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Application of Fluorine-19 labeled RNA in Ligand-binding Studies

19氟标记RNA在配体结合研究中的应用

基本信息

批准号：
7910637
负责人：
Mirko Hennig
金额：
$ 18.98万
依托单位：
MEDICAL UNIVERSITY OF SOUTH CAROLINA
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-08-15 至 2012-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7910637
关键词：
2-Fluoroadenine Affinity Antibiotic Resistance Arts Awareness Binding Binding Sites Biological Assay Biological Models Biological Process Chemicals Complement Complex Development Disease Disease Progression Docking Drug Delivery Systems Drug Interactions Essential Genes Fluorine Gene Expression Regulation Generations Genomics Goals HIV Label Lead Libraries Ligand Binding Ligands Light Major Groove Mediating Methodology Methods Minor Groove Monitor Nuclear Pharmaceutical Preparations Pharmacologic Substance Positioning Attribute Process Proteins Protons RNA RNA Binding RNA Ligase (ATP)Reporter Role Scheme Screening procedure Severe Acute Respiratory Syndrome Solutions Structure-Activity Relationship Tars Techniques Technology Therapeutic Therapeutic Intervention Translations Validation Virus Virus Diseases base chemical property drug discovery flexibility fluoropyrimidine follow-up high throughput screening improved inhibitor/antagonist interest marine natural product novel therapeutic intervention nucleotide analog pathogenic bacteria physical property public health relevance research study small molecule tmRNA

项目摘要

DESCRIPTION (provided by applicant): The increasing awareness of the essential role of RNA in biological processes and progression of diseases, including its involvement in translation, gene regulation, and viral infections, make RNA an interesting target for therapeutic intervention. Riboswitches or the tmRNA-mediated rescue mechanism represent very attractive novel therapeutic approaches against pathogenic bacteria, especially in light of the growing threat of emerging antibiotic resistance. Genomic RNA of pathogenic viruses such as HIV or SARS provides many opportunities for drug discovery. However, the discovery of new RNA-binding drugs has significantly lagged behind their protein targeting counterparts. This can be at least partially attributed to a paucity of state-of-the-art NMR methods to detect such interactions. RNA-ligand recognition typically occurs by conformationally flexible "induced-fit" rather than by rigid "lock-and-key" docking. The resulting greater disorder and the limited chemical diversity in comparison to proteins complicate NMR (and other) studies of RNA and its complexes. It is the goal of our application to establish a combination of NMR methods and RNA labeling techniques to facilitate the identification of specific, high affinity RNA-ligand interactions. A wellstudied example, HIV TAR, will serve as our model system. The Tat-TAR complex formation provides an essential gene regulatory function for HIV. Our specific aims are: 1. To develop sensitive ligand-based NMR screening approaches utilizing 19F-labeled RNA We aim to explore heteronuclear 19F,1H-NOE interactions in NMR-based RNA-ligand binding studies. We propose to utilize chemically induced dynamic nuclear polarization (CIDNP) enhancements to boost sensitivity in this ligand-detected screening method. We will pursue CIDNP to facilitate the NMR analysis of 19F-labeled RNA in general. 2. To optimize and apply SAR by NMR follow-up validation assays using 19F-labeled RNA We will use 5-Fluoropyrimidine (major groove reporter) or 2-Fluoroadenine (minor groove reporter) substituted RNA in follow-up screening assays utilizing extraordinarily sensitive 19F-NMR chemical shift perturbations. We aim to develop and apply segmental isotopic labeling approaches using T4 RNA ligase to overcome potential limitations associated with uniform 19F-labeling schemes. Public Health Relevance Statement: Many RNA molecules are drug targets with great potential for therapeutic treatment of bacterial or viral infections. However, the discovery of new RNA-binding drugs has been lagging because of the chemical properties of RNA and a lack of state-of-the-art technology to identify RNA-drug interactions. If the premises of our proof-of-concept studies are realized, then our approach will greatly facilitate the discovery of RNA-binding therapeutic candidates.

描述（由申请人提供）：人们越来越认识到 RNA 在生物过程和疾病进展中的重要作用，包括其参与翻译、基因调控和病毒感染，使 RNA 成为治疗干预的有趣目标。核糖开关或 tmRNA 介导的救援机制代表了针对病原菌的非常有吸引力的新型治疗方法，特别是考虑到新出现的抗生素耐药性的威胁日益严重。 HIV 或 SARS 等病原病毒的基因组 RNA 为药物发现提供了许多机会。然而，新的 RNA 结合药物的发现明显落后于其蛋白质靶向药物。这至少部分归因于缺乏最先进的核磁共振方法来检测这种相互作用。 RNA配体识别通常通过构象灵活的“诱导配合”而不是通过严格的“锁钥匙”对接来发生。与蛋白质相比，由此产生的更大的无序性和有限的化学多样性使 RNA 及其复合物的 NMR（和其他）研究变得复杂。我们应用的目标是建立 NMR 方法和 RNA 标记技术的组合，以促进特异性、高亲和力 RNA-配体相互作用的识别。 HIV TAR 是一个经过充分研究的例子，将作为我们的模型系统。 Tat-TAR 复合物的形成为 HIV 提供了重要的基因调控功能。我们的具体目标是： 1. 利用 19F 标记的 RNA 开发基于配体的灵敏 NMR 筛选方法我们的目标是在基于 NMR 的 RNA-配体结合研究中探索异核 19F,1H-NOE 相互作用。我们建议利用化学诱导动态核极化（CIDNP）增强来提高这种配体检测筛选方法的灵敏度。我们将追求 CIDNP 以促进 19F 标记 RNA 的 NMR 分析。 2. 使用 19F 标记的 RNA 通过 NMR 后续验证测定来优化和应用 SAR 我们将在利用极其敏感的 19F-NMR 化学位移扰动的后续筛选测定中使用 5-氟嘧啶（大沟报告基因）或 2-氟腺嘌呤（小沟报告基因）取代的 RNA。我们的目标是开发和应用使用 T4 RNA 连接酶的分段同位素标记方法，以克服与统一 19F 标记方案相关的潜在限制。公共卫生相关性声明：许多 RNA 分子是药物靶标，在治疗细菌或病毒感染方面具有巨大潜力。然而，由于 RNA 的化学性质以及缺乏识别 RNA 药物相互作用的最先进技术，新的 RNA 结合药物的发现一直滞后。如果我们的概念验证研究的前提得以实现，那么我们的方法将极大地促进 RNA 结合治疗候选药物的发现。