An L-Aptamer-Displacement Assay for High-Throughput Screening of RNA-Targeted Small Molecule Antivirals

用于高通量筛选 RNA 靶向小分子抗病毒药物的 L 适体置换测定

• 批准号: 10648368
• 负责人: Jonathan Thomas Sczepanski
• 金额: $ 16.71万
• 依托单位: TEXAS A&M UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10648368
• 关键词: 2019-nCoV; Address; Affinity; Antibodies; Antiviral Agents; Area; Attention; Base Pairing; Binding; Biochemical; Biological Assay; COVID-19 pandemic; Cells; Charge; Chemicals; Couples; DNA; Development; Drug Targeting; Drug usage; Elements; Goals; Image; Ligands; Methodology; Molecular Conformation; Nucleic Acids; Nucleotides; Oligonucleotides; Optics; Pharmaceutical Preparations; Process; Productivity; Property; Proteins; Publishing; RNA; RNA Binding; RNA Viruses; RNA-targeting therapy; Research Project Grants; Ribose; SARS-CoV-2 antiviral; SARS-CoV-2 genome; Specificity; Structure; Technology; Time; Untranslated RNA; Vertebral column; Viral; Viral Genome; Viral Physiology; Viral Proteins; Virus; Virus Replication; antiviral drug development; aptamer; betacoronavirus; combat; drug discovery; enantiomer; future outbreak; high throughput screening; novel; screening; small molecule; small molecule inhibitor; small molecule libraries; stereochemistry; variants of concern; viral RNA

Project Summary/Abstract While the vast majority of antiviral efforts to combat severe acute respiratory syndrome coronavirus 2 (SARS- CoV-2) focus on essential viral proteins, emerging evidence shows that conserved viral RNA (vRNA) structural elements are compelling targets with the potential for pan-antiviral activity. Despite this promise, however, selective targeting of RNA using drug-like small molecules remains challenging. In particular, methodologies for screening small molecule libraries against RNA remain underdeveloped, and do not adequately address the central problem of target specificity. As a result, RNA-targeted screens often fail to yield efficacious compounds. The proposed study takes direct aim at this technological gap through the development of a novel RNA-targeted screening technology using L-aptamers composed of mirror-image L-DNA. The PI previously established that L- aptamers can be evolved to bind native D-RNA structures, including SARS-CoV-2 vRNAs, with high affinity and selectivity. He now proposes to develop L-aptamers into RNA-specific competitive displacement probes for identifying small molecules with analogous properties. The general utility of nucleic acid aptamers, combined with the unique RNA-binding properties of L-aptamers, impart the proposed L-aptamer-displacement assay with several advantages over current RNA-centric screening technologies, and is hypothesized to facilitate the discovery of small molecules with unprecedented RNA-binding capabilities. The PI has already prepared an L-aptamer targeting a conserved RNA element with the SARS-CoV-2 genome, which will be developed into a biochemical assay that couples competitive displacement of the L- aptamer from the vRNA target with an optical readout (Aim 1). Using this assay, the PI will initiate a high- throughput screen to identify potent ligands targeting the corresponding vRNA. The most promising lead compounds will be evaluated for antiviral activity against SARS-CoV-2 infected cells (Aim 2). Parallel efforts will be undertaken to generate L-aptamers against additional SARS-CoV-2 RNA structures (Aim 3), which will be shuttled through this same pipeline. Successful completion of this project will signify a major advance in the area of RNA-targeted drug discovery. While combatting SARS-CoV-2 is the immediate goal, technologies developed herein are readily adaptable to target any RNA virus. By targeting essential RNA structures that are conserved across β-coronaviruses, the PI envision that this approach will allow for identification of antiviral compounds with broad-spectrum activity that might quickly pivot to address future outbreaks.

项目总结/摘要 虽然绝大多数对抗严重急性呼吸系统综合征冠状病毒2（SARS- CoV-2）专注于重要的病毒蛋白，新出现的证据表明保守的病毒RNA（vRNA）结构 元素是具有泛抗病毒活性潜力的引人注目的靶标。然而，尽管有这样的承诺， 使用药物样小分子选择性靶向RNA仍然具有挑战性。特别是， 针对RNA筛选小分子文库仍然不发达，并且没有充分解决 目标特异性的核心问题。因此，RNA靶向筛选通常不能产生有效的化合物。 这项拟议的研究通过开发一种新的RNA靶向的技术，直接瞄准了这一技术差距。 使用镜像L-DNA组成的L-适体的筛选技术。PI先前确定L- 适体可以进化为以高亲和力结合天然D-RNA结构，包括SARS-CoV-2 vRNA， 选择性他现在建议将L-适体开发成RNA特异性竞争性置换探针， 识别具有类似性质的小分子。核酸适体的一般效用，组合 由于L-适体的独特RNA结合特性， 与目前的以RNA为中心的筛选技术相比有几个优点，并假设有助于 发现具有前所未有的RNA结合能力的小分子。 PI已经制备了靶向SARS-CoV-2保守RNA元件的L-适体 基因组，这将发展成为一个生化测定，夫妇的竞争性置换的L- 用光学读数从vRNA靶标检测适体（Aim 1）。使用该测定，PI将启动高- 通量筛选以鉴定靶向相应vRNA的有效配体。最有希望的线索 将评价化合物对SARS-CoV-2感染细胞的抗病毒活性（Aim 2）。并行的努力将 进行L-适体产生额外的SARS-CoV-2 RNA结构（目标3），这将是 穿梭于同一条管道 该项目的成功完成将标志着RNA靶向药物发现领域的重大进展。 虽然抗击SARS-CoV-2是近期目标，但本文开发的技术很容易适应于 针对任何RNA病毒。通过靶向在β-冠状病毒中保守的基本RNA结构，PI 设想这种方法将允许鉴定具有广谱活性的抗病毒化合物， 可能会迅速转向应对未来的疫情。