title: Inactivation of prostaglandin endoperoxide synthase by acylating derivatives of indomethacin.
abstract: Derivatives of the potent antiinflammatory agent and cyclooxygenase inhibitor indomethacin were synthesized in which the carboxylic acid moiety was converted into reactive acylating agents. Indomethacin imidazole (indomethacin-IM) and indomethacin N-hydroxysuccinimide (indomethacin-NHS) inactivated both the cyclooxygenase and peroxidase activities when incubated with the apo form of purified prostaglandin endoperoxide synthase (PGH synthase) at a stoichiometry of 1:1. Treatment of the inactivated enzyme with hydroxylamine at neutral pH led to recovery of all peroxidase and about 50% of the cyclooxygenase activity. Hydroxylamine did not regenerate the cyclooxygenase activity of indomethacin-inactivated protein. Reconstitution of the apoprotein with heme protected against inactivation by indomethacin-NHS. Visible spectroscopy established that indomethacin-NHS-inactivated apoenzyme had a reduced capacity to bind heme. Indomethacin-NHS also substantially protected the apoenzyme from cleavage at the trypsin-sensitive Arg277 site. Incubation of [2-14C]indomethacin-NHS with PGH synthase led to incorporation of radioactivity into the protein, but no adduct was detected by reversed-phase HPLC, suggesting it was unstable to the chromatographic conditions. Incubation of indomethacin-NHS with apoprotein followed by HPLC analysis led to the formation of greater amounts of the hydrolysis product indomethacin than did similar treatment of holoprotein. The results suggest that indomethacin-IM and indomethacin-NHS covalently and selectively label PGH synthase near the heme binding site, leading to loss of both catalytic activities of the enzyme.
authors.affiliation: A. B. Hancock, Jr., Memorial Laboratory for Cancer Research, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146.
authors.forename: I
authors.initials: I
authors.lastname: Wells
authors.forename: L J
authors.initials: LJ
authors.lastname: Marnett
mesh_terms: D000215:Acylation; D000818:Animals; D016861:Cyclooxygenase Inhibitors / Q000378:metabolism* / Q000494:pharmacology*; D004789:Enzyme Activation; D006418:Heme / Q000378:metabolism; D019811:Hydroxylamine; D006898:Hydroxylamines / Q000494:pharmacology; D007093:Imidazoles / Q000138:chemical synthesis / Q000494:pharmacology*; D007202:Indicators and Reagents; D007213:Indomethacin / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000494:pharmacology*; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008297:Male; D012669:Seminal Vesicles / Q000201:enzymology; D012756:Sheep; D013388:Succinimides / Q000138:chemical synthesis / Q000494:pharmacology*
chemical_list: D016861:Cyclooxygenase Inhibitors; D006898:Hydroxylamines; D007093:Imidazoles; D007202:Indicators and Reagents; D013388:Succinimides; C080201:N-succinimidyl-1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indole-3-acetate; D019811:Hydroxylamine; D006418:Heme; C080200:1-(4-chlorobenzoyl)-3-(2-(1H-imidazol-1-yl)-2-oxoethyl)-5-methoxy-2-methyl-1H-indole; D007213:Indomethacin