A novel scintillation proximity assay for fatty acid amide hydrolase compatible with inhibitor screening

• Published in: Analytical biochemistry 2006-07, Vol.354 (1), p.35-42
• Main Authors: Wang, Yuren, Xu, Jun, Uveges, Albert, Ramarao, Manjunath K., Rogers, Kathryn E., Jones, Philip G.
• Format: Article
• Language: English
• Subjects: Amidohydrolases - analysis; Amidohydrolases - antagonists & inhibitors; Amidohydrolases - metabolism; Animals; Arachidonic Acids - metabolism; Arachidonic Acids - pharmacology; Benzamides - metabolism; Benzamides - pharmacology; Binding Sites - drug effects; Calcium Channel Blockers - metabolism; Calcium Channel Blockers - pharmacology; Carbamates - metabolism; Carbamates - pharmacology; CHO Cells; Cricetinae; Dose-Response Relationship, Drug; Drug Evaluation, Preclinical - methods; Endocannabinoids; Enzyme binding assay; Enzyme inhibitors; Enzyme Inhibitors - metabolism; Enzyme Inhibitors - pharmacology; Fatty acid amide hydrolase; Humans; Inhibitory Concentration 50; Ligands; Methanandamide; Microsomes - enzymology; Microsomes - metabolism; Polyunsaturated Alkamides; Scintillation Counting; Scintillation proximity assay; Wheat Germ Agglutinins - metabolism; Wheat Germ Agglutinins - pharmacology
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/j.ab.2006.04.005

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 IC 50 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.