Structural Insights into Drug Processing by Human Carboxylesterase 1: Tamoxifen, Mevastatin, and Inhibition by Benzil

Fleming, Christopher D.; Bencharit, Sompop; Edwards, Carol C.; Hyatt, Janice L.; Tsurkan, Lyudmila; Bai, Feng; Fraga, Charles H.; Morton, Christopher L.; Howard-Williams, Escher L.; Potter, Philip M.; Redinbo, Matthew R.

doi:10.1016/j.jmb.2005.07.016

Cited by 130 publications

(144 citation statements)

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“…These space groups have been observed in previous hCE1 structures (25)(26)(27)(28), and correspond either to two (P2 1 , hCE1-soman) or one (P2 1 2 1 2 1 , hCE1-tabun) trimer per asymmetric unit. While the cell constants of the hCE1-soman structure appear to be capable of adapting P2 1 2 1 2 1 space group symmetry, R sym values establish P2 1 as correct for this complex.…”

Section: Crystallographic Analysissupporting

confidence: 66%

“…The active site of the enzyme is located at the interface of the three domains and is composed of the catalytic triad Ser221, His468, and Glu354. The hCE1 trimer exhibits C3 symmetry and is formed largely by contacts between the αβ domains of each monomer (not shown) (25,27,28). A hexamer has also been observed for hCE1 in which two trimers are stacked with their active sites facing in and with the Z-site loops pinterdigitating (26,27); neither of the structures described here form a hexamer, however.…”

Section: Domain Architecture Of Hce1mentioning

confidence: 66%

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Crystal Structures of Human Carboxylesterase 1 in Covalent Complexes with the Chemical Warfare Agents Soman and Tabun^,

et al. 2007

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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 Å resolution crystal structures of the serine hydrolase human carboxylesterase 1 (hCE1), a broad-spectrum drug metabolism enzyme, in covalent acylenzyme 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 P S stereoisomer of soman relative to the P R form. In addition, structural features of the hCE1 active site indicate that the enzyme may be resistant to deadend 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.The organophosphorus (OP) nerve agents sarin, soman, tabun, and VX are among the deadliest man-made chemicals (1). While the military use of OP nerve agents is widely banned, these compounds have been employed in recent decades by rogue states and terrorist groups. In 1988, Iraq employed weaponized sarin against its own Kurdish citizens in Halabja, a town adjacent to the Iranian border, killing an estimated 5,000 (2). Coordinated attacks on the Tokyo subway system by the Japanese Aum Shinrikyo cult in 1995 also employed sarin, killing 12 and injuring † This work was supported, in part, by NIH grants CA98468 and NS58089, and the American Lebanese Syrian Associated Charities. ‡ Protein Data Bank codes are 2HRQ for the hCE1-soman structure and 2HRR for the hCE1-tabun structure. *Corresponding Author: Matthew R. Redinbo, Ph.D., Department of Chemistry, CB #3290, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-3290, (919)843-8910; Fax: (919)962-2388, redinbo@unc.edu. 1 Abbreviations: AcChE, Human acetylcholinesterase; BuChE, Human butyrylcholinesterase; CE, carboxylesterase; hCE1, Human carboxylesterase 1; HI-6, 1-(2-hydroxy-iminomethylpyridinium)-1-(4-carboxyamino)-pyridinium dimethylether dichloride; MuAcChE, Mus musculus acetylcholinesterase; OP, organophosphate; Ortho-7, 1,7-heptylene-bis-N,N′-2-pyridiniumaldoxime dichloride; PON1, human serum paraoxonase 1; rmsd, root mean square deviation; Sarin, methylethyl methylphosphonofluoridate; Soman, Tabun, ethyl N, TcAcChE, Torpedo californica acetylcholinesterase; VX, OP toxicity is thought to be mediated through the inhibition of human acety...

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Section: Crystallographic Analysissupporting

confidence: 66%

Section: Domain Architecture Of Hce1mentioning

confidence: 66%

Crystal Structures of Human Carboxylesterase 1 in Covalent Complexes with the Chemical Warfare Agents Soman and Tabun^,

et al. 2007

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“…These features are similar to those of the purified PMPMEase, which shows a molecular weight on SDS-PAGE of about 57 kDa, and both the rat liver [10] and porcine liver in this study show a high affinity for the hydrophobic PC analog ester substrates. The crystal structure of the human isoform, hCE1, reveals complexes of homotrimers at equilibrium with the homohexamers and the catalytic triad of Ser221-His468-Glu354 inside a deep hydrophobic pocket that has a small rigid subsite and a large flexible region [24,25]. Purification-induced dissociation of these multimeric complexes especially when high-salt concentrations were involved might have resulted in monomeric, enzymatically inactive conformations.…”

Section: Discussionmentioning

confidence: 99%

Liver prenylated methylated protein methyl esterase is the same enzyme as sus scrofa carboxylesterase

Oboh

Lamango

2008

J Biochem & Molecular Tox

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The C-terminal -COOH of prenylated proteins is methylated to -COOCH 3 . The -COOCH 3 ester forms are hydrolyzed by prenylated methylated protein methyl esterase (PMPMEase) to the original acid forms. This is the only reversible step of the prenylation pathway. PMPMEase has not been purified and identified and is therefore understudied. Using a prenylated-L-cysteine methyl ester as substrate, PMPMEase was purified to apparent homogeneity from porcine liver supernatant. SDS-PAGE analysis revealed an apparent mass of 57 kDa. Proteomics analyses identified 17 peptides (242 amino acids). A Mascot database search revealed these as portions of the Sus scrofa carboxylesterase, a 62-kDa serine hydrolase with the C-terminal HAEL endoplasmic reticulum-retention signal. It is at least 71% identical to such mammalian carboxylesterases as human carboxylesterase 1 with affinities toward hydrophobic substrates and known to activate prodrugs, metabolize active drugs, as well as detoxify various substances such as cocaine and food-derived esters. The purified enzyme hydrolyzed benzoyl-Gly-farnesyl-Lcysteine methyl ester and hydrocinamoyl farnesyl-L-cysteine methyl ester with Michaelis-Menten constant (K m ) values of 33 ± 4 and 25 ± 4 µM and V max values of 4.51 ± 0.28 and 6.80 ± 0.51 nmol/min/mg of protein, respectively. It was inhibited by organophosphates, chloromethyl ketones, ebelactone A and B, and phenylmethylsulfonyl fluoride.

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“…Tryptic digests were separated using a Waters NanoAcquity UPLC system equipped with a 5 μm Symmetry C 18 (180 μm × 20 mm) trap column and a 1.8 μm High Strength Silica (HSS-T3) analytical column (75 μm × 150 mm) heated to 35 °C coupled to a picotip emitter (New Objective). Tryptic peptides were loaded onto the trap column over 3 minutes, followed by analytical separation over a 90 minute gradient (3% acetonitrile to 40% acetonitrile over 90 minutes).…”

Section: Mass Spectrometry and Data Analysismentioning

confidence: 99%

“…Human liver carboxylesterase 1 (CES1) is a known activating enzyme of several ester prodrugs, including oseltamivir 5,[15][16][17][18] . The co-expression of multiple carboxylesterase (CES) genes 19,20 and the differential substrate preferences between species complicates direct analysis of contributions from distinct CES enzymes.…”

Section: Introductionmentioning

confidence: 99%

Substrate-competitive activity-based profiling of ester prodrug activating enzymes

Martin

2018

Drug Metabolism and Pharmacokinetics

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Understanding the mechanistic basis of prodrug delivery and activation is critical for establishing species-specific prodrug sensitivities necessary for evaluating pre-clinical animal models and potential drug-drug interactions. Despite significant adoption of prodrug methodologies for enhanced pharmacokinetics, functional annotation of prodrug activating enzymes is laborious and often unaddressed. Activity-based protein profiling (ABPP) describes an emerging chemoproteomic approach to assay active site occupancy within a mechanistically similar enzyme class in native proteomes. The serine hydrolase enzyme family is broadly reactive with reporterlinked fluorophosphonates, which have shown to provide a mechanism-based covalent labeling strategy to assay the activation state and active site occupancy of cellular serine amidases, esterases, and thioesterases. Here we describe a modified ABPP approach using direct substrate competition to identify activating enzymes for an ethyl ester prodrug, the influenza neuraminidase inhibitor oseltamivir. Substrate-competitive ABPP analysis identified carboxylesterase 1 (CES1) as an oseltamivir-activating enzyme in intestinal cell homogenates. Saturating concentrations of oseltamivir lead to a 4-fold reduction in the observed rate constant for CES1 inactivation by fluorophosphonates. WWL50, a reported carbamate inhibitor of mouse CES1, blocked oseltamivir hydrolysis activity in human cell homogenates, confirming CES1 is the primary prodrug activating enzyme for oseltamivir in human liver and intestinal cell lines. The related carbamate inhibitor WWL79 inhibited mouse, but not human CES1, providing a series of probes for analyzing prodrug activation mechanisms in different preclinical models. Overall, we present a substratecompetitive activity-based profiling approach for broadly surveying candidate prodrug hydrolyzing enzymes and outline the kinetic parameters for activating enzyme discovery, ester prodrug design and preclinical development of ester prodrugs. HHS Public Access

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Structural Insights into Drug Processing by Human Carboxylesterase 1: Tamoxifen, Mevastatin, and Inhibition by Benzil

Cited by 130 publications

References 50 publications

Crystal Structures of Human Carboxylesterase 1 in Covalent Complexes with the Chemical Warfare Agents Soman and Tabun^,

Crystal Structures of Human Carboxylesterase 1 in Covalent Complexes with the Chemical Warfare Agents Soman and Tabun^,

Liver prenylated methylated protein methyl esterase is the same enzyme as sus scrofa carboxylesterase

Substrate-competitive activity-based profiling of ester prodrug activating enzymes

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Structural Insights into Drug Processing by Human Carboxylesterase 1: Tamoxifen, Mevastatin, and Inhibition by Benzil

Cited by 130 publications

References 50 publications

Crystal Structures of Human Carboxylesterase 1 in Covalent Complexes with the Chemical Warfare Agents Soman and Tabun,

Crystal Structures of Human Carboxylesterase 1 in Covalent Complexes with the Chemical Warfare Agents Soman and Tabun,

Liver prenylated methylated protein methyl esterase is the same enzyme as sus scrofa carboxylesterase

Substrate-competitive activity-based profiling of ester prodrug activating enzymes

Contact Info

Product

Resources

About

Crystal Structures of Human Carboxylesterase 1 in Covalent Complexes with the Chemical Warfare Agents Soman and Tabun^,

Crystal Structures of Human Carboxylesterase 1 in Covalent Complexes with the Chemical Warfare Agents Soman and Tabun^,