THE INTERACTION OF ARGININE-106 OF HUMAN PROSTAGLANDIN G H SYNTHASE-2WITH INHIBITORS IS NOT A UNIVERSAL COMPONENT OF INHIBITION MEDIATED BY NONSTEROIDAL ANTIINFLAMMATORY DRUGS/

The three-dimensional cocrystal structures of ovine prostaglandin G/H synthase-1 (PGHS-1) with S-flurbiprofen and murine PGHS-2 with S-flurb iprofen and indomethacin reveal that the carboxylate acid groups of th ese nonsteroidal anti-inflammatory drugs (NSAIDs) form a salt bridge w ith the guanidinium group of Arg120 in PGHS-1 and Arg106 in PGHS-2. Mu tagenesis studies confirmed that the Arg120 residue of PGHS-1 is criti cal for binding of substrate and inhibitors through ionic interactions of its guanidinium group with the carboxylate moieties of arachidonic acid and certain NSAIDs. We report here that the analogous R106E subs titution in human PGHS-2 results in a catalytically active enzyme with a 30-fold higher K-m value for arachidonic acid. Comparison of the in hibition of hPGHS-2(R106E) with wild-type hPGHS-2 by 11 structurally d iverse selective and nonselective PGHS inhibitors revealed a 0-1000-fo ld decrease in inhibitory potency on the mutant enzyme. The loss of in hibitory potency of NSAIDs on hPGHS-2(R106E) could not be correlated w ith the presence or absence of a carboxylate functional group in the i nhibitor, as was demonstrated previously for the PGHS-1(R120E) mutant, or with the selective or nonselective nature of the PGHS inhibitor. T he decreases in the inhibitory potencies on hPGHS-2(R106E) by the carb oxylate-containing NSAIDs flurbiprofen, indomethacin, meclofenamic aci d, and diclofenac on hPGHS-2(R106E) were 965-, 48-, 5.5-, and 4.5-fold , respectively. The nonuniversal requirement for interaction of the ca rboxylate group of certain NSAIDs with the Arg106 residue in hPGHS-2 i s supported by the observation that the methyl ester derivative of ind omethacin was a more potent inhibitor than indomethacin on both hPGHS- 2 and hPGHS-2(R106E). The greatest loss of potency for inhibition of h PGHS-2(R106E) was observed with the hPGHS-2-selective sulfonamide-cont aining inhibitors NS-398 and flosulide. The PGHS-2-selective inhibitor DuP697 and a desbromo-sulfonamide analogue of DuP697 displayed equiva lent potency on hPGHS-2(R106E) and hPGHS-2. The change in inhibitory p otency of NS-398 on hPGHS-2(R106E) was due to a difference in the kine tics of inhibition, with NS-398 displaying time-dependent inhibition o f hPGHS-2 but time-independent inhibition of PGHS-2(R106E). The time-d ependent inhibition of hPGHS-2 by DuP697 was not affected by the prese nce of the R106E mutation. We conclude that the Arg106 residue of hPGH S-2 is involved in binding arachidonic acid and certain NSAIDs, but in teractions with Arg106 are not a universal requirement for inhibition by either carboxylate-containing NSAIDs or PGHS-2-selective inhibitors .