Glutamate Vesicular Transport:Pharmacophore Development

描述（由申请人提供）：本竞争性更新申请的主要目标是确定选择性和有效结合谷氨酸突触囊泡转运蛋白（VGLUT）的结构要求，并促进我们对VGLUT结构和功能作用的理解。本研究的长期目标是：（a）开发VGLUT的药效团和结构模型，这将有助于开发可以调节囊泡储存、摄取和释放谷氨酸的选择性分子，以及（B）定义VGLUT和相关囊泡转运蛋白在囊泡功能中的作用和贡献。 所提出的策略将优化VGLUT抑制和结合的选择性，使用我们目前的项目结合新的化学，计算，生物化学和药理学方法的结果。一个并行的战略将寻求获得一个更全面的了解VGLUT的结构和功能。使用综合策略，我们将增加我们的项目活动，并扩大我们的努力，以确定VGLUT的结构，功能和药理作用。在建议的资助期内，我们会致力达致以下目标： 具体目标1：设计和合成选择性结合VGLUT的抑制剂结构。重点将放在开发满足动态药效团模型的元素的抑制剂上，例如，具有区域和立体化学定义的酸和亲脂性取代基的位置的环状平台。 具体目的2：测试SA 1中制备的化合物作为VGLUT抑制剂的活性。还将测定这些化合物结合EAA受体和细胞转运蛋白的能力，以评估VGLUT的特异性。 具体目标3：利用SA 1和2中生成的化学、生物化学和药理学数据生成VGLUT的计算衍生药效团模型。药效团模型将用于改进和验证抑制剂设计（SA 1）、亲和剂（SA 4）、蛋白质探针结构（SA 4和5），并创建与探针和交联结果一致的蛋白质3D结构假设（SA 4和5）。 具体目标4：表征VGLUT的跨膜结构域区域和有助于VGLUT结构和功能的邻近结构。使用N端VGLUT抗体、亲和标签、栓系和交联探针，将通过质谱法分离和鉴定VGLUT残基和VGLUT近端结构。

DESCRIPTION (provided by applicant): The primary goals of this competing renewal application are to define the structural requirements for selective and potent binding to the glutamate synaptic vesicular transporter (VGLUT) and to advance our understanding of the structural and functional roles of VGLUT. The long-range goals of this study are to: (a) develop pharmacophore and structural models of VGLUT that will aid the development of selective molecules that can regulate vesicular storage, uptake and release of glutamate and, (b) define the role and contribution of VGLUTs and related vesicular transporter proteins in vesicle function. The proposed strategy will optimize VGLUT inhibition and binding selectivity using results from our current project combined with novel chemical, computational, biochemical and pharmacological approaches. A concurrent strategy will seek to obtain a more global understanding of VGLUT structure and function. Using integrative strategies, we will increase our project activity and broaden our efforts to define the structural, functional and pharmacological roles of VGLUT. For the proposed grant period, we will address the following objectives: Specific Aim 1: Design and synthesize inhibitor structures that selectively bind VGLUT. Emphasis will be placed on developing inhibitors that satisfy the elements of a dynamic pharmacophore model, for example, a cyclic platform with regio- and stereochemically defined placement of acid and lipophilic substituents. Specific Aim 2: Test the activity of compounds prepared in SA 1 as inhibitors of VGLUT. The ability of these compounds to bind EAA receptors and cellular transporters also will be determined to assess the specificity at VGLUT. Specific Aim 3: Generate a computationally-derived pharmacophore model of VGLUT utilizing the chemical, biochemical and pharmacological data generated in SA's 1 & 2, The pharmacophore model will be used to refine and iterate inhibitor design (SA1), affinity agents (SA4), protein probe structures (SA's 4 and 5), and to create protein 3D structural hypotheses consistent with the probe and cross-linking results (SA4 & 5). Specific Aim 4: Characterize the transmembrane domain regions of VGLUT and neighboring architectures that contribute to VGLUT structure and function. Using N-term VGLUT antibodies, affinity tags, tethered- and cross-linking probes, VGLUT residues and structures proximal to VGLUT will be isolated and identified by mass spectrometry.