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

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.

总结 RNA分子对致病细菌和病毒的生存力和毒力做出重要贡献， 参与与人类疾病有关的最基本的细胞过程。许多RNA含有 结构化区域对功能至关重要，因此代表了有吸引力的药物靶标，特别是对于 蛋白质突变率高的病原体。用小分子干预这些RNA是一种有效的方法。 治疗感染的有效方法。虽然大量的努力集中在确定有效的和具体的 RNA结合剂，这些研究中的大多数都是在体外进行的，将实际的RNA-小分子相互作用留在体外。 体内未经测试，因为在体内捕获和鉴定RNA结合小分子的稳健方法不 存在.缺乏这种方法可能会阻碍候选RNA结合药物的优化， 延迟临床前和临床研究的进展。这项建议的重点是发展一个简便， 捕获和鉴定特异性结合感兴趣RNA的小分子的廉价且稳健的方法 在细菌细胞中。这项拟议中的研究将使用天然的调节性非编码RNA、核糖开关和一种插入蛋白。 体外选择的RNA适体，能够特异性结合细胞小分子和抗生素，作为模型 系统.具体目标1是致力于发展生物化学方法生产RNA 细菌细胞中的物种，通过RNA捕获同源小分子，提取RNA小分子 通过亲和色谱法测定复合物，并通过质谱法鉴定结合的小分子。的 将使用一组识别化学上不同的小分子的RNA对方法进行基准测试。 具体目标2将验证其同源配体不清楚或未知的核糖开关的方法。在 为此，我们期望从人类病原体中鉴定出一个“孤儿”核糖开关的同源配体。 幽门螺杆菌和核糖开关-配体复合物的生化，生物药理学特征， 无论是在体外还是体内。这种核糖开关控制着细菌毒力所必需的基因， 是潜在的药物靶点拟议的办法填补了方法上的空白， 用于推进RNA靶向小分子的命中-先导优化和同源小分子的鉴定， 天然RNA的分子粘合剂。