An unbiased highthroughput behavior based screen for small molecule analgesics

 描述(申请人提供)：疼痛刺激的感觉是生存的关键。然而，由于受伤，急性和慢性疼痛影响着数亿人，在许多疾病中都是一种令人虚弱的症状。不幸的是，目前可用的药物疗法具有许多有害的副作用和/或滥用和成瘾的可能性，同时在治疗持续性疼痛方面也不有效。尽管投入了大量资金，但在开发新型止痛化合物方面取得的成功有限。占主导地位的药物开发模式遵循一条可预测的临床前路径，在该路径中，选定的靶点被服从于高通量的体外筛选，产生先导化合物，最后在疾病模型系统中进行安全性和有效性评估的测试。虽然这种方法可以取得成功，但由于目标选择不当、无法使用体外测试来模拟复杂的疼痛行为和/或在动物模型测试中无效和不可预见的副作用，它经常导致失败。止痛药开发的另一种方法是开发低成本、高通量、无靶向的基于动物的行为屏幕，模拟复杂的伤害性行为，在其中筛选止痛化合物。事实上，最常用的止痛药是在确定目标之前确定的，这是因为它们的止痛特性。在这里，我们建议使用一种替代的止痛药物发现方法，利用一种新的基于斑马鱼的行为测试来进行小分子筛选，以鉴定新的止痛化合物。斑马鱼为研究伤害性感觉提供了一个有趣的模型系统。斑马鱼幼虫的伤害性神经回路与啮齿动物和人类等高等脊椎动物的神经回路高度相似。此外，我们还发现斑马鱼幼体具有不同功能的外周和中枢神经系统，并对有害刺激做出强有力的反应。此外，斑马鱼可以低成本大量生产，其体积小，可以利用现有的高吞吐量平台快速扩大规模，这是其他脊椎动物系统(如啮齿动物)所不可能做到的。我们将利用位置厌恶分析，它可能利用脊髓上伤害性神经元电路，因为幼虫必须选择避免伤害性刺激，以模拟敏感化的热痛觉过敏，这是许多慢性疼痛疾病的症状。在本实验中，单独排列的斑马鱼幼体对有害温度表现出极大的厌恶。证明我们的位置厌恶反映了伤害性行为，具有已知止痛特性的小分子有效地逆转了热厌恶。在这项探索性研究中，利用所描述的方法，我们建议为新的止痛化合物筛选10,000个化合物小分子文库。在未来利用啮齿动物模型系统进行的研究中，将评估识别出的分子的止痛特性以及它们的作用部位和 可能会导致新型止痛药的产生。这项提案的发现可能会为止痛药的发现提供一条前进的道路，这可能会为靶向药物开发提供一条补充的平行途径。

 DESCRIPTION (provided by applicant): Sensation of painful stimuli is critical for the survival. However, acute and chronic pain affects hundreds of millions of people as a result of injury, and is a debilitating symptom in many illnesses. Unfortunately, currently available drug based therapies have numerous deleterious side effects and/or potential for abuse and addiction, while also not being effective in the treatment of persistent pain. Despite massive investment, there has been limited success in the development of novel analgesic compounds. The dominant drug development model follows a predictable preclinical path in which a selected target is subjugated to high-throughput in vitro screens, the generation of lead compounds, and finally testing in disease model systems for safety and efficacy assessments. While this approach can be successful, it often leads to failure due to poor target selection, the inability to model complx pain behaviors using in vitro testing and/or ineffectiveness and unforeseen side effects in animal model testing. An alternative approach to analgesic development would be to develop low cost, high throughput, untargeted animal based behavioral screens that model complex nociceptive behaviors in which to screen for analgesic compounds. In fact the most commonly used analgesics were identified due to their analgesic properties prior to target identification. Here w propose to use an alternative analgesic drug discovery approach by utilizing a novel zebrafish based behavioral assay to conduct a small molecule screen to identify novel analgesic compounds. The zebrafish provides an intriguing model system to study nociception. The neural circuits underling nociception in zebrafish larvae are highly analogous to those found in higher vertebrates such as rodents and humans. Furthermore we've shown that zebrafish larvae have a functionally diverse peripheral and central nervous system and respond robustly to noxious stimuli. Additionally zebrafish can be generated in large numbers at low costs and their small size allows for rapid upscaling using existing high throughput platforms, which is not possible with other vertebrate systems such as rodents. We will utilize a place aversion assay, which likely utilizes supraspinal nociceptive neuronal circuitry since the larvae must choose to avoid the noxious stimulus, to model sensitized thermal hyperalgesia, a symptom of many chronic pain diseases. In this assay individually arrayed zebrafish larvae show profound aversion to noxious temperature. Demonstrating that our place aversion reflects nociceptive behavior, small molecules with known analgesic properties potently reverse thermal aversion. In this exploratory study, utilizing the described assay, we propose to screen a 10,000 compound small molecule library for novel analgesic compounds. Identified molecules will be evaluated for their analgesic properties in future studies utilizing rodent model systems as well as for their site of action and could lead to the generation of novel analgesics. Findings from this proposal could provide a way forward for analgesic discovery that may offer a complementary parallel pathway to target-based drug development.