Inhalational Anesthetic Binding Studies

描述（由申请人提供）： PI寻求持续的资金，以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，以及在脑区域和蛋白质水平上的结合分布。IA与蛋白质的结合依赖于内腔体积、疏水性和弱的货车范德华力，而完全静电降低亲和力。约1000/M的最大缔合常数表明位点的严格性低，因此表明许多广泛分布的靶标。这一点通过脑切片中的光标记和1D SDS-PAGE得到证实。这些数据表明，需要一个药效学作用模型，包括多个目标，但需要进一步的证据。在提出的目标1中，我们将使用系统诱变，光谱学，量热法和晶体学在一个新的替代肽模型中表征结合位点结构。该目的将检验以下假设：最大IA结合亲和力约为1 mM，疏水性（内腔体积）主导结合能，但存在足够的严格性以选择不同类别的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来完善从大鼠和人类CNS组织中寻找结合靶点。这一目标背后的假设是，少数脑蛋白特异性结合IA，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。尽管这些靶点的亲和力相似，但我们假设结合化学计量学变化更大。化学计量可以提供当仅考虑亲和力时所缺失的选择性，即，功能靶可以是结合靶的子集。在对药理学相关性的结合数据进行加权的初步尝试中，我们将定量检查目标2中的蛋白质组的表达模式，这些蛋白质组在暴露于相同IA的动物和细胞模型中特异性结合IA。在这个周期结束时，我们希望知道IA亲和力的结构基础，IA结合位点的分布和可分辨的哺乳动物中枢神经系统蛋白之间，并确定新的IA目标进一步研究。

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.