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.

项目摘要/摘要 尽管绝大多数抗病毒努力应对严重的急性呼吸综合症冠状病毒2（SARS- COV-2）专注于必需病毒蛋白，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目标。尽管有这个承诺，但是 使用类似药物的小分子对RNA的选择性靶向仍然受到挑战。特别是 筛选针对RNA的小分子库仍然不发达，并且不能充分解决 目标特异性的中心问题。结果，靶向RNA的屏幕通常无法产生有效的化合物。 拟议的研究通过开发新的RNA靶向，直接针对这一技术差距 使用由镜像L-DNA组成的L-Aptamers筛选技术。 PI先前确定了L- 可以演变适体以结合具有高亲和力的天然D-RNA结构，包括SARS-COV-2 VRNA， 选择性。他现在提议将L-Appamers开发为RNA特异性竞争位移问题 鉴定具有类似特性的小分子。核酸适体的一般效用，合并 具有L-Appamers独特的RNA结合特性，将提出的L-Appamer-Displacement Assay授予 与当前以RNA为中心的筛选技术相比，有几个优点，并被认为可以促进 发现具有前所未有的RNA结合能力的小分子。 PI已经制备了针对具有SARS-COV-2的配置RNA元件的L-调子仪 基因组将发展为生化测定法，该测定法将L-的竞争位移伴侣 带有光学读数的VRNA目标的apatamer（AIM 1）。使用此测定，PI将启动高 吞吐量屏幕以识别针对相应VRNA的潜在配体。最有希望的领导 将评估化合物对SARS-COV-2感染细胞的抗病毒活性（AIM 2）。平行努力将 为了针对其他SARS-COV-2 RNA结构（AIM 3）产生L-Appamers，这将是 穿过同一条管道。 该项目的成功完成将表示在靶向RNA的药物发现领域的重大进步。 在打击SARS-COV-2是直接目标的同时，此处开发的技术很容易适应 靶向任何RNA病毒。通过靶向跨β-核纳病毒的必需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药化合物 可能会迅速解决未来爆发的问题。