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

基本信息

批准号：
10377569
负责人：
Christian B. Hilty
金额：
$ 30.67万
依托单位：
TEXAS A&M UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-04-01 至 2025-03-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10377569
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 Stimulator of Interferon Genes Structure Surface System Techniques Time Vesicle Water Work base cancer cell cellular targeting chemokine receptor delivery vehicle detection limit 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

项目摘要

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.

抽象的蛋白质 - 配体相互作用在包括细胞信号在内的基本生物学过程中起关键作用和监管。进一步筛选与药物候选化合物和片段的相互作用代表了药物发现中必不可少的一步。经常确定此类互动的传统方法需要高度纯化的蛋白质，特别是在膜蛋白的情况下总是可用的。通过募集多个功能复合物的表征存在相同的局限性膜上或细胞内的成分。尽管如此，目前有60％的药物靶向膜蛋白。在这里，将开发一种方法来确定任意类型的小分子之间的相互作用，直接带有位于表面或细胞内部的受体或其他成分。核磁配体共振（NMR）信号将通过使用溶解来增强几个数量级动态核极化（D-DNP）。该超极化产生的非平衡自旋态技术导致灵敏度提高，从而使纳摩尔浓度范围内的靶标有检测。在同时，超极化在复杂的背景光谱上提供对比度。因此，它可以使标签 - 免费，选择性检测感兴趣的配体，同时保留NMR中可用的化学信息光谱法。在项目的第一个目标中，一种用于检测和表征结合的实验方法将开发含有脂质囊泡中蛋白质的异质模型。核自旋放松竞争性结合以及材料内或间的核核次横向效应（NOE）将提供结合亲和力结合口袋内的测量和结构约束。在第二个目标中，这些方法将是用于表征配体与G蛋白偶联趋化因子受体在表面的结合哺乳动物细胞。用于NMR检测细胞培养物的专用装置，用超极化培养基灌注为此目的开发。第三个目标将把这些方法扩展到访问细胞质内的目标细胞的内膜，这里特别是CGAS刺干扰器的寡聚信号蛋白感应途径。超极化水将自然地在整个细胞膜上运输。然后，超极化将通过质子交换或NOE转移到细胞内成分，从而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。在一起，这些目标将提供一个全面的新型工具集，以表征自然中的蛋白质 - 配体相互作用细胞的环境。