Rapid inactivation of prostaglandin endoperoxide synthases by N-(carboxyalkyl)maleimides

N-(Carboxyalkyl)maleimides were synthesized as potential inhibitors of prostaglandin endoperoxide synthase (PGHS). Inactivation of the cyclooxygenase and peroxidase activities of PGHS occurred in a biphasic manner with extremely rapid inactivation followed by slow, time-dependent inactivation. The carboxylic acid moiety was required for rapid inactivation. Optimal inhibition was observed with N-(carboxyheptyl)maleimide, which inhibited the cyclooxygenase activity of ovine PGHS-1 with an IC50 of 0.1 microM and the peroxidase activity with an IC50 of 3 microM. Inactivation of peroxidase activity was not prevented by pretreating the enzyme with the cyclooxygenase inhibitor indomethacin. N-(Carboxyheptyl)-succinimide inhibited neither enzyme activity, suggesting that covalent modification is critical for rapid as well as time-dependent inactivation. Shortening or increasing the alkyl chain by one methylene unit drastically reduced inhibitory potency. N-(Carboxyalkyl)maleimides also instantaneously inactivated the inducible form of PGHS (PGHS-2) from mouse and human sources but with higher IC50's (4.5 and 14 microM, respectively). N-(Carboxyheptyl)maleimide is the most potent covalent inactivator of PGHS yet described with an inhibitory potency 3-5 orders of magnitude greater than aspirin.