title: Characterization of an AM404 analogue, N-(3-hydroxyphenyl)arachidonoylamide, as a substrate and inactivator of prostaglandin endoperoxide synthase.
abstract: N-(4-Hydroxyphenyl)arachidonoylamide (AM404) is an inhibitor of endocannabinoid inactivation that has been used in cellular and animal studies. AM404 is a derivative of arachidonic acid and has been reported to inhibit arachidonate oxygenation by prostaglandin endoperoxide synthase-1 and -2 (PGHS-1 and -2, respectively). While examining the structural requirements for inhibition of PGHS, we discovered that the meta isomer of AM404, N-(3-hydroxyphenyl)arachidonoylamide (3-HPAA), is a substrate for purified PGHS. PGHS-2 efficiently oxygenated 3-HPAA to prostaglandin and hydroxyeicosatetraenoate products. No oxidation of the phenolamide moiety was observed. 3-HPAA appeared to be converted by PGHS-1 in a similar manner; however, conversion was less efficient than that by PGHS-2. PGHS-2 was selectively, dose-dependently, and irreversibly inactivated in the presence of 3-HPAA. Complete inactivation of PGHS-2 was achieved with 10 muM 3-HPAA. Preliminary characterization revealed that 3-HPAA inactivation did not result from covalent modification of PGHS-2 or damage to the heme moiety. These studies provide additional insight into the structural requirements for substrate metabolism and inactivation of PGHS and report the first metabolism-dependent, selective inactivator of PGHS-2.
authors.affiliation: A. B. Hancock, Jr., Memorial Laboratory for Cancer Research, Department of Biochemistry, Vanderbilt Institute of Chemical Biology, Center in Molecular Toxicology, and Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
authors.forename: Melissa V
authors.initials: MV
authors.lastname: Turman
authors.forename: Philip J
authors.initials: PJ
authors.lastname: Kingsley
authors.forename: Lawrence J
authors.initials: LJ
authors.lastname: Marnett
mesh_terms: D000818:Animals; D001095:Arachidonic Acids / Q000138:chemical synthesis / Q000737:chemistry*; D051545:Cyclooxygenase 1 / Q000737:chemistry*; D051546:Cyclooxygenase 2 / Q000737:chemistry*; D016861:Cyclooxygenase Inhibitors / Q000138:chemical synthesis / Q000737:chemistry*; D007700:Kinetics; D051379:Mice; D012756:Sheep; D013379:Substrate Specificity
chemical_list: D001095:Arachidonic Acids; D016861:Cyclooxygenase Inhibitors; C546271:N-(3-hydroxyphenyl)arachidonoylamide; D051545:Cyclooxygenase 1; D051546:Cyclooxygenase 2; C107594:N-(4-hydroxyphenyl)arachidonylamide