Inhibitors of arachidonoyl ethanolamide hydrolysis.
Arachidonoyl ethanolamide (anandamide) is a naturally occurring brain constituent that binds to a specific brain cannabinoid receptor (CBR1). An amidase activity (anandamide amidase) in membrane fractions of brain and in cultured neuroblastoma cells rapidly degrades anandamide to arachidonic acid (Deutsch, D. G., and Chin, S. (1993) Biochem. Pharmacol. 46, 791-796). In the current study, analogs of anandamide representing three classes of putative transition-state inhibitor (trifluoromethyl ketones, alpha-keto esters, and alpha-keto amides) were synthesized and tested as inhibitors of anandamide hydrolysis in vitro and as ligands for CBR1. The trifluoromethyl ketones and alpha-keto esters showed nearly 100% inhibition of anandamide hydrolysis in vitro at 7.5 microM inhibitor and 27.7 microM anandamide. Arachidonyl trifluoromethyl ketone was the only synthetic compound in the series of fatty acid derivatives able to displace [3H]CP-55940 binding to CBR1 with a Ki of 0.65 microM. It was also the most effective inhibitor in intact neuroblastoma cells, leading to a 12-fold increase of cellular anandamide levels at 12 microM. From the action of these inhibitors on this hydrolytic enzyme, it seems likely that anandamide is cleaved by a mechanism that involves an active-site serine hydroxyl group. These inhibitors may serve as useful tools to elucidate the role anandamide plays in vivo.
Koutek
B
B
Department of Chemistry, State University of New York, Stony Brook 11794.
Prestwich
G D
GD
Howlett
A C
AC
Chin
S A
SA
Salehani
D
D
Akhavan
N
N
Deutsch
D G
DG
D000581:Amidohydrolases / Q000037:antagonists & inhibitors; D000818:Animals; D001095:Arachidonic Acids / Q000138:chemical synthesis / Q000378:metabolism*; D001667:Binding, Competitive; D002186:Cannabinoids / Q000378:metabolism; D003511:Cyclohexanols / Q000378:metabolism; D063388:Endocannabinoids; D004952:Esters / Q000378:metabolism; D005227:Fatty Acids / Q000378:metabolism; D006868:Hydrolysis / Q000187:drug effects; D007659:Ketones / Q000378:metabolism; D053284:Polyunsaturated Alkamides; D051381:Rats; D014407:Tumor Cells, Cultured
D001095:Arachidonic Acids; D002186:Cannabinoids; D003511:Cyclohexanols; D063388:Endocannabinoids; D004952:Esters; D005227:Fatty Acids; D007659:Ketones; D053284:Polyunsaturated Alkamides; C081565:arachidonyltrifluoromethane; C054649:3-(2-hydroxy-4-(1,1-dimethylheptyl)phenyl)-4-(3-hydroxypropyl)cyclohexanol; D000581:Amidohydrolases; C078814:anandamide