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

作者：Lee Kin Sing Stephen; Morisseau Christophe; Yang Jun; Wang Peng; Hwang Sung Hee; Hammock Bruce D^*

来源：Analytical Biochemistry, 2013, 434(2): 259-268.

DOI：10.1016/j.ab.2012.11.015

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

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 (K-1) for these inhibitors using catalytic assay is laborious. In addition, k(off), 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 Forster resonance energy transfer (FRET)-based competitive displacement assay using intrinsic byptophan fluorescence from sEH. In addition, the resulting assay allows us to measure the K-i values of very potent compounds to the picomolar level and to obtain relative k(off) values of the inhibitors. This assay provides additional data to evaluate the potency of sEH inhibitors.

• 出版日期2013-3-15
• 单位中国农业大学