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

Inhibitory neurotransmission mediated by γ-aminobutyric\nacid\n(GABA) plays an important role in maintaining body homeostasis. Disturbances\nin GABA signaling are implicated in a multitude of neurologic and\npsychiatric conditions, including epilepsy, ischemia, anxiety, depression,\ninsomnia, and mood disorders. Clinically relevant increases in GABA\nneurotransmitter level can be achieved by inhibition of its uptake\ninto presynaptic neurons and surrounding glial cells, driven by GABA\ntransporters (GAT1, BGT1, GAT2, and GAT3). Herein, we focused on the\nsearch for inhibitors of the BGT1 transporter which is understudied\nand for which the therapeutic potential of its inhibition is partly\nunknown. We applied multilevel virtual screening to identify compounds\nwith inhibitory properties. Among selected hits, compound <b>9</b> was shown to be a preferential inhibitor of BGT1 (IC<sub>50</sub> 13.9 μM). The compound also revealed some inhibitory activity\nagainst GAT3 (4x lower) while showing no or low activity (IC<sub>50</sub> > 100 μM) toward GAT1 and GAT2, respectively. The predicted\nbinding mode of compound <b>9</b> was confirmed by mutagenesis\nstudies on E52A, E52Y, Q299L, and E52A+Q299L human BGT1 mutants. Subsequent\nevaluation showed that the selected hit displayed no affinity toward\nmajor GABA<sub>A</sub> receptor subtypes. Moreover, it was nontoxic\nwhen tested on normal human astrocytes and even showed some neuroprotective\nactivity in SH-SY5Y cells. Compound <b>9</b> is considered a\npromising candidate for further evaluation of the therapeutic potential\nof BGT1 transporter inhibition and the development of novel inhibitors.