Förster resonance energy transfer competitive displacement assay for human soluble epoxide hydrolase

Kin Sing Stephen Lee, Christophe Morisseau, Jun Yang, Peng Wang, Sung Hee Hwang, Bruce D. Hammock

Research output: Contribution to journalArticle

19 Scopus citations

Abstract

The soluble epoxide hydrolase (sEH), responsible for the hydrolysis of various fatty acid epoxides to their corresponding 1,2-diols, is becoming an attractive pharmaceutical target. These fatty acid epoxides, particularly epoxyeicosatrienoic acids (EETs), play an important role in human homeostatic and inflammation processes. Therefore, inhibition of human sEH, which stabilizes EETs in vivo, brings several beneficial effects to human health. Although there are several catalytic assays available to determine the potency of sEH inhibitors, measuring the in vitro inhibition constant (Ki) for these inhibitors using catalytic assay is laborious. In addition, koff, which has been recently suggested to correlate better with the in vivo potency of inhibitors, has never been measured for sEH inhibitors. To better measure the potency of sEH inhibitors, a reporting ligand, 1-(adamantan-1-yl)-3-(1-(2-(7- hydroxy-2-oxo-2H-chromen-4-yl)acetyl) piperidin-4-yl)urea (ACPU), was designed and synthesized. With ACPU, we have developed a Förster resonance energy transfer (FRET)-based competitive displacement assay using intrinsic tryptophan fluorescence from sEH. In addition, the resulting assay allows us to measure the Ki values of very potent compounds to the picomolar level and to obtain relative koff values of the inhibitors. This assay provides additional data to evaluate the potency of sEH inhibitors.

Original languageEnglish (US)
Pages (from-to)259-268
Number of pages10
JournalAnalytical Biochemistry
Volume434
Issue number2
DOIs
StatePublished - Mar 15 2013

Keywords

  • Binding assay
  • Competitive displacement assay
  • Fluorescent assay
  • Förster resonance energy transfer (FRET)
  • Soluble epoxide hydrolase

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology
  • Cell Biology

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  • Cite this

    Lee, K. S. S., Morisseau, C., Yang, J., Wang, P., Hwang, S. H., & Hammock, B. D. (2013). Förster resonance energy transfer competitive displacement assay for human soluble epoxide hydrolase. Analytical Biochemistry, 434(2), 259-268. https://doi.org/10.1016/j.ab.2012.11.015