High-performance liquid chromatographic assay with fluorescence detection for the evaluation of inhibitors against fatty acid amide hydrolase.
A fluorescent assay for the evaluation of inhibitors of fatty acid amide hydrolase (FAAH) is described. Microsomes from rat brain served as enzyme source. N-(2-Hydroxyethyl)-4-pyren-1-ylbutanamide was designed and synthesized as novel fluorogenic substrate. For substrate solubilization, Triton X-100 was employed. The FAAH activity was determined directly without further sample clean-up by measuring the amount of 4-pyren-1-ylbutanoic acid released by the enzyme with reversed-phase HPLC and fluorescence detection. The known FAAH inhibitors URB597, phenyl hexanoyl oxazolopyridine (PHOP) and [6-(2-methyl-4,5-diphenyl-1H-imidazol-1-yl)hexyl]carbamic acid phenyl ester were used to validate the test assay.
Forster
Laura
L
Institute of Pharmaceutical and Medicinal Chemistry, University of Münster, Hittorfstrasse 58-62, 48149 Münster, Germany.
Schulze Elfringhoff
Alwine
A
Lehr
Matthias
M
D000581:Amidohydrolases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D000818:Animals; D001921:Brain / Q000187:drug effects / Q000201:enzymology; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D005260:Female; D005456:Fluorescent Dyes / Q000032:analysis* / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D015394:Molecular Structure; D051381:Rats; D017207:Rats, Sprague-Dawley; D013379:Substrate Specificity; D000099252:Fatty Acid Amide Hydrolases
D000581:Amidohydrolases; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D000099252:Fatty Acid Amide Hydrolases