O-Acetylsalicylhydroxamic Acid, a Novel Acetylating Inhibitor of Prostaglandin H₂Synthase: Structural and Functional Characterization of Enzyme-Inhibitor Interactions

Abstract

Aspirin is unique among clinically used nonsteroidal antiinflammatory drugs in that it irreversibly inactivates prostaglandin (PG) H2 synthase (PGHS) via acetylation of an active-site serine residue. We report the synthesis and characterization of a novel acetylating agent, O-acetylsalicylhydroxamic acid (AcSHA), which inhibits PGE2 synthesis in vivo and blocks the cyclooxygenase activity of PGHS in vitro. AcSHA requires the presence of the active-site residue Ser-529 to be active against human PGHS-1; the S529A mutant is resistant to inactivation by the inhibitor. Analysis of PGHS inactivation by AcSHA, coupled with the X-ray crystal structure of the complex of ovine PGHS-1 with AcSHA, confirms that the inhibitor elicits its effects via acetylation of Ser-529 in the cyclooxygenase active site. The crystal structure reveals an intact inhibitor molecule bound in the enzyme's cyclooxygenase active-site channel, hydrogen bonding with Arg-119 of the enzyme. The structure-activity profile of AcSHA can be rationalized in terms of the crystal structure of the enzyme-ligand complex. AcSHA may prove useful as a lead compound to facilitate the development of new acetylating inhibitors.

Structural and Functional Basis of Cyclooxygenase Inhibition

“…57,59 The presence of Tyr-385 across the active site from Ser-530 appears to be a critical determinant of acetylation. 41,60 Mutation of Tyr-385 to Phe reduces aspirin acetylation of the serine hydroxyl by 93%.…”

“…Mutation of Arg-120 to Gln or Ala eliminates ion-pairing and hydrogenbonding interactions with the salicylate group and significantly reduces aspirin acetylation of Ser-530. 58 Another aspirin analogue, o-acetylsalicylhydroxamic acid, 59 also binds in the COX-1 active site channel, acetylates Ser-530, and hydrogen-bonds with Arg-120 at the constriction site. The acetyl group on Ser-530 projects into the active site immediately below Tyr-385, closing off the top of the channel and blocking the access of substrate to the catalytic tyrosyl radical.…”

Maintaining Equilibrium by Selective Targeting of Cyclooxygenase Pathways

“…77 In addition to heme oxidation by ROS and NO x species, the COX peroxidase site can oxidize electron donor compounds such as polycyclic aromatic hydrocarbons from cigarette smoke. The latter reaction can lead to polar, highly reactive, and strongly mutagenic intermediates that attack DNA and proteins.…”

Heme catalyzes tyrosine 385 nitration and inactivation of prostaglandin H2 synthase-1 by peroxynitrite

“…This assay monitors the oxidation of TMPD by changes in absorbance at 611 nm after activation (33,34). In some experiments, ONOO 2 was used as the activating species.…”