Accelerating General Anesthetic Discovery and Mechanisms Research with Zebrafish

加速斑马鱼全身麻醉的发现和机制研究

• 批准号: 9983104
• 负责人: STUART A FORMAN
• 金额: $ 37.99万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-08-01 至 2021-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9983104
• 关键词: Academic Medical Centers; Adrenergic Receptor; Affect; Age; Amnesia; Anesthetics; Biological Assay; Boston; CRISPR/Cas technology; Clinical; Computer software; Data; Data Analyses; Development; Disease; Drug Kinetics; Drug Screening; Drug Targeting; Electrophysiology (science); Equipment; Fishes; Gene Mutation; General Anesthesia; General anesthetic drugs; Genetic; Glycine Receptors; Goals; HCN1 gene; Hypnosis; Immobilization; Intravenous; Intravenous Anesthetics; Ion Channel; Knock-in; Knock-out; Knockout Mice; Larva; Lead; Learning; Libraries; Link; Measures; Mediating; Medicine; Modeling; Modern Medicine; Modernization; Molecular; Molecular Structure; Molecular Target; Motion; Mutation; N-Methyl-D-Aspartate Receptors; N-Methylaspartate; Nervous system structure; Neurobiology; Neurons; Pain; Patient Care; Patients; Pharmaceutical Preparations; Pharmacodynamics; Pharmacology; Phenotype; Play; Probability; Procedures; Property; Rattus; Reflex action; Research; Response to stimulus physiology; Risk; Role; Sedation procedure; Series; Site; Sprague-Dawley Rats; Stimulus; Structure; Structure-Activity Relationship; Tadpoles; Testing; Transgenic Organisms; Universities; Variant; Vulnerable Populations; Xenopus; Zebrafish; base; drug mechanism; experimental analysis; experimental study; hypnotic; improved; inhibitor/antagonist; intravenous administration; molecular modeling; mutant; neurobiological mechanism; novel; novel strategies; novel therapeutics; receptor; response; scale up; sedative; side effect; success; tool; voltage clamp

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.

摘要：全身麻醉对于现代手术医学至关重要。 找到当前镇静催眠药的更好替代品，这些药物源自之前确定的一些药物类别 1980 年，并且还造成不良或有害的副作用，特别是在弱势群体中。 发现新麻醉剂的策略基于分子模型，主要是 GABAA 受体， 我们的长期目标是确定一些有效的静脉催眠药。 化学型可以改善患者护理，同时也推进分子和神经生物学研究 我们努力寻找各种未被发现的强效镇静催眠药。 存在化学型，它们可能通过几个已知位点调节 GABAA 受体，或通过其他位点发挥作用。 我们的新颖策略使用最多可达的并发视频运动分析。 96 只斑马鱼幼虫作为高通量无偏刺激响应测试平台，以加速发现 以及浸入非挥发性溶液中的新型强效镇静催眠药的表征。 建立快速稳态药物条件以评估药效学效果。 使用携带突变的新转基因斑马鱼品系的方法可以改变或消除已知和 神经系统中可疑的全身麻醉靶点也将加速分子探索 在目标 1 中，我们将使用视频分析工具来评估基线运动。 我们已经开发出强大的模型，可以同时检测多达 96 只斑马鱼幼虫的光运动反应概率。 筛选新镇静催眠药并确定镇静剂的实验和数据分析方法 和催眠功效（目标 1a）。我们已经在药物中鉴定出几种有效的活性先导化合物。 波士顿大学医学发现中心 (BUCMD) 的图书馆也将测试新的活性线索。 蝌蚪和大鼠的活性/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，能够探索镇静和镇静作用的结构-活性关系 斑马鱼的催眠，如初步数据所示（目标 1c），在目标 2 中，我们将探索分子机制。 在一组异源中使用电压钳电生理学的新型镇静催眠药的机制 表达通道（GABAAR、GlyR、NMDA-R、神经元 nAChR、HCN1 和 TREK-3）或 α2 肾上腺素受体的抗帕美唑拮抗作用（目标 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）。 我们将使用 CRISPR-Cas9 创建转基因斑马鱼品系，用于测试药物靶点在镇静中的作用 我们将首先创建一组 GABAAR 亚基敲除和敲入线（目标 3a；β3 和 δ）。 我们还将对 HCN1（已制成）、TREK-3、K2P 和 α2 AdR（目标 3b）进行敲除。 将在突变型斑马鱼与野生型斑马鱼中比较已知药物和新型药物的镇静和催眠功效。

项目成果

Drug-selective Anesthetic Insensitivity of Zebrafish Lacking γ-Aminobutyric Acid Type A Receptor β3 Subunits.

缺乏γ-氨基丁酸A型受体β3亚基的斑马鱼的药物选择性麻醉不敏感性。

• DOI: 10.1097/aln.0000000000002963
• 发表时间: 2019-12-01
• 期刊: Anesthesiology
• 影响因子: 8.8
• 作者: Xiaoxuan Yang;Y. Jounaidi;Kusumika Mukherjee;Ryan J. Fantasia;E. Liao;Buwei Yu;S. Forman
• 通讯作者: S. Forman