Advanced bioanalytical mass spectrometry methods for investigating the mechanism of protein binding by reactive metabolites

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The main role of drug metabolism is to detoxify the body from xenobiotics, rendering them more polar and thus more easily cleared. Some drugs, however, are bioactivated into reactive metabolites with the potential to covalently bind to proteins. These reactive metabolites can bind to proteins in vivo and cause administration of these drugs to be potentially very dangerous. The goal of our research program is to design new innovative methods to study these toxic interactions. Our focus is to devise a strategy to further understand binding of reactive metabolites to proteins using a combination of modern mass spectrometry tools for studying both small molecules and proteins. Advanced liquid chromatography-mass spectrometry (LC-MS) instrumentation, together with sophisticated data processing tools, will be utilized for the structural elucidation of reactive metabolites and modified proteins. The proposed research program involves the identification of modifications sites on proteins known to be in vivo protein targets of reactive metabolites. Our original approach based on innovative analytical methods including affinity purification of protein targets and selective filtering of mass spectral data. A promising avenue we are currently pursuing for identifying protein targets in complex samples is using drug alkyne analogs for labeling modified proteins followed by tagging and selective purification using click chemistry. We will also be employing stable-isotope labeled drugs to selectively filter complex MS data for specific isotope patterns. Once protein targets are identified, adduct formation will be studied under different conditions in order to better understand the mechanism of protein covalent binding by reactive metabolites. Novel strategies will be devised for studying the mechanisms of protein binding to reactive drug metabolites. A systematic approach will be taken to identify important protein targets and their modification sites as well as elucidate the mechanism of binding. This research should have very important consequences for better understanding drug metabolism and bioactivation as well as idiosyncratic drug reactions (IDRs).