Kinetic basis for selective inhibition of cyclo-oxygenases

Non-steroidal anti-inflammatory drugs (NSAIDs) inhibit the formation of prostaglandins by cyclo-oxygenases (COX). The discovery of a second COX isoform (COX-2) associated with inflammation led to agents that selectively inhibit COX-2, e.g. celecoxib. We evaluated the kinetics of inhibition of celecoxib and several NSAIDs. Celecoxib displays classic competitive kinetics on COX-1 (K-i = 10-16 mu M). An initial competitive interaction with COX-2 can also be discerned with celecoxib (K-i = 11-15 mu M), followed by a time-dependent interaction leading to potent inhibition, characterized as inactivation (K-inact = 0.03-0.5 s(-1)). Half-maximal inhibition (IC50) using endpoint assays reflects the competitive component on COX-1 (IC50 = 4-19 mu M) and the inactivation component on COX-2 (IC50 = 0.003-0.006 mu M). NSAIDs exhibit four distinct modes of COX inhibition based on kinetic behaviour: (1) competitive, e.g. ibuprofen; (2) weak binding, time-dependent, e.g. naproxen, oxicams; (3) tight binding, time-dependent, e.g. indomethacin; (4) covalent, e.g. aspirin. In addition, most NSAIDs display different kinetic behaviour for each isoform. Weakly binding inhibitors show variable behaviour in enzyme assays, with apparent inhibitory activity being markedly influenced by experimental conditions; determination of kinetic constants with this class is unreliable and IC50 values are strongly dependent on assay conditions. Although IC50 determinations are useful for structure/activity analyses, the complex and distinct mechanisms of enzyme inhibition of each COX isoform by the NSAIDs renders comparison of inhibitory activity on COX-I and COX-2 using IC50 ratios of questionable validity.