Publications by authors named "Stefanie Kickinger"

6 Publications

  • Page 1 of 1

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.

Sci Rep 2020 08 3;10(1):12992. Epub 2020 Aug 3.

Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, 2100, Copenhagen, Denmark.

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 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-, exocyclic-N-, and C-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 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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http://dx.doi.org/10.1038/s41598-020-69908-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7400577PMC
August 2020

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

Neurochem Res 2020 Jul 4;45(7):1551-1565. Epub 2020 Apr 4.

Department of Drug Design and Pharmacology, University of Copenhagen, Copenhagen, Denmark.

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 [H]GABA uptake assay at heterologously expressed BGT1, revealed an elusive biphasic inhibition profile with two IC 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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http://dx.doi.org/10.1007/s11064-020-03017-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7297817PMC
July 2020

A widespread role for SLC transmembrane transporters in resistance to cytotoxic drugs.

Nat Chem Biol 2020 04 9;16(4):469-478. Epub 2020 Mar 9.

CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria.

Solute carriers (SLCs) are the largest family of transmembrane transporters in humans and are major determinants of cellular metabolism. Several SLCs have been shown to be required for the uptake of chemical compounds into cellular systems, but systematic surveys of transporter-drug relationships in human cells are currently lacking. We performed a series of genetic screens in a haploid human cell line against 60 cytotoxic compounds representative of the chemical space populated by approved drugs. By using an SLC-focused CRISPR-Cas9 library, we identified transporters whose absence induced resistance to the drugs tested. This included dependencies involving the transporters SLC11A2/SLC16A1 for artemisinin derivatives and SLC35A2/SLC38A5 for cisplatin. The functional dependence on SLCs observed for a significant proportion of the screened compounds suggests a widespread role for SLCs in the uptake and cellular activity of cytotoxic drugs and provides an experimentally validated set of SLC-drug associations for a number of clinically relevant compounds.
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http://dx.doi.org/10.1038/s41589-020-0483-3DOI Listing
April 2020

Structural and molecular aspects of betaine-GABA transporter 1 (BGT1) and its relation to brain function.

Neuropharmacology 2019 12 18;161:107644. Epub 2019 May 18.

University of Copenhagen, Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, 2 Universitetsparken, 2100, Copenhagen, Denmark. Electronic address:

ɣ-aminobutyric-acid (GABA) functions as the principal inhibitory neurotransmitter in the central nervous system. Imbalances in GABAergic neurotransmission are involved in the pathophysiology of various neurological diseases such as epilepsy, Alzheimer's disease and stroke. GABA transporters (GATs) facilitate the termination of GABAergic signaling by transporting GABA together with sodium and chloride from the synaptic cleft into presynaptic neurons and surrounding glial cells. Four different GATs have been identified that all belong to the solute carrier 6 (SLC6) transporter family: GAT1-3 (SLC6A1, SLC6A13, SLC6A11) and betaine/GABA transporter 1 (BGT1, SLC6A12). BGT1 has emerged as an interesting target for treating epilepsy due to animal studies that reported anticonvulsant effects for the GAT1/BGT1 selective inhibitor EF1502 and the BGT1 selective inhibitor RPC-425. However, the precise involvement of BGT1 in epilepsy remains elusive because of its controversial expression levels in the brain and the lack of highly selective and potent tool compounds. This review gathers the current structural and functional knowledge on BGT1 with emphasis on brain relevance, discusses all available compounds, and tries to shed light on the molecular determinants driving BGT1 selectivity. This article is part of the issue entitled 'Special Issue on Neurotransmitter Transporters'.
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http://dx.doi.org/10.1016/j.neuropharm.2019.05.021DOI Listing
December 2019

Structure-Activity Relationship, Pharmacological Characterization, and Molecular Modeling of Noncompetitive Inhibitors of the Betaine/γ-Aminobutyric Acid Transporter 1 (BGT1).

J Med Chem 2017 11 19;60(21):8834-8846. Epub 2017 Oct 19.

Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen , 2100 Copenhagen, Denmark.

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.
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http://dx.doi.org/10.1021/acs.jmedchem.7b00924DOI Listing
November 2017

Development of Non-GAT1-Selective Inhibitors: Challenges and Achievements.

Adv Neurobiol 2017 ;16:315-332

Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, 2100, Copenhagen, Denmark.

γ-Aminobutyric acid (GABA) neurotransmission is terminated by the GABA transporters (GATs) via uptake of GABA into neurons and surrounding glial cells. Four different transporters have been identified: GAT1, GAT2, GAT3, and the betaine/GABA transporter 1 (BGT1). The GAT1 subtype is the most explored transporter due to its high abundance in the brain and the existence of selective and potent GAT1 inhibitors. Consequently, less is known about the role and therapeutic potential of the non-GAT1 subtypes. Emerging pharmacological evidence suggests that some of these transporters pose interesting targets in several brain disorders. Pharmacological non-GAT1-selective tool compounds are important to further investigate the involvement of GATs in different pathological conditions. Extensive medicinal chemistry efforts have been put into the development of subtype-selective inhibitors, but truly selective and potent inhibitors of non-GAT1 subtypes are still limited. This review covers the advances within the medicinal chemistry area and the structural basis for obtaining non-GAT1-selective inhibitors.
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http://dx.doi.org/10.1007/978-3-319-55769-4_16DOI Listing
September 2018