Novel Molecule Exhibiting Selective Affinity for GABAA Receptor Subtypes

Abstract

Aminoquinoline derivatives were evaluated against a panel of receptors/channels/transporters in radioligand binding experiments. One of these derivatives (DCUK-OEt) displayed micromolar affinity for brain γ-aminobutyric acid type A (GABA_A) receptors. DCUK-OEt was shown to be a positive allosteric modulator (PAM) of GABA currents with α1β2γ2, α1β3γ2, α5β3γ2 and α1β3δ GABA_A receptors, while having no significant PAM effect on αβ receptors or α1β1γ2, α1β2γ1, α4β3γ2 or α4β3δ receptors. DCUK-OEt modulation of α1β2γ2 GABA_A receptors was not blocked by flumazenil. The subunit requirements for DCUK-OEt actions distinguished DCUK-OEt from other currently known modulators of GABA function (e.g., anesthetics, neurosteroids or ethanol). Simulated docking of DCUK-OEt at the GABA_A receptor suggested that its binding site may be at the α + β- subunit interface. In slices of the central amygdala, DCUK-OEt acted primarily on extrasynaptic GABA_A receptors containing the α1 subunit and generated increases in extrasynaptic "tonic" current with no significant effect on phasic responses to GABA. DCUK-OEt is a novel chemical structure acting as a PAM at particular GABA_A receptors. Given that neurons in the central amygdala responding to DCUK-OEt were recently identified as relevant for alcohol dependence, DCUK-OEt should be further evaluated for the treatment of alcoholism.

Figure 1

Chemical structure of DCUK compounds. (a) DCUKA (5,7-Dichloro-4-([diphenyl carbamoyl] amino) quinoline-2-carboxylic acid). (b) DCUK-OEt (5,7-Dichloro-4-([diphenyl carbamoyl] amino) quinoline-2-ethyl carboxylate).

Figure 2

DCUK effect on GABA responses. (a) Effect of DCUK compounds on submaximal (EC₁₀) GABA responses of α1β2γ2 GABA_A receptors (n = 4–5 at each concentration of DCUK compound). (b) Effect of DCUK-OEt on submaximal (EC₁₀) GABA responses of α1β3δ GABA_A receptors (n = 5–6 at each concentration of DCUK-OEt). (c) Effect of DCUK-OEt (0.3 µM) and escalating GABA concentrations applied to α1β2γ2 and α1β3δ GABA_A receptors (n = 9 each). GABA concentrations used: 3 and 1 µM for α1β2γ2 and α1β3δ, respectively (~EC₁₀); 30 µM (~EC₆₀); 3 mM (~EC₁₀₀). (d) DCUK-OEt (0.3 μM) effect in the absence and presence of 20 μM flumazenil (Flu) (n = 5 for each condition). Data represent mean ± SEM. **p < 0.01 compared to α1β2γ2 (one-way ANOVA and post-hoc contrasts).

Figure 3

Predicted docking of DCUK-OEt and DCUKA within extracellular domain interfaces of GABA_A receptor subunits. The α subunit is shaded in green, β in cyan and γ in yellow. (a) Extracellular (top down) view of the pentameric GABA_A receptor. The interfaces illustrated are α + β- (alternative site), and α + γ- (benzodiazepine site). (b) DCUK-OEt and (c) DCUKA within the alternative site (α + β-). DCUK-OEt is represented by orange sticks and DCUKA is represented by pink sticks. (d) DCUKA and (e) DCUK-OEt within the benzodiazepine site (α + γ-). Dashed lines indicate predicted non-bond interactions (green = H-bonds, orange = electrostatic or π-cation/anion, magenta = π-π, purple = π-σ, pink = hydrophobic).

Figure 4

DCUK-OEt potentiates tonic currents in medial CeA neurons. (a and b) Focal application of DCUK-OEt (0.5 µM) significantly increased the holding current in medial CeA neurons (*p < 0.05, paired t-test). (c) No change was evident in frequency, amplitude, rise and decay of mIPSPs with focal application of DCUK-OEt. (d) Correlation of magnitude of increase in tonic current produced by 0.5 μM DCUK-OEt with reduction of current by subsequent application of 100 μM gabazine. To demonstrate that changes in holding current were due to increases in tonic signaling, the GABA_A receptor antagonist gabazine (GBZ) (100 μM) was focally applied following DCUK-OEt application. For all graphs, n = 11 cells.