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Development of highly sensitive fluorescent assays for fatty acid amide hydrolase.

Authors
Huang-H; Nishi-K; Tsai-HJ; Hammock-BD
Source
Anal Biochem 2007 Apr; 363(1):12-21
NIOSHTIC No.
20032930
Abstract
Fatty acid amide hydrolase (FAAH) is a pharmaceutical target whose inhibition may lead to valuable therapeutics. Sensitive substrates for high-throughput assays are crucial for the rapid-screening FAAH inhibitors. Here we describe the development of novel and highly sensitive fluorescent assays for FAAH based on substituted aminopyridines. Examining the relationship between the structure and the fluorescence of substituted aminopyridines suggested that a methoxy group in the para position relative to the amino group in aminopyridines greatly increased the fluorescence (i.e., quantum yields approach unity). These novel fluorescent reporters had a high Stokes' shift of 94 nm, and their fluorescence in buffer systems increased with pH values from neutral to basic. Fluorescent substrates with these reporters displayed a very low fluorescent background and high aqueous solubility. Most importantly, fluorescent assays for FAAH based on these substrates were at least 25 times more sensitive than assays using related compounds with published colorimetric or fluorescent reporters. This property results in shorter assay times and decreased protein concentrations in the assays. Such sensitive assays will facilitate distinguishing the relative potency of powerful inhibitors of FAAH. When these fluorescent substrates were applied to human liver microsomes, results suggested that there was at least one amide hydrolase in addition to FAAH that could hydrolyze long-chain fatty acid amides. These results show that these fluorescent substrates are very valuable tools in FAAH activity assays including screening inhibitors by high-throughput assays instead of using the costly and labor-intensive radioactive ligands. Potential applications of novel fluorescent reporters are discussed.
Keywords
Fatty-acids; Pharmaceuticals; Therapeutic-agents; Chelating-agents; Chemical-binding; Amino-compounds; Chemical-analysis; Chemical-composition; Chemical-deposition; Chemical-extraction; Pathology; Pain-tolerance
Contact
Huazhang Huang:Department of Entomology and Cancer Research Center, University of California, Davis, CA 95616, USA
CODEN
ANBCA2
Publication Date
20070401
Document Type
Journal Article
Funding Type
Agriculture; Cooperative Agreement
Fiscal Year
2007
NTIS Accession No.
NTIS Price
Identifying No.
Cooperative-Agreement-Number-U50-OH-007550
Issue of Publication
1
ISSN
0003-2697
Source Name
Analytical Biochemistry
State
CA
Performing Organization
University of California - Davis
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