Molecular Basis for Cyclooxygenase Inhibition by the Non-steroidal Anti-inflammatory Drug Naproxen

Abstract

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...

Pre-existent Asymmetry in the Human Cyclooxygenase-2 Sequence Homodimer

“…S530A/S530A huPGHS-2), significant accumulation of unreacted [1-14 C]AA occurred and was attributable to the binding of unreacted [1-14 C]AA in the COX site of E allo . [1-14 C]AA was displaced from S530A/S530A huPGHS-2 by PA, but PA was unable to displace [1][2][3][4][5][6][7][8][9][10][11][12][13][14] C]AA from Y385F S530A/ Native huPGHS-2. Again, this is consistent with a lack of stimulation of this latter mutant heterodimer by PA. We interpret these results to mean that Ser-530 partners with Tyr-385 in the allosteric regulation of huPGHS-2 by nonsubstrate FAs.…”

“…Various amounts of guanidine hydrochloride were then added, and the samples were incubated at room temperature for 30 min. After centrifugation, the denaturant-treated proteins were incubated again with 1 mM [1][2][3][4][5][6][7][8][9][10][11][12][13][14] C]acetylsalicylate for 30 min at room temperature. Total aspirin acetylation was quantified by liquid scintillation counting as described above.…”

Impact of chirality on the photoinduced charge transfer in linked systems containing naproxen enantiomers

“…Namely, only (S)-NPX is a real inhibitor of arachidonic acid oxygenation 16 (anti-inflammatory effect) but all 2-aryl propionyl derivatives are potent inhibitors of endocannabinoid oxygenation 7 (analgetic effect) and (R)-NPX more actively undergoes chiral metabolism. 8 Our results show the prevailing of the (R,S)-dyad exciplex fluorescence quantum yields (Φexc, up to two times) and the rate constants of the exciplex formation (k4, in half times), as well as the different CIDNP effects of the optical isomers.…”

“…8 For that, according to the results of biochemical research, there is no complete understanding, for example about what kind of physicochemical interactions are responsible for the difference in the action of (S)-and (R)-NPX. 7,16 Taking into consideration the above-mentioned possible involvement of NPX optical isomers in charge transfer processes, it seems promising to use a model one-electron transfer process for studying the chemical nature of the difference between (S)-and (R)-isomers. In this connection, the comparison of (S)-and (R)-NPX reactivity in one of the most universal elementary processes, electron transfer, may have not only of a fundamental but also of a practical interest.…”

“…11,12,14 The stereoselectivity of NSAIDs in biological chemistry is investigated, specifically with respect to their main function of the inhibition of COX 2. 16 The COX 2 enzyme has several active sites: one catalytic site provides cyclization, and another one performs oxidation (involving electron transfer (ET)).…”