Development of functionalized blockers for glutamate transporters

谷氨酸转运体的非转运性阻滞剂是研究突触传递机制不可缺少的工具。DL-苏氨酸-β-苄氧天冬氨酸(DL-TBOA)是兴奋性氨基酸转运体(EAATs)各亚型的强效阻断剂。我们表征了苯环上带有取代基的新型L-TBOA类似物。这些类似物显著抑制标记的谷氨酸摄取，其中以(2S，3S)-3-{3-[4-(trifluoromethyl)benzoylamino]benzyloxy}aspartate(TfB-TboA)作用最强。在瞬时表达EAATs的细胞摄取实验中，TFB-TBOA对EAAT1、EAAT2和EAAT3的IC值分别为22 nM、17 nM和300 nM。TFB-TBOA对EAATI和EAAT2的抑制作用明显强于L-TBOA(对EAATs1-3的IC值分别为33μM、0.2μM和15μM)。电生理分析显示，TBOA类似物阻断了所有五种EAAT亚型的运输相关电流，也阻断了EAAT5的泄漏电流。抑制底物诱导电流的效力的等级顺序与摄取试验观察到的相同。然而，TFB-TBOA与L-TBOA的动力学特征不同，可能是由于TFB-TBOA具有较强的结合亲和力。值得注意的是，TFB-TBOA不影响其他有代表性的神经递质转运体或受体，包括离子型和代谢型谷氨酸受体，这表明它对EAAT具有高度的选择性。此外，脑室内注射TBOA类似物可引起小鼠严重的惊厥行为，这可能归因于谷氨酸的积累。综上所述，新的TBOA类似物，特别是TFB-TBOA，应该成为阐明谷氨酸转运体的生理作用的有用工具。

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.