Inhalational Anesthetic Binding Studies

吸入麻醉剂结合研究

• 批准号: 6876539
• 负责人: Roderic G Eckenhoff
• 金额: $ 29.25万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-08-01 至 2008-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6876539
• 关键词: X ray crystallography; affinity labeling; anesthetics; binding proteins; binding sites; brain cell; calorimetry; chemical binding; chemical structure function; drug receptors; halothane; human tissue; hydropathy; inhalation anesthesia; isoflurane; laboratory rat; matrix assisted laser desorption ionization; pharmacokinetics; protein quantitation /detection; protein structure; proteomics; site directed mutagenesis; spectrometry; stoichiometry; tissue /cell culture; two dimensional gel electrophoresis

DESCRIPTION (provided by applicant): The PI seeks continued funding to improve our understanding of the molecular pharmacology of inhaled anesthetic (IA) action through a detailed characterization of anesthetic binding. We have determined the molecular architecture of three IA binding sites in mammalian proteins, and the distribution of binding at both the brain regional and protein level. IA binding to protein relies on internal cavity volume, hydrophobic and weak van der Waals forces, whereas full electrostatics reduce affinity. Maximum association constants of approximately 1000/M indicate low stringency of sites, and therefore suggests many widely distributed targets. This was confirmed using photolabeling in brain sections and 1D SDS-PAGE. These data suggest the need for a pharmacodynamic model of action that incorporates multiple targets, but further evidence is required. In the proposed aim 1, we will characterize binding site architecture using systematic mutagensis, spectroscopy, calorimetry and crystallography in a new surrogate peptide model. This aim will test the hypothesis that the maximum IA binding affinity is approximately 1mM, that hydrophobicity (internal cavity volume) dominates the binding energetics, but that sufficient stringency exists to select for different classes of IA. These data will then serve as a basis for PDB data mining. The 2nd aim will use IA photolabeling, 2D gels and MALDI-MS to refine the search for binding targets from rat and human CNS tissue. The hypothesis underlying this aim is that a minority of brain proteins bind IA specifically, and that different IAs will exhibit unique selectivity. These experiments will identify binding targets, and provide affinity and stoichiometry values. Although affinity will be similar in these targets, we hypothesize that binding stoichiometry will vary more.. Stoichiometry may provide the selectivity that is missing when considering only affinity, i.e., functional targets may be a subset of binding targets. In an initial attempt to weight the binding data for pharmacological relevance, we will quantitatively examine the expression pattern of that group of proteins from aim 2 that bind IA specifically in animal and cell models exposed to the same IA. At the end of this cycle we expect to know the structural basis for IA affinity, the distribution of IA binding sites within and between resolvable mammalian CNS proteins, and the identification of novel IA targets for further study.

描述（由申请人提供）： PI寻求继续资金，以提高我们对吸入麻醉（IA）作用的分子药理学，通过详细的麻醉结合表征。我们已经确定了哺乳动物蛋白中三个IA结合位点的分子结构，以及在脑区域和蛋白质水平上的结合分布。 IA与蛋白质的结合取决于内部空腔体积，疏水性和弱范德华力的力，而全静电剂会减少亲和力。大约1000/m的最大关联常数表示位点的严格性较低，因此表明许多分布的目标。使用脑切片和1D SDS-PAGE的光标记证实了这一点。这些数据表明需要采用包含多个目标的药效动作模型，但需要进一步的证据。在拟议的目标1中，我们将使用系统的诱变，光谱，量热法和晶体学在新的替代肽模型中表征结合位点结构。该目标将检验以下假设：最大IA结合亲和力约为1mm，疏水性（内腔体积）主导了结合能量，但是存在足够的严格度以选择不同类别的IA类。然后，这些数据将作为PDB数据挖掘的基础。第二个目标将使用IA光标记，2D凝胶和MALDI-MS来完善对大鼠和人CNS组织的结合靶标的搜索。该目标的基本假设是，少数脑蛋白具有特定的结合，并且不同的IAS将表现出独特的选择性。这些实验将识别结合靶标，并提供亲和力和化学计量值。尽管在这些靶标中亲和力将相似，但我们假设结合化学计量法会有所不同。.Stoichiemementry可能会提供仅考虑亲和力时缺少的选择性，即功能目标可能是结合目标的子​​集。在最初尝试加重药理学相关性的结合数据时，我们将定量检查该蛋白的表达模式，该蛋白质的AIM 2中该蛋白的表达模式，这些蛋白质在暴露于相同IA的动物和细胞模型中结合了IA。在此周期结束时，我们期望知道IA亲和力的结构基础，可分解的哺乳动物CNS蛋白内和之间的IA结合位点的分布，以及鉴定新的IA靶标进行进一步研究。