Inhibitors of Vesicular Glutamate Storage and Release

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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.