RT Journal Article SR Electronic T1 A benzodiazepine ligand with improved GABAA-receptor α5-subunit-selectivity driven by interactions with loop C JF Molecular Pharmacology JO Mol Pharmacol FD American Society for Pharmacology and Experimental Therapeutics SP MOLPHARM-AR-2020-000067 DO 10.1124/molpharm.120.000067 A1 Xenia Simeone A1 Filip Koniuszewski A1 Markus Müllegger A1 Andreas Smetka A1 Friederike Steudle A1 Roshan Puthenkalam A1 Margot Ernst A1 Petra Scholze YR 2020 UL http://molpharm.aspetjournals.org/content/early/2020/12/02/molpharm.120.000067.abstract AB The family of GABAA receptors is an important drug target group in the treatment of sleep disorders, anxiety, epileptic seizures and many others. The most frequent GABAA receptor subtype is composed of two α, two β and one γ2-subunit, while the nature of the α-subunit critically determines the properties of the benzodiazepine binding site of those receptors. Nearly all of the clinically relevant drugs target all GABAA receptor subtypes equally. In the past years, however, drug development research has focused on studying α5‑containing GABAA receptors. Beyond the CNS, α5‑containing GABAA receptors in airway smooth muscles are considered as emerging target for bronchial asthma. Here, we investigated a novel compound derived from the previously described imidazobenzodiazepine SH‑053‑2′F‑R‑CH3 (SH53d‑ester). While SH53d‑ester is only moderately selective for α5‑subunit containing GABAAreceptors, the derivative SH53d‑acid shows superior (>40-fold) affinity selectivity, and is a positive modulator. Using two‑electrode voltage clamp electrophysiology in Xenopus laevis oocytes and radioligand displacement assays with HEK 293 cells, we demonstrated that an acid group as substituent on the imidazobenzodiazepine scaffold leads to large improvements of functional and binding selectivity for α5β3γ2 over other αxβ3γ2 GABAA receptors. Atom level structural studies provide hypotheses for the improved affinity to this receptor subtype. Mutational analysis confirmed the hypotheses, indicating that loop C of the GABAA receptor α‑subunit is the dominant molecular determinant of drug selectivity. Thus, we characterize a promising novel α5‑subunit‑selective drug candidate. Significance Statement In the current study we present the detailed pharmacological characterization of a novel compound derived from the previously described imidazobenzodiazepine SH-053-2′F-R-CH3. We describe its superior (>40-fold) affinity selectivity for α5-containing GABAA receptors and show atom level structure predictions to provide hypotheses for the improved affinity to this receptor subtype. Mutational analysis confirmed the hypotheses, indicating that loop C of the GABAA-receptor α-subunit is the dominant molecular determinant of drug selectivity.