The structure of ibuprofen bound to cyclooxygenase-2

Abstract

The cyclooxygenases (COX-1 and COX-2) catalyze the rate-limiting step in the biosynthesis of prostaglandins, and are the pharmacological targets of non-steroidal anti-inflammatory drugs (NSAIDs) and COX-2 selective inhibitors (coxibs). Ibuprofen (IBP) is one of the most commonly available over-the-counter pharmaceuticals in the world. The anti-inflammatory and analgesic properties of IBP are thought to arise from inhibition of COX-2 rather than COX-1. While an X-ray crystal structure of IBP bound to COX-1 has been solved, no such structure exists for the cognate isoform COX-2. We have determined the crystal structure of muCOX-2 with a racemic mixture of (R/S)-IBP. Our structure reveals that only the S-isomer of IBP was bound, indicating that the S-isomer possesses higher affinity for COX-2 than the R-isomer. Mutational analysis of Arg-120 and Tyr-355 at the entrance of the cyclooxygenase channel confirmed their role in binding and inhibition of COX-2 by IBP. Our results provide the first atomic level detail of the interaction between IBP and COX-2.

Keywords: Crystal structure; Cyclooxygenase; Ibuprofen; Nonsteroidal anti-inflammatory drugs; Prostaglandin H(2) synthase.

Copyright © 2014 Elsevier Inc. All rights reserved.

Figures

Figure 1. IBP Bound in the Cyclooxygenase Channel of COX-2

Stereo view of IBP bound within the cyclooxygenase channel of monomer A of the muCOX-2:IBP crystal structure. Fo-Fc simulated annealing omit map electron density (light blue), contoured at 3.5σ, is shown with the final refined model of IBP (pink). Residues lining the cyclooxygenase channel, along with the spatial locations of the channel opening (O), channel apex (A), and COX-2 specific side pocket (S) are labeled accordingly. Carbon atoms of residues lining the channel are colored green, while nitrogen, and oxygen atoms are colored blue and red, respectively.

Figure 2. Inhibition of COX activity by IBP

COX-2 was incubated with 100μM (R/S)-IBP on ice for 30 minutes before assaying residual COX activity at 37°C with a Clark-type oxygen electrode. The reaction cuvette contained 3mL of 100mM Tris (pH 8), 1mM phenol, 5μM Fe3+-PPIX, 100μM AA, and 100μM (R/S)-IBP. Reactions were initiated via injection of 5μg of COX-2, and were repeated in triplicate.