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Matthew R Redinbo - One of the best experts on this subject based on the ideXlab platform.
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human Carboxylesterase 1 stereoselectively binds the nerve agent cyclosarin and spontaneously hydrolyzes the nerve agent sarin
Molecular Pharmacology, 2010Co-Authors: Andrew C. Hemmert, Philip M. Potter, Tamara C. Otto, Monika Wierdl, Carol C. Edwards, Christopher D Fleming, Mary Macdonald, Douglas M Cerasoli, Matthew R RedinboAbstract:Organophosphorus (OP) nerve agents are potent toxins that inhibit cholinesterases and produce a rapid and lethal cholinergic crisis. Development of protein-based therapeutics is being pursued with the goal of preventing nerve agent toxicity and protecting against the long-term side effects of these agents. The drug-metabolizing enzyme human Carboxylesterase 1 (hCE1) is a candidate protein-based therapeutic because of its similarity in structure and function to the cholinesterase targets of nerve agent poisoning. However, the ability of wild-type hCE1 to process the G-type nerve agents sarin and cyclosarin has not been determined. We report the crystal structure of hCE1 in complex with the nerve agent cyclosarin. We further use stereoselective nerve agent analogs to establish that hCE1 exhibits a 1700- and 2900-fold preference for the P R enantiomers of analogs of soman and cyclosarin, respectively, and a 5-fold preference for the P S isomer of a sarin analog. Finally, we show that for enzyme inhibited by racemic mixtures of bona fide nerve agents, hCE1 spontaneously reactivates in the presence of sarin but not soman or cyclosarin. The addition of the neutral oxime 2,3-butanedione monoxime increases the rate of reactivation of hCE1 from sarin inhibition by more than 60-fold but has no effect on reactivation with the other agents examined. Taken together, these data demonstrate that hCE1 is only reactivated after inhibition with the more toxic P S isomer of sarin. These results provide important insights toward the long-term goal of designing novel forms of hCE1 to act as protein-based therapeutics for nerve agent detoxification.
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structural and biochemical analysis reveals the stereoselectivity of inhibition and reactivation of human Carboxylesterase 1 for the nerve agents sarin soman and cyclosarin
2010Co-Authors: Andrew C. Hemmert, Philip M. Potter, Tamara C. Otto, Monika Wierdl, Carol C. Edwards, Mary Macdonald, Douglas M Cerasoli, John R Cashman, Matthew R RedinboAbstract:Departments of Chemistry (M.R.R.) and Biochemistry and Biophysics (A.C.H., C.D.F., M.R.R), University of North Carolina at Chapel Hill, Chapel Hill, North Carolina; U.S. Army Medical Research Institute of Chemical Defense (T.C.O., D.M.C.), Aberdeen Proving Ground, Maryland; Human BioMolecular Research Institute (M.M., J.R.C.), San Diego, California; Department of Molecular Pharmacology (M.W., C.C.E., P.M.P.), St. Jude Children’s Research Hospital, Memphis, Tennessee Molecular Pharmacology Fast Forward. Published on January 5, 2010 as doi:10.1124/mol.109.062356
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Human Carboxylesterase 1 Stereoselectively Binds the Nerve Agent Cyclosarin and Spontaneously Hydrolyzes the Nerve Agent Sarin
2009Co-Authors: Andrew C. Hemmert, Philip M. Potter, Tamara C. Otto, Monika Wierdl, Carol C. Edwards, Christopher D Fleming, Mary Macdonald, Douglas M Cerasoli, Matthew R RedinboAbstract:Organophosphorus (OP) nerve agents are potent toxins that inhibit cholinesterases and produce a rapid and lethal cholin-ergic crisis. Development of protein-based therapeutics is be-ing pursued with the goal of preventing nerve agent toxicity and protecting against the long-term side effects of these agents. The drug-metabolizing enzyme human Carboxylesterase 1 (hCE1) is a candidate protein-based therapeutic because of its similarity in structure and function to the cholinesterase targets of nerve agent poisoning. However, the ability of wild-type hCE1 to process the G-type nerve agents sarin and cyclosarin has not been determined. We report the crystal structure of hCE1 in complex with the nerve agent cyclosarin. We further use ste-reoselective nerve agent analogs to establish that hCE1 exhib-its a 1700- and 2900-fold preference for the PR enantiomers o
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crystal structures of human Carboxylesterase 1 in covalent complexes with the chemical warfare agents soman and tabun
Biochemistry, 2007Co-Authors: Christopher D Fleming, Philip M. Potter, Carol C. Edwards, Douglas M Cerasoli, Stephen D Kirby, Donald M Maxwell, Matthew R RedinboAbstract:The organophosphorus nerve agents sarin, soman, tabun, and VX exert their toxic effects by inhibiting the action of human acetylcholinesterase, a member of the serine hydrolase superfamily of enzymes. The current treatments for nerve agent exposure must be administered quickly to be effective, and they often do not eliminate long-term toxic side effects associated with organophosphate poisoning. Thus, there is significant need for effective prophylactic methods to protect at-risk personnel from nerve agent exposure, and protein-based approaches have emerged as promising candidates. We present the 2.7 A resolution crystal structures of the serine hydrolase human Carboxylesterase 1 (hCE1), a broad-spectrum drug metabolism enzyme, in covalent acyl-enzyme intermediate complexes with the chemical weapons soman and tabun. The structures reveal that hCE1 binds stereoselectively to these nerve agents; for example, hCE1 appears to react preferentially with the 104-fold more lethal PS stereoisomer of soman relative...
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crystal structures of human Carboxylesterase 1 in covalent complexes with the chemical warfare agents soman and tabun
Biochemistry, 2007Co-Authors: Christopher D Fleming, Philip M. Potter, Carol C. Edwards, Douglas M Cerasoli, Stephen D Kirby, Donald M Maxwell, Matthew R RedinboAbstract:The organophosphorus nerve agents sarin, soman, tabun, and VX exert their toxic effects by inhibiting the action of human acetylcholinesterase, a member of the serine hydrolase superfamily of enzymes. The current treatments for nerve agent exposure must be administered quickly to be effective, and they often do not eliminate long-term toxic side effects associated with organophosphate poisoning. Thus, there is significant need for effective prophylactic methods to protect at-risk personnel from nerve agent exposure, and protein-based approaches have emerged as promising candidates. We present the 2.7 A resolution crystal structures of the serine hydrolase human Carboxylesterase 1 (hCE1), a broad-spectrum drug metabolism enzyme, in covalent acyl-enzyme intermediate complexes with the chemical weapons soman and tabun. The structures reveal that hCE1 binds stereoselectively to these nerve agents; for example, hCE1 appears to react preferentially with the 10(4)-fold more lethal PS stereoisomer of soman relative to the PR form. In addition, structural features of the hCE1 active site indicate that the enzyme may be resistant to dead-end organophosphate aging reactions that permanently inactivate other serine hydrolases. Taken together, these data provide important structural details toward the goal of engineering hCE1 into an organophosphate hydrolase and protein-based therapeutic for nerve agent exposure.
Hao Jie Zhu - One of the best experts on this subject based on the ideXlab platform.
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impact of Carboxylesterase 1 genetic polymorphism on trandolapril activation in human liver and the pharmacokinetics and pharmacodynamics in healthy volunteers
Clinical and Translational Science, 2021Co-Authors: Xinwen Wang, Barry E. Bleske, Jian Shi, Lucy Her, Jingcheng Xiao, Hao Jie ZhuAbstract:Trandolapril, an angiotensin-converting enzyme inhibitor prodrug, needs to be activated by Carboxylesterase 1 (CES1) in the liver to exert its intended therapeutic effect. A previous in vitro study demonstrated that the CES1 genetic variant G143E (rs71647871) abolished CES1-mediated trandolapril activation in cells transfected with the variant. This study aimed to determine the effect of the G143E variant on trandolapril activation in human livers and the pharmacokinetics (PKs) and pharmacodynamics (PDs) in human subjects. We performed an in vitro incubation study to assess trandolapril activation in human livers (5 G143E heterozygotes and 97 noncarriers) and conducted a single-dose (1 mg) PK and PD study of trandolapril in healthy volunteers (8 G143E heterozygotes and 11 noncarriers). The incubation study revealed that the mean trandolapril activation rate in G143E heterozygous livers was 42% of those not carrying the variant (p = 0.0015). The clinical study showed that, relative to noncarriers, G143E carriers exhibited 20% and 15% decreases, respectively, in the peak concentration (Cmax ) and area under the curve from 0 to 72 h (AUC0-72 h ) of the active metabolite trandolaprilat, although the differences were not statistically significant. Additionally, the average maximum reductions of systolic blood pressure and diastolic blood pressure in carriers were ~ 22% and 23% less than in noncarriers, respectively, but the differences did not reach a statistically significant level. In summary, the CES1 G143E variant markedly impaired trandolapril activation in the human liver under the in vitro incubation conditions; however, this variant had only a modest impact on the PK and PD of trandolapril in healthy human subjects.
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fracpred 2d prm a fraction prediction algorithm assisted 2d liquid chromatography based parallel reaction monitoring mass spectrometry approach for measuring low abundance proteins in human plasma
Proteomics, 2020Co-Authors: Jian Shi, Xinwen Wang, Lucy Her, Jingcheng Xiao, Matthew J Sorensen, Hao Jie ZhuAbstract:Multidimensional fractionation-based enrichment methods improve the sensitivity of proteomic analysis for low-abundance proteins. However, a major limitation of conventional multidimensional proteomics is the extensive labor and instrument time required for analyzing many fractions obtained from the first dimension separation. Here, a fraction prediction algorithm-assisted 2D LC-based parallel reaction monitoring-mass spectrometry (FRACPRED-2D-PRM) approach for measuring low-abundance proteins in human plasma is presented. Plasma digests are separated by the first dimension high-pH RP-LC with data-dependent acquisition (DDA). The FRACPRED algorithm is then usedto predict the retention times of undetectable target peptides according to those of other abundant plasma peptides during the first dimension separation. Fractions predicted to contain target peptides are analyzed by the second dimension low-pH nano RP-LC PRM. The accuracy and robustness of fraction prediction with the FRACPRED algorithm are demonstrated by measuring two low-abundance proteins, aldolase B and Carboxylesterase 1, in human plasma. The FRACPRED-2D-PRM proteomics approach demonstrates markedly improved efficiency and sensitivity over conventional 2D-LC proteomics assays. It is expected that this approach will be widely used in the study of low-abundance proteins in plasma and other complex biological samples.
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Carboxylesterase 1 and precision pharmacotherapy pharmacogenetics and nongenetic regulators
Drug Metabolism and Disposition, 2020Co-Authors: Lucy Her, Hao Jie ZhuAbstract:Carboxylesterase (CES) 1 is the most abundant drug-metabolizing enzyme in human livers, comprising approximately 1% of the entire liver proteome. CES1 is responsible for 80%-95% of total hydrolytic activity in the liver and plays a crucial role in the metabolism of a wide range of drugs (especially ester-prodrugs), pesticides, environmental pollutants, and endogenous compounds. Expression and activity of CES1 vary markedly among individuals, which is a major contributing factor to interindividual variability in the pharmacokinetics (PK) and pharmacodynamics (PD) of drugs metabolized by CES1. Both genetic and nongenetic factors contribute to CES1 variability. Here, we discuss genetic polymorphisms, including single-nucleotide polymorphisms (SNPs), and copy number variants and nongenetic contributors, such as developmental status, genders, and drug-drug interactions, that could influence CES1 functionality and the PK and PD of CES1 substrates. Currently, the loss-of-function SNP G143E (rs71647871) is the only clinically significant CES1 variant identified to date, and alcohol is the only potent CES1 inhibitor that could alter the therapeutic outcomes of CES1 substrate medications. However, G143E and alcohol can only explain a small portion of the interindividual variability in the CES1 function. A better understanding of the regulation of CES1 expression and activity and identification of biomarkers for CES1 function in vivo could lead to the development of a precision pharmacotherapy strategy to improve the efficacy and safety of many CES1 substrate drugs. SIGNIFICANCE STATEMENT: The clinical relevance of CES1 has been well demonstrated in various clinical trials. Genetic and nongenetic regulators can affect CES1 expression and activity, resulting in the alteration of the metabolism and clinical outcome of CES1 substrate drugs, such as methylphenidate and clopidogrel. Predicting the hepatic CES1 function can provide clinical guidance to optimize pharmacotherapy of numerous medications metabolized by CES1.
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Age-and Sex-Related Expression and Activity of Carboxylesterase 1 and 2 in Mouse and Human Liver
2020Co-Authors: Hao Jie Zhu, David I Appel, Yan Jiang, John S MarkowitzAbstract:ABSTRACT: Carboxylesterase (CES) 1 and CES2 are two major hepatic hydrolases responsible for the metabolism of numerous endogenous and exogenous compounds. In this study, age-and sex-dependent expression and activity of CES1 and CES2 were investigated using both animal models and individual human liver s9 samples. The expression and activity of mouse CES1 (mCES1) and mCES2 in the liver were markedly lower in newborns relative to adults and increased gradually with age, approximating levels of adult animals by age 2 to 4 weeks. Likewise, the average human CES1 (hCES1) expression in the subjects <1 year of age was significantly lower than that of pooled samples. In particular, hCES1 expression in the 13-day and 1-month-old subjects was just 20.3 and 11.1%, respectively, of the pooled sample values. In addition, the subjects <1 year of age exhibited a trend suggestive of low hCES2 expression, but this difference failed to reach statistical significance because of large interindividual variability. The expression and activity of mCES1 and mCES2 were not significantly altered after the animals were treated with human growth hormone, indicating growth hormone may not be associated with the low level of CES expression during early developmental stages. No significant differences of the expression and activity of mCES1 and mCES2 were observed between sexually mature male and female mice. In conclusion, the expression and activity of CES1 and CES2 are age-related but independent of growth hormone level. Sex seems to be an unlikely factor contributing to the regulation of CES1 and CES2. Carboxylesterase (CES) 1 and CES2 are two major hydrolytic enzymes responsible for the metabolism of numerous carboxylic acid esters, carbamates, thioesters, and amide agents. A variety of widely prescribed therapeutic drugs such as methylphenidate (MPH), oseltamivir, and irinotecan are metabolized by CES1 and/or CES2, resulting in the formation of hydrolytic metabolite
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functional study of Carboxylesterase 1 protein isoforms
Proteomics, 2019Co-Authors: Xinwen Wang, Jian Shi, Hao Jie ZhuAbstract:Carboxylesterase 1 (CES1) is a primary human hepatic hydrolase involved in hydrolytic biotransformation of numerous medications. Considerable interindividual variability in CES1 expression and activity has been consistently reported. Four isoforms of the CES1 protein are produced by alternative splicing (AS). In the current study, the activity and expression of each CES1 isoform are examined using transfected cell lines, and CES1 isoform composition and its impact on CES1 activity in human livers are determined. In transfected cells, isoforms 3 and 4 show mRNA and protein expressions comparable to isoforms 1 and 2, but have significantly impaired activity when hydrolyzing enalapril and clopidogrel. In individual human liver samples, isoforms 1 and 2 are the major forms, contributing 73-90% of the total CES1 protein expression. In addition, the protein expression ratios of isoforms 1 and 2 to isoforms 3 and 4 are positively associated with CES1 activity in the liver, suggesting that CES1 isoform composition is a factor contributing to the variability in hepatic CES1 function. Further investigations of the regulation of CES1 AS would improve the understanding of CES1 variability and help develop a strategy to optimize the pharmacotherapy of many CES1 substrate medications.
Philip M. Potter - One of the best experts on this subject based on the ideXlab platform.
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control of rhoa methylation by Carboxylesterase i
Journal of Biological Chemistry, 2013Co-Authors: Ian Cushman, Philip M. Potter, Stephanie M Cushman, Patrick J CaseyAbstract:A number of proteins that play key roles in cell signaling are post-translationally modified by the prenylation pathway. The final step in this pathway is methylation of the carboxyl terminus of the prenylated protein by isoprenylcysteine carboxylmethyltransferase. Due to the impact of methylation on Rho function, we sought to determine if the process was reversible and hence could control Rho function in a dynamic fashion. Elevating isoprenylcysteine carboxylmethyltransferase activity in cells has profound effects on MDA-MB-231 cell morphology, implying the presence of a pool of unmethylated prenyl proteins in these cells under normal conditions. Using a knockdown approach, we identified a specific esterase, Carboxylesterase 1, whose function had a clear impact not only on the methylation status of RhoA but also RhoA activation and cell morphology. These data provide compelling evidence that C-terminal modification of prenyl proteins, rather than being purely a constitutive process, can serve as a point of regulation of function for this important class of protein.
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Covalent inhibition of recombinant human Carboxylesterase 1 and 2 and monoacylglycerol lipase by the carbamates JZL184 and URB597
Biochemical Pharmacology, 2012Co-Authors: J. Allen Crow, Victoria Bittles, Abdolsamad Borazjani, Philip M. Potter, Matthew K. RossAbstract:Abstract Carboxylesterase type 1 (CES1) and CES2 are serine hydrolases located in the liver and small intestine. CES1 and CES2 actively participate in the metabolism of several pharmaceuticals. Recently, carbamate compounds were developed to inhibit members of the serine hydrolase family via covalent modification of the active site serine. URB597 and JZL184 inhibit fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), respectively; however, Carboxylesterases in liver have been identified as a major off-target. We report the kinetic rate constants for inhibition of human recombinant CES1 and CES2 by URB597 and JZL184. Bimolecular rate constants ( k inact / K i ) for inhibition of CES1 by JZL184 and URB597 were similar [3.9 (±0.2) × 10 3 M −1 s −1 and 4.5 (±1.3) × 10 3 M −1 s −1 , respectively]. However, k inact / K i for inhibition of CES2 by JZL184 and URB597 were significantly different [2.3 (±1.3) × 10 2 M −1 s −1 and 3.9 (±1.0) × 10 3 M −1 s −1 , respectively]. Rates of inhibition of CES1 and CES2 by URB597 were similar; however, CES1 and MAGL were more potently inhibited by JZL184 than CES2. We also determined kinetic constants for spontaneous reactivation of CES1 carbamoylated by either JZL184 or URB597 and CES1 diethylphosphorylated by paraoxon. The reactivation rate was significantly slower (4.5×) for CES1 inhibited by JZL184 than CES1 inhibited by URB597. Half-life of reactivation for CES1 carbamoylated by JZL184 was 49 ± 15 h, which is faster than Carboxylesterase turnover in HepG2 cells. Together, the results define the kinetics of inhibition for a class of drugs that target hydrolytic enzymes involved in drug and lipid metabolism.
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Inhibition of recombinant human Carboxylesterase 1 and 2 and monoacylglycerol lipase by chlorpyrifos oxon, paraoxon and methyl paraoxon.
Toxicology and applied pharmacology, 2011Co-Authors: J. Allen Crow, Victoria Bittles, Abdolsamad Borazjani, Philip M. Potter, Katye L. Herring, Matthew K. RossAbstract:Oxons are the bioactivated metabolites of organophosphorus insecticides formed via cytochrome P450 monooxygenase-catalyzed desulfuration of the parent compound. Oxons react covalently with the active site serine residue of serine hydrolases, thereby inactivating the enzyme. A number of serine hydrolases other than acetylcholinesterase, the canonical target of oxons, have been reported to react with and be inhibited by oxons. These off-target serine hydrolases include Carboxylesterase 1 (CES1), CES2, and monoacylglycerol lipase. Carboxylesterases (CES, EC 3.1.1.1) metabolize a number of xenobiotic and endobiotic compounds containing ester, amide, and thioester bonds and are important in the metabolism of many pharmaceuticals. Monoglyceride lipase (MGL, EC 3.1.1.23) hydrolyzes monoglycerides including the endocannabinoid, 2-arachidonoylglycerol (2-AG). The physiological consequences and toxicity related to the inhibition of off-target serine hydrolases by oxons due to chronic, low level environmental exposures are poorly understood. Here, we determined the potency of inhibition (IC(50) values; 15 min preincubation, enzyme and inhibitor) of recombinant CES1, CES2, and MGL by chlorpyrifos oxon, paraoxon and methyl paraoxon. The order of potency for these three oxons with CES1, CES2, and MGL was chlorpyrifos oxon>paraoxon>methyl paraoxon, although the difference in potency for chlorpyrifos oxon with CES1 and CES2 did not reach statistical significance. We also determined the bimolecular rate constants (k(inact)/K(I)) for the covalent reaction of chlorpyrifos oxon, paraoxon and methyl paraoxon with CES1 and CES2. Consistent with the results for the IC(50) values, the order of reactivity for each of the three oxons with CES1 and CES2 was chlorpyrifos oxon>paraoxon>methyl paraoxon. The bimolecular rate constant for the reaction of chlorpyrifos oxon with MGL was also determined and was less than the values determined for chlorpyrifos oxon with CES1 and CES2 respectively. Together, the results define the kinetics of inhibition of three important hydrolytic enzymes by activated metabolites of widely used agrochemicals.
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human Carboxylesterase 1 stereoselectively binds the nerve agent cyclosarin and spontaneously hydrolyzes the nerve agent sarin
Molecular Pharmacology, 2010Co-Authors: Andrew C. Hemmert, Philip M. Potter, Tamara C. Otto, Monika Wierdl, Carol C. Edwards, Christopher D Fleming, Mary Macdonald, Douglas M Cerasoli, Matthew R RedinboAbstract:Organophosphorus (OP) nerve agents are potent toxins that inhibit cholinesterases and produce a rapid and lethal cholinergic crisis. Development of protein-based therapeutics is being pursued with the goal of preventing nerve agent toxicity and protecting against the long-term side effects of these agents. The drug-metabolizing enzyme human Carboxylesterase 1 (hCE1) is a candidate protein-based therapeutic because of its similarity in structure and function to the cholinesterase targets of nerve agent poisoning. However, the ability of wild-type hCE1 to process the G-type nerve agents sarin and cyclosarin has not been determined. We report the crystal structure of hCE1 in complex with the nerve agent cyclosarin. We further use stereoselective nerve agent analogs to establish that hCE1 exhibits a 1700- and 2900-fold preference for the P R enantiomers of analogs of soman and cyclosarin, respectively, and a 5-fold preference for the P S isomer of a sarin analog. Finally, we show that for enzyme inhibited by racemic mixtures of bona fide nerve agents, hCE1 spontaneously reactivates in the presence of sarin but not soman or cyclosarin. The addition of the neutral oxime 2,3-butanedione monoxime increases the rate of reactivation of hCE1 from sarin inhibition by more than 60-fold but has no effect on reactivation with the other agents examined. Taken together, these data demonstrate that hCE1 is only reactivated after inhibition with the more toxic P S isomer of sarin. These results provide important insights toward the long-term goal of designing novel forms of hCE1 to act as protein-based therapeutics for nerve agent detoxification.
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structural and biochemical analysis reveals the stereoselectivity of inhibition and reactivation of human Carboxylesterase 1 for the nerve agents sarin soman and cyclosarin
2010Co-Authors: Andrew C. Hemmert, Philip M. Potter, Tamara C. Otto, Monika Wierdl, Carol C. Edwards, Mary Macdonald, Douglas M Cerasoli, John R Cashman, Matthew R RedinboAbstract:Departments of Chemistry (M.R.R.) and Biochemistry and Biophysics (A.C.H., C.D.F., M.R.R), University of North Carolina at Chapel Hill, Chapel Hill, North Carolina; U.S. Army Medical Research Institute of Chemical Defense (T.C.O., D.M.C.), Aberdeen Proving Ground, Maryland; Human BioMolecular Research Institute (M.M., J.R.C.), San Diego, California; Department of Molecular Pharmacology (M.W., C.C.E., P.M.P.), St. Jude Children’s Research Hospital, Memphis, Tennessee Molecular Pharmacology Fast Forward. Published on January 5, 2010 as doi:10.1124/mol.109.062356
Carol C. Edwards - One of the best experts on this subject based on the ideXlab platform.
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Human Carboxylesterase 1 active site structure.
2013Co-Authors: Andrew C. Hemmert, Tamara C. Otto, Roberto A. Chica, Monika Wierdl, Jonathan S. Edwards, Steven L. Lewis, Carol C. Edwards, Lyudmila Tsurkan, Linn C. Cadieux, Shane A. KastenAbstract:Active site of human Carboxylesterase 1 covalently inhibited via S221 with cyclosarin (magenta) [8]. The other catalytic residues, in addition to S221, are H468 and E354 (yellow), and are surrounded by hydrophobic residues (grey surface) including V146 and L363 (light blue), as well as the oxyanion hole (white).
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Nerve agent hydrolysis activity designed into a human drug metabolism enzyme.
PloS one, 2011Co-Authors: Andrew C. Hemmert, Tamara C. Otto, Roberto A. Chica, Monika Wierdl, Steven L. Lewis, Carol C. Edwards, Lyudmila Tsurkan, Jonathan Edwards, C. Linn Cadieux, Shane A. KastenAbstract:Organophosphorus (OP) nerve agents are potent suicide inhibitors of the essential neurotransmitter-regulating enzyme acetylcholinesterase. Due to their acute toxicity, there is significant interest in developing effective countermeasures to OP poisoning. Here we impart nerve agent hydrolysis activity into the human drug metabolism enzyme Carboxylesterase 1. Using crystal structures of the target enzyme in complex with nerve agent as a guide, a pair of histidine and glutamic acid residues were designed proximal to the enzyme's native catalytic triad. The resultant variant protein demonstrated significantly increased rates of reactivation following exposure to sarin, soman, and cyclosarin. Importantly, the addition of these residues did not alter the high affinity binding of nerve agents to this protein. Thus, using two amino acid substitutions, a novel enzyme was created that efficiently converted a group of hemisubstrates, compounds that can start but not complete a reaction cycle, into bona fide substrates. Such approaches may lead to novel countermeasures for nerve agent poisoning.
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human Carboxylesterase 1 stereoselectively binds the nerve agent cyclosarin and spontaneously hydrolyzes the nerve agent sarin
Molecular Pharmacology, 2010Co-Authors: Andrew C. Hemmert, Philip M. Potter, Tamara C. Otto, Monika Wierdl, Carol C. Edwards, Christopher D Fleming, Mary Macdonald, Douglas M Cerasoli, Matthew R RedinboAbstract:Organophosphorus (OP) nerve agents are potent toxins that inhibit cholinesterases and produce a rapid and lethal cholinergic crisis. Development of protein-based therapeutics is being pursued with the goal of preventing nerve agent toxicity and protecting against the long-term side effects of these agents. The drug-metabolizing enzyme human Carboxylesterase 1 (hCE1) is a candidate protein-based therapeutic because of its similarity in structure and function to the cholinesterase targets of nerve agent poisoning. However, the ability of wild-type hCE1 to process the G-type nerve agents sarin and cyclosarin has not been determined. We report the crystal structure of hCE1 in complex with the nerve agent cyclosarin. We further use stereoselective nerve agent analogs to establish that hCE1 exhibits a 1700- and 2900-fold preference for the P R enantiomers of analogs of soman and cyclosarin, respectively, and a 5-fold preference for the P S isomer of a sarin analog. Finally, we show that for enzyme inhibited by racemic mixtures of bona fide nerve agents, hCE1 spontaneously reactivates in the presence of sarin but not soman or cyclosarin. The addition of the neutral oxime 2,3-butanedione monoxime increases the rate of reactivation of hCE1 from sarin inhibition by more than 60-fold but has no effect on reactivation with the other agents examined. Taken together, these data demonstrate that hCE1 is only reactivated after inhibition with the more toxic P S isomer of sarin. These results provide important insights toward the long-term goal of designing novel forms of hCE1 to act as protein-based therapeutics for nerve agent detoxification.
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Nerve Agent Hydrolysis Activity Designed into a Human Drug Metabolism Enzyme
2010Co-Authors: Andrew C. Hemmert, Tamara C. Otto, Roberto A. Chica, Monika Wierdl, Jonathan S. Edwards, Steven L. Lewis, Carol C. Edwards, Lyudmila Tsurkan, Linn C. Cadieux, Shane A. KastenAbstract:Organophosphorus (OP) nerve agents are potent suicide inhibitors of the essential neurotransmitter-regulating enzyme acetylcholinesterase. Due to their acute toxicity, there is significant interest in developing effective countermeasures to OP poisoning. Here we impart nerve agent hydrolysis activity into the human drug metabolism enzyme Carboxylesterase 1. Using crystal structures of the target enzyme in complex with nerve agent as a guide, a pair of histidine and glutamic acid residues were designed proximal to the enzyme’s native catalytic triad. The resultant variant protein demonstrated significantly increased rates of reactivation following exposure to sarin, soman, and cyclosarin. Importantly, the addition of these residues did not alter the high affinity binding of nerve agents to this protein. Thus, using two amino acid substitutions, a novel enzyme was created that efficiently converted a group of hemisubstrates, compounds that can start but not complete a reaction cycle, into bona fide substrates. Such approaches may lead to novel countermeasures for nerv
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structural and biochemical analysis reveals the stereoselectivity of inhibition and reactivation of human Carboxylesterase 1 for the nerve agents sarin soman and cyclosarin
2010Co-Authors: Andrew C. Hemmert, Philip M. Potter, Tamara C. Otto, Monika Wierdl, Carol C. Edwards, Mary Macdonald, Douglas M Cerasoli, John R Cashman, Matthew R RedinboAbstract:Departments of Chemistry (M.R.R.) and Biochemistry and Biophysics (A.C.H., C.D.F., M.R.R), University of North Carolina at Chapel Hill, Chapel Hill, North Carolina; U.S. Army Medical Research Institute of Chemical Defense (T.C.O., D.M.C.), Aberdeen Proving Ground, Maryland; Human BioMolecular Research Institute (M.M., J.R.C.), San Diego, California; Department of Molecular Pharmacology (M.W., C.C.E., P.M.P.), St. Jude Children’s Research Hospital, Memphis, Tennessee Molecular Pharmacology Fast Forward. Published on January 5, 2010 as doi:10.1124/mol.109.062356
Allen S Yang - One of the best experts on this subject based on the ideXlab platform.
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2 deoxy n4 2 4 nitrophenyl ethoxycarbonyl 5 azacytidine a novel inhibitor of dna methyltransferase that requires activation by human Carboxylesterase 1
Cancer Letters, 2008Co-Authors: Hyangmin Byun, Si Ho Choi, Peter W Laird, B Trinh, Maqbool A Siddiqui, Victor E Marquez, Allen S YangAbstract:2'-Deoxy-N4-[2-(4-nitrophenyl)ethoxycarbonyl]-5-azacytidine (NPEOC-DAC), decitabine with a modification of the N4 position of the azacitidine ring can be used to inhibit DNA methyltransferase. This modification protects the azacitidine ring and can be cleaved by Carboxylesterase to release decitabine. NPEOC-DAC was 23-fold less potent at low doses ( or = 10microM NPEOC-DAC was more effective at inhibiting DNA methylation. Theses differences between decitabine and NPEOC-DAC are dependent on the cleavage of the carboxylester bond, and could be potentially exploited pharmacologically.
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2 deoxy n4 2 4 nitrophenyl ethoxycarbonyl 5 azacytidine a novel inhibitor of dna methyltransferase that requires activation by human Carboxylesterase 1
Cancer Letters, 2008Co-Authors: Hyangmin Byun, Si Ho Choi, Peter W Laird, Maqbool A Siddiqui, Victor E Marquez, Binh N Trinh, Allen S YangAbstract:2’-Deoxy-N4-[2-(4-nitrophenyl) ethoxycarbonyl]-5-azacytidine (NPEOC-DAC), decitabine with a modification of the N4 position of the azacitidine ring can be used to inhibit DNA methyltransferase. This modification protects the azacitidine ring and can be cleaved by Carboxylesterase to release decitabine. NPEOC-DAC was 23-fold less potent at low doses (<10 µM) than decitabine at inhibiting DNA methylation, and was also associated with a 3-day delay in its effect. However, at doses ≥ 10 µM NPEOC-DAC was more effective at inhibiting DNA methylation. Theses differences between decitabine and NPEOC-DAC are dependent on the cleavage of the carboxylester bond, and could be potentially exploited pharmacologically.