title: Förster resonance energy transfer competitive displacement assay for human soluble epoxide hydrolase.
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 (K(i)) 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 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 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.
authors.affiliation: Department of Entomology and UCD Comprehensive Cancer Center, University of California, Davis, CA 95616, USA.
authors.forename: Kin Sing Stephen
authors.initials: KS
authors.lastname: Lee
authors.forename: Christophe
authors.initials: C
authors.lastname: Morisseau
authors.forename: Jun
authors.initials: J
authors.lastname: Yang
authors.forename: Peng
authors.initials: P
authors.lastname: Wang
authors.forename: Sung Hee
authors.initials: SH
authors.lastname: Hwang
authors.forename: Bruce D
authors.initials: BD
authors.lastname: Hammock
mesh_terms: D001667:Binding, Competitive; D001681:Biological Assay / Q000379:methods*; D018360:Crystallography, X-Ray; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D004851:Epoxide Hydrolases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D005453:Fluorescence; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006801:Humans; D020128:Inhibitory Concentration 50; D012995:Solubility; D014364:Tryptophan / Q000737:chemistry
chemical_list: D004791:Enzyme Inhibitors; D014364:Tryptophan; D004851:Epoxide Hydrolases