Fluorescent substrates for soluble epoxide hydrolase and application to inhibition studies

Abstract

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...

Acute augmentation of epoxygenated fatty acid levels rapidly reduces pain-related behavior in a rat model of type I diabetes

“…The cytochrome P450-mediated oxygenation of fatty acids leads to the production of EpFAs, a class of bioactive lipids that have exceedingly short half-lives (33). Levels of these molecules are regulated by synthesis, degradation, and membrane incorporation (17,34,35).…”

Identification of two epoxide hydrolases in Caenorhabditis elegans that metabolize mammalian lipid signaling molecules

“…Assays were performed as described [36] with the following exception. An enzyme concentration of 0.57 μg/mL for CEEH1 was used in the assay.…”

“…[36] In order to compare the CEEH1 to the human enzyme, a limited structure activity experiment was performed using these substrates. All of the substrates were hydrolyzed by CEEH1 (Table 4).…”

Characterization of pyrethroid hydrolysis by the human liver carboxylesterases hCE-1 and hCE-2

“…Thus, the use of pyrethroid-like fluorescent surrogates could be highly useful for the identification and monitoring of enzymes involved in the hydrolysis of pyrethroids. The use of α-cyanoalcoholsas a reporter system offers substantial advantages in the analysis of several enzymes including cytochrome P450s [25], epoxide hydrolases [26] and, as illustrated here, esterases such as hCE-1 and hCE-2. These advantages include high quantum yield, very large Stokes' shifts, low background, hydrolytic stability, and flexibility of structure.…”