Development of highly sensitive fluorescent assays for fatty acid amide hydrolase

Huazhang Huang, Kosuke Nishi, Hsing Ju Tsai, Bruce D. Hammock

Research output: Contribution to journalArticle

19 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)12-21
Number of pages10
JournalAnalytical Biochemistry
Volume363
Issue number1
DOIs
StatePublished - Apr 1 2007

Fingerprint

Assays
Aminopyridines
Fluorescence
Substrates
Amides
High-Throughput Screening Assays
Screening
Throughput
Liver Microsomes
Hydrolases
fatty-acid amide hydrolase
Solubility
Quantum yield
Buffers
Fatty Acids
Liver
Ligands
Personnel
Pharmaceutical Preparations
Proteins

Keywords

  • Aminopyridines
  • FAAH
  • Fluorescence
  • Quantum yields
  • Sensitivity
  • Substrates

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

Development of highly sensitive fluorescent assays for fatty acid amide hydrolase. / Huang, Huazhang; Nishi, Kosuke; Tsai, Hsing Ju; Hammock, Bruce D.

In: Analytical Biochemistry, Vol. 363, No. 1, 01.04.2007, p. 12-21.

Research output: Contribution to journalArticle

Huang, Huazhang ; Nishi, Kosuke ; Tsai, Hsing Ju ; Hammock, Bruce D. / Development of highly sensitive fluorescent assays for fatty acid amide hydrolase. In: Analytical Biochemistry. 2007 ; Vol. 363, No. 1. pp. 12-21.
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