Identification of Orthosteric GABA_B Receptor Ligands by Virtual Screening and <i>In Vitro</i> Validation

The GABAB receptor (GABAB-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 GABAB1a/b 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 in vitro using the functional Hit Hunter cAMP assay in Chinese hamster ovary (CHO)-K1 cells stably overexpressing the human GABAB(1b,2)-R and in wild-type CHO-K1 cells. Based on the initial testing, two compounds were selected for studies in the [35S]GTPγS binding assays and a competition binding assay using the GABAB-R antagonist [3H]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 GABAB-R and are antagonists.