An unbiased highthroughput behavior based screen for small molecule analgesics

 描述（由申请人提供）：疼痛刺激的感觉对生存至关重要。然而，急性和慢性疼痛影响着数亿人的伤害，是许多疾病的衰弱症状。不幸的是，目前可用的基于药物的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中也无效。尽管有大量的投资，但在开发新型镇痛化合物方面取得的成功有限。主导药物开发模型遵循可预测的临床前路径，其中选定的靶标服从于高通量体外筛选，产生先导化合物，并最终在疾病模型系统中进行安全性和有效性评估测试。虽然这种方法可能是成功的，但由于目标选择不佳，无法使用体外测试和/或动物模型测试中的无效性和不可预见的副作用来模拟复杂的疼痛行为，它通常会导致失败。镇痛剂开发的另一种方法是开发低成本、高通量、非靶向的基于动物的行为筛选，其模拟复杂的伤害感受行为，以筛选镇痛剂化合物。事实上，最常用的镇痛药是在靶点确定之前根据其镇痛特性确定的。在这里，我们建议使用一种替代的镇痛药物发现方法，利用一种新的斑马鱼为基础的行为分析进行小分子筛选，以确定新的镇痛化合物。斑马鱼为研究伤害性感受提供了一个有趣的模型系统。斑马鱼幼鱼的伤害感受神经回路与啮齿类和人类等高等脊椎动物的伤害感受神经回路高度相似。此外，我们已经表明，斑马鱼幼虫具有功能多样的外周和中枢神经系统，并对有害刺激做出强烈反应。此外，斑马鱼可以以低成本大量产生，并且它们的小尺寸允许使用现有的高通量平台进行快速放大，这对于其他脊椎动物系统如啮齿动物是不可能的。我们将利用一个位置厌恶试验，这可能利用脊髓上的伤害性神经元回路，因为幼虫必须选择避免有害的刺激，模型致敏热痛觉过敏，许多慢性疼痛疾病的症状。在该试验中，单独排列的斑马鱼幼虫对有害温度表现出深刻的厌恶。证明我们的位置厌恶反映了伤害性行为，具有已知镇痛特性的小分子有效地逆转了热厌恶。在该探索性研究中，利用所描述的测定，我们提出筛选10，000个化合物小分子文库以寻找新型镇痛化合物。在未来的研究中，将利用啮齿动物模型系统评估鉴定的分子的镇痛特性以及它们的作用位点， 可能会导致新的止痛药的产生。这项提案的发现可以为镇痛药的发现提供一种前进的方式，这可能为基于靶点的药物开发提供一种互补的平行途径。

 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.