Pharmacokinetics and antinociceptive effects of the soluble epoxide hydrolase inhibitor t-TUCB in horses with experimentally induced radiocarpal synovitis.

This study determined the pharmacokinetics, antinociceptive, and anti-inflammatory effects of the soluble epoxide hydrolase (sEH) inhibitor t-TUCB (trans-4-{4-[3-(4-Trifluoromethoxy-phenyl)-ureido]-cyclohexyloxy}-benzoic acid) in horses with lipopolysaccharide (LPS)-induced radiocarpal synovitis. A total of seven adult healthy mares (n = 4-6/treatment) were administered 3 µg LPS into one radiocarpal joint and t-TUCB intravenously (i.v.) at 0 (control), 0.03, 0.1, 0.3, and 1 mg/kg in a blinded, randomized, crossover design with at least 3 weeks washout between. Two investigators independently assigned pain scores (at rest, walk and trot) and lameness scores before and up to 48 hr after t-TUCB/LPS. Responses to touching the joint skin to assess tactile allodynia, plasma, and synovial fluid (SF) t-TUCB concentrations were determined before and up to 48 hr after t-TUCB/LPS. Blood and SF were collected for clinical laboratory evaluations before and up to 48 hr after t-TUCB/LPS. Areas under the curves of pain and lameness scores were calculated and compared between control and treatments. Data were analyzed using repeated measures ANOVA with Dunnett or Bonferroni post-test. p < .05 was considered significant. Data are mean ± SEM. Compared to control, pain, lameness, and tactile allodynia were significantly lower with 1 mg/kg t-TUCB, but not the other doses. For 0.1, 0.3, and 1 mg/kg t-TUCB treatments, plasma terminal half-lives were 13 ± 3, 13 ± 0.5, and 24 ± 5 hr, and clearances were 68 ± 15, 48 ± 5, and 14 ± 1 ml hr-1  kg-1 . The 1 mg/kg t-TUCB reached the SF at high concentrations. There were no important anti-inflammatory effects. In conclusion, sEH inhibition with t-TUCB may provide analgesia in horses with inflammatory joint pain.

Dysregulation of soluble epoxide hydrolase and lipidomic profiles in anorexia nervosa.

Individuals with anorexia nervosa (AN) restrict eating and become emaciated. They tend to have an aversion to foods rich in fat. Because epoxide hydrolase 2 (EPHX2) was identified as a novel AN susceptibility gene, and because its protein product, soluble epoxide hydrolase (sEH), converts bioactive epoxides of polyunsaturated fatty acid (PUFA) to the corresponding diols, lipidomic and metabolomic targets of EPHX2 were assessed to evaluate the biological functions of EPHX2 and their role in AN. Epoxide substrates of sEH and associated oxylipins were measured in ill AN, recovered AN and gender- and race-matched controls. PUFA and oxylipin markers were tested as potential biomarkers for AN. Oxylipin ratios were calculated as proxy markers of in vivo sEH activity. Several free- and total PUFAs were associated with AN diagnosis and with AN recovery. AN displayed elevated n-3 PUFAs and may differ from controls in PUFA elongation and desaturation processes. Cytochrome P450 pathway oxylipins from arachidonic acid, linoleic acid, alpha-linolenic acid and docosahexaenoic acid PUFAs are associated with AN diagnosis. The diol:epoxide ratios suggest the sEH activity is higher in AN compared with controls. Multivariate analysis illustrates normalization of lipidomic profiles in recovered ANs. EPHX2 influences AN risk through in vivo interaction with dietary PUFAs. PUFA composition and concentrations as well as sEH activity may contribute to the pathogenesis and prognosis of AN. Our data support the involvement of EPHX2-associated lipidomic and oxylipin dysregulations in AN, and reveal their potential as biomarkers to assess responsiveness to future intervention or treatment.

Modulation of the Ca2 permeable cation channel TRPV4 by cytochrome P450 epoxygenases in vascular endothelium.

TRPV4 is a broadly expressed Ca2+-permeable cation channel in the vanilloid subfamily of transient receptor potential channels. TRPV4 gates in response to a large variety of stimuli, including cell swelling, warm temperatures, the synthetic phorbol ester 4alpha-phorbol 12,13-didecanoate (4alpha-PDD), and the endogenous lipid arachidonic acid (AA). Activation by cell swelling and AA requires cytochrome P450 (CYP) epoxygenase activity to convert AA to epoxyeicosatrienoic acids (EETs) such as 5,6-EET, 8,9-EET, which both act as direct TRPV4 agonists. To evaluate the role of TRPV4 and its modulation by the CYP pathway in vascular endothelial cells, we performed Ca2+ imaging and patch-clamp measurements on mouse aortic endothelial cells (MAECs) isolated from wild-type and TRPV4(-/-) mice. All TRPV4-activating stimuli induced robust Ca2+ responses in wild-type MAECs but not in MAECs isolated from TRPV4(-/-) mice. Upregulation of CYP2C expression by preincubation with nifedipine enhanced the responses to AA and cell swelling in wild-type MAECs, whereas responses to other stimuli remained unaffected. Conversely, inhibition of CYP2C9 activity with sulfaphenazole abolished the responses to AA and hypotonic solution (HTS). Moreover, suppression of EET hydrolysis using 1-adamantyl-3-cyclo-hexylurea or indomethacin, inhibitors of soluble epoxide hydrolases (sEHs), and cyclooxygenases, respectively, enhanced the TRPV4-dependent responses to AA, HTS, and EETs but not those to 4alpha-PDD or heat. Together, our data establish that CYP-derived EETs modulate the activity of TRPV4 channels in endothelial cells and shows the unraveling of novel modulatory pathways via CYP2C modulation and sEH inhibition.

Soluble epoxide hydrolase activity and pharmacologic inhibition in horses with chronic severe laminitis.

BACKGROUND: The roles of soluble epoxide hydrolase and lipid mediators in inflammatory and neuropathic pain could be relevant in laminitis pain management. OBJECTIVES: To determine soluble epoxide hydrolase (sEH) activity in the digital laminae, sEH inhibitor potency in vitro, and efficacy of a sEH inhibitor as an adjunct analgesic therapy in chronic laminitic horses. STUDY DESIGN: In vitro experiments and clinical case series. METHODS: sEH activity was measured in digital laminae from euthanised healthy and laminitic horses (n = 5-6/group). Potency of 7 synthetic sEH inhibitors was determined in vitro using equine liver cytosol. One of them (t-TUCB; 0.1 mg/kg bwt i.v. every 24 h) was selected based on potency and stability, and used as adjunct therapy in 10 horses with severe chronic laminitis (Obel grades 2, one horse; 3-4, nine horses). Daily assessments of forelimb lifts, pain scores, physiologic and laboratory examinations were performed before (baseline) and during t-TUCB treatment. Data are presented as mean ± s.d. and 95% confidence intervals (CI). RESULTS: sEH activity in the digital laminae from laminitic horses (0.9±0.6 nmol/min/mg; 95% CI 0.16-1.55 nmol/min/mg) was significantly greater (P = 0.01) than in healthy horses (0.17±0.09 nmol/min/mg; CI 0.07-0.26 nmol/min/mg). t-TUCB as an adjunct analgesic up to 10 days (4.3±3 days) in laminitic horses was associated with significant reduction in forelimb lifts (36±22%; 95% CI 9-64%) and in pain scores (18±23%; 95% CI 2-35%) compared with baseline (P = 0.04). One horse developed gas colic and another corneal vascularisation in a blind eye during treatment. No other significant changes were observed. MAIN LIMITATIONS: Absence of control group and evaluator blinding in case series. CONCLUSIONS: sEH activity is significantly higher in the digital laminae of actively laminitic compared with healthy horses, and use of a potent inhibitor of equine sEH as adjunct analgesic therapy appears to decrease signs of pathologic pain in laminitic horses.

Soluble epoxide hydrolase regulates hydrolysis of vasoactive epoxyeicosatrienoic acids.

The cytochrome P450-derived epoxyeicosatrienoic acids (EETs) have potent effects on renal vascular reactivity and tubular sodium and water transport; however, the role of these eicosanoids in the pathogenesis of hypertension is controversial. The current study examined the hydrolysis of the EETs to the corresponding dihydroxyeicosatrienoic acids (DHETs) as a mechanism for regulation of EET activity and blood pressure. EET hydrolysis was increased 5- to 54-fold in renal cortical S9 fractions from the spontaneously hypertensive rat (SHR) relative to the normotensive Wistar-Kyoto (WKY) rat. This increase was most significant for the 14,15-EET regioisomer, and there was a clear preference for hydrolysis of 14, 15-EET over the 8,9- and 11,12-EETs. Increased EET hydrolysis was consistent with increased expression of soluble epoxide hydrolase (sEH) in the SHR renal microsomes and cytosol relative to the WKY samples. The urinary excretion of 14,15-DHET was 2.6-fold higher in the SHR than in the WKY rat, confirming increased EET hydrolysis in the SHR in vivo. Blood pressure was decreased 22+/-4 mm Hg (P:<0.01) 6 hours after treatment of SHRs with the selective sEH inhibitor N:, N:'-dicyclohexylurea; this treatment had no effect on blood pressure in the WKY rat. These studies identify sEH as a novel therapeutic target for control of blood pressure. The identification of a potent and selective inhibitor of EET hydrolysis will be invaluable in separating the vascular effects of the EET and DHET eicosanoids.

The cloning and characterization of a soluble epoxide hydrolase in chicken.

The mammalian soluble epoxide hydrolase (sEH) plays a role in the regulation of blood pressure and vascular homeostasis through its hydrolysis of the endothelial-derived messenger molecules, the epoxyeicosatrienoic acids. This study reports the cloning and expression of a sEH homolog from chicken liver. The resulting 63-kDa protein has an isoelectric point of 6.1. The recombinant enzyme displayed epoxide hydrolase activity when assayed with [3H]-trans-1,3-diphenylpropene oxide (t-DPPO), as well as trans-9,10-epoxystearate and the cis-8,9-, 11,12-, and 14,15- epoxyeicosatrienoic acids. The chicken enzyme displayed a lower kcat:Km for t-DPPO than the mammalian enzymes. The enzyme was sensitive to urea-based inhibitors developed for mammalian sEH. Such compounds could be used to study the role of chicken sEH in conditions in which endothelial-derived vasodilation is believed to be impaired, such as pulmonary hypertension syndrome.

Evaluation of fish models of soluble epoxide hydrolase inhibition.

Substituted ureas and carbamates are mechanistic inhibitors of the soluble epoxide hydrolase (sEH). We screened a set of chemicals containing these functionalities in larval fathead minnow (Pimphales promelas) and embryo/larval golden medaka (Oryzias latipes) models to evaluate the utility of these systems for investigating sEH inhibition in vivo. Both fathead minnow and medaka sEHs were functionally similar to the tested mammalian orthologs (murine and human) with respect to substrate hydrolysis and inhibitor susceptibility. Low lethality was observed in either larval or embryonic fish exposed to diuron [N-(3,4-dichlorophenyl), N'-dimethyl urea], desmethyl diuron [N-(3,4-dichlorophenyl), N'-methyl urea], or siduron [N-(1-methylcyclohexyl), N'-phenyl urea]. Dose-dependent inhibition of sEH was a sublethal effect of substituted urea exposure with the potency of siduron < desmethyl diuron = diuron, differing from the observed in vitro sEH inhibition potency of siduron > desmethyl diuron > diuron. Further, siduron exposure synergized the toxicity of trans-stilbene oxide in fathead minnows. Medaka embryos exposed to diuron, desmethyl diuron, or siduron displayed dose-dependent delays in hatch, and elevated concentrations of diuron and desmethyl diuron produced developmental toxicity. The dose-dependent toxicity and in vivo sEH inhibition correlated, suggesting a potential, albeit undefined, relationship between these factors. Additionally, the observed inversion of in vitro to in vivo potency suggests that these fish models may provide tools for investigating the in vivo stability of in vitro inhibitors while screening for untoward effects.

Expression and characterization of the recombinant juvenile hormone epoxide hydrolase (JHEH) from Manduca sexta.

The cDNA of the microsomal Juvenile Hormone Epoxide Hydrolase (JHEH) from Manduca sexta was expressed in vitro in the baculovirus system. In insect cell culture, the recombinant enzyme (Ms-JHEH) was produced at a high level (100 fold over background EH catalytic activity). As expected, Ms-JHEH was localized in the microsomal fraction with a molecular mass of approximately 50 kDa. Ms-JHEH showed a substrate and inhibitor spectrum similar to the wild type JHEH isolated from eggs of M. sexta. Its enzymatic activity was the highest for Juvenile Hormone III. Ms-JHEH hydrolyzed several trans-epoxides faster than cis-epoxides. A putative hydroxyl-acyl enzyme intermediate was isolated suggesting a catalytic mechanism of Ms-JHEH similar to the mammalian EHs.

Differential induction of hepatic drug-metabolizing enzymes by fenvaleric acid in male rats.

Racemic fenvaleric acid [2-(4-chlorophenyl)-3-methyl-butanoic acid], the principal metabolite of fenvalerate, was administrated orally at 0.75, 1.5, and 3.0 mmol/kg body weight/day to Fisher-344 male rats for 7 days. Both pure enantiomers of fenvaleric acid were administered at 1.5 mmol/kg body weight/day; the clofibric acid at the same concentration was used as a positive control. Hepatic enzyme activities were measured. Results obtained clearly show that fenvaleric acid induced numerous hepatic drug metabolism enzymes in F344 rats. The (R) enantiomer of this compound induces a proliferation of peroxisomes, whereas the (S) enantiomer induces CYP2B and mEH activities. Therefore, high exposure to pyrethroid insecticides could interact with the normal metabolism of drugs or xenobiotics.

Microbiological Transformations. 33. Fungal Epoxide Hydrolases Applied to the Synthesis of Enantiopure Para-Substituted Styrene Oxides. A Mechanistic Approach.

The biohydrolysis of differently para-substituted styrene oxide derivatives was studied, using whole cells of the fungi Aspergillus niger or Beauveria sulfurescens. These microorganisms proved to be equipped with epoxide hydrolases which are able to achieve these hydrolyses with high enantioselectivity. This allowed the preparation of the optically active epoxides and of the corresponding vicinal diols which were obtained with good to excellent enantiomeric purity. These two microorganisms proved to be enantiocomplementary. A mechanistic study, carried out using a crude lyophilized enzymatic extract from A.niger, indicated via Hammet coefficient plotting that this hydrolysis is very probably due to a general base-catalyzed process.

Pharmacokinetics and in vivo potency of soluble epoxide hydrolase inhibitors in cynomolgus monkeys.

BACKGROUND AND PURPOSE: Soluble epoxide hydrolase inhibitors (sEHIs) possess anti-inflammatory, antiatherosclerotic, antihypertensive and analgesic properties. The pharmacokinetics (PK) and pharmacodynamics in terms of inhibitory potency of sEHIs were assessed in non-human primates (NHPs). Development of a sEHI for use in NHPs will facilitate investigations on the role of sEH in numerous chronic inflammatory conditions. EXPERIMENTAL APPROACH: PK parameters of 11 sEHIs in cynomolgus monkeys were determined after oral dosing with 0.3 mg·kg(-1). Their physical properties and inhibitory potency in hepatic cytosol of cynomolgus monkeys were examined. Dose-dependent effects of the two inhibitors 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU) and the related acetyl piperidine derivative, 1-trifluoromethoxyphenyl-3-(1-acetylpiperidin-4-yl) urea (TPAU), on natural blood eicosanoids, were determined. KEY RESULTS: Among the inhibitors tested, TPPU and two 4-(cyclohexyloxy) benzoic acid urea sEHIs displayed high plasma concentrations (>10 × IC(50)), when dosed orally at 0.3 mg·kg(-1). Although the 4-(cyclohexyloxy) benzoic acid ureas were more potent against monkey sEH than piperidyl ureas (TPAU and TPPU), the latter compounds showed higher plasma concentrations and more drug-like properties. The C(max) increased with dose from 0.3 to 3 mg·kg(-1) for TPPU and from 0.1 to 3 mg·kg(-1) for TPAU, although it was not linear over this range of doses. As an indication of target engagement, ratios of linoleate epoxides to diols increased with TPPU administration. CONCLUSION AND IMPLICATIONS: Our data indicate that TPPU is suitable for investigating sEH biology and the role of epoxide-containing lipids in modulating inflammatory diseases in NHPs.

Improvement of endothelium-dependent vasodilations by SKA-31 and SKA-20, activators of small- and intermediate-conductance Ca2+ -activated K+ -channels.

AIM: Endothelial membrane hyperpolarization mediated by KCa3.1 and KCa2.3 channels has been demonstrated to initiate endothelium-derived hyperpolarizing factor (EDHF)-type vasodilations. Moreover, pharmacological potentiation of KCa3.1/KCa2.3 channels has been suggested to improve EDHF-type vasodilations. Herein, we determined whether the KCa3.1/KCa2.3 activator SKA-31 and its derivative SKA-20 improve endothelial dysfunction in KCa3.1-/- and NOS3-/- mice. METHODS: Membrane potentials were measured using patch-clamp electrophysiology on carotid artery (CA) endothelial cells (CAEC) from wild-type (wt) and KCa3.1-/- mice. Endothelium-dependent vasodilations were determined by pressure myography in CA. RESULTS: SKA-31 (1 µm) activated KCa3.1 and KCa2.3 channels and induced membrane hyperpolarization in CAEC of wt (ΔMP -45 mV). These responses were significantly reduced in CAEC of KCa3.1-/- (ΔMP -8 mV). SKA-31 (200 nm, 500 nm) and SKA-20 (300 nm) significantly enhanced EDHF vasodilations in wt. SKA-20 also improved vasodilations during NO synthesis. In KCa3.1-/-, the defective EDHF vasodilations were unchanged at 200 nm SKA-31, but were significantly improved at 500 nm. EDHF vasodilations were slightly enhanced at 300 nm SKA-20, but vasodilations during NO synthesis were unchanged. SKA-31 (500 nm) enhanced the impaired endothelium-dependent vasodilation in NOS3-/- mice twofold. Pharmacological inhibition of the soluble epoxide hydrolase by t-AUCB (1 µm) in contrast did not increase ACh-induced EDHF- or NO-mediated vasodilations in wt and KCa3.1-/-. CONCLUSION: Normal and defective endothelium-dependent vasodilations in murine carotid arteries can be improved by pharmacological enhancement of KCa3.1/KCa2.3 functions. These findings further support the concept that pharmacological activation of endothelial KCa2.3/KCa3.1 could offer a novel endothelium-specific antihypertensive strategy.

Enantioselective hydrolysis of p-nitrostyrene oxide by an epoxide hydrolase preparation from Aspergillus niger.

The epoxide hydrolase activity of Aspergillus niger was synthesized during growth of the fungus and was shown to be associated with the soluble cell fraction. An enzyme preparation was worked out which could be used in place of the whole mycelium as biocatalyst for the hydrolysis of epoxides. The effect of four different cosolvents on enzyme activity was investigated. Consequently, dimethylsulfoxide (DMSO) was selected for epoxide solubilization. The effect of temperature on both reaction rate and enzyme stability was studied in the presence of DMSO (0.2 volume ratio). A temperature of 25 degrees C was selected for the reaction of bioconversion. With a substrate concentration of 4.5 mM a batch reactor showed that the enzyme preparation hydrolyzed para-nitrostyrene oxide with very high enantioselectivity. The (S) enantiomer of the epoxide remained in the reaction mixture and showed an enantiomeric excess higher than 99%. The substrate concentration could be increased to 20 mM without affecting the enantiomeric excess and degree of conversion. Therefore, the method is potentially useful for the preparative resolution of epoxides. Application are in the field of chiral synthons which are important building blocks in organic synthesis.

Metabolism of monoepoxides of methyl linoleate: bioactivation and detoxification.

Leukotoxin (ltx) and isoleukotoxin (iltx) methyl esters, are metabolites of methyl linoleic acid, an essential fatty acid. They have been associated with acute respiratory distress syndrome. The observed toxicity of ltx and iltx is, in fact, due to the metabolism of the epoxides to their corresponding diols by soluble epoxide hydrolase (sEH). Herein, we demonstrate that ltx/iltx are toxic in a time-dependent manner to human sEH expressing cells with a LT(50) of 10.6 +/- 0.8 h and that ltx and iltx have K(M) of 6.15 +/- 1.0 and 5. 17 +/- 0.56 microM, respectively, and V(max) of 2.67 +/- 0.04 and 1. 86 +/- 0.06 micromol/min/mg, respectively, which can be inhibited by sEH inhibitors. We show that four major metabolites of ltx/iltx are formed in our system, including ltx/iltx free acid, ltxd/iltxd, free acid, and phosphotidylcholine and phosphotidylethanolamine containing the carboxylic acid forms of both ltx/iltx and ltxd/iltxd, but that the only metabolite associated with toxicity is the carboxylic acid form of ltxd/iltxd, suggesting the involvement of cellular esterases. We demonstrate that a serine esterase inhibitor provides some protection from the toxicity of epoxy fatty esters to sEH expressing cells as do intercellular free sulfhydryls, but that this protection is not due to glutathione conjugation. With these data, we have proposed an extension of the metabolic pathway for ltx/iltx in eukaryotic cells.

Multiple epoxide hydrolases in Alternaria alternata f. sp. lycopersici and their relationship to medium composition and host-specific toxin production.

The production of Alternaria alternata f. sp. lycopersici host-specific toxins (AAL toxins) and epoxide hydrolase (EH) activity were studied during the growth of this plant-pathogenic fungus in stationary liquid cultures. Media containing pectin as the primary carbon source displayed peaks of EH activity at day 4 and at day 12. When pectin was replaced by glucose, there was a single peak of EH activity at day 6. Partial characterization of the EH activities suggests the presence of three biochemically distinguishable EH activities. Two of them have a molecular mass of 25 kDa and a pI of 4.9, while the other has a molecular mass of 20 kDa and a pI of 4.7. Each of the EH activities can be distinguished by substrate preference and sensitivity to inhibitors. The EH activities present at day 6 (glucose) or day 12 (pectin) are concomitant with AAL toxin production.

Mechanism of mammalian soluble epoxide hydrolase inhibition by chalcone oxide derivatives.

A series of substituted chalcone oxides (1,3-diphenyl-2-oxiranyl propanones) and structural analogs was synthesized to investigate the mechanism by which they inhibit soluble epoxide hydrolases (sEH). The inhibitor potency and inhibition kinetics were evaluated using both murine and human recombinant sEH. Inhibition kinetics were well described by the kinetic models of A. R. Main (1982, in Introduction to Biochemical Toxicology, pp. 193-223, Elsevier, New York) supporting the formation of a covalent enzyme-inhibitor intermediate with a half-life inversely proportional to inhibitor potency. Structure-activity relationships describe active-site steric constraints and support a mechanism of inhibition consistent with the electronic stabilization of the covalent enzyme-inhibitor intermediate. The electronic effects induced by altering the ketone functionality and the para-substitution of the phenyl attached to the epoxy C1 (i.e., the alpha-carbon) had the greatest influence on inhibitor potency. The direction of the observed influence was reversed for the inhibitory potency of glycidol (1-phenyl-2-oxiranylpropanol) derivatives. Recent insights into the mechanism of epoxide hydrolase activity are combined with these experimental results to support a proposed mechanism of sEH inhibition by chalcone oxides.

Purification and characterization of a highly enantioselective epoxide hydrolase from Aspergillus niger.

The epoxide hydrolase from Aspergillus niger was purified to homogeneity using a four-step procedure and p-nitrostyrene oxide (pNSO) as substrate. The enzyme was purified 246-fold with 4% activity yield. The protein is a tetramer composed of four identical subunits of molecular mass 45 kDa. Maximum activity was observed at 40 degrees C, pH 7.0, and with dimethylformamide as cosolvent to dissolve pNSO. Hydrolysis of pNSO was highly enantioselective, with an E value (i.e. enantiomeric ratio) of 40 and a high regioselectivity (97%) for the less hindered carbon atom of the epoxide. This enzyme may be a good biocatalyst for the preparation of enantiopure epoxides or diols.

Detoxification of environmental mutagens and carcinogens: structure, mechanism, and evolution of liver epoxide hydrolase.

The crystal structure of recombinant murine liver cytosolic epoxide hydrolase (EC 3.3.2.3) has been determined at 2.8-A resolution. The binding of a nanomolar affinity inhibitor confirms the active site location in the C-terminal domain; this domain is similar to that of haloalkane dehalogenase and shares the alpha/beta hydrolase fold. A structure-based mechanism is proposed that illuminates the unique chemical strategy for the activation of endogenous and man-made epoxide substrates for hydrolysis and detoxification. Surprisingly, a vestigial active site is found in the N-terminal domain similar to that of another enzyme of halocarbon metabolism, haloacid dehalogenase. Although the vestigial active site does not participate in epoxide hydrolysis, the vestigial domain plays a critical structural role by stabilizing the dimer in a distinctive domain-swapped architecture. Given the genetic and structural relationships among these enzymes of xenobiotic metabolism, a structure-based evolutionary sequence is postulated.

Inhibition of microsomal epoxide hydrolases by ureas, amides, and amines.

The microsomal epoxide hydrolase (mEH) plays a significant role in the metabolism of xenobiotics such as polyaromatic toxicants. Additionally, polymorphism studies have underlined a potential role of this enzyme in relation to several diseases, such as emphysema, spontaneous abortion, and several forms of cancer. To provide new tools for studying the function of mEH, inhibition of this enzyme was investigated. Inhibition of recombinant rat and human mEH was achieved using primary ureas, amides, and amines. Several of these compounds are more potent than previously published inhibitors. Elaidamide, the most potent inhibitor that is obtained, has a K(i) of 70 nM for recombinant rat mEH. This compound interacts with the enzyme forming a noncovalent complex, and blocks substrate turnover through an apparent mix of competitive and noncompetitive inhibition kinetics. Furthermore, in insect cell cultures expressing rat mEH, elaidamide enhances the toxicity effects of epoxide-containing xenobiotics. These inhibitors could be valuable tools for investigating the physiological and toxicological roles of mEH.

Biochemical evidence for the involvement of tyrosine in epoxide activation during the catalytic cycle of epoxide hydrolase.

Epoxide hydrolases (EH) catalyze the hydrolysis of epoxides and arene oxides to their corresponding diols. The crystal structure of murine soluble EH suggests that Tyr(465) and Tyr(381) act as acid catalysts, activating the epoxide ring and facilitating the formation of a covalent intermediate between the epoxide and the enzyme. To explore the role of these two residues, mutant enzymes were produced and the mechanism of action was analyzed. Enzyme assays on a series of substrates confirm that both Tyr(465) and Tyr(381) are required for full catalytic activity. The kinetics of chalcone oxide hydrolysis show that mutation of Tyr(465) and Tyr(381) decreases the rate of binding and the formation of an intermediate, suggesting that both tyrosines polarize the epoxide moiety to facilitate ring opening. These two tyrosines are, however, not implicated in the hydrolysis of the covalent intermediate. Sequence comparisons showed that Tyr(465) is conserved in microsomal EHs. The substitution of analogous Tyr(374) with phenylalanine in the human microsomal EH dramatically decreases the rate of hydrolysis of cis-stilbene oxide. These results suggest that these tyrosines perform a significant mechanistic role in the substrate activation by EHs.