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 寻求持续的资助，通过麻醉剂结合的详细表征来提高我们对吸入麻醉剂 (IA) 作用的分子药理学的理解。我们已经确定了哺乳动物蛋白质中三个 IA 结合位点的分子结构，以及结合在大脑区域和蛋白质水平上的分布。 IA 与蛋白质的结合依赖于内腔体积、疏水性和弱范德华力，而完全静电会降低亲和力。大约 1000/M 的最大关联常数表明位点的严格性较低，因此表明有许多广泛分布的目标。使用脑切片中的光标记和一维 SDS-PAGE 证实了这一点。这些数据表明需要一个包含多个靶点的药效作用模型，但还需要进一步的证据。在拟议的目标 1 中，我们将在新的替代肽模型中使用系统诱变、光谱学、量热法和晶体学来表征结合位点结构。该目标将测试以下假设：最大 IA 结合亲和力约为 1mM，疏水性（内腔体积）主导结合能量，但存在足够的严格性来选择不同类别的 IA。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 来完善对大鼠和人类 CNS 组织结合靶标的搜索。这一目标的假设是，少数脑蛋白特异性结合 IA，并且不同的 IA 将表现出独特的选择性。这些实验将鉴定结合靶标，并提供亲和力和化学计量值。尽管这些靶标的亲和力相似，但我们假设结合化学计量会变化更多。化学计量可能提供仅考虑亲和力时所缺少的选择性，即功能性靶标可能是结合靶标的子集。在权衡结合数据的药理学相关性的初步尝试中，我们将定量检查目标 2 中在暴露于相同 IA 的动物和细胞模型中特异性结合 IA 的那组蛋白质的表达模式。在这个周期结束时，我们期望了解 IA 亲和力的结构基础、可解析的哺乳动物 CNS 蛋白内部和之间 IA 结合位点的分布，以及新 IA 靶点的鉴定以供进一步研究。