Molecular tools for GABAA receptors: High affinity ligands for β1-containing subtypes

Abstract

γ-Aminobutyric acid type A (GABAA) receptors are pentameric GABA-gated chloride channels that are, in mammalians, drawn from a repertoire of 19 different genes, namely α1-6, β1-3, γ1-3, δ, ε, θ, π and ρ1-3. The existence of this wide variety of subunits as well as their diverse assembly into different subunit compositions result in miscellaneous receptor subtypes. In combination with the large number of known and putative allosteric binding sites, this leads to a highly complex pharmacology. Recently, a novel binding site at extracellular α+/β− interfaces was described as the site of modulatory action of several pyrazoloquinolinones. In this study we report a highly potent ligand from this class of compounds with pronounced β1-selectivity that mainly lacks α-subunit selectivity. It constitutes the most potent β1-selective positive allosteric modulatory ligand with known binding site. In addition, a proof of concept pyrazoloquinolinone ligand lacking the additional high affinity interaction with the benzodiazepine binding site is presented. Ultimately, such ligands can be used as invaluable molecular tools for the detection of β1-containing receptor subtypes and the investigation of their abundance and distribution.

International Union of Basic and Clinical Pharmacology. CVI: GABAAReceptor Subtype- and Function-selective Ligands: Key Issues in Translation to Humans

“…However, the extent of GABA-induced current modulation would then strongly be influenced also by the desensitization of the receptor, a phenomenon more dominant at higher GABA concentrations and longer measurement times (Jones and Westbrook, 1995). This effect will be even more dramatic with drugs that allosterically accelerate current decay (Dillon et al, 1993(Dillon et al, , 1995Simeone et al, 2017).…”

“…Whereas CGS 9895 and PZ-II-029 preferentially modulate GABA A receptors containing b2 or b3 subunits, some other pyrazoloquinolinones, such as PZ- II-028 (compound 11 of Varagic et al, 2013b), as well as some structural analogs thereof, preferentially or exclusively (DCBS96, Fig. 38) modulate GABA A receptors containing b1 subunits (Simeone et al, 2017). Unfortunately, however, the so far investigated compounds show only limited a selectivity and, thus, can only be used for investigating the effects of b1 subunit-containing receptors irrespective of the associated a subunit type.…”

GABAA Receptor Ligands Often Interact with Binding Sites in the Transmembrane Domain and in the Extracellular Domain—Can the Promiscuity Code Be Cracked?

“…The observed β isoform profile could reflect interactions with either site 2 at the extracellular domain's α+/β− interface or site 3 at the transmembrane domain's β+/α− interface. We thus utilized previously published mutations [18,19] to investigate their impact on modulation. β3N41R is a partial conversion of the ECD site of β3 into the β1 [18], while β2N265S is a conversion at site 3 of the β2 into β1, which is known to reverse the effects of etomidate and loreclezole [20,21].…”

“…Preparation of mRNA for rat α1, β1, β2, β2N265S, β3, β3N41R, and γ2 subunits as well as α1β3γ2 concatenated constructs (α1β3γ2cct) and electrophysiological experiments with Xenopus laevis oocytes were performed as described previously [12,18,19]. α1β3γ2cct was prepared as described previously [37][38][39] and included subunits in the following order: (α1-β3-α1; γ2-β3).…”

“…Rat cerebellar membranes were prepared and radioligand binding assays were performed as described previously [18,40]. In brief, membrane pellets were incubated for 90 min at 4 • C in a total of 500 µL of a solution containing 50 mM Tris/citrate buffer, pH = 7.1, 150 mM NaCl and 2 nM [ 3 H]-flunitrazepam in the absence or presence of either 5 µM diazepam (to determine unspecific binding) or various concentrations of DCBS192 or MTI163 (dissolved in DMSO, final DMSO-concentration 0.5%).…”