Small-molecule probes for study of CLC-2 chloride-channel function in the central nervous system

用于研究中枢神经系统 CLC-2 氯离子通道功能的小分子探针

• 批准号: 10355474
• 负责人: Justin Du Bois
• 金额: $ 55.99万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-03-15 至 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10355474
• 关键词: Action Potentials; Acute; Affect; Antibodies; Biotin; Brain; Carbazoles; Cardiovascular Diseases; Carrier Proteins; Cell membrane; Cell physiology; Central Nervous System Diseases; Chemical Agents; Chemicals; Chloride Channels; Chloride Ion; Chlorides; Collection; Data; Docking; Electrolyte Balance; Electrophysiology (science); Epilepsy; Family; Hippocampus (Brain); Homologous Gene; Human; Image; Inhibitory Synapse; Investigation; Ion Channel; Knockout Mice; Label; Laboratories; Length; Libraries; Ligands; Link; Membrane; Membrane Proteins; Metabolic; Methods; Molecular; Muscle; Musculoskeletal Diseases; Mutation; Nature; Nervous System Physiology; Neuraxis; Neuroglia; Neurons; Neurosciences; Physiological; Physiology; Positioning Attribute; Presynaptic Terminals; Proteins; Publishing; Regulation; Research; Role; Site-Directed Mutagenesis; Slice; Small Interfering RNA; Specificity; Streptavidin; Structure; Structure-Activity Relationship; Synapses; Synaptic Transmission; Synthesis Chemistry; Testing; Thalamic structure; Tissues; Whole-Cell Recordings; Work; base; design; differential expression; efficacy validation; experimental study; extracellular; fluorescence imaging; imaging probe; improved; inhibitor; knock-down; mutant; nervous system disorder; neuronal excitability; neurotransmission; novel; patch clamp; programs; protein function; receptor; response; small molecule; tool; voltage

The CLC chloride channel family is a class of membrane proteins that controls the flux of chloride ions across cell membranes. Nine unique CLC homologs are differentially expressed in mammalian tissue and function in diverse physiological roles, ranging from electrical excitation of muscles and neurons to regulation of electrolyte balance. One subtype, CLC-2, is a voltage-dependent channel expressed broadly in the brain. Although the presence of CLC-2 in the brain has been known for decades, the role of this CLC homolog in neuronal signaling and proper brain function remains poorly understood, in part due to the absence of potent and selective small-molecule tools that enable studies of the molecular physiology of this channel. A recent breakthrough in our laboratories now opens the door to developing small molecule tools specific to CLC-2. Through a compound-library screen, we identified 'hit' compounds that inhibit CLC-2 activity. We developed one of these into a potent and selective CLC-2 inhibitor, FA44, which has an IC50 of 18 nM for CLC-2 and no off-target effects on the closest CLC homolog or on a panel of 65 CNS channels, receptors, and transporters. The efficacy and selectivity of FA44 for CLC-2 is further supported by our electrophysiological recordings of brain slices from wild-type versus CLC-2 knock-out mice. In this project, we will continue our collaborative efforts to develop, characterize, and use chemical tool compounds for studying CLC-2. In Aim 1, we will identify the mechanism of action and molecular determinants for inhibition of CLC-2. In Aim 2, we will develop novel probes, including small-molecule activators and fluorescent imaging probes for localizing channel expression. In Aim 3, we will leverage our tool compounds to query the role of CLC-2 in excitatory synaptic transmission and network excitability in the thalamus and to evaluate the potential causative link between CLC-2 malfunction and epilepsy. Our team's combined expertise in synthetic chemistry (Du Bois), ion-channel structure-function (Maduke), computation (Dror), and cellular neuroscience/epilepsy (Huguenard) ideally positions us to advance this research program.

CLC氯化物通道家族是一类膜蛋白，可控制 跨细胞膜的氯离子。九个独特的CLC同源物在 哺乳动物组织和在各种生理角色中的功能，范围从电激发 肌肉和神经元调节电解质平衡。一种亚型Clc-2是一个在大脑中广泛表达的电压依赖性通道。虽然大脑中CLC-2的存在 数十年来一直闻名，该CLC同源物在神经元信号传导中的作用 大脑功能仍然很少理解，部分原因是缺乏有效和选择性 小分子工具可以研究该通道的分子生理学。最近 现在，我们的实验室突破为开发小分子工具开辟了大门 到Clc-2。通过复合图形屏幕，我们确定了抑制CLC-2的“命中”化合物 活动。我们将其中之一开发为有效的选择性CLC-2抑制剂FA44，该抑制剂 对于CLC-2，IC50为18 nm，对最近的CLC同源物或对 65个CNS通道，受体和转运蛋白的面板。 FA44的功效和选择性 我们的电生理记录进一步支持了野生型脑切片的电生理记录 与CLC-2敲除小鼠相比。在这个项目中，我们将继续我们的合作努力 开发，表征和使用用于研究CLC-2的化学工具化合物。在AIM 1中，我们将 确定抑制Clc-2的作用机理和分子决定因素。在AIM 2中， 我们将开发新的探针，包括小分子激活剂和荧光成像 用于定位通道表达的探针。在AIM 3中，我们将利用我们的工具化合物 查询CLC-2在兴奋性突触传播和网络兴奋性中的作用 丘脑并评估CLC-2故障与癫痫之间的潜在因果关系。 我们团队在合成化学方面的综合专业知识（DU BOIS），离子通道结构功能功能 （Maduke），计算（DROR）和细胞神经科学/癫痫（Huguenard）的位置 我们推进了该研究计划。