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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.

谷氨酸转运体的非转运性阻断剂是研究突触传递机制不可或缺的工具。DL-苏型-β-苄氧基天冬氨酸（DL-TBOA）是兴奋性氨基酸转运蛋白（EAAT）所有亚型的强效阻断剂。我们表征了在其苯环上具有取代基的新型L-TBOA类似物。这些类似物显著抑制标记的谷氨酸摄取，其中（2S，3S）-3-{3-[4-（三氟甲基）苯甲酰氨基]苄氧基}天冬氨酸（TFB-TBOA）是最有效的。在使用瞬时表达EAAT的细胞的摄取测定中，<50>TFB-TBOA对EAAT 1、EAAT 2和EAAT 3的IC_值分别为22 nM、17 nM和300 nM。与L-TBOA相比，TFB-TBOA在抑制EAAT 1和EAAT 2方面显著更有效（<50>对EAAT 1 -3的IC_值分别为33 μM、0.2μM和15 μM）。电生理学分析显示，TBOA类似物阻断所有五种EAAT亚型中的转运相关电流，并且还阻断EAAT 5中的漏电流。抑制底物诱导电流的效价的等级顺序与摄取试验中观察到的相同。然而，TFB-TBOA的动力学特征与L-TBOA不同，这可能是因为TFB-TBOA具有较强的结合亲和力。值得注意的是，TFB-TBOA不影响其他代表性的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，表明其对EAAT具有高度选择性。此外，侧脑室给药TBOA类似物诱导小鼠严重惊厥行为，这可能归因于谷氨酸的积累。总之，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。