Pharmacological Characterization of [³H]ATPCA as a Substrate for Studying the Functional Role of the Betaine/GABA Transporter 1 and the Creatine Transporter

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_A receptors. With the aim of providing new functional insight into BGT1, we here present the synthesis and pharmacological characterization of the tritiated analogue, [³H]­ATPCA. Using traditional uptake assays at recombinant transporters expressed in cell lines, [³H]­ATPCA displayed a striking selectivity for BGT1 among the four GATs (<i>K</i>_m and <i>V</i>_max 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 [³H]­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 [³H]­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

Structure–Activity Relationship, Pharmacological Characterization, and Molecular Modeling of Noncompetitive Inhibitors of the Betaine/γ-Aminobutyric Acid Transporter 1 (BGT1)

<i>N</i>-(1-Benzyl-4-piperidinyl)-2,4-dichloro­benzamide <b>5</b> (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 <b>26m</b> as a more soluble 2,4-Cl substituted 3-pyridine analogue with retained BGT1 activity and an improved off-target profile compared to <b>5</b>. 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

Identification of the First Highly Subtype-Selective Inhibitor of Human GABA Transporter GAT3

Screening a library of small-molecule compounds using a cell line expressing human GABA transporter 3 (hGAT3) in a [³H]­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 <b>20</b> and <b>34</b>) that were superior to the reference hGAT3 inhibitor, (<i>S</i>)-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 <b>20</b> (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