Silencing of spontaneous activity at α4β1/3δ GABAA receptors in hippocampal granule cells reveals different ligand pharmacology

BACKGROUND AND PURPOSE: The δ‐subunit‐containing GABA(A) receptors, α(4)β(1)δ and α(4)β(3)δ, in dentate gyrus granule cells (DGGCs) are known to exhibit both spontaneous channel openings (i.e. constitutive activity) and agonist‐induced current. The functional implications of spontaneous gating are unclear. In this study, we tested the hypothesis that constitutively active α(4)β(1/3)δ receptors limit agonist efficacy. EXPERIMENTAL APPROACH: Whole‐cell electrophysiological recordings of adult male rat and mouse hippocampal DGGCs were used to characterize known agonists and antagonists at δ‐subunit‐containing GABA(A) receptors. To separate constitutive and agonist‐induced currents, different recording conditions were employed. KEY RESULTS: Recordings at either 24°C or 34°C, including the PKC autoinhibitory peptide (19–36) intracellularly, removed spontaneous gating by GABA(A) receptors. In the absence of spontaneous gating, DGGCs responded to the α(4)β(1/3)δ orthosteric agonist Thio‐THIP with a four‐fold increased efficacy relative to recording conditions favouring constitutive activity. Surprisingly, the neutral antagonist gabazine was unable to antagonize the current by Thio‐THIP. Furthermore, a current was elicited by gabazine alone only when the constitutive current was silenced (EC(50) 2.1 μM). The gabazine‐induced current was inhibited by picrotoxin, potentiated by DS2, completely absent in δ(−/−) mice and reduced in β(1) (−/−) mice, but could not be replicated in human α(4)β(1/3)δ receptors expressed heterologously in HEK cells. CONCLUSION AND IMPLICATIONS: Kinase activity infers spontaneous gating in α(4)β(1/3)δ receptors in DGGCs. This significantly limits the efficacy of GABA(A) agonists and has implications in pathologies involving aberrant excitability caused by phosphorylation (e.g. addiction and epilepsy). In such cases, the efficacy of δ‐preferring GABA(A) ligands may be reduced

Structural Determinants for the Mode of Action of the Imidazopyridine DS2 at d-containing g-Aminobutyric Acid Type A (GABAA) Receptors

A SAR study of the delta-selective positive modulators DS2 was performed to assist the quest for the binding site. The modulatory effect was measured using a fluorometric inaging plate reader (FLIPR) membrane potential (FMP) functional assay. Specific positions in the structural scaffold of DS2 was found to severly affect the pharmacological profile. Analogs superior to DS2 were identified displaying higher potency and selectivity for the alfa4beta1delta over alfa4beta1gamma.<br /

Structure–activity studies of 3,9-diazaspiro[5.5]undecane-based γ-aminobutyric acid type A receptor antagonists with immunomodulatory effect

The 3,9-diazaspiro[5.5]undecane-based compounds 2027 and 018 have previously been reported to be potent competitive γ-aminobutyric acid type A receptor (GABAAR) antagonists showing low cellular membrane permeability. Given the emerging peripheral application of GABAAR ligands, we hypothesize 2027 analogs as promising lead structures for peripheral GABAAR inhibition. We herein report a study on the structural determinants of 2027 in order to suggest a potential binding mode as a basis for rational design. The study identified the importance of the spirocyclic benzamide, compensating for the conventional acidic moiety, for GABAAR ligands. The structurally simplified m-methylphenyl analog 1e displayed binding affinity in the high-nanomolar range (Ki = 180 nM) and was superior to 2027 and 018 regarding selectivity for the extrasynaptic α4βδ subtype versus the α1- and α2- containing subtypes. Importantly, 1e was shown to efficiently rescue inhibition of T cell proliferation, providing a platform to explore the immunomodulatory potential for this class of compounds

Molecular Hybridization of Potent and Selective γ‑Hydroxybutyric Acid (GHB) Ligands: Design, Synthesis, Binding Studies, and Molecular Modeling of Novel 3‑Hydroxycyclopent-1-enecarboxylic Acid (HOCPCA) and <i>trans</i>-γ-Hydroxycrotonic Acid (T-HCA) Analogs

γ-Hydroxybutyric acid (GHB) is a neuroactive substance with specific high-affinity binding sites. To facilitate target identification and ligand optimization, we herein report a comprehensive structure–affinity relationship study for novel ligands targeting these binding sites. A molecular hybridization strategy was used based on the conformationally restricted 3-hydroxy­cyclopent-1-enecarboxylic acid (HOCPCA) and the linear GHB analog <i>trans</i>-4-hydroxy­crotonic acid (T-HCA). In general, all structural modifications performed on HOCPCA led to reduced affinity. In contrast, introduction of diaromatic substituents into the 4-position of T-HCA led to high-affinity analogs (medium nanomolar <i>K</i>_i) for the GHB high-affinity binding sites as the most high-affinity analogs reported to date. The SAR data formed the basis for a three-dimensional pharmacophore model for GHB ligands, which identified molecular features important for high-affinity binding, with high predictive validity. These findings will be valuable in the further processes of both target characterization and ligand identification for the high-affinity GHB binding sites