Molecular Sites of Neurosteroid Binding

神经类固醇结合的分子位点

基本信息

批准号：
9913562
负责人：
ALEX S. EVERS
金额：
$ 58.92万
依托单位：
WASHINGTON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-05-01 至 2023-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9913562
关键词：
ART protein Affect Affinity Allopregnanolone Amino Acids Anesthetics Antidepressive Agents Antiepileptic Agents Baculoviruses Behavior Binding Binding Sites Biochemical Biological Assay Biology Brain Cell surface Cells Chloride Channels Data Development Docking GABA Receptor Goals Label Laboratories Libraries Ligand Binding Ligands Mass Spectrum Analysis Measures Mediating Methods Molecular Molecular Target Muscimol Mutate Mutation Neuroprotective Agents Nootropic Agents Outcome Pharmaceutical Chemistry Pharmaceutical Preparations Photoaffinity Labels Positioning Attribute Pregnanolone Production Protein Chemistry Protein Isoforms Proteins Reagent Research Site Sterols Structure Surface Synapses System Techniques Therapeutic Therapeutic Agents Transferase Transmembrane Domain Vertebral column Viral Work analog gamma-Aminobutyric Acid glycosylation insight molecular modeling molecular site mutant nervous system development neuronal excitability neurosteroids novel novel therapeutics pharmacophore positive allosteric modulator pregnenolone sulfate prevent protein expression receptor receptor expression receptor function sedative simulation stoichiometry trafficking

项目摘要

Neurosteroids are important modulators of neuronal excitability and nervous system development with enormous therapeutic potential as anti-depressants, anesthetics and neuro-protectants. The principal molecular target of neurosteroids is the GABAA receptor. We have shown that there are multiple, subunit- specific binding sites for neurosteroids on GABAA receptors, each of which contributes to the effects of neurosteroids on receptor expression and function. This project will use photolabeling techniques to define the precise sites of neurosteroid binding on the most abundant forms of synaptic and extrasynaptic GABAA receptors. Novel neurosteroid analogue photolabeling reagents will be developed and used to determine the number of neurosteroid binding sites on each subunit, to identify the binding sites and to determine the orientation of the neurosteroids in these sites. To achieve these goals we will utilize state-of-the-art protein chemistry and expression techniques in conjunction with cutting edge mass spectrometry (MS) methods, including middle-down, intact protein and native MS. We will then mutate amino acids in each of the identified binding sites and use functional readouts to determine which binding sites mediate the various effects of neurosteroids on GABAA receptor expression and function. The Project has two specific aims: In Aim 1, a viral expression system (BacMam) will be used to express large quantities of synaptic (α1β2γ2) and extrasynaptic (α4β3δ) GABAA receptors and a suite of neurosteroid analogue photolabeling reagents will be synthesized in which the photolabeling moieties are placed at various positions around the neurosteroid backbone. The expressed receptors will be used in conjunction with the photolabeling reagents to determine the number of labeling sites on each subunit using intact protein MS and on each pentameric receptor using native MS. The specific amino acids modified by photolabeling will be identified using middle-down MS. The photolabeling data will then be used in conjunction with molecular modeling and docking to determine the preferred orientation of neurosteroids in each of the binding pockets and to identify critical residues which may be necessary for neurosteroid binding or effect. In Aim 2 receptors will be expressed in which these critical residues are mutagenized. The mutant receptors will be used in functional assays (channel gating, trafficking of receptors to the surface and modulation of orthosteric ligand binding) to determine the contribution of each binding site to neurosteroid action(s). Neurosteroid labeling and orientation will be then be assessed in the mutated receptors (as in Aim 1) to determine how the mutations alter neurosteroid binding and/or effect. The data from these studies will provide insight into the specific molecular interactions underlying neurosteroid actions at each of its binding sites on GABAA receptors. These data will provide a structural template for development of subunit- and function-specific neurosteroid analogues as well as novel insights into neurosteroid biology.

神经类固醇是神经兴奋性和神经系统发育的重要调节剂作为抗抑郁药、麻醉剂和神经保护剂具有巨大的治疗潜力。神经类固醇的分子靶标是 GABAA 受体，我们已经证明有多个亚基。 GABAA 受体上神经类固醇的特异性结合位点，每个位点都有助于该项目将使用光标记技术来定义神经类固醇对受体表达和功能的影响。神经类固醇在最丰富形式的突触和突触外 GABAA 上结合的精确位点新型神经类固醇类似物光标记试剂将被开发并用于确定每个亚基上神经类固醇结合位点的数量，以确定结合位点并确定为了实现这些目标，我们将利用最先进的蛋白质。化学和表达技术与尖端质谱（MS）方法相结合，包括中下完整蛋白质和天然 MS，然后我们将突变每个已识别的氨基酸。结合位点并使用功能读数来确定哪些结合位点介导了各种影响神经类固醇对 GABAA 受体表达和功能的影响该项目有两个具体目标：在目标 1 中，将使用病毒表达系统 (BacMam) 表达大量突触 (α1β2γ2) 和突触外 (α4β3δ) GABAA 受体和一套神经类固醇类似物光标记试剂将被合成，其中光标记部分被放置在神经类固醇周围的不同位置表达的受体将与光标记试剂结合使用来确定。使用完整蛋白质 MS 计算每个亚基上的标记位点数量，使用使用 MS 计算每个五聚体受体上的标记位点数量天然 MS。通过光标记修饰的特定氨基酸将使用中下 MS 进行鉴定。然后，光标记数据将与分子建模和对接结合使用，以确定每个结合袋中神经类固醇的优选方向并确定可能的关键残基对于神经类固醇的结合或作用是必需的，在 Aim 2 受体中将表达这些关键的受体。残基被诱变。突变受体将用于功能测定（通道门控、运输）。受体对表面的影响和正位配体结合的调节）以确定每个的贡献然后将评估神经类固醇作用的结合位点和方向。突变的受体（如目标 1）以确定突变如何改变神经类固醇的结合和/或效果。这些研究的数据将深入了解神经类固醇背后的特定分子相互作用这些数据将为 GABAA 受体的每个结合位点提供结构模板。亚基和功能特异性神经类固醇类似物的开发以及新的见解神经类固醇生物学。