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1
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Liu X, Bian Z, Hu S, Dickinson CF, Benjamin MM, Jia J, Tian Y, Place A, Hanna GS, Luesch H, Croot P, Reddy MM, Thomas OP, Hardiman G, Puglisi MP, Yang M, Zhong Z, Lemasters JJ, Korte JE, Waters AL, Heltzel CE, Williamson RT, Strangman WK, Valeriote F, Tius MA, DiTullio GR, Ferreira D, Alekseyenko A, Wang S, Hamann MT, Wang X. The Chemistry of Phytoplankton. Chem Rev 2024; 124:13099-13177. [PMID: 39571071 PMCID: PMC11638913 DOI: 10.1021/acs.chemrev.4c00177] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/09/2024] [Revised: 08/12/2024] [Accepted: 08/26/2024] [Indexed: 12/12/2024]
Abstract
Phytoplankton have a high potential for CO2 capture and conversion. Besides being a vital food source at the base of oceanic and freshwater food webs, microalgae provide a critical platform for producing chemicals and consumer products. Enhanced nutrient levels, elevated CO2, and rising temperatures increase the frequency of algal blooms, which often have negative effects such as fish mortalities, loss of flora and fauna, and the production of algal toxins. Harmful algal blooms (HABs) produce toxins that pose major challenges to water quality, ecosystem function, human health, tourism, and the food web. These toxins have complex chemical structures and possess a wide range of biological properties with potential applications as new therapeutics. This review presents a balanced and comprehensive assessment of the roles of algal blooms in generating fixed carbon for the food chain, sequestering carbon, and their unique secondary metabolites. The structural complexity of these metabolites has had an unprecedented impact on structure elucidation technologies and total synthesis, which are highlighted throughout this review. In addition, the influence of biogeochemical environmental perturbations on algal blooms and their influence on biospheric environments is discussed. Lastly, we summarize work on management strategies and technologies for the control and treatment of HABs.
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Affiliation(s)
- Xiaoying Liu
- Department
of Pharmacy, Lanzhou University, Lanzhou 730000, Gansu China
| | - Zhiwei Bian
- Department
of Pharmacy, Lanzhou University, Lanzhou 730000, Gansu China
| | - Shian Hu
- Department
of Pharmacy, Lanzhou University, Lanzhou 730000, Gansu China
| | - Cody F. Dickinson
- Department
of Drug Discovery & Biomedical Sciences, Medical University of South Carolina, Charleston, South Carolina 29425, United States
| | - Menny M. Benjamin
- Department
of Drug Discovery & Biomedical Sciences, Medical University of South Carolina, Charleston, South Carolina 29425, United States
| | - Jia Jia
- School
of Life Sciences, Shanghai University, Shanghai 200031, China
| | - Yintai Tian
- Department
of Pharmacy, Lanzhou University, Lanzhou 730000, Gansu China
| | - Allen Place
- Institute
of Marine Biotechnology and Technology, University of Maryland Center for Environmental Science, Baltimore, Maryland 21202, United States
| | - George S. Hanna
- Department
of Drug Discovery & Biomedical Sciences, Medical University of South Carolina, Charleston, South Carolina 29425, United States
| | - Hendrik Luesch
- Department
of Medicinal Chemistry and Center for Natural Products, Drug Discovery
and Development, University of Florida, Gainesville, Florida 32610, United States
- Program
in Cancer and Stem Cell Technology, Duke-NUS
Medical School, Singapore 169857, Singapore
| | - Peter Croot
- Irish
Centre
for Research in Applied Geoscience, Earth and Ocean Sciences and Ryan
Institute, School of Natural Sciences, University
of Galway, Galway H91TK33, Ireland
| | - Maggie M. Reddy
- School
of
Biological and Chemical Sciences, Ryan Institute, University of Galway, H91TK33 Galway, Ireland
| | - Olivier P. Thomas
- School
of
Biological and Chemical Sciences, Ryan Institute, University of Galway, H91TK33 Galway, Ireland
| | - Gary Hardiman
- School of
Biological Sciences Institute for Global Food Security, Queen’s University Belfast, Belfast, Northern Ireland BT7 1NN, U.K.
| | - Melany P. Puglisi
- Department
of Pharmaceutical Sciences, Chicago State
University, Chicago, Illinois 60628, United States
| | - Ming Yang
- Department
of Chemical and Biomolecular Engineering, Clemson University, Clemson, South Carolina 29634, United States
| | - Zhi Zhong
- Department
of Drug Discovery & Biomedical Sciences, Medical University of South Carolina, Charleston, South Carolina 29425, United States
| | - John J. Lemasters
- Department
of Drug Discovery & Biomedical Sciences, Medical University of South Carolina, Charleston, South Carolina 29425, United States
| | - Jeffrey E. Korte
- Department
of Public Health Sciences, College of Medicine, Medical University of South Carolina, Charleston, South Carolina 29425, United States
| | - Amanda L. Waters
- Department
of Chemistry, University of Central Oklahoma, Edmond, Oklahoma 73034, United States
| | - Carl E. Heltzel
- Department
of Drug Discovery & Biomedical Sciences, Medical University of South Carolina, Charleston, South Carolina 29425, United States
| | - R. Thomas Williamson
- Department
of Chemistry and Biochemistry, University
of North Carolina Wilmington, Wilmington, North Carolina 28409, United States
| | - Wendy K. Strangman
- Department
of Chemistry and Biochemistry, University
of North Carolina Wilmington, Wilmington, North Carolina 28409, United States
| | - Fred Valeriote
- Henry
Ford Health Systems, Detroit, Michigan 48202, United States
| | - Marcus A. Tius
- Department
of Chemistry, University of Hawaii, Honolulu, Hawaii 96822, United States
| | - Giacomo R. DiTullio
- Department
of Oceanography, College of Charleston, Charleston, South Carolina 29403, United States
| | - Daneel Ferreira
- Department
of BioMolecular Sciences, Division of Pharmacognosy, University of Mississippi, Oxford, Mississippi 38677, United States
| | - Alexander Alekseyenko
- Department
of Public Health Sciences, College of Medicine, Medical University of South Carolina, Charleston, South Carolina 29425, United States
| | - Shengpeng Wang
- State Key
Laboratory of Quality Research in Chinese Medicine, Institute of Chinese
Medical Sciences, University of Macau, Macau 999078, China
| | - Mark T. Hamann
- Department
of Drug Discovery & Biomedical Sciences, Medical University of South Carolina, Charleston, South Carolina 29425, United States
| | - Xiaojuan Wang
- Department
of Pharmacy, Lanzhou University, Lanzhou 730000, Gansu China
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Hwang J, Lee MJ, Lee SG, Do H, Lee JH. Structural insights into the distinct substrate preferences of two bacterial epoxide hydrolases. Int J Biol Macromol 2024; 264:130419. [PMID: 38423431 DOI: 10.1016/j.ijbiomac.2024.130419] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/20/2023] [Revised: 01/22/2024] [Accepted: 02/22/2024] [Indexed: 03/02/2024]
Abstract
Epoxide hydrolases (EHs), which catalyze the transformation of epoxides to diols, are present in many eukaryotic and prokaryotic organisms. They have recently drawn considerable attention from organic chemists owing to their application in the semisynthesis of enantiospecific diol compounds. Here, we report the crystal structures of BoEH from Bosea sp. PAMC 26642 and CaEH from Caballeronia sordidicola PAMC 26510 at 1.95 and 2.43 Å resolution, respectively. Structural analysis showed that the overall structures of BoEH and CaEH commonly possess typical α/β hydrolase fold with the same ring-opening residues (Tyr-Tyr) and conserved catalytic triad residues (Asp-Asp-His). However, the two enzymes were found to have significantly different sequence compositions in the cap domain region, which is involved in the formation of the substrate-binding site in both enzymes. Enzyme activity assay results showed that BoEH had the strongest activity toward the linear aliphatic substrates, whereas CaEH had a higher preference for aromatic- and cycloaliphatic substrates. Computational docking simulations and tunnel identification revealed important residues with different substrate-binding preferences. Collectively, structure comparison studies, together with ligand docking simulation results, suggested that the differences in substrate-binding site residues were highly correlated with substrate specificity.
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Affiliation(s)
- Jisub Hwang
- Division of Life Sciences, Korea Polar Research Institute, Incheon 21990, Republic of Korea; Department of Polar Sciences, University of Science and Technology, Incheon 21990, Republic of Korea
| | - Min Ju Lee
- Division of Life Sciences, Korea Polar Research Institute, Incheon 21990, Republic of Korea; Synthetic Biology and Bioengineering Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 34141, Republic of Korea
| | - Sung Gu Lee
- Division of Life Sciences, Korea Polar Research Institute, Incheon 21990, Republic of Korea; Department of Polar Sciences, University of Science and Technology, Incheon 21990, Republic of Korea
| | - Hackwon Do
- Division of Life Sciences, Korea Polar Research Institute, Incheon 21990, Republic of Korea; Department of Polar Sciences, University of Science and Technology, Incheon 21990, Republic of Korea.
| | - Jun Hyuck Lee
- Division of Life Sciences, Korea Polar Research Institute, Incheon 21990, Republic of Korea; Department of Polar Sciences, University of Science and Technology, Incheon 21990, Republic of Korea.
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The Multifaceted Role of Epoxide Hydrolases in Human Health and Disease. Int J Mol Sci 2020; 22:ijms22010013. [PMID: 33374956 PMCID: PMC7792612 DOI: 10.3390/ijms22010013] [Citation(s) in RCA: 48] [Impact Index Per Article: 9.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/27/2020] [Revised: 12/18/2020] [Accepted: 12/19/2020] [Indexed: 12/12/2022] Open
Abstract
Epoxide hydrolases (EHs) are key enzymes involved in the detoxification of xenobiotics and biotransformation of endogenous epoxides. They catalyze the hydrolysis of highly reactive epoxides to less reactive diols. EHs thereby orchestrate crucial signaling pathways for cell homeostasis. The EH family comprises 5 proteins and 2 candidate members, for which the corresponding genes are not yet identified. Although the first EHs were identified more than 30 years ago, the full spectrum of their substrates and associated biological functions remain partly unknown. The two best-known EHs are EPHX1 and EPHX2. Their wide expression pattern and multiple functions led to the development of specific inhibitors. This review summarizes the most important points regarding the current knowledge on this protein family and highlights the particularities of each EH. These different enzymes can be distinguished by their expression pattern, spectrum of associated substrates, sub-cellular localization, and enzymatic characteristics. We also reevaluated the pathogenicity of previously reported variants in genes that encode EHs and are involved in multiple disorders, in light of large datasets that were made available due to the broad development of next generation sequencing. Although association studies underline the pleiotropic and crucial role of EHs, no data on high-effect variants are confirmed to date.
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Barnette DA, Schleiff MA, Osborn LR, Flynn N, Matlock M, Swamidass SJ, Miller GP. Dual mechanisms suppress meloxicam bioactivation relative to sudoxicam. Toxicology 2020; 440:152478. [PMID: 32437779 DOI: 10.1016/j.tox.2020.152478] [Citation(s) in RCA: 16] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/22/2020] [Revised: 04/17/2020] [Accepted: 04/24/2020] [Indexed: 01/07/2023]
Abstract
Thiazoles are biologically active aromatic heterocyclic rings occurring frequently in natural products and drugs. These molecules undergo typically harmless elimination; however, a hepatotoxic response can occur due to multistep bioactivation of the thiazole to generate a reactive thioamide. A basis for those differences in outcomes remains unknown. A textbook example is the high hepatotoxicity observed for sudoxicam in contrast to the relative safe use and marketability of meloxicam, which differs in structure from sudoxicam by the addition of a single methyl group. Both drugs undergo bioactivation, but meloxicam exhibits an additional detoxification pathway due to hydroxylation of the methyl group. We hypothesized that thiazole bioactivation efficiency is similar between sudoxicam and meloxicam due to the methyl group being a weak electron donator, and thus, the relevance of bioactivation depends on the competing detoxification pathway. For a rapid analysis, we modeled epoxidation of sudoxicam derivatives to investigate the impact of substituents on thiazole bioactivation. As expected, electron donating groups increased the likelihood for epoxidation with a minimal effect for the methyl group, but model predictions did not extrapolate well among all types of substituents. Through analytical methods, we measured steady-state kinetics for metabolic bioactivation of sudoxicam and meloxicam by human liver microsomes. Sudoxicam bioactivation was 6-fold more efficient than that for meloxicam, yet meloxicam showed a 6-fold higher efficiency of detoxification than bioactivation. Overall, sudoxicam bioactivation was 15-fold more likely than meloxicam considering all metabolic clearance pathways. Kinetic differences likely arise from different enzymes catalyzing respective metabolic pathways based on phenotyping studies. Rather than simply providing an alternative detoxification pathway, the meloxicam methyl group suppressed the bioactivation reaction. These findings indicate the impact of thiazole substituents on bioactivation is more complex than previously thought and likely contributes to the unpredictability of their toxic potential.
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Affiliation(s)
- Dustyn A Barnette
- Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, 4301 W Markham St, Little Rock, AR, 72205, United States
| | - Mary A Schleiff
- Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, 4301 W Markham St, Little Rock, AR, 72205, United States
| | - Laura R Osborn
- Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, 4301 W Markham St, Little Rock, AR, 72205, United States
| | - Noah Flynn
- Department of Pathology and Immunology, 660 S Euclid Ave, Washington University, St. Louis, MO, 63130, United States
| | - Matthew Matlock
- Department of Pathology and Immunology, 660 S Euclid Ave, Washington University, St. Louis, MO, 63130, United States
| | - S Joshua Swamidass
- Department of Pathology and Immunology, 660 S Euclid Ave, Washington University, St. Louis, MO, 63130, United States
| | - Grover P Miller
- Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, 4301 W Markham St, Little Rock, AR, 72205, United States.
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Yamashita S, Kato A, Akatsuka T, Sawada T, Asai T, Koyama N, Okita K. Clinical relevance of increased serum preneoplastic antigen in hepatitis C-related hepatocellular carcinoma. World J Gastroenterol 2020; 26:1463-1473. [PMID: 32308347 PMCID: PMC7152515 DOI: 10.3748/wjg.v26.i13.1463] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/20/2019] [Revised: 03/06/2020] [Accepted: 03/19/2020] [Indexed: 02/06/2023] Open
Abstract
BACKGROUND The prognosis of hepatocellular carcinoma (HCC) patients remains poor despite advances in treatment modalities and diagnosis. It is important to identify useful markers for the early detection of HCC in patients. Preneoplastic antigen (PNA), originally reported in a rat carcinogenesis model, is increased in the tissues and serum of HCC patients.
AIM To determine the diagnostic value of PNA for discriminating HCC and to characterize PNA-positive HCC.
METHODS Patients with hepatitis C virus (HCV)-related hepatic disorders were prospectively enrolled in this study, which included patients with hepatitis, with cirrhosis, and with HCC. A novel enzyme-linked immunosorbent assay was developed to measure serum PNA concentrations in patients.
RESULTS Serum PNA concentrations were measured in 89 controls and 141 patients with HCV infections (50 hepatitis, 44 cirrhosis, and 47 HCC). Compared with control and non-HCC patients, PNA was increased in HCC. On receiver operating characteristic curve analysis, the sensitivity of PNA was similar to the HCC markers des-γ-carboxy-prothrombin (DCP) and α-fetoprotein (AFP), but the specificity of PNA was lower. There was no correlation between PNA and AFP and a significant but weak correlation between PNA and DCP in HCC patients. Importantly, the correlations with biochemical markers were completely different for PNA, AFP, and DCP; glutamyl transpeptidase was highly correlated with PNA, but not with AFP or DCP, and was significantly higher in PNA-high patients than in PNA-low patients with HCV-related HCC.
CONCLUSION PNA may have the potential to diagnose a novel type of HCC in which glutamyl transpeptidase is positively expressed but AFP or DCP is weakly or negatively expressed.
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Affiliation(s)
- Satoyoshi Yamashita
- Department of Gastroenterology and Hepatology, Japan Community Health Care Organization Shimonoseki Medical Center, Shimonoseki, Yamaguchi 7500061, Japan
| | - Akira Kato
- Department of Gastroenterology and Hepatology, Japan Community Health Care Organization Shimonoseki Medical Center, Shimonoseki, Yamaguchi 7500061, Japan
| | - Toshitaka Akatsuka
- Department of Physiology, Faculty of Medicine, Saitama Medical University, Iruma-gun, Saitama 3500495, Japan
| | - Takashi Sawada
- Research and Development Division, Sekisui Medical Company Limited, Ryugasaki, Ibaraki 3010852, Japan
| | - Tomohide Asai
- Research and Development Division, Sekisui Medical Company Limited, Ryugasaki, Ibaraki 3010852, Japan
| | - Noriyuki Koyama
- Clinical Research Department, Eidia Company Limited, Chiyoda-ku, Tokyo 1010032, Japan
- Eisai Company Limited, Shinjuku-ku, Tokyo 1620812, Japan
| | - Kiwamu Okita
- Department of Internal Medicine, Shunan Memorial Hospital, Kudamatsu, Yamaguchi 7440033, Japan
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6
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Barnych B, Singh N, Negrel S, Zhang Y, Magis D, Roux C, Hua X, Ding Z, Morisseau C, Tantillo DJ, Siegel JB, Hammock BD. Development of potent inhibitors of the human microsomal epoxide hydrolase. Eur J Med Chem 2020; 193:112206. [PMID: 32203787 DOI: 10.1016/j.ejmech.2020.112206] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/23/2020] [Revised: 03/03/2020] [Accepted: 03/03/2020] [Indexed: 11/15/2022]
Abstract
Microsomal epoxide hydrolase (mEH) hydrolyzes a wide range of epoxide containing molecules. Although involved in the metabolism of xenobiotics, recent studies associate mEH with the onset and development of certain disease conditions. This phenomenon is partially attributed to the significant role mEH plays in hydrolyzing endogenous lipid mediators, suggesting more complex and extensive physiological functions. In order to obtain pharmacological tools to further study the biology and therapeutic potential of this enzyme target, we describe the development of highly potent 2-alkylthio acetamide inhibitors of the human mEH with IC50 values in the low nanomolar range. These are around 2 orders of magnitude more potent than previously obtained primary amine, amide and urea-based mEH inhibitors. Experimental assay results and rationalization of binding through docking calculations of inhibitors to a mEH homology model indicate that an amide connected to an alkyl side chain and a benzyl-thio function as key pharmacophore units.
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Affiliation(s)
- Bogdan Barnych
- Department of Entomology and Nematology, UCD Comprehensive Cancer Center, University of California Davis, Davis, CA, 95616, United States
| | - Nalin Singh
- Department of Entomology and Nematology, UCD Comprehensive Cancer Center, University of California Davis, Davis, CA, 95616, United States
| | - Sophie Negrel
- Department of Entomology and Nematology, UCD Comprehensive Cancer Center, University of California Davis, Davis, CA, 95616, United States
| | - Yue Zhang
- Department of Chemistry, University of California Davis, Davis, CA, 95616, United States
| | - Damien Magis
- Department of Entomology and Nematology, UCD Comprehensive Cancer Center, University of California Davis, Davis, CA, 95616, United States
| | - Capucine Roux
- Department of Entomology and Nematology, UCD Comprehensive Cancer Center, University of California Davis, Davis, CA, 95616, United States
| | - Xiude Hua
- Department of Entomology and Nematology, UCD Comprehensive Cancer Center, University of California Davis, Davis, CA, 95616, United States; College of Plant Protection, Nanjing Agricultural University, Nanjing, 210095, China
| | - Zhewen Ding
- Department of Entomology and Nematology, UCD Comprehensive Cancer Center, University of California Davis, Davis, CA, 95616, United States
| | - Christophe Morisseau
- Department of Entomology and Nematology, UCD Comprehensive Cancer Center, University of California Davis, Davis, CA, 95616, United States
| | - Dean J Tantillo
- Department of Chemistry, University of California Davis, Davis, CA, 95616, United States
| | - Justin B Siegel
- Department of Chemistry, University of California Davis, Davis, CA, 95616, United States; Department of Biochemistry and Molecular Medicine, University of California Davis, Davis, CA, 95616, United States; Genome Center, University of California Davis, Davis, CA, 95616, United States
| | - Bruce D Hammock
- Department of Entomology and Nematology, UCD Comprehensive Cancer Center, University of California Davis, Davis, CA, 95616, United States.
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Castillo-Peinado L, López-Bascón M, Mena-Bravo A, Luque de Castro M, Priego-Capote F. Determination of primary fatty acid amides in different biological fluids by LC–MS/MS in MRM mode with synthetic deuterated standards: Influence of biofluid matrix on sample preparation. Talanta 2019; 193:29-36. [DOI: 10.1016/j.talanta.2018.09.088] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/10/2018] [Revised: 09/21/2018] [Accepted: 09/24/2018] [Indexed: 12/18/2022]
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Blum M, Dogan I, Karber M, Rothe M, Schunck WH. Chiral lipidomics of monoepoxy and monohydroxy metabolites derived from long-chain polyunsaturated fatty acids. J Lipid Res 2019; 60:135-148. [PMID: 30409844 PMCID: PMC6314268 DOI: 10.1194/jlr.m089755] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/06/2018] [Revised: 11/01/2018] [Indexed: 01/10/2023] Open
Abstract
A chiral lipidomics approach was established for comprehensive profiling of regio- and stereoisomeric monoepoxy and monohydroxy metabolites of long-chain PUFAs as generated enzymatically by cytochromes P450 (CYPs), lipoxygenases (LOXs), and cyclooxygenases (COXs) and, in part, also unspecific oxidations. The method relies on reversed-phase chiral-LC coupled with ESI/MS/MS. Applications revealed partially opposing enantioselectivities of soluble and microsomal epoxide hydrolases (mEHs). Ablation of the soluble epoxide hydrolase (sEH) gene resulted in specific alterations in the enantiomeric composition of endogenous monoepoxy metabolites. For example, the (R,S)/(S,R)-ratio of circulating 14,15-EET changed from 2.1:1 in WT to 9.7:1 in the sEH-KO mice. Studies with liver microsomes suggested that CYP/mEH interactions play a primary role in determining the enantiomeric composition of monoepoxy metabolites during their generation and release from the ER. Analysis of human plasma showed significant enantiomeric excess with several monoepoxy metabolites. Monohydroxy metabolites were generally present as racemates; however, Ca2+-ionophore stimulation of whole blood samples resulted in enantioselective increases of LOX-derived metabolites (12S-HETE and 17S-hydroxydocosahexaenoic acid) and COX-derived metabolites (11R-HETE). Our chiral approach may provide novel opportunities for investigating the role of bioactive lipid mediators that generally exert their physiological functions in a highly regio- and stereospecific manner.
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Affiliation(s)
- Maximilian Blum
- Max Delbrueck Center for Molecular Medicine, Berlin, Germany
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Racovita RC, Jetter R. Composition of the epicuticular waxes coating the adaxial side of Phyllostachys aurea leaves: Identification of very-long-chain primary amides. PHYTOCHEMISTRY 2016; 130:252-261. [PMID: 27402630 DOI: 10.1016/j.phytochem.2016.06.005] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/05/2016] [Revised: 06/05/2016] [Accepted: 06/13/2016] [Indexed: 06/06/2023]
Abstract
The present study presents comprehensive chemical analyses of cuticular wax mixtures of the bamboo Phyllostachys aurea. The epicuticular and intracuticular waxes were sampled selectively from the adaxial side of leaves on young and old plants and investigated by gas chromatography-mass spectrometry and flame ionization detection. The epi- and intracuticular layers on young and old leaves had wax loads ranging from 1.7 μg/cm(2) to 1.9 μg/cm(2). Typical very-long-chain aliphatic wax constituents were found with characteristic chain length patterns, including alkyl esters (primarily C48), alkanes (primarily C29), fatty acids (primarily C28 and C16), primary alcohols (primarily C28) and aldehydes (primarily C30). Alicyclic wax components were identified as tocopherols and triterpenoids, including substantial amounts of triterpenoid esters. Alkyl esters, alkanes, fatty acids and aldehydes were found in greater amounts in the epicuticular layer, while primary alcohols and most terpenoids accumulated more in the intracuticular wax. Alkyl esters occurred as mixtures of metamers, combining C20 alcohol with various acids into shorter ester homologs (C36C40), and a wide range of alcohols with C22 and C24 acids into longer esters (C42C52). Primary amides were identified, with a characteristic chain length profile peaking at C30. The amides were present exclusively in the epicuticular layer and thus at or near the surface, where they may affect plant-herbivore or plant-pathogen interactions.
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Affiliation(s)
- Radu C Racovita
- Department of Chemistry, The University of British Columbia, Vancouver, BC, V6T 1Z1, Canada
| | - Reinhard Jetter
- Department of Chemistry, The University of British Columbia, Vancouver, BC, V6T 1Z1, Canada; Department of Botany, The University of British Columbia, Vancouver, BC, V6T 1Z4, Canada.
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10
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Kitamura S, Hvorecny KL, Niu J, Hammock BD, Madden DR, Morisseau C. Rational Design of Potent and Selective Inhibitors of an Epoxide Hydrolase Virulence Factor from Pseudomonas aeruginosa. J Med Chem 2016; 59:4790-9. [PMID: 27120257 DOI: 10.1021/acs.jmedchem.6b00173] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/08/2023]
Abstract
The virulence factor cystic fibrosis transmembrane conductance regulator (CFTR) inhibitory factor (Cif) is secreted by Pseudomonas aeruginosa and is the founding member of a distinct class of epoxide hydrolases (EHs) that triggers the catalysis-dependent degradation of the CFTR. We describe here the development of a series of potent and selective Cif inhibitors by structure-based drug design. Initial screening revealed 1a (KB2115), a thyroid hormone analog, as a lead compound with low micromolar potency. Structural requirements for potency were systematically probed, and interactions between Cif and 1a were characterized by X-ray crystallography. On the basis of these data, new compounds were designed to yield additional hydrogen bonding with residues of the Cif active site. From this effort, three compounds were identified that are 10-fold more potent toward Cif than our first-generation inhibitors and have no detectable thyroid hormone-like activity. These inhibitors will be useful tools to study the pathological role of Cif and have the potential for clinical application.
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Affiliation(s)
- Seiya Kitamura
- Department of Entomology and Nematology, and UC Davis Comprehensive Cancer Center, University of California, Davis , One Shields Avenue, Davis, California 95616, United States
| | - Kelli L Hvorecny
- Department of Biochemistry, Geisel School of Medicine at Dartmouth , 7200 Vail Building, Hanover, New Hampshire 03755, United States
| | - Jun Niu
- Department of Entomology and Nematology, and UC Davis Comprehensive Cancer Center, University of California, Davis , One Shields Avenue, Davis, California 95616, United States
| | - Bruce D Hammock
- Department of Entomology and Nematology, and UC Davis Comprehensive Cancer Center, University of California, Davis , One Shields Avenue, Davis, California 95616, United States
| | - Dean R Madden
- Department of Biochemistry, Geisel School of Medicine at Dartmouth , 7200 Vail Building, Hanover, New Hampshire 03755, United States
| | - Christophe Morisseau
- Department of Entomology and Nematology, and UC Davis Comprehensive Cancer Center, University of California, Davis , One Shields Avenue, Davis, California 95616, United States
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11
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Floor RJ, Wijma HJ, Jekel PA, Terwisscha van Scheltinga AC, Dijkstra BW, Janssen DB. X-ray crystallographic validation of structure predictions used in computational design for protein stabilization. Proteins 2015; 83:940-51. [PMID: 25739581 DOI: 10.1002/prot.24791] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/09/2014] [Revised: 02/11/2015] [Accepted: 02/14/2015] [Indexed: 11/10/2022]
Abstract
Protein engineering aimed at enhancing enzyme stability is increasingly supported by computational methods for calculation of mutant folding energies and for the design of disulfide bonds. To examine the accuracy of mutant structure predictions underlying these computational methods, crystal structures of thermostable limonene epoxide hydrolase variants obtained by computational library design were determined. Four different predicted effects indeed contributed to the obtained stabilization: (i) enhanced interactions between a flexible loop close to the N-terminus and the rest of the protein; (ii) improved interactions at the dimer interface; (iii) removal of unsatisfied hydrogen bonding groups; and (iv) introduction of additional positively charged groups at the surface. The structures of an eightfold and an elevenfold mutant showed that most mutations introduced the intended stabilizing interactions, and side-chain conformations were correctly predicted for 72-88% of the point mutations. However, mutations that introduced a disulfide bond in a flexible region had a larger influence on the backbone conformation than predicted. The enzyme active sites were unaltered, in agreement with the observed preservation of catalytic activities. The structures also revealed how a c-Myc tag, which was introduced for facile detection and purification, can reduce access to the active site and thereby lower the catalytic activity. Finally, sequence analysis showed that comprehensive mutant energy calculations discovered stabilizing mutations that are not proposed by the consensus or B-FIT methods.
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Affiliation(s)
- Robert J Floor
- Biotransformation and Biocatalysis, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG, Groningen, The Netherlands
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12
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El-Sherbeni AA, El-Kadi AOS. The role of epoxide hydrolases in health and disease. Arch Toxicol 2014; 88:2013-32. [PMID: 25248500 DOI: 10.1007/s00204-014-1371-y] [Citation(s) in RCA: 45] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/11/2014] [Accepted: 09/11/2014] [Indexed: 01/09/2023]
Abstract
Epoxide hydrolases (EH) are ubiquitously expressed in all living organisms and in almost all organs and tissues. They are mainly subdivided into microsomal and soluble EH and catalyze the hydration of epoxides, three-membered-cyclic ethers, to their corresponding dihydrodiols. Owning to the high chemical reactivity of xenobiotic epoxides, microsomal EH is considered protective enzyme against mutagenic and carcinogenic initiation. Nevertheless, several endogenously produced epoxides of fatty acids function as important regulatory mediators. By mediating the formation of cytotoxic dihydrodiol fatty acids on the expense of cytoprotective epoxides of fatty acids, soluble EH is considered to have cytotoxic activity. Indeed, the attenuation of microsomal EH, achieved by chemical inhibitors or preexists due to specific genetic polymorphisms, is linked to the aggravation of the toxicity of xenobiotics, as well as the risk of cancer and inflammatory diseases, whereas soluble EH inhibition has been emerged as a promising intervention against several diseases, most importantly cardiovascular, lung and metabolic diseases. However, there is reportedly a significant overlap in substrate selectivity between microsomal and soluble EH. In addition, microsomal and soluble EH were found to have the same catalytic triad and identical molecular mechanism. Consequently, the physiological functions of microsomal and soluble EH are also overlapped. Thus, studying the biological effects of microsomal or soluble EH alterations needs to include the effects on both the metabolism of reactive metabolites, as well as epoxides of fatty acids. This review focuses on the multifaceted role of EH in the metabolism of xenobiotic and endogenous epoxides and the impact of EH modulations.
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Affiliation(s)
- Ahmed A El-Sherbeni
- Faculty of Pharmacy and Pharmaceutical Sciences, 2142J Katz Group-Rexall Centre for Pharmacy and Health Research, University of Alberta, Edmonton, AB, T6G 2E1, Canada
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13
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Nithipatikom K, Endsley MP, Pfeiffer AW, Falck JR, Campbell WB. A novel activity of microsomal epoxide hydrolase: metabolism of the endocannabinoid 2-arachidonoylglycerol. J Lipid Res 2014; 55:2093-102. [PMID: 24958911 DOI: 10.1194/jlr.m051284] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022] Open
Abstract
Microsomal epoxide hydrolase (EPHX1, EC 3.3.2.9) is a highly abundant α/β-hydrolase enzyme that is known for its catalytical epoxide hydrolase activity. A wide range of EPHX1 functions have been demonstrated including xenobiotic metabolism; however, characterization of its endogenous substrates is limited. In this study, we present evidence that EPHX1 metabolizes the abundant endocannabinoid 2-arachidonoylglycerol (2-AG) to free arachidonic acid (AA) and glycerol. The EPHX1 metabolism of 2-AG was demonstrated using commercially available EPHX1 microsomes as well as PC-3 cells overexpressing EPHX1. Conversely, EPHX1 siRNA markedly reduced the EPHX1 expression and 2-AG metabolism in HepG2 cells and LNCaP cells. A selective EPHX1 inhibitor, 10-hydroxystearamide, inhibited 2-AG metabolism and hydrolysis of a well-known EPHX1 substrate, cis-stilbene oxide. Among the inhibitors studied, a serine hydrolase inhibitor, methoxy-arachidonyl fluorophosphate, was the most potent inhibitor of 2-AG metabolism by EPHX1 microsomes. These results demonstrate that 2-AG is an endogenous substrate for EPHX1, a potential role of EPHX1 in the endocannabinoid signaling and a new AA biosynthetic pathway.
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Affiliation(s)
- Kasem Nithipatikom
- Department of Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee, WI 53226
| | - Michael P Endsley
- Department of Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee, WI 53226
| | - Adam W Pfeiffer
- Department of Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee, WI 53226
| | - John R Falck
- Departments of Biochemistry and Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX 75390
| | - William B Campbell
- Department of Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee, WI 53226
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14
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Design, synthesis and biological evaluation of 4-(1-(4(sulphanilamide)phenyl)-3-(methyl)-1H-pyrazol-5-yl)dine urea and N-acyl derivatives as a soluble epoxide hydrolase inhibitors. Med Chem Res 2013. [DOI: 10.1007/s00044-013-0817-8] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/26/2022]
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15
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Kamita SG, Yamamoto K, Dadala MM, Pha K, Morisseau C, Escaich A, Hammock BD. Cloning and characterization of a microsomal epoxide hydrolase from Heliothis virescens. INSECT BIOCHEMISTRY AND MOLECULAR BIOLOGY 2013; 43:219-228. [PMID: 23276675 PMCID: PMC3577957 DOI: 10.1016/j.ibmb.2012.12.002] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/15/2012] [Revised: 12/07/2012] [Accepted: 12/10/2012] [Indexed: 06/01/2023]
Abstract
Epoxide hydrolases (EHs) are α/β-hydrolase fold superfamily enzymes that convert epoxides to 1,2-trans diols. In insects EHs play critical roles in the metabolism of toxic compounds and allelochemicals found in the diet and for the regulation of endogenous juvenile hormones (JHs). In this study we obtained a full-length cDNA, hvmeh1, from the generalist feeder Heliothis virescens that encoded a highly active EH, Hv-mEH1. Of the 10 different EH substrates that were tested, Hv-mEH1 showed the highest specific activity (1180 nmol min(-1) mg(-1)) for a 1,2-disubstituted epoxide-containing fluorescent substrate. This specific activity was more than 25- and 3900-fold higher than that for the general EH substrates cis-stilbene oxide and trans-stilbene oxide, respectively. Although phylogenetic analysis placed Hv-mEH1 in a clade with some lepidopteran JH metabolizing EHs (JHEHs), JH III was a relatively poor substrate for Hv-mEH1. Hv-mEH1 showed a unique substrate selectivity profile for the substrates tested in comparison to those of MsJHEH, a well-characterized JHEH from Manduca sexta, and hmEH, a human microsomal EH. Hv-mEH1 also showed unique enzyme inhibition profiles to JH-like urea, JH-like secondary amide, JH-like primary amide, and non-JH-like primary amide compounds in comparison to MsJHEH and hmEH. Although Hv-mEH1 is capable of metabolizing JH III, our findings suggest that this enzymatic activity does not play a significant role in the metabolism of JH in the caterpillar. The ability of Hv-mEH1 to rapidly hydrolyze 1,2-disubstituted epoxides suggests that it may play roles in the metabolism of fatty acid epoxides such as those that are commonly found in the diet of Heliothis.
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Affiliation(s)
- Shizuo G. Kamita
- Department of Entomology and UC Davis Comprehensive Cancer Center, University of California, Davis, CA 95616, USA
| | - Kohji Yamamoto
- Department of Entomology and UC Davis Comprehensive Cancer Center, University of California, Davis, CA 95616, USA
| | - Mary M. Dadala
- Department of Entomology and UC Davis Comprehensive Cancer Center, University of California, Davis, CA 95616, USA
| | - Khavong Pha
- Department of Entomology and UC Davis Comprehensive Cancer Center, University of California, Davis, CA 95616, USA
| | - Christophe Morisseau
- Department of Entomology and UC Davis Comprehensive Cancer Center, University of California, Davis, CA 95616, USA
| | - Aurélie Escaich
- Department of Entomology and UC Davis Comprehensive Cancer Center, University of California, Davis, CA 95616, USA
| | - Bruce D. Hammock
- Department of Entomology and UC Davis Comprehensive Cancer Center, University of California, Davis, CA 95616, USA
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16
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Duan H, Yoshimura K, Kobayashi N, Sugiyama K, Sawada JI, Saito Y, Morisseau C, Hammock BD, Akatsuka T. Development of monoclonal antibodies to human microsomal epoxide hydrolase and analysis of "preneoplastic antigen"-like molecules. Toxicol Appl Pharmacol 2012; 260:17-26. [PMID: 22310175 DOI: 10.1016/j.taap.2012.01.023] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/17/2011] [Revised: 01/20/2012] [Accepted: 01/22/2012] [Indexed: 01/13/2023]
Abstract
Microsomal epoxide hydrolase (mEH) is a drug metabolizing enzyme which resides on the endoplasmic reticulum (ER) membrane and catalyzes the hydration of reactive epoxide intermediates that are formed by cytochrome P450s. mEH is also thought to have a role in bile acid transport on the plasma membrane of hepatocytes. It is speculated that efficient execution of such multiple functions is secured by its orientation and association with cytochrome P450 enzymes on the ER membrane and formation of a multiple transport system on the plasma membrane. In certain disease status, mEH loses its association with the membrane and can be detected as distinct antigens in the cytosol of preneoplastic foci of liver (preneoplastic antigen), in the serum in association with hepatitis C virus infection (AN antigen), or in some brain tumors. To analyze the antigenic structures of mEH in physiological and pathological conditions, we developed monoclonal antibodies against different portions of mEH. Five different kinds of antibodies were obtained: three, anti-N-terminal portions; one anti-C-terminal; and one, anti-conformational epitope. By combining these antibodies, we developed antigen detection methods which are specific to either the membrane-bound form or the linearized form of mEH. These methods detected mEH in the culture medium released from a hepatocellular carcinoma cell line and a glioblastoma cell line, which was found to be a multimolecular complex with a unique antigenic structure different from that of the membrane-bound form of mEH. These antibodies and antigen detection methods may be useful to study pathological changes of mEH in various human diseases.
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Affiliation(s)
- Hongying Duan
- Department of Microbiology, Faculty of Medicine, Saitama Medical University, Moroyama-cho, Iruma-gun, Saitama 350-0495, Japan
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17
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Synthesis and structure–activity relationship of acylthiourea derivatives as inhibitors of microsomal epoxide hydrolase. Med Chem Res 2012. [DOI: 10.1007/s00044-011-9953-1] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/14/2022]
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18
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Ahn KC, Kasagami T, Tsai HJ, Schebb NH, Ogunyoku T, Gee SJ, Young TM, Hammock BD. An immunoassay to evaluate human/environmental exposure to the antimicrobial triclocarban. ENVIRONMENTAL SCIENCE & TECHNOLOGY 2012; 46:374-81. [PMID: 22077920 PMCID: PMC3258304 DOI: 10.1021/es202494d] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/05/2023]
Abstract
A sensitive, competitive indirect enzyme-linked immunosorbent assay (ELISA) for the detection of the antimicrobial triclocarban (TCC) was developed. The haptens were synthesized by derivatizing the para position of a phenyl moiety of TCC. The rabbit antisera were screened and the combination of antiserum 1648 and a heterologous competitive hapten containing a piperidine was further characterized. The IC(50) and detection range for TCC in buffer were 0.70 and 0.13-3.60 ng/mL, respectively. The assay was selective for TCC, providing only low cross-reactivity to TCC-related compounds and its major metabolites except for the closely related antimicrobial 3-trifluoromethyl-4,4'-dichlorocarbanilide. A liquid-liquid extraction for sample preparation of human body fluids resulted in an assay that measured low part per billion levels of TCC in small volumes of the samples. The limits of quantification of TCC were 5 ng/mL in blood/serum and 10 ng/mL in urine, respectively. TCC in human urine was largely the N- or N'-glucuronide. TCC concentrations of biosolids measured by the ELISA were similar to those determined by LC-MS/MS. This immunoassay can be used as a rapid, inexpensive, and convenient tool to aid researchers monitoring human/environmental exposure to TCC to better understand the health effects.
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Affiliation(s)
- Ki Chang Ahn
- Department of Entomology and the UCD Cancer Center, University of California Davis, Davis, CA 95616
| | - Takeo Kasagami
- Department of Entomology and the UCD Cancer Center, University of California Davis, Davis, CA 95616
| | - Hsing-Ju Tsai
- Department of Entomology and the UCD Cancer Center, University of California Davis, Davis, CA 95616
| | - Nils Helge Schebb
- Department of Entomology and the UCD Cancer Center, University of California Davis, Davis, CA 95616
| | - Temitope Ogunyoku
- Department of Civil and Environmental Engineering, University of California Davis, Davis, CA 95616
| | - Shirley J. Gee
- Department of Entomology and the UCD Cancer Center, University of California Davis, Davis, CA 95616
| | - Thomas M. Young
- Department of Civil and Environmental Engineering, University of California Davis, Davis, CA 95616
| | - Bruce D. Hammock
- Department of Entomology and the UCD Cancer Center, University of California Davis, Davis, CA 95616
- Corresponding author: telephone (530) 752–7519, fax (530) 752–1537,
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19
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Farrell EK, Chen Y, Barazanji M, Jeffries KA, Cameroamortegui F, Merkler DJ. Primary fatty acid amide metabolism: conversion of fatty acids and an ethanolamine in N18TG2 and SCP cells. J Lipid Res 2011; 53:247-56. [PMID: 22095832 DOI: 10.1194/jlr.m018606] [Citation(s) in RCA: 35] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Primary fatty acid amides (PFAM) are important signaling molecules in the mammalian nervous system, binding to many drug receptors and demonstrating control over sleep, locomotion, angiogenesis, and many other processes. Oleamide is the best-studied of the primary fatty acid amides, whereas the other known PFAMs are significantly less studied. Herein, quantitative assays were used to examine the endogenous amounts of a panel of PFAMs, as well as the amounts produced after incubation of mouse neuroblastoma N(18)TG(2) and sheep choroid plexus (SCP) cells with the corresponding fatty acids or N-tridecanoylethanolamine. Although five endogenous primary amides were discovered in the N(18)TG(2) and SCP cells, a different pattern of relative amounts were found between the two cell lines. Higher amounts of primary amides were found in SCP cells, and the conversion of N-tridecanoylethanolamine to tridecanamide was observed in the two cell lines. The data reported here show that the N(18)TG(2) and SCP cells are excellent model systems for the study of PFAM metabolism. Furthermore, the data support a role for the N-acylethanolamines as precursors for the PFAMs and provide valuable new kinetic results useful in modeling the metabolic flux through the pathways for PFAM biosynthesis and degradation.
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Affiliation(s)
- Emma K Farrell
- Department of Chemistry, University of South Florida, Tampa, FL 33620-5250, USA
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20
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Ponte F, Carvalho F, Porto B. Protective effect of acetyl-l-carnitine and α-lipoic acid against the acute toxicity of diepoxybutane to human lymphocytes. Toxicology 2011; 289:52-58. [PMID: 21807063 DOI: 10.1016/j.tox.2011.07.009] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/08/2011] [Revised: 07/15/2011] [Accepted: 07/16/2011] [Indexed: 12/21/2022]
Abstract
The biotransformation and oxidative stress may contribute to 1,2:3,4-diepoxybutane (DEB)-induced toxicity to human lymphocytes of Fanconi Anemia (FA) patients. Thus, the identification of putative inhibitors of bioactivation, as well as the determination of the protective role of oxidant defenses, on DEB-induced toxicity, can help to understand what is failing in FA cells. In the present work we studied the contribution of several biochemical pathways for DEB-induced acute toxicity in human lymphocyte suspensions, by using inhibitors of epoxide hydrolases, inhibitors of protective enzymes as glutathione S-transferase and catalase, the depletion of glutathione (GSH), and the inhibition of protein synthesis; and a variety of putative protective compounds, including antioxidants, and mitochondrial protective agents. The present study reports two novel findings: (i) it was clearly evidenced, for the first time, that the acute exposure of freshly isolated human lymphocytes to DEB results in severe GSH depletion and loss of ATP, followed by cell death; (ii) acetyl-l-carnitine elicits a significant protective effect on DEB induced toxicity, which was potentiated by α-lipoic acid. Collectively, these findings contribute to increase our knowledge of DEB-induce toxicity and will be very useful when applied in studies with lymphocytes from FA patients, in order to find out a protective agent against spontaneous and DEB-induced chromosome instability.
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Affiliation(s)
- Filipa Ponte
- REQUIMTE, Laboratory of Toxicology, Department of Biological Sciences, Faculty of Pharmacy, University of Porto, Rua Anibal Cunha, 164, 4099-030 Porto, Portugal.
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21
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Morisseau C, Bernay M, Escaich A, Sanborn JR, Lango J, Hammock BD. Development of fluorescent substrates for microsomal epoxide hydrolase and application to inhibition studies. Anal Biochem 2011; 414:154-62. [PMID: 21371418 PMCID: PMC3090455 DOI: 10.1016/j.ab.2011.02.038] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/18/2011] [Revised: 02/23/2011] [Accepted: 02/25/2011] [Indexed: 10/18/2022]
Abstract
The microsomal epoxide hydrolase (mEH) plays a significant role in the metabolism of numerous xenobiotics. In addition, it has a potential role in sexual development and bile acid transport, and it is associated with a number of diseases such as emphysema, spontaneous abortion, eclampsia, and several forms of cancer. Toward developing chemical tools to study the biological role of mEH, we designed and synthesized a series of absorbent and fluorescent substrates. The highest activity for both rat and human mEH was obtained with the fluorescent substrate cyano(6-methoxy-naphthalen-2-yl)methyl glycidyl carbonate (11). An in vitro inhibition assay using this substrate ranked a series of known inhibitors similarly to the assay that used radioactive cis-stilbene oxide but with a greater discrimination between inhibitors. These results demonstrate that the new fluorescence-based assay is a useful tool for the discovery of structure-activity relationships among mEH inhibitors. Furthermore, this substrate could also be used for the screening chemical library with high accuracy and with a Z' value of approximately 0.7. This new assay permits a significant decrease in labor and cost and also offers the advantage of a continuous readout. However, it should not be used with crude enzyme preparations due to interfering reactions.
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Affiliation(s)
- Christophe Morisseau
- Department of Entomology and Cancer Center, University of California - Davis, 95616, USA.
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22
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Shen HC, Ding FX, Wang S, Deng Q, Zhang X, Chen Y, Zhou G, Xu S, Chen HS, Tong X, Tong V, Mitra K, Kumar S, Tsai C, Stevenson AS, Pai LY, Alonso-Galicia M, Chen X, Soisson SM, Roy S, Zhang B, Tata JR, Berger JP, Colletti SL. Discovery of a highly potent, selective, and bioavailable soluble epoxide hydrolase inhibitor with excellent ex vivo target engagement. J Med Chem 2010; 52:5009-12. [PMID: 19645482 DOI: 10.1021/jm900725r] [Citation(s) in RCA: 37] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Abstract
4-Substituted piperidine-derived trisubstituted ureas are reported as highly potent and selective inhibitors for sEH. The SAR outlines approaches to improve activity against sEH and reduce ion channel and CYP liability. With minimal off-target activity and a good PK profile, the benchmark 2d exhibited remarkable in vitro and ex vivo target engagement. The eutomer entA-2d also elicited vasodilation effect in rat mesenteric artery.
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Affiliation(s)
- Hong C Shen
- Merck Research Laboratories, Merck & Co., Inc., Rahway, NJ 07065-0900, USA.
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23
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Shen HC, Ding FX, Deng Q, Xu S, Chen HS, Tong X, Tong V, Zhang X, Chen Y, Zhou G, Pai LY, Alonso-Galicia M, Zhang B, Roy S, Tata JR, Berger JP, Colletti SL. Discovery of 3,3-disubstituted piperidine-derived trisubstituted ureas as highly potent soluble epoxide hydrolase inhibitors. Bioorg Med Chem Lett 2009; 19:5314-20. [DOI: 10.1016/j.bmcl.2009.07.138] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/14/2009] [Revised: 07/28/2009] [Accepted: 07/29/2009] [Indexed: 11/24/2022]
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24
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Falck JR, Kodela R, Manne R, Atcha KR, Puli N, Dubasi N, Manthati VL, Capdevila JH, Yi XY, Goldman DH, Morisseau C, Hammock BD, Campbell WB. 14,15-Epoxyeicosa-5,8,11-trienoic acid (14,15-EET) surrogates containing epoxide bioisosteres: influence upon vascular relaxation and soluble epoxide hydrolase inhibition. J Med Chem 2009; 52:5069-75. [PMID: 19653681 PMCID: PMC2888647 DOI: 10.1021/jm900634w] [Citation(s) in RCA: 75] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/24/2023]
Abstract
All-cis-14,15-epoxyeicosa-5,8,11-trienoic acid (14,15-EET) is a labile, vasodilatory eicosanoid generated from arachidonic acid by cytochrome P450 epoxygenases. A series of robust, partially saturated analogues containing epoxide bioisosteres were synthesized and evaluated for relaxation of precontracted bovine coronary artery rings and for in vitro inhibition of soluble epoxide hydrolase (sEH). Depending upon the bioisostere and its position along the carbon chain, varying levels of vascular relaxation and/or sEH inhibition were observed. For example, oxamide 16 and N-iPr-amide 20 were comparable (ED(50) 1.7 microM) to 14,15-EET as vasorelaxants but were approximately 10-35 times less potent as sEH inhibitors (IC(50) 59 and 19 microM, respectively); unsubstituted urea 12 showed useful activity in both assays (ED(50) 3.5 microM, IC(50) 16 nM). These data reveal differential structural parameters for the two pharmacophores that could assist the development of potent and specific in vivo drug candidates.
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Affiliation(s)
- J R Falck
- Departments of Biochemistry and Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
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25
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Barbosa-Sicard E, Frömel T, Keserü B, Brandes RP, Morisseau C, Hammock BD, Braun T, Krüger M, Fleming I. Inhibition of the soluble epoxide hydrolase by tyrosine nitration. J Biol Chem 2009; 284:28156-28163. [PMID: 19704161 DOI: 10.1074/jbc.m109.054759] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022] Open
Abstract
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 (Tyr(383) and Tyr(466)) 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 Tyr(383) and Tyr(466). 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.
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Affiliation(s)
- Eduardo Barbosa-Sicard
- Institute for Vascular Signalling, Johann Wolfgang Goethe University, D-60590 Frankfurt am Main, Germany
| | - Timo Frömel
- Institute for Vascular Signalling, Johann Wolfgang Goethe University, D-60590 Frankfurt am Main, Germany
| | - Benjamin Keserü
- Institute for Vascular Signalling, Johann Wolfgang Goethe University, D-60590 Frankfurt am Main, Germany
| | - Ralf P Brandes
- Institut für Kardiovaskuläre Physiologie, Johann Wolfgang Goethe University, D-60590 Frankfurt am Main, Germany
| | | | - Bruce D Hammock
- Department of Entomology, University of California, Davis, California 95616
| | - Thomas Braun
- Department of Cardiac Development and Remodeling, Max-Planck-Institut for Heart and Lung Research, D-61231 Bad Nauheim, Germany
| | - Marcus Krüger
- Department of Cardiac Development and Remodeling, Max-Planck-Institut for Heart and Lung Research, D-61231 Bad Nauheim, Germany
| | - Ingrid Fleming
- Institute for Vascular Signalling, Johann Wolfgang Goethe University, D-60590 Frankfurt am Main, Germany.
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Shen HC, Ding FX, Wang S, Xu S, Chen HS, Tong X, Tong V, Mitra K, Kumar S, Zhang X, Chen Y, Zhou G, Pai LY, Alonso-Galicia M, Chen X, Zhang B, Tata JR, Berger JP, Colletti SL. Discovery of spirocyclic secondary amine-derived tertiary ureas as highly potent, selective and bioavailable soluble epoxide hydrolase inhibitors. Bioorg Med Chem Lett 2009; 19:3398-404. [PMID: 19481932 DOI: 10.1016/j.bmcl.2009.05.036] [Citation(s) in RCA: 36] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/17/2009] [Revised: 05/07/2009] [Accepted: 05/11/2009] [Indexed: 10/20/2022]
Abstract
Spirocyclic secondary amine-derived trisubstituted ureas were identified as highly potent, bioavailable and selective soluble epoxide hydrolase (sEH) inhibitors. Despite good oral exposure and excellent ex vivo target engagement in blood, one such compound, rac-1a, failed to lower blood pressure acutely in spontaneously hypertensive rats (SHRs). This study posed the question as to whether sEH inhibition provides a robust mechanism leading to a significant antihypertensive effect.
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Affiliation(s)
- Hong C Shen
- Department of Medicinal Chemistry, Merck Research Laboratories, PO Box 2000, Rahway, NJ 07065-0900, USA.
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27
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Decker M, Arand M, Cronin A. Mammalian epoxide hydrolases in xenobiotic metabolism and signalling. Arch Toxicol 2009; 83:297-318. [PMID: 19340413 DOI: 10.1007/s00204-009-0416-0] [Citation(s) in RCA: 153] [Impact Index Per Article: 9.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/11/2009] [Accepted: 02/16/2009] [Indexed: 12/14/2022]
Abstract
Epoxide hydrolases catalyse the hydrolysis of electrophilic--and therefore potentially genotoxic--epoxides to the corresponding less reactive vicinal diols, which explains the classification of epoxide hydrolases as typical detoxifying enzymes. The best example is mammalian microsomal epoxide hydrolase (mEH)-an enzyme prone to detoxification-due to a high expression level in the liver, a broad substrate selectivity, as well as inducibility by foreign compounds. The mEH is capable of inactivating a large number of structurally different, highly reactive epoxides and hence is an important part of the enzymatic defence of our organism against adverse effects of foreign compounds. Furthermore, evidence is accumulating that mammalian epoxide hydrolases play physiological roles other than detoxification, particularly through involvement in signalling processes. This certainly holds true for soluble epoxide hydrolase (sEH) whose main function seems to be the turnover of lipid derived epoxides, which are signalling lipids with diverse functions in regulatory processes, such as control of blood pressure, inflammatory processes, cell proliferation and nociception. In recent years, the sEH has attracted attention as a promising target for pharmacological inhibition to treat hypertension and possibly other diseases. Recently, new hitherto uncharacterised epoxide hydrolases could be identified in mammals by genome analysis. The expression pattern and substrate selectivity of these new epoxide hydrolases suggests their participation in signalling processes rather than a role in detoxification. Taken together, epoxide hydrolases (1) play a central role in the detoxification of genotoxic epoxides and (2) have an important function in the regulation of physiological processes by the control of signalling molecules with an epoxide structure.
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Affiliation(s)
- Martina Decker
- Institute of Pharmacology and Toxicology, University of Zürich, Winterthurer Str. 190, 8057 Zurich, Switzerland
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28
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Kasagami T, Kim IH, Tsai HJ, Nishi K, Hammock BD, Morisseau C. Salicylate-urea-based soluble epoxide hydrolase inhibitors with high metabolic and chemical stabilities. Bioorg Med Chem Lett 2009; 19:1784-9. [PMID: 19216074 PMCID: PMC2900318 DOI: 10.1016/j.bmcl.2009.01.069] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/16/2008] [Revised: 01/20/2009] [Accepted: 01/21/2009] [Indexed: 10/21/2022]
Abstract
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.
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Affiliation(s)
| | | | - Hsing-Ju Tsai
- Department of Entomology and University of California Davis Cancer Center, University of California, One Shields Avenue, Davis, CA 95616, USA
| | | | - Bruce D. Hammock
- Department of Entomology and University of California Davis Cancer Center, University of California, One Shields Avenue, Davis, CA 95616, USA
| | - Christophe Morisseau
- Department of Entomology and University of California Davis Cancer Center, University of California, One Shields Avenue, Davis, CA 95616, USA
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29
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Hu G, Shi Z, Hu J, Zou G, Peng G, Ran P. Association between polymorphisms of microsomal epoxide hydrolase and COPD: results from meta-analyses. Respirology 2009; 13:837-50. [PMID: 18811882 DOI: 10.1111/j.1440-1843.2008.01356.x] [Citation(s) in RCA: 33] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
BACKGROUND AND OBJECTIVE COPD is a complex polygenic disease in which gene-environment interactions are very important. The gene encoding microsomal epoxide hydrolase (EPHX1) is one of several candidate loci for COPD pathogenesis and is highly polymorphic. Based chi on the polymorphisms of EPHX1 gene (tyrosine/histidine 113, histidine/arginine 139), the population can be classified into four groups of putative EPHX1 phenotypes (fast, normal, slow and very slow). A number of studies have investigated the association between the genotypes and phenotypes of EPHX1 and COPD susceptibility in different populations, with inconsistent results. A systematic review and meta-analysis of the published data was performed to gain a clearer understanding of this association. METHODS The MEDLINE database was searched for case-control studies published from 1966 to August 2007. Data were extracted and pooled odds ratios (OR) with 95% confidence intervals (CI) were calculated. RESULTS Sixteen eligible studies, comprising 1847 patients with COPD and 2455 controls, were included in the meta-analysis. The pooled result showed that the EPHX1 113 mutant homozygote was significantly associated with an increased risk of COPD (OR 1.59, 95% CI: 1.14-2.21). Subgroup analysis supported the result in the Asian population, but not in the Caucasian population. When the analysis was limited to only the larger-sample-size studies, studies in which controls were in Hardy-Weinberg equilibrium and studies in which controls were smokers/ex-smokers, the pooled results supported the conclusion. The EPHX1 139 heterozygote protected against the development of COPD in the Asian population, but not in the Caucasian population. The other gene types of EPHX1 113 and EPHX1 139 were not associated with an increased risk of COPD. The slow activity phenotype of EPHX1 was associated with an increased risk of COPD. The fast activity phenotype of EPHX1 was a protective factor for developing COPD in the Asian population, but not in the Caucasian population. However, the very slow activity phenotype of EPHX1 was a risk for developing COPD in the Caucasian population, but not in the Asian population. CONCLUSIONS The polymorphisms of EPHX1 113 and EPHX1 139 are genetic contributors to COPD susceptibility in Asian populations. The phenotypes of EPHX1 were contributors to overall COPD susceptibility.
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Affiliation(s)
- Guoping Hu
- Guangzhou Institute of Respiratory Diseases, State Key Lab of Respiratory Disease, The First Affiliated Hospital, Guangzhou Medical College, Guangzhou, Guangdong, China
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Farrell EK, Merkler DJ. Biosynthesis, degradation and pharmacological importance of the fatty acid amides. Drug Discov Today 2008; 13:558-68. [PMID: 18598910 PMCID: PMC2667908 DOI: 10.1016/j.drudis.2008.02.006] [Citation(s) in RCA: 97] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/01/2007] [Revised: 01/29/2008] [Accepted: 02/18/2008] [Indexed: 01/08/2023]
Abstract
The identification of two biologically active fatty acid amides, N-arachidonoylethanolamine (anandamide) and oleamide, has generated a great deal of excitement and stimulated considerable research. However, anandamide and oleamide are merely the best-known and best-understood members of a much larger family of biologically occurring fatty acid amides. In this review, we will outline which fatty acid amides have been isolated from mammalian sources, detail what is known about how these molecules are made and degraded in vivo, and highlight their potential for the development of novel therapeutics.
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Affiliation(s)
- Emma K Farrell
- Department of Chemistry, University of South Florida, Tampa, FL 33620, USA
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31
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Morisseau C, Hammock BD. Gerry Brooks and epoxide hydrolases: four decades to a pharmaceutical. PEST MANAGEMENT SCIENCE 2008; 64:594-609. [PMID: 18383502 DOI: 10.1002/ps.1583] [Citation(s) in RCA: 39] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/26/2023]
Abstract
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.
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Affiliation(s)
- Christophe Morisseau
- Department of Entomology and UCD, Cancer Center, University of California, Davis, CA 95616, USA.
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32
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Morisseau C, Newman JW, Wheelock CE, Hill III T, Morin D, Buckpitt AR, Hammock BD. Development of Metabolically Stable Inhibitors of Mammalian Microsomal Epoxide Hydrolase. Chem Res Toxicol 2008; 21:951-7. [DOI: 10.1021/tx700446u] [Citation(s) in RCA: 31] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Affiliation(s)
- Christophe Morisseau
- Department of Entomology, U. C. Cancer Center, and Department of Molecular Biosciences, School of Veterinary Medicine, University of California, Davis, California 95616
| | - John W. Newman
- Department of Entomology, U. C. Cancer Center, and Department of Molecular Biosciences, School of Veterinary Medicine, University of California, Davis, California 95616
| | - Craig E. Wheelock
- Department of Entomology, U. C. Cancer Center, and Department of Molecular Biosciences, School of Veterinary Medicine, University of California, Davis, California 95616
| | - Thomas Hill III
- Department of Entomology, U. C. Cancer Center, and Department of Molecular Biosciences, School of Veterinary Medicine, University of California, Davis, California 95616
| | - Dexter Morin
- Department of Entomology, U. C. Cancer Center, and Department of Molecular Biosciences, School of Veterinary Medicine, University of California, Davis, California 95616
| | - Alan R. Buckpitt
- Department of Entomology, U. C. Cancer Center, and Department of Molecular Biosciences, School of Veterinary Medicine, University of California, Davis, California 95616
| | - Bruce D. Hammock
- Department of Entomology, U. C. Cancer Center, and Department of Molecular Biosciences, School of Veterinary Medicine, University of California, Davis, California 95616
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33
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Chiamvimonvat N, Ho CM, Tsai HJ, Hammock BD. The soluble epoxide hydrolase as a pharmaceutical target for hypertension. J Cardiovasc Pharmacol 2007; 50:225-37. [PMID: 17878749 DOI: 10.1097/fjc.0b013e3181506445] [Citation(s) in RCA: 142] [Impact Index Per Article: 7.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/26/2023]
Abstract
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.
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Affiliation(s)
- Nipavan Chiamvimonvat
- Division of Cardiovascular Medicine, Department of Internal Medicine, University of California, Davis, CA 95616, USA
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34
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Fleming I, Rueben A, Popp R, Fisslthaler B, Schrodt S, Sander A, Haendeler J, Falck JR, Morisseau C, Hammock BD, Busse R. Epoxyeicosatrienoic acids regulate Trp channel dependent Ca2+ signaling and hyperpolarization in endothelial cells. Arterioscler Thromb Vasc Biol 2007; 27:2612-8. [PMID: 17872452 DOI: 10.1161/atvbaha.107.152074] [Citation(s) in RCA: 150] [Impact Index Per Article: 8.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
OBJECTIVE An initial step in endothelium-derived hyperpolarizing factor-mediated responses is endothelial cell hyperpolarization. Here we address the mechanisms by which cytochrome P450 (CYP)-derived epoxyeicosatrienoic acids (EETs) contribute to this effect in native and cultured endothelial cells. METHODS AND RESULTS In native CYP2C-expressing endothelial cells, bradykinin elicited a Ca(2+) influx that was potentiated by the soluble epoxide hydrolase inhibitor, 1-adamantyl-3-cyclohexylurea (ACU), and attenuated by CYP inhibition. Similar effects were observed in cultured endothelial cells overexpressing CYP2C9, but not in CYP2C9-deficient cells, and were prevented by the EET antagonist 14,15-epoxyeicosa-5(Z)-enoic acid as well as by the cAMP antagonist, Rp-cAMPS. The effects on Ca(2+) were mirrored by prolongation of the bradykinin-induced hyperpolarization. Ruthenium red and the combination of charybdotoxin and apamin prevented the latter effect, suggesting that Trp channel activation increases Ca(2+) influx and prolongs the activation of Ca(2+)-dependent K(+) (K(Ca)) channels. Indeed, overexpression of CYP2C9 enhanced the agonist-induced translocation of a TrpC6-V5 fusion protein to caveolin-1-rich areas of the endothelial cell membrane, which was prevented by Rp-cAMPS and mimicked by 11,12-EET. CONCLUSIONS Elevated EET levels regulate Ca(2+) influx into endothelial cells and the subsequent activation of K(Ca) channels, via a cAMP/PKA-dependent mechanism that involves the intracellular translocation of Trp channels.
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Affiliation(s)
- Ingrid Fleming
- Vascular Signalling Group, Institut für Kardiovaskuläre Physiologie, Johann Wolfgang Goethe-Universität, Theodor-Stern-Kai 7, D-60590 Frankfurt am Main, Germany.
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35
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Chung JK, Yuan W, Liu G, Zheng J. Investigation of bioactivation and toxicity of styrene in CYP2E1 transgenic cells. Toxicology 2006; 226:99-106. [PMID: 16872732 DOI: 10.1016/j.tox.2006.06.001] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/22/2006] [Revised: 05/17/2006] [Accepted: 06/03/2006] [Indexed: 10/24/2022]
Abstract
Styrene has been found to be toxic to the respiratory system, and the toxicity of styrene is metabolism-dependent. CYP2E1 is suggested to be one of the cytochrome P450 enzymes responsible for the bioactivation of styrene. Our work focused on the roles of CYP2E1 and epoxide, a metabolite of styrene epoxidation, in the cytotoxicity of styrene. Styrene was found to be more toxic to h2E1 cells than to the wild type, while there was no difference found when styrene oxide was administered. Both soluble and microsomal epoxide hydrolase inhibitors dramatically enhanced styrene toxicity. Glutathione and glutathione ethyl ester showed protection against styrene cytotoxicity. Cytotoxicity of a selection of styrene analogues, such as ethylbenzene, vinylcyclohexane, and ethylcyclohexane, was assessed to determine if unsaturation is required for styrene toxicity. Ethylbenzene and vinylcyclohexane were found to be as toxic as styrene to h2E1 cells, whereas little toxicity of ethylcyclohexane to h2E1 cells was observed. This indicates the importance of vinyl group of styrene in its cytotoxicity, but saturation of the vinyl group does not necessarily eliminate styrene toxicity. An N-acetylcysteine conjugate derived from styrene oxide was identified by LC/MS/MS in the sample obtained from the incubation of h2E1 cell lysate with styrene in the presence of N-acetylcysteine. Formation of the N-acetylcysteine conjugate was found to be NADPH-dependent. These studies provided strong evidence in support of toxic role of styrene epoxide metabolite in styrene toxicity.
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Affiliation(s)
- Jou-Ku Chung
- Department of Pharmaceutical Sciences, School of Pharmacy, Northeastern University, Boston, MA 02115, USA
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36
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Roy U, Joshua R, Stark R, Balazy M. Cytochrome P450/NADPH-dependent biosynthesis of 5,6-trans-epoxyeicosatrienoic acid from 5,6-trans-arachidonic acid. Biochem J 2006; 390:719-27. [PMID: 15916533 PMCID: PMC1199666 DOI: 10.1042/bj20050681] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/17/2022]
Abstract
5,6-trans-AA (5,6-TAA, where TAA stands for trans-arachidonic acid) is a recently identified trans fatty acid that originates from the cis-trans isomerization of AA initiated by the NO2 radical. This trans fatty acid has been detected in blood circulation and we suggested that it functions as a lipid mediator of the toxic effects of NO2. To understand its role as a lipid mediator, we studied the metabolism of 5,6-TAA by liver microsomes stimulated with NADPH. Profiling of metabolites by liquid chromatography/MS revealed a complex mixture of oxidized products among which were four epoxides, their respective hydrolysis products (dihydroxyeicosatrienoic acids), and several HETEs (hydroxyeicosatetraenoic acids) resulting from allylic, bis-allylic and (omega-1)/(omega-2) hydroxylations. We found that the C5-C6 trans bond competed with the three cis bonds for oxidative metabolism mediated by CYP (cytochrome P450) epoxygenase and hydroxylase. This was evidenced by the detection of 5,6-trans-EET (where EET stands for epoxyeicosatrienoic acid), 5,6-erythro-dihydroxyeicosatrienoic acid and an isomer of 5-HETE. A standard of 5,6-trans-EET obtained by iodolactonization of 5,6-TAA was used for the unequivocal identification of the unique microsomal epoxide in which the oxirane ring was of trans configuration. Additional lipid products originated from the metabolism involving the cis bonds and thus these metabolites had the trans C5-C6 bond. The 5,6-trans-isomers of 18- and 19-HETE were likely to be products of the CYP2E1, because a neutralizing antibody partially inhibited their formation without having an effect on the formation of the epoxides. Our study revealed a novel pathway of microsomal oxidative metabolism of a trans fatty acid in which both cis and trans bonds participated. Of particular significance is the detection of the trans-epoxide of AA, which may be involved in the metabolic activation of such trans fatty acids and probably contribute to their biological activity. Unlike its cis-isomer, 5,6-trans-EET was significantly more stable and resisted microsomal hydrolysis and conjugation with glutathione catalysed by hepatic glutathione S-transferase.
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Affiliation(s)
- Uzzal Roy
- Department of Pharmacology, New York Medical College, Valhalla, NY 10595, U.S.A
| | - Robert Joshua
- Department of Pharmacology, New York Medical College, Valhalla, NY 10595, U.S.A
| | - Russell L. Stark
- Department of Pharmacology, New York Medical College, Valhalla, NY 10595, U.S.A
| | - Michael Balazy
- Department of Pharmacology, New York Medical College, Valhalla, NY 10595, U.S.A
- To whom correspondence should be addressed (email )
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Abstract
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.
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Affiliation(s)
- Christophe Morisseau
- Department of Entomology and U.C. Davis Cancer Center, University of California, Davis, California 95616, USA
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38
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Paulsson B, Rannug A, Henderson AP, Golding BT, Törnqvist M, Warholm M. In vitro studies of the influence of glutathione transferases and epoxide hydrolase on the detoxification of acrylamide and glycidamide in blood. MUTATION RESEARCH-GENETIC TOXICOLOGY AND ENVIRONMENTAL MUTAGENESIS 2005; 580:53-9. [PMID: 15668107 DOI: 10.1016/j.mrgentox.2004.11.006] [Citation(s) in RCA: 31] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/10/2004] [Revised: 11/09/2004] [Accepted: 11/10/2004] [Indexed: 11/20/2022]
Abstract
Enzymes involved in the metabolism of xenobiotic substances are often polymorphic in humans. Such genetic polymorphisms may result in inter-individual differences in detoxification of certain chemicals, and as a consequence, possibly affect health-risk assessments. This present work concerns studies of the influence of polymorphic enzymes in the detoxification of acrylamide and its metabolite glycidamide. Enzymes that enhance conjugation with glutathione (GSH), the glutathione transferases (GSTs), may influence the detoxification of both acrylamide and glycidamide, whereas the enzyme epoxide hydrolase (EH) should only catalyse the hydrolysis of glycidamide. In this study, the doses of acrylamide or glycidamide measured as specific adducts to hemoglobin (Hb) were analysed in blood samples after in vitro incubation with these compounds. Blood samples from individuals with different genotypes for GSTT1 and GSTM1 were studied. No significant differences in adduct levels depending on genotype were noted. In a parallel experiment, incubation with ethylene oxide was used as positive control. In this experiment individuals carrying GSTT1 showed lower adduct level increments from ethylene oxide than individuals lacking GSTT1. Furthermore, addition of ethacrynic acid or laurylamine, compounds which inhibit GST and EH, respectively, did not affect the adduct levels. These results suggest that neither GSTs nor EH have any significant effect on the blood dose, measured as Hb-adducts over time, after exposure to acrylamide or glycidamide.
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Affiliation(s)
- Birgit Paulsson
- Department of Environmental Chemistry, Stockholm University, SE-106 91 Stockholm, Sweden
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39
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Newman JW, Morisseau C, Hammock BD. Epoxide hydrolases: their roles and interactions with lipid metabolism. Prog Lipid Res 2005; 44:1-51. [PMID: 15748653 DOI: 10.1016/j.plipres.2004.10.001] [Citation(s) in RCA: 342] [Impact Index Per Article: 17.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/15/2022]
Abstract
The epoxide hydrolases (EHs) are enzymes present in all living organisms, which transform epoxide containing lipids by the addition of water. In plants and animals, many of these lipid substrates have potent biologically activities, such as host defenses, control of development, regulation of inflammation and blood pressure. Thus the EHs have important and diverse biological roles with profound effects on the physiological state of the host organisms. Currently, seven distinct epoxide hydrolase sub-types are recognized in higher organisms. These include the plant soluble EHs, the mammalian soluble epoxide hydrolase, the hepoxilin hydrolase, leukotriene A4 hydrolase, the microsomal epoxide hydrolase, and the insect juvenile hormone epoxide hydrolase. While our understanding of these enzymes has progressed at different rates, here we discuss the current state of knowledge for each of these enzymes, along with a distillation of our current understanding of their endogenous roles. By reviewing the entire enzyme class together, both commonalities and discrepancies in our understanding are highlighted and important directions for future research pertaining to these enzymes are indicated.
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Affiliation(s)
- John W Newman
- Department of Entomology, UCDavis Cancer Center, University of California, One Shields Avenue, Davis, CA 95616, USA
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40
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Fang X, Weintraub NL, McCaw RB, Hu S, Harmon SD, Rice JB, Hammock BD, Spector AA. Effect of soluble epoxide hydrolase inhibition on epoxyeicosatrienoic acid metabolism in human blood vessels. Am J Physiol Heart Circ Physiol 2004; 287:H2412-20. [PMID: 15284062 DOI: 10.1152/ajpheart.00527.2004] [Citation(s) in RCA: 65] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
We investigated the effects of soluble epoxide hydrolase (sEH) inhibition on epoxyeicosatrienoic acid (EET) metabolism in intact human blood vessels, including the human saphenous vein (HSV), coronary artery (HCA), and aorta (HA). When HSV segments were perfused with 2 micromol/l 14,15-[3H]EET for 4 h, >60% of radioactivity in the perfusion medium was converted to 14,15-dihydroxyeicosatrienoic acid (DHET). Similar results were obtained with endothelium-denuded vessels. 14,15-DHET was released from both the luminal and adventitial surfaces of the HSV. When HSVs were incubated with 14,15-[3H]EET under static (no flow) conditions, formation of 14,15-DHET was detected within 15 min and was inhibited by the selective sEH inhibitors N,N'-dicyclohexyl urea and N-cyclohexyl-N'-dodecanoic acid urea (CUDA). Similarly, CUDA inhibited the conversion of 11,12-[3H]EET to 11,12-DHET by the HSV. sEH inhibition enhanced the uptake of 14,15-[3H]EET and facilitated the formation of 10,11-epoxy-16:2, a beta-oxidation product. The HCA and HA converted 14,15-[3H]EET to DHET, and this also was inhibited by CUDA. These findings in intact human blood vessels indicate that conversion to DHET is the predominant pathway for 11,12- and 14,15-EET metabolism and that sEH inhibition can modulate EET metabolism in vascular tissue.
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MESH Headings
- 8,11,14-Eicosatrienoic Acid/analogs & derivatives
- 8,11,14-Eicosatrienoic Acid/pharmacology
- Cells, Cultured
- Cyclohexanes/pharmacology
- Endothelium, Vascular/cytology
- Endothelium, Vascular/metabolism
- Epoxide Hydrolases/antagonists & inhibitors
- Epoxide Hydrolases/metabolism
- Epoxy Compounds/metabolism
- Humans
- Hydroxyeicosatetraenoic Acids/pharmacokinetics
- Lauric Acids/pharmacology
- Lipid Metabolism
- Muscle, Smooth, Vascular/cytology
- Muscle, Smooth, Vascular/metabolism
- Oxidation-Reduction
- Saphenous Vein/drug effects
- Saphenous Vein/enzymology
- Solubility
- Tritium
- Vasodilator Agents/pharmacology
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Affiliation(s)
- Xiang Fang
- Dept. of Biochemistry, 4-403 BSB, Univ. of Iowa College of Medicine, Iowa City, IA 52242, USA.
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McElroy NR, Jurs PC, Morisseau C, Hammock BD. QSAR and classification of murine and human soluble epoxide hydrolase inhibition by urea-like compounds. J Med Chem 2003; 46:1066-80. [PMID: 12620084 DOI: 10.1021/jm020269o] [Citation(s) in RCA: 62] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
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.
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Affiliation(s)
- Nathan R McElroy
- Department of Chemistry, 152 Davey Laboratory, The Pennsylvania State University, University Park, Pennsylvania 16802, USA
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42
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Severson TF, Goodrow MH, Morisseau C, Dowdy DL, Hammock BD. Urea and amide-based inhibitors of the juvenile hormone epoxide hydrolase of the tobacco hornworm (Manduca sexta: Sphingidae). INSECT BIOCHEMISTRY AND MOLECULAR BIOLOGY 2002; 32:1741-1756. [PMID: 12429126 DOI: 10.1016/s0965-1748(02)00115-7] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/24/2023]
Abstract
A new class of inhibitors of juvenile hormone epoxide hydrolase (JHEH) of Manduca sexta and further in vitro characterization of the enzyme are reported. The compounds are based on urea and amide pharmacophores that were previously demonstrated as effective inhibitors of mammalian soluble and microsomal epoxide hydrolases. The best inhibitors against JHEH activity so far within this class are N-[(Z)-9-octadecenyl]-N'-propylurea and N-hexadecyl-N'-propylurea, which inhibited hydrolysis of a surrogate substrate (t-DPPO) with an IC(50) around 90 nM. The importance of substitution number and type was investigated and results indicated that N, N'-disubstitution with asymmetric alkyl groups was favored. Potencies of pharmacophores decreased as follows: amide>urea>carbamate>carbodiimide>thiourea and thiocarbamate for N, N'-disubstituted compounds with symmetric substituents, and urea>amide>carbamate for compounds with asymmetric N, N'-substituents. JHEH hydrolyzes t-DPPO with a K(m) of 65.6 microM and a V(max) of 59 nmol min(-1) mg(-1) and has a substantially lower K(m) of 3.6 microM and higher V(max) of 322 nmol min(-1) mg(-1) for JH III. Although none of these compounds were potent inhibitors of hydrolysis of JH III by JHEH, they are the first leads toward inhibitors of JHEH that are not potentially subject to metabolism through epoxide degradation.
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Affiliation(s)
- Tonya F Severson
- Department of Entomology and Cancer Research Center, University of California, Davis, 95616, USA
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