Identification of Orthosteric GABA Receptor Ligands by Virtual Screening and Validation.

The GABA receptor (GABA-R) is a heterodimeric class C G-protein coupled receptor (GPCR) associated with numerous neurological and neuropsychiatric disorders and is an interesting target for drug development. Each subunit has an extracellular part called the Venus flytrap domain (VFT), and the VFT of the GABA subunit contains the orthosteric γ-aminobutyric acid (GABA) binding site. In the present study, we have used a combined ligand- and structure-based virtual screening (VS) campaign to identify putative compounds binding to the orthosteric binding site. Based on the VS, 34 ligands were purchased and tested using the functional Hit Hunter cAMP assay in Chinese hamster ovary (CHO)-K1 cells stably overexpressing the human GABA-R and in wild-type CHO-K1 cells. Based on the initial testing, two compounds were selected for studies in the [S]-GTPγS binding assays and a competition binding assay using the GABA-R antagonist [H]-CGP54626 as the radioligand. In addition, their effects on the dose-response curve of GABA were further evaluated in the Hit Hunter cAMP assay. The experimental testing confirmed that both compounds bind to the orthosteric site of GABA-R and are antagonists.