A fluorescence-based assay for fatty acid amide hydrolase compatible with high-throughput screening.
A novel fluorescent assay to continuously monitor fatty acid amide hydrolase (FAAH) activity that is simple, sensitive, and amenable to high-throughput screening (HTS) of compound libraries is described in this article. Stable Chinese hamster ovary (CHO) cell lines expressing either human FAAH or an inactive mutant, FAAH-S241A, were established. Arachidonyl 7-amino, 4-methyl coumarin amide (AAMCA), a novel fluorogenic substrate for FAAH, was designed and synthesized. FAAH catalyzes the hydrolysis of AAMCA to generate arachidonic acid and a highly fluorescent 7-amino, 4-methyl coumarin (AMC). The assay was done at 25 degrees C by incubating whole cell or microsomal preparations from FAAH-expressing cells with AAMCA. Release of AMC was monitored continuously using a fluorometer. Microsomal FAAH catalyzed the hydrolysis of AAMCA with an apparent K(m) of 0.48muM and V(max) of 58pmolmin(-1)mgprotein(-1). The assay is specific for FAAH given that microsomes prepared from cells expressing FAAH-S241A or vector alone had no significant activity against AAMCA. Furthermore, the activity was inhibited by URB-597, an FAAH-specific inhibitor, in a concentration-dependent manner with an IC(50) of 33.5nM. The assay was optimized for HTS and had a Z' value ranging from 0.7 to 0.9. The assay is also compatible with ex vivo analysis of FAAH activity.
Ramarao
Manjunath K
MK
Inflammation Department, Wyeth Research, 200 Cambridge Park Drive, Cambridge, MA 02140, USA. mramarao@wyeth.com
Murphy
Elizabeth A
EA
Shen
Marina W H
MW
Wang
Yuren
Y
Bushell
Kristen N
KN
Huang
Nelson
N
Pan
Ning
N
Williams
Cara
C
Clark
James D
JD
D000581:Amidohydrolases / Q000737:chemistry* / Q000235:genetics; D000818:Animals; D001095:Arachidonic Acids / Q000138:chemical synthesis / Q000737:chemistry*; D001549:Benzamides / Q000494:pharmacology; D001681:Biological Assay / Q000379:methods*; D016466:CHO Cells; D002219:Carbamates / Q000494:pharmacology; D003374:Coumarins / Q000138:chemical synthesis / Q000737:chemistry*; D006224:Cricetinae; D003412:Cricetulus; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry*; D006801:Humans; D008861:Microsomes / Q000201:enzymology*; D000099252:Fatty Acid Amide Hydrolases
D000581:Amidohydrolases; D001095:Arachidonic Acids; D001549:Benzamides; D002219:Carbamates; D003374:Coumarins; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D000099252:Fatty Acid Amide Hydrolases; C504580:arachidonyl 7-amino, 4-methyl coumarin amide; C500528:cyclohexyl carbamic acid 3'-carbamoylbiphenyl-3-yl ester; C030123:coumarin