Resveratrol is a peroxidase-mediated inactivator of COX-1 but not COX-2 - A mechanistic approach to the design of COX-1 selective agents

Resveratrol ( 3,4', 5- trihydroxy- trans- stilbene) is a phytoalexin found in grapes that has anti- inflammatory, cardiovascular protective, and cancer chemopreventive properties. It has been shown to target prostaglandin H-2 synthase ( COX)- 1 and COX- 2, which catalyze the first committed step in the synthesis of prostaglandins via sequential cyclooxygenase and peroxidase reactions. Resveratrol discriminates between both COX isoforms. It is a potent inhibitor of both catalytic activities of COX- 1, the desired drug target for the prevention of cardiovascular disease, but only a weak inhibitor of the peroxidase activity of COX- 2, the isoform target for nonsteroidal anti- inflammatory drugs. We have investigated the unique inhibitory properties of resveratrol. We find that it is a potent peroxidase- mediated mechanism-based inactivator of COX- 1 only ( k(inact) = 0.069 +/- 0.004 s(-1), K-i( inact) = 1.52 +/- 0.15 muM), with a calculated partition ratio of 22. Inactivation of COX- 1 was time- and concentration- dependent, it had an absolute requirement for a peroxide substrate, and it was accompanied by a concomitant oxidation of resveratrol. Resveratrol-inactivated COX- 1 was devoid of both the cyclooxygenase and peroxidase activities, neither of which could be restored upon gel- filtration chromatography. Inactivation of COX- 1 by [ H-3] resveratrol was not accompanied by stable covalent modification as evident by both SDS-PAGE and reverse phase- high performance liquid chromatography analysis. Structure activity relationships on methoxy- resveratrol analogs showed that the m- hydroquinone moiety was essential for irreversible inactivation of COX- 1. We propose that resveratrol inactivates COX- 1 by a hit- and- run mechanism, and offers a basis for the design of selective COX- 1 inactivators that work through a mechanism- based event at the peroxidase active site.