Discovery of a subtype selective inhibitor of the human betaine/GABA transporter 1 (BGT-1) with a non-competitive pharmacological profile

Kragholm, Bolette; Kvist, Trine; Madsen, Karsten K.; Jørgensen, Lars; Vogensen, Stine B.; Schousboe, Arne; Clausen, Rasmus P.; Jensen, Anders A.; Bräuner‐Osborne, Hans

doi:10.1016/j.bcp.2013.06.007

Cited by 30 publications

(34 citation statements)

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“…CHO Flp-In and tsA201 cells were cultured at 37 °C in a humidified atmosphere of 95% O 2 and 5% CO 2 . As previously described 38 , 63 , CHO Flp-In cells stably expressing the hGATs were cultured in Ham’s F12 Nutrient Mix supplemented with 10% FBS, 1% P/S and plasmocin (5 µg/mL) under the selection pressure of hygromycin B (200 µg/mL). DMEM with GlutaMAX-I supplemented with 10% FBS and 1% P/S was used as growth medium for the tsA201 cells, which were transfected with DNA constructs using PolyFect according to the manufacturer’s specifications (Qiagen, West Sussex, UK), but with half of the recommended reagent volumes.…”

Section: Methodsmentioning

confidence: 99%

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Exploring the molecular determinants for subtype-selectivity of 2-amino-1,4,5,6-tetrahydropyrimidine-5-carboxylic acid analogs as betaine/GABA transporter 1 (BGT1) substrate-inhibitors

Kickinger

Al-Khawaja

Haugaard

et al. 2020

Sci Rep

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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.

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Section: Methodsmentioning

confidence: 99%

“…Uptake assays were performed in PDL-coated white 96-well polystyrene cell culture microplates (PerkinElmer, Boston, MA, USA) as previously described 38 , 63 . Approximately 16–20 h after cell plating (~ 50.000 cells/well), cells were incubated with 30 nM of [ 3 H]GABA with and without varying concentrations of ligands for 3 min at 37 °C.…”

Section: Methodsmentioning

confidence: 99%

Exploring the molecular determinants for subtype-selectivity of 2-amino-1,4,5,6-tetrahydropyrimidine-5-carboxylic acid analogs as betaine/GABA transporter 1 (BGT1) substrate-inhibitors

Kickinger

Al-Khawaja

Haugaard

et al. 2020

Sci Rep

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“…1-[3-(9H-Carbazol-9-yl)propyl]-4-(2-methoxyphenyl)-4-piperidinol (NNC 05-2090) and A 804598 were purchased from Tocris Bioscience (Bristol, UK). [2, cyclohexane-1-carboxylic acid ((1R,2R)-4a), (1S,2R)-2-((4,4-bis(3-methylthiophen-2-yl)but-3-en-1-yl)(methyl) amino)cyclohexane-1-carboxylic acid ((1S,2R)-5a), (1S,2R)-2-((3-(10,11-dihydro-5H-dibenzo[a,d] [7] annulen-5-ylidene)propyl)(methyl)amino)cyclohexane-1-carboxylic acid ((1S,2R)-5c), and N-(1-benzyl-4-piperidinyl)-2,4-dichlorobenzamide (BPDBA) were synthesized in-house as previously described [23,30,31]. The Madin-Darby canine kidney (MDCK) II cell line was kindly provided by Dr. Hanne Borger Rasmussen (University of Copenhagen, Denmark).…”

Section: Methodsmentioning

confidence: 99%

“…The HEK-293 and Flp-In CHO cell lines stably expressing mouse (m) and human (h) GATs, respectively, have been described previously and were cultured accordingly [31][32][33]. The tsA201 cell line was used for transient expression while the MDCK II cell line express BGT1 endogenously [34].…”

Section: Cell Culturing and Transient Gat Expressionmentioning

confidence: 99%

“…(R)-EF1502 was also included in the detailed pharmacological characterization, with no prior notion of a biphasic inhibition profile, as it is structurally highly similar to SBV2-114. Finally, two BGT1 inhibitors belonging to other compound classes namely BPDBA [31] and NNC 05-2090 [44] were included (Fig. 1).…”

Section: Several Structurally Related Sbv2-114 Analogues Also Inhibitmentioning

confidence: 99%

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Pharmacological Characterization of a Betaine/GABA Transporter 1 (BGT1) Inhibitor Displaying an Unusual Biphasic Inhibition Profile and Anti-seizure Effects

et al. 2020

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Focal epileptic seizures can in some patients be managed by inhibiting γ-aminobutyric acid (GABA) uptake via the GABA transporter 1 (GAT1) using tiagabine (Gabitril®). Synergistic anti-seizure effects achieved by inhibition of both GAT1 and the betaine/GABA transporter (BGT1) by tiagabine and EF1502, compared to tiagabine alone, suggest BGT1 as a target in epilepsy. Yet, selective BGT1 inhibitors are needed for validation of this hypothesis. In that search, a series of BGT1 inhibitors typified by (1R,2S)-2-((4,4-bis(3-methylthiophen-2-yl)but-3-en-yl)(methyl)amino)cyclohexanecarboxylic acid (SBV2-114) was developed. A thorough pharmacological characterization of SBV2-114 using a cell-based [ 3 H]GABA uptake assay at heterologously expressed BGT1, revealed an elusive biphasic inhibition profile with two IC 50 values (4.7 and 556 μM). The biphasic profile was common for this structural class of compounds, including EF1502, and was confirmed in the MDCK II cell line endogenously expressing BGT1. The possibility of two binding sites for SBV2-114 at BGT1 was assessed by computational docking studies and examined by mutational studies. These investigations confirmed that the conserved residue Q299 in BGT1 is involved in, but not solely responsible for the biphasic inhibition profile of SBV2-114. Animal studies revealed anti-seizure effects of SBV2-114 in two mouse models, supporting a function of BGT1 in epilepsy. However, as SBV2-114 is apparent to be rather non-selective for BGT1, the translational relevance of this observation is unknown. Nevertheless, SBV2-114 constitutes a valuable tool compound to study the molecular mechanism of an emerging biphasic profile of BGT1-mediated GABA transport and the putative involvement of two binding sites for this class of compounds.

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γ‐Aminobutyric Acid and Glycine Neurotransmitter Transporters

Wellendorph

Jacobsen

Skovgaard-Petersen

et al. 2017

Methods and Principles in Medicinal Chemistry

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Discovery of a subtype selective inhibitor of the human betaine/GABA transporter 1 (BGT-1) with a non-competitive pharmacological profile

Cited by 30 publications

References 34 publications

Exploring the molecular determinants for subtype-selectivity of 2-amino-1,4,5,6-tetrahydropyrimidine-5-carboxylic acid analogs as betaine/GABA transporter 1 (BGT1) substrate-inhibitors

Exploring the molecular determinants for subtype-selectivity of 2-amino-1,4,5,6-tetrahydropyrimidine-5-carboxylic acid analogs as betaine/GABA transporter 1 (BGT1) substrate-inhibitors

Pharmacological Characterization of a Betaine/GABA Transporter 1 (BGT1) Inhibitor Displaying an Unusual Biphasic Inhibition Profile and Anti-seizure Effects

γ‐Aminobutyric Acid and Glycine Neurotransmitter Transporters

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