Identification of the Protein Targets of the Reactive Metabolite of Teucrin A in Vivo in the Rat

Abstract

Covalent modification of proteins is associated with the toxicity of many electrophiles, and the identification of relevant in vivo protein targets is a desirable but challenging goal. Here, we describe a strategy for the enrichment of adducted proteins utilizing single-chain fragment variable (ScFv) antibodies selected using phage-display technology. Teucrin A is a furan-containing diterpenoid found in the herb germander that is primarily responsible for the herb's hepatotoxicity in rodents and humans following metabolic activation by cytochrome P450 enzymes. Conjugates of the 1,4-enedial derivative of teucrin A, its presumed toxic metabolite, with lysine- and cysteine-containing peptides were synthesized and used to select ScFvs from a rodent phage-displayed library, which recognized the terpenoid moiety of the teucrin-derived adducts. Immunoaffinity isolation of adducted proteins from rat liver homogenates following administration of a toxic dose of teucrin A afforded a family of proteins that were identified by liquid chromatography/tandem mass spectrometry. Of the 46 proteins identified in this study, most were of mitochondrial and endoplasmic reticulum origin. Several cytosolic proteins were found, as well as four peroxisomal and two secreted proteins. Using Ingenuity Pathway Analysis software, two significant networks involving the target genes were identified that had major functions in gene expression, small molecule biochemistry, and cellular function and maintenance. These included proteins involved in lipid, amino acid, and drug metabolism. This study illustrates the utility of chemically synthesized biological conjugates of reactive intermediates and the potential of the phage display technology for the generation of affinity reagents for the isolation of adducted proteins.