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.

Idiosyncratic Adverse Drug Reactions: Current Concepts

“…In fact, these observations have resulted in the development and use of chemical reactivity measurements for the predictive identification of skin sensitizing chemicals (Divkovic et al, 2005;Gerberick et al, 2007Gerberick et al, , 2008. The development of sophisticated protein mass spectrometry methods to measure the binding of sensitizing chemicals to protein has greatly assisted analysis of chemical protein interactions (Jenkins et al, 2008). Protein binding has been found to vary in terms of protein and amino acid specificity, reaction mechanisms, and rates of reaction .…”

Multiple reaction monitoring for quantitative biomarker analysis in proteomics and metabolomics☆

“…The pharmaceutical industry have adopted small molecule trapping agents such as glutathione to study adduct formation following bioactivation of new drugs [121], but a much more realistic indication of covalent modification may be achieved if modification of proteins can be measured. We utilised MRM-MS to analyse the modification of human glutathione-Stransferase pi with the CRM of paracetamol, and were able to show that adducts could be detected when the protein was exposed in vitro to a molar ratio of drug to protein of 1:10,000, comparable to in vivo levels of exposure [38]. Other studies using this approach for the detection of chemical adducts are as yet rare, but they include assessment of exposure to agents of biological warfare [122] and industrial chemicals [123], and mapping of the covalent modification of proteins with the lipid peroxidation product 4-hydroxy-2-nonenol [124].…”

“…These approaches provide the opportunity not only to measure protein levels but also to quantify specific protein post-translational modifications (PTM) [28][29][30][31][32][33][34][35][36]. We and other groups have also exploited these approaches in order to quantify non-physiological modification of proteins, for example by drugs or their metabolites, which may provide toxicological alerts during pre-clinical pharmaceutical safety testing [37][38][39][40]. These techniques are proving to be very useful for the discovery of potential biomarkers, but in order for the target to be detected with high specificity and sensitivity in clinical samples that exhibit a high degree of matrix noise, more sophisticated MS approaches are now being considered.…”

Adduct Formation and Context Factors in Drug Hypersensitivity: Insight from Proteomic Studies

“…The detoxifying protein GSTP1-1, which contains several reactive cysteines, has been frequently used as a model for exploring adduct formation in vitro and in cells [41,64], although the direct involvement of drug-modified GSTP1-1 in hypersensitivity reactions is not established. In particular, GSTP1-1 has been shown to form adducts with diclofenac and its metabolite 5'-hydroxydiclofenac at cys14 and 47 [65] and with the toxic reactive metabolite of acetaminophen Nacetyl-p-benzoquinoneimine at cys47 [64]. In this last work, a modified iTRAQ method is described to allow a fine quantitative detection of adduct formation that can be correlated with the functional outcome.…”

“…The binding capacity of a nitrosylated form of sulfamethoxazole has been analyzed by in vitro incubations with the synthetic peptide "DS3", observing that this compound reacts mainly with the cysteine residue [63]. The detoxifying protein GSTP1-1, which contains several reactive cysteines, has been frequently used as a model for exploring adduct formation in vitro and in cells [41,64], although the direct involvement of drug-modified GSTP1-1 in hypersensitivity reactions is not established. In particular, GSTP1-1 has been shown to form adducts with diclofenac and its metabolite 5'-hydroxydiclofenac at cys14 and 47 [65] and with the toxic reactive metabolite of acetaminophen Nacetyl-p-benzoquinoneimine at cys47 [64].…”