Inhibition of soluble epoxide hydrolase by fulvestrant and sulfoxides

The soluble epoxide hydrolase (sEH) is a key enzyme in the metabolism of epoxy-fatty acids, signaling molecules involved in numerous biologies. Toward finding novel inhibitors of sEH, a library of known drugs was tested for inhibition of sEH. We found that fulvestrant, an anticancer agent, is a potent (K_I = 26 nM) competitive inhibitor of sEH. From this observation, we found that alkyl-sulfoxides represent a new kind of pharmacophore for the inhibition of sEH.     

Preparation and evaluation of soluble epoxide hydrolase inhibitors with improved physical properties and potencies for treating diabetic neuropathic pain

Soluble epoxide hydrolase (sEH), a novel therapeutic target for neuropathic pain, is a largely cytosolic enzyme that degrades epoxy-fatty acids (EpFAs), an important class of lipid signaling molecules. Many inhibitors of sEH have been reported, and to date, the 1,3-disubstituted urea has the highest affinity reported for the sEH among the central pharmacophores evaluated. An earlier somewhat water soluble sEH inhibitor taken to the clinic for blood pressure control had mediocre potency (both affinity and kinetics) and a short in vivo half-life. We undertook a study to overcome these difficulties, but the sEH inhibitors carrying a 1,3-disubstituted urea often suffer poor physical properties that hinder their formulation. In this report, we described new strategies to improve the physical properties of sEH inhibitors with a 1,3-disubstituted urea while maintaining their potency and drug-target residence time (a complementary in vitro parameter) against sEH. To our surprise, we identified two structural modifications that substantially improve the potency and physical properties of sEH inhibitors carrying a 1,3-disubstituted urea pharmacophore. Such improvements will greatly facilitate the movement of sEH inhibitors to the clinic.     

Effect of soluble epoxide hydrolase polymorphism on substrate and inhibitor selectivity and dimer formation

Epoxy FAs (EpFAs) are important lipid mediators that are mainly metabolized by soluble epoxide hydrolase (sEH). Thus, sEH inhibition is a promising therapeutic target to treat numerous ailments. Several sEH polymorphisms result in amino acid substitutions and alter enzyme activity. K55R and R287Q are associated with inflammatory, cardiovascular, and metabolic diseases. R287Q seems to affect sEH activity through reducing formation of a catalytically active dimer. Thus, understanding how these SNPs affect the selectivity of sEH for substrates and inhibitors is of potential clinical importance. We investigated the selectivity of four sEH SNPs toward a series of EpFAs and inhibitors. We found that the SNPs alter the catalytic activity of the enzyme but do not alter the relative substrate and inhibitor selectivity. We also determined their dimer/monomer constants (K_D/M). The WT sEH formed a very tight dimer, with a K_D/M in the low picomolar range. Only R287Q resulted in a large change of the K_D/M. However, human tissue concentrations of sEH suggest that it is always in its dimer form independently of the SNP. These results suggest that the different biologies associated with K55R and R287Q are not explained by alteration in dimer formation or substrate selectivity.     

Exploring the size of the lipophilic unit of the soluble epoxide hydrolase inhibitors

Soluble epoxide hydrolase (sEH) inhibitors are potential drugs for several diseases. Adamantyl ureas are excellent sEH inhibitors but have limited metabolic stability. Herein, we report the effect of replacing the adamantane group by alternative polycyclic hydrocarbons on sEH inhibition, solubility, permeability and metabolic stability. Compounds bearing smaller or larger polycyclic hydrocarbons than adamantane yielded all good inhibition potency of the human sEH (0.4 ≤ IC₅₀ ≤ 21.7 nM), indicating that sEH is able to accommodate inhibitors of very different size. Human liver microsomal stability of diamantane containing inhibitors is lower than that of their corresponding adamantane counterparts.     

Isolation and characterization of Xenopus soluble epoxide hydrolase

Soluble epoxide hydrolase (sEH) contributes to cell growth, but the contribution of sEH to embryonic development is not well understood. In this study, Xenopus sEH cDNA was isolated from embryos of Xenopus laevis. The Xenopus sEH was expressed in Escherichia coli and was purified. The epoxide hydrolase and phosphatase activities of purified sEH were investigated. The Xenopus sEH did not show phosphatase activity toward 4-methylumbelliferyl phosphate or several lysophosphatidic acids although it had EH activity. The amino acid sequence of Xenopus sEH was compared with that reported previously. We found amino acid substitutions of the 29th Thr to Asn and the 146th Arg to His and prepared a sEH mutant (N29T/H146R), designed as mutant 1. Neither wild-type sEH nor mutant 1 had phosphatase activity. Additional substitution of the 11th Gly with Asp was found by comparison with human sEH which has phosphatase activity, but the Xenopus sEH mutant G11D prepared as mutant 2 did not have phosphatase activity. The epoxide hydrolase activity of sEH seemed to be similar to that of human sEH, while Xenopus sEH did not have phosphatase activity toward several substrates that human sEH metabolizes.     

Human Valacyclovir Hydrolase/Biphenyl Hydrolase-Like Protein Is a Highly Efficient Homocysteine Thiolactonase

Homocysteinylation of lysine residues by homocysteine thiolactone (HCTL), a reactive homocysteine metabolite, results in protein aggregation and malfunction, and is a well-known risk factor for cardiovascular, autoimmune and neurological diseases. Human plasma paraoxonase-1 (PON1) and bleomycin hydrolase (Blmh) have been reported as the physiological HCTL detoxifying enzymes. However, the catalytic efficiency of HCTL hydrolysis by Blmh is low and not saturated at 20 mM HCTL. The catalytic efficiency of PON1 for HCTL hydrolysis is 100-fold lower than that of Blmh. A homocysteine thiolactonase (HCTLase) was purified from human liver and identified by mass spectrometry (MS) as the previously described human biphenyl hydrolase-like protein (BPHL). To further characterize this newly described HCTLase activity, BPHL was expressed in Escherichia coli and purified. The sequence of the recombinant BPHL (rBPHL) and hydrolytic products of the substrates HCTL and valacyclovir were verified by MS. We found that the catalytic efficiency (k_cat/K_m) of rBPHL for HCTL hydrolysis was 7.7 × 10⁴ M⁻¹s⁻¹, orders of magnitude higher than that of PON1 or Blmh, indicating a more significant physiological role for BPHL in detoxifying HCTL.     

Identification and optimization of soluble epoxide hydrolase inhibitors with dual potency towards fatty acid amide hydrolase

Multi-target inhibitors have become increasing popular as a means to leverage the advantages of poly-pharmacology while simplifying drug delivery. Here, we describe dual inhibitors for soluble epoxide hydrolase (sEH) and fatty acid amide hydrolase (FAAH), two targets known to synergize when treating inflammatory and neuropathic pain. The structure activity relationship (SAR) study described herein initially started with t-TUCB (trans-4-[4-(3-trifluoromethoxyphenyl-l-ureido)-cyclohexyloxy]-benzoic acid), a potent sEH inhibitor that was previously shown to weakly inhibit FAAH. Inhibitors with a 6-fold increase of FAAH potency while maintaining high sEH potency were developed by optimization. Interestingly, compared to most FAAH inhibitors that inhibit through time-dependent covalent modification, t-TUCB and related compounds appear to inhibit FAAH through a time-independent, competitive mechanism. These inhibitors are selective for FAAH over other serine hydrolases. In addition, FAAH inhibition by t-TUCB appears to be higher in human FAAH over other species; however, the new dual sEH/FAAH inhibitors have improved cross-species potency. These dual inhibitors may be useful for future studies in understanding the therapeutic application of dual sEH/FAAH inhibition.     

In vitro inhibitory effects of ethanol extract of Danshen (Salvia miltiorrhiza) and its components on the catalytic activity of soluble epoxide hydrolase

Background: Soluble epoxide hydrolase (sEH) has been demonstrated to be a key enzyme involved in the pathologic development of several cardiovascular diseases and inflammation, and inhibition of sEH is therefore very helpful or crucial for the treatment of ischemia-reperfusion injury, cardiac hypertrophy, hypertension and inflammation. Danshen, the dried root of Salvia miltiorrhiza (Fam. Labiatae), has been used for the treatment of cardiovascular and cerebrovascular diseases in China and other countries for hundreds of years. Recent studies indicated that Danshen and its preparations also have potential for the management of inflammation. However, little information is available about the possibility of Danshen and its components on sEH inhibition.Purpose and methods: Danshen extracts and its constituents were tested for sEH inhibition using its physiological substrate, 8,9-EET, based on a LC–MS/MS assay in this study.Results: Among the tested 15 compounds, tanshinone IIA and cryptotanshinone were found to be the potent (K_i = 0.87 μM) and medium (K_i = 6.7 μM) mixed-type inhibitors of sEH, respectively. Salvianolic acid C (K_i = 8.6 μM) was proved to be a moderate noncompetitive sEH inhibitor. In consistent with the inhibition results of the pure compounds, the 75% ethanol extract of Danshen (EE, IC₅₀ = 86.5 μg/ml) which contained more tanshinone IIA and cryptotanshinone exhibited more potent inhibition on sEH than the water extract (WE, IC₅₀ > 200 μg/ml) or 1 M NaHCO₃ (BE, IC₅₀ > 200 μg/ml) extract.Conclusion: These data indicated that using the ethanol fraction of Danshen and increasing the amounts of tanshinone IIA, cryptotanshinone and salvianolic acid C, especially the contents of tanshinone IIA in Danshen extract or preparations to enhance the inhibitory effects on sEH might be efficient ways to improve its cardiovascular protective and anti-inflammatory effects, and that herbal medicines could be an untapped reservoir for sEH-inhibition agents and developing sEH inhibitors from the cardiovascular protective and anti-inflammatory herbs is a promising approach.     

Cholesteryl ester hydrolase activity is abolished in HSL macrophages but unchanged in macrophages lacking KIAA1363

Cholesteryl ester (CE) accumulation in macrophages represents a crucial event during foam cell formation, a hallmark of atherogenesis. Here we investigated the role of two previously described CE hydrolases, hormone-sensitive lipase (HSL) and KIAA1363, in macrophage CE hydrolysis. HSL and KIAA1363 exhibited marked differences in their abilities to hydrolyze CE, triacylglycerol (TG), diacylglycerol (DG), and 2-acetyl monoalkylglycerol ether (AcMAGE), a precursor for biosynthesis of platelet-activating factor (PAF). HSL efficiently cleaved all four substrates, whereas KIAA1363 hydrolyzed only AcMAGE. This contradicts previous studies suggesting that KIAA1363 is a neutral CE hydrolase. Macrophages of KIAA1363^−/− and wild-type mice exhibited identical neutral CE hydrolase activity, which was almost abolished in tissues and macrophages of HSL^−/− mice. Conversely, AcMAGE hydrolase activity was diminished in macrophages and some tissues of KIAA1363^−/− but unchanged in HSL^−/− mice. CE turnover was unaffected in macrophages lacking KIAA1363 and HSL, whereas cAMP-dependent cholesterol efflux was influenced by HSL but not by KIAA1363. Despite decreased CE hydrolase activities, HSL^−/− macrophages exhibited CE accumulation similar to wild-type (WT) macrophages. We conclude that additional enzymes must exist that cooperate with HSL to regulate CE levels in macrophages. KIAA1363 affects AcMAGE hydrolase activity but is of minor importance as a direct CE hydrolase in macrophages.     

Soluble epoxide hydrolase contamination of specific catalase preparations inhibits epoxyeicosatrienoic acid vasodilation of rat renal arterioles

Cytochrome P-450 metabolites of arachidonic acid, the epoxyeicosatrienoic acids (EETs) and hydrogen peroxide (H(2)O(2)), are important signaling molecules in the kidney. In renal arteries, EETs cause vasodilation whereas H(2)O(2) causes vasoconstriction. To determine the physiological contribution of H(2)O(2), catalase is used to inactivate H(2)O(2). However, the consequence of catalase action on EET vascular activity has not been determined. In rat renal afferent arterioles, 14,15-EET caused concentration-related dilations that were inhibited by Sigma bovine liver (SBL) catalase (1,000 U/ml) but not Calbiochem bovine liver (CBL) catalase (1,000 U/ml). SBL catalase inhibition was reversed by the soluble epoxide hydrolase (sEH) inhibitor tAUCB (1 μM). In 14,15-EET incubations, SBL catalase caused a concentration-related increase in a polar metabolite. Using mass spectrometry, the metabolite was identified as 14,15-dihydroxyeicosatrienoic acid (14,15-DHET), the inactive sEH metabolite. 14,15-EET hydrolysis was not altered by the catalase inhibitor 3-amino-1,2,4-triazole (3-ATZ; 10-50 mM), but was abolished by the sEH inhibitor BIRD-0826 (1-10 μM). SBL catalase EET hydrolysis showed a regioisomer preference with greatest hydrolysis of 14,15-EET followed by 11,12-, 8,9- and 5,6-EET (V(max) = 0.54 ± 0.07, 0.23 ± 0.06, 0.18 ± 0.01 and 0.08 ± 0.02 ng DHET·U catalase(-1)·min(-1), respectively). Of five different catalase preparations assayed, EET hydrolysis was observed with two Sigma liver catalases. These preparations had low specific catalase activity and positive sEH expression. Mass spectrometric analysis of the SBL catalase identified peptide fragments matching bovine sEH. Collectively, these data indicate that catalase does not affect EET-mediated dilation of renal arterioles. However, some commercial catalase preparations are contaminated with sEH, and these contaminated preparations diminish the biological activity of H(2)O(2) and EETs.     

Epoxide hydrolase activities and epoxy fatty acids in the mosquito Culex quinquefasciatus

Culex mosquitoes have emerged as important model organisms for mosquito biology, and are disease vectors for multiple mosquito-borne pathogens, including West Nile virus. We characterized epoxide hydrolase activities in the mosquito Culex quinquefasciatus, which suggested multiple forms of epoxide hydrolases were present. We found EH activities on epoxy eicosatrienoic acids (EETs). EETs and other eicosanoids are well-established lipid signaling molecules in vertebrates. We showed EETs can be synthesized in vitro from arachidonic acids by mosquito lysate, and EETs were also detected in vivo both in larvae and adult mosquitoes by LC-MS/MS. The EH activities on EETs can be induced by blood feeding, and the highest activity was observed in the midgut of female mosquitoes. The enzyme activities on EETs can be inhibited by urea-based inhibitors designed for mammalian soluble epoxide hydrolases (sEH). The sEH inhibitors have been shown to play diverse biological roles in mammalian systems, and they can be useful tools to study the function of EETs in mosquitoes. Besides juvenile hormone metabolism and detoxification, insect epoxide hydrolases may also play a role in regulating lipid signaling molecules, such as EETs and other epoxy fatty acids, synthesized in vivo or obtained from blood feeding by female mosquitoes.     

Inhibition of the Soluble Epoxide Hydrolase by Tyrosine Nitration

Inhibition of the soluble epoxide hydrolase (sEH) has beneficial effects on vascular inflammation and hypertension indicating that the enzyme may be a promising target for drug development. As the enzymatic core of the hydrolase domain of the human sEH contains two tyrosine residues (Tyr³⁸³ and Tyr⁴⁶⁶) that are theoretically crucial for enzymatic activity, we addressed the hypothesis that the activity of the sEH may be affected by nitrosative stress. Epoxide hydrolase activity was detected in human and murine endothelial cells as well in HEK293 cells and could be inhibited by either authentic peroxynitrite (ONOO⁻) or the ONOO⁻ generator 3-morpholino-sydnonimine (SIN-1). Protection of the enzymatic core with 1-adamantyl-3-cyclohexylurea in vitro decreased sensitivity to SIN-1. Both ONOO⁻ and SIN-1 elicited the tyrosine nitration of the sEH protein and mass spectrometry analysis of tryptic fragments revealed nitration on several tyrosine residues including Tyr³⁸³ and Tyr⁴⁶⁶. Mutation of the latter residues to phenylalanine was sufficient to abrogate epoxide hydrolase activity. In vivo, streptozotocin-induced diabetes resulted in the tyrosine nitration of the sEH in murine lungs and a significant decrease in its activity. Taken together, these data indicate that the activity of the sEH can be regulated by the tyrosine nitration of the protein. Moreover, nitrosative stress would be expected to potentiate the physiological actions of arachidonic acid epoxides by preventing their metabolism to the corresponding diols.Over the last decade, a great deal has been discovered about the physiological role of cytochrome P450-derived epoxides, such as those generated from arachidonic and linoleic acid, in the regulation of vascular homeostasis (1). For example, CYP2C- and CYP2J-derived epoxyeicosatrienoic acids (EETs)³ can acutely regulate vascular tone by inducing endothelial and smooth muscle cell hyperpolarization in the systemic circulation while promoting constriction in pulmonary circulation. EETs also stimulate a number of endothelial signaling cascades to promote angiogenesis (2).The arachidonic acid epoxides (apart from 5,6-EET) are chemically stable, and their intracellular level is tightly regulated by a number of different mechanisms including β-oxidation (3), chain elongation (4), and hydration. However, of these regulatory mechanisms, it appears that the physiologically most important enzyme for the intracellular regulation of EET levels is the soluble epoxide hydrolase (sEH) (5). The dihydroxyeicosatrienoic acids (DHETs) generated from the EETs by sEH are biologically active, although generally less so than the parent epoxides (for review, see Ref. 6). Indeed, when the EETs are converted to the more polar DHETs, they are not as readily incorporated into membrane lipids (7, 8) and rapidly leave cells as diols or as still more polar conjugates.Surprisingly little is known about the mechanisms that regulate sEH activity, and although there have been a number of studies linking changes in sEH expression with inflammatory or hormonal stimuli (9, 10), nothing is known about the regulation of sEH by post-translational modification. Given that two tyrosine residues (Tyr³⁸³ and Tyr⁴⁶⁶) in the active site of the hydrolase are reportedly essential for enzyme activity (11), we determined whether or not the sEH could be regulated by tyrosine nitration.     

Deletion of soluble epoxide hydrolase suppressed chronic kidney disease-related vascular calcification by restoring Sirtuin 3 expression

Vascular calcification is common in chronic kidney disease (CKD) and contributes to cardiovascular disease (CVD) without any effective therapies available up to date. The expression of soluble epoxide hydrolase (sEH) is different in patients with and without vascular calcification. The present study investigates the role of sEH as a potential mediator of vascular calcification in CKD. Both Ephx2^−/− and wild-type (WT) mice fed with high adenine and phosphate (AP) diet were used to explore the vascular calcification in CKD. Compared with WT, deletion of sEH inhibited vascular calcification induced by AP. sEH deletion also abolished high phosphorus (Pi)-induced phenotypic transition of vascular smooth muscle cells (VSMCs) independent of its epoxyeicosatrienoic acids (EETs) hydrolysis. Further gene expression analysis identified the potential role of Sirtuin 3 (Sirt3) in the sEH-regulated VSMC calcification. Under high Pi treatment, sEH interacted with Sirt3, which might destabilize Sirt3 and accelerate the degradation of Sirt3. Deletion of sEH may preserve the expression of Sirt3, and thus maintain the mitochondrial adenosine triphosphate (ATP) synthesis and morphology, significantly suppressing VSMC calcification. Our data supported that sEH deletion inhibited vascular calcification and indicated a promising target of sEH inhibition in vascular calcification prevention.Subject terms: Chronic kidney disease, Calcification     

Symmetric adamantyl-diureas as soluble epoxide hydrolase inhibitors

A series of inhibitors of the soluble epoxide hydrolase (sEH) containing two urea groups has been developed. Inhibition potency of the described compounds ranges from 2.0 μM to 0.4 nM. 1,6-(Hexamethylene)bis[(adamant-1-yl)urea] (3b) was found to be a potent slow tight binding inhibitor (IC₅₀ = 0.5 nM) with a strong binding to sEH (K_i = 3.1 nM) and a moderately long residence time on the enzyme (k_off = 1.05 × 10⁻³ s⁻¹; t_1/2 = 11 min).     

Effects of human soluble epoxide hydrolase polymorphisms on isoprenoid phosphate hydrolysis

Soluble epoxide hydrolase (sEH) is highly expressed in human liver and contains a C-terminal epoxide hydrolase activity and an N-terminal phosphatase activity. Endogenous C-terminal hydrolase substrates include arachidonic acid epoxides, however, data are limited regarding possible endogenous substrates for the N-terminal phosphatase. Possible sEH N-terminal substrates include isoprenoid phosphate precursors of cholesterol biosynthesis and protein isoprenylation. Here, we report the kinetic analysis for a range of sEH isoprenoid substrates. We also provide an analysis of the effects of human sEH polymorphisms on isoprenoid hydrolysis. Interestingly, the Arg287Gln polymorphism recently suggested to be involved in hypercholesterolemia was found to possess a higher isoprenoid phosphatase activity than the wild type sEH. Consistent with the finding of isoprenoid phosphates as substrates for sEH, we identified isoprenoid-derived N-terminal inhibitors with IC50 values ranging from 0.84 (+/-0.9) to 55.1 (+/-30.7) microM. Finally, we evaluated the effects of the different isoprenoid compounds on the C-terminal hydrolase activity.     

Kinetic and Thermodynamic Characterization of Glucoamylase from Colletotrichum sp. KCP1

Extracellular glucoamylase of Colletotrichum sp. KCP1 produced through solid state fermentation was purified by two steps purification process comprising ammonium sulphate precipitation followed by gel permeation chromatography (GPC). The Recovery of glucoamylase after GPC was 50.40 % with 19.3-fold increase in specific activity. The molecular weight of enzyme was found to be 162.18 kDa by native-PAGE and was dimeric protein of two sub-units with molecular weight of 94.62 and 67.60 kDa as determined by SDS-PAGE. Activation energy for starch hydrolysis was 26.45 kJ mol⁻¹ while temperature quotient (Q₁₀) was found to be 1.9. The enzyme was found to be stable over wide pH range and thermally stable at 40–50 °C up to 120 min while exhibited maximum activity at 50 °C with pH 5.0. The pKa₁ and pKa₂ of ionisable groups of active site controlling V_max were 3.5 and 6.8, respectively. V_max, K_m and K_cat for starch hydrolysis were found to be 58.82 U ml⁻¹, 1.17 mg (starch) ml⁻¹ and 449 s⁻¹, respectively. Activation energy for irreversible inactivation (E_a(d)) of glucoamylase was 74.85 kJ mol⁻¹. Thermodynamic parameters of irreversible inactivation of glucoamylase and starch hydrolysis were also determined.     

Discovery of potent non-urea inhibitors of soluble epoxide hydrolase

Soluble epoxide hydrolase (sEH) is a novel target for the treatment of hypertension and vascular inflammation. A new class of potent non-urea sEH inhibitors was identified via high throughput screening (HTS) and chemical modification. IC₅₀s of the most potent compounds range from micromolar to low nanomolar.     

Purification and properties of S-adenosyl-L-homocysteine hydrolase from leaves of spinach beet.

S-Adenosyl-l-homocysteine hydrolase (EC 3.3.1.1) has been isolated from spinach-beet leaves and purified 100-fold. The enzyme catalyzes both the hydrolysis of S-adenosyl-l-homocysteine to adenosine and l-homocysteine and its synthesis from these compounds. The equilibrium constant for the reaction is 1.8 × 10^?6 in relation to hydrolysis. The enzyme shows optimum activity at pH 8.5. Enzyme preparations were stabilized by the addition of bovine serum albumin. The K_m for S-adenosylhomocysteine was 41 μm in the hydrolysis reaction and for adenosine, dl-homocysteine, and l-homocysteine it was 13 μm, 2.2 mm, and 1.2 mm, respectively.The enzyme was inhibited by S-adenosylmethionine, homocysteine, and adenine. These inhibitions and the K_m values determined are discussed in relation to the regulation of the enzyme in vivo and especially its effect on methylation reactions using S-adenosylmethionine as methyl donor.