Excitatory amino acid transporter-1/2/3 (EAAT-1/2/3)

TFB-TBOA (CF3-Bza-TBOA) lowers synaptically activated transporter currents (STCs) to about 10% of the control at 100 nM. It does this in a dose-dependent manner in astrocytes in the stratum radiatum in rat hippocampus slices, with an IC50 of 13 nM[2]. Based on the amplitude of the Na+i response, TFB-TBOA exhibits concentration-dependent inhibition of the Na+i response elicited by 200 µM glutamate, with an IC50 value of 43 nM[1].

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Nontransportable blockers of the glutamate transporters are important tools for investigating mechanisms of synaptic transmission. DL-threo-β-Benzyloxyaspartate (DL-TBOA) is a potent blocker of all subtypes of the excitatory amino acid transporters (EAATs). We characterized novel L-TBOA analogs possessing a substituent on their respective benzene rings. The analogs significantly inhibited labeled glutamate uptake, the most potent of which was (2S,3S)-3-{3-[4-(trifluoromethyl)benzoylamino]benzyloxy}aspartate (TFB-TBOA). In an uptake assay using cells transiently expressing EAATs, the IC50 values of TFB-TBOA for EAAT1, EAAT2, and EAAT3 were 22, 17, and 300 nM, respectively. TFB-TBOA was significantly more potent at inhibiting EAAT1 and EAAT2 compared with L-TBOA (IC50 values for EAAT1-3 were 33, 6.2, and 15 μM, respectively). Electrophysiological analyses revealed that TBOA analogs block the transport-associated currents in all five EAAT subtypes and also block leak currents in EAAT5. The rank order of the analogs for potencies at inhibiting substrate-induced currents was identical to that observed in the uptake assay. However, the kinetics of TFBTBOA differed from the kinetics of L-TBOA, probably because of the strong binding affinity. Notably, TFB-TBOA did not affect other representative neurotransmitter transporters or receptors, including ionotropic and metabotropic glutamate receptors, indicating that it is highly selective for EAATs. Moreover, intracerebroventricular administration of the TBOA analogs induced severe convulsive behaviors in mice, probably because of the accumulation of glutamate. Taken together, these findings indicate that novel TBOA analogs, especially TFB-TBOA, should serve as useful tools for elucidating the physiological roles of the glutamate transporters. [1]

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Here, we characterized the effects of (2S, 3S)-3-[3-[4-(trifluoromethyl)benzoylamino]benzyloxy]aspartate (TFB-TBOA), a recently developed inhibitor of the glutamate transporter on mouse cortical astrocytes in primary culture. The glial Na(+)-glutamate transport system is very efficient and its activation by glutamate causes rapid intracellular Na(+) concentration (Na(+)(i)) changes that enable real time monitoring of transporter activity. Na(+)(i) was monitored by fluorescence microscopy in single astrocytes using the fluorescent Na(+)-sensitive probe sodium-binding benzofuran isophtalate. When applied alone, TFB-TBOA, at a concentration of 1 microM, caused small alterations of Na(+)(i). TFB-TBOA inhibited the Na(+)(i) response evoked by 200 microM glutamate in a concentration-dependent manner with IC(50) value of 43+/-9 nM, as measured on the amplitude of the Na(+)(i) response. The maximum inhibition of glutamate-evoked Na(+)(i) increase by TFB-TBOA was >80%, but was only partly reversible. The residual response persisted in the presence of the AMPA/kainate receptor antagonist CNQX. TFB-TBOA also efficiently inhibited Na(+)(i) elevations caused by the application of d-aspartate, a transporter substrate that does not activate non-NMDA ionotropic receptors. TFB-TBOA was found not to influence the membrane properties of cultured cortical neurons recorded in whole-cell patch clamp. Thus, TFB-TBOA, with its high potency and its apparent lack of neuronal effects, appears to be one of the most useful pharmacological tools available so far for studying glial glutamate transporters [3].

Glutamate transporters rapidly take up synaptically released glutamate and maintain the glutamate concentration in the synaptic cleft at a low level. (2S, 3S)-3-[3-[4-(trifluoromethyl)benzoylamino]benzyloxy]aspartate (TFB-TBOA) is a novel glutamate transporter blocker that potently suppresses the activity of glial transporters. TFB-TBOA inhibited synaptically activated transporter currents (STCs) in astrocytes in the stratum radiatum in rat hippocampal slices in a dose-dependent manner with an IC50 of 13 nM, and reduced them to approximately 10% of the control at 100 nM. We investigated the effects of TFB-TBOA on glutamatergic synaptic transmission and cell excitability in CA1 pyramidal cells. TFB-TBOA (100 nM) prolonged the decay of N-methyl-D-aspartic acid receptor (NMDAR)-mediated excitatory postsynaptic currents (EPSCs), whereas it prolonged that of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR)-mediated EPSCs only when the desensitization of AMPARs was reduced by cyclothiazide (CTZ). Furthermore, long-term application of TFB-TBOA induced spontaneous epileptiform discharges with a continuous depolarization shift of membrane potential. These epileptiform activities were mainly attributed to NMDAR activation. Even after pharmacological block of NMDARs, however, TFB-TBOA induced similar changes by activating AMPARs in the presence of CTZ. Thus, the continuous uptake of synaptically released glutamate by glial transporters is indispensable for protecting hippocampal neurons from glutamate receptor-mediated hyperexcitabilities [2].

Whole-cell electrophysiological recordings in neurons [3]
Whole-cell voltage-clamp recordings were made with borosilicate glass pipettes with a resistance of 5.5–8 MΩ. In voltage-clamp mode, the clamp potential was set at − 70 mV. Recordings were made with an Axopatch 200A amplifier. Current were filtered at 1 kHz. Data were acquired with a Digidata 1440A, at 10 kHz sampling rate, controlled with Pclamp 10 software and analyzed with Clampfit software. A period of 5 min was routinely allowed after establishment of the whole-cell configuration. The patch-clamp intracellular solution contained (in mM) K-gluconate 130, NaCl 5, Na-phosphocreatine 10, MgCl2 1, EGTA 0.02, HEPES 10, Mg-ATP 2, and Na3-GTP 0.5, pH 7.3 (adjusted with KOH). Experiments were performed using an open perfusion chamber. Control extracellular solutions and solutions containing the tested drugs were gravity fed at 600 μl/min and 35 °C on the cultured cells.

Hippocampal slices were prepared from 15- to 21-day-old Wistar rats. Rats of either sex were deeply anesthetized with isoflurane and sacrificed by decapitation. The hippocampi were rapidly removed and placed in an ice-cold sucrose Ringer solution containing (in mM): 234 sucrose, 2.5 KCl, 1.25 NaH2PO4, 10.0 MgSO4, 0.5 CaCl2, 26 NaHCO3, and 11 glucose, saturated with 95% O2 and 5% CO2. After 5 min incubation in the ice-cold sucrose solution, the hippocampi were glued on the stage of a microslicer with an agar block, and immersed in an ice-cold, oxygenated sucrose Ringer solution. Frontal hippocampal slices 300 μm in thickness were cut with the microslicer and incubated in the control external solution at room temperature for 1 h and maintained for up to 9 h. For recording, the slices were transferred to a 2.5-ml recording chamber mounted on the stage of an upright microscope and perfused at a rate of 2 ml min−1 with external solution maintained at 32 °C with a solution in-line heater.[2]

[1]. Characterization of novel L-threo-beta-benzyloxyaspartate derivatives, potent blockers of the glutamate transporters. Mol Pharmacol. 2004;65(4):1008-1015.

[2]. Effects of a novel glutamate transporter blocker, (2S, 3S)-3-[3-[4-(trifluoromethyl)benzoylamino]benzyloxy]aspartate (TFB-TBOA), on activities of hippocampal neurons. Neuropharmacology. 2005;48(4):479-491.

[3]. Inhibitory effects of (2S, 3S)-3-[3-[4-(trifluoromethyl)benzoylamino]benzyloxy]aspartate (TFB-TBOA) on the astrocytic sodium responses to glutamate. Brain Res. 2010;1316:27-34.

In this study, the TFB-TBOA-induced continuous depolarization with a burst of spike discharges was mimicked by activation of AMPARs when CTZ was co-applied. As described above, CTZ not only increases the affinity of glutamate for AMPARs, but also markedly reduces the desensitization of AMPARs. This indicates that an elevation in the concentration of extracellular glutamate due to a dysfunction of transporters preferentially activates receptors with a high affinity for the agonist and a weak desensitization property. Thus, NMDARs play a more crucial role than AMPARs in epileptogenesis. Shimamoto et al. (2004) have shown that intracerebroventricular injection of TFB-TBOA elicited convulsive seizures within several minutes after the injection in mice. Moreover, we found that intrahippocampal injection of TFB-TBOA elicited similar convulsive seizures in rats (Tsukada et al., unpublished data). These findings are consistent with the present results obtained in hippocampal slices, and indicate directly that the dysfunction of glial glutamate transporters causes convulsive seizures. However, it is presently unknown what extent of blockage of glutamate transporters actually elicits seizures. Obviously, much remains to be done to clarify the involvement of dysfunction of glial glutamate transporters in epileptogenesis in the whole animal. TFB-TBOA will be useful as a pharmacological tool for elucidating the mechanisms for epileptogenesis both in vivo and in vitro experiments. [2]

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Selective inhibition of the EAAT2 (GLT-1) subtype is classically achieved with dihydrokainate, which, however, possesses a fairly low affinity for the transporter (Bridges and Esslinger, 2005) and causes complex effects on astrocytes in situ (Bernardinelli and Chatton, 2008). A very recent report described the first selective inhibitor of EAAT1, UCPH-101, with an IC50 of ∼1 μM and > 400-fold selectivity over EAAT2 and EAAT3 (Jensen et al., 2009). With TFB-TBOA acting on both glial isoforms, the latter pharmacological tool could represent an interesting complement for the functional studies of glutamate transport. Taken together, the present study showed that TFB-TBOA is able to inhibit Na+-dependent glutamate transport in astrocytes with high potency. Despite a partial reversibility of inhibitory effects that have to be taken into account in experimental designs, TFB-TBOA is to be considered as an extremely valuable tool to study glutamate transport and neuron–glia interactions.[3]

C19H17F3N2O6

426.34

426.104

C, 53.53; H, 4.02; F, 13.37; N, 6.57; O, 22.52

480439-73-4

52941382

White to off-white solid powder

3.112

4

10

8

30

622

2

C1=CC(=CC(=C1)NC(=O)C2=CC=C(C=C2)C(F)(F)F)CO[C@@H]([C@@H](C(=O)O)N)C(=O)O

LPWONNPEPDHEAI-GJZGRUSLSA-N

InChI=1S/C19H17F3N2O6/c20-19(21,22)12-6-4-11(5-7-12)16(25)24-13-3-1-2-10(8-13)9-30-15(18(28)29)14(23)17(26)27/h1-8,14-15H,9,23H2,(H,24,25)(H,26,27)(H,28,29)/t14-,15-/m0/s1

(2S,3S)-2-amino-3-[[3-[[4-(trifluoromethyl)benzoyl]amino]phenyl]methoxy]butanedioic acid

TFB-TBOA; 480439-73-4; (3S)-3-[[3-[[4-(TRIFLUOROMETHYL)BENZOYL]AMINO]PHENYL]METHOXY]-L-ASPARTIC ACID; (2S,3S)-2-amino-3-[[3-[[4-(trifluoromethyl)benzoyl]amino]phenyl]methoxy]butanedioic acid; CHEMBL1257519; (2S,3S)-2-amino-3-(3-(4-(trifluoromethyl)benzamido)benzyloxy)succinic acid; CF3-Bza-TBOA; (2~{s},3~{s})-2-Azanyl-3-[[3-[[4-(Trifluoromethyl)phenyl]carbonylamino]phenyl]methoxy]butanedioic Acid;

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 (117.28 mM)

Solubility in Formulation 1: ≥ 1.25 mg/mL (2.93 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 12.5 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: ≥ 1.25 mg/mL (2.93 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 12.5 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: ≥ 1.25 mg/mL (2.93 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 12.5 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.)