The mechanism of allosteric modulation of glutamate transporters

谷氨酸转运蛋白变构调节机制

基本信息

批准号：
9916361
负责人：
Olga Boudker
金额：
$ 60.26万
依托单位：
DREXEL UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-12-01 至 2024-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9916361
关键词：
Acute Address Affect Allosteric Site American Amino Acid Transporter Antibodies Binding Biological Assay Brain Carrier Proteins Central Nervous System Diseases Chemicals Chronic Communication Complex Computer Models Cryoelectron Microscopy Crystallization Development Drug Targeting Elevator Enhancers Epilepsy Excision Excitatory Amino Acid Transporter 1 Excitatory Amino Acids Fluorescence Resonance Energy Transfer Future Glutamate Transporter Glutamates Goals Homologous Gene Human Injury Knowledge Lead Measurement Mediating Methods Microscopy Modernization Molecular Molecular Conformation Movement Mutagenesis Neurodegenerative Disorders Neuronal Injury Neurotransmitters Pain Peripheral Pharmaceutical Chemistry Pharmacology Phylogenetic Analysis Play Population Process Property Proteins Research Resolution Role Sampling Specificity Stroke Structure Synaptic Cleft Techniques Technology Testing Transmembrane Transport Traumatic Brain Injury Variant Vesicle X-Ray Crystallography base biophysical analysis clinically relevant conformational conversion design drug discovery excitotoxicity extracellular improved in vitro Model in vivo Model multidisciplinary nanobodies neuron loss neurotoxic novel strategies painful neuropathy particle programs scaffold single molecule small molecule therapy development thermostability three dimensional structure virtual screening

项目摘要

Abstract Excitatory amino acid transporters (EAATs) remove synaptically released glutamate and maintain extracellular glutamate concentrations below neurotoxic levels. Particularly the glial glutamate transporter EAAT2 plays a major role in glutamate clearance in synaptic clefts. Removal of excess cellular glutamate is strongly implicated as a clinically relevant means to treat neurodegenerative diseases where excitotoxicity due to excess glutamate contributes to neuronal injury and death. We have designed a unique research program to understand the molecular mechanisms of allosteric modulation of glutamate transporters by recently discovered small-compound molecules, including long-sought activators of transport. To reach this goal, we will obtain and integrate multidisciplinary knowledge of the transport function, 3D structures, and single-molecule dynamics of the transporters and their complexes with the allosteric compounds. In turn, this knowledge will aid understanding the basic membrane transport mechanisms of the most important excitatory neurotransmitter in the human brain. We will pursue the following aims: Aim 1: Elucidate the molecular determinants within the human EAATs that are important for allosteric modulator activity. We will use functional studies and computational approaches to define the allosteric site within EAAT2 that mediate the effects of the compounds. Aim 2: Determine the structures of human EAATs in complex with allosteric modulators. We will determine the three-dimensional structures of EAATs in complex with positive and negative allosteric modulators to unravel the atomic details of their coordination. Aim 3: Establish whether allosteric modulators modulate the function of EAATs though altering the rates of conformational transitions underlying transport. We will examine the conformational dynamics and its modulation by allosteric modulators using single-molecule FRET and other spectroscopic techniques and couple these studies with single-vesicle/single-transporter assays to determine the effects of allosteric modulators on turnover rates and the timing of transport cycles. IMPACT: Information generated in this research program will open new avenues for drug discovery as these transporters serve as important drug targets for many severe debilitating CNS conditions, such as traumatic brain injury, stroke, epilepsy, ALS and neuropathic pain, that collectively affect nearly 5% of the American population.

抽象的兴奋性氨基酸转运蛋白（EAATS）去除突触释放的谷氨酸并保持细胞外谷氨酸浓度低于神经毒性水平。特别是神经胶质谷氨酸转运蛋白EAAT2播放A 突触裂隙中谷氨酸清除中的主要作用。去除过多的细胞谷氨酸是强烈暗示的作为治疗神经退行性疾病的一种临床相关手段，而由于谷氨酸过多而引起的兴奋性毒性导致神经元损伤和死亡。我们设计了一个独特的研究计划，以了解变构调节的分子机制最近发现的小型分子的谷氨酸转运蛋白，包括长期追求的激活剂运输。为了实现这一目标，我们将获得并整合有关运输功能的多学科知识，转运蛋白的3D结构和单分子动力学及其复合物具有变构型化合物。反过来，这些知识将有助于了解该知识的基本膜运输机制人脑中最重要的兴奋性神经递质。我们将追求以下目标：目的1：阐明人类老鹰中对变构重要的分子决定因素调节器活动。我们将使用功能研究和计算方法来定义变构位点在EAAT2中介导化合物的作用。 AIM 2：确定与变构调节剂复杂的人类EAAT的结构。我们将确定 EAAT的三维结构，具有正分和负变构调节剂，以解开它们协调的原子细节。目标3：确定变构调节器是否在改变速率的情况下调节EAAT的功能构象转换的基础运输。我们将检查构象动力及其使用单分子fret和其他光谱技术和夫妇对变构调节剂进行调节这些对单维/单转运蛋白测定的研究，以确定变构调节剂对周转率和运输周期的时机。影响：本研究计划中产生的信息将为这些毒品发现开放新途径转运蛋白作为许多严重使人衰弱的中枢神经系统条件（例如创伤性）的重要药物靶标脑损伤，中风，癫痫，ALS和神经性疼痛，这些疼痛总共影响了近5％的美国人人口。