Inhibitors of Vesicular Glutamate Storage and Release

描述（申请者提供）：谷氨酸在突触囊泡中的积累是谷氨酸传递的第一个重要步骤。尽管各种证据表明谷氨酸异常传递参与中枢神经系统（CNS）的多种病理生理，并且人们已经努力开发针对各种谷氨酸受体的药物，但相对较少关注帮助开发针对谷氨酸囊泡储存的中枢神经系统药物。我们最近获得的证据表明，一种膜渗透荧光素类似物是一种有效的谷氨酸囊泡摄取抑制剂（IC50 = 37 nM），不仅能抑制谷氨酸囊泡摄取到孤立的突触囊泡，还能抑制谷氨酸囊泡摄取到位于突触体内的突触囊泡（被挤压的神经末段制备），导致胞外分泌释放的谷氨酸量减少。最近的初步研究表明，其不带电的内酯（称为“内酯化合物”）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方面更有效。很可能内酯化合物能够以比带电前体更高的效率穿透质膜。本研究的总体目标是验证中性内酯化合物比阴离子前体更有效地抑制谷氨酸释放的假设，通过抑制谷氨酸在突触体中积聚到突触囊泡中，并了解其抑制谷氨酸囊泡摄取的机制。希望该内酯类化合物能作为原型先导化合物，为某些涉及谷氨酸异常突触传递的中枢神经系统疾病开发有效药物。我们计划通过以下目标来实现这一目标：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孤立突触囊泡的最有效抑制剂，并定义一个有效的抑制剂药效团。2)内酯化合物在减少(a)突触小体中囊泡[3H]谷氨酸含量和(b)胞外释放[3HJ]谷氨酸的量方面是否比前体更有效，远比从孤立突触小泡摄取谷氨酸的IC50差异所预期的有效，因为它很可能容易穿透突触小体质膜？3)确定内酯化合物、前体和一些弱效荧光素类似物抑制作用的神经递质转运体特异性。4)阐明内酯类化合物抑制谷氨酸囊泡摄取的机制。5)提供额外的证据，以支持玫瑰内酯诱导的谷氨酸释放减少是通过抑制谷氨酸在突触体中积聚到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.