Advanced bioanalytical mass spectrometry methods for investigating protein covalent 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，因此很容易清除。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。这种结合被认为是特异性药物反应(IDR)的重要原因。因此，反应性代谢物的形成对药物化合物来说是一个重大的负担。这些反应性代谢产物在体内能与蛋白质共价结合，并引起显著的毒性，但其确切机制仍不清楚。我们研究计划的目标是设计新的方法来研究这些有毒的相互作用。这项研究的主要重点是设计新的策略，以结合使用小分子和基于蛋白质组的质谱学工具来进一步了解反应性代谢物与蛋白质的共价结合，目标如下：1)Drugs的代谢谱2)评估蛋白质与特定蛋白质的结合3)在体外识别蛋白质靶标，以及4)进行体内实验，以测量观察到的加合物的生物相关性，并有可能发现反应性代谢物介导的毒性的特定生物标志物。科学方法：先进的LC-MS/MS分析，结合复杂的数据处理工具，将被用于代谢物和修饰蛋白质的结构阐明。基于我们以前的研究，我们的目标是通过研究反应性代谢物-蛋白质结合的新策略来扩展这项工作。这将涉及使用两种互补的方法从体外和体内模型中识别蛋白质靶标及其修饰位置。第一种方法涉及使用与HR-MS/MS联用的多维色谱，第二种方法将使用选定的炔烃类似物，使用我们研究组开发的基于树脂的点击化学方法来选择性纯化修饰目标。如果可能，两种方法的结果将进行比较，以确定给定反应性代谢物的蛋白质靶标。一旦确定了蛋白质靶标，我们就可以研究不同条件下(体外和体内)加合物的形成，以便更好地了解这些反应性代谢产物与蛋白质共价结合的机制。为此，我们将开发新的方法来测量与选定的感兴趣蛋白质的结合程度。影响：将设计新的策略来研究反应性药物代谢物修饰蛋白质的机制。利用已知的参与蛋白质共价结合的药物，将采取系统的方法来识别蛋白质靶标以及阐明结合的确切位置。这项研究对于更好地理解药物生物激活及其后果，从而对人类健康产生直接影响，应该具有非常重要的意义。

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.