Novel approach to identify RNA-bound small molecules in vivo

体内鉴定 RNA 结合小分子的新方法

基本信息

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
项目类别：
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

项目摘要

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 分子对病原细菌和病毒的活力和毒力做出了重要贡献，参与与人类疾病有关的最基本的细胞过程。对功能至关重要的结构化区域，因此代表有吸引力的药物靶点，特别是对于用小分子干预这些蛋白质突变率较高的病原体。尽管大量努力集中在确定有效且特异性的治疗感染的方法上。 RNA结合剂，大多数这些研究都是在体外进行的，将实际的RNA-小分子相互作用留在体内体内未经测试，因为在体内捕获和识别 RNA 结合小分子的稳健方法并没有缺乏这种方法可能会无意中阻碍候选 RNA 结合药物的优化。该提案的重点是开发一种简便的、捕获和识别特异性结合感兴趣的 RNA 的小分子的廉价且可靠的方法拟议的研究将使用天然调节非编码 RNA、核糖开关和 in 细菌细胞。体外选择的RNA适体，能够与细胞小分子和抗生素特异性结合，作为模型具体目标 1 致力于开发生产 RNA 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小分子通过亲和色谱法分析复合物，并通过质谱法鉴定结合的小分子。该方法将使用一组识别化学上不同的小分子的 RNA 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。为了实现这一目标，我们期望从人类病原体中识别出“孤儿”核糖开关的同源配体幽门螺杆菌并从生化、生物物理和角度表征核糖开关-配体复合物该核糖开关在遗传、体外和体内控制对细菌毒力至关重要的基因。代表了一个潜在的药物靶点，所提出的方法填补了方法学上的空白，并且是至关重要的。用于推进 RNA 靶向小分子的 hit-to-lead 优化和同源小分子的鉴定天然RNA的分子结合剂。