Novel approach to identify RNA-bound small molecules in vivo

体内鉴定 RNA 结合小分子的新方法

• 批准号: 10646626
• 负责人: Giulio Quarta
• 金额: $ 25.43万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-01-19 至 2024-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10646626
• 关键词: Address; Affinity; Affinity Chromatography; Aminoglycoside Antibiotics; Antibiotics; Bacillus subtilis; Benchmarking; Binding; Biochemical; Biological Assay; Biological Models; Biophysics; Cell Extracts; Cell physiology; Cells; Chemicals; Clinical; Clinical Research; Complex; Data; Development; Drug Targeting; Elements; Ensure; Escherichia coli; Fishes; Gene Expression; Genes; Goals; Helicobacter pylori; Human; In Vitro; Infection; Infectious Agent; Isotope Labeling; Ligand Binding; Ligands; Mammalian Cell; Mass Spectrum Analysis; Medicine; Messenger RNA; Metabolic; Methodology; Methods; Mutation; Oligonucleotides; Orphan; Pharmaceutical Preparations; Play; Procedures; Process; Production; Proteins; RNA; RNA Binding; RNA Viruses; RNA purification; Research Personnel; Ribosomal RNA; Role; Small RNA; Staphylococcus aureus; Structure; Therapeutic; Time; Untranslated RNA; Virulence; Virus; aptamer; complex data; efficacy evaluation; experimental study; genetic information; high throughput screening; human disease; human pathogen; in vivo; interest; lead optimization; novel; novel strategies; pathogen; pathogenic bacteria; pathogenic virus; preclinical study; protein complex; small molecule; success; transcriptome; viral RNA; Address; Affinity; Affinity Chromatography; Aminoglycoside Antibiotics; Antibiotics; Bacillus subtilis; Benchmarking; Binding; Biochemical; Biological Assay; Biological Models; Biophysics; Cell Extracts; Cell physiology; Cells; Chemicals; Clinical; Clinical Research; Complex; Data; Development; Drug Targeting; Elements; Ensure; Escherichia coli; Fishes; Gene Expression; Genes; Goals; Helicobacter pylori; Human; In Vitro; Infection; Infectious Agent; Isotope Labeling; Ligand Binding; Ligands; Mammalian Cell; Mass Spectrum Analysis; Medicine; Messenger RNA; Metabolic; Methodology; Methods; Mutation; Oligonucleotides; Orphan; Pharmaceutical Preparations; Play; Procedures; Process; Production; Proteins; RNA; RNA Binding; RNA Viruses; RNA purification; Research Personnel; Ribosomal RNA; Role; Small RNA; Staphylococcus aureus; Structure; Therapeutic; Time; Untranslated RNA; Virulence; Virus; aptamer; complex data; efficacy evaluation; experimental study; genetic information; high throughput screening; human disease; human pathogen; in vivo; interest; lead optimization; novel; novel strategies; pathogen; pathogenic bacteria; pathogenic virus; preclinical study; protein complex; small molecule; success; transcriptome; viral RNA

Summary RNA molecules make an important contribution to viability and virulence of pathogenic bacteria and viruses, participating in the most fundamental cellular processes implicated in human disease. Many RNAs contain structured regions that are critical to function and therefore represent attractive drug targets, especially for pathogens with high mutation rates in proteins. Intervening against these RNAs with small molecules is a powerful way to treat infections. Although significant efforts are concentrated on identifying potent and specific RNA binders, most of these studies are done in vitro, leaving the actual RNA-small molecule interactions in vivo untested because a robust method to capture and identify RNA-bound small molecules in vivo does not exist. The lack of such a method could hinder optimization of candidate RNA-binding drugs and inadvertently delay the progress to preclinical and clinical studies. This proposal is focused on developing a facile, inexpensive, and robust approach to capture and identify small molecules specifically binding RNAs of interest in bacterial cells. The proposed study will use natural regulatory non-coding RNAs, riboswitches, and an in vitro-selected RNA aptamer, capable of specific binding to cellular small molecules and antibiotics, as model systems. Specific Aim 1 is devoted to the development of the biochemical approach for producing RNA species in bacterial cells, capturing cognate small molecules by RNA, extracting RNA-small molecule complexes by affinity chromatography, and identifying bound small molecules by mass spectrometry. The methodology will be benchmarked using a panel of RNAs recognizing chemically diverse small molecules. Specific Aim 2 will validate the approach on riboswitches whose cognate ligands are unclear or unknown. In this aim, we anticipate to identify a cognate ligand for an “orphan” riboswitch from the human pathogen Helicobacter pylori and characterize the riboswitch-ligand complex biochemically, biophysically, and genetically, in vitro and in vivo. This riboswitch controls genes that are essential for bacterial virulence and thus represents a potential drug target. The proposed approach is filling a methodological gap and will be essential for advancing hit-to-lead optimization of the RNA-targeting small molecules and identification of cognate small molecule binders for natural RNAs.

概括 RNA分子对致病细菌和病毒的生存能力和病毒做出了重要贡献， 参加人类疾病实施的最基本的细胞过程。许多RNA包含 对功能至关重要的结构化区域，因此代表有吸引力的药物目标，尤其是 蛋白质中突变率高的病原体。用小分子介入这些RNA是一个 尽管重大努力集中在确定潜力和特定的努力上 RNA粘合剂，大多数研究是在体外进行的，在实际RNA-MALL分子相互作用中进行 体内未经测试，因为在体内捕获和识别结合RNA结合的小分子的强大方法不会 存在。缺乏这种方法可能会阻碍候选RNA结合药物的优化和无意中的 延迟临床前和临床研究的进展。该建议的重点是开发一个便利， 捕获和识别小分子特异性结合RNA的廉价且健壮的方法 在细菌细胞中。拟议的研究将使用自然调节性非编码RNA，核糖开关和AN 体外选择的RNA APATMER，能够特异性与细胞小分子和抗生素的结合为模型 系统。具体目标1致力于开发生化方法的生化方法 细菌细胞中的物种，通过RNA捕获同源小分子，提取RNA-MALL分子 通过亲和色谱法进行复合物，并通过质谱鉴定结合的小分子。 方法将使用识别化学多样的小分子的RNA面板进行基准测试。 具体目标2将验证核糖开关的方法，其同源配体尚不清楚或未知。在 这个目标，我们预计将确定人类病原体“孤儿”核糖开关的同源配体 幽门螺杆菌的幽门螺杆菌并以生物物理和生物物质和 从遗传上，体外和体内。这种核糖开关控制对细菌毒力必不可少的基因，因此 代表潜在的药物靶标。拟议的方法是填补方法论上的差距，将是必不可少的 为了推进靶向RNA的小分子的命中率优化，并鉴定了相关小分子 天然RNA的分子粘合剂。