Rational Search for Betaine/GABA Transporter 1 Inhibitors─<i>In Vitro</i> Evaluation of Selected Hit Compound

Inhibitory neurotransmission mediated by γ-aminobutyric acid (GABA) plays an important role in maintaining body homeostasis. Disturbances in GABA signaling are implicated in a multitude of neurologic and psychiatric conditions, including epilepsy, ischemia, anxiety, depression, insomnia, and mood disorders. Clinically relevant increases in GABA neurotransmitter level can be achieved by inhibition of its uptake into presynaptic neurons and surrounding glial cells, driven by GABA transporters (GAT1, BGT1, GAT2, and GAT3). Herein, we focused on the search for inhibitors of the BGT1 transporter which is understudied and for which the therapeutic potential of its inhibition is partly unknown. We applied multilevel virtual screening to identify compounds with inhibitory properties. Among selected hits, compound <b>9</b> was shown to be a preferential inhibitor of BGT1 (IC₅₀ 13.9 μM). The compound also revealed some inhibitory activity against GAT3 (4x lower) while showing no or low activity (IC₅₀ > 100 μM) toward GAT1 and GAT2, respectively. The predicted binding mode of compound <b>9</b> was confirmed by mutagenesis studies on E52A, E52Y, Q299L, and E52A+Q299L human BGT1 mutants. Subsequent evaluation showed that the selected hit displayed no affinity toward major GABA_A receptor subtypes. Moreover, it was nontoxic when tested on normal human astrocytes and even showed some neuroprotective activity in SH-SY5Y cells. Compound <b>9</b> is considered a promising candidate for further evaluation of the therapeutic potential of BGT1 transporter inhibition and the development of novel inhibitors.