Fluorescence quenching analysis of the association and dissociation of a diarylheterocycle to cyclooxygenase-1 and cyclooxygenase-2: dynamic basis of cyclooxygenase-2 selectivity.
Cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) are the enzymes responsible for the biosynthesis of the precursor to the biologically active prostaglandins, prostacyclin, and thromboxane and are the molecular targets for nonsteroidal antiinflammatory drugs (NSAIDs). Selective COX-2 inhibitors are antiinflammatory and analgesic but lack gastrointestinal toxicity, an undesirable side effect attributed to COX-1 inhibition. Crystallographic analysis of selective COX inhibitors complexed with either isoform provides some information about the molecular determinants of selectivity but does not provide information about the dynamics of inhibitor association/dissociation. We employed rapid-mixing techniques and fluorescence quenching to monitor the association and dissociation of a selective COX-2 inhibitor to COX-1 or COX-2. The association of the fluorescent diaryloxazole, SC299, with both enzymes occurs in a time-dependent fashion. Its binding to COX-2 occurs in three kinetically distinct steps whereas its binding to COX-1 occurs in two steps. In contrast to the relatively rapid association of SC299 with both enzymes, its dissociation from COX-2 is quite slow and occurs over several hours whereas the dissociation from COX-1 is complete in less than 1 min. The selectivity of SC299 as a COX-2 inhibitor correlates to its relative rates of dissociation from the two COX isoforms. A model is proposed for diarylheterocycle binding to COX's that integrates these kinetic data with available structural information.
Lanzo
C A
CA
Departments of Biochemistry and Chemistry, Center in Molecular Toxicology, Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
Sutin
J
J
Rowlinson
S
S
Talley
J
J
Marnett
L J
LJ
D000818:Animals; D001667:Binding, Competitive; D051545:Cyclooxygenase 1; D051546:Cyclooxygenase 2; D052246:Cyclooxygenase 2 Inhibitors; D016861:Cyclooxygenase Inhibitors / Q000737:chemistry*; D004735:Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry; D005480:Flurbiprofen / Q000737:chemistry; D007527:Isoenzymes / Q000737:chemistry*; D007700:Kinetics; D008297:Male; D008565:Membrane Proteins; D051379:Mice; D010080:Oxazoles / Q000737:chemistry*; D011451:Prostaglandin-Endoperoxide Synthases / Q000737:chemistry*; D011485:Protein Binding; D012756:Sheep; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics
D052246:Cyclooxygenase 2 Inhibitors; D016861:Cyclooxygenase Inhibitors; D005456:Fluorescent Dyes; D007527:Isoenzymes; D008565:Membrane Proteins; D010080:Oxazoles; C409370:SC 299; D005480:Flurbiprofen; D051545:Cyclooxygenase 1; D051546:Cyclooxygenase 2; D011451:Prostaglandin-Endoperoxide Synthases; C496512:Ptgs1 protein, mouse