hGAT-3 (IC50 = 5 μM); rGAT-2 (IC50 = 21 μM)

Molecular cloning has revealed the presence of four high-affinity GABA transporters in the brain. The existence of three of these sites, GAT-2, GAT-3, and BGT-1, was unknown prior to their cloning and almost nothing is known of the role they play in regulating GABAergic transmission. In large measure our paucity of knowledge is attributable to the lack of specific inhibitors for these sites. In the present communication we describe the cloning and expression of the human homologue of GAT-3, and the identification of an inhibitor, (S)-SNAP-5114, with selectivity for this site. ((S)-SNAP-5114 displays an IC50 of 5 microM at GAT-3, 21 microM at GAT-2, and > or = 100 microM at GAT-1 and BGT-1. Due to its lipophilicity, (S)-SNAP-5114 is also expected to cross the blood-brain-barrier and therefore, should be an important tool for evaluating the role of GAT-3 in neural function. [1]

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The most interesting compound to result from this line of investigation was the nipecotic acid derivative (S)-SNAP-5114, the structure of which is shown in Fig. 1. As shown in Table 3, (S)-SNAP-5114 is most potent at GAT-3 (ic50 = 5 μM), and about 4-fold less potent at GAT-2 (ic50 ∼ 21 μM). In contrast, (S)-SNAP-5114 is a much weaker inhibitor of BGT-1 (ic50 ∼ 140 μM), and is weakest at GAT-1 (ic50 ∼ 400 μM). Interestingly, (S)-SNAP-5114 is more potent than the (R) isomer (Dhar et al., 1994), in contrast to nipecotic acid in which most of the activity resides within the (R) isomer (Borden et al., 1994b). To our knowledge, (S)-SNAP-5114 is the first inhibitor with selectivity for GAT-3.
Due to its lipophilic methoxyphenyl substituents, (S)-SNAP-5114 is expected to effectively cross the blood-brain barrier. Further, since it shows 75-fold selectivity for GAT-3 over GAT-1, it should be possible to selectively inhibit GAT-3 (and possibly GAT-2) in vivo, while sparing the more prevalent GAT-1. Thus, (S)-SNAP-5114 should serve as an important tool for determining the functional role of GAT-2 and GAT-3 in the central nervous system, and it is hoped that further refinements in its structure will lead to compounds with increased potency at, and selectivity for, these sites [2].

Brain ischemia triggers excitotoxicity and cell death, yet no neuroprotective drugs have made it to the clinic. While enhancing GABAergic signaling to counterbalance excitotoxicity has shown promise in animal models, clinical studies have failed. Blockade of GABA transporters (GATs) offers an indirect approach to increase GABA inhibition to lower the excitation threshold of neurons. Among the GATs, GAT1 is known to promote neuroprotection, while the protective role of the extrasynaptic transporters GAT3 and BGT1 is elusive. A focal lesion was induced in the motor cortex in two to four-month-old C57BL/6 J male mice by photothrombosis. The GAT1 inhibitor, tiagabine (1 and 10 mg/kg), the GAT2/3 inhibitor, (S)-SNAP-5114 (5 and 30 mg/kg) and the GAT1/BGT1 inhibitor, EF-1502 (1 and 10 mg/kg) were given i.p. 1 and 6 h post-stroke to assess their impact on infarct volume and motor performance seven days post-stroke. One mg/kg tiagabine improved motor performance, while 10 mg/kg tiagabine, (S)-SNAP-5114 and EF-1502 had no effect. None of the compounds affected infarct volume. Interestingly, treatment with tiagabine induced seizures and (S)-SNAP-5114 led to increased mortality. Although we show that tiagabine can promote protection, our findings indicate that caution should be had when using GAT1 and GAT3 inhibitors for conditions of brain ischemia [3].

Compound administration [3]
Tiagabine and (S)-SNAP-5114 and (R,S)-EF-1502 (EF-1502) were used. Tiagabine (1 mg/kg and 10 mg/kg) and EF-1502 (1 mg/kg and 5 mg/kg) were dissolved in sterile isotonic saline with 2% DMSO. (S)-SNAP-5114 (5 mg/kg and 30 mg/kg) was dissolved in sterile isotonic saline with 10% DMSO due to its low solubility. All animals were randomly assigned to a treatment group post-stroke, to ensure that all animals in any given cage received a different treatment. The compounds were administrated at a low and high dose based on previously published EC50 values obtained in other disease models with no notation of seizure-inducing effects. While control animals were injected with 2% DMSO in isotonic saline to serve as controls for tiagabine and EF-1502, 10% DMSO in isotonic saline was injected to serve as controls for (S)-SNAP-5114. Each compound or vehicle were administered i.p. (12 µL/g of body mass) 1 and 6 h post-stroke, as all compounds have been shown to modulate brain excitability after i.p. administration at doses in the same range as used in the present study. Mice were then returned to their home cage after each injection and observed for 5-10 min per hour for the first 2 h post-compound administration for signs of abnormal behaviors.

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Infarct size [3]
The mice were deeply anesthetized with pentobarbital (University of Otago, Animal Welfare Office, New Zealand) and transcardially perfused with 4% paraformaldehyde (PFA) seven days post-stroke. The brains were removed and post-fixed for 1 h in 4% PFA before being transferred to 30% sucrose. The brains were cut on a sliding freezing stage microtome in six coronal parallel sets in sections of 40 µm thickness and kept in cryoprotectant at −20℃. Infarct volume was determined by histological assessment using cresyl violet staining according to a previously published protocol. Infarct volume was quantified using Image J (National Institutes of Health, USA) by an observer blind as to the treatment groups, and is based on obtaining measurements from every 6th section through the entire infarct (area in mm2), and infarct volume was quantified as follows: infarct volume mm3 = √𝑎𝑟𝑒𝑎⁢𝑚𝑚2×𝑠𝑒𝑐𝑡𝑖𝑜𝑛⁢𝑡ℎ𝑖𝑐𝑘𝑛𝑒𝑠𝑠×𝑠𝑒𝑐𝑡𝑖𝑜𝑛⁢𝑖𝑛𝑡𝑒𝑟𝑣𝑎𝑙. A total of six mice had stroke volumes less than 0.6 mm3 and were subsequently excluded on the basis that the stroke induction was incomplete (1 from the 10 mg/kg tiagabine group; 3 from the 5 mg /kg EF-1502 group; 2 from the 10% DMSO vehicle group). One and five mice from the 5 mg/kg and 30 mg/kg (S)-SNAP-5114 groups, respectively, were found dead the day after the compound had been administered, and therefore the final number of mice for infarct and behavioral analysis were: 2% DMSO vehicle, n = 10; 10% DMSO vehicle, n = 8; 1 mg/kg tiagabine, n = 6; 10 mg/kg tiagabine, n = 8; 1 mg/kg EF-1502, n = 8; 10 mg/kg EF-1502, n = 8; 5 mg/kg (S)-SNAP-5114  n = 7; 30 mg/kg (S)-SNAP-5114, n = 6.

[1]. Cloning of the human homologue of the GABA transporter GAT-3 and identification of a novel inhibitor with selectivity for this site. Receptors Channels. 1994;2(3):207-13.

[2]. GABA transporter heterogeneity: pharmacology and cellular localization. Neurochem Int. 1996 Oct;29(4):335-56.

[3]. Inhibition of GABA transporters fails to afford significant protection following focal cerebral ischemia. J Cereb Blood Flow Metab. 2018 Jan;38(1):166-173.

Gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the mammalian brain. GABA is cleared from the synaptic cleft by specific high-affinity sodium and chloride-dependent transporters thought to be located on presynaptic terminals and surrounding glial cells. Early studies suggested differences in GABA transport between neurons and glial cells, but molecular cloning revealed the presence of genes for four distinct GABA transporters (named GAT-1, GAT-2, GAT-3, and BGT-1), revealing greater heterogeneity than previously thought. Heterologous expression allowed researchers to characterize the pharmacological properties of these transporters in detail and discovered that GAT-1 is the site of action for the anticonvulsant tiagabe. In situ hybridization and immunocytochemistry experiments showed that each transporter has a unique regional distribution in the brain. Combined with cell culture experiments, researchers are elucidating the localization of various transporters in neurons and glial cells. Future research will focus on identifying the role of each transporter in the regulation of GABAergic transmission and designing more subtype-specific inhibitors that are expected to become novel therapeutic agents for the treatment of neuropsychiatric disorders. [2] These studies have shown that the neuroprotective effect of inhibiting GATs in the acute phase of focal ischemic stroke is limited, especially when the inhibitor is given immediately at 1 hour and 6 hours after the stroke. Although GAT1 inhibition can promote protection, its potential for translational application in stroke patients is limited because tiagabin can induce post-stroke seizures (presumably mediated by excessive tonic inhibition). In order to investigate the neuroprotective potential of inhibiting only GAT3 or BGT1 in acute stroke, it is necessary to develop inhibitors with better subtype selectivity for these two transporters, easier crossing of the blood-brain barrier, and greater potency. Finally, given the unexplained deaths during treatment with the GAT3 inhibitor (S)-SNAP-5114, caution should be exercised when developing new drugs and conducting clinical trials of GAT inhibitors for the treatment of brain injury. Given the differences between different animal stroke models and species, more research is needed to assess the correlation between IC10 and IC50 concentrations and changes in tonic inhibition and neuroprotection, especially in cases where GAT function may or may not be impaired after stroke. [3]

C30H35NO6

505.61

505.246

C, 71.27; H, 6.98; N, 2.77; O, 18.99

157604-55-2

10458835

White to off-white solid powder

1.175g/cm3

643.9ºC at 760mmHg

343.2ºC

0mmHg at 25°C

1.571

4.755

1

7

11

37

620

1

COC1=CC=C(C=C1)C(C2=CC=C(C=C2)OC)(C3=CC=C(C=C3)OC)OCCN4CCC[C@@H](C4)C(=O)O

VDLDUZLDZBVOAS-QFIPXVFZSA-N

InChI=1S/C30H35NO6/c1-34-26-12-6-23(7-13-26)30(24-8-14-27(35-2)15-9-24,25-10-16-28(36-3)17-11-25)37-20-19-31-18-4-5-22(21-31)29(32)33/h6-17,22H,4-5,18-21H2,1-3H3,(H,32,33)/t22-/m0/s1

(3S)-1-[2-[tris(4-methoxyphenyl)methoxy]ethyl]piperidine-3-carboxylic acid

SNAP5114; SNAP 5114; 157604-55-2; (S)-SNAP 5114; (3S)-1-{2-[tris(4-methoxyphenyl)methoxy]ethyl}piperidine-3-carboxylic acid; DTXSID40440224; (3S)-1-[2-[tris(4-methoxyphenyl)methoxy]ethyl]piperidine-3-carboxylic acid; (3S)-1-(2-(tris(4-methoxyphenyl)methoxy)ethyl)piperidine-3-carboxylic acid; DTXCID00391046; SNAP-5114

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 : ~100 mg/mL (~197.78 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.94 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 (4.94 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 (4.94 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.)