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.
Highlights
The cyclooxygenases (COX-1 and COX-2) convert arachidonic acid (AA)2 to prostaglandin H2 [1]
COX-1 preferentially oxygenates AA, whereas COX-2 efficiently oxygenates a broad spectrum of fatty acid, ester, and amide substrates, including the endocannabinoids 1-arachidonoyl glycerol (1-AG), 2-arachidonoyl glycerol, and anandamide [3,4,5,6,7,8]. 2-Arachidonoyl glycerol and anandamide are widely distributed in mammalian tissues and were the first characterized endogenous ligands for the cannabinoid receptors CB1 and CB2 [9]
Endocannabinoid substrates bind in a similar fashion to AA in the cyclooxygenase active site [16], endocannabinoid oxygenation is sensitive to peroxide tone and requires more turnover of hydroperoxide to sustain cyclooxygenase catalysis, suggesting that the branched chain mechanism is less efficient with endocannabinoid substrates [17]
Summary
Materials—Meclofenamic acid sodium salt, diclofenac sodium salt, AA, 1-AG, and R-flurbiprofen were purchased from Cayman Chemical Co. (Ann Arbor, MI). To generate the remaining complexes, Co3ϩ-protoporphyrin IX reconstituted huCOX-2 at 4 mg/ml was incubated with a 5-fold molar excess of inhibitor followed by crystallization using the sitting drop vapor diffusion method. All four huCOX-2 inhibitor complexes crystallized with two molecules in the asymmetric unit in orthorhombic space group I222, the unit cell parameters for crystals grown in polyacrylic acid 5100 and PEG 400 differ significantly (Table 1) Given these observations, difference Fourier maps were not utilized to generate initial phases, and extensive care was taken to remove model bias during structure solution. UV-Visible Spectroscopy—To determine the effect that tolfenamic acid had on the formation of heme oxidation states, wild type huCOX-2 (3 M monomer) in 100 mM Tris, pH 8.0, was complexed with 3 M Fe3ϩ-protoporphyrin IX in a stirred quartz cuvette at 25 °C. Quency, 2G modulation amplitude, and a time constant of 10.24 ms
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