Accelerating General Anesthetic Discovery and Mechanisms Research with Zebrafish

摘要：全身麻醉是现代手术医学的基础。迫切需要 找到比目前的镇静催眠药更好的上级替代品，这些替代品来自以前确定的几类药物 1980年，也造成不良或有害的副作用，特别是在弱势群体中。到目前为止， 发现新麻醉剂的策略是基于分子模型，主要是GABAA受体， 几种强效静脉注射催眠药的靶点我们的长期目标是找出新的镇静催眠药 化学型，可以改善病人的护理，同时也推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 存在化学型。这些可以通过几个已知的位点调节GABAA受体，或通过其他途径起作用。 已知或未知的机制。我们的新策略使用并发视频运动分析高达 96斑马鱼幼虫作为高通量无偏刺激反应测试平台，以加速发现 和新型强效镇静催眠药的特性。斑马鱼幼虫浸泡在非挥发性的 药物快速建立用于评估药效学效应的稳态条件。组合该 方法与新的转基因斑马鱼线窝藏突变，改变或消除已知的， 神经系统中疑似全身麻醉靶点的发现也将加速对分子麻醉的探索。 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基线运动 和光运动反应概率在多达96斑马鱼幼虫同时。我们开发了强大的 实验和数据分析的方法来筛选新的镇静催眠药和确定镇静剂 催眠作用（Aim 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 Boston University Center for Medical Discovery（BUCMD）还将测试新的有源电极导线 在蝌蚪和大鼠中的活性/效力（目标1b）。BUCMD的合作者还将合成结构 所选先导化合物的变体，能够探索镇静和 催眠在斑马鱼，如初步数据所示（目标1c）。在目标2中，我们将探索分子 在一组异源性神经元中使用电压钳电生理学研究新镇静催眠药的机制 表达的离子通道（GABAAR、GlyR、NMDA-R、神经元nAChR、HCN 1和TREK-3）或 α2肾上腺素能受体拮抗剂（Aim 2a）。增强GABAAR的镇静催眠药， 还将在一系列受体突变体中进行测试，以评估对已知亚位点的选择性（目标2b）。在目标3中， 我们将使用CRISPR-Cas9来创建转基因斑马鱼系，以测试药物靶点在镇静中的作用。 还有催眠我们将首先建立一组GABAAR亚基敲除和敲入系（Aim 3a; β3和δ 制造科斯）。我们还将敲除HCN 1（已制备）、TREK-3、K2 P和α2 AdR（Aim 3b）。 将在突变型与野生型斑马鱼中比较已知和新型药物的镇静和催眠效力。

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.