title: A novel scintillation proximity assay for fatty acid amide hydrolase compatible with inhibitor screening.
abstract: A binding assay for human fatty acid amide hydrolase (FAAH) using the scintillation proximity assay (SPA) technology is described. This SPA uses the specific interactions of [3H]R(+)-methanandamide (MAEA) and FAAH expressing microsomes to evaluate the displacement activity of FAAH inhibitors. We observed that a competitive nonhydrolyzed FAAH inhibitor, [3H]MAEA, bound specifically to the FAAH microsomes. Coincubation with an FAAH inhibitor, URB-597, competitively displaced the [3H]MAEA on the FAAH microsomes. The released radiolabel was then detected through an interaction with the SPA beads. The assay is specific for FAAH given that microsomes prepared from cells expressing the inactive FAAH-S241A mutant or vector alone had no significant ability to bind [3H]MAEA. Furthermore, the binding of [3H]MAEA to FAAH microsomes was abolished by selective FAAH inhibitors in a dose-dependent manner, with IC50 values comparable to those seen in a functional assay. This novel SPA has been validated and demonstrated to be simple, sensitive, and amenable to high-throughput screening.
authors.affiliation: Neuroscience Discovery Research, Wyeth Research, Princeton, NJ 08543, USA. wangy4@wyeth.com
authors.forename: Yuren
authors.initials: Y
authors.lastname: Wang
authors.forename: Jun
authors.initials: J
authors.lastname: Xu
authors.forename: Albert
authors.initials: A
authors.lastname: Uveges
authors.forename: Manjunath K
authors.initials: MK
authors.lastname: Ramarao
authors.forename: Kathryn E
authors.initials: KE
authors.lastname: Rogers
authors.forename: Philip G
authors.initials: PG
authors.lastname: Jones
mesh_terms: D000581:Amidohydrolases / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000378:metabolism; D000818:Animals; D001095:Arachidonic Acids / Q000378:metabolism / Q000494:pharmacology; D001549:Benzamides / Q000378:metabolism / Q000494:pharmacology; D001665:Binding Sites / Q000187:drug effects; D016466:CHO Cells; D002121:Calcium Channel Blockers / Q000378:metabolism / Q000494:pharmacology; D002219:Carbamates / Q000378:metabolism / Q000494:pharmacology; D006224:Cricetinae; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical / Q000379:methods; D063388:Endocannabinoids; D004791:Enzyme Inhibitors / Q000378:metabolism / Q000494:pharmacology*; D006801:Humans; D020128:Inhibitory Concentration 50; D008024:Ligands; D008861:Microsomes / Q000201:enzymology / Q000378:metabolism; D053284:Polyunsaturated Alkamides; D012588:Scintillation Counting; D014909:Wheat Germ Agglutinins / Q000378:metabolism / Q000494:pharmacology; D000099252:Fatty Acid Amide Hydrolases
chemical_list: D000581:Amidohydrolases; D001095:Arachidonic Acids; D001549:Benzamides; D002121:Calcium Channel Blockers; D002219:Carbamates; D063388:Endocannabinoids; D004791:Enzyme Inhibitors; D008024:Ligands; D053284:Polyunsaturated Alkamides; D014909:Wheat Germ Agglutinins; D000099252:Fatty Acid Amide Hydrolases; C500528:cyclohexyl carbamic acid 3'-carbamoylbiphenyl-3-yl ester; C088155:methanandamide; C078814:anandamide