真实生物体系中靶蛋白-配体动态结合模式及结合位点的全景式研究

Conventional methodologies that can probe ligand-target protein interactions and characterize their binding sites usually rely on recombinant protein systems. However, this ignores the differences of exposed sites and topological structures of the recombinant protein from those of the native form present in biological systems. Such differences can result in discrepancies between ligand-target protein interactions in the recombinant and native biological systems, and ultimately lead to failures of drug discovery. Consequently, we sought to tackle this problem by investigating ligand-protein interactions in native biological systems with new analytical techniques. Using PKM2 and FXR as target proteins, we propose to firstly employ native MS to detect ligand-target protein complexes in native cellular environment and identify ligands of high affinity in a high throughput, followed by identifying those ligands that interact with the target protein in a specific manner via the newly established Thermal shift-induced Native Denatured Exchange Mass Spectrometry (TiNDX-MS) approach. Subsequently, we aim to design and synthesize versatile cell membrane-permeable Photoactivatable Probes (PP). Using hybrid PP combinations, the target protein can be labeled in a significantly improved coverage directly from native biological systems. Next, since ligand-target binding would impair the ability of PP to label certain sites of the target protein due to steric hindrance, we can probe ligand-protein interactions and identify the corresponding binding sites in a panoramic manner with the aid of quantitative proteomics techniques. In summary, this project aims to establish new techniques to probe dynamic ligand-target protein interactions and identify their binding sites in native biological systems. This analytical platform will be widely applicable to target-oriented drug design and screening, and is expected to improve the successful rate and efficiency of the drug R&D process.

目前针对靶蛋白与药物分子结合模式与位点分析多依赖于重组蛋白，忽略了单一蛋白体系与真实生物环境中蛋白构象、结合位点暴露的差异，无法模拟天然的配体-靶标结合状态，这是导致创新药物研发失败的重要原因。为突破这一瓶颈，本项目创新性探索配体与靶蛋白在真实生物体系中动态结合模式在线分析新技术。拟以丙酮酸激酶、法尼醇受体为靶蛋白，首先基于原态质谱技术检测化合物在自然细胞环境中与靶蛋白结合情况，高通量筛选出亲和力强的配体；再建立热偏移诱导的原态-变性转换质谱技术，在线筛选出特异性结合的配体；最后，设计并合成可穿透细胞膜的光激活探针阵列，在细胞、组织中优化探针组合，达到深度覆盖靶蛋白，定量分析与配体结合后标记效率发生显著性变化的位点，揭示配体的结合域或诱导的变构模式。本项目通过建立真实生物环境中靶蛋白与配体结合模式与位点动态分析新技术，为新药设计与筛选提供具有普适性的方法，提高创新药物研发成功率及效率。

本研究致力于解决基于重组蛋白获取的配体筛选和互作结构表征的结果无法完全反映真实生理环境中靶蛋白存在的状态，影响针对靶蛋白的药物筛选和优化在细胞和体内模型的实际药效这一科学问题；开发了结合原态质谱（native MS）与穿透细胞膜的化学探针和光激活探针阵列的新标记技术，探索配体与靶蛋白在真实生物体系中结合的动态模式。研究首先建立了原态-变性质谱切换离子源(Native Denatured Exchange)-质谱（MS）技术，通过改变电离液滴中变性试剂的比例，实现在线获得配体-靶蛋白复合物在自然的细胞环境中从原态到变性状态的变化情况，通过其亲和力Holo/Apo Ratio的动态变化，实现高通量、高效筛选高亲和力、特异性强的配体。进一步，在基于NDX-MS技术具有获取在全蛋白质组水平发现配体与靶蛋白复合物形成的基础上，开发了结合定量蛋白组学技术和还原烷基化标记反应AcH Labeling的技术，探测蛋白各活性赖氨酸残基在配体结合后的化学可及性变化谱，解析在真实体系中配体与靶蛋白结合位点的全景；最后，我们设计并合成可穿透细胞膜的光激活卡宾探针阵列，基于探针产生的卡宾自由基离子的高反应活性对靶蛋白进行全覆盖的标记，再结合离子淌度技术，区分探针对各个氨基酸残基标记产生的位置异构体，首次实现结构质谱技术在亚残基水平对靶蛋白及其与配体结合域的全景覆盖这一突破。综上，通过本项目的实施所建立的真实生物环境中靶蛋白与配体结合模式与位点动态分析的技术体系，已经初步展现配体-靶蛋白复合物在单纯的孵育体系与复杂的细胞环境中存在显著的亲和力和结合位点的差异；研究提示针对靶点的创新药物的筛选与设计必须考虑生理学相关的拓扑结构状态，并且捕捉真实生物体系中的配体-靶蛋白相互作用是提高创新药物研发成功率及效率的关键。