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 microm), 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 [(3)H]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.
Szewczuk
Lawrence M
LM
Department of Biochemistry & Biophysics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA.
Forti
Luca
L
Stivala
Lucia A
LA
Penning
Trevor M
TM
D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000494:pharmacology; D016718:Arachidonic Acid / Q000494:pharmacology; D001665:Binding Sites; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D051545:Cyclooxygenase 1; D051546:Cyclooxygenase 2; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006873:Hydroquinones / Q000737:chemistry; D020128:Inhibitory Concentration 50; D007527:Isoenzymes / Q000378:metabolism*; D007700:Kinetics; D008565:Membrane Proteins; D008956:Models, Chemical; D010100:Oxygen / Q000378:metabolism; D009195:Peroxidase / Q000737:chemistry; D010544:Peroxidases / Q000737:chemistry / Q000378:metabolism*; D011451:Prostaglandin-Endoperoxide Synthases / Q000378:metabolism*; D011453:Prostaglandins / Q000378:metabolism; D000077185:Resveratrol; D013053:Spectrophotometry; D013267:Stilbenes / Q000494:pharmacology*; D013329:Structure-Activity Relationship; D013997:Time Factors
D000894:Anti-Inflammatory Agents, Non-Steroidal; D006873:Hydroquinones; D007527:Isoenzymes; D008565:Membrane Proteins; D011453:Prostaglandins; D013267:Stilbenes; D016718:Arachidonic Acid; D010544:Peroxidases; D009195:Peroxidase; D051545:Cyclooxygenase 1; D051546:Cyclooxygenase 2; C496511:PTGS1 protein, human; C496540:PTGS2 protein, human; D011451:Prostaglandin-Endoperoxide Synthases; D000077185:Resveratrol; D010100:Oxygen; C031927:hydroquinone