Site Of COX-2 Research Articles

Bromoacetamido analogs of indomethacin and mefenamic acid as affinity-labeling agents and mechanistic probes for prostaglandin H2 synthase.

Affinity-labeling agents, 1-[4-(bromoacetamido)benzyl]-5-methoxy-2-methylindole-3-acetic acid (I) and 4-(bromoacetamido)-N-(2,3-dimethylphenyl)anthranilic acid (II), were synthesized on the basis of their respective nonsteroidal anti-inflammatory drugs (NSAIDs), indomethacin and mefenamic acid [Askonas & Penning (1991) Biochemistry 30, 11553-11560]. Compounds I and II are now shown to inhibit homogeneous ram seminal vesicle prostaglandin H2 (PGH2) synthase by two kinetically distinct complexes. They are competitive inhibitors versus arachidonic acid via the formation of high-affinity E.I complexes, and they cause time-dependent inactivation of the holoenzyme via low-affinity E.I complexes. Compounds I and II, unlike classical NSAIDs, were found to inactivate both the cyclooxygenase and peroxidase reactions of the synthase in a parallel manner. Inactivation was accompanied by the incorporation of 2 mol of either radiolabeled I or II per synthase monomer. The covalent bonds that result were stable to boiling in SDS, indicating that I and II offer alternatives to aspirin in locating NSAID binding sites. Incubation of aspirin-treated PGH2 synthase with radiolabeled I reduced the stoichiometry of incorporation to 1.0, suggesting that one of the sites modified corresponds to the cyclooxygenase site. By saturating the cyclooxygenase site with mefenamic acid, I and II only abolished the peroxidase activity of the enzyme, suggesting that the second site of modification corresponds to the peroxidase site. When PGH2 synthase was incubated with mefenamic acid and I or II, only the peroxidase activity was inactivated. Subsequent removal of all drugs by dialysis gave a preparation of PGH2 synthase that could perform the cyclooxygenase reaction, but lacked the ability to cleave ethyl hydroperoxide to ethanol and water.(ABSTRACT TRUNCATED AT 250 WORDS)

Nonsteroidal antiinflammatory agents, XVIII: C-5 functionalized 6,7-diphenyl-2,3-dihydro-1H-pyrrolizines as inhibitors of bovine cyclooxygenase and 5-lipoxygenase.

6-(4-Chlorophenyl)-7-phenyl-2,3-dihydro-1H-pyrrolizines with functional groups at position 5 of the heterocyclic moiety were synthesized and tested. To determine their antiinflammatory activity bovine blood was used as enzyme source for the cyclooxygenase and 5-lipoxygenase, respectively. The iminoxy acetic acid derivative and the iminotetrazole selectively inhibit the 5-lipoxygenase, all the other compounds show medium or low affinity to the active sites of cyclooxygenase and 5-lipoxygenase. In general all compounds inhibit 5-lipoxygenase more effectively than cyclooxygenase. Concerning the inhibition of 5-lipoxygenase the most active compounds found are equipotent to the corresponding propionic acid compounds, but they aren't well balanced dual inhibitors as shown for the carboxylic derivatives. A structure activity relationship and the enzyme selectivity are discussed.

Target size analysis of prostaglandin endoperoxide synthase. Radiation inactivation of both cyclooxygenase and peroxidase correlated with the monomer of 72 kDa.

To determine the size of the functional catalytic unit of prostaglandin endoperoxide (prostaglandin H) synthase, radiation inactivation experiments were performed. Both microsomes from ovine seminal vesicles and purified enzyme were irradiated with 10 MeV electrons. The enzymic activities of prostaglandin H synthase, cyclooxygenase and peroxidase, showed mono-exponential inactivation curves dependent on radiation dose, indicating molecular masses of approximately 72 kDa. The enzyme in microsomes, in its native environment, as well as in its purified state after solubilisation with nonionic detergent showed identical molecular masses. The results clearly demonstrate that the monomer of the enzyme with an apparent molecular mass of 72 kDa (SDS/PAGE) is the functional unit for catalysis of both activities. Hence the two active sites of cyclooxygenase and peroxidase reside on the same polypeptide chain.

Action of some salicylate derivatives on in vitro platelet aggregation. Inhibitory and inhibition antagonistic effects

Twenty salicylate derivatives were tested for their antagonistic activity on the inhibitory effect of aspirin on platelet aggregation. The blocking effect was not limited to the salicylate but also characterised some of its substituted compounds. The substituant influence did not seem to be related to electronic or size parameters. This antagonistic activity of these derivatives decreased as concentrations increased, owing to the emergence of their own inhibitory activity: several salicylate derivatives showed dual inhibitory and inhibition antagonistic activity, with both properties present at the same concentration. A mechanism involving dissociated activities on the two enzymatic sites of cyclooxygenase is proposed.

Synthesis and Structure–Activity Relationships of Anti-inflammatory 9,lO-Dihydro-9-oxo-2-Acridine-alkanoic Acids and 4-(2-Carboxyphenyl)aminobenzenealkanoic Acids

Eighteen test compounds, in three chemical series, were prepared as potential anti-inflammatory agents and evaluated by the rat hindpaw carrageenan-induced edema assay. The compounds, isosteric with known anti-inflammatory and antiallergic cyclo-oxygenase and lipoxygenase inhibitors, are 10-methyl-9, 10-dihydro-9-oxo-2-acridinealkanoic acids, 9, 10-dihydro-9-oxo-2-acridinealkanoic acids, and 4-(2-carboxyphenyl)aminobenzenealkanoic acids. Compounds within each of these series differ in the structure of the alkanoic acid side chain. Compounds containing the acetic acid and the branched 2-propionic acid side chain showed inhibition of carrageenan-induced edema. The activity of compounds with these side chains and the inactivity of those with carboxy, oxyacetic, thioacetic, and 3-propionic acid side chains is in accordance with the proposed template model of Appleton and Brown for the active site of cyclo-oxygenase, rather than with the alternative active site model proposed by Gund and Shen. One compound, (±)-2-[N-(2-carboxyphenyl)-4-aminophenyl]propionic acid (3c), showed edema inhibition at 50 mg/kg po, comparable to that of an equivalent dose level of (+)-naproxen. Compounds 4a and 5a, which contain a carboxylic acid side chain, exhibited inhibition of soybean 12-lipoxygenase with IC50 values of 17.2 and 8.4 μM, respectively. The inhibition observed for the control drug, naproxen, was 24 μM.

Tolerance to acetylsalicylic acid (ASA) induced in ASA-sensitive asthmatics does not depend on initial adverse reaction.

A state of tolerance to aspirin (ASA) was induced in 10 aspirin-sensitive patients by daily administration of incremental doses of ASA. No adverse reactions were reported. The initial dose (from 5 to 60 mg) was gradually increased each day up to 300 mg and then doubled. 50 mg indomethacin given the day after administration of 600 mg ASA did not elicit any symptom of intolerance. The authors discuss a possible mechanism of tolerance to aspirin in ASA-sensitive asthmatics after ASA administration, suggesting that it might be connected either with inhibition of the lipooxygenetic pathway of arachidonic acid metabolism or with blockade of the cyclooxygenase supplementary binding site by salicylic acid, a product of acetylsalicylic acid hydrolysis. This would prevent aspirin from binding with the catalytic cyclooxygenase site.

Isolation of the cDNA for human prostaglandin H synthase

Prostaglandin H Synthase (PGHS, cyclooxygenase) is a 67 kd protein which catalyzes the first step in prostaglandin systhesis. The primary amino acid sequence and the molecular mechanisms regulating expression are unknown. We report here isolation of a cDNA clone for the enzyme from human vascular endothelial cells for use in such studies. High titre, polyclonal antiserum against PGHS was developed in rabbits. The antiserum was monospecific, reacted with cyclooxygenase on Western blots at a limiting dilution of 1:500, 000 and immunoprecipitated cyclooxygenase synthesized by in vitro translation of PGHS messenger RNA. It was used to screen a lambda gt11 cDNA expression library from human endothelial cells. Three positive clones wre isolated. Following plaque purification, one clone reacted strongly with two other polyclonal antisera independently raised against highly purified cyclooxygenase and the aspirin-acetylated enzyme. Western blot analysis confirmed production of a large ▪ 180 kd fusion protein of cyclooxygenase and beta-galactosidase. The cDNA insert of approximately 2.2 kilo base pairs was excised and subcloned into plasmid pUC8. A 24 nucleotide DNA probe, synthesized according to the amino acid sequence of the aspirin-acetylation site of cyclooxygenase, hybridized strongly with the 2.2 kbp cDNA insert. It is concluded that the 2.2 kbp cDNA insert represents a cDNA clone for human cyclooxygenate, which also expresses the aspirin-acetylation site. This is the first reported isolation of the cDNA for this enzyme, and will facilitate further studies on the primary sequence and on the regulation of the enzyme at the molecular level.

Uptake and subcellular distribution of [3H]arachidonic acid in murine fibrosarcoma cells measured by electron microscope autoradiography.

We have used quantitative electron microscope autoradiography to study uptake and distribution of arachidonate in HSDM1C1 murine fibrosarcoma cells and in EPU-1B, a mutant HSDM1C1 line defective in high affinity arachidonate uptake. Cells were labeled with [3H]arachidonate for 15 min, 40 min, 2 h, or 24 h. Label was found almost exclusively in cellular phospholipids; 92-96% of incorporated radioactivity was retained in cells during fixation and tissue processing. All incorporated radioactivity was found to be associated with cellular membranes. Endoplasmic reticulum (ER) contained the bulk of [3H]arachidonate at all time points in both cell types, while mitochondria, which contain a large portion of cellular membrane, were labeled slowly and to substantially lower specific activity. Plasma membrane (PM) also labeled slowly, achieving a specific activity only one-sixth that of ER at 15 min in HSDM1C1 cells (6% of total label) and one-third of ER in EPU-1B (10% of total label). Nuclear membrane (NM) exhibited the highest specific activity of labeling at 15 min in HSDM1C1 cells (twice that of ER) but was not preferentially labeled in the mutant. Over 24 h, PM label intensity increased to that of ER in both cell lines. However, NM activity diminished in HSDM1C1 cells by 24 h to a small fraction of that in ER. In response to agonists, HSDM1C1 cells release labeled arachidonate for eicosanoid synthesis most readily when they have been labeled for short times. Our results therefore suggest that NM and ER, sites of cyclooxygenase in murine fibroblasts, are probably sources for release of [3H]arachidonate, whereas PM and mitochondria are unlikely to be major sources of eicosanoid precursors.

Effects of nonsteroid antiinflammatory drugs on the specific binding of platelet activating factor to membrane preparations of rabbit platelets

The inhibitory effects of several antiinflammatory agents on the specific binding of tritiated 1-O-alkyl-2-O-acetyl-sn-glycero-3-phosphorylcholine, (platelet activating factor, PAF), with its receptor on isolated rabbit platelet plasma membranes were investigated. Several potent cyclooxygenase inhibitors do not inhibit 3H-PAF binding to its receptor sites. Yet, three others, indomethacin, phenylbutazone and sulfinpyrazone, as well as three non-cyclooxygenase inhibitors, the 3′,4′-dimethoxy analog of indomethacin, the prodrug sulindac and its sulfone metabolite, are moderately active at relatively high concentrations (50 - 100 μM). Parallel inhibitions of 3H-PAF binding and PAF-induced platelet aggregation by derivatives of these antiinflammatory agents suggest that these inhibitors are probably interacting with the functional binding sites of PAF. The results clearly indicate that the configuration of PAF binding site is very different from the inhibitory site of cyclooxygenase. A preference for oxygenated substituents in these hydrophobic molecules to inhibit the PAF-receptor binding is noted. Some binding characteristics of the receptor are briefly discussed.

Structural requirements for preventing the aspirin- and the arachidonate-induced inactivation of platelet cyclo-oxygenase: Additional evidence for distinct enzymatic sites

2-Hydroxybenzoic acid (salicylic acid) prevents the inhibition by aspirin (ASA) of platelet aggregation and of the generation of thromboxane A 2 from arachidonic acid (AA). We studied the ability of 2-hydroxybenzoic acid analogues to block ASA and to prevent the platelet desensitization due to a first exposure to AA. Inactivation was prevented when exposure to AA was done in the presence of reversible inhibitors of cyclo-oxygenase. Phenol, methyl salicylate and L8027 were thus strong inhibitors of AA-induced platelet activation and desensitization. The minimal structural requirement for inhibition of thromboxane A 2 generation from AA was a phenol group as benzoic acid was fully inactive. 2-Hydroxybenzoic acid, and to some extent 2,6-dihydroxybenzoic acid were effective against ASA, the most active substances being methyl salicylate and L8027. The minimal structural requirement for blocking ASA was that 2-hydroxybenzoic acid, 2-methoxybenzoic acid should be devoid of activity, which highlights the fact that the hydroxyl group must be available. Our work favours the hypothesis that non-steroidal anti-inflammatory drugs react with two sites of cyclo-oxygenase, which were named the supplementary and the catalytic sites. The interaction of 2-hydroxybenzoic acid and of its analogues with the supplementary site is necessary but not sufficient for the efficacy of these compounds as cyclo-oxygenase inhibitors. The intensity of interaction with the supplementary site and the modifications of the catalytic site determine the potency of these compounds as cyclo-oxygenase inhibitors. For preventing ASA inactivation, an interaction with the supplementary sites is always necessary, but furthermore an appropriate group, preferentially in the position ortho to the hydroxyl, is needed.

Zero extracellular K + and prostaglandin E release in the guinea-pig taenia coli

Prostaglandin E release rates from isolated strips of guinea-pig taenia coli increased during exposure to zero K + bathing fluid, from control values of 0.78 ± 0.11 ng/g per min to levels as high as 29.2 ng/per min. Release rates increased for 40–50 min and then remained constant or fell despite progressive increases in intracellular sodium [Na i +] or fall in intracellular potassium [K i +]. Readmittance of K + to the bathing solution resulted in rapid reversal of elevated prostaglandin E release rates. [Na + i] and [K + i] were markedly more abnormal in strips exposed to zero K + for 70–201 min compared to 30-min exposures. Upon the readdition of K + after long zero K + exposure, the rate of prostaglandin E release fell long before [Na + i] and [K + i] returned to control levels. After K + was readded to the bathing solution, the ion concentration of tissues exposed to zero K + for 30 min returned to normal much more quickly than did those of tissues exposed for the longer time periods, yet the exponential rate constants for fall of prostaglandin E release rate after K + was added were not significantly different after short or long zero K + exposure. Thus there was a dissociation between the return of [Na + i] and [K + i] and the fall of prostaglandin E release rate to control levels. Ouabain augmented prostaglandin E release under conditions where [K + i] could not fall. Addition of known neurotransmitters present in this tissue to the bathing fluid did not augment prostaglandin E release. Guinea-pig taenia coli strips that had been incubated with [ 3H]arachidonic acid, constantly released [ 3H]arachidonic acid and [ 3H]prostaglandin E and a prostaglandin which cochromatographed with prostaglandin E but could not be converted to prostaglandin B by alkali and was shown to be 6-ketoprostaglandin F 1α. Release of [ 3H]arachidonic acid and [ 3H]prostaglandin E plus 6-[ 3H]ketoprostaglandin F 1α was increased when strips were exposed to zero K +. Data obtained in this study suggest the augmented prostaglandin E release seen during zero K + or ouabain is related to increased availability of unbound arachidonic acid at the site of cyclooxygenase in the cell. Augmented prostaglandin E release is apparently not related to alterations in intracellular electrolyte concentrations or release of known neurotransmitters.