title: Substrate-selective Inhibition of Cyclooxygeanse-2 by Fenamic Acid Derivatives Is Dependent on Peroxide Tone.
abstract: Cyclooxygenase-2 (COX-2) catalyzes the oxygenation of arachidonic acid (AA) and endocannabinoid substrates, placing the enzyme at a unique junction between the eicosanoid and endocannabinoid signaling pathways. COX-2 is a sequence homodimer, but the enzyme displays half-of-site reactivity, such that only one monomer of the dimer is active at a given time. Certain rapid reversible, competitive nonsteroidal anti-inflammatory drugs (NSAIDs) have been shown to inhibit COX-2 in a substrate-selective manner, with the binding of inhibitor to a single monomer sufficient to inhibit the oxygenation of endocannabinoids but not arachidonic acid. The underlying mechanism responsible for substrate-selective inhibition has remained elusive. We utilized structural and biophysical methods to evaluate flufenamic acid, meclofenamic acid, mefenamic acid, and tolfenamic acid for their ability to act as substrate-selective inhibitors. Crystal structures of each drug in complex with human COX-2 revealed that the inhibitor binds within the cyclooxygenase channel in an inverted orientation, with the carboxylate group interacting with Tyr-385 and Ser-530 at the top of the channel. Tryptophan fluorescence quenching, continuous-wave electron spin resonance, and UV-visible spectroscopy demonstrate that flufenamic acid, mefenamic acid, and tolfenamic acid are substrate-selective inhibitors that bind rapidly to COX-2, quench tyrosyl radicals, and reduce higher oxidation states of the heme moiety. Substrate-selective inhibition was attenuated by the addition of the lipid peroxide 15-hydroperoxyeicosatertaenoic acid. Collectively, these studies implicate peroxide tone as an important mechanistic component of substrate-selective inhibition by flufenamic acid, mefenamic acid, and tolfenamic acid.
authors.affiliation: From the Department of Structural Biology, The State University of New York at Buffalo and.
authors.forename: Benjamin J
authors.initials: BJ
authors.lastname: Orlando
authors.affiliation: From the Department of Structural Biology, The State University of New York at Buffalo and the Hauptman-Woodward Medical Research Institute, Buffalo, New York 14203 mgm22@buffalo.edu.
authors.forename: Michael G
authors.initials: MG
authors.lastname: Malkowski
mesh_terms: D019943:Amino Acid Substitution; D001665:Binding Sites; D018360:Crystallography, X-Ray; D051546:Cyclooxygenase 2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D052246:Cyclooxygenase 2 Inhibitors / Q000737:chemistry* / Q000494:pharmacology*; D063388:Endocannabinoids / Q000378:metabolism; D054361:Fenamates / Q000737:chemistry / Q000494:pharmacology*; D006418:Heme / Q000737:chemistry; D006801:Humans; D066298:In Vitro Techniques; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010545:Peroxides / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D014364:Tryptophan / Q000737:chemistry
chemical_list: D052246:Cyclooxygenase 2 Inhibitors; D063388:Endocannabinoids; D054361:Fenamates; D010545:Peroxides; D011994:Recombinant Proteins; D006418:Heme; D014364:Tryptophan; D051546:Cyclooxygenase 2; C496540:PTGS2 protein, human
keywords: crystal structure; cyclooxygenase (COX); electron paramagnetic resonance (EPR); endocannabinoid; nonsteroidal anti-inflammatory drugs; structural biology