Naproxen ((S)-6-methoxy-␣-methyl-2-naphthaleneacetic acid)is a powerful non-selective non-steroidal anti-inflammatory drug that is extensively used as a prescription and over-thecounter medication. Naproxen exhibits gastrointestinal toxicity, but its cardiovascular toxicity may be reduced compared with other drugs in its class. Despite the fact that naproxen has been marketed for many years, the molecular basis of its interaction with cyclooxygenase (COX) enzymes is unknown. We performed a detailed study of naproxen-COX-2 interactions using site-directed mutagenesis, structure-activity analysis, and x-ray crystallography. The results indicate that each of the pendant groups of the naphthyl scaffold are essential for COX inhibition, and only minimal substitutions are tolerated. Mutation of Trp-387 to Phe significantly reduced inhibition by naproxen, a result that appears unique to this inhibitor. Substitution of S or CH 2 for the O atom of the p-methoxy group yielded analogs that were not affected by the W387F substitution and that exhibited increased COX-2 selectivity relative to naproxen. Crystallization and x-ray analysis yielded structures of COX-2 complexed to naproxen and its methylthio analog at 1.7 and 2.3 Å resolution, respectively. The combination of mutagenesis, structure analysis, and x-ray crystallography provided comprehensive information on the unique interactions responsible for naproxen binding to COX-2. Cyclooxygenase (COX)4 enzymes are the targets for inhibition by a diverse array of non-steroidal anti-inflammatory drugs (NSAIDs), which contain functional groups, such as arylacetic acids, arylpropionic acids, ␤-ketoenols, and diarylheterocycles. Investigation of the molecular determinants of inhibition by different classes of compounds reveals that the protein residues in the active site maintain similar orientations and that each chemical class forms distinct sets of interactions within the active site (1). Compounds with nanomolar binding affinity (and, in many cases, COX-2 selectivity) have been successfully designed for multiple chemical series, despite their diverse binding modes.Naproxen is one of the oldest and largest selling NSAIDs (Fig. 1). It was introduced in prescription form as Naprosyn in 1976 and as the over-the-counter drug Aleve in 1994. It exhibits analgesic, anti-pyretic, and anti-inflammatory activity and was recently reported to be effective in the prevention of bladder cancer progression even when administered several weeks after the tumor-initiating agent (2). Naproxen is a non-selective NSAID that inhibits both COX-1 and COX-2 with comparable IC 50 values (3). It exhibits significant gastrointestinal side effects, but recent mounting evidence suggests that it does not exert cardiovascular side effects when administered in the higher doses that provide sustained inhibition of platelet COX-1 throughout the dosing interval (e.g. Ն500 mg twice daily) (4 -6). This latter property has taken on increasing importance because evolving data suggest that the cardiovascular toxicit...

Cyclooxygenase-2 (COX-2) catalyzes the oxygenation of arachidonic acid and the endocannabinoids, 2-arachidonoylglycerol and arachidonoylethanolamide. Evaluation of a series of COX-2 inhibitors revealed that many weak, competitive inhibitors of arachidonic acid oxygenation are potent inhibitors of endocannabinoid oxygenation. (R)-Enantiomers of ibuprofen, naproxen, and flurbiprofen, which are considered to be inactive as COX-2 inhibitors, are potent “substrate-selective inhibitors” of endocannabinoid oxygenation. Crystal structures of the COX-2-(R)-naproxen and COX-2-(R)-flurbiprofen complexes verified this unexpected binding and defined the orientation of the (R)-enantiomers relative to (S)-enantiomers. (R)-Profens selectively inhibited endocannabinoid oxygenation by lipopolysaccharide-stimulated dorsal root ganglion cells. Substrate-selective inhibition provides novel tools for investigating the role of COX-2 in endocannabinoid oxygenation and a possible explanation for the ability of (R)-profens to maintain endocannabinoid tone in models of neuropathic pain.

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Cyclooxygenase enzymes house spatially distinct cyclooxygenase- and peroxidase-active sites. The two-electron reduction of peroxides to their corresponding alcohols by the heme bound in the peroxidase-active site converts the heme to a ferryloxoprotoporyphrin cation radical, with a reductant providing the two electrons necessary to bring the heme back to its resting state. The ferryloxoprotoporyphrin cation radical can abstract a hydrogen atom from a tyrosine residue in the cyclooxygenase-active site, activating the oxygenase functionality. The tyrosyl radical subsequently abstracts a hydrogen atom from the cyclooxygenase substrate, arachidonic acid, leading to its oxygenation and the formation of a hydroperoxy endoperoxide intermediate, PGG(2). The peroxidase functionality reduces PGG(2) to the hydroxy endoperoxide, PGH(2), which serves as the precursor to downstream prostaglandins and thromboxane. The peroxidase activity of cycloxygenase enzymes can be assayed by quantifying the oxidation of a peroxidase reductant or the reduction of a hydroperoxide substrate. Here we describe a spectrophotometric assay used to measure the oxidation of a reductant, 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), as well as a high-performance liquid chromatography method for the measurement of the conversion of 5-phenyl-4-pentyl hydroperoxide (PPHP) to its corresponding alcohol. The first provides a continuous but indirect assay of peroxidase activity, whereas the second provides a discontinuous but direct assay.