Glutathione S-transferase pi trapping method for generation and characterization of drug–protein adducts in human liver microsomes using liquid chromatography–tandem mass spectrometry

Yukinaga, Hideo; Iwabuchi, Haruo; Okazaki, Osamu; Izumi, Tohru

doi:10.1016/j.jpba.2012.04.035

Cited by 13 publications

(17 citation statements)

References 28 publications

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“…Although GST P1-1 is endowed with four cysteine 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 19 residues (cys-14, cys-47, cys-101 and cys-169), adducts of DF were only observed to cys-14 and cys-47. 24,[41][42][43] In the present investigation we identified cys-47 as the single target of an o-imine-methide metabolite formed by P450-mediated decarboxylation of DF ( Figure 4). 35 In addition, its localization near the GSH-binding site makes cys-47 solvent accessible.…”

Section: Discussionmentioning

confidence: 64%

Mass Spectrometric Characterization of Protein Adducts of Multiple P450-Dependent Reactive Intermediates of Diclofenac to Human Glutathione-S-transferase P1-1

Boerma

Dragović

Vermeulen

et al. 2012

Chem. Res. Toxicol.

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Use of the nonsteroidal anti-inflammatory drug diclofenac (DF) is associated with serious idiosyncratic hepatotoxicity. Covalent binding of reactive intermediates of DF to proteins is considered to initiate the process leading to this severe side-effect. The aim of this study was to characterize the nature of covalent protein modifications by reactive metabolites of DF which result from bioactivation by cytochrome P450. DF and its major monohydroxylated metabolites 4'-hydroxydiclofenac (4'-OH-DF) and 5-hydroxydiclofenac (5-OH-DF) were bioactivated using a highly active P450 BM3 mutant (CYP102A1M11H) in the presence of the model target protein human glutathione-S-transferase P1-1 (hGST P1-1). Protein-adducts were subsequently identified by LC-MS/MS analysis of tryptic digests of hGST P1-1. In total, 10 different peptide adducts were observed which result from modifications of Cys-47 and Cys-14 of hGST P1-1. The majority of the protein thiol modifications appeared to be derived from 5-OH-DF, which produced seven different peptide adducts with mass increments of 289.0, 309.0, and 339.0 Da. Remarkably, no peptide adducts were observed upon the bioactivation of 4'-OH-DF. Incubations of P450 BM3 with DF also showed the peptide adducts derived from 5-OH-DF and peptide adducts that are not derived from quinone imine. A peptide adduct with a mass increment of 249.0 Da most likely results from the o-imine methide formed by oxidative decarboxylation of DF. In addition, a peptide adduct was observed with a mass increment of 259.0 Da, which corresponds to the substitution of one of the chlorine atoms of DF by protein thiol. A corresponding GSH-conjugate with a similar mass increment was only observed if incubations of DF with P450 and GSH were supplemented by human GST P1-1. The results of this study not only confirm the importance of 5-OH-DF in covalent protein-binding but also suggest that the nature of protein adduction is not necessarily reflected by chemical conjugation with GSH.

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Section: Discussionmentioning

confidence: 64%

Mass Spectrometric Characterization of Protein Adducts of Multiple P450-Dependent Reactive Intermediates of Diclofenac to Human Glutathione-S-transferase P1-1

Boerma

Dragović

Vermeulen

et al. 2012

Chem. Res. Toxicol.

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“…In a follow-up study Boerma et al (2011) confirmed also diclofenac-related binding to Cys15 and 48. Using GSTP1 in a protein trapping study, binding of the reactive diquinone methide of raloxifene was also observed to Cys48 (Yukinaga et al, 2012). Here, we detected modified Cys48 and additional target sites of NAPQI in GSTP1 by using pepsin as an additional protease with different cleavage specificity than trypsin.…”

Section: Discussionmentioning

confidence: 99%

Liquid Chromatography-Tandem Mass Spectrometry Analysis of Acetaminophen Covalent Binding to Glutathione S-Transferases

et al. 2019

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Acetaminophen (APAP)-induced hepatotoxicity is the most common cause of acute liver failure in the Western world. APAP is bioactivated to N -acetyl p -benzoquinone imine (NAPQI), a reactive metabolite, which can subsequently covalently bind to glutathione and protein thiols. In this study, we have used liquid chromatography-tandem mass spectrometry (LC-MS/MS) to characterize NAPQI binding to human glutathione S -transferases (GSTs) in vitro . GSTs play a crucial role in the detoxification of reactive metabolites and therefore are interesting target proteins to study in the context of APAP covalent binding. Recombinantly-expressed and purified GSTs were used to assess NAPQI binding in vitro . APAP biotransformation to NAPQI was achieved using rat liver microsomes or human cytochrome P450 Supersomes in the presence of GSTA1, M1, M2, or P1. Resulting adducts were analyzed using bottom-up proteomics, with or without LC fractionation prior to LC-MS/MS analysis on a quadrupole-time-of-flight instrument with data-dependent acquisition (DDA). Targeted methods using multiple reaction monitoring (MRM) on a triple quadrupole platform were also developed by quantitatively labeling all available cysteine residues with a labeling reagent yielding isomerically-modified peptides following enzymatic digestion. Seven modified cysteine sites were confirmed, including Cys112 in GSTA1, Cys78 in GSTM1, Cys115 and 174 in GSTM2, as well as Cys15, 48, and 170 in GSTP1. Most modified peptides could be detected using both untargeted (DDA) and targeted (MRM) approaches, however the latter yielded better detection sensitivity with higher signal-to-noise and two sites were uniquely found by MRM.

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“…8,14−19 The majority of the efforts have been focused on the interaction of soft electrophiles such as epoxides, quinones, quinoneiminies etc. with proteins, [9][10][11][12]14,15,17,18 but the reactivity of hard electrophiles like iminium ions and aldehydes with proteins has been less extensively studied. 6,7,16,20 Electrochemistry (EC) is a useful tool to generate several different types of metabolites.…”

Section: ■ Introductionmentioning

confidence: 99%

Use of Electrochemical Oxidation and Model Peptides To Study Nucleophilic Biological Targets of Reactive Metabolites: The Case of Rimonabant

Thorsell

Isin

Jurva

2014

Chem. Res. Toxicol.

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Electrochemical oxidation of drug molecules is a useful tool to generate several different types of metabolites. In the present study we developed a model system involving electrochemical oxidation followed by characterization of the oxidation products and their propensity to modify peptides. The CB1 antagonist rimonabant was chosen as the model drug. Rimonabant has previously been shown to give high covalent binding to proteins in human liver microsomes and hepatocytes and the iminium ion and/or the corresponding aminoaldehyde formed via P450 mediated α-carbon oxidation of rimonabant was proposed to be a likely contributor. This proposal was based on the observation that levels of covalent binding were significantly reduced when iminium species were trapped as cyanide adducts but also following addition of methoxylamine expected to trap aldehydes. Incubation of electrochemically oxidized rimonabant with peptides resulted in peptide adducts to the N-terminal amine with a mass increment of 64 Da. The adducts were shown to contain an addition of C5H4 originating from the aminopiperidine moiety of rimonabant. Formation of the peptide adducts required further oxidation of the iminium ion to short-lived intermediates, such as dihydropyridinium species. In addition, the metabolites and peptide adducts generated in human liver microsomes were compared with those generated by electrochemistry. Interestingly, the same peptide modification was found when rimonabant was coincubated with one of the model peptides in microsomes. This clearly indicated that reactive metabolite(s) of rimonabant identical to electrochemically generated species are also present in the microsomal incubations. In summary, electrochemical oxidation combined with peptide trapping of reactive metabolites identified a previously unobserved bioactivation pathway of rimonabant that was not captured by traditional trapping agents and that may contribute to the in vitro covalent binding.

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Glutathione S-transferase pi trapping method for generation and characterization of drug–protein adducts in human liver microsomes using liquid chromatography–tandem mass spectrometry

Cited by 13 publications

References 28 publications

Mass Spectrometric Characterization of Protein Adducts of Multiple P450-Dependent Reactive Intermediates of Diclofenac to Human Glutathione-S-transferase P1-1

Mass Spectrometric Characterization of Protein Adducts of Multiple P450-Dependent Reactive Intermediates of Diclofenac to Human Glutathione-S-transferase P1-1

Liquid Chromatography-Tandem Mass Spectrometry Analysis of Acetaminophen Covalent Binding to Glutathione S-Transferases

Use of Electrochemical Oxidation and Model Peptides To Study Nucleophilic Biological Targets of Reactive Metabolites: The Case of Rimonabant

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