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

药物新陈代谢的主要作用是使身体免受外来物质的毒害，使它们更具极性，因此更容易清除。然而，一些药物被生物激活为反应性代谢物，有可能与蛋白质共价结合。这些反应性代谢物可以与体内的蛋白质结合，导致服用这些药物具有潜在的非常危险。我们研究计划的目标是设计新的创新方法来研究这些有毒的相互作用。我们的重点是设计一种策略，利用研究小分子和蛋白质的现代质谱学工具的组合，进一步了解反应性代谢物与蛋白质的结合。 先进的液质联用(LC-MS)仪器和先进的数据处理工具将被用于活性代谢物和修饰蛋白质的结构鉴定。拟议的研究计划涉及识别已知为体内活性代谢物靶标的蛋白质上的修饰位点。我们最初的方法基于创新的分析方法，包括亲和纯化蛋白质目标和选择性过滤质谱学数据。我们目前正在探索的一种在复杂样本中识别蛋白质靶标的有希望的方法是使用药物炔类似物来标记修饰的蛋白质，然后使用点击化学来标记和选择性纯化。我们还将使用稳定同位素标记的药物来选择性地过滤特定同位素模式的复杂MS数据。一旦确定了蛋白质靶标，将研究不同条件下加合物的形成，以更好地了解蛋白质与反应性代谢产物共价结合的机制。 将设计新的策略来研究蛋白质与反应性药物代谢物结合的机制。将采取系统的方法来确定重要的蛋白质靶点及其修饰位置，并阐明结合的机制。这项研究对于更好地理解药物代谢和生物激活以及特殊的药物反应(IDR)应该有非常重要的结果。

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).