Delineation of the functional properties and the mechanism of action of AA29504, an allosteric agonist and positive allosteric modulator of GABAA receptors

Abstract

The retigabine analog 2-amino-4-[(2,4,6-trimethylbenzylamino)-phenyl]-carbamic acid ethyl ester (AA29504) is a positive allosteric modulator (PAM) of γ-aminobutyric acid receptors (GABARs), and the modulator has been used in ex vivo/in vivo studies to probe the physiological roles of native δ-containing GABARs. In this study, the functional properties and mode of action of AA29504 were investigated at human GABARs expressed in Xenopus oocytes by two-electrode voltage clamp electrophysiology. AA29504 was found to be an allosteric GABAR agonist displaying low intrinsic activities at 3-30 μM. AA29504 was essentially equipotent as a PAM at the 13 GABAR subtypes tested (EC: 0.45-5.2 μM), however GABA EC-evoked currents through αβδ subtypes were modulated to substantially higher levels than those through αβγ subtypes (relative to GABA I). While the δ/γ-difference clearly was key for this differential GABA efficacy modulation, studies of the AA29504-mediated modulation of different α-containing αβ, αβγ and αβδ GABARs revealed the α-subunit identity to be another important determinant. Based on its functional properties at numerous mutant GABARs and on in silico analysis of its low-energy conformations, AA29504 is proposed to act through an allosteric site in the transmembrane β/α interface in the GABAR also targeted by etomidate and several other modulators. In contrast to these modulators, however, AA29504 did not display substantial β/β-over-β GABAR preference, which challenges the notion of ligands targeting this site always possessing this subtype-selectivity profile. Hence, the detailed pharmacological profiling of AA29504 both highlights the complexity of allosteric GABAR modulation and provides valuable information about this modulator as a pharmacological tool.

Molecular determinants underlying DS2 activity at δ-containing GABAAreceptors

“…Additionally, AA29504 showed similar potentiation at the mutants and WT receptors (Fig. S6, Table S7), indicating that it does not mediate its effect through the same site as DS2, correlating with a proposed binding site for AA29504 in the TMD β (+) α (-) interface (36).…”

“…To confirm that the mutant receptors with altered DS2 sensitivity were not overall compromised in their general PAM responsiveness, we tested etomidate (50) and AA29504 (36, 51) at both the WT and the single mutants α 4 (S303L)β 1 δ and α 4 β 1 (I289Q)δ. In the FMP assay both compounds showed intact positive modulation of both mutants compared to WT.…”

“…This low-affinity diazepam binding site is located in the same pocket as the binding site for the general anesthetics (e.g. etomidate) (35) which is also the proposed binding site for AA29504 (36). Additionally, similar sites are suggested to be present in all five inter-subunit interfaces in the TMD (37–39).…”

Molecular Determinants Underlying Delta Selective Compound 2 Activity at δ-Containing GABAA Receptors

“…To confirm that the mutant receptors with altered DS2 sensitivity were not overall compromised in their general PAM responsiveness, we tested etomidate (Hill-Venning et al, 1997) andAA29504 (Hoestgaard-Jensen et al, 2010;Olander et al, 2018) at both WT and the single mutants  4 (S303L) 1  and  4  1 (I289Q). In the FMP assay both compounds showed intact positive modulation of both mutants compared to WT.…”

Alcohol-Responsive Hyperkinetic Movement Disorders—a Mechanistic Hypothesis

“…Identification of the specific binding sites in these subunits may be relevant for designing drugs with selective EtOH effects and to explain variable responses to GABA receptor ligands related to specific polymorphisms in genes encoding for these subunits, as previously shown [ 10 ]. For example, the compound AA29504 has been reported to have specific affinity for the αβδ receptors [ 11 ]. On the other side, Purkinje cells are instead enriched with α1β2/3γ2 GABA receptors [ 12 ].…”