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Erratum to “3-D QSAR Analysis of Inhibition of Murine Soluble Epoxide Hydrolase (MsEH) by Benzoylureas, Arylureas, and their Analogues”
Abstract
Two hundred and seventy-one compounds including benzoylureas, arylureas and related compounds were assayed using recombinant murine soluble epoxide hydrolase (MsEH) produced from a baculovirus expression system. Among all the insect growth regulators assayed, 18 benzoylphenylurea congeners showed weak activity against MsEH. Newly synthesized cyclohexylphenylurea, 1-benzyl-3-phenylurea, and 1,3-dibenzylurea analogues were rather potent. The introduction of a methyl group at the para-position of the phenyl ring of cyclohexylphenylurea enhanced the activity 6-fold, though similar substituent effects were not seen for any of the benzoylphenylureas. The activities of these compounds, including several previously reported compounds, such as dicyclohexylurea, diphenylurea, and their related analogues (Morisseau et al., Proc. Natl. Acad. Sci., 1999, 96, 8849), were quantitatively analyzed using comparative molecular field analysis (CoMFA), a three-dimensional quantitative structure-activity relationship (3-D QSAR) method. Both steric and electrostatic factors contributing to variations in the activity were visualized using CoMFA. CoMFA results showed that one side of the cyclohexylurea moiety having a trans-amide conformation (A-ring moiety) is surrounded by large sterically unfavorable fields, while the other side of A-ring moiety and the other cyclohexyl group (B-ring moiety) is encompassed by sterically favored fields. Electrostatically negative fields were scattered around the entire molecule, and a positive field surrounds the carbon of the carbonyl group. Hydrophobic fields were visualized using Kellogg's hydropathic interaction (HINT) in conjunction with CoMFA. Hydrophobically favorable fields appeared beside the 4- and 4'-carbon atoms of the cyclohexyl groups, and hydrophobically unfavorable fields surrounded the urea bridge. The addition of the molecular hydrophobicity, log P [corrected], to CoMFA did not improve the correlation significantly. The ligand-binding interactions shown by X-ray crystallographic data were rationalized using the results of the CoMFA and HINT analyses, and the essential physicochemical parameters for the design of new MsEH inhibitors were disclosed.
... 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. ...
... Speci®cally , substitution at the X or Y atom retained maximal potency for the human and murine enzymes, respectively. Inhibition of sEH is enhanced by the presence of two hydrophobic groups (R and R H ) on both sides of the central pharmacophore in which case one side (R H ) could be larger than the other (R) [21,32]. For both enzymes studied, the best inhibition is obtained with a cyclohexyl or an adamantyl appendage on the small side (R). ...
The soluble epoxide hydrolase (sEH) is involved in the metabolism of arachidonic, linoleic, and other fatty acid epoxides, endogenous chemical mediators that play an important role in blood pressure regulation and inflammation. 1,3-Disubstituted ureas, carbamates, and amides are new potent and stable inhibitors of sEH. However, the poor solubility of the lead compounds limits their use. Inhibitor structure-activity relationships were investigated to better define the structural requirements for inhibition and to identify points in the molecular topography that could accept polar groups without diminishing inhibition potency. Results indicate that lipophilicity is an important factor controlling inhibitor potency. Polar groups could be incorporated into one of the alkyl groups without loss of activity if they were placed at a sufficient distance from the urea function. The resulting compounds had a 2-fold higher water solubility. These findings will facilitate the rational design and optimization of sEH inhibitors with better physical properties.
... Further urea, carbamate & amide derivatives appeared to be good inhibitor of the enzyme and noticeably these compounds showed satisfactory in vivo activity 6 . With the help of ligand and structure based drug design technique the chemical structure of these compounds were further modified to produce more potent compounds [7][8][9][10] . Esters and salts of adamantane -1-yl-ureido]-dodecanoic acid (AUDA) have been found to be good inhibitor of sEH but its clinical use has been restricted due to metabolic instability & limited solubility in water and many organic solvents 7,10,11 . ...
In view of the role of sEH (soluble epoxide hydrolase) in hypertension, we have developed a rigorously validated pharmacophore model containing one HBA (Hydrogen Bond Acceptor), two HY (Hydrophobic) and one RA (Ring Aromatic) features. The model was used as a query to search the NCI (National Cancer Institute) and Maybridge database leading to retrieval of many compounds which were sorted on the basis of predicted activity, fit value and Lipinski's violation. The selected compounds were docked into the active site of enzyme soluble epoxide hydrolase. Potential interactions were observed between the features of the identified hits and the amino acids present in the docking site. The three selected compounds were subjected to in vitro evaluation using enzyme- based assay and the isolated rat aortic model followed by cytotoxicity studies. The results demonstrate that the identified compounds are potent, safe and novel soluble epoxide hydrolase inhibitors.
... sEH Inhibitors from P. brazzeana Synthesis of ureas 1,3-Dibenzylurea (BBU) was previously synthesized [27]. Fig. 1 shows the synthetic scheme for the other 2 ureas described in this study. ...
We describe here three urea-based soluble epoxide hydrolase (sEH) inhibitors from the root of the plant Pentadiplandra brazzeana. The concentration of these ureas in the root was quantified by LC-MS/MS, showing that 1, 3-bis (4-methoxybenzyl) urea (MMU) is the most abundant (42.3 μg/g dry root weight). All of the ureas were chemically synthesized, and their inhibitory activity toward recombinant human and recombinant rat sEH was measured. The most potent compound, MMU, showed an IC50 of 92 nM via fluorescent assay and a Ki of 54 nM via radioactivity-based assay on human sEH. MMU effectively reduced inflammatory pain in a rat nociceptive pain assay. These compounds are among the most potent sEH inhibitors derived from natural sources. Moreover, inhibition of sEH by these compounds may mechanistically explain some of the therapeutic effects of P. brazzeana.
... The availability of several X-ray structures has dramatically helped the structure-activity studies aimed toward development of potent inhibitors of the enzyme. Using classical quantitative structure activity relationship (QSAR), 3D-QSAR, and medicinal chemistry approaches , the structure of these inhibitors were improved to yield compounds that have orders of magnitude better inhibition potency than first generation compounds [66,686970. The key to developing effective in vivo inhibitors is to optimize the absorption, distribution, metabolism, and excretion (ADME) as well as ease of formulation . ...
Cardiovascular disease remains one of the leading causes of death in the Western societies. Heart failure (HF) is due primarily to progressive myocardial dysfunction accompanied by myocardial remodeling. Once HF develops, the condition is, in most cases, irreversible and is associated with a very high mortality rate. Soluble epoxide hydrolase (sEH) is an enzyme that catalyzes the hydrolysis of epoxyeicosatrienoic acids (EETs), which are lipid mediators derived from arachidonic acid through the cytochrome P450 epoxygenase pathway. EETs have been shown to have vasodilatory, antiinflammatory, and cardioprotective effects. When EETs are hydrolyzed by sEH to corresponding dihydroxyeicosatrienoic acids, their cardioprotective activities become less pronounced. In line with the recent genetic study that has identified sEH as a susceptibility gene for HF, the sEH enzyme has received considerable attention as an attractive therapeutic target for cardiovascular diseases. Indeed, sEH inhibition has been demonstrated to have antihypertensive and antiinflammatory actions, presumably due to the increased bioavailability of endogenous EETs and other epoxylipids, and several potent sEH inhibitors have been developed and tested in animal models of cardiovascular disease including hypertension, cardiac hypertrophy, and ischemia/reperfusion injury. sEH inhibitor treatment has been shown to effectively prevent pressure overload- and angiotensin II-induced cardiac hypertrophy and reverse the pre-established cardiac hypertrophy caused by chronic pressure overload. Application of sEH inhibitors in several cardiac ischemia/reperfusion injury models reduced infarct size and prevented the progressive cardiac remodeling. Moreover, the use of sEH inhibitors prevented the development of electrical remodeling and ventricular arrhythmias associated with cardiac hypertrophy and ischemia/reperfusion injury. The data published to date support the notion that sEH inhibitors may represent a promising therapeutic approach for combating detrimental cardiac remodeling and HF.
... Expression of sEH is well known to be increased by peroxisome proliferator-activated receptor (PPAR)-alpha agonists, such as fibrates commonly used as hypolipidemic drugs or phthalates commonly used as plasticizers (Grant et al., 1994). Further, the sEH activity is inhibited by urea-containing compounds, such as the herbicide Siduron or the topical antiseptic trichlorocarban (Morisseau et al., 1999;McElroy et al., 2003) as well as some benzoylurea insecticides (Nakagawa et al., 2000). Taken together, these findings underline how environmental contaminants could influence human health through induction or inhibition of sEH. ...
The human soluble epoxide hydrolase (sEH; EC 3.3.3.2) is the product of the EXPH2 gene. The sEH catalyzes the addition of a water molecule to an epoxide, resulting in the corresponding diol. Early work suggested a role of sEH in detoxifying a wide array of xenobiotic epoxides; however, recent findings clearly implicate the sEH in the regulation of blood pressure, pain, and inflammation through the hydrolysis of endogenous epoxy fatty acids such as epoxyeicosatrienoic acids (EETs). Both expression and activity of sEH are influenced by a wide array of xenobiotics, underlying how environmental contaminants could influence human health through sEH. This unit describes radiometric, fluorimetric, and mass spectrometric assays for measuring the activity of sEH and its inhibition. Curr. Protoc. Toxicol. 33:4.23.1-4.23.18. © 2007 by John Wiley & Sons, Inc.
... QSAR (quantitative structureactivity relation) techniques [12][13][14], especially 3D-QSAR-CoMFA (Comparative Molecular Field Analysis) or 3D-QSAR-CoMSIA (Comparative Molecular Similarity Analysis) [15][16][17][18][19][20] proved to be very useful when the HIV-1 PR inhibitors potency are to be predicted. In the last years, a good correlation between the predicted and the observed biological activity for the HIV-1 PR cyclic urea inhibitors was reported [16,20,21]. In a previous work [20] we established a QSAR by the CoMFA method for a series of 45 cyclic urea derivatives used as HIV-1 PR inhibitors. ...
The predicted inhibition constant (Ki) and the predicted inhibitor concentration (IC90) of the HIV-1 protease (HIV- 1 PR) inhibitors: symmetric and nonsymmetric - benzyl, ketone, oxime, pyrazole, imidazole, and triazole cyclic urea derivatives, were obtained by the 3D-CoMFA (Comparative Molecular Field Analysis) method. The CoMFA statistical parameters: cross-validate correlation coefficient (q2), higher than 0.5, and the fitted correlation coefficient (r2), higher than 0.90 validated the predicted biological activities. The best predictions were found for the trifluoromethyl ketoxime derivative (log 1/Ki predict = 8.42), the m-pyridineCH2 pyrazole derivative (log 1/Ki predict = 9.77) and the 1,2,3 triazole derivative (log 1/Ki predict = 7.03). We attempted to design a new potent HIV-1 protease inhibitor by addition of o-benzyl to the (p-HOPhCH2) pyrazole 12f derivative inhibitor. A favorable steric area surrounded the o-benzyl, suggesting a possible new potent HIV-1 protease inhibitor.
... Interestingly, because of the presence of a methionine residue (Met 337 ) pointing into the catalytic cavity, the orientation of the urea inhibitors is reversed in the human sEH compared with the mouse enzyme (87). Using classical quantitative structure activity relationship (QSAR), 3-D-QSAR, and medicinal chemistry approaches, the structure of these inhibitors were improved to yield compounds that have an order of magnitude better inhibition potency (116)(117)(118)(119). This new generation of sEH inhibitors display on one side of the urea functionality secondary and tertiary pharmacophores at 5 and 11 atoms away from the urea carbonyl group, respectively (119). ...
Organisms are exposed to epoxide-containing compounds from both exogenous and endogenous sources. In mammals, the hydration of these compounds by various epoxide hydrolases (EHs) can not only regulate their genotoxicity but also, for lipid-derived epoxides, their endogenous roles as chemical mediators. Recent findings suggest that the EHs as a family represent novel drug discovery targets for regulation of blood pressure, inflammation, cancer progression, and the onset of several other diseases. Knowledge of the EH mechanism provides a solid foundation for the rational design of inhibitors, and this review summarizes the current understanding of the catalytic mechanism of the EHs. Although the overall EH mechanism is now known, the molecular basis of substrate selectivity, possible allosteric regulation, and many fine details of the catalytic mechanism remain to be solved. Finally, recent development in the design of EH inhibitors and the EH biological role are discussed.
... Quantitative structure–activity relationships (QSAR) point to further ambiguities in the binding of inhibitors to human sEH and murine sEH. For instance, QSAR analysis of dialkylurea inhibitors yields equations for murine sEH and human sEH with r 2 values of 0.955 and 0.616, respectively (Nakagawa et al. 2000; McElroy et al. 2003), suggesting that the binding of dialkylurea inhibitors to the human enzyme is not as readily predictable as the binding of inhibitors to the murine enzyme. To further probe the complexities of dialkylurea inhibition we now report the X-ray crystal structures of human sEH complexed with four dialkylurea inhibitors bearing pendant carboxylate " tails " of varying lengths: ...
X-ray crystal structures of human soluble epoxide hydrolase (sEH) complexed with four different dialkylurea inhibitors bearing pendant carboxylate "tails" of varying length have been determined at 2.3-3.0 A resolution. Similarities among inhibitor binding modes reinforce the proposed roles of Y381 and/or Y465 as general acids that protonate the epoxide ring of the substrate in concert with nucleophilic attack of D333 at the electrophilic epoxide carbon. Additionally, the binding of these inhibitors allows us to model the binding mode of the endogenous substrate 14,15-epoxyeicosatrienoic acid. Contrasts among inhibitor binding modes include opposite orientations of inhibitor binding in the active-site hydrophobic tunnel. Alternative binding orientations observed for this series of inhibitors to human sEH, as well as the binding of certain dialkylurea inhibitors to human sEH and murine sEH, complicate the structure-based design of human sEH inhibitors with potential pharmaceutical applications in the treatment of hypertension. Thus, with regard to the optimization of inhibitor designs targeting human sEH, it is critical that human sEH and not murine sEH be utilized for inhibitor screening, and it is critical that structures of human sEH-inhibitor complexes be determined to verify inhibitor binding orientations that correlate with measured affinities.
... Highly potent and selective inhibitors for sEH were developed in our laboratory [13][14][15][16][17][18] and used to describe the biology associated with sEH [5,19]. Although medicinal chemistry approaches were used to increase their inhibitory activity and solubility [16][17][18], the latest compounds reported still do not possess physical properties to produce a successful *Corresponding ...
Mammalian soluble epoxide hydrolase (sEH) represents a highly promising new target for drug development. Chemical inhibition of this enzyme in animal models was shown to treat hypertension and vascular inflammation as well as related syndromes. Existing sEH inhibitors are relatively potent and specific. However, the low solubility and relatively fast metabolism of described sEH inhibitors make them less than therapeutically efficient, stating the need for novel inhibitor structures. Therefore, a series of alpha-cyanoester and alpha-cyanocarbonate epoxides were evaluated as potential human sEH (HsEH) substrates for the high-throughput screen (HTS) of compound libraries. (3-Phenyl-oxiranyl)-acetic acid cyano-(6-methoxy-naphthalen-2-yl)-methyl ester (PHOME), which displayed the highest aqueous stability and solubility, was selected for the development of an HTS assay with long incubation times at room temperature. Concentrations of HsEH and PHOME were optimized to ensure assay sensitivity, reliability, and reproducibility. Assay validation, which employed these optimized concentrations, resulted in good accuracy (60-100%) and high precision (<7% relative standard deviation). In addition, an overall Z' value of 0.7 proved the system's robustness and potential for HTS. The developed assay system will be a valuable tool to discover new structures for the therapeutic inhibition of sEH to treat various cardiovascular diseases.
Recently, dibenzylurea-based potent soluble epoxide hydrolase (sEH) inhibitors were identified in Pentadiplandra brazzeana, a plant in the order Brassicales. In an effort to generalize the concept, we hypothesized that plants that produce benzyl glucosinolates and corresponding isothiocyanates also produce these dibenzylurea derivatives. Our overall aim here was to examine the occurrence of urea derivatives in Brassicales, hoping to find biologically active urea derivatives from plants. First, plants in the order Brassicales were analyzed for the presence of 1, 3-dibenzylurea (compound 1), showing that three additional plants in the order Brassicales produce the urea derivatives. Based on the hypothesis, three dibenzylurea derivatives with sEH inhibitory activity were isolated from maca (Lepidium meyenii) roots. Topical application of one of the identified compounds (compound 3, human sEH IC50 = 222 nM) effectively reduced pain in rat inflammatory pain model, and this compound was bioavailable after oral administration in mice. The biosynthetic pathway of these urea derivatives was investigated using papaya (Carica papaya) seed as a model system. Finally, a small collection of plants from the Brassicales order was grown, collected, extracted and screened for sEH inhibitory activity. Results show that several plants of the Brassicales order could be potential sources of urea-based sEH inhibitors.
Bayesian models constructed from structure-derived fingerprints have been a popular and useful method for drug discovery research when applied to bioactivity measurements that can be effectively classified as active or inactive. The results can be used to rank candidate structures according to their probability of activity, and this ranking benefits from the high degree of interpretability when structure-based fingerprints are used, making the results chemically intuitive. Besides selecting an activity threshold, building a Bayesian model is fast and requires few or no parameters or user intervention. The method also does not suffer from such acute overtraining problems as quantitative structure activity relationships or quantitative structure property relationships (QSAR/QSPR). This makes it an approach highly suitable for automated workflows that are independent of user expertise or prior knowledge of the training data. We now describe a new method for creating a composite group of Bayesian models to extend the method to work with multiple states, rather than just binary. Incoming activities are divided into bins, each covering a mutually exclusive range of activities. For each of these bins, a Bayesian model is created to model whether or not the compound belongs in the bin. Analyzing putative molecules using the composite model involves making a prediction for each bin, and examining the relative likelihood for each assignment, e.g. highest value wins. The method has been evaluated on a collection of hundreds of datasets extracted from ChEMBL v20 and validated datasets for ADME/Tox and bioactivity.
Drug discovery is a lengthy and costly process which aims at bringing in a novel therapeutic molecule for the treatment of various diseases. In the present study, a novel series of eighty chalcone derivatives [(4-substituted)- (4'-substituted)-3' substituted sulphonyl (2E)- 1,3-diphenylprop-2-en-1-one] were designed to inhibit soluble epoxide hydrolase enzyme (sEH). Lipinski's rule of 5 and absorption, distribution, metabolism, elimination and toxicity (ADMET) properties of the compounds were calculated using Molinspiration server and Accord for excel software respectively. All 80 compounds have passed the Lipinski's rule of 5 and only 20 compounds showed considerable ADMET properties. These 20 compounds were subjected to molecular docking studies using AutoDock 4.2 in order to rationalize the possible interactions between test compounds and the active site of human soluble epoxide hydrolase enzyme (1ZD3). Binding energy, intermolecular energy and inhibition constant were the main parameters taken into consideration in this study. The binding energies ranged from -6.07 to -7.89 kcal/mol, the inhibition constant ranging from 1.64 μΜ to 35.45 μΜ and intermolecular energy ranging between -9.38 kcal/mol to -6.97 kcal/mol. Hence, further pharmacophore optimization and in vivo studies are necessary to develop potent chemical entities that could inhibit the sEH enzyme.
The synthesis of two carbamoylmethylphosphate (CMP)‐ and ‐phosphine oxide (CMPO)‐tetrafunctionalized cavitands with a propyl spacer between the cavitand platform and the chelating sites is described. Liquid–liquid extractions with CMP(O)‐and N‐acyl(thio)urea‐tetrafunctionalized cavitands were performed from aqueous nitric acid into dichloromethane or o‐nitrophenyl hexyl ether (NPHE). The extraction of americium (III) and europium (III) from aqueous nitric acid into NPHE was studied in both the absence and presence of Br6‐COSAN as a synergist. CMP(O) cavitands are more efficient extractants than the corresponding N‐acyl(thio)urea derivatives, whereas the latter ones show an interesting selectivity for americium (III) over europium (III) in the presence of Br6‐COSAN. The introduction of a propyl chain between the cavitand platform and the chelating groups increases the extraction efficiency of the ionophores (8, 11, and 12). A high selectivity was achieved by N‐acylthiourea cavitand 12 at pH 3 using Br6‐COSAN as a synergist.
Comparative Molecular Field Analysis (CoMFA) is a popular Three-Dimensional Quantitative Structure–Activity Relationship (3D-QSAR) method. The effect of varying molecular fields [CoMFA vs. Comparative Molecular Similarity Indices Analysis (CoMSIA)], lattice spacing and analysis options on predictive performance was assessed based on cross validated R2 values of 30 datasets taken from the literature. The CoMFA method results in statistically significantly higher cross validated R2 values compared to CoMSIA when only steric and electrostatic fields are used. When the hydrophobic molecular field is included in the CoMSIA analysis (as is most commonly the case) the difference between CoMFA and CoMSIA is no longer statistically significant. Addition of hydrogen bond field does not improve CoMFA predictivity. Although there was a trend towards increased predictivity with decreased lattice spacing, this was not statistically significant. Altering the default filtering criteria and cut-off values for Lennard Jones and Coulomb fields also did not generally result in a statistically significant effect on predictive performance.
Introduction Methods Results Discussion References
IntroductionApproaches to Modeling Enzymes, Transporters, Channels and Receptors Classical QSARPharmacophore ModelsHomology ModelsTransporter Modeling Applications of TransportersThe Human Small Peptide Transporter, hPEPT1The Apical Sodium-Dependent Bile Acid TransporterP-GlycoproteinVitamin TransportersOrganic Cation TransporterOrganic Anion TransporterNucleoside TransporterBreast Cancer Resistance ProteinSodium Taurocholate Transporting PolypeptideEnzymes Cytochrome P450sEpoxide HydrolaseMonoamine OxidasesFlavin Containing MonooxygenaseSulfotransferasesGlucuronosyltransferasesGlutathione S-TransferasesChannels Human Ether-a-gogo Related GeneReceptors Pregnane X-ReceptorConstitutive Androstane Receptor Classical QSARPharmacophore ModelsHomology Models Applications of TransportersThe Human Small Peptide Transporter, hPEPT1The Apical Sodium-Dependent Bile Acid TransporterP-GlycoproteinVitamin TransportersOrganic Cation TransporterOrganic Anion TransporterNucleoside TransporterBreast Cancer Resistance ProteinSodium Taurocholate Transporting Polypeptide Cytochrome P450sEpoxide HydrolaseMonoamine OxidasesFlavin Containing MonooxygenaseSulfotransferasesGlucuronosyltransferasesGlutathione S-Transferases Human Ether-a-gogo Related Gene Pregnane X-ReceptorConstitutive Androstane Receptor
Introduction QSAR for Enzymes Limitations Conclusions References
The synthesis and binding properties of resorcinarene-based cavitands functionalized with N-acylthiourea moieties towards different cations are described. Extraction studies with metal (pb(2+), Cu2+, Ag+, Hg2+, Cd2+, Eu3+, Fe3+, K+, Na+, and Ca2+) picrates and the incorporation in ion selective electrodes (ISEs), show that there is more than a 40% increase of the Ag+ extraction for N-acylthiourea ionophores (2. 3, and 8) in comparison with N-benzoyl-N'-benzylthiourea (9). Ionophore 8, which has a C-3 chain between the platform and the ionophore, extracts two times more Cu2+ than the more rigid one (2). Stoichiometry studies showed for ligand 2 a ligand/metal ratio of 1:1, while for model compound 9 a ratio of 1:2 was found. Potentiometric studies of electrodes revealed that cavitands 2, 3, and 8 induce a significantly different selectivity pattern compared to the cation-exchanger used, as well as model compound 9. Especially, a considerable enhancement of the selectivity towards Ag+ and Ph2+ over K+, Ca2+, and Na 21 ions was observed.
The inhibition of soluble epoxide hydrolase (sEH) by small molecules has been shown to increase the levels of fatty acid epoxides such as epoxyeicosatrienoic acids (EETs), and decrease the levels of the corresponding fatty acid diols including dihydroxyeicosatrienoic acids (DHETs). Due to numerous beneficial effects of EETs, the elevation of their levels, as a result of sEH inhibition, may eventually lead to new therapeutic approaches for multiple diseases. Specifically, it has been shown, largely in rodent models, that sEH inhibitors can be used to treat heart failure, hypertension, diabetes, stroke, dyslipidemia, inflammatory and neuropathic pain, immunological disorders, eye diseases, neurological diseases, and other indications. Medicinal chemistry strategies, key biological data of benchmark compounds, and possible paths for sEH inhibitors to reach the clinic will be reviewed.
Substituted urea compounds are well-known as potent inhibitors of juvenile hormone epoxide hydrolase (JHEH) of the tobacco hornworm Manduca sexta. Docking simulations of 47 derivatives inside JHEH were performed to gain insight into the structural characteristics of these complexes. The obtained orientations show a strong similitude with the observed in the known X-ray crystal structures of human soluble epoxide hydrolase (sEH) complexed with dialkylurea inhibitors. In addition, the predicted inhibitor concentration (IC₅₀) of the above-mentioned compounds as JHEH inhibitors were obtained by a quantitative structure-activity relationship (QSAR) method by using comparative molecular field analysis (CoMFA) applied to aligned dataset. The best models included steric and electrostatic fields and had adequate predictive abilities. In addition, these models were used to predict the activity of an external test set of compounds that was not used for building the model. Furthermore, plots of the CoMFA fields allowed conclusions to be drawn for the choice of suitable inhibitors.
We present the strong fluorescence effect, a new 392 nm emission peak appearing after binding of a naphtol-urea inhibitor XIIa to the enzyme epoxide hydrolase (EH), along with the quenching of the EH tryptophan fluorescence. We have studied the quenching of the 392-nm peak (attributed to XIIa bound inside the active center of the enzyme) of the mixture EH +XIIa by various strong transparent inhibitors (competing with XIIa for binding to EH), and measured the corresponding values of the Stern-Volmer constants, K(mix)(SV). Strong EH inhibitors demonstrate different replacement behavior which can be used to distinguish them. We further demonstrate a novel fluorescent assay which allows to distinguish highly potent inhibitors and to visualize the strongest among them. We generated our assay calibration curve based on the quenching data, by plotting quenching strength K(mix)(SV) versus inhibiting strength, IC(50) values. We used moderate inhibitors for the assay plot generation. We then applied this curve to determine IC(50) values for several highly potent inhibitors, with IC(50) values at the limit of the IC(50) detection sensitivity by colorimetric enzyme assay. IC(50) values determined from our quenching assay show correlation with IC(50) values determined in the literature by more sensitive radioactive-based assay and allow differentiating the inhibitors potency in this group. To our knowledge, this is the first inhibitor assay of such kind. Chemical inhibition of EH is an important technology in the treatment of various cardiovascular diseases, therefore, this tool may play a crucial role in discovering new inhibitor structures for therapeutic EH inhibition.
We investigated N-adamantyl-N'-phenyl urea derivatives as simple sEH inhibitors. Salicylate ester derivatives have high inhibitory activities against human sEH, while the free benzoic acids are less active. The methyl salicylate derivative is a potent sEH inhibitor, which also has high metabolic and chemical stabilities; suggesting that such inhibitors are potential lead molecule for bioactive compounds acting in vivo.
There have been several recent advances in the area of biocatalysed hydrolytic kinetic resolution of epoxides using 'newly discovered' enzymes (i.e. epoxide hydrolases). These biocatalysts, two of which will become commercially available in the near future, appear to be highly promising tools for fine organic synthesis, as they enable the preparation of various epoxides and/or their corresponding diols in enantiopure form.
A data set of 348 urea-like compounds that inhibit the soluble epoxide hydrolase enzyme in mice and humans is examined. Compounds having IC(50) values ranging from 0.06 to >500 microM (murine) and 0.10 to >500 microM (human) are categorized as active or inactive for classification, while quantitation is performed on smaller compound subsets ranging from 0.07 to 431 microM (murine) and 0.11 to 490 microM (human). Each compound is represented by calculated structural descriptors that encode topological, geometrical, electronic, and polar surface features. Multiple linear regression (MLR) and computational neural networks (CNNs) are employed for quantitative models. Three classification algorithms, k-nearest neighbor (kNN), linear discriminant analysis (LDA), and radial basis function neural networks (RBFNN), are used to categorize compounds as active or inactive based on selected data split points. Quantitative modeling of human enzyme inhibition results in a nonlinear, five-descriptor model with root-mean-square errors (log units of IC(50) [microM]) of 0.616 (r(2) = 0.66), 0.674 (r(2) = 0.61), and 0.914 (r(2) = 0.33) for training, cross-validation, and prediction sets, respectively. The best classification results for human and murine enzyme inhibition are found using kNN. Human classification rates using a seven-descriptor model for training and prediction sets are 89.1% and 91.4%, respectively. Murine classification rates using a five-descriptor model for training and prediction sets are 91.5% and 88.6%, respectively.
An overview is given of the CoMFA (comparative molecular field analysis) and CoMSIA (comparative molecular similarity indices analysis) methodologies that are established ligand‐based molecular design tools widely used by medicinal and pesticide chemists. In the absence of a three‐dimensional structure of the target biopolymer, CoMFA and CoMSIA often provide a practical solution to an otherwise intractable problem of proper characterization of ligand–receptor interactions. These techniques are especially important in agrochemistry, where the number of known molecular structures of pesticide targets is limited. The use of CoMFA and CoMSIA in the agrochemical field for modelling the interactions of insecticides, fungicides, herbicides and herbicide safeners with their target binding sites is illustrated by using some selected published work. The CoMFA and CoMSIA models developed have been used successfully to map the properties of unknown receptors, construct hypotheses for ligand–receptor interactions, optimize lead structures, design novel active compounds, and predict biological activities. The application of CoMFA by the present authors for deriving a binding site hypothesis for dichloroacetamide‐type herbicide safeners is described in somewhat more detail.
© 2003 Society of Chemical Industry
The enantioselective ring-opening catalyzed by epoxide hydrolases originating from seven different sources of a series of 2,2-disubstituted oxiranes containing alkyl chains of different lengths, unsaturated (alkenyl, alkinyl) and aromatic groups as well as electronegative heteroatoms at various positions within the side chain was analyzed by quantitative structure-activity relationships. Models for the enantioselectivity were derived with the aid of multiple linear regression analysis (MLR) using several steric and electronic (quantum chemical) descriptors. On the basis of the models derived by MLR nonlinear modeling with artificial neural networks (ANN) was also done. Good predictive performance was observed for both modeling approaches. The models also indicate that different steric and/or electronic features account for the enantioselectivities observed for the individual epoxide hydrolases.
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 alpha-cyanoester and alpha-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-methoxy-naphthalen-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.
Drug metabolism information is a necessary component of drug discovery and development. The key issues in drug metabolism include identifying: the enzyme(s) involved, the site(s) of metabolism, the resulting metabolite(s), and the rate of metabolism. Methods for predicting human drug metabolism from in vitro and computational methodologies and determining relationships between the structure and metabolic activity of molecules are also critically important for understanding potential drug interactions and toxicity. There are numerous experimental and computational approaches that have been developed in order to predict human metabolism which have their own limitations. It is apparent that few of the computational tools for metabolism prediction alone provide the major integrated functions needed to assist in drug discovery. Similarly the different in vitro methods for human drug metabolism themselves have implicit limitations. The utilization of these methods for pharmaceutical and other applications as well as their integration is discussed as it is likely that hybrid methods will provide the most success.
The soluble epoxide hydrolase appears to be a promising target for the development of antihypertensive therapies based on a previously unexplored mechanism of action. Epoxide hydrolases are enzymes that add water to three membered cyclic ethers known as epoxides. The soluble epoxide hydrolase in mammalian systems (sEH) is a member of the alpha/beta-hydrolase fold family of enzymes and it shows a high degree of selectivity for epoxides of fatty acids. The regioisomeric epoxides of arachidonic acid or epoxyeicosanoids (EETs) are particularly good substrates. These EETs appear to be major components of the endothelium-derived hyperpolarizing factors (EDHFs). As such, EETs cause vasodilation and reduce blood pressure. The EETs also are strongly anti-inflammatory and analgesic. By inhibiting sEH, the increase in circulating EETs leads to a reduction in blood pressure in a number of animal models. Potent transition state mimic inhibitors have been developed for the sEH. Some of these sEH inhibitors (sEHIs) show nanomolar to picomolar potency and good pharmacokinetic properties. Because of their unique mode of action they show promise in treating hypertension while reducing problems with end organ failure, vascular inflammation and diabetes. Indeed, the anti-inflammatory properties of the sEHI may make them particularly suitable for treating hypertension in patients with other concomitant metabolic syndromes. They are more potent on a molar basis than most nonsteroidal anti-inflammatory drugs (NSAIDs) in reducing PGE2 in inflammation models, they strongly synergize with NSAIDs, and appear to ameliorate apparently unfavorable eicosanoid profiles associated with some cyclo-oxygenase-2 inhibitors.
The pioneering work of Gerry Brooks on cyclodiene insecticides led to the discovery of a class of enzymes known as epoxide hydrolases. The results from four decades of work confirm Brooks' first observations that the microsomal epoxide hydrolase is important in foreign compound metabolism. Brooks and associates went on to be the first to carry out a systematic study of the inhibition of this enzyme. A second role for this enzyme family was in the degradation of insect juvenile hormone (JH). JH epoxide hydrolases have now been cloned and expressed from several species, and there is interest in developing inhibitors for them. Interestingly, the distantly related mammalian soluble epoxide hydrolase has emerged as a promising pharmacological target for treating hypertension, inflammatory disease and pain. Tight-binding transition-state inhibitors were developed with good ADME (absorption, distribution, metabolism and excretion). These compounds stabilize endogenous epoxides of fatty acids, including arachidonic acid, which have profound therapeutic effects. Now EHs from microorganisms and plants are used in green chemistry. From his seminal work, Dr Brooks opened the field of epoxide hydrolase research in many directions including xenobiotic metabolism, insect physiology and human health, as well as asymmetric organic synthesis.
A microsomal preparation from larval stages of the brine shrimp Artemia salina was found to catalyze the transfer of N-acetyl-D-glucosamine from UDP-N-acetylglucosamine to an endogenous acceptor. The product was identified as chitin by its resistance to extraction with alkali and high concentrations of urea and the liberation of chito-oligosaccharides by treatment with purified chitinases. The enzyme requires Mg2+ for activity and is inhibited by UDP and diflubenzuron, but not by Polyoxin D. The pH optimum is 7.0. The enzyme is not significantly activated by N-acetyl-D-glucosamine nor by trypsin treatment. Incorporation of radioactivity into endogenous acceptor is inhibited by chitodextrins which appear to serve as alternate acceptors. The crustacean enzyme can also utilize exogenous, macromolecular chitin as acceptor. The enzyme, which was partially purified by sucrose step-gradient ultracentrifugation, appears maximally active after 72 h of larval growth.
ヨトウガ終令幼虫を供試して, diflubenzuron およびポリオキシンDのキチン合成阻害作用を検討した結果, 両薬剤ともキチン合成を強く阻害することが, in vivo, in vitro ともに認められ, in vitro でのID50はそれぞれ8.9×10-9Mおよび8.8×10-8Mであった. 14C-glucosamine を終令幼虫に注射し, 体中で生成された 14C-uridinediphospho-N-acetylglucosamine (14C-UDP-AGA) を分析した結果, diflubenzuron 処理虫では無処理虫に比し約1.5倍の14C-UDP-AGAの蓄積が認められた. このことから, diflubenzuron はUDP-AGAからキチンへの step を阻害すると結論された. ヨトウガ幼虫から得られたキチン合成酵素に及ぼす両薬剤の影響を調べた結果, ポリオキシンDはこの酵素を強く阻害するが, diflubenzuron はこの酵素を in vitro では阻害しない. しかし, diflubenzuron を in vivo で処理した幼虫から得られた酵素の活性は, 無処理虫の酵素活性の約1/10にすぎなかった.
The larvicidal activity of benzoylphenylureas with various multiple substitution patterns at the anilide moiety including chlorfluazuron and teflubenzuron was estimated against the rice stem borer, Chilo suppressalis, under conditions in which the oxidative metabolism was eliminated as far as possible by using piperonyl butoxide. The activity was quantitatively analyzed using physicochemical parameters of substituents at each of the anilide aromatic positions. The results indicated that the total hydrophobicity (with an optimum) and electron-withdrawing ability of substituents are favorable to the activity. Small size of substituents was advantageous but the size effect was specific to their positions. The molecular mode of interaction with the possible receptor was suggested so that the compounds may recognize the possible receptor wall from the side of the vicinal pair of ortho and meta positions occupied by smaller substituents, and the receptor site corresponding to the ortho substituent is hydrophilic and constrained.
Benzoylurea-type compounds are a new class of powerful insecticides/growth regulators known to cause chitin-synthesis inhibition in vivo. We have found by using the isolated integument from the freshly molted American cockroach that diflubenzuron (Dimilin®) and its active analogs cause a rise in cAMP-dependent protein kinase activity, both in intact and homogenized (cell free) systems. Such a cAMP-dependent protein kinase has been found to be particularly active in the integument during molting. The effect of diflubenzuron in isolated intact epidermis was observed at the 10 nM range, occurring with only active diflubenzuron analogs. At the same time, DFB was found to cause changes in protein phosphorylation on a few specific proteins both in situ and in cell free conditions in the concentration range of 10 nM to 10 μM. The possibility that the action of diflubenzuron on chitin biosynthesis is directly or indirectly mediated through modulation of cAMP-dependent protein kinase system was discussed.
The partition coefficient P in the 1-octanol/water system was analyzed for a great number of multisubstituted benzenes of ecotoxicological importance consisting of acetanilides, benzamides, nitrobenzenes, and anisoles having various substitution patterns in terms of the Δ log P (log P – log P[unsubstituted benzene]) quantitatively with free energy-related physicochemical substituent parameters. The analyses showed that the stereoelectronic effects of ortho, meta, and para substituents on the relative solvation of individual polar groups capable of hydrogen bonding with the partitioning solvents are very important in determining the variations in the log P value. The effects were additive in the set of complicated multisubstituted benzenes, leading to a correlation equation represented by a linear combination of terms of hydrophobic, electronic, and steric parameters summed up over substituents. It was suggested that the procedure be extended to analyze and predict the log P value of any multiply substituted benzenes.
To study the mechanism of action of diflubenzuron (DFB) and other benzoylphenylureas, we have initially hypothesized that their action may be related to exocytosis: to test the hypothesis, we obtained an intracellular vesicle preparation from the homogenate of integument of newly molted American cockroachs (Periplaneta americana L.) in 10 mM MES buffer containing 250 mM sucrose (isotonic) and 2.5 mM MgSO4, at pH 6.6. By studying DFB's effect on various ion transporting activities, we demonstrated that calcium uptake in this intracellular particulate preparation was significantly inhibited by DFB at low concentrations (e.g., 10−8 M). Such an inhibitory effect of DFB on Ca2+ uptake was eliminated by the addition of ionophores or membrane disruptors, as well as the sonication of vesicle preparation. On the other hand, oligomycin, protein phosphorylation modulators, Na+, and Li+ did not affect the calcium uptake. Among ionophores, agents disrupting H+ gradients (e.g. FCCP and NEM) totally eliminated 45Ca uptaking activity by vesicles as well as the inhibitory effect of DFB. Among calcium ion modulators, calmodulin inhibitors such as calmidazolium and trifluoperazine decreased the Ca2+-uptake, whereas membrane calcium channel blocker, verapamil, did not. ATP and γ-S-GTP stimulated Ca2+ uptake. However, the former increased only the DFB insensitive portion and the latter largely the DFB sensitive part of Ca2+. Together these data support the hypothesis that the action site of DFB in this preparation is the GTP-dependent Ca2+ transport process which is coupled to vacuolar type intracellular vesicles in the integument cells.
The larvicidal activity of analogs of a recently developed larvicide, chlorfluazuron, without 3,5-dichloro substituents at the phenyl moiety, i.e., of 2,6-difluorobenzoyl-4-(substituted 2-pyridyloxy)phenylurea, was estimated against the rice stem borer, Chilo suppressalis, under conditions in which oxidative metabolism was eliminated by the use of piperonyl butoxide. Results were analyzed quantitatively by physicochemical substituent parameters and regression analysis. The activity of some newly synthesized 2,6-difluorobenzoyl-4-substituted phenylureas with substituents at the para position that are electron-withdrawing, hydrophobic, and thin, such as Ph(4-Br). O(CH2)3Ph, and (Z)CHCHPh, were also measured against C. suppressalis and analyzed quantitatively. The results indicated that structural requirements for the activity of the whole series of compounds were basically identical with those for 2,6-difluorobenzoylphenylurea derivatives with simpler substituents.
The larvicidal activity of a series of N-2,6-difluoro- and N-2,6-dichlorobenzoyl-N′-(4-substituted phenyl)ureas against nondiapause larvae of the rice stem borer, Chilo suppressalis Walker, was measured by topical application and oral administration methods under conditions with and without piperonyl butoxide as an inhibitor of oxidative metabolism. The effects of substituents at the anilide moiety on the larvicidal activity were analyzed quantitatively using physicochemical substituent parameters and regression analysis. The results indicate that the oxidative metabolism in the larval body which is favored by electron-donating substituents is significant in determining the activity. The activity, when the metabolic factor is eliminated, is enhanced by electron-with-drawing and hydrophobic substituents and lowered by bulky groups. The inhibitory activity against new cuticle formation of the same series of compounds was also measured using cultured integument of the rice stem borer diapause larva. The comparison of the quantitative analyses between larvicidal and integument-level activities shows that inhibition of cuticular development is the most important factor governing larvicidal activity.
We identified a 39 kilodalton (kDa) protein from newly molted American cockroaches (Periplaneta americana), isolated from a 100 000 g precipitate of a 1000 g supernatant from an integument homogenate, which exhibited increased phosphorylation following diflubenzuron (DFB) treatment. This 39 kDa phosphoprotein was partially purified from an intracellular membrane vesicle containing fractions obtained by discontinuous sucrose density centrifugation. Both the interfaces of the 30/40% and 40/50% sucrose density layers were found to be enriched in the 39 kDa phosphoprotein. Treatments with various chemicals such as the ionophores valinomycin and A23187, the protonophore carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP), and the vacuolar H+-ATPase inhibitor N-ethylmaleimide (NEM), like DFB, were also found to stimulate phosphorylation of the 39 kDa protein. These results suggest that by disrupting the normal pH gradient occurring in certain acidic vacuolar-type membrane vesicles, phosphorylation of the 39 kDa protein will be increased. In addition, we found that by decreasing the external pH to 5.0 from about neutral, it was possible to stimulate the phosphorylation activity of this particular protein, and thus simulate the action of DFB. We concluded that DFB's action is very probably related to its ability to disrupt the normal ionic balance of these vacuolar-type vesicles, leading to eventual disruption of the proton gradient within the vesicles.
The larvicidal activity of 2-substituted- and 2,6-disubstituted-(4-chlorophenyl)ureas against rice stem borers (Chilo suppressalis) and silkworms (Bombyx mori) was measured by topical application under conditions in which oxidative metabolism was eliminated using piperonyl butoxide. The substituent effects at the benzoyl moiety on the activity were analyzed quantitatively using physicochemical parameters and regression analysis. The substituent parameter for the electron withdrawal from the side chain was estimated taking into consideration the steric inhibition of the resonance of ortho substituents. The results indicated that the larvicidal activity was enhanced by electron-withdrawing and hydrophobic substituents and lowered by bulky groups. The effect of substituents at the benzoyl moiety on activity was about the same for rice stem borers and silkworms. In vitro inhibition of the same series of compounds against new cuticle formation was also measured using cultured pieces of integument of diapause larvae of the rice stem borer. In vitro activity was linearly related to in vivo activity after allowing for the hydrophobic factor that may contribute to transport.
The larvicidal activity of a series of N-2,6-difluorobenzoyl-N′-[4-(substituted benzyloxy)-phenyl]-ureas against nondiapause larvae of the rice stem borer, Chilo suppressalis Walker, was measured by a topical application method under conditions in which oxidative metabolism was inhibited by piperonyl butoxide. The effects of the substituted-benzyloxy moiety on variations in the activity were analyzed quantitatively using physicochemical substituent parameters and regression analysis. Results were compared with those found previously for N-2,6-difluorobenzoyl-N′-(4-substituted phenyl)-ureas, indicating that the electron-withdrawing property of the anilide substituents participates in determining the activity through the inductive effect. The hydrophobicity of the total anilide substituents favors activity, whereas the steric dimension in terms of the width lowers it. Although inhibition of new cuticle formation on cultured integument of diapausing larva could not be determined accurately for most of the compounds because of their limited solubility in the assay medium, inhibitory activity seemed related to larvicidal activity, as was the case for previously investigated simpler congeners.
Seventy novel benzoylphenylurea compounds were synthesized and their antitumor activities were examined in vivo against P388 leukemia. N-(2-Nitrobenzoyl)-N'-[4-(2-pyrimidinyloxy)phenyl]ureas showed the highest antitumor activities when dosed intraperitoneally or orally. Their structure-activity relationships were examined with particular focus on the position and the variety of substituent on each aryl ring.
A novel antitumor compound, N-[4-(5-bromo-2-pyrimidinyloxy)-3-chlorophenyl]-N'-(2-nitrobenzoyl ) urea (HO-221) was evaluated for its antitumor activity in experimental tumor models. HO-221 preparation was given orally to tumor-bearing animals. The compound exhibited significant effects against various tumors such as P388 and L1210 leukemias; M5076 reticulum-cell sarcoma; colon 38 carcinoma; human xenografts MX-1, LX-1, GA-1, and Co-1; Lewis lung carcinoma; sarcoma 180; and Walker 256 carcinosarcoma and was especially effective against solid tumors. However, its effect on murine B16 melanoma was moderate. Intermittent administration of HO-221 produced better results. The effects of HO-221 on human tumor xenografts were compared with those of other antitumor agents. HO-221 showed activity against LX-1 lung and Co-1 gastrointestinal tumor and was also effective against advanced-stage L1210 leukemia and Lewis lung carcinoma. Furthermore, the effect of HO-221 on drug-resistant tumors was examined using murine leukemias L1210 and P388. It showed no cross-resistance with the known antitumor agents Adriamycin (ADM), daunomycin (DM), vincristine (VCR), mitomycin C (MMC), cisplatin (CDDP), 5-fluorouracil (5-FU), cytosine arabinoside (Ara-C), methotrexate (MTX), cyclophosphamide (CPA), or carboquone (CQ), and collateral sensitivity to HO-221 was found in MMC-, CDDP-, and CPA-resistant sublines. HO-221 exhibits significant reproducible, broad-spectrum antitumor activity against experimental tumors as well as human neoplasms.
The antitumor action of HO-221, a novel benzoylphenylurea derivative, was studied. The in vitro cytotoxic strength of HO-221 was investigated, as measured by IC50 values, compared with those of other drugs with different action mechanisms, using Chinese hamster lung (CHL) cells, mouse leukemia L1210 cells and human promyelocytic leukemia HL-60 cells. Morphological alterations following treatment were observed under a phase contrast microscope, and the mitotic index was determined at regular intervals to check for accumulation of metaphase cells. HO-221 was found to have a very strong toxic effect on all cell types, equal to that of the spindle poisons used as controls. HO-221 also produced the same specific morphological changes as the spindle poisons, with a significant accumulation of metaphase cells. A chromosome analysis of treated cells showed that HO-221 frequently induced polyploid and aneuploid cells, but without accompanying chromosome-breaking activity. An in vivo mouse bone marrow micronucleus assay was also carried out. The assay allowed the in vivo identification of a chromosome breaker or a spindle poison through the measurement of the relative sizes of micronuclei produced and erythrocytes. HO-221 was found frequently to induce relatively large micronuclei, an action regarded as specific to spindle poisons. It was thus demonstrated that HO-221 acts as a spindle poison both in vitro and in vivo. In order to investigate the mechanism of this action, a study of tubulin assembly using purified calf brain tubulin was carried out, which demonstrated clearly that HO-221 inhibits microtubule assembly. A detailed investigation of the action mechanism of HO-221 as a spindle poison is now called for.
The partition coefficient (P) of some mono- and di-ortho-substituted aromatic compounds was measured in a 1-octanol/water system. For each series of compounds with the same functional group, the pi value (the difference in the log P value between the substituted and unsubstituted compound) was analyzed on the same basis as the values of meta- and para-substituted isomers by an extended Hammett-Taft procedure. In the procedure, we considered the intramolecular electronic and steric effects, operating between substituents and governing the relative hydrogen-bonding solvation with partitioning solvents for solutes in which internal hydrogen-bond formation can be ignored. The pi value for mono- and di-ortho-substituted derivatives was adequately included in the correlation equation for the values of the meta- and para-substituted derivatives in each series. The effect of di-ortho substituents on partition behaviors could be roughly expressed by the sum of the effects of the 2- and 6-position substituents.
A protein mediating hepatocyte sodium-dependent bile acid transport across the sinusoidal plasma membrane has been purified by immunoprecipitation with monoclonal antibody (MAb) 25D-1, which specifically recognizes this protein on the surface of intact hepatocytes (Ananthanarayanan et al. J. Biol. Chem. 263: 8338-8343, 1988). The function of this protein was further established by proteoliposome reconstitution (von Dippe et al. J. Biol. Chem. 265: 14812-14816, 1990). NH2-terminal amino acid sequence analysis and amino acid composition revealed this protein to be closely related to the enzyme microsomal epoxide hydrolase (mEH). Both proteins exhibited the same elution times on a reverse-phase high-pressure liquid chromatography column, comigrated with an apparent molecular weight of 49,000 as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and possessed identical isoelectric points of 8.2. The MAb was capable of immunoprecipitating chromatographically purified mEH, as well as a protein derived from the sinusoidal plasma membrane that exhibited mEH activity comparable to that of the protein isolated from the endoplasmic reticulum. The subtilisin fragmentation patterns derived from chromatographically purified mEH and the MAb-precipitated plasma membrane protein were also identical. Hydropathy profile analysis of the amino acid sequence of mEH suggested the presence of four transmembrane domains. The results of these studies indicate that a protein that is involved in mediating sodium-dependent bile acid transport is closely related to mEH.
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