Vesicular glutamate transporters (VGLUT1-3) mediate the uptake of glutamate into synaptic vesicles. VGLUTs are pivotal actors of excitatory transmission and of almost all brain functions. Their implication in various pathologies has been clearly documented. Despite their functional importance, the pharmacology of VGLUTs is limited to a few dyes such as Trypan Blue, Rose Bengal or Brilliant Yellow type. Here, we report the design and evaluation of new potent analogs based on Trypan Blue scaffold. Our best compound, named LSP5-2157, has an EC50 of 50 nM on glutamate vesicular uptake. Using a 3D homology model of VGLUT1 and docking experiments, we determined its putative binding subdomains within vesicular glutamate transporters and validated the structural requirement for VGLUT inhibition. To better estimate the specificity and potency of LSP5-2157, we also investigated its ability to block glutamatergic transmission in autaptic hippocampal cells. Neither glutamate receptors nor GABAergic transmission or transmission machinery were affected by LSP5-2157. Low doses of compound reversibly reduce glutamatergic neurotransmission in hippocampal autpases. LSP5-2157 had a low and depressing effect on synaptic efficacy in hippocampal slice. Furthermore, LSP5-2157 had no effect on NMDA-R- mediated fEPSP but reduce synaptic plasticity induced by 3 trains of 100 Hz. Finally, LSP5-2157 had the capacity to inhibit VGLUT3-dependent auditory synaptic transmission in the guinea pig cochlea. In this model, it abolished the compound action potential of auditory nerve at high concentration showing the limited permeation of LSP5-2157 in an in-vivo model. In summary, the new ligand LSP5-2157, has a high affinity and specificity for VGLUTs and shows some permeability in isolated neuron, tissue preparations or in vivo in the auditory system. These findings broaden the field of VGLUTs inhibitors and open the way to their use to assess glutamatergic functions in vitro and in vivo.

中文翻译：

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LSP5-2157是水泡谷氨酸转运蛋白的新抑制剂。

囊泡谷氨酸转运蛋白（VGLUT1-3）介导谷氨酸向突触小泡的摄取。VGLUT是兴奋性传递和几乎所有脑功能的关键参与者。它们在各种病理学中的含义已被明确记录。尽管具有功能重要性，但VGLUT的药理作用仅限于几种染料，例如锥虫蓝，玫瑰红或艳黄型。在这里，我们报告基于台盼蓝支架的新型有效类似物的设计和评估。我们最好的化合物LSP5-2157对谷氨酸的水泡摄取EC50为50 nM。使用VGLUT1的3D同源性模型和对接实验，我们确定了其在囊状谷氨酸转运蛋白内的推定结合亚域，并验证了对VGLUT抑制的结构要求。为了更好地估计LSP5-2157的特异性和效力，我们还研究了其在自闭性海马细胞中阻断谷氨酸能传递的能力。LSP5-2157既不影响谷氨酸受体也不影响GABA能传递或传播机制。低剂量的化合物可逆地减少海马Autpases中的谷氨酸能神经传递。LSP5-2157对海马切片的突触功效具有较低的抑制作用。此外，LSP5-2157对NMDA-R介导的fEPSP没有影响，但会降低3列100 Hz诱导的突触可塑性。最后，LSP5-2157具有抑制豚鼠耳蜗中依赖VGLUT3的听觉突触传递的能力。在该模型中，它消除了高浓度听神经的复合动作电位，显示了在体内模型中LSP5-2157的渗透受限。总而言之，新的配体LSP5-2157 对VGLUT具有高亲和力和特异性，并且在分离的神经元，组织制剂或听觉系统的体内显示出一定的通透性。这些发现拓宽了VGLUTs抑制剂的领域，并为它们在体外和体内评估谷氨酸能功能开辟了道路。