Multiple sites on prostaglandin cyclooxygenase are determinants in the action of nonsteroidal antiinflammatory agents.

Abstract

Evidence is presented to show that nonsteroidal antiinflammatory drugs react with two sites on the cyclooxygenase (8,11,14-eicosatrienoate, hydrogen-donor:oxygen oxidoreductase, EC 1.14.99.1). Although the degree of interaction with the catalytic site determines the potency of such compounds, interaction with the supplementary site is also obligatory for efficacy as cyclooxygenase inhibitors and may explain the selectivity of such drugs in inhibiting the cyclooxygenase but not the lipoxygenase pathway. Drugs that interact more effectively with the supplementary site than with the catalytic site-i.e., those of weak to moderate activity as cyclooxygenase inhibitors-are shown to prevent inhibition ofthe enzyme by indomethacin. Compounds in this class are also capable of blocking the ulcerogenic action of indomethacin, which suggests that this antiulcerogenic property stems from a direct action at the level of the cyclooxygenase in the stomach.There are a number of reports indicating that the ulcerogenic action of high doses of nonsteroidal antiinflammatory drugs (NSAIDs) is secondary to a depression of prostaglandin (PG) synthesis in the stomach (1, 2). The most convincing evidence in support of this concept is the finding that coadministration of PGs prevents induction of lesions by such drugs (2). Oral administration of salicylic acid-a weak inhibitor of cyclooxygenase (8,11,14-eicosatrienoate, hydrogen-donor:oxygen oxidoreductase, EC 1.14.99.1), the initial step in PG biosynthesis and the target of most NSAIDs-has been found to diminish gastric lesions resulting from high levels of indomethacin and other NSAIDs in rats (3). A similar action of 5-(2,4-difluorophenyl)-salicylic acid (diflunisal), an inhibitor ofcyclooxygenase of intermediate potency, has been reported (4). In view of the requirement for endogenous synthesis of PGs for homeostasis ofthe stomach, these findings with both salicylate and diflunisal could be due to their ability, after oral administration, to prevent the local transport of indomethacin to the active site on stomach cyclooxygenase or due to direct competition with indomethacin for a common site on the enzyme. The recent finding that salicylic acid also blunts the ulcerogenic action of indomethacin when administered parenterally may be interpreted to support this latter possibility (5). To explore this question and its possible relation to ulcerogenicity, a number of NSAIDs were examined for their ability to counter the effect of indomethacin in inhibiting cyclooxygenase activity in vitro and to suppress indomethacin-induced gastric lesions in vivo. MATERIALS AND METHODSPG Cyclooxygenase. The NSAIDs and surfactant S10-7 (6) were obtained from N. Jensen and R. W. Egan, respectively, of these laboratories. Arachidonic acid was purchased from Sigma. Other materials were obtained from standard suppliers.Microsomes containing cyclooxygenase activity were prepared from ram seminal vesicles as described (7). The NSAIDs were incubated for 2 min at 30'C with approximately 3 mg of micro...

Aspirin Inhibition and Acetylation of the Plant Cytochrome P450, Allene Oxide Synthase, Resembles that of Animal Prostaglandin Endoperoxide H Synthase

“…4D). This is comparable with the effect aspirin has on PGHS, where covalent acetylation of the protein occurs within 15 min of exposure to aspirin (31)(32)(33) and suggests that the irreversible inhibition observed with AOS might also be because of a covalent modification. To test this, quantities of purified AOS were incubated with 14 C-acetate-labeled aspirin for 1 or 2 h. AOS was then precipitated, repeatedly washed, and subjected to SDS-PAGE autoradiography.…”

Effects of salicylic and acetylsalicylic acid alone and in combination on platelet aggregation and prostanoid synthesis in man.

“…Although salicylate has been shown to be similarly potent as an analgesic and anti-inflammatory drug (Atkinson & Collier, 1980; Rainsford, 1984), it has almost no effect on prostanoid formation of gastric mucosa (Ligumsky et al, 1982;Whittle et al, 1980), macrophages (Kuehl et al, 1980), ram seminal vesicle microsomes (Humes et al, 1981), or thrombocytes (Roberts et al, 1984;Smith & Willis, 1971;Vargaftig, 1978a). Platelet function is not affected by salicylate as well (O'Brien, 1968;Roberts et al, 1984;Smith & Willis, 1971;Weiss et al, 1968;Zucker & Peterson, 1970).…”

Interactions of inhibitors of the lipoxygenase and cyclo‐oxygenase pathways with a supplementary binding site on soybean lipoxygenase

“…Surveying our results and the data provided by Kuehl et al (1981) and Humes et al (1981) we suppose that cyclo-oxygenases as well as lipoxygenases contain additional, perhaps rather similar, supplementary binding sites in addition to the wellrecognized, rather different, catalytic sites. In such a model the pattern of interaction with both sites determines the final pharmacological profile of an individual agent resulting in either selective inhibition of cyclo-oxygenases, lipoxygenases or of both enzymes.…”

“…Recently, Kuehl et al (1981) and Humes et al (1981) demonstrated that certain analgesicantipyretic agents apparently interact with two sites on the cyclo-oxygenase. The interaction of these inhibitors with the proposed supplementary binding site was suggested to explain the interactions of weak and potent cyclo-oxygenase inhibitors: drugs interacting more effectively with the supplementary site, which is obligatory for inhibitory action, than with the catalytic site, i.e.…”

“…In contrast to Kuehl et al (1981) and Humes et al (1981) Acetonylacetone bisphenylhydrazone and acetone phenylhydrazone were synthesised in our laboratory from reagent grade materials according to standard procedures as recently described (Baumann & Wurm, 1982 1% (v/v) in the assay system. Appropriate blanks (heat-denaturated enzyme) and controls (ethanolic buffer instead of test drugs) were run through the same procedures.…”