Glutathione-S-transferase pi as a model protein for the characterisation of chemically reactive metabolites.
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.
Jenkins
Rosalind E
RE
Department of Pharmacology and Therapeutics, School of Biomedical Sciences, University of Liverpool, Liverpool, UK. R.Jenkins@liv.ac.uk
Kitteringham
Neil R
NR
Goldring
Christopher E P
CE
Dowdall
Samantha M J
SM
Hamlett
Jane
J
Lane
Catherine S
CS
Boerma
Jan-Simon
JS
Vermeulen
Nico P E
NP
Park
B Kevin
BK
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
D016227:Benzoquinones; D007097:Imines; D000082:Acetaminophen; D051549:Glutathione S-Transferase pi; C028473:N-acetyl-4-benzoquinoneimine; D003545:Cysteine