Accelerating General Anesthetic Discovery and Mechanisms Research with Zebrafish

摘要：全身麻醉是现代程序医学的重要组成部分。迫切需要 寻找当前镇静催眠药的更好替代品，这些镇静催眠药源于之前确定的几种药物类别 而且还会造成不良或有害的副作用，特别是在脆弱人群中。到目前为止， 发现新麻醉药的策略是基于分子模型，主要是GABAA受体，即 几种强力静脉注射催眠药的靶标。我们的长期目标是寻找新的镇静催眠药 化学类型可能改善患者护理，同时也促进分子和神经生物学研究 麻醉机制。我们假设有多种未被发现的强效镇静催眠药 存在化学类型。它们可能通过几个已知的位点来调节GABAA受体，或者通过其他 已知或未知的机制。我们的新策略使用并发视频运动分析， 96条斑马鱼幼虫作为高通量无偏刺激反应测试平台加速发现 以及新型强效镇静催眠药的特性。斑马鱼幼体浸泡在非挥发性溶液中 药物迅速建立评估药效学效果的稳态条件。将这一点结合起来 利用含有突变的新转基因斑马鱼品系来改变或消除已知和 神经系统中可疑的全身麻醉靶点也将加速分子的探索 和神经生物学机制。在目标1中，我们将使用视频分析工具来评估两个基线运动 和多达96条斑马鱼幼体的光运动反应概率。我们已经开发出了强大的 筛选新镇静催眠药和确定镇静剂的实验和数据分析方法 和催眠能力(目标1a)。我们已经在一种药物中发现了几种有效的活性先导化合物 波士顿大学医学发现中心(BUCMD)的图书馆。还将测试新的有效销售线索 用于蝌蚪和大鼠的活动/效力(目标1b)。BUCMD的合作者还将合成结构 选定的先导化合物的变体，使探索镇静和 斑马鱼的催眠作用，如初步数据(目标1c)所示。在目标2中，我们将探索分子 新型镇静催眠药的异种电压钳电生理机制 表达的离子通道(GABAARs、GlyRs、NMDA-Rs、神经元nAChRs、HCN1和Trek-3)或 α-2肾上腺素能受体的安替帕美唑拮抗作用(目标2a)。镇静剂--能增强GABA的催眠药， 还将在一系列受体突变体中进行测试，以评估已知亚基的选择性(目标2b)。在《目标3》中， 我们将使用CRISPR-Cas9创建转基因斑马鱼品系，以测试药物靶标在镇静中的作用 还有催眠。我们将首先创建一组GABAAR亚基敲除和敲入线(目标3a；β3和δ KO是制造的)。我们还将为HN1(已经制造)、Trek-3、K2P和α2 ADR(目标3b)进行基因敲除。 已知和新药的镇静和催眠效力将在突变斑马鱼和野生型斑马鱼中进行比较。

ABSTRACT: General anesthesia is essential for modern procedural medicine. There is a compelling need to find superior alternatives to current sedative-hypnotics, which derive from a few drug classes identified before 1980, and also cause undesirable or harmful side effects, especially in vulnerable populations. To date, strategies to discover new anesthetics are based on molecular models, mostly GABAA receptors, that are targets for a few potent intravenous hypnotics. Our long-term goals are to identify new sedative-hypnotic chemotypes that may improve patient care, while also advancing molecular and neurobiological research on anesthetic mechanisms. We hypothesize that a wide variety of undiscovered potent sedative-hypnotic chemotypes exists. These may modulate GABAA receptors through several known sites, or act through other known or perhaps unknown mechanisms. Our novel strategy uses concurrent video motion analysis of up to 96 zebrafish larvae as a high-throughput un-biased stimulus-response test platform to accelerate discovery and characterization of new potent sedative-hypnotics. Zebrafish larvae immersed in solutions of non-volatile drugs rapidly establishes steady-state conditions for assessing pharmacodynamic effects. Combining this approach with new transgenic zebrafish lines harboring mutations that alter or eliminate known and suspected general anesthetic targets in the nervous system will also accelerate exploration of molecular and neurobiological mechanisms. In Aim 1, we will use video analysis tools to assess both baseline motion and photomotor response probability in up to 96 zebrafish larvae simultaneously. We have developed robust experimental and data analysis approaches to both screen for new sedative-hypnotics and determine sedative and hypnotic potencies (Aim 1a). We already have identified several potent active lead compounds in a drug library from the Boston University Center for Medical Discovery (BUCMD). New active leads will also be tested for activity/potency in both tadpoles and rats (Aim 1b). Collaborators at BUCMD will also synthesize structural variants of selected lead compounds, enabling exploration of structure-activity relationships for sedation and hypnosis in zebrafish, as illustrated in preliminary data (Aim 1c). In Aim 2, we will explore the molecular mechanisms of new sedative-hypnotics using voltage-clamp electrophysiology in a panel of heterologously expressed ion channels (GABAARs, GlyRs, NMDA-Rs, neuronal nAChRs, HCN1 and TREK-3) or antipamezole antagonism of α2 adrenergic receptors (Aim 2a). Sedative-hypnotics that potentiate GABAARs, will also be tested in a series of receptor mutants to assess selectivity for known subsites (Aim 2b). In Aim 3, we will use CRISPR-Cas9 to create transgenic zebrafish lines for testing the roles of drug targets in sedation and hypnosis. We will first create a panel of GABAAR subunit knock-out and knock-in lines (Aim 3a; β3 and δ KOs are made). We will also make knockouts for HCN1 (already made), TREK-3, K2P and α2 AdRs (Aim 3b). Sedative and hypnotic potencies of known and novel drugs will be compared in mutant vs. wild-type zebrafish.