Excitatory amino acid transporters (EAATs) are important regulators of amino acid transport and in particular glutamate. Recently, more interest has arisen in these transporters in the context of neurodegenerative diseases. This calls for ways to modulate these targets to drive glutamate transport, EAAT2 and EAAT3 in particular. Several inhibitors (competitive and noncompetitive) exist to block glutamate transport; however, activators remain scarce. Recently, GT949 was proposed as a selective activator of EAAT2, as tested in a radioligand uptake assay. In the presented research, we aimed to validate the use of GT949 to activate EAAT2-driven glutamate transport by applying an innovative, impedance-based, whole-cell assay (xCELLigence). A broad range of GT949 concentrations in a variety of cellular environments were tested in this assay. As expected, no activation of EAAT3 could be detected. Yet, surprisingly, no biological activation of GT949 on EAAT2 could be observed in this assay either. To validate whether the impedance-based assay was not suited to pick up increased glutamate uptake or if the compound might not induce activation in this setup, we performed radioligand uptake assays. Two setups were utilized; a novel method compared to previously published research, and in a reproducible fashion copying the methods used in the existing literature. Nonetheless, activation of neither EAAT2 nor EAAT3 could be observed in these assays. Furthermore, no evidence of GT949 binding or stabilization of purified EAAT2 could be observed in a thermal shift assay. To conclude, based on experimental evidence in the present study GT949 requires specific assay conditions, which are difficult to reproduce, and the compound cannot simply be classified as an activator of EAAT2 based on the presented evidence. Hence, further research is required to develop the tools needed to identify new EAAT modulators and use their potential as a therapeutic target.

中文翻译：

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仍在寻找 EAAT 激活剂：GT949 在阻抗和放射性配体摄取测定中不会激活 EAAT2 或 EAAT3

兴奋性氨基酸转运蛋白（EAAT）是氨基酸转运的重要调节剂，特别是谷氨酸转运蛋白。最近，人们对神经退行性疾病中的这些转运蛋白产生了更多兴趣。这就需要找到调节这些目标以驱动谷氨酸转运的方法，特别是 EAAT2 和 EAAT3。存在多种抑制剂（竞争性和非竞争性）来阻止谷氨酸转运；然而，活化剂仍然稀缺。最近，GT949 被提议作为 EAAT2 的选择性激活剂，并在放射性配体摄取测定中进行了测试。在本研究中，我们旨在通过应用创新的、基于阻抗的全细胞测定 (xCELLigence) 来验证 GT949 激活 EAAT2 驱动的谷氨酸转运的用途。该测定测试了各种细胞环境中的广泛 GT949 浓度。正如预期的那样，没有检测到 EAAT3 的激活。然而，令人惊讶的是，在该测定中也没有观察到 GT949 对 EAAT2 的生物激活。为了验证基于阻抗的测定是否不适合检测增加的谷氨酸摄取，或者该化合物是否可能不会在此设置中诱导激活，我们进行了放射性配体摄取测定。使用了两种设置；与之前发表的研究相比，这是一种新颖的方法，并且以可重复的方式复制了现有文献中使用的方法。尽管如此，在这些测定中均未观察到 EAAT2 和 EAAT3 的激活。此外，在热位移测定中没有观察到 GT949 结合或纯化的 EAAT2 稳定的证据。总之，根据本研究中的实验证据，GT949 需要特定的测定条件，而这些条件很难重现，并且不能根据现有证据简单地将化合物归类为 EAAT2 激活剂。因此，需要进一步研究来开发识别新 EAAT 调节剂所需的工具并利用其作为治疗靶点的潜力。