A series of substituted 1-hydroxypyrazole analogues of the GABA(A) receptor partial agonist 5-(4-piperidyl)-3-isoxazolol (4-PIOL) have been synthesized and pharmacologically characterized. Several of the analogues displayed K(i) in the low nanomolar range at the native GABA(A) receptors and potent antagonism of the alpha(1)beta(2)gamma(2) receptor. It appears that several regions situated in proximity to the core of the orthosteric binding site of the GABA(A) receptor are able to accommodate large hydrophobic substituents.

The isoxazol-3-one tautomer of the bicyclic isoxazole, 5,6,7,8-tetrahydro-4H-isoxazolo[4,5-d]azepin-3-ol (THAZ), has previously been shown to be a weak GABA(A) and glycine receptor antagonist. In the present study, the potential in this scaffold has been explored through the synthesis and pharmacological characterization of a series of N- and O-substituted THAZ analogues. The analogues N-Bn-THAZ (3d) and O-Bn-THAZ (4d) were found to be potent agonists of the human 5-HT(2A) and 5-HT(2C) receptors. Judging from an elaborate pharmacological profiling at numerous other CNS targets, the 3d analogue appears to be selective for the two receptors. Administration of 3d substantially improved the cognitive performance of mice in a place recognition Y-maze model, an effect fully reversible by coadministration of the selective 5-HT(2C) antagonist SB242084. In conclusion, as novel bioavailable cognitive enhancers that most likely mediate their effects through 5-HT(2A) and/or 5-HT(2C) receptors, the isoxazoles 3d and 4d constitute interesting leads for further medicinal chemistry development.

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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 trans-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 K) 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.

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Background and Purpose The δ‐subunit‐containing GABAA receptors, α4β1δ and α4β3δ, 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 α4β1/3δ 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 GABAA 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 GABAA receptors. In the absence of spontaneous gating, DGGCs responded to the α4β1/3δ 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 (EC50 2.1 μM). The gabazine‐induced current was inhibited by picrotoxin, potentiated by DS2, completely absent in δ−/− mice and reduced in β1−/− mice, but could not be replicated in human α4β1/3δ receptors expressed heterologously in HEK cells. Conclusion and Implications Kinase activity infers spontaneous gating in α4β1/3δ receptors in DGGCs. This significantly limits the efficacy of GABAA 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 GABAA ligands may be reduced.

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In the present study, the orthosteric GABA A receptor (GABA A R) ligand 4,5,6,7-tetrahydroisothiazolo[5,4-c]pyridin-3-ol (Thio-THIP) was found to possess a highly interesting functional profile at recombinant human GABA A Rs and native rat GABA A Rs. Whereas Thio-THIP displayed weak antagonist activity at ␣ 1,2,5 ␤ 2,3 ␥ 2S and 1 GABA A Rs and partial agonism at ␣ 6 ␤ 2,3 ␦ GABA A Rs expressed in Xenopus oocytes, the pronounced agonism exhibited by the compound at ␣ 4 ␤ 1 ␦ and ␣ 4 ␤ 3 ␦ GABA A Rs was contrasted by its negligible activity at the ␣ 4 ␤ 2 ␦ subtype. To elucidate to which extent this in vitro profile translated into functionality at native GABA A Rs, we assessed the effects of 100 M Thio-THIP at synaptic and extrasynaptic receptors in principal cells of four different brain regions by slice electrophysiology. In concordance with its ␣ 6 ␤ 2,3 ␦ agonism, Thio-THIP evoked robust currents through extrasynaptic GABA A Rs in cerebellar granule cells. In contrast, the compound did not elicit significant currents in dentate gyrus granule cells or in striatal medium spiny neurons (MSNs), indicating predominant expression of extrasynaptic ␣ 4 ␤ 2 ␦ receptors in these cells. Interestingly, Thio-THIP evoked differential degrees of currents in ventrobasal thalamus neurons, a diversity that could arise from differential expression of extrasynaptic ␣ 4 ␤␦ subtypes in the cells. Finally, whereas 100 M Thio-THIP did not affect the synaptic currents in ventrobasal thalamus neurons or striatal MSNs, it reduced the current amplitudes recorded from dentate gyrus granule cells, most likely by targeting perisynaptic ␣ 4 ␤␦ receptors expressed at distal dendrites of these cells. Being the first published ligand capable of discriminating between ␤ 2 -and ␤ 3 -containing receptor subtypes, Thio-THIP could be a valuable tool in explorations of native ␣ 4 ␤␦ GABA A Rs.