Molecular Determinants and Pharmacological Analysis for a Class of Competitive Non-transported Bicyclic Inhibitors of the Betaine/GABA Transporter BGT1
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 res...
Autores principales: | , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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Frontiers Media S.A.
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8476755/ https://www.ncbi.nlm.nih.gov/pubmed/34595152 http://dx.doi.org/10.3389/fchem.2021.736457 |
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author | Kickinger, Stefanie Lie, Maria E. K. Suemasa, Akihiro Al-Khawaja, Anas Fujiwara, Koichi Watanabe, Mizuki Wilhelmsen, Kristine S. Falk-Petersen, Christina B. Frølund, Bente Shuto, Satoshi Ecker, Gerhard F. Wellendorph, Petrine |
author_facet | Kickinger, Stefanie Lie, Maria E. K. Suemasa, Akihiro Al-Khawaja, Anas Fujiwara, Koichi Watanabe, Mizuki Wilhelmsen, Kristine S. Falk-Petersen, Christina B. Frølund, Bente Shuto, Satoshi Ecker, Gerhard F. Wellendorph, Petrine |
author_sort | Kickinger, Stefanie |
collection | PubMed |
description | 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 [(3)H]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 (pK (B) value of 6.4). To further elaborate on the selectivity profile both compounds were tested at recombinant α(1)β(2)γ(2) GABA(A) receptors. Whereas bicyclo-GABA showed low micromolar agonistic activity, the N-methylated 2 was completely devoid of activity at GABA(A) 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. |
format | Online Article Text |
id | pubmed-8476755 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
spelling | pubmed-84767552021-09-29 Molecular Determinants and Pharmacological Analysis for a Class of Competitive Non-transported Bicyclic Inhibitors of the Betaine/GABA Transporter BGT1 Kickinger, Stefanie Lie, Maria E. K. Suemasa, Akihiro Al-Khawaja, Anas Fujiwara, Koichi Watanabe, Mizuki Wilhelmsen, Kristine S. Falk-Petersen, Christina B. Frølund, Bente Shuto, Satoshi Ecker, Gerhard F. Wellendorph, Petrine Front Chem Chemistry 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 [(3)H]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 (pK (B) value of 6.4). To further elaborate on the selectivity profile both compounds were tested at recombinant α(1)β(2)γ(2) GABA(A) receptors. Whereas bicyclo-GABA showed low micromolar agonistic activity, the N-methylated 2 was completely devoid of activity at GABA(A) 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. Frontiers Media S.A. 2021-09-14 /pmc/articles/PMC8476755/ /pubmed/34595152 http://dx.doi.org/10.3389/fchem.2021.736457 Text en Copyright © 2021 Kickinger, Lie, Suemasa, Al-Khawaja, Fujiwara, Watanabe, Wilhelmsen, Falk-Petersen, Frølund, Shuto, Ecker and Wellendorph. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. |
spellingShingle | Chemistry Kickinger, Stefanie Lie, Maria E. K. Suemasa, Akihiro Al-Khawaja, Anas Fujiwara, Koichi Watanabe, Mizuki Wilhelmsen, Kristine S. Falk-Petersen, Christina B. Frølund, Bente Shuto, Satoshi Ecker, Gerhard F. Wellendorph, Petrine Molecular Determinants and Pharmacological Analysis for a Class of Competitive Non-transported Bicyclic Inhibitors of the Betaine/GABA Transporter BGT1 |
title | Molecular Determinants and Pharmacological Analysis for a Class of Competitive Non-transported Bicyclic Inhibitors of the Betaine/GABA Transporter BGT1 |
title_full | Molecular Determinants and Pharmacological Analysis for a Class of Competitive Non-transported Bicyclic Inhibitors of the Betaine/GABA Transporter BGT1 |
title_fullStr | Molecular Determinants and Pharmacological Analysis for a Class of Competitive Non-transported Bicyclic Inhibitors of the Betaine/GABA Transporter BGT1 |
title_full_unstemmed | Molecular Determinants and Pharmacological Analysis for a Class of Competitive Non-transported Bicyclic Inhibitors of the Betaine/GABA Transporter BGT1 |
title_short | Molecular Determinants and Pharmacological Analysis for a Class of Competitive Non-transported Bicyclic Inhibitors of the Betaine/GABA Transporter BGT1 |
title_sort | molecular determinants and pharmacological analysis for a class of competitive non-transported bicyclic inhibitors of the betaine/gaba transporter bgt1 |
topic | Chemistry |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8476755/ https://www.ncbi.nlm.nih.gov/pubmed/34595152 http://dx.doi.org/10.3389/fchem.2021.736457 |
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