Advanced bioanalytical mass spectrometry methods for investigating protein covalent 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 easily cleared. Some drugs, however, are bioactivated into reactive metabolites with the potential to covalently bind to proteins. This binding is considered to be an important cause of idiosyncratic drug reactions (IDR). As a result, the formation of reactive metabolites represents a significant liability for pharmaceutical compounds. These reactive metabolites can covalently bind to proteins in vivo and cause significant toxicity, however the exact mechanisms are still quite unclear. The goal of our research program is to design new methods to study these toxic interactions. The primary focus of this research is devising novel strategies to further understand reactive metabolite covalent binding to proteins using a combination of small molecule and proteomic-based mass spectrometry tools with the following objectives:1) Metabolic profiling of drugs2) Assess protein binding to specific proteins3) Identify protein targets in vitro, and4) Perform in vivo experiments to measure the biological relevance of adducts observed with the possibility of discovering specific biomarkers for reactive metabolite-mediated toxicity.Scientific approach: Advanced HPLC-MS/MS analysis, combined with sophisticated data processing tools, will be employed for the structural elucidation of metabolites and modified proteins. Based on our previous research, we are aiming to expand on this work with new strategies for studying reactive metabolite-protein binding. This will involve identifying protein targets and their modification sites from both in vitro and in vivo models using two complementary approaches. The first involves using multidimensional chromatography coupled to HR-MS/MS and the second approach will use selected alkyne analogs of drugs for selective purification of modified targets using a resin-based click chemistry method developed in our research group. When possible, results from both methods will be compared for confirming protein targets of a given reactive metabolite. Once protein targets are identified, we can examine adduct formation under different conditions (in vitro and in vivo) in order to better understand the mechanism of protein covalent binding by these reactive metabolites. For this, we will develop novel methods for measuring the extent of binding to selected proteins of interest.Impact: New strategies will be devised for studying the mechanisms of protein modifications by reactive drug metabolites. Using drugs known to be involved in protein covalent binding, a systematic approach will be taken to identify protein targets as well as elucidate the exact site of binding. This research should have very important consequences for better understanding drug bioactivation and its consequences, and therefore a direct impact on human health.