow fluorescence enhancement is associated with selective COX-2 inhibition. Site-directed mutagenesis indicated that residues in the carboxylate-binding region of the COX-2 active site (Arg-120, Tyr-355, and Glu-524) are critical for the binding of the indomethacin conjugates that leads to slow fluorescence enhancement and cyclooxygenase inhibition. The indomethacin conjugates described herein represent powerful tools for the investigation of a novel class of selective inhibitors of COX-2.

Cyclooxygenase inhibition studies with novel indomethacin alkanolamides demonstrate the potential for dramatic differences in inhibitor properties conferred by subtle structural modifications. The transformation of non-selective alpha-(S)-substituted indomethacin ethanolamides to potent, COX-2 selective inhibitors by simple stereocenter inversion highlights this property.

Background: Atherosclerosis has features of an inflammatory disease. Because cyclooxygenase (COX)-2 is expressed in atherosclerotic lesions and promotes inflammation, we tested the hypotheses that selective COX-2 inhibition would reduce early lesion formation in LDL receptor-deficient (LDLR-/-) mice and that macrophage COX-2 expression contributes to atherogenesis in LDLR-/- mice., Methods and Results: Treatment of male LDLR-/- mice fed the Western diet with rofecoxib or indomethacin for 6 weeks resulted in significant reductions in atherosclerosis in the proximal aorta (25% and 37%) and in the aorta en face (58% and 57%), respectively. Rofecoxib treatment did not inhibit platelet thromboxane production, a COX-1-mediated process, but it significantly reduced the urinary prostacyclin metabolite 2,3-dinor-6-keto-PGF1alpha. Fetal liver cell transplantation was used to generate LDLR-/- mice null for expression of the COX-2 gene by macrophages. After 8 weeks on the Western diet, COX-2-/- --> LDLR-/- mice developed significantly less (33% to 39%) atherosclerosis than control COX-2+/+ --> LDLR-/- mice. In both the inhibitor studies and the transplant studies, serum lipids did not differ significantly between groups., Conclusions: The present studies provide strong pharmacological and genetic evidence that COX-2 promotes early atherosclerotic lesion formation in LDLR-/- mice in vivo. These results support the potential of anti-inflammatory approaches to the prevention of atherosclerosis.

This paper describes SAR studies involved in the transformation of the NSAID meclofenamic acid into potent and selective cyclooxygenase-2 (COX-2) inhibitors via neutralization of the carboxylate moiety in this nonselective COX inhibitor.

Prostaglandin synthesis by cyclooxygenases-1 and -2 (COX-1 and COX-2) involves an initial oxygenation of arachidonic acid at C-11, followed by endoperoxide and cyclopentane ring formation, and then a second reaction with molecular oxygen in the S configuration at C-15. The resulting 15S-hydroxyl group of prostaglandins is crucial for their bioactivity. Using human COX-1 and human and murine COX-2, we have identified two amino acids located in the oxygenase active site that control the stereochemistry at C-15. The most crucial determinant is Ser-530, the residue that is acetylated by aspirin. In COX-2, site-directed mutagenesis of Ser-530 to methionine, threonine, or valine produced highly active enzymes that formed 82-95% 15R-configuration prostaglandins; these have the opposite stereochemistry at C-15 to the natural products. In COX-1, the corresponding Ser-530 mutations inactivated the enzyme. The second residue, Val-349, exerts a more subtle influence. When Val-349 was replaced by isoleucine, the mutant COX-1 and COX-2 enzymes formed 41 and 65% 15R-prostaglandins, respectively. This change was highly specific for isoleucine, as mutations of Val-349 to alanine, leucine, asparagine, or threonine did not alter or only slightly altered (< or =13%) the S-configuration at C-15. These results establish a previously unrecognized role for Ser-530 and Val-349 in maintaining the correct S stereochemistry of the carbon-15 hydroxyl group during prostaglandin synthesis. The findings may also explain the absolute conservation of Ser-530, the target of aspirin, throughout the families of cyclooxygenase enzymes.

Disease prevention is one area that both public and governmental agencies strongly support owing to its potential for an improved lifestyle and a reduction in health care costs. In this review, we focus on the clinical development of one target for cancer prevention, the COX-2 enzyme. This provides an excellent example of how basic research in biochemistry and pharmacology can lead to translational studies and eventually to approval of a drug by the FDA for use as a chemopreventive agent in humans. It is hoped that, as the genome sequence is understood more clearly, other targets will emerge that will provide even more effective drugs for future cancer prevention.

Scientists working in the field of cyclooxygenase enzymes have witnessed several major advances in the past two years. Crystal structures of fatty acid substrate and prostaglandin product complexes have been elucidated. Elegant site-directed mutagenesis studies have pinpointed the roles of key amino acids within the active site. Together, these results have provided key insights into the overall reaction mechanism. Detailed kinetics, spectroscopic and crystallographic studies have shed new light on the complex mechanism of inhibition of these fascinating enzymes. Finally, novel substrates of cyclooxygenase-2 have been identified.

Cyclooxygenases (COX) catalyse the oxygenation of arachidonic acid to prostaglandin (PG) endoperoxides. Activity of one of the COX isoforms, COX-2, results in production of prostaglandin E(2) (PGE(2)) via the endoperoxide PGH(2). COX-2 has been implicated in the pathogenesis of colorectal cancer. Malondialdehyde (MDA) is a mutagen produced by spontaneous and enzymatic breakdown of PGH(2). MDA reacts with DNA to form adducts, predominantly the pyrimidopurinone adduct of deoxyguanosine (M(1)G). Here the hypothesis was tested that COX-2 activity in human colon cells results in formation of MDA and generation of M(1)G adducts. M(1)G was detected in basal cultures of human non-malignant colon epithelial (HCEC) and malignant SW48, SW480, HT29 and HCA-7 colon cells, at levels from 77 to 148 adducts/10(8) nucleotides. Only HCA-7 and HT29 cells expressed COX-2 protein. Levels of M(1)G correlated significantly (r = 0.98, P < 0.001) with those of intracellular MDA determined colorimetrically in the four malignant cell types, but neither parameter correlated with expression of COX-2 or PG biosynthesis. Induction of COX-2 expression by phorbol 12-myristate 13-acetate in HCEC cells increased PGE(2) production 20-fold and MDA concentration 3-fold. Selective inhibition of COX-2 activity in HCA-7 cells by NS-398 significantly inhibited PGE(2) production, but altered neither MDA nor M(1)G levels. Malondialdehyde treatment of HCEC cells resulted in a doubling of M(1)G levels. These results show for the first time in human colon cells that COX-2 activity is associated with formation of the endogenous mutagen, MDA. Moreover, they demonstrate the correlation between MDA concentration and M(1)G adduct levels in malignant cells.

The endocannabinoid, 2-arachidonylglycerol (2-AG), is an endogenous ligand for the central (CB1) and peripheral (CB2) cannabinoid receptors and has been shown to be efficiently and selectively oxygenated by cyclooxygenase (COX)-2. We have investigated 2-AG/COX-2 interactions through site-directed mutagenesis. An evaluation of more than 20 site-directed mutants of murine COX-2 has allowed for the development of a model of 2-AG binding within the COX-2 active site. Most strikingly, these studies have identified Arg-513 as a critical determinant in the ability of COX-2 to efficiently generate prostaglandin H(2) glycerol ester, explaining, in part, the observed isoform selectivity for this substrate. Mutational analysis of Leu-531, an amino acid located directly across from Arg-513 in the COX-2 active site, suggests that 2-AG is shifted in the active site away from this hydrophobic residue and toward Arg-513 relative to arachidonic acid. Despite this difference, aspirin-treated COX-2 oxygenates 2-AG to afford 15-hydroxyeicosatetraenoic acid glycerol ester in a reaction analogous to the C-15 oxygenation of arachidonic acid observed with acetylated COX-2. Finally, the differences in substrate binding do not alter the stereospecificity of the cyclooxygenase reaction; 2-AG-derived and arachidonic acid-derived products share identical stereochemistry.

Prostate cancer continues to be the most frequently diagnosed cancer in men in the United States. Despite aggressive intervention, a significant number of men with prostate cancer will not be cured of their disease and will face the possibility of metastatic disease. Thus, development of potent prevention strategies to diminish or eliminate this threat is in order. Cellular exposure to chronic oxidative stress may be 1 possible etiologic factor in the development of many cancers, including prostate cancer. Oxygen radicals can attack DNA directly and result in the accumulation of potentially promutagenic oxidized DNA bases such as 8-hydroxydeoxyguanosine. In addition, chronic oxidant stress may also result in lipid peroxidation and the subsequent generation of a range of reactive products that can damage DNA. Disruption of certain genes may result in cellular tolerance to oxidative genomic injury. GSTP1 is an enzyme that helps catalyze the conjugation reaction between potentially damaging electrophiles and glutathione. Inactivation of GSTP1 has been documented to occur in nearly 100% of human prostate cancers; it is also frequently inactivated in prostatic intraepithelial neoplasia lesions. This inactivation may leave the cell vulnerable to oxidative DNA damage and/or tolerant to accumulation of oxidized DNA base adducts. These base adducts can be measured by several quantitative methods, such as gas chromatography-mass spectrometry with selected ion monitoring. These sophisticated methods can be readily integrated into prostate cancer chemoprevention studies of new and developing prevention agents by providing quantitative assessment of oxidative DNA damage before and after administration of these candidate chemopreventive drugs. The combination of genetic information, state-of-the-art assessment tools, and novel agents will allow rational, directed prostate cancer chemoprevention studies to be performed and, together, will help determine the role of chronic oxidative stress in the carcinogenic process of prostate cancer.

Cyclooxygenase is involved in the biosynthesis and function of prostaglandins. It is a glycoprotein located in the endoplasmic reticulum and in the nuclear envelope, and it has been found to have two isoforms termed COX-1 and COX-2. This paper reports on the glycosylation site analysis of recombinant COX-2 using matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) and nanoelectrospray (nanoESI) quadrupole-TOF (Q-TOF) MS. The nanoESI MS analysis of COX-2 revealed the presence of three glycoforms at average molecular masses of 71.4, 72.7, and 73.9 kDa. Each glycoform contained a number of peaks differing by 162 Da indicating heterogeneity and suggesting the presence of high-mannose sugars. The masses of the glycoforms indicate that oligosaccharides occupy two to four sites and a single N-acetylglucosamine (GlcNAc) residue occupied up to two sites. The MALDI MS analysis of a tryptic digest of the protein showed a number of potential glycopeptides. The peptides differed by 162 Da which further suggested high-mannose sugars. Nanoelectrospray MS/MS experiments confirmed glycosylation at the Asn 53 and Asn 130 sites and confirmed the presence of the peptides Asn 396-Arg 414 + GlcNAc and Thr 576-Arg 587 + GlcNAc containing Asn 580. It was not possible to conclusively determine whether the Asn 396 site was glycosylated via an MS/MS experiment, so the tryptic digest was deglycosylated to confirm the presence of the glycopeptides. Finally, a non-glycosylated tryptic peptide was observed containing the Asn 592.

Cyclooxygenases (COX) play an important role in lipid signaling by oxygenating arachidonic acid to endoperoxide precursors of prostaglandins and thromboxane. Two cyclooxygenases exist which differ in tissue distribution and regulation but otherwise carry out identical chemical functions. The neutral arachidonate derivative, 2-arachidonylglycerol (2-AG), is one of two described endocannabinoids and appears to be a ligand for both the central (CB1) and peripheral (CB2) cannabinoid receptors. Here we report that 2-AG is a substrate for COX-2 and that it is metabolized as effectively as arachidonic acid. COX-2-mediated 2-AG oxygenation provides the novel lipid, prostaglandin H(2) glycerol ester (PGH(2)-G), in vitro and in cultured macrophages. PGH(2)-G produced by macrophages is a substrate for cellular PGD synthase, affording PGD(2)-G. Pharmacological studies reveal that macrophage production of PGD(2)-G from endogenous sources of 2-AG is calcium-dependent and mediated by diacylglycerol lipase and COX-2. These results identify a distinct function for COX-2 in endocannabinoid metabolism and in the generation of a new family of prostaglandins derived from diacylglycerol and 2-AG.

Recent studies from our laboratory have shown that derivatization of the carboxylate moiety in substrate analogue inhibitors, such as 5,8,11,14-eicosatetraynoic acid, and in nonsteroidal antiinflammatory drugs (NSAIDs), such as indomethacin and meclofenamic acid, results in the generation of potent and selective cyclooxygenase-2 (COX-2) inhibitors (Kalgutkar et al. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 925-930). This paper summarizes details of the structure-activity studies involved in the transformation of the arylacetic acid NSAID, indomethacin, into a COX-2-selective inhibitor. Many of the structurally diverse indomethacin esters and amides inhibited purified human COX-2 with ICo5 values in the low-nanomolar range but did not inhibit ovine COX-1 activity at concentrations as high as 66 microM. Primary and secondary amide analogues of indomethacin were more potent as COX-2 inhibitors than the corresponding tertiary amides. Replacement of the 4-chlorobenzoyl group in indomethacin esters or amides with the 4-bromobenzyl functionality or hydrogen afforded inactive compounds. Likewise, exchanging the 2-methyl group on the indole ring in the ester and amide series with a hydrogen also generated inactive compounds. Inhibition kinetics revealed that indomethacin amides behave as slow, tight-binding inhibitors of COX-2 and that selectivity is a function of the time-dependent step. Conversion of indomethacin into ester and amide derivatives provides a facile strategy for generating highly selective COX-2 inhibitors and eliminating the gastrointestinal side effects of the parent compound.

Cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) are the enzymes responsible for the biosynthesis of the precursor to the biologically active prostaglandins, prostacyclin, and thromboxane and are the molecular targets for nonsteroidal antiinflammatory drugs (NSAIDs). Selective COX-2 inhibitors are antiinflammatory and analgesic but lack gastrointestinal toxicity, an undesirable side effect attributed to COX-1 inhibition. Crystallographic analysis of selective COX inhibitors complexed with either isoform provides some information about the molecular determinants of selectivity but does not provide information about the dynamics of inhibitor association/dissociation. We employed rapid-mixing techniques and fluorescence quenching to monitor the association and dissociation of a selective COX-2 inhibitor to COX-1 or COX-2. The association of the fluorescent diaryloxazole, SC299, with both enzymes occurs in a time-dependent fashion. Its binding to COX-2 occurs in three kinetically distinct steps whereas its binding to COX-1 occurs in two steps. In contrast to the relatively rapid association of SC299 with both enzymes, its dissociation from COX-2 is quite slow and occurs over several hours whereas the dissociation from COX-1 is complete in less than 1 min. The selectivity of SC299 as a COX-2 inhibitor correlates to its relative rates of dissociation from the two COX isoforms. A model is proposed for diarylheterocycle binding to COX's that integrates these kinetic data with available structural information.

Prostaglandin H(2) synthesis by prostaglandin endoperoxide synthase (PGHS) requires the heme-dependent activation of the protein's cyclooxygenase activity. The PGHS heme participates in cyclooxygenase activation by accepting an electron from Tyr385 located in the cyclooxygenase active site. Two mechanisms have been proposed for the oxidation of Tyr385 by the heme iron: (1) ferric enzyme oxidizes a hydroperoxide activator and the incipient peroxyl radical oxidizes Tyr385, or (2) ferric enzyme reduces a hydroperoxide activator and the incipient ferryl-oxo heme oxidizes Tyr385. The participation of ferrous PGHS in cyclooxygenase activation was evaluated by determining the reduction potential of PGHS-2. Under all conditions tested, this potential (<-135 mV) was well below that required for reactions leading to cyclooxygenase activation. Substitution of the proximal heme ligand, His388, with tyrosine was used as a mechanistic probe of cyclooxygenase activation. His388Tyr PGHS-2, expressed in insect cells and purified to homogeneity, retained cyclooxygenase activity but its peroxidase activity was diminished more than 300-fold. Concordant with this poor peroxidase activity, an extensive lag in His388Tyr cyclooxygenase activity was observed. Addition of hydroperoxides resulted in a concentration-dependent decrease in lag time consistent with each peroxide's ability to act as a His388Tyr peroxidase substrate. However, hydroperoxide treatment had no effect on the maximal rate of arachidonate oxygenation. These data imply that the ferryl-oxo intermediates of peroxidase catalysis, but not the Fe(III)/Fe(II) couple of PGHS, are essential for cyclooxygenase activation. In addition, our findings are strongly supportive of a branched-chain mechanism of cyclooxygenase catalysis in which one activation event leads to many cyclooxygenase turnovers.

Cyclooxygenases are bifunctional enzymes that catalyse the first committed step in the synthesis of prostaglandins, thromboxanes and other eicosanoids. The two known cyclooxygenases isoforms share a high degree of amino-acid sequence similarity, structural topology and an identical catalytic mechanism. Cyclooxygenase enzymes catalyse two sequential reactions in spatially distinct, but mechanistically coupled active sites. The initial cyclooxygenase reaction converts arachidonic acid (which is achiral) to prostaglandin G2 (which has five chiral centres). The subsequent peroxidase reaction reduces prostaglandin G2 to prostaglandin H2. Here we report the co-crystal structures of murine apo-cyclooxygenase-2 in complex with arachidonic acid and prostaglandin. These structures suggest the molecular basis for the stereospecificity of prostaglandin G2 synthesis.

The two isoforms of cyclooxygenase, COX-1 and COX-2, are acetylated by aspirin at Ser-530 and Ser-516, respectively, in the cyclooxygenase active site. Acetylated COX-2 is essentially a lipoxygenase, making 15-(R)-hydroxyeicosatetraenoic acid (15-HETE) and 11-(R)-hydroxyeicosatetraenoic acid (11-HETE), whereas acetylated COX-1 is unable to oxidize arachidonic acid to any products. Because the COX isoforms are structurally similar and share approximately 60% amino acid identity, we postulated that differences within the cyclooxygenase active sites must account for the inability of acetylated COX-1 to make 11- and 15-HETE. Residues Val-434, Arg-513, and Val-523 were predicted by comparison of the COX-1 and -2 crystal structures to account for spatial and flexibility differences observed between the COX isoforms. Site-directed mutagenesis of Val-434, Arg-513, and Val-523 in mouse COX-2 to their COX-1 equivalents resulted in abrogation of 11- and 15-HETE production after aspirin treatment, confirming the hypothesis that these residues are the major isoform selectivity determinants regulating HETE production. The ability of aspirin-treated R513H mCOX-2 to make 15-HETE, although in reduced amounts, indicates that this residue is not an alternate binding site for the carboxylate of arachidonate and that it is not the only specificity determinant regulating HETE production. Further experiments were undertaken to ascertain whether the steric bulk imparted by the acetyl moiety on Ser-530 prevented the omega-end of arachidonic acid from binding within the top channel cavity in mCOX-2. Site-directed mutagenesis was performed to change Val-228, which resides at the junction of the main cyclooxygenase channel and the top channel, and Gly-533, which is in the top channel. Both V228F and G533A produced wild type-like product profiles, but, upon acetylation, neither was able to make HETE products. This suggests that arachidonic acid orientates in a L-shaped binding configuration in the production of both prostaglandin and HETE products.

All nonsteroidal antiinflammatory drugs (NSAIDs) inhibit the cyclooxygenase (COX) isozymes to different extents, which accounts for their anti-inflammatory and analgesic activities and their gastrointestinal side effects. We have exploited biochemical differences between the two COX enzymes to identify a strategy for converting carboxylate-containing NSAIDs into selective COX-2 inhibitors. Derivatization of the carboxylate moiety in moderately selective COX-1 inhibitors, such as 5,8,11,14-eicosatetraynoic acid (ETYA) and arylacetic and fenamic acid NSAIDs, exemplified by indomethacin and meclofenamic acid, respectively, generated potent and selective COX-2 inhibitors. In the indomethacin series, esters and primary and secondary amides are superior to tertiary amides as selective inhibitors. Only the amide derivatives of ETYA and meclofenamic acid inhibit COX-2; the esters are either inactive or nonselective. Inhibition kinetics reveal that indomethacin amides behave as slow, tight-binding inhibitors of COX-2 and that selectivity is a function of the time-dependent step. Site-directed mutagenesis of murine COX-2 indicates that the molecular basis for selectivity differs from the parent NSAIDs and from diarylheterocycles. Selectivity arises from novel interactions at the opening and at the apex of the substrate-binding site. Lead compounds in the present study are potent inhibitors of COX-2 activity in cultured inflammatory cells. Furthermore, indomethacin amides are orally active, nonulcerogenic, anti-inflammatory agents in an in vivo model of acute inflammation. Expansion of this approach can be envisioned for the modification of all carboxylic acid-containing NSAIDs into selective COX-2 inhibitors.

The recent marketing of two selective cyclooxygenase 2 (COX-2) inhibitors climaxes the first phase of an exciting and fast-paced effort to exploit a novel molecular target for nonsteroidal anti-inflammatory drugs (NSAIDs). Much has been written in the lay and scientific press about the potential of COX-2 inhibitors as anti-inflammatory and analgesic agents that lack the gastrointestinal side-effects of traditional NSAIDs. Although research on COX-2 inhibitors has focussed mainly on inflammation and pain, experimental and epidemiological data suggest that COX-2 inhibitors could be used in the treatment or prevention of a broader range of diseases. In this review, some key points and unresolved issues related to the discovery of COX-2 inhibitors, the kinetic and structural basis for their selectivity, and possible complications in their development and use will be discussed.

Cyclooxygenase-2 (COX-2) inhibitors reduce angiogenic responses to a variety of stimuli, suggesting that products of COX-2 may mediate critical steps. Here, we show that thromboxane A2 (TXA2) is one of several eicosanoid products generated by activated human microvascular endothelial cells. Selective COX-2 antagonists inhibit TXA2 production, endothelial migration, and fibroblast growth factor-induced corneal angiogenesis. Endothelial migration and corneal angiogenesis are similarly inhibited by a TXA2 receptor antagonist, SQ29548. A TXA2 agonist, U46619, reconstitutes both migration and angiogenesis responses under COX-2-inhibited conditions. These findings identify TXA2 as a COX-2 product that functions as a critical intermediary of angiogenesis.

The chemical mandates for arachidonic acid conversion to prostaglandin G(2) within the cyclooxygenase (COX) active site predict that the substrate will orient in a kinked or L-shaped conformation. Molecular modeling of arachidonic acid in sheep COX-1 confirms that this L-shaped conformation is possible, with the carboxylate moiety binding to Arg-120 and the omega-end positioned above Ser-530 in a region termed the top channel. Mutations of Gly-533 to valine or leucine in the top channel of mCOX-2 abolished the conversion of arachidonic acid to prostaglandin G(2), presumably because of a steric clash between the omega-end of the substrate and the introduced side chains. A smaller G533A mutant retained partial COX activity. The loss of COX activity with these mutants was not the result of reduced peroxidase activity, because the activity of all mutants was equivalent to the wild-type enzyme and the addition of exogenous peroxide did not restore full COX activity to any of the mutants. However, the Gly-533 mutants were able to oxidize the carbon 18 fatty acid substrates linolenic acid and stearidonic acid, which contain an allylic carbon at the omega-5 position. In contrast, linoleic acid, which is like arachidonic acid in that its most omega-proximal allylic carbon is at the omega-8 position, was not oxidized by the Gly-533 mutants. Finally, the ability of Gly-533 mutants to efficiently process omega-5 allylic substrates suggests that the top channel does not serve as a product exit route indicating that oxygenated substrate diffuses from the cyclooxygenase active site in a membrane proximal direction.

All of the selective COX-2 inhibitors described to date inhibit the isoform by binding tightly but noncovalently at the substrate binding site. Recently, we reported the first account of selective covalent modification of COX-2 by a novel inactivator, 2-acetoxyphenyl hept-2-ynyl sulfide (70) (Science 1998, 280, 1268-1270). Compound 70 selectively inactivates COX-2 by acetylating the same serine residue that aspirin acetylates. This paper describes the extensive structure-activity relationship (SAR) studies on the initial lead compound 2-acetoxyphenyl methyl sulfide (36) that led to the discovery of 70. Extension of the S-alkyl chain in 36 with higher alkyl homologues led to significant increases in inhibitory potency. The heptyl chain in 2-acetoxyphenyl heptyl sulfide (46) was optimum for COX-2 inhibitory potency, and introduction of a triple bond in the heptyl chain (compound 70) led to further increments in potency and selectivity. The alkynyl analogues were more potent and selective COX-2 inhibitors than the corresponding alkyl homologues. Sulfides were more potent and selective COX-2 inhibitors than the corresponding sulfoxides or sulfones or other heteroatom-containing compounds. In addition to inhibiting purified COX-2, 36, 46, and 70 also inhibited COX-2 activity in murine macrophages. Analogue 36 which displayed moderate potency and selectivity against purified human COX-2 was a potent inhibitor of COX-2 activity in the mouse macrophages. Tryptic digestion and peptide mapping of COX-2 reacted with [1-14C-acetyl]-36 indicated that selective COX-2 inhibition by 36 also resulted in the acetylation of Ser516. That COX-2 inhibition by aspirin resulted from the acetylation of Ser516 was confirmed by tryptic digestion and peptide mapping of COX-2 labeled with [1-14C-acetyl]salicyclic acid. The efficacy of the sulfides in inhibiting COX-2 activity in inflammatory cells, our recent results on the selectivity of 70 in attenuating growth of COX-2-expressing colon cancer cells, and its selectivity for inhibition of COX-2 over COX-1 in vivo indicate that this novel class of covalent modifiers may serve as potential therapeutic agents in inflammatory and proliferative disorders.

The discovery of a second isoform of cyclooxygenase (cyclooxygenase-2) that is expressed in inflammatory cells and the central nervous system, but not in the gastric mucosa, offers the possibility of developing anti-inflammatory and analgesic agents that lack the gastrointestinal side effects of currently available nonsteroidal anti-inflammatory drugs. Lead compounds from several different structural classes have been identified and shown to be slow, tight-binding inhibitors that express their selectivity for cyclooxygenase-2 in the time-dependent step. The determination of structures of enzyme-inhibitor co-crystals along with site-directed mutagenesis experiments reveal the molecular basis for selectivity of some, but not all, inhibitors. Preclinical and clinical studies suggest cyclooxygenase-2 inhibitors are highly promising new agents for the treatment of pain and inflammation, and for the prevention of cancer.

Many of aspirin's therapeutic effects arise from its acetylation of cyclooxygenase-2 (COX-2), whereas its antithrombotic and ulcerogenic effects result from its acetylation of COX-1. Here, aspirin-like molecules were designed that preferentially acetylate and irreversibly inactivate COX-2. The most potent of these compounds was o-(acetoxyphenyl)hept-2-ynyl sulfide (APHS). Relative to aspirin, APHS was 60 times as reactive against COX-2 and 100 times as selective for its inhibition; it also inhibited COX-2 in cultured macrophages and colon cancer cells and in the rat air pouch in vivo. Such compounds may lead to the development of aspirin-like drugs for the treatment or prevention of immunological and proliferative diseases without gastrointestinal or hematologic side effects.

Prostaglandin endoperoxide synthase (PGHS) is a heme protein that catalyzes the committed step in prostaglandin and thromboxane biosynthesis. Two isoforms of PGHS exist, a constitutive form termed PGHS-1 and an inducible form termed PGHS-2. We report here fluorescence resonance energy transfer analysis of isoform-selective inhibitors interacting with PGHS-1 and PGHS-2. By measuring fluorescence quenching due to the energy transfer of the inhibitor fluorescence to the heme prosthetic group of PGHS, we determined these inhibitors bind in the arachidonic acid substrate access channel with an R0 of 35 A for PGHS-1 with the PGHS-1 inhibitor and an R0 of 21 A for PGHS-2 with the PGHS-2 inhibitor. The observed fluorescence quenching is completely dynamic and dominated by quenching by the heme. Time-resolved results combined with molecular modeling determine the distance from the inhibitor to the heme moiety to be 20 A in PGHS-1 and 18 A in PGHS-2. Preliminary stopped-flow kinetic studies reveal that the rate of quenching is limited by a first-order protein transition, which is slow, and that bound inhibitor undergoes rapid exchange.

Site-directed mutants of prostaglandin-endoperoxide synthase-2 (PGHS-2) with changes in the peroxidase active site were prepared by mutagenesis, expressed in Sf-9 cells, and purified to homogeneity. The distal histidine, His193, was mutated to alanine and the distal glutamine, Gln189, was changed to asparagine, valine, and arginine. The guaiacol peroxidase activities of H193A, Q189V, and Q189R were drastically reduced to levels observed in the absence of protein; only Q189N retained wild-type PGHS-2 (wtPGHS-2) activity. The mechanism of hydroperoxide reduction by the PGHS-2 mutants was investigated using 15-hydroperoxyeicosatetraenoic acid (15-HPETE), a diagnostic probe of hydroperoxide reduction pathways. The hydroperoxide reduction activity of Q189V and Q189R was reduced to that of free Fe(III) protoporphyrin IX levels, whereas Q189N catalyzed more reduction events than wtPGHS-2. The percentage of two-electron reduction events was identical for wtPGHS-2 and Q189N. The number of hydroperoxide reductions catalyzed by H193A was reduced to approximately 60% of wtPGHS-2 activity, but the majority of products were the one-electron reduction products, 15-KETE and epoxyalcohols. Thus, mutation of the distal histidine to alanine leads to a change in the mechanism of hydroperoxide reduction. Reaction of wtPGHS-2, Q189N, and H193A with varying concentrations of 15-HPETE revealed a change in product profile that suggests that 15-HPETE can compete with the reducing substrate for oxidation by the peroxidase higher oxidation state, compound I. The ability of the PGHS-2 proteins to catalyze two-electron hydroperoxide reduction correlated with the activation of cyclooxygenase activity. The reduced ability of H193A to catalyze two-electron hydroperoxide reduction resulted in a substantial lag phase in the cyclooxygenase assay. The addition of 2-methylimidazole chemically reconstituted the two-electron hydroperoxide reduction activity of H193A and abolished the cyclooxygenase lag phase. These observations are consistent with the involvement of the two-electron oxidized peroxidase intermediate, compound I, as the mediator of the activation of the cyclooxygenase of PGHS.

Prostaglandin endoperoxide synthase (PGH synthase) is responsible for converting arachdonic acid to PGH2, the common precursor of prostaglandins. It has been shown previously that phorbol ester-induced differentiation of human promonocytic leukemia cell lines is accompanied by induction of PGH synthase enzyme and enhanced capacity to produce prostaglandins. However, the identity of the isoform of PGH synthase, i.e., PGH synthase-1 or -2, that is induced under these conditions has not been established. Northern and Western analyses revealed a dramatic increase in levels of PGH synthase-1 mRNA and protein levels within 24 hr after treatment of THP-1 cells with phorbol ester. No significant increase in PGH synthase-2 mRNA or protein was observed. The increases in PGH synthase-1 were accompanied by an enhanced capacity of the cells to produce PGE2. The current findings indicate that expression of PGH synthase-1 is greatly enhanced in a promonocytic cell line by treatment with an agent that induces differentiation.

Human liver microsomes oxidized 7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene [B(a)P-7,8-diol] to products that yield DNA adduct formation and umu gene expression in the tester system Salmonella typhimurium TA1535/pSK1002. The umu response is correlated to levels of microsomal cytochrome P-450NF (P-450NF) and nifedipine oxidation in different human liver samples used for activation, and both the (+)- and (-)-enantiomers of B(a)P-7,8-diol gave similar results in these and other assays. The microsomal umu response was inhibited by antibodies raised against P-450NF. 7,8-Benzoflavone stimulated the B(a)P-7,8-diol-dependent umu response observed with purified P-450NF and human liver and lung microsomes. Thus, P-450NF appears to be the major enzyme involved in the activation of B(a)P-7,8-diol in human liver and possibly lung. Similar results were obtained for the activation of trans-9,10-dihydroxy-9,10-dihydrobenzo(b)fluoranthene and trans-3,4-dihydroxy-3,4-dihydro-7,12-dimethylbenz(a)anthracene, compounds that are known to form highly tumorigenic diol-epoxides. The major product of the oxidation of (+)-B(a)P-7,8-diol was the cis-syn isomer of benzo(a)pyrene-7,8,9,10-tetraol[7 beta, 8 alpha, 9 beta, 10 beta-tetrahydroxy-7,8,9,10-tetrahydrobenzo(a)pyrene]. Studies on the nature of the human liver enzymes involved in the formation of B(a)P-7,8-diol [from benzo(a)pyrene] indicate that neither P-450NF, P-450PA, P-450j, P-450DB, nor P-450MP is involved. The correlation of 7,8-diol formation with phenacetin O-deethylation in a set of liver samples and the partial inhibition of the reaction by 7,8-benzoflavone and anti-rat P-450 beta NF-B suggest that the enzyme involved may be P1-450, the human ortholog of rat P-450 beta NF-B, which catalyzes both the formation of B(a)P-7,8-diol and its subsequent oxidation in tissues of polycyclic hydrocarbon-treated rats. The differential effects of inhibitors indicate that benzo(a)pyrene 3-hydroxylation, 4,5-epoxidation, and 9,10-epoxidation are catalyzed by an enzyme(s) distinct from that which forms the 7,8-epoxide. The roles of the human P-450 enzymes differ from the rodent orthologs in the paradigm for bioactivation of polycyclic hydrocarbons; further, flavones appear to have opposing effects on diol formation and further epoxidation in both human liver and lung.