ORCID Profile
0000-0003-1640-7940
Current Organisation
IT University of Copenhagen
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Publisher: Springer Science and Business Media LLC
Date: 04-04-2020
DOI: 10.1007/S11064-020-03017-Y
Abstract: 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 (1 R ,2 S )-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.
Publisher: Springer Science and Business Media LLC
Date: 16-08-2023
DOI: 10.1007/S00044-023-03126-7
Abstract: The GABA transporter 3 (GAT3) is a member of the GABA transporter (GAT) family proposed to have a role in regulating tonic inhibition. The GAT3-preferring substrate ( S )-isoserine has shown beneficial effects in a mouse model of stroke accompanied by an increased GAT3 expression, indicating a molecular mechanism mediated by GAT3. However, ( S )-isoserine is not ideally suited for in vivo studies due to a lack of selectivity and brain permeability. To elucidate the structural determinants of ( S )-isoserine for GAT3 inhibition, and to optimize and inform further ligand development, we here present the design, synthesis and pharmacological evaluation of a series of conformationally constrained isoserine analogs with defined stereochemistry. Using [ 3 H]GABA uptake assays at recombinant human GAT3, we identified the azetidine and pyrrolidine analogs (2 S ,2´ S )- 6 and (2 S ,2´ S )- 7 as the most potent inhibitors. To further elaborate on the selectivity profile both compounds were tested at all GATs, the taurine transporter (TauT) and GABA A receptors. Although (2 S ,2´ S )- 6 and (2 S ,2´ S )- 7 are comparable to ( S )-isoserine with respect to potency, the selectivity vs. the taurine transporter was significantly improved (at least 6 and 53 times more activity at hGAT3, respectively). A subsequent comprehensive structure-activity study showed that different connectivity approaches, stereochemical variations, simple or larger α- and N -substituents, and even minor size enlargement of the heterocyclic ring all abrogated GAT3 inhibition, indicating very strict stereochemical and size requirements. The observed structure activity relationships may guide future ligand optimization and the novel ligands ((2 S ,2´ S )- 6 and (2 S ,2´ S )- 7 ) can serve as valuable tools to validate the proposed GAT3-mediated effect of ( S )-isoserine such as in functional recovery after stroke and thus help corroborate the relevance of targeting GAT3 and tonic inhibition in relevant brain pathologies.
Publisher: Springer Science and Business Media LLC
Date: 26-10-2023
No related grants have been discovered for Stefanie Kickinger.