A high throughput fluorescent assay for measuring the activity of fatty acid amide hydrolase.
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.
Kage
Karen L
KL
Neuroscience Research, Global Pharmaceutical Research Division, Abbott Laboratories, Abbott Park, IL 60064, USA. Karen.Kage@Abbott.com
Richardson
Paul L
PL
Traphagen
Linda
L
Severin
Jean
J
Pereda-Lopez
Ana
A
Lubben
Thomas
T
Davis-Taber
Rachel
R
Vos
Melissa H
MH
Bartley
Diane
D
Walter
Karl
K
Harlan
John
J
Solomon
Larry
L
Warrior
Usha
U
Holzman
Thomas F
TF
Faltynek
Connie
C
Surowy
Carol S
CS
Scott
Victoria E
VE
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
D000581:Amidohydrolases; D003374:Coumarins; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D000099252:Fatty Acid Amide Hydrolases; C028743:7-amino-4-methylcoumarin