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 timedependent 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 noncovalent complex, in the case of aspirin, and the "high-affinity" binding 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 noncovalent enzyme−inhibitor complex. As a corollary, we previously hypothesized that the flexibility associated with Leu-531 contributes to the binding of arachidonic acid (AA) to acetylated COX-2 to generate 15R-hydroxyeicosatetraenoic acid (15R-HETE). We determined the X-ray crystal structure of AA bound to Co 3+protoporphyrin IX-reconstituted V349I murine COX-2 (muCOX-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.

Targeting Lipid Peroxidation for Cancer Treatment

“…Its implication in cancer has been associated with COX inhibition via acetylation of the active site, where AA binds. This accounts for COX-1 isozyme inhibition, while in COX-2, aspirin binding produces a structural rearrangement shifting the cyclooxygenase towards the lipoxygenase activity [86,87]. Therefore, AA oxygenation and cyclization to form a 15R-Prostaglandin endoperoxide is promoted, which favors the production of Prostaglandin D 2 (PGD 2 ) (with has a suggested function in inflammation resolution) instead of PGE 2 [88].…”

Structural and Chemical Biology of the Interaction of Cyclooxygenase with Substrates and Non-Steroidal Anti-Inflammatory Drugs

“… 136 However, the presence of the extra methyl group in threonine as opposed to serine must influence the substrate’s conformation in such a way as to result in a change in the orientation of oxygen addition at carbon-15. Furthermore, the presence of the larger side chain of Thr-530 is predicted to alter the binding pose of AA in such a way as to require movement of Leu-531 in order to accommodate the C-terminal end of the substrate, as was observed in the crystal structure of a complex of V349I COX-2 with AA 56 (see Section 4.1.2 ). To test this hypothesis, the effects of L531F and L531N mutations on products formed following aspirin acetylation were explored.…”

“…In an attempt to explain the change in stereochemistry observed with the Ser-530 COX-2 mutant enzymes, which is also observed upon aspirin acetylation of COX-2 (see Section 5.2.10 ), a 2.2 Å resolution crystal structure of V349I COX-2 in complex with Co 3+ -protoporphyrin IX and AA (PDB 6OFY) was obtained. 56 This enzyme was selected due to its production of mostly 15( R )-HETE and 15( R )-PGH 2 as products. 47 The findings revealed that AA is bound in an inverted, unproductive conformation in one subunit and a productive conformation in the second.…”

“… 137 Support for this hypothesis was provided through site-directed mutation studies that revealed an increase in sensitivity to aspirin in R513H COX-2 as compared to the wild-type enzyme. 56 …”

“…These observations were explained on the basis of a postulated interference by the substituted side chains on the initial binding interaction of aspirin in the cyclooxygenase active site. 56 …”