Structural refinement of inhibitors of urea-based soluble epoxide hydrolases
Introduction
EHs (EC 3.3.2.3) catalyze the hydrolysis of epoxides or arene oxides to their corresponding diols by the addition of water [1]. In mammals, the soluble and microsomal EH forms are known to complement each other in detoxifying a wide array of mutagenic, toxic, and carcinogenic xenobiotic epoxides [2], [3]. sEH is also involved in the metabolism of arachidonic [4] and linoleic [5] acid epoxides, which are endogenous chemical mediators [6]. Epoxides of arachidonic acid (epoxyeicosatrienoic acids or EETs) are known effectors of blood pressure regulation [7], modulating vascular permeability [8]. The vasodilatory properties of EETs are associated with an increased open-state probability of calcium-activated potassium channels leading to hyperpolarization of the vascular smooth muscle [9]. Hydrolysis of the epoxides by sEH diminishes this activity [7]. sEH hydrolysis of EETs also regulates their incorporation into coronary endothelial phospholipids, suggesting a regulation of endothelial function by sEH [10]. In keeping with this hypothesis, treatment of spontaneous hypertensive rats (SHRs) with selective sEH inhibitors significantly reduces their blood pressure [11]. Additionally, male knockout sEH mice have significantly lower blood pressure than wild-type mice [12], further supporting the role of sEH in blood pressure regulation.
The EETs have also demonstrated some anti-inflammatory properties in endothelial cells [13], [14]. In contrast, diols derived from epoxy-linoleate (leukotoxin) perturb membrane permeability and calcium homeostasis [5], which results in inflammation that is modulated by nitric oxide synthase and endothelin-1 [15], [16]. Micromolar concentrations of leukotoxin reported in association with inflammation and hypoxia [17] depress mitochondrial respiration in vitro[18] and cause mammalian cardio-pulmonary toxicity in vivo[15], [19], [20]. Leukotoxin toxicity presents symptoms suggestive of multiple organ failure and acute respiratory distress syndrome (ARDS) [17]. In both cellular and organismal models, leukotoxin-mediated toxicity is dependent upon epoxide hydrolysis [5], [21], suggesting a role for sEH in the regulation of inflammation. The bioactivity of these epoxy-fatty acids suggests that inhibition of vicinal-dihydroxy-lipid biosynthesis may have therapeutic value, making sEH a promising pharmacological target.
We recently described 1,3-disubstituted ureas, carbamates, and amides (Fig. 1) as new potent and stable inhibitors of sEH. These compounds are competitive tight-binding inhibitors with nanomolar KI values that interact stoichiometrically with purified recombinant sEH [21]. From crystal structure determination, it was shown that the urea inhibitors established hydrogen bonding and salt bridges between the urea function of the inhibitor and residues of the sEH active site, mimicking features encountered in the reaction coordinate of epoxide ring opening by this enzyme [22], [23]. These inhibitors efficiently reduced epoxide hydrolysis in several in vitro and in vivo models [11], [21], [24]. However, these dialkyl-ureas have high crystal lattice energies as indicated by their high melting points, and also have limited solubility in water [11], which probably affects their in vivo efficacy. Therefore, we investigated the effect of changes in the structure of disubstituted ureas (Fig. 1) in relation to their potency as sEH inhibitors to design powerful inhibitors with improved physical properties.
Section snippets
Reagents and apparatus
Compound (7) was obtained from Aldrich. The synthesis and characterization of compound (27) were described in a report by Argiriadi et al. [23]. For the other compounds described for this study, reaction yield, melting point, and mass spectroscopy data are given in Table 1. Other chemicals and reagents were purchased from Aldrich or Sigma and were used without further purification. Melting points were determined with a Thomas-Hoover apparatus (A.H. Thomas Co.) and are uncorrected. Infrared (IR)
Results
In previous studies [21], [32], a spectrophotometric assay [29] was used to determine the potency of sEH inhibitors. While this assay allows rapid, accurate, and precise screening of numerous compounds in a 96-well plate format, it does not permit the segregation of very potent compounds, due to its low sensitivity. For example, as the effective concentration of a candidate inhibitor approaches the concentration of the target enzyme used in the assay, the ability to distinguish among the
Discussion
Over the past 30 years, sEH has been studied mainly for its role in hepatic xenobiotic detoxification [3]. We recently showed that sEH present in kidneys and coronary endothelial cells plays an important role in EET metabolism [11], [33]. EETs possess important vasodilating and anti-inflammatory properties [6], [7]. The bioactivity of these epoxy-fatty acids suggests that inhibition of vicinal-dihydroxy-lipid biosynthesis may have therapeutic value, making sEH a promising pharmacological
Acknowledgements
The authors thank Dr. A.D. Jones (Pennsylvania State University) for mass spectra analyses, and Dr. Yoshiaki Nakagawa (Kyoto University) for his helpful comments and assistance in the QSAR multiple regression analysis. This work was supported, in part, by NIEHS Grant R01-ES02710, NIEHS Center for Environmental Health Sciences 1P30-ES05707, and the NIH/NIEHS Superfund Basic Research program P42-ES04699.
References (35)
- et al.
Epoxide hydrolases: biochemistry and molecular biology
Chem. Biol. Interact.
(2000) - et al.
Regio- and enantiofacial selectivity of epoxyeicosatrienoic acid hydration by cytosolic epoxide hydrolase
J. Biol. Chem.
(1993) - et al.
Cytochrome P450 and arachidonic acid bioactivation: molecular and functional properties on the arachidonate monooxygenase
J. Lipid Res.
(2000) - et al.
Targeted disruption of soluble epoxide hydrolase reveals a role in blood pressure regulation
J. Biol. Chem.
(2000) New role for epoxyeicosatrienoic acids as anti-inflammatory mediators
Trends Pharmacol. Sci.
(2000)- et al.
Lipid oxidation products in ischemic porcine heart tissue
Chem. Phys. Lipids
(1996) - et al.
Binding of alkylurea inhibitors to epoxide hydrolase implicates active site tyrosines in substrate activation
J. Biol. Chem.
(2000) - et al.
Molecular cloning and expression of murine liver soluble epoxide hydrolase
J. Biol. Chem.
(1993) - et al.
cDNA cloning and expression of a soluble epoxide hydrolase from human liver
Arch. Biochem. Biophys.
(1993) - et al.
Affinity purification of cytosolic epoxide hydrolase using derivatized epoxy-activated Sepharose gels
Anal. Biochem.
(1988)
Improved radiolabeled substrates for soluble epoxide hydrolase
Anal. Biochem.
Spectrophotometric substrates for cytosolic epoxide hydrolase
Anal. Biochem.
3-D QSAR analysis of inhibition of murine soluble epoxide hydrolase (MsEH) by benzoylureas, arylureas, and their analogues
Bioorg. Med. Chem.
Pathways of epoxyeicosatrienoic acid metabolism in endothelial cells: implication for the vascular effects of soluble epoxide hydrolase inhibition
J. Biol. Chem.
The reaction of arachidonic acid epoxides (epoxyeicosatrienoic acids) with a cytosolic epoxide hydrolase
Arch. Biochem. Biophys.
Biochemical evidence for the involvement of tyrosine in epoxide activation during the catalytic cycle of epoxide hydrolase
J. Biol. Chem.
Mammalian epoxide hydrolases: inducible enzymes catalyzing the inactivation of carcinogenic and cytotoxic metabolites derived from aromatic and olefinic compounds
Xenobiotica
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