RNA Aptamers Selective for TPRV Channels

描述(由申请人提供)：该项目的总体目标是利用一种新颖的、新兴的组合化学技术来识别和开发与选定的TRPV通道靶点具有高亲和力和特异性的小RNA分子，作为治疗伤害性行为的潜在治疗剂。Trp通道香草素受体亚家族的两个亚型，TRPV1(辣椒素受体)和TRPV4(渗透压受体)，表现出对多种伤害性刺激敏感的多模式门控行为。这两种通道广泛分布于背根和三叉神经节的中小型神经元。然而，识别特定拮抗剂的传统药物发现工作相对缓慢，取得的成功有限，尽管开发工作正在加紧。最近发现的小RNA分子可以折叠成独特的三维结构，与蛋白质结合结构域具有高亲和力和特异性，类似于单抗，这为开发一种新的、强大的药物治疗剂打开了大门。本研究的目的是制备与TRPV1和/或TRPV4亚型高亲和力的选择性RNA适配子，并作为药物治疗的拮抗剂。为了实现这一目标，我们提出了三个具体目标：1.制备高亲和力的针对TRPV通道异构体TRPV1和TRPV4的RNA适配子。通过系统进化、扩增和浓缩高亲和力RNA配体(适配子)，将从大量随机RNA分子中鉴定出RNA适配子。2.筛选高亲和力的RNA适配子作为TRPV亚型的潜在拮抗剂。对通过TRPV1或TRPV4通道钙内流的已鉴定RNA适配子进行高通量、高含量、动态筛选，以评估每个已鉴定适配子作为TRPV1和TRPV4功能拮抗剂的潜力。3.评价已鉴定的RNA适配子作为药理工具/治疗剂的潜在功能。从动力学筛选中确定的RNA适配子将作为药物治疗工具进行评估，使用膜片钳分析直接评估适配子对通道功能的影响。在已建立的TRPV1和TRPV4依赖的疼痛行为模型中，有希望的适配子将在体内进一步评估治疗潜力。这些研究将在为识别和开发RNA适配子作为药物治疗剂提供基础，为伤害性行为的特定治疗提供新的治疗工具，以及为与TRPV离子通道相关的许多其他病理生理状况提供深远的影响。与公共卫生相关：该项目的目标是使用一种新的、新兴的方法来产生小的RNA分子(RNA适配子，3-D折叠)，以高亲和力和特异性(10；26)结合到与感知疼痛相关的特定通道蛋白(TRPV通道)上的靶点(40；57；61)。选择性地阻断TRPV通道功能并因此阻断痛觉的RNA分子将成为特定的目标。生成这些分子将为开发这些和其他化合物作为潜在的药理工具和治疗剂打开一个新的平台，用于治疗与有害刺激或炎症状态(例如，压力、高温、神经损伤/牙齿腐烂、痛觉过敏和接触有害化学物质)相关的广泛痛苦状况(12；13；17；52；78)。

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).