This proposal evaluates the hypothesis that N-(3-hydroxyphenyl)arachidonylamide (3-HPA) is a selective, mechanism-based inactivator of COX-2. COX-2 has important implications in inflammatory processes, cancer, and endocannabinoid metabolism. Metabolism of 3-HPA by COX-1 and COX-2 may provide insight into selectivity of endocannabinoid oxygenation, as well as the selectivity of COX-2 inactivation by 3-HPA. This will involve product characterization by tandem mass spectrometry and structure determination by x-ray crystallography. 3-HPA also represents the first metabolism-dependent, selective inactivator of COX-2. Quantification of 3-HPA oxidation by high performance liquid chromatography with UV detection, analysis of protein inactivation by thin layer chromatography, and determination of the site of modification by proteomic approaches involving electrospray ionization mass spectrometry will be important in characterizing the mechanism of inactivation. ? ?
