Hyperpolarization Assisted and Structure Based Screening of Protein-Ligand Interactions in Live Cells

摘要 蛋白质-配体相互作用在包括细胞信号在内的基本生物过程中起着关键作用。 和监管。进一步筛选与药物候选化合物和片段相互作用的实验 代表着药物发现中不可或缺的一步。确定这种相互作用的传统方法通常 需要高度纯化的蛋白质，特别是在膜蛋白的情况下，这并不总是可用的。 同样的限制也存在于表征由多个 膜上或细胞内的成分。尽管如此，目前60%的药物针对的是膜蛋白。 这里，将开发一种确定任意类型小分子之间相互作用的方法， 直接与位于细胞表面或细胞内的受体或其他成分结合。核磁 利用溶解作用，配体的核磁共振信号将增强几个数量级 动态核极化(D-DNP)。这种超极化产生的非平衡自旋态 这项技术带来了灵敏度增益，能够检测到纳摩尔浓度范围内的目标。在 同时，超极化在复杂的背景光谱上提供了对比度。因此，它使标签- 对感兴趣的配体进行自由、选择性的检测，同时保留核磁共振中可用的化学信息 光谱学。在该项目的第一个目标中，一种检测和表征结合的实验方法 在脂泡中含有蛋白质的异质模型将被开发出来。原子核的自旋驰豫 竞争性结合和配基内或配基间的核Overhauser效应(NOE)将提供结合亲和力 捆绑袋内的测量和结构约束。在第二个目标中，这些方法将是 应用于表征配体与细胞表面G蛋白偶联趋化因子受体的结合 哺乳动物细胞。一种用于核磁共振检测灌流超极化介质的细胞培养的专用设备将 是为此目的而开发的。第三个目标是将这些方法扩展到访问胞质中的目标或 细胞内膜，这里特别是来自cGAS刺激性干扰素的寡聚信号蛋白 诱导途径。超极化的水会自然地穿过细胞膜。 然后，超极化将通过质子交换或NOE转移到细胞内成分，导致 细胞内未标记或选择性同位素标记配体的可检测信号变化。加在一起，这些 AIMS将提供一个全面的、新颖的工具集来表征天然的蛋白质-配体相互作用 细胞的环境。

Abstract Protein-ligand interactions play a pivotal role in fundamental biological processes including cellular signaling and regulation. Experimental screening for interactions with drug candidate compounds and fragments further represents an indispensable step in drug discovery. Traditional methods for determining such interactions often require highly purified proteins, which in particular are not always available in the case of membrane proteins. The same limitations exist for the characterization of functional complexes formed by recruitment of multiple constituents on a membrane or within a cell. Notwithstanding, 60% of current drugs target membrane proteins. Here, a method will be developed for determining interactions between small molecules of arbitrary type, directly with receptors or other components located on the surface or within a cell. Nuclear magnetic resonance (NMR) signals of ligands will be enhanced by several orders of magnitude using dissolution dynamic nuclear polarization (D-DNP). The non-equilibrium spin states produced by this hyperpolarization technique results in sensitivity gains enabling detection to targets in the nanomolar concentration range. At the same time, hyperpolarization provides contrast over complicated background spectra. It thus enables the label- free, selective detection of the ligand of interest, while retaining the chemical information available in NMR spectroscopy. In a first aim of the project, an experimental method for detecting and characterizing binding in heterogeneous models containing proteins in lipid vesicles will be developed. Nuclear spin relaxation under competitive binding, and intra- or interligand nuclear Overhauser effect (NOE) will provide binding affinity measurements and structural constraints within the binding pocket. In a second aim, these methods will be applied to characterize the binding of ligands to a G-protein coupled chemokine receptor on the surface of mammalian cells. A dedicated device for NMR detection of cell cultures perfused with hyperpolarized media will be developed for this purpose. A third aim will extend these methods to access targets within the cytosol or an internal membrane of the cell, here specifically an oligomeric signaling protein from the cGAS-STING interferon induction pathway. Hyperpolarized water will be naturally transported across the cell membrane. Hyperpolarization will then transfer to intracellular components by proton exchange or NOE, resulting in detectable signal changes of unlabeled or selectively isotope labeled ligands within the cell. Together, these aims will provide a comprehensive, novel toolset to characterize protein-ligand interactions in the natural environment of the cell.