A novel scintillation proximity assay for fatty acid amide hydrolase compatible with inhibitor screening.
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.
Wang
Yuren
Y
Neuroscience Discovery Research, Wyeth Research, Princeton, NJ 08543, USA. wangy4@wyeth.com
Xu
Jun
J
Uveges
Albert
A
Ramarao
Manjunath K
MK
Rogers
Kathryn E
KE
Jones
Philip G
PG
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
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