Peptide-Based In Vitro Assay for the Detection of Reactive Metabolites

Mitchell, Mark B.; Elrick, Mollisa M.; Walgren, Jennie; Mueller, Richard A.; Morris, Dale L.; Thompson, David C.

doi:10.1021/tx700344m

Cited by 33 publications

(29 citation statements)

References 52 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…A different approach was presented by Lim et al in their work of minimizing bioactivation of a drug candidate, in which S9 incubation in the presence of GSH and CN, along with their stable isotope analogues, was subject to HR-MS and isotope pattern dependent MS n acquisition on Orbitrap MS [11]. Mitchell et al used a peptide consisting of eleven amino acids including a terminus GSH and additional nucleophilic residues (e.g., lysine) for simultaneously trapping of both soft and hard electrophiles [31]. The assay utilized a surfaceenhanced laser detection/ionization (SELDI)-TOF MS system without chromatography separation.…”

Section: Introductionmentioning

confidence: 99%

Simultaneous Screening of Glutathione and Cyanide Adducts Using Precursor Ion and Neutral Loss Scans-Dependent Product Ion Spectral Acquisition and Data Mining Tools

Jian

Liu²,

Zhao

et al. 2012

J. Am. Soc. Mass Spectrom.

View full text Add to dashboard Cite

Drugs can be metabolically activated to soft and hard electrophiles, which are readily trapped by glutathione (GSH) and cyanide (CN), respectively. These adducts are often detected and structurally characterized using separate tandem mass spectrometry methods. We describe a new method for simultaneous screening of GSH and CN adducts using precursor ion (PI) and neutral loss (NL) scansdependent product ion spectral acquisition and data mining tools on an triple quadrupole linear ion trap mass spectrometry. GSH, potassium cyanide, and their stable isotope labeled analogues were incubated with liver microsomes and a test compound. Negative PI scan of m/z 272 for detection of GSH adducts and positive NL scans of 27 and 29 Da for detection of CN adducts were conducted as survey scans to trigger acquisition of enhanced resolution (ER) spectrum and subsequent enhanced product ion (EPI) spectrum. Post-acquisition data mining of EPI data set using NL filters of 129 and 27 Da was then performed to reveal the GSH adducts and CN adducts, respectively. Isotope patterns and EPI spectra of the detected adducts were utilized for identification of their molecular weights and structures. The effectiveness of this method was evaluated by analyzing reactive metabolites of nefazodone formed from rat liver microsomes. In addition to known GSH-and CN-trapped reactive metabolites, several new CN adducts of nefazodone were identified. The results suggested that current approach is highly effective in the analysis of both soft and hard reactive metabolites and can be used as a high-throughput method in drug discovery.

show abstract

Section: Introductionmentioning

confidence: 99%

Simultaneous Screening of Glutathione and Cyanide Adducts Using Precursor Ion and Neutral Loss Scans-Dependent Product Ion Spectral Acquisition and Data Mining Tools

Jian

Liu²,

Zhao

et al. 2012

J. Am. Soc. Mass Spectrom.

View full text Add to dashboard Cite

show abstract

“…1 Of particular concern are lowincidence, idiosyncratic events that are not detected in preclinical animal testing or in clinical trials with inadequate numbers of test subjects. 2 Although the mechanisms are not fully understood, idiosyncratic toxicity is thought to involve the initial formation of reactive metabolites from the parent drug followed by covalent binding to macromolecules, leading to the formation of peptide/protein-compound conjugates. [3][4][5] Subsequent proteolytic processing of drug-modified proteins may lead to the generation of peptides that are recognized as foreign and consequently produce either tolerance or initiation of an active immune response in vivo.…”

Section: Introductionmentioning

confidence: 99%

Screening and Characterization of Reactive Compounds with In Vitro Peptide-Trapping and Liquid Chromatography/High-Resolution Accurate Mass Spectrometry

et al. 2014

View full text Add to dashboard Cite

The present study describes a novel methodology for the detection of reactive compounds using in vitro peptidetrapping and liquid chromatography-high-resolution accurate mass spectrometry (LC-HRMS). Compounds that contain electrophilic groups can covalently bind to nucleophilic moieties in proteins and form adducts. Such adducts are thought to be associated with drug-mediated toxicity and therefore represent potential liabilities in drug discovery programs. In addition, reactive compounds identified in biological screening can be associated with data that can be misinterpreted if the reactive nature of the compound is not appreciated. In this work, to facilitate the triage of hits from high-throughput screening (HTS), a novel assay was developed to monitor the formation of covalent peptide adducts by compounds suspected to be chemically reactive. The assay consists of in vitro incubations of test compounds (under conditions of physiological pH) with synthetically prepared peptides presenting a variety of nucleophilic moieties such as cysteine, lysine, histidine, arginine, serine, and tyrosine. Reaction mixtures were analyzed using full-scan LC-HRMS, the data were interrogated using postacquisition data mining, and modified amino acids were identified by subsequent LC-HRMS/mass spectrometry. The study demonstrated that in vitro nucleophilic peptide trapping followed by LC-HRMS analysis is a useful approach for screening of intrinsically reactive compounds identified from HTS exercises, which are then removed from follow-up processes, thus obviating the generation of data from biochemical activity assays.

show abstract

“…The differences in isomer formation encountered with this system suggest that care must be taken in using the HRP assay as a model for hepatic oxidative metabolism. However, the HRP assay may be a relevant model for endogenous peroxidases localized to individual tissue types other than liver 31,125 , and further studies examining the possible formation of 2-hydroxylated cocaine metabolites by tissue-specific peroxidases may validate this system as a predictor of non-hepatic in vivo metabolism.…”

Section: Cocaine Metabolism Discussionmentioning

confidence: 99%

“…30 Other researchers have employed metabolic assays in pharmaceutical protein binding studies. 31 While the assays used for these purposes are numerous and varied, toxicologists must balance the effectiveness, applicability, ease of use, and overall cost of each assay to determine the optimal system for their individual …”

Section: In Vitro Metabolic Assaysmentioning

confidence: 99%

See 1 more Smart Citation

Covalent Protein Adduction by Drugs of Abuse

Schneider¹

View full text Add to dashboard Cite

Recreational abuse of the drugs cocaine, methamphetamine, and morphine continues to be prevalent in the United States of America and around the world. While numerous methods of detection exist for each drug, they are generally limited by the lifetime of the parent drug and its metabolites in the body. However, the covalent modification of endogenous proteins by these drugs of abuse may act as biomarkers of exposure and allow for extension of detection windows for these drugs beyond the lifetime of parent molecules or metabolites in the free fraction. Additionally, existence of covalently bound molecules arising from drug ingestion can offer insight into downstream toxicities associated with each of these drugs.This research investigated the metabolism of cocaine, methamphetamine, and morphine in common in vitro assay systems, specifically focusing on the generation of reactive intermediates and metabolites that have the potential to form covalent protein adducts. Results demonstrated the formation of covalent adduction products between biological cysteine thiols and reactive moieties on cocaine and morphine metabolites. Rigorous mass spectrometric analysis in conjunction with in vitro metabolic activation, pharmacogenetic reaction phenotyping, and computational modeling were utilized to characterize structures and mechanisms of formation for each resultant thiol adduction product. For cocaine, data collected demonstrated the formation of adduction products from a reactive arene epoxide intermediate, designating a novel metabolic pathway for cocaine. In the case of morphine, data expanded on known adduct-forming pathways using sensitive and selective analysis techniques, following the known reactive metabolite, morphinone, and a proposed novel metabolite, morphine quinone methide. Data collected in this study describe novel metabolic events for multiple important drugs of abuse, culminating v in detection methods and mechanistic descriptors useful to both medical and forensic investigators when examining the toxicology associated with cocaine, methamphetamine, and morphine.

show abstract

Peptide-Based In Vitro Assay for the Detection of Reactive Metabolites

Cited by 33 publications

References 52 publications

Simultaneous Screening of Glutathione and Cyanide Adducts Using Precursor Ion and Neutral Loss Scans-Dependent Product Ion Spectral Acquisition and Data Mining Tools

Simultaneous Screening of Glutathione and Cyanide Adducts Using Precursor Ion and Neutral Loss Scans-Dependent Product Ion Spectral Acquisition and Data Mining Tools

Screening and Characterization of Reactive Compounds with In Vitro Peptide-Trapping and Liquid Chromatography/High-Resolution Accurate Mass Spectrometry

Covalent Protein Adduction by Drugs of Abuse

Contact Info

Product

Resources

About