一种基于相变的高通量筛选蛋白互作调控物的新方法探究

Protein-protein interactions (PPIs) play a central role in virtually all biological processes. Aberrant PPIs can cause diseases and thus exploring modulators to correct aberrant PPIs is an important way to combat the underlying diseases. To develop PPI modulators relies heavily on high-throughput screening (HTS) approaches. Commonly used HTS methods have various limitations. Therefore, this research proposal is to establish a novel HTS technique based on phase transition. "Phase transition" is a new biological concept and the phase transition-related research has become a hot topic in biological field. The phase transition-based HTS technique could convert microscopic PPI modulations to readily detectible fluorescence signal change within phase-separated protein-rich compartments. By now the novel system has been established and preliminary experiments indicated that it is effective. In this study, we will use this method to screen for inhibitors of P53/MDM2 interaction from a small molecule library composed of known drugs including potent P53/MDM2 inhibitors. In this process, we seek to further optimize the method and to establish a generic screening and data analysis protocol, moreover, we will analyze the results extensively to further verify the feasibility of the method. The method is sensitive, easy to implement, low-cost; its signal is readily detectible; and its protein-rich compartments resemble the environment in vivo. As such, we believe that our successful demonstration of the method will provide a novel alternative to various HTS projects and the prevalent utilization of it is anticipated in the near future.

蛋白互作在生命活动中发挥重要作用，互作异常往往导致疾病的发生，发掘蛋白互作调控物以纠正异常互作已成为疾病治疗的一个重要途径。对互作调控物的发掘依赖于高通量筛选技术，目前常用的筛选技术各有不足，本项目拟建立一种基于相变的高通量筛选方法。“相变”是个全新的生物学概念，目前广受关注，相变生物学已成为新的生物学研究领域和热点。本项目拟创建的方法可将微观的蛋白互作及其调控等生化过程转化为直观的荧光信号强度变化。目前该方法已初步通过了可行性验证。在此基础上，本研究拟以筛选P53/MDM2的互作抑制剂为例，对该方法进行优化以建立普遍适用的筛选程序和数据分析方法，并通过分析筛选结果进一步论证该方法的可行性。该方法具有灵敏度高、操作简单、快捷直观、成本低廉、接近体内真实环境等优势。相信该方法在本项目的成功运用能够为鉴定蛋白互作和高通量筛选互作调控物提供一种新的策略，并会在今后的高通量筛选中得到广泛运用。

蛋白互作异常往往导致疾病发生，发掘互作调控物以纠正异常互作已成为疾病治疗的一个重要途径。对互作调控物的发掘依赖于高通量筛选技术，传统筛选技术各有不足，本项目旨在建立一种基于相变的高通量筛选方法。“相变”是个全新的生物学概念，目前广受关注，相变生物学已成为新的生物学研究领域和热点。目前，本项目完成了既定目标，成功开发了一套基于相变的鉴定蛋白互作及高通量筛选互作调控物的新技术，命名为CEBIT。该技术工作原理为，将一对互作分子的受体与相变骨架蛋白结合，使该受体聚集于相变液滴中；同时将其配体进行荧光标记，由于分子间的互作，荧光标记的配体会被招募到相变液滴中。当加入待测调控物时，通过检测相变液滴中表征配体的荧光强度变化，确定待测物对分子互作是否具有调控作用以及具有何种（抑制或促进）调控作用。该系统不仅适用于蛋白互作抑制物的筛选，而且适用于检测化学修饰的生物大分子（包括DNA、RNA、蛋白质）与配体的互作，也适用于相关修饰酶的活性检测。该成果发表于Journal of Biological Chemistry期刊。CEBIT技术适用于体外筛选。在此基础上，我们还开发了一套细胞水平鉴定蛋白互作及高通量筛选互作调控物的新技术，命名为CoPIC。CoPIC与CEBIT工作原理相同，只是蛋白的表达和互作发生在细胞内。我们将CoPIC技术应用于新冠病毒SARS-CoV-2的蛋白互作研究，绘制了病毒复制相关蛋白的互作图谱，并对其中的多元互作进行了直接/间接互作鉴定。该成果发表于Cell Reports期刊，为项目计划外成果。这两套系统将微观的生物大分子互作及其调控等生化过程转化为直观的荧光信号强度变化，覆盖了体外和细胞层面的各类大分子互作及调控物检测，适用广泛，且灵敏度高、操作简单、快捷直观、成本低廉，弥补了传统筛选技术的不足，在药物筛选方面极具应用前景，可助力药物开发和基础医学研究。