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J Biol Chem. 2016 Jul 15;291(29):15069-81. doi: 10.1074/jbc.M116.725713. Epub 2016 May 20.

Substrate-selective Inhibition of Cyclooxygeanse-2 by Fenamic Acid Derivatives Is Dependent on Peroxide Tone.

Author information

1
From the Department of Structural Biology, The State University of New York at Buffalo and.
2
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.

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.

KEYWORDS:

crystal structure; cyclooxygenase (COX); electron paramagnetic resonance (EPR); endocannabinoid; nonsteroidal anti-inflammatory drugs; structural biology

PMID:
27226593
PMCID:
PMC4946924
DOI:
10.1074/jbc.M116.725713
[Indexed for MEDLINE]
Free PMC Article

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