The γ-aminobutyric acid (GABA) transporters (GATs) are key membrane transporter proteins involved in the termination of GABAergic signaling at synapses in the mammalian brain and proposed drug targets in neurological disorders such as epilepsy. To date, four different GAT subtypes have been identified: GAT1, GAT2, GAT3 and the betaine/GABA transporter 1 (BGT1). Owing to the lack of potent and subtype selective inhibitors of the non-GAT1 GABA transporters, the physiological role and therapeutic potential of these transporters remain to be fully understood. Based on bioisosteric replacement of the amino group in β-alanine or GABA, a series of compounds was generated, and their pharmacological activity assessed at human GAT subtypes. Using a cell-based [(3)H]GABA uptake assay, several selective inhibitors at human BGT1 were identified. The guanidine-containing compound 9 (2-amino-1,4,5,6-tetrahydropyrimidine-5-carboxylic acid hydrochloride) displayed more than 250 times greater potency than the parent compound β-alanine at BGT1 and is thus the most potent inhibitor reported to date for this subtype (IC50 value of 2.5 µM). In addition, compound 9 displayed about 400, 16 and 40 times lower inhibitory potency at GAT1, GAT2 and GAT3, respectively. Compound 9 was shown to be a substrate for BGT1 and to have an overall similar pharmacological profile at the mouse orthologue. Compound 9 constitutes an interesting pharmacological tool for specifically investigating the cellular pharmacology of BGT1 and is the first small-molecule substrate identified with such a high selectivity for BGT1 over the three other GAT subtypes.

Screening a library of small-molecule compounds using a cell line expressing human GABA transporter 3 (hGAT3) in a [(3)H]GABA uptake assay identified isatin derivatives as a new class of hGAT3 inhibitors. A subsequent structure-activity relationship (SAR) study led to the identification of hGAT3-selective inhibitors (i.e., compounds 20 and 34) that were superior to the reference hGAT3 inhibitor, (S)-SNAP-5114, in terms of potency (low micromolar IC50 values) and selectivity (>30-fold selective for hGAT3 over hGAT1/hGAT2/hBGT1). Further pharmacological characterization of compound 20 (5-(thiophen-2-yl)indoline-2,3-dione) revealed a noncompetitive mode of inhibition at hGAT3. This suggests that this compound class, which has no structural resemblance to GABA, has a binding site different from the substrate, GABA. This was supported by a molecular modeling study that suggested a unique binding site that matched the observed selectivity, inhibition kinetics, and SAR of the compound series. These compounds are the most potent GAT3 inhibitors reported to date that provide selectivity for GAT3 over other GABA transporter subtypes.

The betaine/γ-aminobutyric acid (GABA) transporter 1 (BGT1) is one of the four GABA transporters (GATs) involved in the termination of GABAergic neurotransmission. Although suggested to be implicated in seizure management, the exact functional importance of BGT1 in the brain is still elusive. This is partly owing to the lack of potent and selective pharmacological tool compounds that can be used to probe its function. We previously reported the identification of 2-amino-1,4,5,6-tetrahydropyrimidine-5-carboxylic acid (ATPCA), a selective substrate for BGT1 over GAT1/GAT3, but also an agonist for GABA receptors. With the aim of providing new functional insight into BGT1, we here present the synthesis and pharmacological characterization of the tritiated analogue, [H]ATPCA. Using traditional uptake assays at recombinant transporters expressed in cell lines, [H]ATPCA displayed a striking selectivity for BGT1 among the four GATs ( K and V values of 21 μM and 3.6 nmol ATPCA/(min × mg protein), respectively), but was also found to be a substrate for the creatine transporter (CreaT). In experiments with mouse cortical cell cultures, we observed a Na-dependent [H]ATPCA uptake in neurons, but not in astrocytes. The neuronal uptake could be inhibited by GABA, ATPCA, and a noncompetitive BGT1-selective inhibitor, indicating functional BGT1 in neurons. In conclusion, we report [H]ATPCA as a novel radioactive substrate for both BGT1 and CreaT. The dual activity of the radioligand makes it most suitable for use in recombinant studies.

N-(1-Benzyl-4-piperidinyl)-2,4-dichlorobenzamide 5 (BPDBA) is a noncompetitive inhibitor of the betaine/GABA transporter 1 (BGT1). We here report the synthesis and structure-activity relationship of 71 analogues. We identify 26m as a more soluble 2,4-Cl substituted 3-pyridine analogue with retained BGT1 activity and an improved off-target profile compared to 5. We performed radioligand-based uptake studies at chimeric constructs between BGT1 and GAT3, experiments with site-directed mutated transporters, and computational docking in a BGT1 homology model based on the newly determined X-ray crystal structure of the human serotonin transporter (hSERT). On the basis of these experiments, we propose a binding mode involving residues within TM10 in an allosteric site in BGT1 that corresponds to the allosteric binding pocket revealed by the hSERT crystal structure. Our study provides first insights into a proposed allosteric binding pocket in BGT1, which accommodates the binding site for a series of novel noncompetitive inhibitors.

Imbalances in GABA-mediated tonic inhibition are involved in several pathophysiological conditions. A classical way of controlling tonic inhibition is through pharmacological intervention with extrasynaptic GABA receptors that sense ambient GABA and mediate a persistent GABAergic conductance. An increase in tonic inhibition may, however, also be obtained indirectly by inhibiting glial GABA transporters (GATs). These are sodium-coupled membrane transport proteins that normally act to terminate GABA neurotransmitter action by taking up GABA into surrounding astrocytes. The aim of the review is to provide an overview of glial GATs in regulating tonic inhibition, especially in epilepsy and stroke. This entails a comprehensive summary of changes known to occur in GAT expression levels and signalling following epileptic and ischemic insults. Further, we discuss the accumulating pharmacological evidence for targeting GATs in these diseases.

γ-Aminobutyric acid (GABA) neurotransmission is terminated by the GABA transporters (GATs) via uptake of GABA into neurons and surrounding glial cells. Four different transporters have been identified: GAT1, GAT2, GAT3, and the betaine/GABA transporter 1 (BGT1). The GAT1 subtype is the most explored transporter due to its high abundance in the brain and the existence of selective and potent GAT1 inhibitors. Consequently, less is known about the role and therapeutic potential of the non-GAT1 subtypes. Emerging pharmacological evidence suggests that some of these transporters pose interesting targets in several brain disorders. Pharmacological non-GAT1-selective tool compounds are important to further investigate the involvement of GATs in different pathological conditions. Extensive medicinal chemistry efforts have been put into the development of subtype-selective inhibitors, but truly selective and potent inhibitors of non-GAT1 subtypes are still limited. This review covers the advances within the medicinal chemistry area and the structural basis for obtaining non-GAT1-selective inhibitors.

We have previously identified 2-amino-1,4,5,6-tetrahydropyrimidine-5-carboxylic acid (ATPCA) as the most potent substrate-inhibitor of the betaine/GABA transporter 1 (BGT1) (IC 50 2.5 µM) reported to date. Herein, we characterize the binding mode of 20 novel analogs and propose the molecular determinants driving BGT1-selectivity. A series of N 1 -, exocyclic- N -, and C 4 -substituted analogs was synthesized and pharmacologically characterized in radioligand-based uptake assays at the four human GABA transporters (hGATs) recombinantly expressed in mammalian cells. Overall, the analogs retained subtype-selectivity for hBGT1, though with lower inhibitory activities (mid to high micromolar IC 50 values) compared to ATPCA. Further characterization of five of these BGT1-active analogs in a fluorescence-based FMP assay revealed that the compounds are substrates for hBGT1, suggesting they interact with the orthosteric site of the transporter. In silico-guided mutagenesis experiments showed that the non-conserved residues Q299 and E52 in hBGT1 as well as the conformational flexibility of the compounds potentially contribute to the subtype-selectivity of ATPCA and its analogs. Overall, this study provides new insights into the molecular interactions governing the subtype-selectivity of BGT1 substrate-inhibitors. The findings may guide the rational design of BGT1-selective pharmacological tool compounds for future drug discovery.

The betaine/GABA transporter 1 (BGT1) is a member of the GABA transporter (GAT) family with still elusive function, largely due to a lack of potent and selective tool compounds. Based on modeling, we here present the design, synthesis and pharmacological evaluation of five novel conformationally restricted cyclic GABA analogs related to the previously reported highly potent and selective BGT1 inhibitor (1S,2S,5R)-5-aminobicyclo[3.1.0]hexane-2-carboxylic acid (bicyclo-GABA). Using [3H]GABA radioligand uptake assays at the four human GATs recombinantly expressed in mammalian cell lines, we identified bicyclo-GABA and its N-methylated analog (2) as the most potent and selective BGT1 inhibitors. Additional pharmacological characterization in a fluorescence-based membrane potential assay showed that bicyclo-GABA and 2 are competitive inhibitors, not substrates, at BGT1, which was validated by a Schild analysis for bicyclo-GABA (pKB value of 6.4). To further elaborate on the selectivity profile both compounds were tested at recombinant α1β2γ2 GABAA receptors. Whereas bicyclo-GABA showed low micromolar agonistic activity, the N-methylated 2 was completely devoid of activity at GABAA receptors. To further reveal the binding mode of bicyclo-GABA and 2 binding hypotheses of the compounds were obtained from in silico-guided mutagenesis studies followed by pharmacological evaluation at selected BGT1 mutants. This identified the non-conserved BGT1 residues Q299 and E52 as the molecular determinants driving BGT1 activity and selectivity. The binding mode of bicyclo-GABA was further validated by the introduction of activity into the corresponding GAT3 mutant L314Q (38 times potency increase cf. wildtype). Altogether, our data reveal the molecular determinants for the activity of bicyclic GABA analogs, that despite their small size act as competitive inhibitors of BGT1. These compounds may serve as valuable tools to selectively and potently target BGT1 in order to decipher its elusive pharmacological role in the brain and periphery such as the liver and kidneys.