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

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

  • ACS Chem Neurosci. 2024 Apr 3;15(7):1424-1431. doi: 10.1021/acschemneuro.3c00731.
Lieve van Veggel 1 2 3,  Tamara A M Mocking 4,  Hubert J Sijben 4,  Rongfang Liu 4,  Marina Gorostiola González 4,  Majlen A Dilweg 4,  Jeroen Royakkers 5,  Anna Li 6,  Vijay Kumar 6,  Yin Yao Dong 7,  Alex Bullock 6,  David B Sauer 6,  Hanne Diliën 5,  Gerard J P van Westen 4,  Rudy Schreiber 8,  Laura H Heitman 4 9,  Tim Vanmierlo 1 2 3
Affiliations

Affiliations

  • 1 Department of Neuroscience, BIOMED Biomedical Research Institute, Faculty of Medicine and Life Sciences, Hasselt University, 3590 Hasselt, Belgium.
  • 2 Department of Psychiatry and Neuropsychology, Division of Translational Neuroscience, European Graduate School of Neuroscience, School for Mental Health and Neuroscience, Maastricht University, 6200 Maastricht, The Netherlands.
  • 3 University MS Center (UMSC), 3900 Hasselt-Pelt, Belgium.
  • 4 Leiden Academic Centre for Drug Research (LACDR), Division of Drug Discovery and Safety, Leiden University, 2333 Leiden, The Netherlands.
  • 5 Sensor Engineering Department, Faculty of Science and Engineering, Maastricht University, 6200 Maastricht, The Netherlands.
  • 6 Centre for Medicines Discovery, Nuffield Department of Medicine, University of Oxford, OX3 7BN Oxford, U.K.
  • 7 Nuffield Department of Clinical Neurosciences, Weatherall Institute of Molecular Medicine, University of Oxford, OX3 7BN Oxford, U.K.
  • 8 Section of Psychopharmacology, Neuropsychology and Psychopharmacology, Faculty of Psychology and Neuroscience, Maastricht University, 6200 Maastricht, The Netherlands.
  • 9 Oncode Institute, Einsteinweg 55, 2333 Leiden, The Netherlands.
PMID: 38478848 DOI: 10.1021/acschemneuro.3c00731
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.

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