基于蛋白质沉淀的药物靶点筛选方法的研究进展

Recent advances in protein precipitation-based methods for drug-target screening

药物靶点是体内与药物分子结合的生物大分子。因此药物靶点的系统鉴定对于充分认识药物作用机制、疗效以及可能的副作用至关重要;同时全面的靶点识别可以加速药物发现及候选药物筛选的进程。生物质谱具有采集速度快、分辨率和灵敏度高等特点,是蛋白质组深度鉴定和准确定量的关键工具;基于质谱的蛋白质组学技术有助于系统性鉴定与小分子药物结合的蛋白质,阐述蛋白质-药物相互作用,在药物靶点筛选领域大显身手。常用的先进行标记再结合亲和富集的药物靶点表征方法需要对药物分子化学衍生,这既耗时也可能会改变药物的亲和力,同时衍生基团产生的空间效应可能会阻断药物和靶点相互作用,加大了靶点鉴定的难度。因此,无需衍生的药物靶点鉴定方法受到广泛关注。蛋白质受到温度、pH、变性剂以及机械力等条件的影响会发生变性、展开和沉淀。小分子药物与蛋白质结合可能改变蛋白质折叠平衡,稳定靶蛋白的构象,与游离蛋白相比,更能抵抗不同条件诱导的蛋白质沉淀。基于以上原理,联合蛋白质组学和生物质谱技术,目前已经发展了多种基于蛋白质沉淀的药物靶点规模化鉴定方法,包括热蛋白质组分析方法、溶剂诱导蛋白质沉淀方法、机械应力诱导蛋白质沉淀方法、pH依赖性蛋白质沉淀方法等,用于蛋白质组范围内的药物靶点筛选。本综述介绍了该领域近十年报道的药物靶点发现和药物-蛋白质相互作用研究方法,总结了这些方法的特点及其在药物疗效评价、药物发现等方面的发展前景。

Drug targets are biological macromolecules that bind drug molecules in vivo.Therefore,the system-wide identification of drug targets plays a vital role in fully understanding the mechanism of drug action,efficacy,and side effects.The unbiased screening of drug targets may accelerate the process of drug discovery and candidate screening.Mass spectrometry is a key tool for large-scale protein identification and accurate quantification owing to its high acquisition speed,resolution,and sensitivity.Mass spectrometry-based proteomics has been widely used for drug-target screening.It can systematically identify the protein-target landscape of a drug and elucidate drug-protein interactions.Commonly used drug-target characterization methods,such as labeling-based affinity enrichment,require the chemical derivatization of drug molecules,which is not only time-consuming but may also affect the affinity of the drug towards its targets.Furthermore,the spatial effects of the derivatization groups may block interactions between the drug and its targets.Considering the disadvantages of affinity-enrichment methods,strategies that do not require chemical derivatization have received widespread attention.Proteins may undergo denaturation,unfolding,and precipitation under different conditions such as high temperatures,extreme pH,denaturants,and mechanical stress.Binding to small-molecule drugs may alter the folding balance of target proteins.The conformational stability of target proteins can be stabilized by binding with drugs,and protein-drug complexes are more resistant than free proteins to the precipitation induced by different conditions.Based on this mechanism,various large-scale drug-target identification methods using protein precipitation have been developed by combining proteomics and mass spectrometry analysis,including thermal proteome profiling and solvent-,mechanical stress-,and pH-induced protein precipitation.These methods have been successfully applied to the characterization of small-molecule drug targets.In this review,we describe the protein precipitation-based methods used for the high-throughput discovery of drug targets and elucidation of the interactions between drugs and proteins in the past decade.We also summarize the characteristics of each method and discuss their application potential in drug-efficacy evaluation and drug discovery.