Inhibitors of Vesicular Glutamate Storage and Release

描述（由申请人提供）：谷氨酸积聚到突触囊泡中是谷氨酸传递的第一个重要步骤。尽管有各种证据表明，异常谷氨酸传输参与了中枢神经系统（CNS）的许多类型的病理生理学，并作出了巨大的努力，以开发针对各种谷氨酸受体的药物，相对较少的关注，以帮助开发针对囊泡谷氨酸储存的CNS药物。我们最近已经获得的证据表明，膜渗透性荧光素类似物是一种有效的抑制剂（IC 50 = 37 nM）的囊泡谷氨酸摄取不仅到孤立的突触囊泡，但也到突触囊泡内位于突触体（夹断神经末梢准备），导致减少量olgiutamate释放的胞吐。最近的初步研究表明，它的内酯（称为“内酯化合物”），这是不带电荷的，是更有效的比其母体化合物（称为“前体”）在抑制谷氨酸摄取到孤立的突触囊泡。可能的是，内酯化合物能够以比带电前体更高的效率穿透质膜。这项拟议研究的总体目标是测试的假设，中性内酯化合物抑制，更有力地比阴离子前体，谷氨酸释放通过抑制谷氨酸积累到突触囊泡中的突触体，以及了解其抑制作用的机制囊泡谷氨酸摄取。希望该内酯化合物将作为原型先导化合物，用于开发针对涉及异常谷氨酸突触传递的某些CNS疾病的有效药物。我们计划追求这一目标，其目的如下：1）确定内酯化合物是目前可用的荧光素类似物中谷氨酸摄取到分离的突触囊泡中的最有效的抑制剂，并定义有效的抑制剂药效团。2）内酯化合物在减少（a）突触体中囊泡[3 H]谷氨酸含量和（B）细胞外释放的[3 H]谷氨酸的量方面是否比前体有效得多，比从谷氨酸摄取到分离的突触囊泡中的IC 50差异所预期的有效得多，因为它可能容易穿透突触体质膜？3)为了确定内酯化合物的抑制作用的神经递质转运蛋白特异性，和一些效力中等的荧光素类似物。4）阐明内酯化合物抑制囊泡谷氨酸摄取的机制。5）提供额外的证据支持虎红内酯-谷氨酸释放的诱导减少是通过抑制谷氨酸积聚到突触体中的突触囊泡中来介导的。

DESCRIPTION (provided by applicant): Glutamate accumulation into synaptic vesicles is the first important step in glutamate transmission. Despite a variety of evidence suggesting that abnormal glutamate transmission is involved in many types of pathophysiology in the central nervous system (CNS), and enormous efforts made to develop drugs targeting various glutamate receptors, relatively little attention has been paid to help develop CNS drugs aiming at vesicular glutamate storage. We recently have obtained evidence that a membranepermeant fluorescein analog is a potent inhibitor (IC50 = 37 nM) of vesicular glutamate uptake not only into isolated synaptic vesicles, but also into synaptic vesicles situated inside the synaptosome (pinched-off nerve ending preparation), leading to a reduction in the amount olgiutamate released by exocytosis. More recent preliminary studies suggest that its lactone (referred to as "the lactone compound"), which is uncharged, is more potent than its parent compound (referred to as "the precursor") in inhibiting glutamate uptake into isolated synaptic vesicles. It is likely that the lactone compound is capable of penetrating the plasma membrane with higher efficiency than does the charged precursor. The overall goal of this proposed research is to test the hypothesis that the neutral lactone compound inhibits, more potently than does the anionic precursor, glutamate release through inhibition of glutamate accumulation into synaptic vesicles in the synaptosome, as well as to understand the mechanism of its inhibitory action on vesicular glutamate uptake. It is hoped that the lactone compound will serve as a prototype lead compound for developing effective drugs for certain CNS diseases which involve aberrant glutamate synaptic transmission. We plan to pursue this goal with the following aims:1) To establish that the lactone compound is the most potent inhibitor of glutamate uptake into isolated synaptic vesicles among currently available fluorescein analogs, and to define a potent inhibitor pharmacophore.2) Is the lactone compound far more effective than the precursor in reducing (a) vesicular [3H] glutamate content in the synaptosome and (b) the amount of exocytotically released [3HJ glutamate, far more effective than expected from the difference in IC50 for glutamate uptake into isolated synaptic vesicles, because it is likely to penetrate the synaptosomal plasma membrane with ease?3) To determine neurotransmitter transporter specificity of the inhibitory action of the lactone compound, the precursor, andsome of the moderately less potent fluorescein analogs.4) To elucidate the mechanism of vesicular glutamate uptake inhibition by the lactonc compound.5) To provide additional evidence in support of the notion that the Rose Bengal lactone-induced reduction of glutamate release is mediated by inhibition of glutamate accumulation into synaptic vesicles in the synaptosome.