Characterizing Neurosteroid Binding in the GABA(A) Receptor Using Top-Down Mass Spectrometry

使用自上而下质谱法表征 GABA(A) 受体中的神经类固醇结合

• 批准号: 9431581
• 负责人: Wayland Wing-Lun Cheng
• 金额: $ 18.12万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-20 至 2021-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9431581
• 关键词: Address; Affinity; Amino Acid Sequence; Anesthesiology; Anesthetics; Binding; Binding Sites; Biophysics; Brain; Cells; Chimera organism; Coupled; Dependence; Drug Design; Environment; Escherichia coli; Extracellular Domain; Funding; GABA-A Receptor; Gated Ion Channel; General anesthetic drugs; Goals; Heterogeneity; Human; Hydrophobicity; Integral Membrane Protein; Ion Channel; Ion Channel Gating; Knowledge; Label; Lead; Libraries; Ligand Binding; Ligands; Maps; Mass Spectrum Analysis; Measurement; Membrane Proteins; Mental disorders; Mentorship; Methods; Modeling; Modification; Molecular; Mutation; National Institute of General Medical Sciences; Peptide Sequence Determination; Pharmacology; Phosphorylation; Photoaffinity Labels; Positioning Attribute; Post-Translational Protein Processing; Proteins; Reagent; Research; Research Personnel; Resolution; Resources; Sensory Receptors; Site; Specificity; Stereoisomer; Steroids; Sterols; Structure; Study models; System; Techniques; Technology; Testing; Time; Transmembrane Domain; United States National Institutes of Health; Universities; Variant; Vertebral column; Washington; Work; analog; base; design; experimental study; insight; interest; mass spectrometer; milligram; mutant; nervous system disorder; neurosteroids; novel; pharmacophore; receptor; small molecule; stereochemistry; stoichiometry; tool

Dr. Wayland Cheng is a cardiothoracic anesthesiologist with the long-term goal of being an independent investigator in anesthetic pharmacology. His research background is in the field of ion channel biophysics, and his interest is in the interaction of anesthetics with pentameric ligand gated ion channels (pLGICs) with a focus on neurosteroids. Neurosteroids are endogenous brain sterols that potently modulate GABAA receptors (GABAARs), and are being developed as anesthetics or treatments of neurologic and psychiatric disorders. Although neurosteroid modulation of the GABAAR has been well characterized, neurosteroid binding to the GABAAR is not well understood. A detailed understanding of the stoichiometry/site(s), orientation and pharmacology of neurosteroid binding to the GABAAR is essential for structure-based drug design. To address this deficiency, the candidate will apply photo-affinity labeling with an existing library of neurosteroid photolabeling reagents, and top-down mass spectrometry (MS), a technique that analyzes intact membrane proteins, to characterize neurosteroid photolabeling of the GABAAR. Aim 1 is to determine the stoichiometry/site(s), orientation and pharmacophore of neurosteroid photolabeling of ELIC-GABAAR chimeras (tractable model proteins where the transmembrane domain consists of GABAAR subunits). Aim 2 is to determine the stoichiometry of neurosteroid photolabeling of α1β3γ2 GABAARs proteoforms (intact proteins and all associated modifications), and to test whether certain proteoforms are preferentially photolabeled by neurosteroids. This work will yield novel insights into the structural basis of neurosteroid binding to the GABAAR. It will also establish a generalizable method using top-down MS for characterizing small molecule photolabeling of membrane proteins, and is anticipated to lead to sustainable R01-level funding. This research will be conducted under the mentorship of Dr. Alex Evers, an expert in anesthetic pharmacology, photo-affinity labeling and bottom-up mass spectrometry. The candidate will also be working closely with Dr. Michael Gross and Dr. Julian Whitelegge, both renowned leaders in top-down MS of membrane proteins. The Anesthesiology Department at Washington University provides a robust environment for research in molecular pharmacology. Furthermore, the candidate utilizes an Elite mass spectrometer owned by Dr. Evers, and has full access to the Washington University NIH NIGMS Mass Spectrometry Research Resource led by Dr. Gross, which provides state-of-the-art MS technology for high mass and high resolution measurements.

Wayland Cheng博士是一名心胸麻醉师，其长期目标是成为一名独立的麻醉师。 麻醉药理学研究者。他的研究背景是离子通道生物物理学领域， 他的兴趣是麻醉剂与五聚体配体门控离子通道（pLGIC）的相互作用， 神经类固醇神经甾体是内源性脑甾醇，其有效地调节GABAA受体 （GABAAR），并且正在开发作为麻醉剂或神经和精神障碍的治疗。 虽然GABAAR的神经类固醇调节已经被很好地表征，但是神经类固醇与GABAAR的结合仍然是一个未知的问题。 GABAAR不太清楚。详细了解化学计量/位点、取向和 神经类固醇与GABAAR结合的药理学对于基于结构的药物设计是必不可少的。解决 这一缺陷，候选人将应用光亲和标记与现有库的神经类固醇 光标记试剂和自上而下的质谱（MS），一种分析完整膜的技术， 蛋白质，以表征GABAAR的神经类固醇光标记。目标1是确定 ELIC-GABAAR嵌合体的神经类固醇光标记的化学计量/位点、方向和药效团 （易处理的模型蛋白，其中跨膜结构域由GABAAR亚基组成）。目标二是 确定α1β3γ2 GABAAR蛋白形式（完整蛋白）的神经类固醇光标记的化学计量 和所有相关的修饰），并测试某些蛋白形式是否优先被 神经类固醇这项工作将产生新的见解神经类固醇结合的结构基础， 加巴建立了一种可推广的自顶向下质谱表征小分子的方法 该项目的主要目标是对膜蛋白进行光标记，预计将导致可持续的R 01级供资。本研究 将在亚历克斯·埃弗斯博士的指导下进行，他是麻醉药理学、光亲和性 标记和自下而上质谱法。候选人还将与迈克尔·格罗斯博士密切合作 和Julian Whitelegge博士，他们都是膜蛋白质自上而下MS的著名领导者。麻醉学 华盛顿大学的药理学系为分子药理学的研究提供了一个强有力的环境。 此外，候选人使用埃弗斯博士拥有的Elite质谱仪，并可以完全访问 由Gross博士领导的华盛顿大学NIH NIGMS质谱研究资源， 最先进的MS技术，用于高质量和高分辨率测量。