1 2 Förster resonance energy transfer competitive displacement assay for human 3 soluble epoxide hydrolase 4 Kin Sing Stephen Lee a , Christophe Morisseau a , Jun Yang a , Peng Wang a,b , Sung Hee Hwang a , 5 Bruce D. Hammock a,⇑ 6 a Department of Entomology and UCD Comprehensive Cancer Center, University of California, Davis, CA 95616, USA 7 b Department of Applied Chemistry, China Agricultural University, Beijing 100193, People’s Republic of China 8 10 article info 11 Article history: 12 Available online xxxx 13 Keywords: 14 Soluble epoxide hydrolase 15 Binding assay 16 Competitive displacement assay 17 Förster resonance energy transfer (FRET) 18 Fluorescent assay 19 20 abstract 21 The soluble epoxide hydrolase (sEH), responsible for the hydrolysis of various fatty acid epoxides to their 22 corresponding 1,2-diols, is becoming an attractive pharmaceutical target. These fatty acid epoxides, par- 23 ticularly epoxyeicosatrienoic acids (EETs), play an important role in human homeostatic and inflamma- 24 tion processes. Therefore, inhibition of human sEH, which stabilizes EETs in vivo, brings several beneficial 25 effects to human health. Although there are several catalytic assays available to determine the potency of 26 sEH inhibitors, measuring the in vitro inhibition constant (K i ) for these inhibitors using catalytic assay is 27 laborious. In addition, k off , which has been recently suggested to correlate better with the in vivo potency 28 of inhibitors, has never been measured for sEH inhibitors. To better measure the potency of sEH inhibi- 29 tors, a reporting ligand, 1-(adamantan-1-yl)-3-(1-(2-(7-hydroxy-2-oxo-2H-chromen-4-yl)acetyl) piperi- 30 din-4-yl)urea (ACPU), was designed and synthesized. With ACPU, we have developed a Förster 31 resonance energy transfer (FRET)-based competitive displacement assay using intrinsic tryptophan 32 fluorescence from sEH. In addition, the resulting assay allows us to measure the K i values of very potent 33 compounds to the picomolar level and to obtain relative k off values of the inhibitors. This assay provides 34 additional data to evaluate the potency of sEH inhibitors. 35 Ó 2012 Elsevier Inc. All rights reserved. 36 37 38 In mammals, the soluble epoxide hydrolase (sEH) 1 (EC 3.3.2.10) 39 metabolizes important signaling epoxy fatty acids to the correspond- 40 ing 1,2-diols [1]. These epoxy fatty acids, particularly epoxyeicosatri- 41 enoic acids (EETs) from arachidonic acid and epoxides from omega-3 42 fatty acids, have been demonstrated to maintain blood pressure in 43 several animal models, to resolve inflammatory disorders and reduce 44 pain, and generally to maintain homeostasis [2–9]. Therefore, stabil- 45 ization of EETs through inhibition of sEH could be beneficial to 46 human health. Over the past decade, significant progress has been 47 made toward the clinical development of sEH inhibitors [10]. 48 Catalytic assays for sEH have been instrumental in obtaining 49 improved sEH inhibitors. Over the years, numerous substrate- 50 based assays have been developed and used [11–20]. Existing 51 sEH assays are able to distinguish among inhibitors of varying po- 52 tency down to low nanomolar. However, as the inhibitor concen- 53 tration approaches the enzyme concentration, the assays cannot 54 distinguish among the most potent inhibitors. Several laboratories 55 have reached this sensitivity limit with compounds they have 56 made [5,10,21], so an assay capable of distinguishing among highly 57 potent inhibitors would be attractive. 58 Catalytic assays for enzymes and their inhibitors are attractive 59 for many mechanistic reasons; however, binding assays lend them- 60 selves to high-throughput formats easier than enzymatic assays. In 61 general, the different sEH catalytic assays rank compounds in sim- 62 ilar order, but there are notable exceptions. A binding assay with 63 the enzyme in which catalysis plays no role could simplify data 64 interpretation. Several binding assays have been described for 65 sEH [13,16]. In 2009, Eldrup and coworkers reported the use of a 66 tetramethyl rhodamine-labeled probe to measure human or rat 67 sEH inhibition [13]. However, such an assay requires spectrome- 68 ters that can measure fluorescence polarization in order to distin- 69 guish the bound probe from background. In a similar approach, 70 urea-based inhibitors containing aryl groups are able to quench 0003-2697/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ab.2012.11.015 ⇑ Corresponding author. Fax: +1 530 752 1537. E-mail address: bdhammock@ucdavis.edu (B.D. Hammock). 1 Abbreviations used: sEH, soluble epoxide hydrolase; EET, epoxyeicosatrienoic acid; IC 50 , half-maximal inhibitory concentration; FRET, Förster resonance energy transfer; 14,15-EET, 14,15-epoxyeicosatrienoic acid; PTU, 1-(piperidine-4-yl)-3-(4- (trifluoromethyl)phenyl)urea; DMSO, dimethyl sulfoxide; ACPU, 1-(adamantan-1-yl)- 3-(1-(2-(7-hydroxy-2-oxo-2H-chromen-4-yl)acetyl)piperidin-4-yl)urea; EDCI, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; DMAP, 4-dimethylaminopyridine; rt, room temperature; DMF, dimethylformamide; hsEH, human sEH; LC–MS/MS, liquid chromatography–tandem mass spectrometry; PB buffer, sodium phosphate buffer; t-DPPO, trans-diphenyl-propene oxide; CMNPC, cyano(6-methoxy-naphtha- len-2-yl)methyl trans-[(3-phenyloxyran-2-yl)methyl] carbonate; msEH, mouse sEH. Analytical Biochemistry xxx (2012) xxx–xxx Contents lists available at SciVerse ScienceDirect Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio YABIO 11168 No. of Pages 10, Model 5G 28 December 2012 Please cite this article in press as: K.S.S. Lee et al., Förster resonance energy transfer competitive displacement assay for human soluble epoxide hydrolase, Anal. Biochem. (2012), http://dx.doi.org/10.1016/j.ab.2012.11.015