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

活细胞中蛋白质-配体相互作用的超极化辅助和基于结构的筛选

• 批准号: 9986546
• 负责人: Christian B. Hilty
• 金额: $ 21.25万
• 依托单位: TEXAS A&M UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-04-01 至 2020-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9986546
• 关键词: 2,4-Dinitrophenol; Affinity; B-Lymphocytes; Binding; Biochemical Process; Biochemistry; Biological; Biological Assay; Biological Process; Breast Cancer Cell; CXCR4 gene; Cell Culture Techniques; Cell membrane; Cell physiology; Cell surface; Cells; Chemicals; Chemistry; Competitive Binding; Complex; Coupled; Cytosol; Detection; Development; Devices; Drug Targeting; Environment; Epitopes; Equipment; G-Protein-Coupled Receptors; GTP-Binding Protein alpha Subunits, Gs; GTP-Binding Proteins; Goals; Integral Membrane Protein; Interferons; Investigation; Isotope Labeling; Label; Laboratories; Lead; Ligand Binding; Ligands; Lipids; Liposomes; Liquid substance; Mammalian Cell; Measurement; Mediating; Membrane; Membrane Proteins; Methods; Modeling; Molecular Conformation; NMR Spectroscopy; Nuclear; Nuclear Magnetic Resonance; Pathway interactions; Pharmaceutical Preparations; Pharmacologic Substance; Play; Preparation; Process; Proteins; Protons; Regulation; Relaxation; Research; Research Personnel; Research Project Grants; Role; Sampling; Signal Pathway; Signal Transduction; Signaling Protein; Specificity; Structure; Surface; System; Techniques; Time; Vesicle; Water; Work; base; cancer cell; cellular targeting; chemokine receptor; detector; drug candidate; drug discovery; experience; experimental study; extracellular; innovation; interest; nanomolar; novel; overexpression; protein complex; protein purification; receptor; recruit; screening; small molecule; structural biology; tool; transmission process

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.

摘要 蛋白质-配体相互作用在包括细胞信号传导在内的基本生物学过程中起着关键作用 和监管。与药物候选化合物和片段的相互作用的实验筛选进一步 是药物研发中不可或缺的一步用于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。 同样的限制存在于表征通过募集多个聚乙二醇形成的功能复合物中。 细胞膜上或细胞内的成分。尽管如此，目前60%的药物靶向膜蛋白。 在这里，将开发用于确定任意类型的小分子之间的相互作用的方法， 直接与位于细胞表面或细胞内的受体或其它成分结合。核磁 使用溶解，配体的共振（NMR）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 该技术导致灵敏度增益，使得能够检测纳摩尔浓度范围内的靶。在 同时，超偏振提供了复杂背景光谱的对比度。因此，它使标签- 自由、选择性地检测目标配体，同时保留NMR中可用的化学信息 谱在该项目的第一个目标中，提供了一种用于检测和表征结合的实验方法， 将开发在脂质囊泡中含有蛋白质的异质模型。核自旋弛豫 竞争性结合和配体内或配体间的核Overhauser效应（NOE）将提供结合亲和力 约束袋内的尺寸和结构约束。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注有超极化介质的细胞培养物的专用装置将 为此目的而开发。第三个目标是将这些方法扩展到细胞质内的靶点， 细胞的内膜，这里具体地是来自cGAS-STING干扰素的寡聚信号传导蛋白 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转移到细胞内组分，导致 细胞内未标记或选择性同位素标记的配体的可检测信号变化。所有这些 aims将提供一个全面的，新颖的工具集，以表征蛋白质-配体相互作用的自然 细胞的环境。