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异生物质中解毒，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反应性代谢物，具有与蛋白质共价结合的潜力。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对药物化合物来说是一个重大的责任。这些反应性代谢物可以在体内与蛋白质共价结合并引起显着的毒性，但确切的机制仍不清楚。我们研究计划的目标是设计新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策略，结合使用小分子和基于蛋白质组的质谱工具进一步了解反应性代谢物与蛋白质的共价结合，其目标如下：1) 药物的代谢分析2) 评估蛋白质与特定蛋白质的结合3 ) 体外识别蛋白质靶标，4) 进行体内实验，测量观察到的加合物的生物学相关性，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：高级HPLC-MS/MS 分析与复杂的数据处理工具相结合，将用于代谢物和修饰蛋白质的结构阐明。基于我们之前的研究，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点。第一种方法涉及使用与 HR-MS/MS 联用的多维色谱法，第二种方法将使用我们研究小组开发的基于树脂的点击化学方法，使用选定的药物炔类似物选择性纯化修饰的靶标。如果可能，将比较两种方法的结果，以确认给定反应性代谢物的蛋白质靶标。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对蛋白质修饰的机制。使用已知参与蛋白质共价结合的药物，将采用系统方法来识别蛋白质靶标并阐明结合的确切位点。这项研究应该对更好地了解药物生物活化及其后果产生非常重要的影响，从而对人类健康产生直接影响。