Identification and quantification of drug-protein adducts by mass spectrometry

非预期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， 共价加合物与蛋白质，但身份和程度的蛋白质加合物形成往往是难以预测的 和特征。尽管进行了数十年的研究，但目前的方法无法产生全面和全面的结果。 异生物质蛋白质加合物的定量目录。这阻碍了优化新药安全性的进展 并限制了我们确定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 已经开发出创新的蛋白质组学方法，用于发现、表征和定量蛋白质 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， 快速、可靠和定量鉴定和表征药物-蛋白质加合物， 为影响药物安全性评估的人类内收神经节研究定制这些方法。我们将专注于 由药物代谢氧化活化引起的共价蛋白质修饰。我们将使用一组模型 已知在患者中引起不良事件并形成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，我们将测试修饰质量和化学性质的假设， 重组酶系统中活性代谢产物形成的蛋白质加合物的特性预测加合物 在更复杂的系统如肝微粒体和S9组分中形成。通过这项工作， 优化我们的蛋白质组学方法，用于从个体供体制备复杂的人类肝脏。在目标2中， 测试加合物形成在个体之间定量变化的假设， 代谢活性和个体基因型。在这个目标中，我们将建立定量的内收肌， 供体，并定义药物-蛋白质加合物形成的个体间变异性的基础。在目标3中，我们将测试 假设暴露于反应性代谢物的人肝细胞原位分泌蛋白质， 被反应性中间体加合。在这个目标中，我们将建立体外关系， 肝细胞内收体负荷和内收蛋白分泌作为生物标志物。建成后 拟议的研究将是变革性的整合新套件的尖端工具和高维 蛋白质组学数据，以表征导致药物不良反应的关键药物的深内收体特征 反应.所开发的方法将能够评估和量化范围广泛的加合物 在人类蛋白质组中。这些结果将对酶的作用机制产生前所未有的洞察力。 失活，暴露于反应性代谢物后形成的肝内收体和定量关系 加合物的形成和肝脏中的代谢活性之间的关系。这些结果可能最终导致新的药物 降低由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靶向治疗干预措施。

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.