Application of CYP102A1M11H as a tool for the generation of protein adducts of reactive drug metabolites.

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Boerma JS, Vermeulen NP, Commandeur JN

Application of CYP102A1M11H as a tool for the generation of protein adducts of reactive drug metabolites.

Chem Res Toxicol. 2011 Aug 15;24(8):1263-74. doi: 10.1021/tx2001515. Epub 2011 Jun 16.

PubMed ID

21639118 [ View in PubMed

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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.

DrugBank Data that Cites this Article

Drug Targets

Drug	Target	Kind	Organism	Pharmacological Action	Actions
Clozapine	Glutathione S-transferase P	Protein	Humans	Unknown	Not Available	Details
Troglitazone	Glutathione S-transferase P	Protein	Humans	Unknown	Not Available	Details

Drug Enzymes

Drug	Enzyme	Kind	Organism	Pharmacological Action	Actions
Acetaminophen	Glutathione S-transferase P	Protein	Humans	Unknown	Substrate	Details