Mechanism of selective inhibition of the inducible isoform of prostaglandin G/H synthase.
Selective inhibition of the inducible isoform of prostaglandin G/H synthase (cyclooxygenase-2; COX2; EC 1.14.99.1) can be achieved with compounds of the general form of aryl methyl sulfonyls and aryl methyl sulfonamides. DuP 697 and NS-398 are representative examples of these compounds. Both inhibit the constitute (COX1) and inducible (COX2) isoforms of the enzyme with equal potency shortly after mixing, but their potencies increase with time for COX2 selectively. This time-dependent inhibition follows first-order kinetics, and the rate constant for inactivation of COX2 is dose dependent for both compounds. Kinetic analysis allows us to determine KI and kinact (the maximal rate of inactivation) for each inhibitor. The potency of both compounds is substrate concentration dependent, as expected for time-dependent competitive inhibitors. COX2 that has been incubated with these inhibitors, and then extensively dialyzed against buffer, shows no recovery of enzyme activity, while complete recovery of activity is seen for COX1. Thus, these inhibitors irreversibly inactivate COX2 with time, while showing minimal reversible inhibition of COX1. We isolated these inhibitors after long incubation with excess enzyme and subsequent denaturation of the enzyme. Both inhibitors showed no loss of potency resulting from interactions with COX2, suggesting that inhibition is not mediated by covalent modification of the enzyme. These data suggest that binding of these inhibitors to COX2 induces a slow structural transition of the enzyme that results in its selective inactivation.
DuPont Merck Research Laboratories, Wilmington, DE 19880-0400.
R A
RA
Copeland
J M
JM
Williams
J
J
Giannaras
S
S
Nurnberg
M
M
Covington
D
D
Pinto
S
S
Pick
J M
JM
Trzaskos
D000818:Animals; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000737:chemistry / Q000494:pharmacology*; D001665:Binding Sites; D016861:Cyclooxygenase Inhibitors / Q000737:chemistry; D004790:Enzyme Induction; D006801:Humans; D066298:In Vitro Techniques; D007527:Isoenzymes / Q000037:antagonists & inhibitors; D007700:Kinetics; D009578:Nitrobenzenes / Q000737:chemistry / Q000494:pharmacology*; D011451:Prostaglandin-Endoperoxide Synthases / Q000378:metabolism*; D011994:Recombinant Proteins; D012756:Sheep; D013449:Sulfonamides / Q000737:chemistry / Q000494:pharmacology*; D013876:Thiophenes / Q000494:pharmacology*
D000894:Anti-Inflammatory Agents, Non-Steroidal; D016861:Cyclooxygenase Inhibitors; D007527:Isoenzymes; D009578:Nitrobenzenes; D011994:Recombinant Proteins; D013449:Sulfonamides; D013876:Thiophenes; C080955:N-(2-cyclohexyloxy-4-nitrophenyl)methanesulfonamide; C064874:DuP 697; D011451:Prostaglandin-Endoperoxide Synthases