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. 2010 Mar 23;107(12):5411-6.
doi: 10.1073/pnas.0913377107. Epub 2010 Mar 9.

Molecular Basis of Cyclooxygenase Enzymes (COXs) Selective Inhibition

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Free PMC article

Molecular Basis of Cyclooxygenase Enzymes (COXs) Selective Inhibition

Vittorio Limongelli et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

The widely used nonsteroidal anti-inflammatory drugs block the cyclooxygenase enzymes (COXs) and are clinically used for the treatment of inflammation, pain, and cancers. A selective inhibition of the different isoforms, particularly COX-2, is desirable, and consequently a deeper understanding of the molecular basis of selective inhibition is of great demand. Using an advanced computational technique we have simulated the full dissociation process of a highly potent and selective inhibitor, SC-558, in both COX-1 and COX-2. We have found a previously unreported alternative binding mode in COX-2 explaining the time-dependent inhibition exhibited by this class of inhibitors and consequently their long residence time inside this isoform. Our metadynamics-based approach allows us to illuminate the highly dynamical character of the ligand/protein recognition process, thus explaining a wealth of experimental data and paving the way to an innovative strategy for designing new COX inhibitors with tuned selectivity.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
The FES of the dissociation process as a function of the distance and dihedral CVs is shown at the bottom using isosurfaces of 2 kcal/mol. The four main energy basins AD found during the metadynamics simulations are highlighted in the FES graph. The four snapshots of the complex SC-558/COX-2 displayed in the surrounding boxes represent the following binding poses: basin A, crystallographic pose; basin B, alternative pose; basin C, poses at gate site; basin D, external pose. The ligand and the main interacting residues are displayed as licorice, while the protein is represented as a green cartoon with the α-helices forming the gate colored in orange. The interacting waters are shown as spheres, while hydrogens are not displayed for clarity. On the basis of the information derived from the FES, many aspects of the complex inhibition kinetics of COX-2 selective inhibitors can be elucidated. In fact, the presence of the second deep minimum (B) can explain the time-dependent behavior of many COX-2 selective inhibitors and, consequently, their long residence time inside the protein. In this respect we have been encouraged by the interpretation of flourescence quenching experiments of Lanzo et al., who have suggested for a SC-558 analogue an in and out movement from the selectivity pocket (8). They proposed that while in COX-1 the binding is a two-step process (see Fig. 4), in COX-2 a further not yet identified step takes place. Our calculations reveal the nature of this additional step, which considerably slows the off rate.
Fig. 4.
Fig. 4.
The FES of the undocking process of SC-558 in COX-1 as a function of the distance and dihedral CVs using isosurfaces of 2 kcal/mol. Only an internal and an external energy minimum have been found. Once this inner minimum has been filled, SC-558 moves in the direction of the helices that form the exit door. Close to the minimum a shallow wide basin is to be found whose conformation differs very slightly from that of the lowest minimum. When the ligand is in between the two helices and the gate is in the open conformation, it leaves the binding site moving to the external pose.
Fig. 2.
Fig. 2.
Schematic representation of the main binding sites found during our metadynamics simulations. The common and the selectivity pocket represent the SC-558 binding site in the crystallographic pose (basin A in Fig. 1), while the same common pocket with the side pocket represents the site for the alternative pose (basin B in Fig. 1). The gate site (basin C in Fig. 1) is when the ligand is in proximity of the protein gate assuming several similar conformations. Finally, the lobby-like site represents the binding site of SC-558 in its external pose (basin D in Fig. 1).
Fig. 3.
Fig. 3.
Comparison between (A) the alternative binding pose of SC-558 in COX-2 found during metadynamics simulations and (B) the x-ray binding conformation of ibuprofen in complex with COX-1 (PDB ID code 1eqg). The ligands and the interacting residues are represented as licorice, while the protein is represented as green cartoon with the α-helices forming the gate colored in orange. The hydrogens are not displayed for clarity.

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