Identification of botanical hHv1 channel blockers as analgesics for neuropathic pain

项目摘要 与公共卫生的相关性。小胶质细胞，大脑和脊髓中的炎性细胞，参与了 神经损伤后通过释放活性氧（ROS）和 促炎细胞因子NeuP影响超过1600万美国人，与质量严重下降有关 生命NeuP通常对常规镇痛剂难治，并且阿片类药物仅具有有限的功效， 滥用的风险。通常不用作镇痛剂的药物通常是NeuP的一线治疗。怎么-- 曾经，<50%的患者报告当前治疗的疼痛缓解，并且副作用很常见。我们提供证据 抑制小胶质细胞中的电压门控质子通道（Hv 1）可以减少炎症介质的释放 并减弱NeuP。我们建议通过高通量筛选（HTS）鉴定小分子Hv 1阻断剂 植物提取物和植物化合物作为一个步骤，向新的药物NeuP。 简要背景。该应用基于：（i）我们的出版物，即Hv 1控制ROS和促炎性细胞因子， 小胶质细胞中细胞因子的释放，并有助于NeuP的发展，（ii）我们的初步发现，C6， Hv 1的设计肽阻断剂，抑制小胶质细胞释放ROS和促炎细胞因子， 评估小鼠外周神经损伤后的NeuP，以及（iii）我们成功地对一种小分子化合物进行了初步筛选 库和八个范例植物提取物使用一种新的活细胞，荧光为基础的HTS测定。HTS检测 使用与人Hv 1通道遗传连接的pH敏感性荧光蛋白（hHv 1-VFP-H148 G）， 允许实时监测Hv 1的运行和识别Hv 1阻断剂。 独特的功能和创新。我们的目标选择是创新的，因为没有临床批准的药物， 选择性靶向小胶质细胞，Hv 1缺乏有效和特异性的小分子阻断剂。我们的HTS检测是创新的 因为没有报道用于质子通道的HTS方法。我们确定了独特的试剂：C6（第一个 Hv 1的特异性阻断剂）、C6变体、F6（在HTS中抑制Hv 1的小分子）和熏衣草（一种植物 抑制HTS中Hv 1的提取物），以促进HTS测定的开发、改进和验证。 我们将采用HTS测定来筛选具有> 1，500个物种（具有> 15，000个esti）的独特植物提取物文库。 配合化合物），其中许多在历史上用于抗炎和止痛。 三个具体目标。(1)优化HTS测定（R61阶段）旨在改进和验证HTS测定 用于植物提取物和植物化合物。(2)从独特的植物提取物库中选择先导化合物（R33阶段） 旨在筛选植物提取物库，并确定有效和特定的植物Hv 1阻断剂。(3)学习支持 用已鉴定的植物Hv 1阻断剂表达小胶质细胞炎症反应和NeuP（R33期） 旨在验证已鉴定的植物Hv 1阻断剂在小胶质细胞和NeuP小鼠模型中的功效。 意义这项工作解决了NeuP治疗的未满足的医疗需求，并具有更广泛的影响， 这是因为小胶质细胞中的Hv 1参与了其他炎症性疾病，如缺血性中风。

PROJECT SUMMARY Relevance to public health. Microglia, the inflammatory cells in the brain and spinal cord, participate in the development of neuropathic pain (NeuP) after nerve injury by releasing reactive oxygen species (ROS) and proinflammatory cytokines. NeuP affects >16 million Americans, is associated with a severe reduction in quality of life. NeuP is often refractory to conventional analgesics, and opioids have only limited efficacy increasing the risk of their misuse. Medications not typically used as analgesics are often the first-line treatment of NeuP. How- ever, <50% of patients report pain relief with current treatment, and side effects are common. We offer evidence that suppressing the voltage-gated proton channel (Hv1) in microglia can reduce inflammatory mediators release and attenuate NeuP. We propose to identify small-molecule Hv1 blockers by high-throughput screening (HTS) of plant extracts and botanical compounds as a step toward new drugs for NeuP. Brief background. This application builds on: (i) our publication that Hv1 controls ROS and proinflammatory cytokines release in microglia and contributes to the development of NeuP, (ii) our preliminary finding that C6, a designer peptide blocker of Hv1, suppresses ROS and proinflammatory cytokine release by microglia and atten- uates NeuP after peripheral nerve injury in mice, and (iii) our successful pilot screening of a small compound library and eight exemplar plant extracts using a novel live-cell, fluorescence-based HTS assay. The HTS assay employs a pH-sensitive fluorescent protein genetically linked to the human Hv1 channel (hHv1-VFP-H148G), allowing real-time monitoring of Hv1 operation and the identification of Hv1 blockers. Unique features and innovation. Our target choice is innovative because no clinically approved drugs se- lectively target microglia and Hv1 lacks potent and specific small-molecule blockers. Our HTS assay is innovative because there is no reported HTS method for proton channels. We identified unique reagents: C6 (the first specific blocker of Hv1), C6 variants, F6 (a small molecule that inhibits Hv1 in the HTS), and Lavender (a plant extract that inhibits Hv1 in the HTS) to facilitate the development, refinement, and validation of the HTS assay. We will employ the HTS assay to screen a unique plant extract library with >1,500 species (with >15,000 esti- mated compounds), many of which have been used historically for anti-inflammation and pain-relief. Three specific aims. (1) Optimize the HTS assay (R61 phase) seeks to improve and validate the HTS assay for plant extracts and botanical compounds. (2) Select leads from a unique library of plant extracts (R33 phase) seeks to screen the plant extracts library and identify potent and specific botanical Hv1 blockers. (3) Study sup- pression of the microglial inflammatory response and NeuP with identified botanical Hv1 blockers (R33 phase) seeks to validate the efficacy of identified botanical Hv1 blockers in microglia cells and a mouse model of NeuP. Significance. This work addresses an unmet medical need for NeuP therapeutics and has a broader influ- ence because Hv1 in microglia is complicit in additional inflammatory disorders, such as ischemic stroke.