Excitatory amino acid transporter 2 (EAAT2) (positive allosteric modulator; EC50 = 0.26 ± 0.03 nM for the racemic mixture in COS-7 cells expressing EAAT2) [1]

- GT949 (racemic mixture) stimulated EAAT2-mediated glutamate transport in COS-7 cells with an EC50 of 0.26 ± 0.03 nM, increasing the transport rate by approximately 70%. [1]
- The enantiomers of GT949 (GT949A and GT949B) showed differential potency: GT949A had an EC50 of 0.041 ± 0.01 nM, while GT949B had an EC50 of 0.89 ± 0.42 nM, making GT949A approximately 22-fold more potent than GT949B. [1]
- GT949 demonstrated selectivity for EAAT2, with no significant effect on glutamate transport mediated by EAAT1 or EAAT3 in COS-7 cells. [1]
- Kinetic analysis revealed that GT949 (1 nM) increased the Vmax of EAAT2-mediated glutamate transport from 270.3 ± 15 to 399.3 ± 28 pmol/mg/min, with no significant change in KM (43.3 ± 7.6 μM vs. 61.6 ± 13 μM), indicating a noncompetitive, positive allosteric mechanism of action. [1]
- In cultured astrocytes, GT949 (racemic mixture) stimulated glutamate uptake with an EC50 of 1 ± 0.07 nM, achieving approximately 58% increase in transport. The enantiomers showed EC50 values of 0.5 ± 0.04 nM (GT949A) and 15 ± 1.3 nM (GT949B). [1]
- GT949 did not affect the activity of human serotonin (hSERT), noradrenaline (hNET), or dopamine (hDAT) transporters at concentrations up to 1 mM (IC50 > 1 mM). [1]
- GT949 did not modulate NMDA receptor activity, as it had no effect on NMDA-induced calcium influx in cultured cortical neurons. [1]
- Immunoblotting analysis showed that GT949 (1 μM, 24 h) did not alter GLT-1 (EAAT2) protein expression in glial cells, confirming its activity as a direct positive allosteric modulator rather than a transcriptional upregulator. [1]

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Glutamate transport is improved by GT 949 (GT949), which has an EC50 of 0.26 ± 0.03 nM. Additionally, GT 949 is selective for EAAT2 and does not affect glutamate activity mediated by EAAT1 or EAAT3 [1]. It was also investigated how GT 949 affected the rates of glutamate uptake in EAAT2-transfected cells. By improving glutamate transport in a non-competitive way, GT 949 raises Vmax by about 47% [1].

- In Silico Docking: Molecular docking studies were performed to predict the binding mode of GT949 at the allosteric site of EAAT2. The docking program GOLD was used with 20 independent runs per ligand. Protein-ligand complexes were scored using a two-tiered scheme: first with default Goldscore, then with a customized knowledge-based scoring method that weighted positive interactions (e.g., aromatic stacking, hydrogen bonding, hydrophobic interactions) and penalized negative interactions with residues lining the allosteric pocket. Docking studies suggested that residues M86, L295, S465, and W472 contribute directly to the binding of GT949. [1]

- Glutamate Uptake Assay in COS-7 Cells: COS-7 cells were transiently transfected with EAAT2 cDNA. Two days post-transfection, cells were washed and preincubated with varying concentrations of GT949 (0.01–100 nM) for 10 min at 37°C. Uptake was initiated by adding 50 nM ³H-L-glutamate and allowed to proceed for 10 min. Reactions were terminated by removing the solution and washing with buffer. Cells were lysed, and radioactivity was quantified by scintillation counting. Results were normalized to vehicle control. [1]
- Kinetic Assay in COS-7 Cells: Cells expressing EAAT2 were preincubated with vehicle or 1 nM GT949. Uptake was initiated with a range of L-glutamate concentrations (1–1000 μM, 99% unlabeled, 1% labeled) for 10 min. KM and Vmax values were calculated assuming Michaelis-Menten kinetics. [1]
- Glutamate Uptake Assay in Cultured Astrocytes: Astrocytes cultured for 14 days in vitro (DIV) were washed and preincubated with vehicle or varying concentrations of GT949 (0.01 nM to 1 μM) for 10 min at 37°C. Uptake was initiated by adding 50 nM ³H-L-glutamate for 10 min at room temperature. Non-specific uptake was determined in the presence of 10 μM DL-TBOA. Radioactivity was measured by scintillation counting. [1]
- Monoamine Transporter Assays: COS-7 cells transiently transfected with hSERT, hNET, or hDAT were incubated with varying concentrations of GT949 (up to 1 mM) for 10 min. Uptake was initiated by adding the appropriate radiolabeled substrate (³H-serotonin, ³H-norepinephrine, or ³H-dopamine) and allowed to proceed for 10 min. Radioactivity was quantified to assess compound effects. [1]
- Calcium Imaging in Cortical Neurons: Cultured cortical neurons were loaded with Fura-2 calcium dye. Cells were continuously perfused with Tyrode’s solution containing tetrodotoxin. NMDA (100 μM) was applied in the presence of 3 μM glycine and absence of Mg²⁺ to induce calcium responses. The NMDA receptor antagonist AP-V (40 μM) was used as a control. GT949 was applied, and calcium changes were recorded by fluorescence ratio (340/380 nm excitation). [1]
- Immunoblotting: Glial cell samples incubated with 1 μM GT949 for 24 h were lysed and subjected to SDS-PAGE and Western blot analysis using anti-GLT-1 (EAAT2) and anti-β-actin antibodies to assess protein expression levels. [1]

[1]. Identification of Novel Allosteric Modulators of Glutamate Transporter EAAT2. ACS Chem Neurosci. 2018 Mar 21;9(3):522-534.

- Background & Mechanism: GT949 is a positive allosteric modulator (PAM) of EAAT2, identified through a hybrid structure-based virtual screening approach targeting the interface between the trimerization and transport domains of EAAT2. It enhances glutamate transport by increasing Vmax without affecting substrate affinity (KM), indicating a noncompetitive allosteric mechanism. This mechanism of action is distinct from transcriptional upregulators such as ceftriaxone. [1]
- Structural Determinants: Site-directed mutagenesis studies confirmed that residues M86 (TM2), L295 (TM5), S465 (TM8), and W472 (TM8) are critical for GT949 activity. Mutations of these residues (M86V, L295A, S465L, W472I) resulted in loss of stimulation or significantly reduced potency, consistent with docking predictions. [1]
- Enantioselectivity: The significant difference in potency between the GT949A and GT949B enantiomers (22-fold) provides strong evidence that GT949 interacts with the chiral environment of the allosteric binding site. [1]
- Selectivity Profile: GT949 shows high selectivity for EAAT2 over EAAT1 and EAAT3, as well as over monoamine transporters (hSERT, hNET, hDAT) and NMDA receptors, suggesting a favorable off-target safety profile. [1]
- Therapeutic Potential: As a direct positive allosteric modulator of EAAT2, GT949 represents a novel class of compounds with potential for treating conditions involving glutamate excitotoxicity, including acute pathologies such as traumatic brain injury and stroke, as well as chronic neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, and ALS. [1]

C30H37N7O2

527.660485982895

527.3

C, 68.29; H, 7.07; N, 18.58; O, 6.06

460330-27-2

3195790

Off-white to light yellow solid powder

4

1

7

8

39

835

0

O=C1C(C(N2CCN(C3CCCCC3)CC2)C2N(CCC3C=CC=CC=3)N=NN=2)=CC2C(=CC=C(C=2)OC)N1

ZVWPOIUAPXDLMB-UHFFFAOYSA-N

InChI=1S/C30H37N7O2/c1-39-25-12-13-27-23(20-25)21-26(30(38)31-27)28(36-18-16-35(17-19-36)24-10-6-3-7-11-24)29-32-33-34-37(29)15-14-22-8-4-2-5-9-22/h2,4-5,8-9,12-13,20-21,24,28H,3,6-7,10-11,14-19H2,1H3,(H,31,38)

3-[(4-cyclohexylpiperazin-1-yl)-[1-(2-phenylethyl)tetrazol-5-yl]methyl]-6-methoxy-1H-quinolin-2-one

GT 949; GT949; 460330-27-2; 3-((4-Cyclohexylpiperazin-1-yl)(1-phenethyl-1H-tetrazol-5-yl)methyl)-6-methoxyquinolin-2(1H)-one; CHEMBL5178416; GT-949

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~50 mg/mL (~94.76 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.74 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (4.74 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (4.74 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)