The development of a higher throughput reactive intermediate screening assay incorporating micro-bore liquid chromatography–micro-electrospray ionization–tandem mass spectrometry and glutathione ethyl ester as an in vitro conjugating agent

Soglia, John R.; Harriman, Shawn; Zhao, Sabrina X.; Barberia, John; Cole, Mark J.; Boyd, James G.; Contillo, Leonard G.

doi:10.1016/j.jpba.2004.04.019

Cited by 105 publications

(85 citation statements)

References 21 publications

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“…In particular, dehydration of m/z 322.1 was observed to occur from a 30 eV collision energy (laboratory frame). The MS/MS behavior of the protonated hydroxylamine 1b greatly differs from fragmentation patterns described for several glutathione conjugates, characterized in positive ion mode tandem mass spectrometry by the loss of the 129 Da pyroglutamic acid neutral [13][14][15][16][17]. This suggests that a nitroso function on the thiol moiety would completely modify dissociation of the ion in the gas-phase.…”

Section: Structural Characterization By Mass Spectrometrymentioning

confidence: 81%

“…In particular, in the positive ion mode, a main dissociation reaction consists of the elimination of a 129 Da neutral corresponding to a pyroglutamic acid molecule. As a result, detection of glutathione conjugates is often performed using a 129 Da neutral loss MS/MS experiments [13][14][15][16][17]. Nevertheless, different classes of glutathione conjugates, such as aliphatic thioether adducts ionized in the positive ion mode, were shown to behave differently upon collisioninduced dissociation (CID) [18,19].…”

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confidence: 99%

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Nucleophile addition of reduced glutathione on 2-methyl-2-nitroso compound: A combined electron paramagnetic resonance spectroscopy and electrospray tandem mass spectrometry study

Triquigneaux

Tuccio

Lauricella

et al. 2009

J. Am. Soc. Mass Spectrom.

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Mass spectrometry (MS) was used in conjunction with electron paramagnetic resonance (EPR) to characterize products arising from reactions between reduced glutathione (GSH) and 2-methyl 2-nitroso propane (MNP) in an oxidative medium, to evaluate the reactivity of this tripeptide as a nucleophile toward a nitroso compound. Depending on the experimental conditions, different radical species could be detected by EPR, which allowed some structural assumptions. These samples were then submitted to electrospray ionization, in both positive and negative ion modes, for structural elucidation in tandem mass spectrometry. Although the primary nitroxide products could not be detected in MS, structurally related compounds such as hydroxylamine and O-methyl hydroxylamine could be fully characterized. In the absence of light, a S-adduct was formed via a Forrester-Hepburn reaction, that is, a nucleophile addition of MNP onto the thiol function in reduced glutathione to yield a hydroxylamine intermediate, further oxidized into nitroxide. In contrast, irradiating the reaction medium with visible light could allow an inverted spin trapping reaction to take place, involving the oxidation of both MNP and GSH before the nucleophilic addition of the sulfenic acid function onto the nitrogen of MNP, yielding a so-called O-adduct. It was also found that dilution of the reaction medium with methanol for the purpose of electrospray ionization could allow nitroxides to be indirectly observed either as hydroxylamine or O-methyl hydroxylamine species. (J Am Soc

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Section: Structural Characterization By Mass Spectrometrymentioning

confidence: 81%

mentioning

confidence: 99%

Nucleophile addition of reduced glutathione on 2-methyl-2-nitroso compound: A combined electron paramagnetic resonance spectroscopy and electrospray tandem mass spectrometry study

Triquigneaux

Tuccio

Lauricella

et al. 2009

J. Am. Soc. Mass Spectrom.

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“…GSHEE was used for these studies since the presence of the less polar ethyl ester moiety on the glycine molecule has been reported to make GSHEE more sensitive to MS detection (Soglia et al, 2004). GSHEE adducts were observed using LC/MS for all five of the MBI-positive diaziridines 1 to 5, which indicated that an electrophilic reactive intermediate was indeed generated during the metabolism of these compounds by P450 2B6.…”

Section: Discussionmentioning

confidence: 99%

“…The reaction mixtures contained 0.25 nmol of purified P450 2B6, 0.5 nmol of reductase, 120 units of catalase, and 10 mM GSHEE (Soglia et al, 2004) in a total volume of 0.125 ml of 50 mM potassium phosphate buffer, pH 7.4. Each compound tested was added from a stock solution prepared in DMSO.…”

Section: -(4-chlorophenyl)-3-(trifluoromethyl)diaziridine (8mentioning

confidence: 99%

“…The electrophilic reactive metabolites that were generated by incubating compounds 1 to 5 with recombinant 2B6 in the reconstituted system in the presence of NADPH were trapped using the nucleophilic trapping reagent, GSHEE, and analyzed by LC/MS (Soglia et al, 2004). GSHEE has been reported to be a more useful trapping reagent than glutathione for monitoring trapped adducts of FIG.…”

Section: Determinants For 2b6 Inactivation By Phenyl Diaziridinesmentioning

confidence: 99%

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Structure-Activity Relationship and Elucidation of the Determinant Factor(s) Responsible for the Mechanism-Based Inactivation of Cytochrome P450 2B6 by Substituted Phenyl Diaziridines

et al. 2006

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ABSTRACT:It has been demonstrated previously that several 3-trifluoromethyl-3-(4-alkoxyphenyl)diaziridines inhibit the 7-ethoxy-4-(trifluoroethyl)coumarin (7-EFC) O-deethylation activity of P450 2B6 in a mechanism-based manner. In contrast, 3-trifluoromethyl-3-(4-methylthio)phenyl)diaziridine did not have any effect on the activity of P450 2B6. It is interesting that both the alkoxy and the thiophenyl compounds were metabolized by P450 2B6. In this report, the structure-activity relationships for the mechanism-based inactivation of cytochrome P450 2B6 by a series of aryl diaziridines were investigated. Three diaziridines that did not contain a 4-alkoxysubstituent on their phenyl ring, namely, 3-trifluoromethyl-3-(3-methoxyphenyl)diaziridine, 3-trifluoromethyl-3-phenyl diaziridine, and 3-trifluoromethyl-3-(4-chlorophenyl)diaziridine had no effect on the P450 2B6 7-EFC activity. Another analog that did not contain a diaziridine substructure, 3-trifluoromethyl-3-(4-methoxyphenyl)ethanone, also had no effect on the activity of P450 2B6. Glutathione ethyl ester adducts of the phenyldiaziridine reactive intermediates were isolated from reaction mixtures of the inactivated samples and analyzed by liquid chromatography-tandem mass spectrometry. The structures of the conjugates suggested that the electrophilic reactive intermediate in each case was a quinone methide (quinomethane), 4-ethylidene-cyclohexa-2,5-dienone, generated from the 4-alkoxyphenyldiaziridines by removal of both of the diaziridine and the 4-alkyl groups. In conclusion, the determinant factor for the mechanism-based inactivator activity of the aryl diaziridines seems to be the formation of the reactive quinomethane intermediate, which is generated from the 4-alkoxyphenyl diaziridines by a cytochrome P450-catalyzed metabolic reaction.

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Minimizing the Potential for Drug Bioactivation of Drug Candidates to Success in Clinical Development

Nassar

2008

Drug Metabolism Handbook

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The development of a higher throughput reactive intermediate screening assay incorporating micro-bore liquid chromatography–micro-electrospray ionization–tandem mass spectrometry and glutathione ethyl ester as an in vitro conjugating agent

Cited by 105 publications

References 21 publications

Nucleophile addition of reduced glutathione on 2-methyl-2-nitroso compound: A combined electron paramagnetic resonance spectroscopy and electrospray tandem mass spectrometry study

Nucleophile addition of reduced glutathione on 2-methyl-2-nitroso compound: A combined electron paramagnetic resonance spectroscopy and electrospray tandem mass spectrometry study

Structure-Activity Relationship and Elucidation of the Determinant Factor(s) Responsible for the Mechanism-Based Inactivation of Cytochrome P450 2B6 by Substituted Phenyl Diaziridines

Minimizing the Potential for Drug Bioactivation of Drug Candidates to Success in Clinical Development

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