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A fluorescence-based assay for fatty acid amide hydrolase compatible with high-throughput screening
Manjunath K Ramarao1, Elizabeth A Murphy, Marina W H Shen
1Inflammation Department, Wyeth Research, 200 Cambridge Park Drive, Cambridge, MA 02140, USA. mramarao@wyeth.com
Analytical Biochemistry
|July 16, 2005
Summary
A new fluorescent assay effectively monitors fatty acid amide hydrolase (FAAH) activity. This simple and sensitive method is suitable for high-throughput screening of potential drug compounds.
Area of Science:
- Biochemistry
- Enzymology
- Assay Development
Background:
- Fatty acid amide hydrolase (FAAH) plays a crucial role in regulating endocannabinoid signaling.
- Developing efficient assays for FAAH activity is essential for drug discovery targeting neurological and inflammatory disorders.
Purpose of the Study:
- To develop a novel, simple, sensitive, and high-throughput screening (HTS)-amenable fluorescent assay for monitoring fatty acid amide hydrolase (FAAH) activity.
- To characterize the kinetic properties and specificity of the developed assay.
Main Methods:
- Established stable Chinese hamster ovary (CHO) cell lines expressing human FAAH or an inactive mutant (FAAH-S241A).
- Designed and synthesized a novel fluorogenic substrate, Arachidonyl 7-amino, 4-methyl coumarin amide (AAMCA).
- Monitored the continuous release of the fluorescent product (AMC) upon FAAH-catalyzed hydrolysis of AAMCA using a fluorometer.
Main Results:
- The assay demonstrated high sensitivity and specificity for FAAH activity, with minimal background from inactive mutant or vector controls.
- Kinetic analysis revealed an apparent K(m) of 0.48 μM and V(max) of 58 pmol/min/mg protein for microsomal FAAH.
- The assay showed concentration-dependent inhibition by the FAAH-specific inhibitor URB-597 (IC(50) = 33.5 nM) and achieved excellent Z' values (0.7–0.9) for HTS.
- The assay proved compatible with ex vivo analysis of FAAH activity.
Conclusions:
- A robust and sensitive fluorescent assay for continuous monitoring of FAAH activity has been successfully developed.
- The assay is suitable for high-throughput screening of compound libraries and ex vivo analysis, facilitating the discovery of novel FAAH inhibitors.
- This assay represents a valuable tool for research into the physiological roles of FAAH and the development of therapeutics targeting FAAH-related pathways.