RNA Aptamers Selective for TPRV Channels

针对 TPRV 通道的选择性 RNA 适体

基本信息

批准号：
7656649
负责人：
ROGER Gordon O'NEIL
金额：
$ 18.75万
依托单位：
UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-08-01 至 2011-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7656649
关键词：
3-Dimensional Acidity Affinity Agonist Antibodies Behavior Binding Binding Sites Biological Assay Biological Availability Calcium Capsaicin Cells Chemicals Chili Pepper Classification Clinical Trials Cloning Dental caries Development Drug Delivery Systems Drug Kinetics Esthesia Event Evolution Exposure to Foundations Ganglia Goals Heating High temperature of physical object Hyperalgesia Image Imaging Techniques In Situ Inflammatory Ion Channel Kinetics Knockout Mice Libraries Ligands Mechanical Stress Modeling Modification Molecular Conformation Monoclonal Antibodies Mus Myxoid cyst Natural regeneration Nature Nerve Nerve Fibers Neurons Nociception Pain Pathway interactions Procedures Process Property Protein Binding Domain Protein Isoforms Proteins RNA Role Screening procedure Seminal Site Small RNA Specificity Stimulus Structure of trigeminal ganglion TRP channel TRPV channel TRPV1 gene Technology Testing Therapeutic Therapeutic Agents Time Trigeminal System Vanilloid Vertebral column Water afferent nerve aptamer base capsaicin receptor combinatorial chemistry drug candidate drug discovery in vivo interest member methyl group mouse model novel novel strategies novel therapeutics nuclease numb protein osmoreceptor pain behavior patch clamp pressure public health relevance spinal nerve posterior root success three dimensional structure tool

项目摘要

DESCRIPTION (provided by applicant): The overall goal of the project is to employ a novel, emerging combinatorial chemistry technology to identify and develop small RNA molecules that bind with high affinity and specificity to selected TRPV channel target sites as potential therapeutic agents for treatment of nociceptive behavior. Two isoforms of the vanilloid receptor subfamily of TRP channels, TRPV1 (the capsaicin receptor) and TRPV4 (an osmoreceptor), display polymodal gating behavior with sensitivity to numerous noxious stimuli. Both channels are widely distributed in small- to medium-sized neurons of the dorsal root and trigeminal ganglia. However, traditional drug discovery efforts to identify specific antagonist are relatively slow and have had limited success, although development efforts are intensifying. The recent discovery of small RNA molecules that fold into unique 3-D structures that bind with high affinity and specificity to protein binding domains, akin to monoclonal antibodies, has opened the door to development of a new, powerful, class of pharmacotherapeutic agent. The purpose of the present study is to generate selective RNA aptamers that bind with high affinity to TRPV1 and/or TRPV4 isoforms and act as antagonist for pharmacotherapeutic applications. Three specific aims are proposed to accomplish this goal: 1. To generate high affinity RNA aptamers with specificity for TRPV channel isoforms, TRPV1 and TRPV4. RNA aptamers will be identified from a large pool of random RNA molecules by systemic evolution, amplification, and enrichment of high affinity RNA ligands (aptamers). 2. To screen the high-affinity RNA aptamers for function as potential antagonist of the TRPV isoforms. High throughput, high-content, kinetic screening of identified RNA aptamers on calcium influx through TRPV1 or TRPV4 channels will be assessed to evaluate the potential of each identified aptamer as functional antagonist of TRPV1 and TRPV4. 3. To evaluate the potential function of the identified RNA aptamers as pharmacological tools/therapeutic agents. RNA aptamers identified from the kinetic screens will be evaluated as a pharmacotherapeutic tools using patch clamp analysis to directly assess aptamer effects on channel function. Promising aptamers will be further evaluated for therapeutic potential, in vivo, in established models of TRPV1- and TRPV4-dependent pain behavior. The studies will have far reaching impact both in providing a foundation for identification and development RNA aptamers as pharmacotherapeutic agents, and in providing new therapeutic tools for the specific treatment of nociceptive behavior, as well as for numerous other pathophysiological conditions associated with the TRPV ion channels. PUBLIC HEALTH RELEVANCE: The goal of the project is to use a novel, emerging, approach to generate small RNA molecules (RNA aptamers, 3-D folded) that bind with high affinity and specificity (10; 26) to target sites on specific channel proteins that are associated with sensing pain (TRPV channels) (40; 57; 61). RNA molecules that selectively block the function of the TRPV channels and, therefore, the pain sensation, would be specifically targeted. Generating such molecules would open a new platform for development of these, and other, compounds as potential pharmacological tools and therapeutic agents for the treatment of a broad range of painful conditions associated with noxious stimuli or inflammatory states (e.g., pressure, high temperature, nerve damage/tooth decay, hyperalgesia, and exposure to noxious chemicals) (12; 13; 17; 52; 78).

描述（由申请人提供）：该项目的总体目标是采用一种新颖的、新兴的组合化学技术来识别和开发小RNA分子，这些分子以高亲和力和特异性结合选定的TRPV通道靶位点，作为治疗以下疾病的潜在治疗剂：伤害性行为。 TRP 通道香草酸受体亚家族的两种亚型 TRPV1（辣椒素受体）和 TRPV4（渗透压感受器）表现出多模式门控行为，对多种有害刺激敏感。这两个通道广泛分布于背根和三叉神经节的中小型神经元中。然而，尽管开发工作正在加强，但识别特定拮抗剂的传统药物发现工作相对缓慢且取得的成功有限。最近发现的小RNA分子可以折叠成独特的3D结构，以高亲和力和特异性与蛋白质结合域结合，类似于单克隆抗体，为开发一类新的、强大的药物治疗剂打开了大门。本研究的目的是生成选择性 RNA 适体，以高亲和力与 TRPV1 和/或 TRPV4 亚型结合，并作为药物治疗应用的拮抗剂。为了实现这一目标，提出了三个具体目标： 1. 生成对 TRPV 通道亚型、TRPV1 和 TRPV4 具有特异性的高亲和力 RNA 适体。通过系统进化、扩增和富集高亲和力 RNA 配体（适体），可以从大量随机 RNA 分子中鉴定出 RNA 适体。 2.筛选高亲和力RNA适体作为TRPV亚型的潜在拮抗剂。将评估通过 TRPV1 或 TRPV4 通道钙流入的已鉴定 RNA 适体的高通量、高内涵、动力学筛选，以评估每个已鉴定适体作为 TRPV1 和 TRPV4 功能性拮抗剂的潜力。 3. 评估已鉴定的RNA适体作为药理学工具/治疗剂的潜在功能。从动力学筛选中鉴定出的RNA适体将被评估为药物治疗工具，使用膜片钳分析直接评估适体对通道功能的影响。将在已建立的 TRPV1 和 TRPV4 依赖性疼痛行为模型中进一步评估有前途的适体的体内治疗潜力。这些研究将产生深远的影响，既为鉴定和开发作为药物治疗剂的RNA适体奠定了基础，又为伤害性行为的特异性治疗以及与TRPV离子通道相关的许多其他病理生理状况提供新的治疗工具。。公共健康相关性：该项目的目标是使用一种新颖的、新兴的方法来生成小 RNA 分子（RNA 适体，3-D 折叠），以高亲和力和特异性 (10; 26) 结合到特定通道上的目标位点与感知疼痛相关的蛋白质（TRPV 通道）（40；57；61）。选择性阻断 TRPV 通道功能并因此阻断痛觉的 RNA 分子将成为专门针对的目标。生成此类分子将为开发这些化合物和其他化合物开辟一个新平台，作为潜在的药理学工具和治疗剂，用于治疗与有害刺激或炎症状态（例如压力、高温、神经）相关的各种疼痛状况。损伤/蛀牙、痛觉过敏和接触有毒化学物质）（12；13；17；52；78）。