Structure and Function of AMPA subtype ionotropic glutamate receptors

AMPA 亚型离子型谷氨酸受体的结构和功能

基本信息

批准号：
10197227
负责人：
MARIA G KURNIKOVA
金额：
$ 50.13万
依托单位：
COLUMBIA UNIVERSITY HEALTH SCIENCES
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-09-30 至 2023-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10197227
关键词：
AMPA Receptors Adamantane Alzheimer&apos s Disease Animal Model Antiepileptic Agents Behavior Binding Sites Biochemical Biophysics Cell Line Cells Color Complement Complex Cryoelectron Microscopy Crystallization Dementia Development Disease Drug Design Drug Targeting Electrophysiology (science)Epilepsy Epileptogenesis Eukaryotic Cell Family Fluorescence Future Glutamate Receptor Goals Homo Individual Insecta Ion Channel Ischemia Knowledge Learning Length Maps Mediating Memory Mental disorders Methodology Methods Modeling Modernization Molecular Molecular Structure Monitor Mutagenesis Neuraxis Neurodegenerative Disorders Pharmaceutical Preparations Pharmacotherapy Play Polyamines Positioning Attribute Primary Lateral Sclerosis Proteins Protocols documentation Regulation Research Resolution Seizures Structural Models Structure Techniques Testing Therapeutic Toxin Transfection Viral base biophysical model cognitive process design drug candidate drug development drug testing experimental study expression vector human disease human model improved in silico inhibitor/antagonist ion channel blocker kinetic model molecular dynamics molecular modeling nervous system disorder neurotransmission next generation novel therapeutics patch clamp promoter receptor expression receptor structure function scaffold small molecule

项目摘要

AMPA receptors mediate fast excitatory neurotransmission, contribute to high cognitive processes such as learning and memory and are implicated in numerous psychiatric and neurodegenerative diseases. In particular, AMPA receptors play a key role in epileptogenesis and seizure spread and, thus, have recently emerged as one of the most promising targets for epilepsy therapy. However, development of drugs targeting AMPA receptors has been stalled because of the lack of knowledge about AMPA receptor structure and function. For example, only structures of homotetrameric intact AMPA receptors have been determined, while the overwhelming majority of AMPA receptors in the central nervous system are heterotetramers. A number of noncompetitive inhibitors and ion channel blockers have been identified as promising candidates for drug development but structural mechanisms of their action on AMPA receptors remain largely unexplored. This missing information is absolutely critical for the future structure-based rational drug design. We plan to study structure and function of AMPA receptors using a combination of biophysical and biochemical approaches, including modern crystallographic and cryo-electron microscopy (cryo-EM) techniques, fluorescence-based methods, electrophysiology, kinetic and molecular modeling. Our specific aims are to (1) obtain structures of heteromeric AMPA receptors, (2) establish the molecular mechanism of noncompetitive inhibition, and (3) build a structural model of ion channel block. To reach our goals, we will optimize AMPA receptor constructs for crystallization and cryo-EM experiments, develop protocols of their expression and purification and solve structures of heterotetrameric AMPA receptors and AMPA receptors in complex with noncompetitive inhibitors and ion channel blockers. To improve our structural models, we will use new methods of structural refinement combined with molecular dynamics (MD) simulations. We will also test our models using a combination of experimental and in silico mutagenesis, whole-cell patch-clamp recordings and MD simulations. To understand the molecular mechanisms of AMPA receptor heteromeric assembly, noncompetitive inhibition and ion channel block, we will perform extensive MD simulations of homo- and heteromeric AMPA receptors in different activation states and in the presence or absence of noncompetitive inhibitors and ion channel blockers. We will combine the results of structural, computational, functional and mutagenesis experiments to propose molecular models of AMPA receptor heteromeric assembly, noncompetitive inhibition and ion channel block. Reaching our research goals will provide molecular level knowledge essential to greatly facilitate design of new molecules that will have a potential to become safe and more efficacious drugs to treat epilepsy and other disorders related to excitatory neurotransmission.

AMPA受体介导快速兴奋性神经传递，有助于高认知过程学习和记忆，并与许多精神病和神经退行性疾病有关。在特别是，AMPA受体在癫痫发生和癫痫发作中起关键作用，因此最近具有成为癫痫疗法最有希望的靶标之一。但是，靶向药物的开发由于缺乏有关AMPA受体结构和功能。例如，仅确定了同型完整AMPA受体的结构，而中枢神经系统中绝大多数AMPA受体是异驱精。许多非竞争性抑制剂和离子通道阻滞剂已被确定为有前途的药物候选者开发但其对AMPA受体的作用的结构机制在很大程度上尚未得到探索。这缺少信息对于将来基于结构的理性药物设计至关重要。我们计划学习使用生物物理和生化方法的组合AMPA受体的结构和功能，包括现代晶体学和冷冻电子显微镜（Cryo-EM）技术，基于荧光的技术方法，电生理学，动力学和分子建模。我们的具体目的是（1）获得杂体AMPA受体，（2）建立非竞争性抑制的分子机制，（3）构建离子通道块的结构模型。为了实现我们的目标，我们将优化AMPA受体构建体结晶和冷冻EM实验，开发其表达和纯化的方案，并解决与非竞争性抑制剂复合物中的异端AMPA受体和AMPA受体的结构和离子通道阻滞剂。为了改善我们的结构模型，我们将使用新的结构改进方法结合分子动力学（MD）模拟。我们还将结合使用在实验和计算机诱变中，全细胞斑块钳记录和MD模拟。理解 AMPA受体异质组装，非竞争性抑制和离子通道的分子机制块，我们将对不同的同型和异源AMPA受体进行广泛的MD模拟激活状态以及存在或不存在非竞争性抑制剂和离子通道阻滞剂。我们将结合结构，计算，功能和诱变实验的结果以提出分子 AMPA受体异质组装，非竞争性抑制和离子通道阻滞的模型。到达我们的研究目标将为大大促进新的设计提供分子级知识分子有可能成为安全，更有效的药物来治疗癫痫和其他药物与兴奋性神经传递有关的疾病。