title: Kinetic basis for selective inhibition of cyclo-oxygenases.
abstract: 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 (Ki=10-16 microM). An initial competitive interaction with COX-2 can also be discerned with celecoxib (Ki=11-15 microM), followed by a time-dependent interaction leading to potent inhibition, characterized as inactivation (Kinact=0.03-0.5 s-1). Half-maximal inhibition (IC50) using end-point assays reflects the competitive component on COX-1 (IC50=4-19 microM) and the inactivation component on COX-2 (IC50=0.003-0.006 microM). 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-1 and COX-2 using IC50 ratios of questionable validity.
authors.affiliation: Monsanto/Searle Discovery Research, 700 Chesterfield Parkway North, Chesterfield, MO 63198, USA. James.K.Gierse@Monsanto.Com
authors.forename: J K
authors.initials: JK
authors.lastname: Gierse
authors.forename: C M
authors.initials: CM
authors.lastname: Koboldt
authors.forename: M C
authors.initials: MC
authors.lastname: Walker
authors.forename: K
authors.initials: K
authors.lastname: Seibert
authors.forename: P C
authors.initials: PC
authors.lastname: Isakson
mesh_terms: D000818:Animals; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000378:metabolism* / Q000494:pharmacology; D001667:Binding, Competitive; D000068579:Celecoxib; D051545:Cyclooxygenase 1; D051546:Cyclooxygenase 2; D052246:Cyclooxygenase 2 Inhibitors; D016861:Cyclooxygenase Inhibitors / Q000378:metabolism* / Q000494:pharmacology; D015232:Dinoprostone / Q000378:metabolism; D006801:Humans; D007052:Ibuprofen / Q000494:pharmacology; D007213:Indomethacin / Q000494:pharmacology; D020128:Inhibitory Concentration 50; D007527:Isoenzymes / Q000378:metabolism; D007700:Kinetics; D008297:Male; D008565:Membrane Proteins; D051379:Mice; D009288:Naproxen / Q000494:pharmacology; D010100:Oxygen / Q000378:metabolism; D011451:Prostaglandin-Endoperoxide Synthases / Q000378:metabolism*; D011720:Pyrazoles; D012756:Sheep; D013329:Structure-Activity Relationship; D013449:Sulfonamides / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D013772:Tetramethylphenylenediamine / Q000378:metabolism; D013816:Thermodynamics
chemical_list: D000894:Anti-Inflammatory Agents, Non-Steroidal; D052246:Cyclooxygenase 2 Inhibitors; D016861:Cyclooxygenase Inhibitors; D007527:Isoenzymes; D008565:Membrane Proteins; D011720:Pyrazoles; D013449:Sulfonamides; D009288:Naproxen; D051545:Cyclooxygenase 1; D051546:Cyclooxygenase 2; C496511:PTGS1 protein, human; C496540:PTGS2 protein, human; D011451:Prostaglandin-Endoperoxide Synthases; C496512:Ptgs1 protein, mouse; D000068579:Celecoxib; D015232:Dinoprostone; D013772:Tetramethylphenylenediamine; D010100:Oxygen; D007052:Ibuprofen; D007213:Indomethacin