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.
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.
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.
Given
the heterogeneity within the γ-aminobutyric acid (GABA) receptor
and transporter families, a detailed insight into the pharmacology
is still relatively sparse. To enable studies of the physiological
roles governed by specific receptor and transporter subtypes, a series
of GABA analogues comprising five-membered nitrogen- and sulfur-containing
heterocycles as amine bioisosteres were synthesized and pharmacologically
characterized at native and selected recombinant GABAA receptors
and GABA transporters. The dihydrothiazole and imidazoline analogues, 5–7, displayed moderate GAT activities
and GABAA receptor binding affinities in the mid-nanomolar
range (K
i, 90–450 nM). Moreover,
they exhibited full and equipotent agonist activity compared to GABA
at GABAA-αβγ receptors but somewhat lower
potency as partial agonists at the GABAA-ρ1 receptor. Stereoselectivity was observed for compounds 4 and 7 for the GABAA-αβγ
receptors but not the GABAA-ρ1 receptor.
This study illustrates how subtle differences in these novel amino
GABA bioisosteres result in diverse pharmacological profiles in terms
of selectivity and efficacy.
Delta selective compound 2 (DS2) is one of the most widely used tools to study selective actions mediated by subunit-containing GABA A receptors. DS2 was discovered over 10 years ago, but despite great efforts, the precise molecular site of action has remained elusive.Using a combination of computational modeling, site-directed mutagenesis and cell-based pharmacological assays, we probed three potential binding sites for DS2 and analogs at 4 1 receptors: an 4 (+) (-) interface site in the extracellular domain (ECD), equivalent to the This article has not been copyedited and formatted. The final version may differ from this version.
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