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.