Dataset Information

Comparison of the Structure and Activity of Glycosylated and Aglycosylated Human Carboxylesterase 1.

ABSTRACT: Human Carboxylesterase 1 (hCES1) is the key liver microsomal enzyme responsible for detoxification and metabolism of a variety of clinical drugs. To analyse the role of the single N-linked glycan on the structure and activity of the enzyme, authentically glycosylated and aglycosylated hCES1, generated by mutating asparagine 79 to glutamine, were produced in human embryonic kidney cells. Purified enzymes were shown to be predominantly trimeric in solution by analytical ultracentrifugation. The purified aglycosylated enzyme was found to be more active than glycosylated hCES1 and analysis of enzyme kinetics revealed that both enzymes exhibit positive cooperativity. Crystal structures of hCES1 a catalytically inactive mutant (S221A) and the aglycosylated enzyme were determined in the absence of any ligand or substrate to high resolutions (1.86 Å, 1.48 Å and 2.01 Å, respectively). Superposition of all three structures showed only minor conformational differences with a root mean square deviations of around 0.5 Å over all C? positions. Comparison of the active sites of these un-liganded enzymes with the structures of hCES1-ligand complexes showed that side-chains of the catalytic triad were pre-disposed for substrate binding. Overall the results indicate that preventing N-glycosylation of hCES1 does not significantly affect the structure or activity of the enzyme.

SUBMITTER: Arena de Souza V

PROVIDER: S-EPMC4676782 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Comparison of the Structure and Activity of Glycosylated and Aglycosylated Human Carboxylesterase 1.

Arena de Souza Victoria V Scott David J DJ Nettleship Joanne E JE Rahman Nahid N Charlton Michael H MH Walsh Martin A MA Owens Raymond J RJ

PloS one 20151211 12

Human Carboxylesterase 1 (hCES1) is the key liver microsomal enzyme responsible for detoxification and metabolism of a variety of clinical drugs. To analyse the role of the single N-linked glycan on the structure and activity of the enzyme, authentically glycosylated and aglycosylated hCES1, generated by mutating asparagine 79 to glutamine, were produced in human embryonic kidney cells. Purified enzymes were shown to be predominantly trimeric in solution by analytical ultracentrifugation. The pu ...[more]

PMID: 26657071

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Project description:Human carboxylesterase 1 (hCE1), one of the most important serine hydrolases distributed in liver and adipocytes, plays key roles in endobiotic homeostasis and xenobiotic metabolism. This study aimed to find potent and selective inhibitors against hCE1 from phytochemicals and their derivatives. To this end, a series of natural triterpenoids were collected and their inhibitory effects against human carboxylesterases (hCEs) were assayed using D-Luciferin methyl ester (DME) and 6,8-dichloro-9,9-dimethyl-7-oxo-7,9-dihydroacridin-2-yl benzoate (DDAB) as specific optical substrate for hCE1, and hCE2, respectively. Following screening of a series of natural triterpenoids, oleanolic acid (OA), and ursolic acid (UA) were found with strong inhibitory effects on hCE1 and relative high selectivity over hCE2. In order to get the highly selective and potent inhibitors of hCE1, a series of OA and UA derivatives were synthesized from OA and UA by chemical modifications including oxidation, reduction, esterification, and amidation. The inhibitory effects of these derivatives on hCEs were assayed and the structure-activity relationships of tested triterpenoids as hCE1 inhibitors were carefully investigated. The results demonstrated that the carbonyl group at the C-28 site is essential for hCE1 inhibition, the modifications of OA or UA at this site including esters, amides and alcohols are unbeneficial for hCE1 inhibition. In contrast, the structural modifications on OA and UA at other sites, such as converting the C-3 hydroxy group to 3-O-?-carboxypropionyl (compounds 20 and 22), led to a dramatically increase of the inhibitory effects against hCE1 and very high selectivity over hCE2. 3D-QSAR analysis of all tested triterpenoids including OA and UA derivatives provide new insights into the fine relationships linking between the inhibitory effects on hCE1 and the steric-electrostatic properties of triterpenoids. Furthermore, both inhibition kinetic analyses and docking simulations demonstrated that compound 22 was a potent competitive inhibitor against hCE1-mediated DME hydrolysis. All these findings are very helpful for medicinal chemists to design and develop highly selective and more potent hCE1 inhibitors for biomedical applications.

Project description:Carboxylesterases are enzymes that hydrolyze a broad suite of endogenous and exogenous ester-containing compounds to the corresponding alcohol and carboxylic acid. These enzymes metabolize a number of therapeutics including the anti-tumor agent CPT-11, the anti-viral drug oseltamivir, and the anti-thrombogenic agent clopidogrel as well as many agrochemicals. In addition, carboxylesterases are involved in lipid homeostasis, including cholesterol metabolism and transport with a proposed role in the development of atherosclerosis. Several different scaffolds capable of inhibiting carboxylesterases have been reported, including organophosphates, carbamates, trifluoromethyl ketone-containing structures (TFKs), and aromatic ethane-1,2-diones. Of these varied groups, only the 1,2-diones evidence carboxylesterase isoform-selectivity, which is an important characteristic for therapeutic application and probing biological mechanisms. This study constructed a series of classical and 3D-QSAR models to examine the physiochemical parameters involved in the observed selectivity of three mammalian carboxylesterases: human intestinal carboxylesterase (hiCE), human carboxylesterase 1 (hCE1), and rabbit carboxylesterase (rCE). CoMFA-based models for the benzil-analogs described 88%, 95% and 76% of observed activity for hiCE, hCE1 and rCE, respectively. For TFK-containing compounds, two distinct models were constructed using either the ketone or gem-diol form of the inhibitor. For all three enzymes, the CoMFA ketone models comprised more biological activity than the corresponding gem-diol models; however the differences were small with described activity for all models ranging from 85-98%. A comprehensive model incorporating both benzil and TFK structures described 92%, 85% and 87% of observed activity for hiCE, hCE1 and rCE, respectively. Both classical and 3D-QSAR analysis showed that the observed isoform-selectivity with the benzil-analogs could be described by the volume parameter. This finding was successfully applied to examine substrate selectivity, demonstrating that the relative volumes of the alcohol and acid moieties of ester-containing substrates were predictive for whether hydrolysis was preferred by hiCE or hCE1. Based upon the integrated benzil and TFK model, the next generation inhibitors should combine the A-ring and the 1,2-dione of the benzil inhibitor with the long alkyl chain of the TFK-inhibitor in order to optimize selectivity and potency. These new inhibitors could be useful for elucidating the role of carboxylesterase activity in fatty acid homeostasis and the development of atherosclerosis as well as effecting the controlled activation of carboxylesterase-based prodrugs in situ.

Project description:Two major isoforms of human carboxylesterases (CEs) are found in metabolically active tissues, CES1 and CES2. These hydrolytic enzymes are involved in xenobiotic and endobiotic metabolism. CES1 is abundantly expressed in human liver and monocytes/macrophages, including the THP1 cell line; CES2 is expressed in liver but not in monocytes/macrophages. The cholesteryl ester hydrolysis activity in human macrophages has been attributed to CES1. Here, we report the direct inhibitory effects of several endogenous oxysterols and fatty acids on the CE activity of THP1 monocytes/macrophages and recombinant human CES1 and CES2. Using THP1 whole-cell lysates we found: (1) 27-hydroxycholesterol (27-HC) is a potent inhibitor of carboxylesterase activity (IC50=33 nM); (2) 24(S),25-epoxycholesterol had moderate inhibitory activity (IC(50)=8.1 microM); and (3) cholesterol, 7-ketocholesterol, 22(R)-hydroxycholesterol, 24(S)-hydroxycholesterol, and 25-hydroxycholesterol each had little inhibitory activity. 27-HC was a partially noncompetitive inhibitor of recombinant CES1 (K(iapp)=10 nM) and impaired intracellular CES1 activity following treatment of intact THP1 cells. In contrast, recombinant CES2 activity was not inhibited by 27-HC, suggesting isoform-selective inhibition by 27-HC. Furthermore, unsaturated fatty acids were better inhibitors of CES1 activity than saturated fatty acids, while CES2 activity was unaffected by any fatty acid. Arachidonic acid (AA) was the most potent fatty acid inhibitor of recombinant CES1 and acted by a noncompetitive mechanism (K(iapp)=1.7 microM); when not complexed to albumin, exogenous AA penetrated intact THP1 cells and inhibited CES1. Inhibition results are discussed in light of recent structural models for CES1 that describe ligand binding sites separate from the active site. In addition, oxysterol-mediated inhibition of CES1 activity was demonstrated by pretreatment of human liver homogenates or intact THP1 cells with exogenous 27-HC, which resulted in significantly reduced hydrolysis of the pyrethroid insecticide bioresmethrin, a CES1-specific xenobiotic substrate. Collectively, these findings suggest that CE activity of recombinant CES1, cell lysates, and intact cells can be impaired by naturally occurring lipids, which may compromise the ability of CES1 to both detoxify environmental pollutants and metabolize endogenous compounds in vivo.

Dataset Information

Comparison of the Structure and Activity of Glycosylated and Aglycosylated Human Carboxylesterase 1.

Publications

Comparison of the Structure and Activity of Glycosylated and Aglycosylated Human Carboxylesterase 1.

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