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)
Tang
M S
MS
Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia 19104-6084.
Askonas
L J
LJ
Penning
T M
TM
D000345:Affinity Labels / Q000737:chemistry*; D000818:Animals; D002384:Catalysis; D016861:Cyclooxygenase Inhibitors / Q000737:chemistry*; D007213:Indomethacin / Q000031:analogs & derivatives*; D008297:Male; D008528:Mefenamic Acid / Q000031:analogs & derivatives*; D010544:Peroxidases / Q000037:antagonists & inhibitors; D011451:Prostaglandin-Endoperoxide Synthases / Q000378:metabolism*; D012669:Seminal Vesicles / Q000201:enzymology; D012756:Sheep
D000345:Affinity Labels; D016861:Cyclooxygenase Inhibitors; D008528:Mefenamic Acid; D010544:Peroxidases; D011451:Prostaglandin-Endoperoxide Synthases; D007213:Indomethacin