ORCID Profile
0000-0002-1947-9125
Current Organisation
IT University of Copenhagen
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Publisher: Wiley
Date: 25-06-2020
DOI: 10.1002/GLIA.23872
Abstract: Synaptic transmission is closely linked to brain energy and neurotransmitter metabolism. However, the extent of brain metabolism of the inhibitory neurotransmitter γ-aminobutyric acid (GABA), and the relative metabolic contributions of neurons and astrocytes, are yet unknown. The present study was designed to investigate the functional significance of brain GABA metabolism using isolated mouse cerebral cortical slices and slices of neurosurgically resected neocortical human tissue of the temporal lobe. By using dynamic isotope labeling, with [
Publisher: SAGE Publications
Date: 17-11-2017
Abstract: Brain ischemia triggers excitotoxicity and cell death, yet no neuroprotective drugs have made it to the clinic. While enhancing GABAergic signaling to counterbalance excitotoxicity has shown promise in animal models, clinical studies have failed. Blockade of GABA transporters (GATs) offers an indirect approach to increase GABA inhibition to lower the excitation threshold of neurons. Among the GATs, GAT1 is known to promote neuroprotection, while the protective role of the extrasynaptic transporters GAT3 and BGT1 is elusive. A focal lesion was induced in the motor cortex in two to four-month-old C57BL/6 J male mice by photothrombosis. The GAT1 inhibitor, tiagabine (1 and 10 mg/kg), the GAT2/3 inhibitor, ( S)-SNAP-5114 (5 and 30 mg/kg) and the GAT1/BGT1 inhibitor, EF-1502 (1 and 10 mg/kg) were given i.p. 1 and 6 h post-stroke to assess their impact on infarct volume and motor performance seven days post-stroke. One mg/kg tiagabine improved motor performance, while 10 mg/kg tiagabine, ( S)-SNAP-5114 and EF-1502 had no effect. None of the compounds affected infarct volume. Interestingly, treatment with tiagabine induced seizures and ( S)-SNAP-5114 led to increased mortality. Although we show that tiagabine can promote protection, our findings indicate that caution should be had when using GAT1 and GAT3 inhibitors for conditions of brain ischemia.
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 International Publishing
Date: 2017
DOI: 10.1007/978-3-319-55769-4_7
Abstract: Imbalances in GABA-mediated tonic inhibition are involved in several pathophysiological conditions. A classical way of controlling tonic inhibition is through pharmacological intervention with extrasynaptic GABA
Publisher: American Chemical Society (ACS)
Date: 23-07-2015
DOI: 10.1021/ACSCHEMNEURO.5B00150
Abstract: Screening a library of small-molecule compounds using a cell line expressing human GABA transporter 3 (hGAT3) in a [(3)H]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 20 and 34) that were superior to the reference hGAT3 inhibitor, (S)-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 20 (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.
Publisher: Wiley
Date: 17-05-2021
DOI: 10.1111/EJN.15267
Abstract: γ‐Aminobutyric acid (GABA) acting through heteropentameric GABA A receptors plays a pivotal role in the sleep‐promoting circuitry. Whereas the role of the different GABA A receptor α‐subunits in sleep regulation and in mediating the effect of benzodiazepines for treatment of insomnia is well‐described, the β‐subunits are less studied. Here we report the first study characterizing sleep in mice lacking the GABA A receptor β 1 ‐subunit (β 1 −/− mice). We show that β 1 −/− mice have a distinct and abnormal sleep phenotype characterized by increased delta power in non‐rapid eye movement (NREM) sleep and decreased theta activity in rapid eye movement (REM) sleep compared to β 1 +/+ mice, without any change in the overall sleep‐wake architecture. From GABA A receptor‐specific autoradiography, it is further demonstrated that functional β 1 ‐subunit‐containing GABA A receptors display the highest binding levels in the hippoc us and frontal cortex. In conclusion, this study suggests that the GABA A receptor β 1 ‐subunit does not play an important role in sleep initiation or maintenance but instead regulates the power spectrum and especially the expression of theta rhythm. This provides new knowledge on the complex role of GABA A receptor subunits in sleep regulation. In addition, β 1 −/− mice could provide a useful mouse model for future studies of the physiological role of delta and theta rhythms during sleep.
Publisher: Elsevier BV
Date: 02-2020
Publisher: Springer Science and Business Media LLC
Date: 23-05-2014
DOI: 10.1007/S11064-014-1336-9
Abstract: The γ-aminobutyric acid (GABA) transporters (GATs) are key membrane transporter proteins involved in the termination of GABAergic signaling at synapses in the mammalian brain and proposed drug targets in neurological disorders such as epilepsy. To date, four different GAT subtypes have been identified: GAT1, GAT2, GAT3 and the betaine/GABA transporter 1 (BGT1). Owing to the lack of potent and subtype selective inhibitors of the non-GAT1 GABA transporters, the physiological role and therapeutic potential of these transporters remain to be fully understood. Based on bioisosteric replacement of the amino group in β-alanine or GABA, a series of compounds was generated, and their pharmacological activity assessed at human GAT subtypes. Using a cell-based [(3)H]GABA uptake assay, several selective inhibitors at human BGT1 were identified. The guanidine-containing compound 9 (2-amino-1,4,5,6-tetrahydropyrimidine-5-carboxylic acid hydrochloride) displayed more than 250 times greater potency than the parent compound β-alanine at BGT1 and is thus the most potent inhibitor reported to date for this subtype (IC50 value of 2.5 µM). In addition, compound 9 displayed about 400, 16 and 40 times lower inhibitory potency at GAT1, GAT2 and GAT3, respectively. Compound 9 was shown to be a substrate for BGT1 and to have an overall similar pharmacological profile at the mouse orthologue. Compound 9 constitutes an interesting pharmacological tool for specifically investigating the cellular pharmacology of BGT1 and is the first small-molecule substrate identified with such a high selectivity for BGT1 over the three other GAT subtypes.
Publisher: Springer Science and Business Media LLC
Date: 03-08-2020
DOI: 10.1038/S41598-020-69908-W
Abstract: 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.
Publisher: Frontiers Media SA
Date: 14-09-2021
DOI: 10.3389/FCHEM.2021.736457
Abstract: 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 (1 S ,2 S ,5 R )-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 (p K 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.
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: SAGE Publications
Date: 21-11-2017
Abstract: Ischemic stroke triggers an elevation in tonic GABA inhibition that impairs the ability of the brain to form new structural and functional cortical circuits required for recovery. This stroke-induced increase in tonic inhibition is caused by impaired GABA uptake via the glial GABA transporter GAT3, highlighting GAT3 as a novel target in stroke recovery. Using a photothrombotic stroke mouse model, we show that GAT3 protein levels are decreased in peri-infarct tissue from 6 h to 42 days post-stroke. Prior studies have shown that GAT substrates can increase GAT surface expression. Therefore, we aimed to assess whether the GAT3 substrate, L-isoserine, could increase post-stroke functional recovery. L-Isoserine (38 µM or 380 µM) administered directly into the infarct from day 5 to 32 post-stroke, significantly increased motor performance in the grid-walking and cylinder tasks in a concentration-dependent manner, without affecting infarct volumes. Additionally, L-isoserine induced a lasting increase in GAT3 expression in peri-infarct regions accompanied by a small decrease in GFAP expression. This study is the first to show that a GAT3 substrate can increase GAT3 expression and functional recovery after focal ischemic stroke following a delayed long-term treatment. We propose that enhancing GAT3-mediated uptake d ens tonic inhibition and promotes functional recovery after stroke.
Publisher: Springer Science and Business Media LLC
Date: 26-10-2023
No related grants have been discovered for Maria Elena Klibo Lie.