Application of CYP102A1M11H as a Tool for the Generation of Protein Adducts of Reactive Drug Metabolites

Abstract

Covalent binding of reactive metabolites (RMs) to proteins is considered to be one of the important mechanisms by which drugs can cause tissue damage. To facilitate the study of drug-protein adducts, we developed a potentially generic method for producing high levels of covalently modified proteins. A highly active drug metabolizing P450 BM3 mutant (CYP102A1M11H) is used for drug bioactivation. Because of its His-tag, CYP102A1M11H is easily removed by nickel affinity chromatography, facilitating subsequent characterization of the modified target protein. The applicability of our procedure is demonstrated by the trapping of RMs of acetaminophen (APAP), clozapine (CLOZ), and troglitazone (TGZ) with human glutathione-S-transferase P1-1 (hGST P1-1) as the model target protein. Tryptic digests of hGST P1-1 were subjected to analysis by LC-MS/MS and modified peptides identified by the comparative analysis of tryptic peptides of adducted and nonadducted hGST P1-1. Characteristic MS/MS ions of drug-modified peptides were identified by first searching for expected adduct-masses. Unanticipated drug-peptide adducts were subsequently identified in an unbiased manner by screening for diagnostic MS/MS ions of modified peptides. Reactive intermediates of APAP and CLOZ adducted to cysteine-47 and mass shifts corresponded to the alkylation of N-acetyl-p-benzoquinone imine (NAPQI) and the CLOZ nitrenium ion, respectively. Adduction of TGZ appeared more complex, yielding three different types of adducts to cysteine-47, two adducts to cysteine-14, and a single adduct to cysteine-101. Together, these findings show that P450 BM3 mutants with high capacity to activate drugs into relevant RMs can be employed to produce protein adducts to study the nucleophilic selectivity of highly reactive electrophiles.

Glutathione S-transferase pi trapping method for generation and characterization of drug–protein adducts in human liver microsomes using liquid chromatography–tandem mass spectrometry

“…Electrochemistry can generate large amounts of RMs without biological crude matrices, however, it cannot mimic all CYPs-mediated metabolic reactions due to the lack of interactions between enzymes and substrates [20,21]. In addition, some studies have reported the characterization of drug-protein adducts using the bioengineered mutant cytochrome P450 BM3 (CYP102A1) system coupled with MS analysis [12,13]. CYP102A1 mutants appeared to produce high levels of human relevant RMs of several drugs [22].…”

“…CYP102A1 mutants appeared to produce high levels of human relevant RMs of several drugs [22]. However, in the case of other drugs, the CYP102A1 mutant system might be less useful or non-relevant RMs might be generated [13]. Since HLMs are a predictive in vitro system for human drug bioactivation, it is valuable to use HLMs as a bioactivation system for the method to generate and characterize drug-protein adducts.…”

“…The results suggest that Cys-47 of hGSTP could be a highly reactive target of RMs as expected from its lower pKa value [15]. The covalent modification of hGSTP by NAPQI was previously investigated [12,13]. They used authentic NAPQI standard or bioactivation of APAP for hGSTP modification.…”

“…As described in other papers [12,13], human glutathione S-transferase pi (hGSTP) is a useful model target protein. The protein has actually shown to be a target protein of acetaminophen (APAP) in mice [14].…”

Mass Spectrometric Characterization of Protein Adducts of Multiple P450-Dependent Reactive Intermediates of Diclofenac to Human Glutathione-S-transferase P1-1

“…[19][20][21][22] Recently, the first mass spectrometric analysis of DF-protein adducts reported modification of the single free cysteine residue in β-lactoglobulin upon electrochemical oxidation of DF. 24 CYP102A1M11H has been shown previously to produce high levels of human-relevant RMs of several drugs, including DF, 4'-OH-DF and 5-OH-DF. 10,23 In the present study, a recently developed method is used to characterize the P450dependent covalent protein modification by DF metabolites by mass spectrometry of tryptic digests and by using purified CYP102A1M11H as bioactivation system.…”