Structural basis of enantioselective inhibition of cyclooxygenase-1 by S-alpha-substituted indomethacin ethanolamides.
The modification of the nonselective nonsteroidal anti-inflammatory drug, indomethacin, by amidation presents a promising strategy for designing novel cyclooxygenase (COX)-2-selective inhibitors. A series of alpha-substituted indomethacin ethanolamides, which exist as R/S-enantiomeric pairs, provides a means to study the impact of stereochemistry on COX inhibition. Comparative studies revealed that the R- and S-enantiomers of the alpha-substituted analogs inhibit COX-2 with almost equal efficacy, whereas COX-1 is selectively inhibited by the S-enantiomers. Mutagenesis studies have not been able to identify residues that manifest the enantioselectivity in COX-1. In an effort to understand the structural impact of chirality on COX-1 selectivity, the crystal structures of ovine COX-1 in complexes with an enantiomeric pair of these indomethacin ethanolamides were determined at resolutions between 2.75 and 2.85 A. These structures reveal unique, enantiomer-selective interactions within the COX-1 side pocket region that stabilize drug binding and account for the chiral selectivity observed with the (S)-alpha-substituted indomethacin ethanolamides. Kinetic analysis of binding demonstrates that both inhibitors bind quickly utilizing a two-step mechanism. However, the second binding step is readily reversible for the R-enantiomer, whereas for the S-enantiomer, it is not. These studies establish for the first time the structural and kinetic basis of high affinity binding of a neutral inhibitor to COX-1 and demonstrate that the side pocket of COX-1, previously thought to be sterically inaccessible, can serve as a binding pocket for inhibitor association.
Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824, USA.
Christine A
CA
Harman
Melissa V
MV
Turman
Kevin R
KR
Kozak
Lawrence J
LJ
Marnett
William L
WL
Smith
R Michael
RM
Garavito
D000577:Amides / Q000737:chemistry; D000818:Animals; D001665:Binding Sites; D018360:Crystallography, X-Ray / Q000379:methods; D051545:Cyclooxygenase 1 / Q000737:chemistry* / Q000502:physiology*; D004305:Dose-Response Relationship, Drug; D007213:Indomethacin / Q000737:chemistry*; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D008968:Molecular Conformation; D016296:Mutagenesis; D011485:Protein Binding; D012756:Sheep; D013237:Stereoisomerism
D000577:Amides; D051545:Cyclooxygenase 1; D007213:Indomethacin