DESIGN, SYNTHESIS, AND BIOCHEMICAL EVALUATION OF N-SUBSTITUTED MALEIMIDES AS INHIBITORS OF PROSTAGLANDIN ENDOPEROXIDE SYNTHASES

N-(Carboxyalkyl)maleimides are rapid as well as time-dependent inhibit ors of prostaglandin endoperoxide synthase (PGHS). The corresponding N -alkylmaleimides were only time-dependent inactivators of PGHS, sugges ting that the carboxylate is critical for rapid inhibition. Several N- substituted maleimide analogs containing structural features similar t o those of the nonsteroidal anti-inflammatory drug aspirin were synthe sized and evaluated as inhibitors of PGHS. Most of the aspirin-like ma leimides inactivated the cyclooxygenase activity of purified ovine PGH S-1 in a time- and concentration-dependent manner similar to that of a spirin. The peroxidase activity of PGHS was also inactivated by the ma leimide analogs. The cyclooxygenase activity of the inducible isozyme, i.e., PGHS-2, was also inhibited by these compounds. The correspondin g succinimide analog of N-5-maleimido-2-acetoxy-1-benzoic acid did not inhibit either enzyme activity, suggesting that inactivation was due to covalent modification of the protein. The mechanism of inhibition o f PGHS-1 by N-(carboxyheptyl)maleimide was investigated. Incubation of apoPGHS-1 with 2 equiv of N-(carboxyheptyl)[3,4-C-14]maleimide led to the incorporation of radioactivity in the protein, but no adduct was detected by reversed-phase HPLC, suggesting that it was unstable to th e chromatographic conditions. Furthermore, hematin-reconstituted PGHS- 1, which was rapidly inhibited by N-(carboxyheptyl)maleimide, displaye d spontaneous regeneration of about 50% of the cyclooxygenase and pero xidase activities, suggesting that the adduct responsible for the inhi bition breaks down to regenerate active enzyme. ApoPGHS-1, inhibited b y N-(carboxyheptyl)maleimide, did not display regeneration of enzyme a ctivity, but addition of hematin to the inhibited apoenzyme led to spo ntaneous recovery of about 50% of cyclooxygenase activity. These resul ts suggest that addition of heme leads to a conformational change in t he protein which increases the susceptibility of the adduct toward hyd rolytic cleavage. ApoPGHS-1, pretreated with N-(carboxyheptyl)maleimid e, was resistant to trypsin cleavage, suggesting that the carboxylate functionality of the maleimide binds in the cyclooxygenase channel. A model for the interaction of N-(carboxyheptyl)maleimide in the cycloox ygenase active site is proposed.