Abstract
Prostaglandin-H synthase-1, the rate-limiting enzyme in prostaglandin synthesis, has both cyclooxygenase (CO) and peroxidase (PO) activities. While most nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit only the CO activity, we describe an inhibitor, tepoxalin, that inhibits both the CO (IC50 = 0.1 microM) and the PO (IC50 = 4 microM) activities. Unlike many NSAIDs which are competitive inhibitors of CO, tepoxalin is a noncompetitive inhibitor of CO and its inhibitory effect on PO but not CO is reversed by excess heme. Moreover, inhibition of the PO activity by tepoxalin is not dependent on the enzymatic turnover of the CO activity. The hydroxamic acid of tepoxalin is responsible for the PO inhibition since a carboxylic acid derivative of tepoxalin retains full CO but not PO inhibition. We postulated that the hydroxamic group might confer the ability to inhibit PO on conventional CO inhibitors. This idea was supported by the observation that naproxen hydroxamic acid, but not naproxen showed PO inhibition. Furthermore, tepoxalin's carboxylic acid analogue and naproxen each competitively relieved PO inhibition by their respective hydroxamic acids. The intracellular activity of PO as monitored by the release of reactive oxygen species was also inhibited by both tepoxalin and naproxen hydroxamic acid. These observations suggest a strategy for design of novel compounds to inhibit prostaglandin synthase PO. The therapeutic implications of these novel PO inhibitors are discussed.
Highlights
In order to compare the CO and PO activities of PGHS1 under similar assay conditions, their activities were measured by monitoring the oxidation of tetramethyl-p-phenylenediamine dihydrochloride (TMPD) using arachidonic acid (AA) or H20 2 as substrates, respectively
To ensure that the CO activity measured under these conditions was comparable to published results, initial studies were conducted with an 02 consumption assay to validate the AAlTMPD assay
Tepoxalin's inhibitory effect on PGHSI was not altered in the presence of different concentrations of cosubstrates, suggesting that tepoxalin is not competing with cosubstrates in the peroxidase reaction
Summary
The intracellular activity of PO as monitored by the release of reactive oxygen species was inhibited by both tepoxalin and naproxen hydroxamic acid These observations suggest a strategy for design ofnovel compounds to inhibit prostaglandin synthase PO. The heme containing enzyme prostaglandin-H synthase-l (PGHSl, EC 1.14.99.1) catalyzes the first committed step in the biosynthesis of prostaglandins, thromboxanes, and prostacyclins (Smith and Marnett, 1991; DeWitt, 1991; Smith et al, 1991) It exhibits two distinct enzymatic activities: cyclooxygenase (CO) activity which converts arachidonic acid (AA) to the hydroperoxide, prostaglandin G2 (PGG2), and a peroxidase (PO) activity which reduces PGG2 to the alcohol PGH2 through a heme-mediated, two-electron transfer process (Marnett and Maddipati, 1991; Lambeir et al, 1985). This model accounts for the stimulation of PGHSI CO activity by exogenous hydroperoxides and the inhibition by hydroperoxide scavengers
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