Development of functionalized blockers for glutamate transporters

谷氨酸转运蛋白功能化阻断剂的开发

基本信息

批准号：
13680681
负责人：
SHIMAMOTO Keiko
金额：
$ 2.62万
依托单位：
Suntory Institute for Bioorganic Research
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2003
项目状态：
已结题

项目摘要

Non-transportable blockers for the glutamate transporters are indispensable tools for investigating mechanisms of synaptic transmission. DL-threo-β-benzyloxyaspartate (DL-TBOA) is a potent blocker of all subtypes of the excitatory amino acid transporters (EAATs). We characterized novel L-TBOA analogs possessing a substituent on its benzene ring. The analogs significantly inhibited labeled glutamate uptake with (2S,3S)-3-{3-[4-(trifluoromethyl)benzoylamino]benzyloxy}aspartate (TFB-TBOA) being the most potent among them. In the uptake assay using cells transiently expressing EAATs, the IC_<50> values ties of TFB-TBOA for EAATI, EAAT2, and EAAT3 were 22 nM, 17 nM, and 300 nM, respectively. TFB-TBOA was significantly more potent at inhibiting EAATI and EAAT2 compared with L-TBOA (IC_<50> values for EAATs1-3 were 33 μM, 0.2μM, and 15 μM, respectively). Electrophysiological analyses revealed that TBOA analogs block the transport-associated currents in all five EAAT subtypes and also block leak currents in EAAT5. The rank order of the potency for inhibiting substrate-induced currents was identical to that observed with the uptake assay. However, the kinetic character of TFB-TBOA was different from that of L-TBOA probably because of the strong binding affinity. Notably, TFB-TBOA did not affect other representative neurotransmitter transporters or receptors, including ionotropic and metabotropic glutamate receptors, indicating that it is highly selective for EAATs. Moreover, intracerebroventricular administration of the TBOA analogs induced severe convulsive behaviors in mice, which would be attributed to the accumulation of glutamate. Taken together, new TBOA analogs, especially TFB-TBOA, should serve as useful tools for elucidating the physiological roles of the glutamate transporters.

谷氨酸转运蛋白的非转运性阻断剂是研究突触传递机制不可或缺的工具，我们对兴奋性氨基酸转运蛋白（EAAT）的所有亚型进行了有效的阻断。 L-TBOA 类似物在其苯环上具有取代基，该类似物显着抑制标记的谷氨酸的摄取。 (2S,3S)-3-{3-[4-(三氟甲基)苯甲酰氨基]苯甲氧基}天冬氨酸(TFB-TBOA)是其中最有效的。在使用瞬时表达EAAT的细胞的摄取测定中，IC_ 50 值。 TFB-TBOA与EAATI、EAAT2和EAAT3的关系分别为22nM、17nM和300nM。与 L-TBOA 相比，TFB-TBOA 抑制 EAATI 和 EAAT2 的能力明显更强（EAATs1-3 的 IC_<50> 值分别为 33 μM、0.2μM 和 15 μM）。电生理学分析表明 TBOA 类似物具有阻断作用。所有五种 EAAT 亚型中的传输相关电流，并且还阻断 EAAT5 中的泄漏电流抑制底物诱导的效力的排序。电流与摄取测定中观察到的电流相同，但是，TFB-TBOA 的动力学特征与 L-TBOA 不同，可能是因为其强结合亲和力。值得注意的是，TFB-TBOA 不影响其他代表性神经递质转运蛋白或受体。，包括离子型和代谢型谷氨酸受体，表明它对 EAAT 具有高度选择性。此外，脑室内施用 TBOA 类似物会引起严重的惊厥行为。总而言之，新的 TBOA 类似物，特别是 TFB-TBOA，应该作为阐明谷氨酸转运蛋白的生理作用的有用工具。