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 [3H]GABA uptake assay at heterologously expressed BGT1, revealed an elusive biphasic inhibition profile with two IC50 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.

Highlights

  • Introduction γAminobutyric acid (GABA) is the main inhibitory neurotransmitter in the central nervous system (CNS) and is critical for maintaining normal neurotransmission

  • This study revealed that SBV2-114 inhibited mBGT1 and hBGT1 in an unusual biphasic manner with ­IC50 values in the low (m/hBGT1: 3.8/4.7 μM) and high μM range (m/hBGT1: 402.7/555.9 μM) corresponding to the high and low affinity components, respectively (Fig. 2a and Table 1)

  • We have demonstrated an apparently novel biphasic inhibition profile of SBV2-114, which is most evident at BGT1

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Summary

Introduction

Introduction γAminobutyric acid (GABA) is the main inhibitory neurotransmitter in the central nervous system (CNS) and is critical for maintaining normal neurotransmission. SBV2-114 displayed a biphasic inhibition profile at hGAT3, and with a reduced Frac value compared to hBGT1-tsA, and in one out of four experiments at GAT2 (Supplemental Table 3).

Results
Conclusion

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