Identification and quantification of drug-protein adducts by mass spectrometry

意想不到的药物不良反应仍然是药物上市后停药的主要原因 限制新药的使用。药物不良反应通常是由活性代谢物引起的 与蛋白质的共价加合物，但蛋白质加合物形成的一致性和程度通常很难预测 并刻画出。尽管进行了几十年的研究，但目前的方法无法产生全面和 外源蛋白加合物定量目录。这阻碍了在优化新药安全性方面的进展 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。为了弥合这一差距，我们 开发了发现、表征和量化蛋白质的创新蛋白质组学方法 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来 快速、可靠和定量地鉴定和表征药物-蛋白质加合物，并且唯一 为影响药物安全性评估的人类内收手术研究定制这些方法。我们将重点关注 药物代谢氧化活化引起的共价蛋白质修饰。我们将使用一套模型 已知会导致患者不良事件并形成活性代谢物的化合物 在蛋白质加合物中。在我们的目标1中，我们将检验这样的假设：修饰质量和化学物质 重组酶系统中反应性代谢产物形成的蛋白质加合物的特性预测加合物 在更复杂的系统中形成，如肝微粒体和S9组分。通过这项工作，我们将 优化我们的蛋白质组学方法，从个人捐赠者那里获得复杂的人类肝脏制剂。在《目标2》中我们将 检验加合物形成因个体不同而不同的假设 代谢活动和个体的基因型别。在这一目标中，我们将为个人建立定量的内收运算 并确定药物-蛋白质加合物形成过程中个体间差异的基础。在目标3中，我们将测试 假设暴露在反应性代谢物中的人肝细胞产生原位分泌的蛋白质， 已被活性中间体加合。在这一目标中，我们将建立体外关系 肝细胞内收负荷和外收蛋白的分泌作为生物标志物。完成后， 拟议的研究将在集成新型尖端工具和高维 用蛋白质组学数据表征导致药物不良反应的关键药物的深层内收图谱 反应。开发的方法将能够对广泛的加合物进行评估和量化 横跨人类蛋白质组。这一结果将为酶的作用机制带来前所未有的洞察力 活性代谢物暴露后形成的失活、肝内收瘤和数量关系 肝脏中加合物的形成和代谢活动之间的关系。这一结果可能最终导致新药的问世。 发现降低加合物形成引起的药物不良反应风险的方法 开发旨在治疗药物不良反应的针对性治疗干预措施。

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.