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Development of Highly Sensitive Fluorescent Assays for Fatty Acid Amide Hydrolase



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  • Personal Author:
  • Description:
    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. [Description provided by NIOSH]
  • Subjects:
  • Keywords:
  • ISSN:
    0003-2697
  • Document Type:
    Text
  • Funding:
    Cooperative Agreement
  • Genre:
    Journal Article
  • Place as Subject:
  • CIO:
    National Institute for Occupational Safety and Health (NIOSH)
  • Topic:
    Workplace Safety & Health
  • Location:
    North America
  • Pages in Document:
    12-21
  • Volume:
    363
  • Issue:
    1
  • NIOSHTIC Number:
    nn:20032930
  • Citation:
    Anal Biochem 2007 Apr; 363(1):12-21
  • Contact Point Address:
    Huazhang Huang:Department of Entomology and Cancer Research Center, University of California, Davis, CA 95616, USA
  • Federal Fiscal Year:
    2007
  • Performing Organization:
    University of California - Davis
  • Peer Reviewed:
    True
  • Start Date:
    20010930
  • Source Full Name:
    Analytical Biochemistry
  • End Date:
    20270929
  • Collection(s):
  • Main Document Checksum:
    urn:sha-512:2798cd8ae1b8baebe074839a5c635a25482ffd11e9a7ab3cd092a3e60ff50c4a95fc31e69de058ebfae4e68a4cf2491423d1772ccca29b674467bf6fa1c5ad90
  • Download URL:
  • File Type:
    Filetype[PDF - 233.70 KB ]
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