Identification and quantification of drug-protein adducts by mass spectrometry

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Unanticipated adverse drug reactions remain a major cause of post marketing withdrawals of drugs and of restricted access to new medications. Adverse drug reactions are often caused by reactive metabolites that form covalent adducts with proteins, but the identity and extent of protein adducts formed is often difficult to predict and characterize. Despite decades of research, current methods are unable to produce a comprehensive and quantitative catalog of xenobiotic protein adducts. This hinders progress in optimizing safety of novel medications and limits our ability to define mechanisms of clinically observed adverse drug reactions. To bridge this gap, we have developed innovative proteomic methods for discovery, characterization and quantification of protein adducts in simple and complex biological matrices. The goal of this proposal is to establish these methods for rapid, reliable and quantitative identification and characterization of drug-protein adducts, and uniquely customize these methods for human adductomics research impacting drug safety assessment. We will focus on covalent protein modifications resulting from metabolic oxidative activation of drugs. We will use a set of model compounds that are known to cause adverse events in patients and form reactive metabolites that likely result in protein adducts. In our aim 1 we will test the hypothesis that the modification masses and chemical characteristics of protein adducts formed by reactive metabolites in recombinant enzyme systems predict adduct formation in more complex systems such as liver microsomes and S9 fractions. Through this work we will optimize our proteomics methods for complex human liver preparations from individual donors. In aim 2 we will test the hypothesis that adduct formation varies quantitatively between individuals and due to differences in metabolic activity and individual genotype. In this aim we will establish quantitative adductomics for individual donors and define the basis for inter-individual variability in drug-protein adduct formation. In aim 3 we will test the hypothesis that human hepatocytes exposed to reactive metabolites generated in situ secrete proteins that have been adducted by the reactive intermediates. In this aim we will establish the in vitro relationship between hepatocyte adductomic burden and secretion of adducted proteins as biomarkers. When completed, the proposed studies will be transformative in integrating novel suite of cutting-edge tools and high-dimensional proteomics data to characterize the deep adductomic profiles of key drugs resulting in adverse drug reactions. The methods developed will enable the assessment and quantification of a broad range of adducts across the human proteome. The results will generate unprecedented insight into mechanisms of enzyme inactivation, liver adductomes formed after exposure to reactive metabolites and quantitative relationships between adduct formation and metabolic activity in the liver. The results may ultimately lead to novel drug discovery approaches that mitigate risk of adverse drug reactions resulting from adduct formation and to development of targeted therapeutic interventions designed to treat adverse drug reactions.