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.
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