Spectrophotometric Substrates for Cytosolic Epoxide Hydrolase

Dietze, Eric C.; Kuwano, Eiichi; Hammock, Bruce D.

doi:10.1006/abio.1994.1023

Cited by 42 publications

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“…The latter substrates provide a higher sensitivity with increased maximal velocities when compared with trans-stilbene oxide. Several additional specific sEH substrates have been developed and characterized [54], including a series of epoxy esters and carbonate-derivatives that cyclize spontaneously upon hydrolysis of the epoxide, releasing an alcohol. These substrates, therefore, enable rapid spectrophotometric analysis of sEH activity without the use of radiolabeled probes.…”

Section: Substrates and Inhibitorsmentioning

confidence: 99%

Epoxide hydrolases: biochemistry and molecular biology

Fretland

Omiecinski

2000

Chemico-Biological Interactions

271

185

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Epoxides are organic three-membered oxygen compounds that arise from oxidative metabolism of endogenous, as well as xenobiotic compounds via chemical and enzymatic oxidation processes, including the cytochrome P450 monooxygenase system. The resultant epoxides are typically unstable in aqueous environments and chemically reactive. Biol. Chem. 260 (1985) 1630-1640). Therefore, it is of vital importance for the biological organism to regulate levels of these reactive species. The epoxide hydrolases (E.C. 3.3.2.3) belong to a sub-category of a broad group of hydrolytic enzymes that include esterases, proteases, dehalogenases, and lipases Beetham et al. (DNA Cell Biol. 14 (1995) 61 -71). In particular, the epoxide hydrolases are a class of proteins that catalyze the hydration of chemically reactive epoxides to their corresponding dihydrodiol products. Simple epoxides are hydrated to their corresponding vicinal dihydrodiols, and arene oxides to trans-dihydrodiols. In general, this hydration leads to more stable and less reactive intermediates, however exceptions do exist. In mammalian species, there are at least five epoxide hydrolase forms, microsomal cholesterol 5,6-oxide hydrolase, hepoxilin A 3 hydrolase, leukotriene A 4 hydrolase, soluble, and microsomal epoxide hydrolase. Each of these enzymes is distinct chemically and immunologically. Table 1 illustrates some general properties for each of these classes of hydrolases. Fig. 1 provides an overview of selected model substrates for each class of epoxide hydrolase.

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Section: Substrates and Inhibitorsmentioning

confidence: 99%

Epoxide hydrolases: biochemistry and molecular biology

Fretland

Omiecinski

2000

Chemico-Biological Interactions

271

185

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“…Evaluation of these inhibitors typically begins with in vitro assays using purified recombinant sEH prior to in vivo studies. Traditionally, we have used racemic 4-nitrophenyl-trans-2,3-epoxy-3-phenylpropyl carbonate (NEPC) as a substrate for a continuous kinetic assay to determine inhibitor potency [14,19]. As the potency of our inhibitors increased (lower IC 50 ), we found that the NEPC absorption-based assay was not sensitive enough to separate structurally different inhibitors on the basis of IC 50 values.…”

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confidence: 98%

“…Although these methods detect epoxide hydrolase activity effectively, they require high concentrations of substrate (millimolar) and rely on external chemical modification steps. An alternate strategy was presented by our previously described substrate, NEPC, which undergoes an intramolecular cyclization liberating the highly colored 4-nitrophenolate anion following hydrolysis of the epoxide moiety [19].We recently reported that α-cyanoesters [24,25] and α-cyanoethers [26] are effective fluorescent substrates for the detection and quantitation of esterase and P450 activities, respectively. In the case of the α-cyanoesters, O-deacylation liberates a cyanohydrin intermediate that rapidly decomposes to the highly fluorescent 6-methoxy-2-naphthaldehyde (1).…”

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confidence: 99%

“…It was hypothesized that replacement of the 4-nitrophenoxide portion of NEPC with an α-cyanoester or a carbonate would result in an acceptable fluorescent substrate for sEH. Because it has been shown previously [19] that there is little difference between (+)-NEPC and (−)-NEPC, we opted for a racemic synthesis. In addition, to maintain consistency with Dietze and coworkers' study [19], we chose to incorporate only trans-epoxides into the substrates described.…”

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confidence: 99%

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Fluorescent substrates for soluble epoxide hydrolase and application to inhibition studies

Jones

Wolf

Morisseau

et al. 2005

Analytical Biochemistry

146

233

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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...

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“…These solutions were used to measure inhibitor potency directly on purified recombinant mouse and human sEH, 11 using a spectrophotometric substrate in a 96-well microplate assay. 12 We first optimized the distance between the urea function and the first amino acid using a subset of amino acids and three (3-cyclohexyl-ureido)-alkanoic acids of different length (Table 1). For the mouse sEH, good inhibition is obtained for both inhibitor series with the 4-and 6-carbon carboxylate tails, suggesting that a 3-carbon spacer (i.e., n = 3) is sufficient for this enzyme.…”

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Peptidyl-urea based inhibitors of soluble epoxide hydrolases

Morisseau

Newman

Tsai

et al. 2006

Bioorganic & Medicinal Chemistry Letters

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We prepared a series of amino acid derived cyclohexyl and adamantyl ureas and tested them as inhibitors of the human soluble epoxide hydrolase, and obtained very potent compounds (K I = 15 nM) that are >10-fold more soluble than previously described sEH inhibitors. While our lead compound 2 showed low apparent bioavailability in dogs and rats, this series of compounds revealed that sEH inhibitor structures could accept large groups that could lead to better orally available drugs. Keywords Cardiovascular diseases; Soluble epoxide hydrolase inhibitorsSoluble epoxide hydrolase (sEH; EC 3.3.3.2) catalyzes the addition of a water molecule to an epoxide resulting in the corresponding diol. 1 Endogenous substrates for sEH include epoxyeicosatrienoic acids (EETs), which are known for their vasodilatory effects as well as for their anti-inflammatory actions. 2 Hydrolysis of the epoxides by sEH diminished this activity. 3 The inhibition of sEH led to the accumulation of EETs and other lipid epoxides in the organism. 4 Furthermore, sEH inhibition in rodent models can successfully treat hypertension, 4b,5 and inflammatory diseases, 6 as well as protect against renal damage caused by hypertension. 7The development in our laboratory of novel, stable, highly potent, and selective inhibitors for sEH 4a has allowed the elucidation of the biology associated with sEH. 1,2d Crystal structure determinations show that the urea inhibitors establish hydrogen bonding and salt bridges between the urea function of the inhibitor and residues of the sEH active site. 8 However, these dialkyl-ureas have high crystal lattice energies as indicated by their high melting point, and also have limited solubility in water, 4 likely affecting their in vivo efficacy. While medicinal chemistry approaches were used to increase their inhibitory activity and solubility, 9 the latest compounds reported may not possess physical properties consistent with successful pharmaceutical candidates. 10 Therefore, there remains a need for novel sEH inhibitor structures with improved biological availability and stability for possible future orally administered in vivo drugs. There is also the need for synthetic methods amenable to high throughput combinatorial or parallel methods.As shown previously, if placed at an appropriate distance from the urea moiety polar groups can be incorporated into one of the alkyl groups of the dialkyl-urea sEH inhibitors without loss of activity. 9 Such modifications give the new inhibitors better solubility and availability; however, only straight alkyl chain derivatives such as 12-(3-adamantyl-ureido)-dodecanoic acid (AUDA; see Fig. 1 that while the catalytic tunnel is restricted around the catalytic residues it enlarges as one moves away from the site of reaction to yield relatively large cavities. These structural features suggest that the enzyme could accommodate branches on the main alkyl chain. To evaluate a wide variety of side chains, we used a semi-combinatorial approach. Because amino acids are simple bifunctional s...

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Spectrophotometric Substrates for Cytosolic Epoxide Hydrolase

Cited by 42 publications

References 0 publications

Epoxide hydrolases: biochemistry and molecular biology

Epoxide hydrolases: biochemistry and molecular biology

Fluorescent substrates for soluble epoxide hydrolase and application to inhibition studies

Peptidyl-urea based inhibitors of soluble epoxide hydrolases

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