MECHANISM OF SELECTIVE-INHIBITION OF THE INDUCIBLE ISOFORM OF PROSTAGLANDIN G H SYNTHASE/

Selective inhibition of the inducible isoform of prostaglandin G/H syn thase (cyclooxygenase-2; COX2; EC 1.14.99.1) can be achieved with comp ounds of the general form of aryl methyl sulfonyls and aryl methyl sul fonamides. DuP 697 and NS-398 are representative examples of these com pounds. Both inhibit the constitutive (COX1) and inducible (COX2) isof orms 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 in activation of COX2 is dose dependent for both compounds. Kinetic analy sis allows us to determine K-I and k(inact) (the maximal rate of inact ivation) for each inhibitor. The potency of both compounds is substrat e concentration dependent, as expected for time-dependent competitive inhibitors. COX2 that has been incubated with these inhibitors, and th en extensively dialyzed against buffer, shows no recovery of enzyme ac tivity, while complete recovery of activity is seen for COX1. Thus, th ese inhibitors irreversibly inactivate COX2 with time, while showing m inimal reversible inhibition of COX1. We isolated these inhibitors aft er long incubation with excess enzyme and subsequent denaturation of t he enzyme, Both inhibitors showed no loss of potency resulting from in teractions with COX2, suggesting that inhibition is not mediated by co valent modification of the enzyme. These data suggest that binding of these inhibitors to COX2 induces a slow structural transition of the e nzyme that results in its selective inactivation.