Identification of botanical hHv1 channel blockers as analgesics for neuropathic pain

植物 hHv1 通道阻滞剂作为神经性疼痛镇痛药的鉴定

• 批准号: 10728526
• 负责人: Steve A N Goldstein
• 金额: $ 25.64万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-06-08 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10728526
• 关键词: Address; Affect; Affinity; American; Analgesics; Astrocytes; Attenuated; Automobile Driving; Biological Assay; Botanicals; Brain; Cells; Central Nervous System; Clinical; Data; Development; Disease; Drug Kinetics; Drug Targeting; Electrophysiology (science); Fluorescence; Goals; Hour; Human; Immune; In Vitro; Inflammation; Inflammation Mediators; Inflammatory; Inflammatory Response; Intrathecal Injections; Ischemic Stroke; Knockout Mice; Lavandula; Lead; Libraries; Link; Mediating; Mediator; Medical; Medicine; Methods; Microglia; Mus; Natural Compound; Neurotoxins; Opioid; Pain; Patients; Peptides; Performance; Peripheral nerve injury; Pharmaceutical Preparations; Pharmacology; Phase; Phytochemical; Plant Extracts; Plants; Production; Proteins; Protons; Public Health; Publications; Quality of life; Rapid screening; Reactive Oxygen Species; Reagent; Recording of previous events; Refractory; Reporting; Research; Research Personnel; Risk; Role; Route; Specificity; Spinal Cord; Spinal cord injury; Spinal nerve structure; Syndrome; Therapeutic; Tissues; Traditional Medicine; Trauma; Validation; Variant; Work; channel blockers; chronic pain; chronic painful condition; cytokine; design; efficacy evaluation; efficacy validation; high throughput screening; improved; in vivo; in vivo Model; inhibitor; innovation; intraperitoneal; link protein; marine; mouse model; nerve injury; nerve transection; neuroinflammation; neuronal excitability; neutrophil; novel; novel therapeutics; operation; pain relief; painful neuropathy; pharmacologic; real time monitoring; receptor; response; screening; side effect; small molecule; small molecule therapeutics; success; temporal measurement; therapeutic target; therapeutically effective; treatment effect; voltage

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.

项目概要 与公共卫生的相关性。小胶质细胞是大脑和脊髓中的炎症细胞，参与 神经损伤后释放活性氧 (ROS) 和神经病理性疼痛 (NeuP) 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 的生活。 NeuP 通常对传统镇痛药无效，而阿片类药物在增加镇痛方面的功效有限。 滥用的风险。通常不用作镇痛药的药物通常是 NeuP 的一线治疗药物。如何- 迄今为止，<50% 的患者表示目前的治疗可缓解疼痛，而且副作用也很常见。我们提供证据 抑制小胶质细胞中的电压门控质子通道（Hv1）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) 来鉴定小分子 Hv1 阻断剂 植物提取物和植物化合物作为 NeuP 新药的一步。 简要背景。该应用程序建立在：(i) 我们的出版物中，Hv1 控制 ROS 和促炎症 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 Hv1 的设计肽阻断剂，抑制小胶质细胞释放 ROS 和促炎细胞因子，并注意 评估小鼠周围神经损伤后的 NeuP，以及 (iii) 我们成功地初步筛选了一种小化合物 使用新型活细胞、基于荧光的 HTS 测定法构建了文库和八种示例性植物提取物。 HTS 测定 采用与人类 Hv1 通道基因相关的 pH 敏感荧光蛋白 (hHv1-VFP-H148G)， 允许实时监控 Hv1 运行并识别 Hv1 阻断剂。 独特的功能和创新。我们的靶标选择具有创新性，因为尚无临床批准的药物 选择性靶向小胶质细胞，而 Hv1 缺乏有效且特异性的小分子阻断剂。我们的 HTS 测定是创新的 因为没有报道质子通道的高温超导方法。我们确定了独特的试剂：C6（第一个 Hv1 的特异性阻断剂）、C6 变体、F6（一种在 HTS 中抑制 Hv1 的小分子）和薰衣草（一种植物） 抑制 HTS 中 Hv1 的提取物），以促进 HTS 测定的开发、完善和验证。 我们将采用 HTS 测定来筛选一个独特的植物提取物库，其中包含超过 1,500 个物种（超过 15,000 个估计物种） 配合化合物），其中许多历史上曾用于抗炎和止痛。 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 相） 旨在筛选植物提取物库并鉴定有效且特异性的植物 Hv1 阻断剂。 (3) 学习支持 使用已鉴定的植物 Hv1 阻滞剂抑制小胶质细胞炎症反应和 NeuP（R33 期） 旨在验证已鉴定的植物 Hv1 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 疗法未满足的医疗需求，并具有更广泛的影响 这是因为小胶质细胞中的 Hv1 与其他炎症性疾病（例如缺血性中风）共谋。