title: A high throughput fluorescent assay for measuring the activity of fatty acid amide hydrolase.
abstract: Fatty acid amide hydrolase (FAAH) is the enzyme responsible for the rapid degradation of fatty acid amides such as the endocannabinoid anandamide. Inhibition of FAAH activity has been suggested as a therapeutic approach for the treatment of chronic pain, depression and anxiety, through local activation of the cannabinoid receptor CB1. We have developed a high throughput screening assay for identification of FAAH inhibitors using a novel substrate, decanoyl 7-amino-4-methyl coumarin (D-AMC) that is cleaved by FAAH to release decanoic acid and the highly fluorescent molecule 7-amino-4-methyl coumarin (AMC). This assay gives an excellent signal window for measuring FAAH activity and, as a continuous assay, inherently offers improved sensitivity and accuracy over previously reported endpoint assays. The assay was validated using a panel of known FAAH inhibitors and purified recombinant human FAAH, then converted to a 384 well format and used to screen a large library of compounds (>600,000 compounds) to identify FAAH inhibitors. This screen identified numerous novel FAAH inhibitors of diverse chemotypes. These hits confirmed using a native FAAH substrate, anandamide, and had very similar rank order potency to that obtained using the D-AMC substrate. Collectively these data demonstrate that D-AMC can be successfully used to rapidly and effectively identify novel FAAH inhibitors for potential therapeutic use.
authors.affiliation: Neuroscience Research, Global Pharmaceutical Research Division, Abbott Laboratories, Abbott Park, IL 60064, USA. Karen.Kage@Abbott.com
authors.forename: Karen L
authors.initials: KL
authors.lastname: Kage
authors.forename: Paul L
authors.initials: PL
authors.lastname: Richardson
authors.forename: Linda
authors.initials: L
authors.lastname: Traphagen
authors.forename: Jean
authors.initials: J
authors.lastname: Severin
authors.forename: Ana
authors.initials: A
authors.lastname: Pereda-Lopez
authors.forename: Thomas
authors.initials: T
authors.lastname: Lubben
authors.forename: Rachel
authors.initials: R
authors.lastname: Davis-Taber
authors.forename: Melissa H
authors.initials: MH
authors.lastname: Vos
authors.forename: Diane
authors.initials: D
authors.lastname: Bartley
authors.forename: Karl
authors.initials: K
authors.lastname: Walter
authors.forename: John
authors.initials: J
authors.lastname: Harlan
authors.forename: Larry
authors.initials: L
authors.lastname: Solomon
authors.forename: Usha
authors.initials: U
authors.lastname: Warrior
authors.forename: Thomas F
authors.initials: TF
authors.lastname: Holzman
authors.forename: Connie
authors.initials: C
authors.lastname: Faltynek
authors.forename: Carol S
authors.initials: CS
authors.lastname: Surowy
authors.forename: Victoria E
authors.initials: VE
authors.lastname: Scott
mesh_terms: D000581:Amidohydrolases / Q000378:metabolism*; D001331:Automation / Q000379:methods; D001681:Biological Assay / Q000379:methods*; D003374:Coumarins / Q000493:pharmacokinetics; D005456:Fluorescent Dyes / Q000032:analysis* / Q000737:chemistry; D006801:Humans; D007202:Indicators and Reagents / Q000493:pharmacokinetics; D015203:Reproducibility of Results; D000099252:Fatty Acid Amide Hydrolases
chemical_list: D000581:Amidohydrolases; D003374:Coumarins; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D000099252:Fatty Acid Amide Hydrolases; C028743:7-amino-4-methylcoumarin