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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.

摘要