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

用于研究反应性代谢物的蛋白质结合机制的先进生物分析质谱方法

• 批准号: 355933-2011
• 负责人: Sleno, Lekha
• 金额: $ 2.55万
• 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: 2015
• 资助国家: 加拿大
• 起止时间: 2015-01-01 至 2016-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=572698
• 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 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).

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。这些反应性代谢物可与体内蛋白质结合，并导致这些药物的给药具有潜在的非常危险性。我们研究计划的目标是设计新的创新方法来研究这些毒性相互作用。我们的重点是设计一种策略，以进一步了解结合的反应性代谢产物的蛋白质使用现代质谱工具研究小分子和蛋白质。 先进的液相色谱-质谱（LC-MS）仪器，以及先进的数据处理工具，将用于活性代谢物和修饰蛋白质的结构解析。拟议的研究计划包括确定已知为活性代谢物体内蛋白质靶点的蛋白质上的修饰位点。我们的原始方法基于创新的分析方法，包括蛋白质靶点的亲和纯化和质谱数据的选择性过滤。我们目前正在寻求一种有前途的途径来识别复杂样品中的蛋白质靶点，即使用药物炔类似物标记修饰的蛋白质，然后使用点击化学进行标记和选择性纯化。我们还将采用稳定同位素标记的药物来选择性地过滤复杂的MS数据，以获得特定的同位素模式。一旦确定了蛋白质靶点，将在不同条件下研究加合物的形成，以更好地了解反应性代谢产物与蛋白质共价结合的机制。 新的策略将被设计用于研究蛋白质与活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点，并阐明结合机制。这项研究应该有非常重要的后果，更好地了解药物代谢和生物活化，以及特异质药物反应（IDR）。