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

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 (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.

Molecular Determinants and Pharmacological Analysis for a Class of Competitive Non-transported Bicyclic Inhibitors of the Betaine/GABA Transporter BGT1

“…Using computationally guided mutagenesis studies we reveal that interactions with the residues Q299 and E52 drive activity and selectivity of bicyclo-GABA and 2 . Since these two residues were already identified as relevant for other compound classes ( Jørgensen et al, 2017 ; Kickinger et al, 2020 ; Lie et al, 2020 ), we conclude that hydrogen bonding between the protonated nitrogen moiety of BGT1 inhibitors and Q299 and E52 is a key feature for selective BGT1 inhibition. Although the synthetic chemistry for producing bicyclo-GABA and 2 is challenging, both compounds constitute relevant tool compounds for further pharmacological investigations, potentially including conversion into (radio)labeled ligands for BGT1 to determine affinities and/or visualize regional and cellular BGT1 localization.…”

“…In a previous study, we have already identified Q299 and E52 in BGT1 as part of the molecular determinants driving activity and selectivity of a series of cyclic 2-amino-tetrahydropyrimidine/pyridine compounds ( Kickinger et al, 2020 ). We therefore conclude that the interaction between the amino moiety of different BGT1 compound classes with Q299 and E52 is a general key feature for selective BGT1 inhibition ( Jørgensen et al, 2017 ; Kickinger et al, 2020 ; Lie et al, 2020 ).…”

“…Based on the binding hypothesis of bicyclo-GABA and 1 , we visually analyzed the available space in the binding pocket to rationalize possible sites for chemical derivatization. The aim of the derivatization was twofold: 1) to facilitate blood-brain-barrier permeability by introducing lipophilic substituents; a strategy successfully applied to several known GAT inhibitors such as tiagabine, EF1502 and RPC-425 ( Skovstrup et al, 2010 ; Vogensen et al, 2013 ; Lie et al, 2018 , 2020 ; Kickinger et al, 2019 ), and 2) to identify positions for derivatization that would reduce off-target effects at GABA A receptors, most particularly α 1 β 2 γ 2 receptors, the major GABA A receptor subtype in the brain ( Olsen and Sieghart, 2009 ).…”

“…Data presented is the pooled data of at least three independent experiments with three technical replicates if not stated otherwise in figure legends. Concentration–response curves were fitted by non-linear regression to the sigmoidal concentration-response model with GraphPad Prism (version 9.0.0, GraphPad Software, San Diego, CA, United States) as described ( Lie et al, 2020 ).…”

“…Based on the binding hypothesis of bicyclo-GABA and 1, we visually analyzed the available space in the binding pocket to rationalize possible sites for chemical derivatization. The aim of the derivatization was twofold: 1) to facilitate blood-brain-barrier permeability by introducing lipophilic substituents; a strategy successfully applied to several known GAT inhibitors such as tiagabine, EF1502 and RPC-425 (Skovstrup et al, 2010;Vogensen et al, 2013;Lie et al, 2018Lie et al, , 2020Kickinger et al, 2019), and 2) to identify positions for derivatization that would reduce off-target effects at GABA A receptors, most particularly α 1 β 2 γ 2 receptors, the major GABA A receptor subtype in the brain (Olsen and Sieghart, 2009). According to our analysis based on the docking results, the carbon 6 (C6) position of bicyclo-GABA (Figure 1) was identified as the most promising position for derivatization since it is located next to the extracellular lid (Y133, F293) (Figure 2E), which would allow bulky substituents to expand into the extracellular vestibule.…”