VESICULAR GLUTAMATE TRANSPORT--PHARMACOPHORE ELUCIDATION

本申请的主要目标是获得对选择性和有效结合谷氨酸突触囊泡转运体（GVT）蛋白的结构要求的详细理解。 本研究的长期目标是开发谷氨酸囊泡转运蛋白的药效团模型，并利用这些信息来调节囊泡储存，摄取和释放谷氨酸或其他感兴趣的化合物。 虽然我们对谷氨酸神经递质系统的整体知识在过去的二十年中有了显着的进步，特别是在受体的药效团开发，但令人惊讶的是，对谷氨酸突触囊泡转运蛋白知之甚少，并且已经确定了很少的竞争性抑制剂。 为了更好地理解GVT的结构、功能和调节，需要有效的和选择性的抑制剂。 本申请的主要策略是将已知抑制剂的关键结构和官能团元素与天然底物谷氨酸合并，以设计和合成新的抑制剂分子。 一旦确定，新的抑制剂库将用于开发药效团模型和制备转运蛋白的亲和配体。 提出了以下目标：目标1：一个系统设计的库取代喹啉二酸，萘胺[二]磺酸和混合喹啉/萘胺分子将被合成，模拟谷氨酸负责结合到谷氨酸囊泡转运蛋白的关键构象。 目的2：测试目的1中制备的化合物作为GVT抑制剂和底物的活性。 将测定这些化合物结合EAA受体和细胞转运蛋白的能力，以评估作用的特异性。 目的3：进行结构-活性和分子模拟研究，以生成谷氨酸囊泡转运蛋白的药效团模型。 本节中获得的结果将用于细化抑制剂结构，为目标1提供反馈，并开发越来越详细的GVT结合结构域模型。 目的4：制备具有光亲和活性的配体，用于共价修饰GVT以探测其结构和功能。 目标5：目标2中鉴定和表征的抑制剂将用于开始阐明囊泡转运在调节囊泡释放的谷氨酸水平中的作用。

The primary goal of this application is to obtain a detailed understanding of the structural requirements for selective and potent binding to the glutamate synaptic vesicular transporter (GVT) protein. The long-range goal of this study is to develop a pharmacophore model of the glutamate vesicular transporter protein and to utilize this information to regulate vesicular storage, uptake and release of glutamate or other compounds of interest. Although our overall knowledge of the glutamate neurotransmitter system has advanced significantly over the past two decades, particularly pharmacophore development at the receptors, surprisingly little is known about the glutamate synaptic vesicular transporter and very few competitive inhibitors have been identified. Potent and selective inhibitors are needed to better understand the GVT structure, function and regulation. The main strategy of this application is to merge key structural and functional group elements of known inhibitors with the natural substrate glutamate to design and synthesize new inhibitor molecules. Once identified, the new inhibitor library will be used to develop a pharmacophore model and prepare affinity ligands of the transporter. The following objectives are proposed: OBJECTIVE 1: A systematically designed library of substituted quinoline diacids, naphthylamine [di]sulfonic acids and hybrid quinoline/naphthylamine molecules will be synthesized that simulate key conformations of glutamate responsible for binding to the glutamate vesicular transporter. OBJECTIVE 2: To test the activity of compounds prepared in Objective 1 as inhibitors and substrates of the GVT. The ability of these compounds to bind EAA receptors and cellular transporters will be determined to assess the specificity of the action. OBJECTIVE 3: To conduct structure-activity and molecular modeling studies to generate a pharmacophore model of the glutamate vesicular transporter. Results obtained in this section will be used to refine inhibitor structure, provide feedback to for Objective 1 and develop an increasingly detailed model of the GVT binding domain. OBJECTIVE 4: To prepare reactive photoaffinity ligands that will be used to covalently modify the GVT to probe structure and function. OBJECTIVE 5: Inhibitors identified and characterized in Objective 2 will be used to begin to elucidate the role of vesicular transport in regulating levels of vesicularly released glutamate.