Identification and quantification of drug-protein adducts by mass spectrometry

通过质谱法鉴定和定量药物-蛋白质加合物

基本信息

批准号：
10687252
负责人：
Nina Isoherranen
金额：
$ 41.19万
依托单位：
UNIVERSITY OF WASHINGTON
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-09-01 至 2026-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10687252
关键词：
Accounting Address Adverse event Amino Acids Biochemical Biological Biological Markers Calibration Catalogs Cells Characteristics Chemicals Chlorine Clinical Complex Computer software Cytochrome P450 Data Detection Development Discipline Early identification Enzymes Event Exposure to Fractionation Gases Genotype Goals Hepatocyte Human In Situ In Vitro Incubated Individual Insecta Isotope Labeling Isotopes Label Life Liver Liver Microsomes Mass Spectrum Analysis Membrane Metabolic Metabolic Activation Methods Modeling Modification Pathway interactions Patients Peptides Performance Pharmaceutical Preparations Phase Post-Translational Protein Processing Preparation Protein Secretion Protein Structure Initiative Proteins Proteome Proteomics Recombinants Reference Standards Research Resolution Safety Sampling Software Validation Spectrometry System Technology Testing Toxic effect Translating Translational Research Variant Withdrawal Work Xenobiotics access restrictions adduct adverse drug reaction clinically relevant design drug development drug discovery drug withdrawal enzyme mechanism high dimensionality innovation insight inter-individual variation ion mobility medication safety novel novel therapeutics post-market prevent rapid technique risk mitigation safety assessment targeted treatment therapy design tool toxicant

项目摘要

Unanticipated adverse drug reactions remain a major cause of post marketing withdrawals of drugs and of restricted access to new medications. Adverse drug reactions are often caused by reactive metabolites that form covalent adducts with proteins, but the identity and extent of protein adducts formed is often difficult to predict and characterize. Despite decades of research, current methods are unable to produce a comprehensive and quantitative catalog of xenobiotic protein adducts. This hinders progress in optimizing safety of novel medications and limits our ability to define mechanisms of clinically observed adverse drug reactions. To bridge this gap, we have developed innovative proteomic methods for discovery, characterization and quantification of protein adducts in simple and complex biological matrices. The goal of this proposal is to establish these methods for rapid, reliable and quantitative identification and characterization of drug-protein adducts, and uniquely customize these methods for human adductomics research impacting drug safety assessment. We will focus on covalent protein modifications resulting from metabolic oxidative activation of drugs. We will use a set of model compounds that are known to cause adverse events in patients and form reactive metabolites that likely result in protein adducts. In our aim 1 we will test the hypothesis that the modification masses and chemical characteristics of protein adducts formed by reactive metabolites in recombinant enzyme systems predict adduct formation in more complex systems such as liver microsomes and S9 fractions. Through this work we will optimize our proteomics methods for complex human liver preparations from individual donors. In aim 2 we will test the hypothesis that adduct formation varies quantitatively between individuals and due to differences in metabolic activity and individual genotype. In this aim we will establish quantitative adductomics for individual donors and define the basis for inter-individual variability in drug-protein adduct formation. In aim 3 we will test the hypothesis that human hepatocytes exposed to reactive metabolites generated in situ secrete proteins that have been adducted by the reactive intermediates. In this aim we will establish the in vitro relationship between hepatocyte adductomic burden and secretion of adducted proteins as biomarkers. When completed, the proposed studies will be transformative in integrating novel suite of cutting-edge tools and high-dimensional proteomics data to characterize the deep adductomic profiles of key drugs resulting in adverse drug reactions. The methods developed will enable the assessment and quantification of a broad range of adducts across the human proteome. The results will generate unprecedented insight into mechanisms of enzyme inactivation, liver adductomes formed after exposure to reactive metabolites and quantitative relationships between adduct formation and metabolic activity in the liver. The results may ultimately lead to novel drug discovery approaches that mitigate risk of adverse drug reactions resulting from adduct formation and to development of targeted therapeutic interventions designed to treat adverse drug reactions.

意外的药物不良反应仍然是药物上市后撤回的主要原因限制获得新药物。药物不良反应通常是由形成反应性代谢物引起的与蛋白质共价加合物，但形成的蛋白质加合物的身份和程度通常难以预测并表征。尽管经过了数十年的研究，目前的方法仍无法产生全面且可靠的结果。外源蛋白加合物的定量目录。这阻碍了优化新药安全性的进展并限制了我们定义临床观察到的药物不良反应机制的能力。为了弥补这一差距，我们开发了创新的蛋白质组学方法，用于蛋白质的发现、表征和定量简单和复杂生物基质中的加合物。本提案的目标是建立这些方法药物-蛋白质加合物的快速、可靠和定量鉴定和表征，以及独特的为影响药物安全评估的人类加成组学研究定制这些方法。我们将重点关注药物代谢氧化激活导致的共价蛋白质修饰。我们将使用一组模型已知会引起患者不良事件并形成可能导致的反应性代谢物的化合物在蛋白质加合物中。在我们的目标 1 中，我们将检验修饰质量和化学物质的假设重组酶系统中反应性代谢物形成的蛋白质加合物的特征预测加合物在更复杂的系统中形成，例如肝微粒体和 S9 组分。通过这项工作我们将优化我们针对来自个体捐献者的复杂人类肝脏制剂的蛋白质组学方法。在目标 2 中，我们将检验以下假设：加合物的形成在个体之间存在数量上的差异，并且由于代谢活动和个体基因型。为此，我们将为个体建立定量加合组学供体并定义药物-蛋白质加合物形成的个体间变异的基础。在目标 3 中我们将测试假设人类肝细胞暴露于原位产生的反应性代谢物，会分泌蛋白质，已被反应中间体加合。为此，我们将建立体外关系肝细胞内合负荷和加合蛋白的分泌作为生物标志物。完成后，将拟议的研究将在整合新颖的尖端工具套件和高维蛋白质组学数据可表征导致不良药物的关键药物的深层内合特征反应。开发的方法将能够对广泛的加合物进行评估和定量跨越人类蛋白质组。这些结果将对酶的机制产生前所未有的见解失活、暴露于反应性代谢物后形成的肝加合物和定量关系加合物形成和肝脏代谢活动之间的关系。结果可能最终导致新药的诞生降低加合物形成引起的药物不良反应风险的发现方法开发旨在治疗药物不良反应的有针对性的治疗干预措施。