Biomimetic trapping cocktail to screen reactive metabolites: use of an amino acid and DNA motif mixture as light/heavy isotope pairs differing in mass shift
Candidate drugs that can be metabolically transformed into reactive electrophilic products, such as epoxides, quinones, and nitroso compounds, are of special concern because subsequent covalent binding to bio-macromolecules can cause adverse drug reactions, such as allergic reactions, hepatotoxicity, and genotoxicity. Several strategies have been reported for screening reactive metabolites, such as a covalent binding assay with radioisotope-labeled drugs and a trapping method followed by LC–MS/MS analyses. Of these, a trapping method using glutathione is the most common, especially at the early stage of drug development. However, the cysteine of glutathione is not the only nucleophilic site in vivo; lysine, histidine, arginine, and DNA bases are also nucleophilic. Indeed, the glutathione trapping method tends to overlook several types of reactive metabolites, such as aldehydes, acylglucuronides, and nitroso compounds. Here, we introduce an alternate way for screening reactive metabolites as follows: A mixture of the light and heavy isotopes of simplified amino acid motifs and a DNA motif is used as a biomimetic trapping cocktail. This mixture consists of [2H0]/[2H3]-1-methylguanidine (arginine motif, Δ 3 Da), [2H0]/[2H4]-2-mercaptoethanol (cysteine motif, Δ 4 Da), [2H0]/[2H5]-4-methylimidazole (histidine motif, Δ 5 Da), [2H0]/[2H9]-n-butylamine (lysine motif, Δ 9 Da), and [13C0,15N0]/[13C1,15N2]-2′-deoxyguanosine (DNA motif, Δ 3 Da). Mass tag triggered data-dependent acquisition is used to find the characteristic doublet peaks, followed by specific identification of the light isotope peak using MS/MS. Forty-two model drugs were examined using an in vitro microsome experiment to validate the strategy.
KeywordsMass spectrometry Activated drug Trapping reagent Cocktail reagent
This work was supported in part by a Grant-in-Aid for Challenging Exploratory Research (to T.O., no. 25670008 for 2013–2014) from Japan Society for the Promotion of Science (JSPS) and TaNeDS 2014 (to T.O., for 2014–2017) from Daiichi Sankyo Co., Ltd. The authors thank Astellas Pharma Inc. (Tsukuba, Japan) for donating a used LCQ DECA. The authors also thank Mr. Reona Yoshiizumi of our laboratory for help in preparing KP-NHS.
Compliance with ethical standards
Conflict of interest
The authors declare that there are no conflicts of interest.
- 3.Albano E, Rundgren M, Harvison PJ, Nelson SD, Moldéus P. Mechanisms of N-acetyl-p-benzoquinone imine cytotoxicity. Mol Pharmacol. 1985;28:306–311. doi: not available (http://molpharm.aspetjournals.org/content/28/3/306).
- 4.Callan HE, Jenkins RE, Maggs JL, Lavergne SN, Clarke SE, Naisbitt DJ, et al. Multiple adduction reactions of nitroso sulfamethoxazole with cysteinyl residues of peptides and proteins: implications for hapten formation. Chem Res Toxicol. 2009;22:937–48. https://doi.org/10.1021/tx900034r.CrossRefPubMedGoogle Scholar
- 5.Srivastava A, Maggs JL, Antoine DJ, Williams DP, Smith DA, Park BK. Role of reactive metabolites in drug-induced hepatotoxicity. Handb Exp Pharmacol. 2010;196:165–194. doi: not available (ISBN: 978–3–642-00662-3, Print, 978-3-642-00663-0, Online).Google Scholar
- 9.Sridar C, Kenaan C, Hollenberg PF. Inhibition of bupropion metabolism by selegiline: mechanism-based inactivation of human CYP2B6 and characterization of glutathione and peptide adducts. Drug Metab Dispos. 2012;40:2256–66. https://doi.org/10.1124/dmd.112.046979.CrossRefPubMedPubMedCentralGoogle Scholar
- 10.Henne KR, Tran TB, VandenBrink BM, Rock DA, Aidasani DK, Subramanian R, et al. Sequential metabolism of AMG 487, a novel CXCR3 antagonist, results in formation of quinone reactive metabolites that covalently modify CYP3A4 Cys239 and cause time-dependent inhibition of the enzyme. Drug Metab Dispos. 2012;40:1429–40. https://doi.org/10.1124/dmd.112.045708.CrossRefPubMedGoogle Scholar
- 11.Kalgutkar AS, Dalvie DK, Aubrecht J, Smith EB, Coffing SL, Cheung JR, et al. Genotoxicity of 2-(3-chlorobenzyloxy)-6-(piperazinyl)pyrazine, a novel 5-hydroxytryptamine2c receptor agonist for the treatment of obesity: role of metabolic activation. Drug Metab Dispos. 2007;35:848–58. https://doi.org/10.1124/dmd.106.013649.CrossRefPubMedGoogle Scholar
- 15.Mutlib A, Lam W, Atherton J, Chen H, Galatsis P, Stolle W. Application of stable isotope labeled glutathione and rapid scanning mass spectrometers in detecting and characterizing reactive metabolites. Rapid Commun Mass Spectrom. 2005;19:3482–92. https://doi.org/10.1002/rcm.2223.CrossRefPubMedGoogle Scholar
- 17.Argoti D, Liang L, Conteh A, Chen L, Bershas D, Yu CP, et al. Cyanide trapping of iminium ion reactive intermediates followed by detection and structure identification using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Chem Res Toxicol. 2005;18:1537–44. https://doi.org/10.1021/tx0501637.CrossRefPubMedGoogle Scholar
- 19.Laine JE, Auriola S, Pasanen M, Juvonen RO. d-isomer of gly-tyr-pro-cys-pro-his-pro peptide: a novel and sensitive in vitro trapping agent to detect reactive metabolites by electrospray mass spectrometry. Toxicol in Vitro. 2011;25:411–25. https://doi.org/10.1016/j.tiv.2010.11.002.CrossRefPubMedGoogle Scholar
- 24.Miyagi M, Wan Q, Ahmad MF, Gokulrangan G, Tomechko SE, Bennett B, et al. Histidine hydrogen-deuterium exchange mass spectrometry for probing the microenvironment of histidine residues in dihydrofolate reductase. PLoS One. 2011;6:e17055. https://doi.org/10.1371/journal.pone.0017055.CrossRefPubMedPubMedCentralGoogle Scholar
- 25.Casasnovas R, Adrover M, Ortega-Castro J, Frau J, Donoso J, Muñoz F. C–H activation in pyridoxal-5′-phosphate Schiff bases: the role of the imine nitrogen. A combined experimental and computational study. J Phys Chem B. 2012;116:10665–−10675. https://doi.org/10.1021/jp303678n.CrossRefPubMedGoogle Scholar
- 33.Nakayama S, Takakusa H, Watanabe A, Miyaji Y, Suzuki W, Sugiyama D, et al. Combination of GSH trapping and time-dependent inhibition assays as a predictive method of drugs generating highly reactive metabolites. Drug Metab Dispos. 2011;39:1247–54. https://doi.org/10.1124/dmd.111.039180.CrossRefPubMedGoogle Scholar
- 41.Yu LJ, Chen Y, DeNinno MP, O’Connell TN, Hop CECA. Identification of a novel glutathione adduct of diclofenac, 4′-hydroxy-2′-glutathion-deschloro-diclofenac, upon incubation with human liver microsomes. Drug Metab Dispos. 2005;33:484–8. https://doi.org/10.1124/dmd.104.002840.CrossRefPubMedGoogle Scholar
- 42.Kent UM, Lin H, Mills DE, Regal KA, Hollenberg PF. Identification of 17-α-ethynylestradiol-modified active site peptides and glutathione conjugates formed during metabolism and inactivation of P450s 2B1 and 2B6. Chem Res Toxicol. 2006;19:279–87. https://doi.org/10.1021/tx050256o.CrossRefPubMedPubMedCentralGoogle Scholar
- 44.Williams DP, Antoine DJ, Butler PJ, Jones R, Randle L, Payne A, et al. The metabolism and toxicity of furosemide in the Wistar rat and CD-1 mouse: a chemical and biochemical definition of the toxicophore. J Pharmacol Exp Ther. 2007;322:1208–20. https://doi.org/10.1124/jpet.107.125302.CrossRefPubMedGoogle Scholar
- 46.Ju C, Uetrecht JP. Oxidation of a metabolite of indomethacin (desmethyldeschlorobenzoylindomethacin) to reactive intermediates by activated neutrophils, hypochlorous acid, and the myeloperoxidase system. Drug Metab Dispos. 1998;26:676–680. doi: not available (http://dmd.aspetjournals.org/content/dmd/26/7/676.full.pdf).
- 48.Gardner I, Zahid N, Maccrimmon D, Uetrecht JP. A comparison of the oxidation of clozapine and olanzapine to reactive metabolites and the toxicity of these metabolites to human leukocytes. Mol Pharmacol. 1998;53:991–998. doi: not available (http://molpharm.aspetjournals.org/content/molpharm/53/6/991.full.pdf).
- 49.Baughman TM, Graham RA, Wells-Knecht K, Silver IS, Tyler LO, Wells-Knecht M, et al. Metabolic activation of pioglitazone identified from rat and human liver microsomes and freshly isolated hepatocytes. Drug Metab Dispos. 2005;33:733–8. https://doi.org/10.1124/dmd.104.002683.CrossRefPubMedGoogle Scholar
- 50.Uetrecht JP. Reactivity and possible significance of hydroxylamine and nitroso metabolites of procainamide. J Pharmacol Exp Ther. 1985;232:420–425. doi: not available (http://jpet.aspetjournals.org/content/232/2/420).
- 51.Sasame HA, Liberato DJ, Gillette JA. The formation of a glutathione conjugate derived from propranolol. Drug Metab Dispos. 1987;15:349–355. doi: not available (http://dmd.aspetjournals.org/content/dmd/15/3/349.full.pdf).
- 52.Alvarez-Sánchez R, Montavon F, Hartung T, Pähler A. Thiazolidinedione bioactivation: a comparison of the bioactivation potentials of troglitazone, rosiglitazone, and pioglitazone using stable isotope-labeled analogues and liquid chromatography tandem mass spectrometry. Chem Res Toxicol. 2006;19:1106–16. https://doi.org/10.1021/tx050353h.CrossRefPubMedGoogle Scholar
- 53.Cribb AE, Miller M, Leeder JS, Hill J, Spielberg SP. Reactions of the nitroso and hydroxylamine metabolites of sulfamethoxazole with reduced glutathione. Implications for idiosyncratic toxicity. Drug Metab Dispos. 1991;19:900–906. doi: not available (http://dmd.aspetjournals.org/content/dmd/19/5/900.full.pdf).
- 56.Chen Q, Doss GA, Tung EC, Liu W, Tang YS, Braun MP, et al. Evidence for the bioactivation of zomepirac and tolmetin by an oxidative pathway: identification of glutathione adducts in vitro in human liver microsomes and in vivo in rats. Drug Metab Dispos. 2006;34:145–51. https://doi.org/10.1124/dmd.105.004341.CrossRefPubMedGoogle Scholar