Inhibitors of Vesicular Glutamate Storage and Release

描述(申请人提供)：谷氨酸积累到突触小泡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的异常传递参与了中枢神经系统(CNS)的多种病理生理过程，而且针对各种谷氨酸受体的药物的开发也付出了巨大的努力，但对于帮助开发针对囊泡性谷氨酸储存的CNS药物的研究相对较少。我们最近获得的证据表明，一种膜上标记的荧光素类似物是一种有效的抑制剂(IC50=37 nM)，不仅可以抑制囊泡谷氨酸摄取到分离的突触小泡，还可以摄取到突触体内的突触小泡(夹断神经末梢制剂)，导致胞吐释放的谷氨酸减少。最近的初步研究表明，其不带电荷的内酯(称为内酯化合物)在抑制谷氨酸摄取到分离的突触小泡方面比其母化合物(称为前体)更有效。很可能内酯化合物能够比带电前体更有效地穿透质膜。这项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，这是通过抑制谷氨酸聚集到突触小体的突触小泡中来实现的，并了解其抑制囊泡谷氨酸摄取的机制。内酯化合物有望成为开发治疗某些涉及谷氨酸突触异常传递的中枢神经系统疾病的有效药物的原型先导化合物。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最有效的谷氨酸摄取到分离的突触小泡的抑制剂，并定义一个有效的抑制药效团2)内酯化合物在减少(A)突触体内的囊泡[~3H]谷氨酸含量和(B)胞外释放的[3HJ谷氨酸]量方面比前体更有效吗？3)为了确定内酯化合物抑制突触小泡摄取谷氨酸的神经递质转运体的特异性，4)阐明内酯化合物抑制囊泡摄取谷氨酸的机制。5)提供额外的证据，支持孟加拉内酯诱导的谷氨酸释放减少是通过抑制谷氨酸聚集到突触小体的突触小泡中来实现的。

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.