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Pohgek Forkert - One of the best experts on this subject based on the ideXlab platform.

  • involvement of cyp2e1 and Carboxylesterase enzymes in vinyl carbamate metabolism in human lung microsomes
    Drug Metabolism and Disposition, 2001
    Co-Authors: Pohgek Forkert, Ken R Reid
    Abstract:

    Previous studies have shown that CYP2E1 and Carboxylesterase enzymes contributed to vinyl carbamate (VC) metabolism in murine lung. Moreover, these studies have implicated CYP2E1 and the Carboxylesterases in bioactivation and detoxication, respectively. Here we have tested the hypothesis that CYP2E1 and Carboxylesterase enzymes are involved also in VC metabolism in human lung. Demethylation of N -nitrosodimethylamine (NDMA) is an enzyme activity associated with CYP2E1, and was used as a catalytic marker for this P450 in human lung microsomes. NDMA demethylase activity in lung microsomes from 10 patients ranged from 36.9 ± 1.0 to 82.4 ± 2.4 pmol/mg protein/min. Significant decreases (40–65%) in demethylase activity were detected in lung microsomes incubated with VC and NADPH, compared with the controls in which incubations were performed with only VC or only NADPH. Preincubation with the CYP2E1 inhibitor diallyl sulfone also significantly decreased demethylase activity, and abrogated the VC-induced effect. Similarly, preincubation of lung microsomes with a human CYP2E1 inhibitory monoclonal antibody ameliorated the VC-induced reduction in demethylase activity. Microsomal Carboxylesterase activity in lung microsomes from 10 patients ranged from 19.02 ± 2.28 to 48.18 ± 4.34 nmol/mg protein/min, and was significantly decreased (25–45%) in microsomes incubated with phenylmethylsulfonyl fluoride, an inhibitor of the Carboxylesterase enzyme. Preincubation of lung microsomes with phenylmethylsulfonyl fluoride and subsequent incubation with VC and NADPH exacerbated the reduction (60–80%) in demethylase activity evoked by reaction with VC and NADPH. These results are consistent with a role for the CYP2E1 enzyme and microsomal Carboxylesterases in VC metabolism.

  • strain related differences in bioactivation of vinyl carbamate and formation of dna adducts in lungs of a j cd 1 and c57bl 6 mice
    Toxicological Sciences, 2001
    Co-Authors: Paul A Titis, Pohgek Forkert
    Abstract:

    Inbred strains of mice exhibit differing susceptibilities to formation of lung tumors induced by procarcinogens including ethyl carbamate (EC) and vinyl carbamate (VC). Strain A/J mice are susceptible, whereas C57BL/6 mice are resistant to lung tumor development. In this study, we tested the hypothesis that differential susceptibilities of A/J, CD-1, and C57BL/6 mice to lung tumor development are associated, in part, with their capacities for VC bioactivation and with the extents of DNA adduct formation. Previous studies have shown that the P450 isozyme CYP2E1 and microsomal Carboxylesterases are involved in activation and detoxication of VC, respectively. Bioactivation capacity, as estimated by ratios of p-nitrophenol hydroxylase, a CYP2E1 catalytic marker, to Carboxylesterase activities, was greater in control A/J (1.32 6 0.18 3 10 ‐6 ) and CD-1 (1.25 6 0.29 3 10 ‐6 ) mice than in control C57BL/6 (0.69 6 0.12 3 10 ‐6 ) mice. The ratios were

  • Strain-Related Differences in Bioactivation of Vinyl Carbamate and Formation of DNA Adducts in Lungs of A/J, CD-1, and C57BL/6 Mice
    Toxicological Sciences, 2001
    Co-Authors: A. Paul Titis, Pohgek Forkert
    Abstract:

    Inbred strains of mice exhibit differing susceptibilities to formation of lung tumors induced by procarcinogens including ethyl carbamate (EC) and vinyl carbamate (VC). Strain A/J mice are susceptible, whereas C57BL/6 mice are resistant to lung tumor development. In this study, we tested the hypothesis that differential susceptibilities of A/J, CD-1, and C57BL/6 mice to lung tumor development are associated, in part, with their capacities for VC bioactivation and with the extents of DNA adduct formation. Previous studies have shown that the P450 isozyme CYP2E1 and microsomal Carboxylesterases are involved in activation and detoxication of VC, respectively. Bioactivation capacity, as estimated by ratios of p-nitrophenol hydroxylase, a CYP2E1 catalytic marker, to Carboxylesterase activities, was greater in control A/J (1.32 6 0.18 3 10 ‐6 ) and CD-1 (1.25 6 0.29 3 10 ‐6 ) mice than in control C57BL/6 (0.69 6 0.12 3 10 ‐6 ) mice. The ratios were

William F. Bosron - One of the best experts on this subject based on the ideXlab platform.

  • hydrolysis of capecitabine to 5 deoxy 5 fluorocytidine by human Carboxylesterases and inhibition by loperamide
    Journal of Pharmacology and Experimental Therapeutics, 2005
    Co-Authors: Sara K. Quinney, Sonal P. Sanghani, Wilhelmina I. Davis, Zejin Sun, Daryl J. Murry, Thomas D Hurley, William F. Bosron
    Abstract:

    Capecitabine is an oral prodrug of 5-fluorouracil that is indicated for the treatment of breast and colorectal cancers. A three-step in vivo-targeted activation process requiring Carboxylesterases, cytidine deaminase, and thymidine phosphorylase converts capecitabine to 5-fluorouracil. Carboxylesterases hydrolyze capecitabine's carbamate side chain to form 5'-deoxy-5-fluorocytidine (5'-DFCR). This study examines the steady-state kinetics of recombinant human Carboxylesterase isozymes Carboxylesterase (CES) 1A1, CES2, and CES3 for hydrolysis of capecitabine with a liquid chromatography/mass spectroscopy assay. Additionally, a spectrophotometric screening assay was utilized to identify drugs that may inhibit Carboxylesterase activation of capecitabine. CES1A1 and CES2 hydrolyze capecitabine to a similar extent, with catalytic efficiencies of 14.7 and 12.9 min(-1) mM(-1), respectively. Little catalytic activity is detected for CES3 with capecitabine. Northern blot analysis indicates that relative expression in intestinal tissue is CES2 > CES1A1 > CES3. Hence, intestinal activation of capecitabine may contribute to its efficacy in colon cancer and toxic diarrhea associated with the agent. Loperamide is a strong inhibitor of CES2, with a K(i) of 1.5 muM, but it only weakly inhibits CES1A1 (IC(50) = 0.44 mM). Inhibition of CES2 in the gastrointestinal tract by loperamide may reduce local formation of 5'-DFCR. Both CES1A1 and CES2 are responsible for the activation of capecitabine, whereas CES3 plays little role in 5'-DFCR formation.

  • hydrolysis of irinotecan and its oxidative metabolites 7 ethyl 10 4 n 5 aminopentanoic acid 1 piperidino carbonyloxycamptothecin and 7 ethyl 10 4 1 piperidino 1 amino carbonyloxycamptothecin by human Carboxylesterases ces1a1 ces2 and a newly expresse
    Drug Metabolism and Disposition, 2004
    Co-Authors: Sonal P. Sanghani, Wilhelmina I. Davis, Sara K. Quinney, Tyler B. Fredenburg, Daryl J. Murry, William F. Bosron
    Abstract:

    Carboxylesterases metabolize ester, thioester, carbamate, and amide compounds to more soluble acid, alcohol, and amine products. They belong to a multigene family with about 50% sequence identity between classes. CES1A1 and CES2 are the most studied human isoenzymes from class 1 and 2, respectively. In this study, we report the cloning and expression of a new human isoenzyme, CES3, that belongs to class 3. The purified recombinant CES3 protein has Carboxylesterase activity. Carboxylesterases metabolize the carbamate prodrug 7-ethyl-10-[4-(1-piperidino)-1-piperidino] carbonyloxycamptothecin (CPT-11; irinotecan) to its active metabolite 7-ethyl-10-hydroxycamptothecin (SN-38), a potent topoisomerase I inhibitor. CYP3A4 oxidizes CPT-11 to two major oxidative metabolites, 7-ethyl-10-[4-N-(5-aminopentanoic acid)-1-piperidino] carbonyloxycamptothecin (APC) and 7-ethyl-10-[4-(1-piperidino)-1-amino]-carbonyloxycamptothecin (NPC). In this study, we investigate whether these oxidative metabolites, NPC and APC, can be metabolized to SN-38 by purified human Carboxylesterases, CES1A1, CES2, and CES3. We find that CPT-11, APC, and NPC can all be metabolized by Carboxylesterases to SN-38. CES2 has the highest catalytic activity of 0.012 min-1 μM-1 among the three Carboxylesterases studied for hydrolysis of CPT-11. NPC was an equally good substrate of CES2 in comparison to CPT-11, with a catalytic efficiency of 0.005 min-1 μM-1. APC was a very poor substrate for all three isoenzymes, exhibiting a catalytic activity of 0.015 × 10-3 min-1 μM-1 for CES2. Catalytic efficiency of CES3 for CPT-11 hydrolysis was 20- to 2000-fold less than that of CES1A1 and CES2. The relative activity of the three isoenzymes was CES2 > CES1A1 >> CES3, for all three substrates.

  • Carboxylesterases expressed in human colon tumor tissue and their role in CPT-11 hydrolysis.
    Clinical cancer research : an official journal of the American Association for Cancer Research, 2003
    Co-Authors: Sonal P. Sanghani, Wilhelmina I. Davis, Sara K. Quinney, Tyler B. Fredenburg, Zejin Sun, Daryl J. Murry, Oscar W. Cummings, David E. Seitz, William F. Bosron
    Abstract:

    Purpose: The purpose is to develop new analytical methods to study the expression profile of CPT-11 Carboxylesterases and topoisomerase I in colon tumor samples and understand the impact of their expression on CPT-11 metabolism in chemotherapy. Experimental Design: We investigated 24 colon tumors for expression of Carboxylesterases CES1A1 , CES2 , CES3 , hBr-3 , and topoisomerase I genes by real-time PCR and correlated the gene expression with activity assays. The relative abundance of the Carboxylesterase isoenzymes and topoisomerase I genes was determined by real-time PCR. Activity assays performed on colon tumor extracts included CPT-11 hydrolase, 4-methylumbelliferyl acetate hydrolase, and topoisomerase I activity assays. Additionally, nondenaturing activity gel electrophoresis with activity staining showed the distribution of Carboxylesterases. Results: We detect the expression of CES1A1 , CES2 , and CES3 Carboxylesterase genes in human colon tumors. We were unable to detect the hBr-3 (also called hCE-3 ) in human liver, colon, or brain. We find large interindividual variation, ≥150-fold, for both CES1A1 and CES3 genes, 23-fold for CES2 , and 66-fold for topoisomerase I. Only CES2 gene expression correlated with the Carboxylesterase activity assays ( P P Conclusions: We conclude that CES2 is the most abundant Carboxylesterase in colon tumors that is responsible for CPT-11 hydrolysis. This pilot study reinforces the hypothesis that there is a large interindividual variation in expression of Carboxylesterases that may contribute to variation in therapeutic outcome and/or toxicity of CPT-11 therapy for colon cancer.

  • Identification of microsomal rat liver Carboxylesterases and their activity with retinyl palmitate
    European journal of biochemistry, 2002
    Co-Authors: Sonal P. Sanghani, Wilhelmina I. Davis, Natividad Dumaual, Alan M. Mahrenholz, William F. Bosron
    Abstract:

    Retinyl esters are a major endogenous storage source of vitamin A in vertebrates and their hydrolysis to retinol is a key step in the regulation of the supply of retinoids to all tissues. Some members of nonspecific Carboxylesterase family (EC 3.1.1.1) have been shown to hydrolyze retinyl esters. However, the number of different isoenzymes that are expressed in the liver and their retinyl palmitate hydrolase activity is not known. Six different Carboxylesterases were identified and purified from rat liver microsomal extracts. Each isoenzyme was identified by mass spectrometry of its tryptic peptides. In addition to previously characterized rat liver Carboxylesterases ES10, ES4, ES3, the protein products for two cloned genes, AB010635 and D50580 (GenBank accession numbers), were also identified. The sixth isoenzyme was a novel Carboxylesterase and its complete cDNA was cloned and sequenced (AY034877). Three isoenzymes, ES10, ES4 and ES3, account for more than 95% of rat liver microsomal Carboxylesterase activity. They obey Michaelis-Menten kinetics for hydrolysis of retinyl palmitate with Km values of about 1 micro m and specific activities between 3 and 8 nmol.min-1.mg-1 protein. D50580 and AY034877 also hydrolyzed retinyl palmitate. Gene-specific oligonucleotide probing of multiple-tissue Northern blot indicates differential expression in various tissues. Multiple genes are highly expressed in liver and small intestine, important tissues for retinoid metabolism. The level of expression of any one of the six different Carboxylesterase isoenzymes will regulate the metabolism of retinyl palmitate in specific rat cells and tissues.

  • purification and cloning of a broad substrate specificity human liver Carboxylesterase that catalyzes the hydrolysis of cocaine and heroin
    Journal of Biological Chemistry, 1997
    Co-Authors: Evgenia V Pindel, Natalia Y Kedishvili, Trent L Abraham, Monica R Brzezinski, Jing Zhang, Robert A Dean, William F. Bosron
    Abstract:

    A human liver Carboxylesterase (hCE-2) that catalyzes the hydrolysis of the benzoyl group of cocaine and the acetyl groups of 4-methylumbelliferyl acetate, heroin, and 6-monoacetylmorphine was purified from human liver. The purified enzyme exhibited a single band on SDS-polyacrylamide gel electrophoresis with a subunit mass of approximately 60 kDa. The native enzyme was monomeric. The isoelectric point of hCE-2 was approximately 4.9. Treatment with endoglycosidase H caused an increase in electrophoretic mobility indicating that the liver Carboxylesterase was a glycoprotein of the high mannose type. The complete cDNA nucleotide sequence was determined. The authenticity of the cDNA was confirmed by a perfect sequence match of 78 amino acids derived from the hCE-2 purified from human liver. The mature 533-amino acid enzyme encoded by this cDNA shared highest sequence identity with the rabbit liver Carboxylesterase form 2 (73%) and the hamster liver Carboxylesterase AT51p (67%). Carboxylesterases with high sequence identity to hCE-2 have not been reported in mouse and rat liver. hCE-2 exhibited different drug ester substrate specificity from the human liver Carboxylesterase called hCE-1, which hydrolyzes the methyl ester of cocaine. hCE-2 had higher catalytic efficiencies for hydrolysis of 4-methylumbelliferyl acetate, heroin, and 6-monoacetylmorphine and greater inhibition by eserine than hCE-1. hCE-2 may play an important role in the degradation of cocaine and heroin in human tissues.

Shai Morin - One of the best experts on this subject based on the ideXlab platform.

  • organophosphates resistance in the b biotype of bemisia tabaci hemiptera aleyrodidae is associated with a point mutation in an ace1 type acetylcholinesterase and overexpression of Carboxylesterase
    Insect Biochemistry and Molecular Biology, 2008
    Co-Authors: Michal Alon, Fishel Alon, Ralf Nauen, Shai Morin
    Abstract:

    Organophosphate (OP) insecticides are inhibitors of the enzyme acetylcholinesterase (AChE), which terminates nerve impulses by catalyzing the hydrolysis of the neurotransmitter acetylcholine. Previous biochemical studies in Bemisia tabaci (Hemiptera: Aleyrodidae) proposed the existence of two molecular mechanisms for OPs' resistance: Carboxylesterase- (COE) mediated hydrolysis or sequestration and decreased sensitivity of AChE. Here, two acetylcholinesterase genes, ace1 and ace2, have been fully cloned and sequenced from an OP-resistant strain and an OP-susceptible strain of B. tabaci. Comparison of nucleic acid and deduced amino acid sequences revealed only silent nucleotide polymorphisms in ace2, and one mutation, Phe392Trp (Phe331 in Torpedo californica), in ace1 of the resistant strain. The Phe392Trp mutation is located in the acyl pocket of the active site gorge and was recently shown to confer OP insensitivity in Culex tritaeniorhynchus. In addition, we also report on the isolation of two Carboxylesterase genes (coe1 and coe2) from B. tabaci, the first Carboxylesterases to be reported from this species. We show that one of the genes, coe1, is overexpressed (∼4-fold) in the OP-resistant strain, and determine, by quantitative PCR, that the elevated expression is not related to gene amplification but probably to modified transcriptional control. Lastly, we bring new biochemical evidence that support the involvement of both AChE insensitivity and COE metabolism in resistance to OP insecticides in the resistant strain.

  • organophosphates resistance in the b biotype of bemisia tabaci hemiptera aleyrodidae is associated with a point mutation in an ace1 type acetylcholinesterase and overexpression of Carboxylesterase
    Insect Biochemistry and Molecular Biology, 2008
    Co-Authors: Michal Alon, Fishel Alon, Ralf Nauen, Shai Morin
    Abstract:

    Organophosphate (OP) insecticides are inhibitors of the enzyme acetylcholinesterase (AChE), which terminates nerve impulses by catalyzing the hydrolysis of the neurotransmitter acetylcholine. Previous biochemical studies in Bemisia tabaci (Hemiptera: Aleyrodidae) proposed the existence of two molecular mechanisms for OPs' resistance: Carboxylesterase- (COE) mediated hydrolysis or sequestration and decreased sensitivity of AChE. Here, two acetylcholinesterase genes, ace1 and ace2, have been fully cloned and sequenced from an OP-resistant strain and an OP-susceptible strain of B. tabaci. Comparison of nucleic acid and deduced amino acid sequences revealed only silent nucleotide polymorphisms in ace2, and one mutation, Phe392Trp (Phe331 in Torpedo californica), in ace1 of the resistant strain. The Phe392Trp mutation is located in the acyl pocket of the active site gorge and was recently shown to confer OP insensitivity in Culex tritaeniorhynchus. In addition, we also report on the isolation of two Carboxylesterase genes (coe1 and coe2) from B. tabaci, the first Carboxylesterases to be reported from this species. We show that one of the genes, coe1, is overexpressed ( approximately 4-fold) in the OP-resistant strain, and determine, by quantitative PCR, that the elevated expression is not related to gene amplification but probably to modified transcriptional control. Lastly, we bring new biochemical evidence that support the involvement of both AChE insensitivity and COE metabolism in resistance to OP insecticides in the resistant strain.

Philip M Potter - One of the best experts on this subject based on the ideXlab platform.

  • Carboxylesterase inhibitors.
    Expert opinion on therapeutic patents, 2011
    Co-Authors: M Jason Hatfield, Philip M Potter
    Abstract:

    Carboxylesterases play major roles in the hydrolysis of numerous therapeutically active compounds. This is, in part, due to the prevalence of the ester moiety in these small molecules. However, the impact these enzymes may play on drug stability and pharmacokinetics is rarely considered prior to molecule development. Therefore, the application of selective inhibitors of this class of proteins may have utility in modulating the metabolism, distribution and toxicity of agents that are subjected to enzyme hydrolysis. This review details the development of all such compounds dating back to 1986, but principally focuses on the very recent identification of selective human Carboxylesterases inhibitors. The implementation of Carboxylesterase inhibitors may significantly revolutionize drug discovery. Such molecules may allow for improved efficacy of compounds inactivated by this class of enzymes and/or reduce the toxicity of agents that are activated by these proteins. Furthermore, since lack of Carboxylesterase activity appears to have no obvious biological consequence, these compounds could be applied in combination with virtually any esterified drug. Therefore, inhibitors of these proteins may have utility in altering drug hydrolysis and distribution in vivo. The characteristics, chemical and biological properties and potential uses of such agents are discussed here.

  • comparison of benzil and trifluoromethyl ketone tfk mediated Carboxylesterase inhibition using classical and 3d quantitative structure activity relationship analysis
    Bioorganic & Medicinal Chemistry, 2009
    Co-Authors: Toshiyuki Harada, Philip M Potter, Yoshiaki Nakagawa, Randy M Wadkins, Craig E Wheelock
    Abstract:

    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.

  • intracellular inhibition of Carboxylesterases by benzil modulation of cpt 11 cytotoxicity
    Molecular Cancer Therapeutics, 2006
    Co-Authors: Janice L Hyatt, Lyudmila Tsurkan, Monika Wierdl, Carol C Edwards, Mary K Danks, Philip M Potter
    Abstract:

    Carboxylesterases are ubiquitous proteins responsible for the detoxification of xenobiotics. However, these enzymes also activate prodrugs, such as the anticancer agents capecitabine and CPT-11. As a consequence, overexpression of Carboxylesterases within tumor cells sensitizes these cells to CPT-11. We have recently identified two classes of Carboxylesterase inhibitors based on either a benzil (diphenylethane-1,2-dione) or a benzene sulfonamide scaffold and showed that these compounds inhibit Carboxylesterases with K is in the low nanomolar range. Because both classes of inhibitors show reversible enzyme inhibition, conventional in vitro biochemical assays would not accurately reflect the in situ levels of Carboxylesterase activity or inhibition. Therefore, we have developed a novel assay for the determination of intracellular Carboxylesterase activity using 4-methylumbelliferone as a substrate. These studies show that benzil and a dimethylbenzil analogue efficiently enter cells and inhibit human intestinal Carboxylesterase and rabbit liver Carboxylesterase intracellularly. This inhibition results in reduced cytotoxicity to CPT-11 due to the lack of Carboxylesterase-mediated conversion of the prodrug to SN-38. These results suggest that intracellular modulation of Carboxylesterase activity with benzil or its analogues may be applied to minimize the toxicity of normal cells to CPT-11. [Mol Cancer Ther 2006;5(9):2281–8]

  • Characterization of inhibitors of specific Carboxylesterases: Development of Carboxylesterase inhibitors for translational application
    Molecular cancer therapeutics, 2004
    Co-Authors: Kyoung Jin P. Yoon, Janice L Hyatt, Philip M Potter, Christopher L. Morton, Richard E. Lee, Mary K Danks
    Abstract:

    Carboxylesterases, expressed at high levels in human liver and intestine, are thought to detoxify xenobiotics. The anticancer prodrug 7-ethyl-10-[4-1-piperidino)-1-piperidino]carbonyloxycamptothecin (CPT-11) is also metabolized by Carboxylesterases to produce the active drug 7-ethyl-10-hydroxycamptothecin. Activation of CPT-11 by human intestinal Carboxylesterase (hiCE) in the human intestine may contribute to delayed onset diarrhea, a dose-limiting side effect of this drug. The goal of this study was to develop small molecule inhibitors selective for hiCE to circumvent or treat the toxic side effects of CPT-11. A secondary goal was to develop molecules that specifically inhibit activation of CPT-11 by a rabbit liver Carboxylesterase (rCE). rCE is the most efficient CPT-11–activating enzyme thus far identified, and this enzyme is being developed for viral-directed enzyme prodrug therapy applications. Based on in vitro assays with partially purified hiCE and rCE proteins and on growth inhibition assays using U373MG human glioma cells transfected to express hiCE or rCE (U373pIREShiCE or U373pIRESrCE), we identified specific inhibitors of each enzyme. Lead compounds are derivatives of nitrophenol having 4-(furan-2-carbonyl)-piperazine-1-carboxylic acid or 4-[(4-chlorophenyl)-phenylmethyl]-piperazine-1-carboxylic acid substitutions in the p position. Kinetic analysis of each compound for hiCE compared with rCE showed that the K i values of the most selective of these inhibitors differed by 6- to 10-fold. In growth inhibition assays, nontoxic, low micromolar concentrations of these inhibitors increased the EC50 of CPT-11 for U373pIREShiCE or U373pIRESrCE cells by 13- to >1,500-fold. The four compounds characterized in this study will serve as lead compounds for a series of inhibitors to be constructed using a combinatorial approach.

  • comparison of activation of cpt 11 by rabbit and human Carboxylesterases for use in enzyme prodrug therapy
    Clinical Cancer Research, 1999
    Co-Authors: Mary K Danks, Christopher L. Morton, Erik J Krull, Pamela J Cheshire, Lois B Richmond, Clayton W Naeve, Cynthia A Pawlik, Peter J Houghton, Philip M Potter
    Abstract:

    Several recent studies have examined the possibility of producing tumor-specific cytotoxicity with various enzyme/prodrug combinations. The enzymes are targeted to tumor cells either with antibodies (ADEPT, antibody directed enzyme prodrug therapy) or with viruses (VDEPT). The goal of the present study was to identify an appropriate enzyme for use in activating the prodrug 7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin (CPT-11). In this study, we compared the efficiency of CPT-11 metabolism by rabbit and human Carboxylesterases in in vitro and in situ assays. Although the rabbit and human enzymes are very similar (81% identical; 86% homologous) and the active site amino acids are 100% identical, the rabbit enzyme was 100-1000-fold more efficient at converting CPT-11 to SN-38 in vitro and was 12–55-fold more efficient in sensitizing transfected cells to CPT-11. In vivo , Rh30 rhabdomyosarcoma cells expressing the rabbit Carboxylesterase and grown as xenografts in immune-deprived mice were also more sensitive to CPT-11 than were control xenografts or xenografts expressing the human enzyme. Each of the three types of xenografts regressed when the mice were treated with CPT-11 given i.v. at 2.5 mg of CPT-11/kg/daily for 5 days/week for 2 weeks [(dx5)2] (one cycle of therapy), repeated every 21 days for a total of three cycles. However, following cessation of treatment, recurrent tumors were detected in seven of seven mice bearing control Rh30 xenografts and in two of seven mice bearing Rh30 xenografts that expressed the human enzyme. No tumors recurred in mice bearing xenografts that expressed the rabbit Carboxylesterase. We conclude that rabbit Carboxylesterase/CPT-11 may be a useful enzyme/prodrug combination.

Nannan Liu - One of the best experts on this subject based on the ideXlab platform.

  • Functional Analyses of House Fly Carboxylesterases Involved in Insecticide Resistance
    Frontiers in physiology, 2020
    Co-Authors: Xuechun Feng, Nannan Liu
    Abstract:

    Carboxylesterase-mediated metabolism is one of major mechanisms involved in insecticide resistance. Our previous study has identified multiple Carboxylesterase genes with their expression levels were significantly upregulated in pyrethroid resistant house flies. To further explore their metabolic functions, we used insect Spodoptera frugiperda (Sf9) cells to express these Carboxylesterases in vitro and measure their hydrolytic activities toward esterase substrates. Our results indicated that these Carboxylesterases can efficiently hydrolyze α-naphthyl acetate rather than β- naphthyl acetate. A cell based MTT cytotoxicity assay indicated that Carboxylesterase-expressing cells show enhanced tolerance to permethrin, suggesting important roles of these Carboxylesterases in metabolizing permethrin and thereby protecting cells from permethrin treatments. Later on, the metabolic functions of Carboxylesterases were further verified by conducting in vitro metabolism studies toward permethrin and its potential metabolites 3-phenoxybenzyl alcohol and 3-phenoxybenzaldehyde, which not only suggested the potential metabolic pathway of permethrin in insects, but also important roles of these candidate Carboxylesterases in metabolizing permethrin and conferring resistance in house flies. Homology modeling and docking were finally conducted to reflect interactions between permethrin ligand and Carboxylesterase proteins, visually confirming the metabolic functions of Carboxylesterases to insecticides in house flies.

  • Functional Characterization of Carboxylesterases in Insecticide Resistant House Flies, Musca Domestica.
    Journal of visualized experiments : JoVE, 2018
    Co-Authors: Xuechun Feng, Nannan Liu
    Abstract:

    Carboxylesterase-mediated metabolism is thought to play a major role in insecticide resistance in various insects. Several Carboxylesterase genes were found up-regulated in the resistant house fly strain, whereas their roles in conferring insecticide resistance remained to be explored. Here, we designed a protocol for the functional characterization of Carboxylesterases. Three example experiments are presented: (1) expression and isolation of Carboxylesterase proteins through a baculovirus-mediated insect Spodoptera frugiperda (Sf9) cell expression system; (2) a cell-based MTT (3-[4, 5-dimethykthiazol-2-yl]-2, 5-diphenyltetrazolium bromide) cytotoxicity assay to measure the tolerance of insect cells to different permethrin treatments; and (3) in vitro metabolic studies to explore the metabolic capabilities of Carboxylesterases toward permethrin. The Carboxylesterase gene MdαE7 was cloned from a resistant house fly strain ALHF and used to construct a recombinant baculovirus for Sf9 cells infection. The cell viabilities against different permethrin treatments were measured with the MTT assay. The enhanced cell tolerance of the experimental group (MdαE7-recombinant baculovirus infected cells) compared with those of the control groups (CAT-recombinant baculovirus infected cells and GFP-recombinant baculovirus infected cells) to permethrin treatments suggested the capabilities of MdαE7 in metabolizing insecticides, thereby protecting cells from chemical damages. Besides that, Carboxylesterase proteins were expressed in insect Sf9 cells and isolated to conduct an in vitro metabolic study. Our results indicated a significant in vitro metabolic efficiency of MdαE7 toward permethrin, directly indicating the involvement of Carboxylesterases in metabolizing insecticides and thus conferring insecticide resistance in house flies.

  • Carboxylesterase genes in pyrethroid resistant house flies, Musca domestica.
    Insect biochemistry and molecular biology, 2017
    Co-Authors: Xuechun Feng, Nannan Liu
    Abstract:

    Carboxylesterases are one of the major enzyme families involved in the detoxification of pyrethroids. Up-regulation of Carboxylesterase genes is thought to be a major component of insecticide resistant mechanisms in insects. Based on the house fly transcriptome and genome database, a total of 39 Carboxylesterase genes of different functional clades have been identified in house flies. In this study, eleven of these genes were found to be significantly overexpressed in the resistant ALHF house fly strain compared with susceptible aabys and wild-type CS strains. Eight up-regulated Carboxylesterase genes with their expression levels were further induced to a higher level in response to permethrin treatments, indicating that constitutive and inductive overexpression of Carboxylesterases are co-responsible for the enhanced detoxification of insecticides. Spatial expression studies revealed these up-regulated genes to be abundantly distributed in fat bodies and genetically mapped on autosome 2 or 3 of house flies, and their expression could be regulated by factors on autosome 1, 2 and 5. Taken together, these results demonstrate that multiple Carboxylesterase genes are co-upregulated in resistant house flies, providing further evidence for their involvement in the detoxification of insecticides and development of insecticide resistance.