Inhibition of the mammalian soluble epoxide hydrolase (sEH) is a promising new therapy in the treatment of disorders resulting from hypertension and vascular inflammation. A spectrophotometric assay (4-nitrophenyl-trans-2,3-epoxy-3-phenylpropyl carbonate, NEPC) is currently used to screen libraries of chemicals; however this assay lacks the required sensitivity to differentiate the most potent inhibitors. A series of fluorescent α-cyanoester and α-cyanocarbonate epoxides that produce a strong fluorescent signal on epoxide hydrolysis by both human and murine sEH were designed as potential substrates for an in vitro inhibition assay. The murine enzyme showed a broad range of specificities, whereas the human enzyme showed the highest specificity for cyano(6-methoxynaphthalen-2-yl)methyl trans- [(3-phenyloxiran-2-yl) methyl] carbonate. An in vitro inhibition assay was developed using this substrate and recombinant enzyme. The utility of the fluorescent assay was confirmed by determining the IC 50 values for a series of known inhibitors. The new IC 50 values were compared with those determined by spectrophotometric NEPC and radioactive tDPPO assays. The fluorescent assay ranked these inhibitors on the basis of IC 50 values, whereas the NEPC assay did not. The ranking of inhibitor potency generally agreed with that determined using the tDPPO assay. These results show that the fluorescence-based assay is a valuable tool in the development of sEH inhibitors by revealing structure-activity relationships that previously were seen only by using the costly and labor-intensive radioactive tDPPO assay.
KeywordsSoluble epoxide hydrolase; α-Cyanoester; α-Cyanocarbonate; Kinetic assay; Fluorescent substrateThe soluble epoxide hydrolase (sEH, EC 3.3.2.3) 1 is a member of the α/β-hydrolase fold family of enzymes [1] and catalyzes the hydrolysis of an epoxide to its corresponding diol through the catalytic addition of a water molecule [2]. The endogenous substrates for the sEH include epoxides of arachidonic acid [3,4] and linoleic acid [5,6]. Arachidonic acid epoxides (epoxyeicosatrienoic acid epoxides, EETs) are known modulators of blood pressure [7,8] and vascular permeability [9,10]. It has been shown that sEH inhibition not only lowers blood * 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 epoxides; NEPC, 4-nitrophenyl-trans-2,3-epoxy-3-phenylpropyl carbonate; TEA, triethylamine; TLC, thin-layer chromatography; TMS, tetramethylsilane; ppm, parts per million; oa-TOF, orthogonal acceleration time-of-flight; THF, tetrahydrofuran; m-CPBA, m-chloroperbenzoic acid; BSA, bovine serum albumin; RFU, relative fluorescent units; OD, optical density; DMSO, dimethyl sulfoxide; EDCI, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; tDPPO, [ 3 H] pressure in rodent models but also offers protection against hypertension-related renal damage [4,11,12].For the past 6 years, we have investi...