Advanced bioanalytical mass spectrometry methods for investigating protein covalent binding by reactive metabolites

用于研究反应性代谢物的蛋白质共价结合的先进生物分析质谱方法

• 批准号: RGPIN-2016-06034
• 负责人: Sleno, Lekha
• 金额: $ 2.62万
• 依托单位: Université du Québec à Montréal
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=748715
• 关键词: Advanced; bioanalytical; mass; spectrometry; methods

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.

药物代谢的主要作用是使身体从外源性物质中解毒，使它们更具极性，从而更容易清除。然而，一些药物被生物活化成具有与蛋白质共价结合的潜力的活性代谢物。这种结合被认为是特异质药物反应（IDR）的重要原因。因此，反应性代谢物的形成代表了药物化合物的重大责任。这些反应性代谢物可以在体内与蛋白质共价结合并产生显着毒性，但具体机制仍不清楚。我们研究计划的目标是设计新的方法来研究这些毒性相互作用。本研究的主要重点是设计新的策略，以进一步了解反应性代谢物共价结合蛋白质使用的小分子和蛋白质组学为基础的质谱工具的组合，具有以下目标：1）药物的代谢谱2）评估蛋白质结合到特定蛋白质3）确定蛋白质靶点在体外，和4）进行体内实验，以测量观察到的加合物的生物相关性与发现反应性代谢物介导的毒性的特定生物标志物的可能性。科学方法：先进的HPLC-MS/MS分析，结合先进的数据处理工具，将用于代谢产物和修饰蛋白质的结构解析。基于我们以前的研究，我们的目标是扩大这项工作的新策略，研究反应性代谢产物-蛋白质结合。这将涉及使用两种互补方法从体外和体内模型中鉴定蛋白质靶点及其修饰位点。第一种方法涉及使用多维色谱与HR-MS/MS耦合，第二种方法将使用选定的药物炔类似物，使用我们研究小组开发的基于树脂的点击化学方法选择性纯化修饰的靶标。如可能，将比较两种方法的结果，以确认给定反应性代谢产物的蛋白质靶点。一旦确定了蛋白质靶点，我们就可以在不同条件下（体外和体内）检查加合物的形成，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结合到选定的蛋白质的interests.Impact的程度：新的策略将被设计用于研究反应性药物代谢产物的蛋白质修饰的机制。使用已知参与蛋白质共价结合的药物，将采取系统的方法来鉴定蛋白质靶点以及阐明结合的确切位点。这项研究对于更好地理解药物的生物活性及其后果，从而对人类健康产生直接影响，具有非常重要的意义。