Identifying Protein–Drug Interactions in Cell Lysates Using Histidine Hydrogen Deuterium Exchange

Identifying the targets of a drug is critical to understand the mechanism of action and predicts possible side effects. The conventional approach is capturing interacting proteins by affinity purification. However, it requires drugs to be immobilized to a solid support or derivatized with chemical moieties used for pulling down interacting proteins. Such covalent modifications to drugs may mask a critical recognition site for or alter the binding affinity to their targets. To overcome the drawback, several methods that do not require covalent modifications to drugs have been developed. These methods identify targets by detecting proteins whose thermodynamic stability is enhanced in the presence of drugs. Although the utility of these methods has been demonstrated, the difficulty in identifying low abundant targets is the common problem of these methods. We have developed a new target identification method that increases the likelihood of identifying low abundant targets. The method uses histidine-hydrogen deuterium exchange (His-HDX) as a readout technique to probe the changes in protein stability induced by drugs. The workflow involves incubating cell lysates in various concentrations of a protein denaturant in the presence and absence of a drug in D2O followed by digestion of the proteins, enrichment of His-containing peptides, and analysis of the enriched His-peptides by liquid chromatography–tandem mass spectrometry (LC–MS/MS). The developed method was successfully applied to identify the interaction between endogenously expressed MAPK14 and its inhibitor in HEK293 cell lysates. The implementation of selective enrichment of histidine-containing peptides in the workflow was a key that enabled identifying the MAPK14–inhibitor interaction.确定药物作用靶点，对于阐明其作用机制、预测潜在不良反应至关重要。传统方法通过亲和纯化捕获与药物相互作用的蛋白质，但该方法需将药物固定于固相载体，或通过用于富集相互作用蛋白的化学基团对药物进行衍生化修饰。此类对药物的共价修饰，可能会遮蔽其关键识别位点，或改变其与靶点的结合亲和力。为克服该缺陷，学界已开发出多种无需对药物进行共价修饰的靶点鉴定方法。此类方法通过检测药物存在下热力学稳定性提升的蛋白质，来鉴定药物靶点。尽管此类方法的应用价值已得到验证，但鉴定低丰度靶点仍是其普遍存在的难题。本团队开发了一种可提升低丰度靶点鉴定成功率的新型药物靶点鉴定方法，该方法以组氨酸-氢氘交换（histidine-hydrogen deuterium exchange, His-HDX）作为读出技术，用于探测药物诱导的蛋白质稳定性变化。该实验流程包括：在含有重水（D2O）的体系中，分别添加与不添加药物，将细胞裂解液与不同浓度的蛋白质变性剂共同孵育；随后对蛋白质进行酶解、富集含组氨酸的肽段，并通过液相色谱-串联质谱（liquid chromatography–tandem mass spectrometry, LC–MS/MS）对富集得到的组氨酸肽段进行分析。该方法已成功应用于HEK293细胞裂解液中，鉴定内源性表达的丝裂原活化蛋白激酶14（MAPK14）与其抑制剂的相互作用。实验流程中引入的含组氨酸肽段选择性富集步骤，是成功鉴定MAPK14-抑制剂相互作用的关键环节。