| Summary: | The cyclooxygenase enzymes (COX-1 and COX-2) catalyze the conversion arachidonic acid (AA) to prostaglandin H2 (PGH2), which is the precursor to biologically active prostanoids. The primary mechanism of action of non-steroidal anti-inflammatory drugs (NSAIDs) is the inhibition of prostaglandin biosynthesis by binding within the active site of the COX enzymes. Naproxen, a non-selective NSAID, has been marketed as analgesic and anti-inflammatory agent for over thirty years. In the work presented herein, the structure and dynamics of naproxen binding to COX is elucidated. We determined a 1.7 Å crystal structure of naproxen complexed to COX-2, which indicates that naproxen is bound similarly to other arylpropionic acid inhibitors with the carboxylate moiety making critical interactions at the base of the active site. Interestingly, we identified a novel interaction at the top of the active site between Trp-387 and the p-methoxy moiety of naproxen. Each of the major functional groups of naproxen is required for inhibitory activity suggesting that the development of more potent and/or COX-2 preferring naproxen analogs may be difficult. However, we have synthesized derivatives of naproxen with single atom modifications at the 6-position resulting in COX-2-preferring inhibitors. The crystal structure of one of these analogs, p-methylthio naproxen, bound to COX-2 suggests that the analogs occupy relatively the same conformation as naproxen within the active site.
COX-2 has the ability to metabolize alternative fatty acid substrates in addition to AA including the endocannabinoids, 2-arachidonoylglycerol (2-AG) and anandamide (AEA). Previous studies have shown that ibuprofen and mefenamic acid are weak, competitive inhibitors of COX-2 mediated AA metabolism, but potent, non-competitive inhibitors of 2-AG oxygenation; this phenomenon was dubbed substrate-selective inhibition. In the present work, we demonstrate that a series of reversible inhibitors are significantly more potent inhibitors of 2-AG oxygenation compared to AA whereas a series of tight-binding inhibitors block the oxygenation of both substrates by COX-2 with comparable potency. Furthermore, (R)-arylpropionates, which were previously thought to lack COX inhibitory activity, are potent inhibitors of COX-2-mediated 2-AG oxygenation. A highly substrate-selective inhibitor may represent a novel analgesic agent that lacks the deleterious side effects associated with the use of traditional NSAIDs.
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