Engineered Flumazenil Recognition Site Provides Mechanistic Insight Governing Benzodiazepine Modulation in GABA_A Receptors

The anxiolytic, anticonvulsant, muscle-relaxant, and sedative-hypnotic effects of benzodiazepine site ligands are mainly elicited by allosteric modulation of GABA_A receptors via their extracellular αx+/γ2– (<i>x</i> = 1, 2, 3, 5) interfaces. In addition, a low affinity binding site at the homologous α+/β– interfaces was reported for some benzodiazepine site ligands. Classical benzodiazepines and pyrazoloquinolinones have been used as molecular probes to develop structure–activity relationship models for benzodiazepine site activity. Considering all possible α+/β– and α+/γ– interfaces, such ligands potentially interact with as many as 36 interfaces, giving rise to undesired side effects. Understanding the binding modes at their binding sites will enable rational strategies to design ligands with desired selectivity profiles. Here, we compared benzodiazepine site ligand interactions in the high affinity α1+/γ2– site with the homologous α1+/β3– site using a successive mutational approach. We incorporated key amino acids known to contribute to high affinity benzodiazepine binding of the γ2– subunit into the β3– subunit, resulting in a quadruple mutant β3­(4mut) with high affinity flumazenil (Ro 15-1788) binding properties. Intriguingly, some benzodiazepine site ligands displayed positive allosteric modulation in the tested recombinant α1β3­(4mut) constructs while diazepam remained inactive. Consequently, we performed <i>in silico</i> molecular docking in the wildtype receptor and the quadruple mutant. The results led to the conclusion that different benzodiazepine site ligands seem to use distinct binding modes, rather than a common binding mode. These findings provide structural hypotheses for the future optimization of both benzodiazepine site ligands, and ligands that interact with the homologous α+/β– sites

Design and Synthesis of Novel Deuterated Ligands Functionally Selective for the γ‑Aminobutyric Acid Type A Receptor (GABA_AR) α6 Subtype with Improved Metabolic Stability and Enhanced Bioavailability

Recent reports indicate that α6β2/3γ2 GABA_AR selective ligands may be important for the treatment of trigeminal activation-related pain and neuropsychiatric disorders with sensori-motor gating deficits. Based on 3 functionally α6β2/3γ2 GABA_AR selective pyrazoloquinolinones, 42 novel analogs were synthesized, and their in vitro metabolic stability and cytotoxicity as well as their in vivo pharmacokinetics, basic behavioral pharmacology, and effects on locomotion were investigated. Incorporation of deuterium into the methoxy substituents of the ligands increased their duration of action via improved metabolic stability and bioavailability, while their selectivity for the GABA_AR α6 subtype was retained. <b>8b</b> was identified as the lead compound with a substantially improved pharmacokinetic profile. The ligands allosterically modulated diazepam insensitive α6β2/3γ2 GABA_ARs and were functionally silent at diazepam sensitive α1β2/3γ2 GABA_ARs, thus no sedation was detected. In addition, these analogs were not cytotoxic, which render them interesting candidates for treatment of CNS disorders mediated by GABA_AR α6β2/3γ2 subtypes