Arg-513 and Leu-531 Are Key Residues Governing Time-Dependent Inhibition of Cyclooxygenase-2 by Aspirin and Celebrex.

Abstract

Aspirin and Celebrex are well-known time-dependent inhibitors of the Cyclooxygenases (COX). Molecular dynamics simulations suggest that Arg-513 and Leu-531 contribute to the structural mechanisms of COX inhibition. We used mutagenesis and functional analyses to characterize how substitutions at these positions influence time-dependent inhibition by aspirin and Celebrex. We show that substitutions of Leu-531 with asparagine and phenylalanine significantly attenuate time-dependent inhibition of COX-2 by these drugs. The introduction of side chain bulk, rigidity, and charge would disrupt the formation of the initial non-covalent complex, in the case of aspirin, and the high-affinity biding state, in the case of Celebrex. Substitution of Arg-513 with histidine (the equivalent residue in COX-1) resulted in a 2-fold potentiation of aspirin inhibition, in support of the hypothesis that the presence of histidine in COX-1 lowers the activation barrier associated with the formation of the initial non-covalent enzyme-inhibitor complex. As a corollary, we previously hypothesized that the flexibility associated with Leu-531 contributes to the binding of AA to acetylated COX-2 to generate 15(R)-hydroxyeicosatetraenoic acid (15R-HETE). We determined the X-ray crystal structure of AA bound to Co3+-protoporphyrin IX reconstituted V349I murine COX-2. V349I muCOX-2 was utilized as a surrogate to trap AA in a conformation leading to 15R-HETE. AA binds in a C-shaped pose, facilitated by the rotation of the Leu-531 side chain. Ile-349 is positioned to sterically shield antarafacial oxygen addition at carbon-15 in a manner similar to that proposed for the acetylated Ser-530 side chain.