Still in Search for an EAAT Activator: GT949 Does Not Activate EAAT2, nor EAAT3 in Impedance and Radioligand Uptake Assays

Abstract

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.

Keywords: EAAT2; GT949; glutamate; modulation; radioligand uptake; transport.

Figure 1

(A) Representative time trace of HEK293-JumpIn-EAAT2 cells following growth, starvation with PBS, pretreatment, and l-glutamate stimulation as measured by xCELLigence. Data were analyzed by taking the AUC over the first 2 h after stimulation. (B) EAAT2 or (C) EAAT3 mediated uptake of l-glutamate and l-cysteine after pretreated with vehicle or GT949 (1 μM) as measured by xCELLigence. Several starvation methods were used before the cells were pretreated with GT949 and stimulated, such as medium (left), serum free medium (middle), and PBS (right). Data are shown as the mean ± SEM of three individual experiments each performed in duplicate. *p < 0.05, one-way ANOVA with Dunnett’s posthoc test.

Figure 2

Dose response curve of the effects of GT949 pretreatment on the l-glutamate and l-cysteine response in EAAT2 expressing cells. Data are shown as the mean ± SEM of three individual experiments each performed in duplicate. One-way ANOVA with Dunnett’s posthoc test.

Figure 3

Radioligand uptake assay of EAAT2 (A) and EAAT3 (B). Dose response curves (DRCs) are shown. Dotted-line represent basal uptake by uninduced HEK-JumpIn cells. Data are shown as the mean ± SEM of two to four individual experiments each performed in at least duplicate. *p < 0.05, **p < 0.001, ***p < 0.005, ****p < 0.0001, one-way ANOVA with Tukey’s multiple comparison test.

Figure 4

[³H]-l-glutamate uptake assay of HEK293-JumpIN-EAAT2 (A) and HEK-JumpIn-EAAT3 (B) cells showing the effect of GT949. Cells were induced with doxycycline to expressed the transporters. The dotted line represents the observed basal uptake of L-glutamate in uninduced cells (EAAT2: 0.65 ± 0.10, EAAT3: 0.68 ± 0.13). Data are shown as the mean ± SEM of three individual experiments each performed in at least duplicate. *p < 0.05, **p < 0.001, ***p < 0.005, ****p < 0.0001, one-way ANOVA with Tukey’s multiple comparison test.

Figure 5

EAAT2 melting temperature with glutamate, GT949, and TFB-TBOA. Data are shown as mean ± SEM from three biological replicates. * p < 0.05, **** p < 0.0001, one-way ANOVA with Tukey’s multiple comparison test.