The GABA_AReceptor α+β− Interface: A Novel Target for Subtype Selective Drugs

Abstract

GABA A receptorsmediatetheactionofmanyclinicallyimportantdrugsinteractingwithdifferentbindingsites.Forsomepotentialbindingsites, no interacting drugs have yet been identified. Here, we established a steric hindrance procedure for the identification of drugs acting at the extracellular ␣1ϩ␤3Ϫ interface, which is homologous to the benzodiazepine binding site at the ␣1ϩ␥2Ϫ interface. On screening of Ͼ100 benzodiazepine site ligands, the anxiolytic pyrazoloquinoline 2-p-methoxyphenylpyrazolo[4,3Ϫc]quinolin-3(5H)-one (CGS 9895) was able to enhance GABA-induced currents at ␣1␤3 receptors from rat. CGS 9895 acts as an antagonist at the benzodiazepine binding site at nanomolar concentrations, but enhances GABA-induced currents via a different site present at ␣1␤3␥2 and ␣1␤3 receptors. By mutating pocket-forming amino acid residues at the ␣1ϩ and the ␤3Ϫ side to cysteines, we demonstrated that covalent labeling of these cysteines by the methanethiosulfonate ethylamine reagent MTSEA-biotin was able to inhibit the effect of CGS 9895. The inhibition was not caused by a general inactivation of GABA A receptors, because the GABA-enhancing effect of ROD 188 or the steroid ␣-tetrahydrodeoxycorticosterone was not influenced by MTSEA-biotin. Other experiments indicated that the CGS 9895 effect was dependent on the ␣ and ␤ subunit types forming the interface. CGS 9895thusrepresentsthefirstprototypeofdrugsmediatingbenzodiazepine-likemodulatoryeffectsviathe␣ϩ␤ϪinterfaceofGABA A receptors. Sincesuchbindingsitesarepresentat␣␤,␣␤␥,and␣␤␦receptors,suchdrugswillhaveamuchbroaderactionthanbenzodiazepinesandmight become clinical important for the treatment of epilepsy.

Identification of novel positive allosteric modulators and null modulators at the GABAA receptor α+β− interface

“…At the a1+b2/3-interface (Figure 1), it acts as a positive allosteric modulator. At the high affinity benzodiazepine binding site -the a1+g2-interface -it is a high affinity null modulator (Ramerstorfer et al, 2011). To identify further compounds possibly mediating their effects via the a1+b3-interface and to exclude effects mediated via the high affinity benzodiazepine binding site at the a+g-interface, we investigated 32 additional pyrazoloquinolinones/pyrazolopyridinones that are structurally analogous to CGS 9895 (compound 3) (Table 1), at receptors composed of a1 and b3 subunits ( Figure 1B).…”

“…In analogy to the previous study, we employed the substituted cysteine accessibility method to introduce a steric hindrance into the a1+b3-interface of a1b3 receptors (Ramerstorfer et al, 2011). The point mutations a1V211C (loop C of the a1+side) and b3Q64C (loop D of the b3-side) have been shown previously to not significantly change the potency or efficacy of GABA for enhancing GABA-induced currents (Ramerstorfer et al, 2011) at a1b3 or a1b3g2 receptors. These mutations, however, partially reduced the effects of compound 11 in the absence of MTSEAbiotin.…”

“…Very recently, it was demonstrated that the high affinity benzodiazepine binding site ligand CGS 9895 behaves as a null modulator via this site, and in addition, exerts a low potency positive modulatory action at GABAA receptors via a newly discovered drug binding site at the extracellular a+b-interface (Ramerstorfer et al, 2011). A low affinity flurazepam binding site has been identified previously at this interface (Baur et al, 2008).…”

“…Here, we investigated 32 structural analogues of CGS 9895 for their ability to mediate their effects via the a+b-interface (Ramerstorfer et al, 2011). We identified compounds displaying not only higher efficacy and apparent potency than CGS 9895, but also for the first time discovered compounds that act as null modulators at the a+b-binding site.…”

(Z)-4,6-Dichloro-N-(4-chlorophenyl)quinoline-3-carbimidoyl chloride

“…Additionally, they act as allosteric modulators via the +-interface (Ramerstorfer et al, 2011;Varagic et al, 2013). In the context of this research, we obtained the title compound, (III), as a by-product during the synthesis of ethyl 4,6-dichloroquinoline-3-carboxylate, (II) (Fig.…”