title: Glutathione-S-transferase pi as a model protein for the characterisation of chemically reactive metabolites.
abstract: Chemically reactive metabolites (CRMs) are thought to be responsible for a number of adverse drug reactions through modification of critical proteins. Methods that defined the chemistry of protein modification at an early stage would provide invaluable tools for drug safety assessment. Here, human GST pi (GSTP) was exploited as a model target protein to determine the chemical, biochemical and functional consequences of exposure to the hepatotoxic CRM of paracetamol (APAP), N-acetyl-p-benzoquinoneimine (NAPQI). Site-specific, dose-dependent modification of Cys47 in native and His-tagged GSTP was revealed by MS, and correlated with inhibition of glutathione (GSH) conjugating activity. In addition, the adaptation of iTRAQ labelling technology to define precisely the quantitative relationship between covalent modification and protein function is described. Multiple reaction monitoring (MRM)-MS of GSTP allowed high sensitivity detection of modified peptides at physiological levels of exposure. Finally, a bioengineered mutant cytochrome P450 with a broad spectrum of substrate specificities was used in an in vitro reaction system to bioactivate APAP: in this model, GSTP trapped the CRM and exhibited both reduced enzyme activity and site-specific modification of the protein. These studies provide the foundation for the development of novel test systems to predict the toxicological potential of CRMs produced by new therapeutic agents.
authors.affiliation: Department of Pharmacology and Therapeutics, School of Biomedical Sciences, University of Liverpool, Liverpool, UK. R.Jenkins@liv.ac.uk
authors.forename: Rosalind E
authors.initials: RE
authors.lastname: Jenkins
authors.forename: Neil R
authors.initials: NR
authors.lastname: Kitteringham
authors.forename: Christopher E P
authors.initials: CE
authors.lastname: Goldring
authors.forename: Samantha M J
authors.initials: SM
authors.lastname: Dowdall
authors.forename: Jane
authors.initials: J
authors.lastname: Hamlett
authors.forename: Catherine S
authors.initials: CS
authors.lastname: Lane
authors.forename: Jan-Simon
authors.initials: JS
authors.lastname: Boerma
authors.forename: Nico P E
authors.initials: NP
authors.lastname: Vermeulen
authors.forename: B Kevin
authors.initials: BK
authors.lastname: Park
mesh_terms: D000082:Acetaminophen / Q000378:metabolism / Q000494:pharmacology; D000595:Amino Acid Sequence; D016227:Benzoquinones / Q000494:pharmacology; D002478:Cells, Cultured; D003545:Cysteine / Q000737:chemistry; D051549:Glutathione S-Transferase pi / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006801:Humans; D007097:Imines / Q000494:pharmacology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D040901:Proteomics / Q000379:methods
chemical_list: D016227:Benzoquinones; D007097:Imines; D000082:Acetaminophen; D051549:Glutathione S-Transferase pi; C028473:N-acetyl-4-benzoquinoneimine; D003545:Cysteine