GABAA Ligands Research Articles

Design, Synthesis and Anticonvulsant Activity of Cinnamoyl Derivatives of 3,4,6,7,8,9-hexahydrodibenzo[b,d]furan-1-(2H)-one Oxime.

Epilepsy continues to be a significant global health problem and the search for new drugs for its treatment remains an urgent task. 5-HT2 and GABAA-receptors are among promising biotargets for the search for new anticonvulsants. New potential 5-HT2 and GABAA ligands in the series of substituted cinnamoyl derivatives of 3,4,6,7,8,9-hexahydrodibenzo[b,d]furan-1-(2H)-one oxime were designed using pharmacophore model and molecular docking analysis. The synthesis of new compounds was carried out from 3,4,6,7,8,9-hexahydrodibenzo[b,d]furan-1(2H)-one oxime and substituted cinnamoyl chlorides. The anticonvulsant activity of new substances has been established using the maximal electroshock seizure test. Several synthesized substituted cinnamoyl derivatives of 3,4,6,7,8,9-hexahydrodibenzo [b,d]furan-1-(2H)-one oxime significantly reduced the severity of convulsive manifestations and completely prevented the death of animals after MES. The structure-activity relationship was investigated. The most effective compound was found to be GIZH-348 (1g) (3,4,6,7,8,9-hexahydrodibenzo[ b,d]furan-1(2Н)-one О-(4-chlorophenyl)acryloyl)oxime) at the doses of 10-20 mg/kg. Molecular and pharmacophore modelling methods allowed us to create a new group of substituted cinnamoyl derivatives of 3,4,6,7,8,9-hexahydrodibenzo[b,d]furan-1-(2H)-one oxime with anticonvulsant activity.

Novel synthetic procedures for C2 substituted imidazoquinolines as ligands for the α/β-interface of the GABAA-receptor

A series of substituted imidazoquinolines, a structurally related chemotype to pyrazoloquinolinones, a well-known class of GABAA ligands, was prepared via two synthetic procedures and the efficiency of these procedures were compared. One method relies on classical heterocyclic synthesis, the other one aims at late-stage decoration of a truncated scaffold via direct C–H functionalization. A pharmacological evaluation disclosed that one of the synthesized derivatives showed interesting activity on a α1β3 containing receptor subtype.Graphical abstract

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

The δ-subunit-containing GABAA receptors, α4 β1 δ and α4 β3 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 α4 β1/3 δ receptors limit agonist efficacy. 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. 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 α4 β1/3 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 (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 α4 β1/3 δ receptors expressed heterologously in HEK cells. Kinase activity infers spontaneous gating in α4 β1/3 δ 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.

Discriminative Stimulus Effects and Metabolism of Ethanol in Rhesus Monkeys.

Animal models are an essential feature of drug and pharmacotherapy development for treating alcohol use disorders (AUDs). The rhesus macaque is a robust animal model for many aspects of AUDs particularly in exploiting individual differences in oral self-administration of ethanol (EtOH), endocrine orchestration of stress response, and menstrual cycle characteristics. However, the clearance rates of EtOH have not been reported in this species, and the GABAA and N-methyl-D-aspartate (NMDA) receptor involvement in EtOH's discriminative stimulus effects has not been fully characterized. EtOH clearance rates following 2 doses of EtOH on separate days (0.5 and 1.0g/kg, i.g.) were determined in 8 young adult male rhesus macaques. The EtOH was given by nasogastric gavage, and repeated blood samples were taken over 5hours without sedation. Next, all subjects were trained on a 2-choice 1.0g/kg EtOH (i.g.) versus water discrimination with a 60-minutes pretreatment period to capture peak blood EtOH concentration (BEC). Substitution testing was conducted with GABAA ligands pentobarbital (i.g. and i.m.) and midazolam (i.g.), as well as NMDA antagonist MK-801 (i.m.). Peak BECs were 34 and 87mg/dl for 0.5 and 1.0g/kg doses, respectively, and occurred at 66 and 87minutes following gavage. All GABAA and NMDA ligands tested resulted in responding on the EtOH-appropriate lever with the potency ranking of MK-801 (ED50 : 0.017mg/kg)>midazolam (ED50 : 1.6mg/kg)>pentobarbital (ED50 : 3.7mg/kg)>EtOH (ED50 : 700mg/kg, or 0.7g/kg) in these subjects. These results suggest that the compound discriminative stimulus effects of EtOH are highly consistent across species, providing further support for the rhesus macaque as strong model for pharmacotherapy development for AUD.

Development of Subtype Selective GABAA Modulators: A Review Article

GABAA receptors are Cl- channels that can be opened by GABA and are the major inhibitory neurotransmitter receptors in the Central Nervous System. A variety of pharmacologically important drugs, such as benzodiazepines, barbiturates, neuroactive steroids, general anesthetics and convulsants, produce at least part of their clinically relevant effects by interacting with distinct allosteric binding sites on GABAA receptors. GABAA receptors are involved in anxiety, feeding and drinking behavior, circadian rhythm, cognition, vigilance, and learning and memory. Different subtype mediates different functions. Neurons expressing a1GABAA receptors have been found to mediate sedation, whereas those expressing a2GABAA receptors mediate anxiolysis. Furthermore, associative temporal and spatial memory can be regulated by modulating the activity of hippocampal pyramidal cells via extrasynaptic a5GABAA receptors. In addition, neurons expressing a3GABAA receptors are instrumental in the processing of sensory motor information related to a schizophrenia endophenotype. The phenotypic analysis of transgenic knock-in and knock-out mice in which particular GABAA receptors were rendered insensitive to the effects of BZ while others were unaffected confirmed this speculation. Subsequently, subtype-specific GABAA ligands were developed that, for example, retained the anxiolytic effects of BZs but were devoid of their sedative effects. Therefore, it may be possible to develop effective anxiolytic compounds that have a much reduced side-effect profile compared with existing drug.

Palladium (II) Acetate/Base‐Catalyzed Intermolecular Coupling of Imidazo[1, 2‐a]pyridine Esters with Aryl Halides

AbstractThe combination of Cs2CO3 and Pd(OAc)2 enables the coupling of imidazo[1, 2‐a]pyridine esters and aryl halides, allowing an easy access to generate 3‐aryl imidazo[1, 2‐a]pyridine. The reaction pathway involves a Cs2CO3‐mediated ester hydrolysis followed by a palladium (II) acetate‐catalyzed decarboxylative arylation. The method is comparable with direct arylation of imidazo[1, 2‐a]pyridine and aryl halides. The synthetic utility of this relay reaction resulted in the generation of novel GABAA ligand and potent kinase inhibitor in high overall yields.

PP273—Effect of the gabaa ligands clobazam and clonazepam on the modulation of pain transmission in humans: A pk-pd study

PP273—Effect of the gabaa ligands clobazam and clonazepam on the modulation of pain transmission in humans: A pk-pd study

Use of the beam walking and drug discrimination models for determining intrinsic efficacy and selectivity of subtype selective GABA-A ligands in rodents

Use of the beam walking and drug discrimination models for determining intrinsic efficacy and selectivity of subtype selective GABA-A ligands in rodents

Neuroprotective effect of ceftriaxone on the penumbra in a rat venous ischemia model

ObjectiveGlutamate transporter-1 (GLT-1) maintains low concentrations of extracellular glutamate by removing glutamate from the extracellular space. It is controversial, however, whether upregulation of GLT-1 is neuroprotective under all ischemic/hypoxic conditions. Recently, a neuroprotective effect of preconditioning with a β-lactam antibiotic ceftriaxone (CTX) that increases expression of GLT-1 has been reported in animal models of focal ischemia. On the other hand, it is said that CTX does not play a neuroprotective role in an in vitro study. Thus, we examined the effect of CTX on ischemic injury in a rat model of two-vein occlusion (2VO). This model mimics venous ischemia during, e.g. tumor surgery, a clinical situation that is best suitable for pretreatment with CTX. MethodsCTX (100mg/kg, 200mg/kg per day) or vehicle (0.9% NaCl) was intraperitoneally injected into Wistar rats for 5days before venous ischemia (n=57). Then, animals were prepared for occlusion of two adjacent cortical veins (2VO) by photothrombosis with rose bengal that was followed by KCl-induced cortical spreading depression (CSD). Infarct volume was evaluated with hematoxylin and eosin (H&E) staining 2days after venous occlusion. [3H]MK-801, [3H]AMPA and [3H]Muscimol ligand binding were examined autoradiographically in additional two groups without 2VO (n=5/group). Animals were injected either with NaCl (vehicle) or CTX 200mg/kg for 5days in order to evaluate whether NMDA, AMPA and GABAA ligand binding densities were affected. ResultsCTX pretreatment reduced infarct volume compared to vehicle pretreatment (p<0.05). The effect of CTX pretreatment was attenuated by administration of the GLT-1 inhibitor, dihydrokainate (DHK) 30min before 2VO. CTX had no effect on the number of spontaneous spreading depressions after 2VO. Analysis of quantitative receptor autoradiography showed no statistically significant difference between rats after administration with CTX compared to control rats. ConclusionsPretreatment with CTX has neuroprotective potential without effect on NMDA, AMPA and GABAA receptor density and spontaneous spreading depression. This effect can be abolished by GLT-1 inhibition, indicating that upregulation of GLT-1 is an important mechanism for neuroprotective action in penumbra-like conditions, e.g. if neurosurgeons plan to occlude cerebral veins during tumor surgery.

A GABA-A ligand Gated ion Channel Screen - results and Best Practices

The GABAA receptors belong to a family of ligand-gated ion channels mediating fast synaptic transmission. They are drawing great attention in pharmaceutical field to their potential roles in the development of new therapeutics affecting anxiety, sleep disorders, and muscle relaxation. However, ligand gated ion channel screening has been hampered by the lack of suitable high throughput electrophysiology platforms. While some studies have shown that it is possible to record the GABA tail current and use that information during a screen, such methods have and inherently lower signal to noise ratio and cannot be used on faster desensitizers. Here we present a the use of a novel electrophysiology screening platform integrating a microfluidics network for the study of GABAAreceptor pharmacology. This platform features fast (<100ms) solution exchange coupled with simultaneous data recording. A novel assay could monitor GABA response in real time, and obtain a 3 point EC50 dose curve within 1 minute. The GABAA α1β3γ2 expressing HEK cells from Millipore were used for this study. The channel was targeted with agonists, including GABA and muscimol, inhibitors (picrotoxin, bicuculline, and gabazine), and positive modulators, including diazepam, zolpidem and chlordiazepoxide. The positive modulators produced concentration dependent augmentation of the GABA EC20 response. The pharmacology data determined using this method was consistent with the literature values obtained using other platforms. Statistical data for inter and intra-plate reproducibility, current stability, and Z-values, is used to validate this approach.

An investigation of non-selective and selective GABAA ligands using the beam walking assay in rats and mice

An investigation of non-selective and selective GABAA ligands using the beam walking assay in rats and mice

Novel GABAa ligands, inspired by nature

The major inhibitory neurotransmitter γ-amino butyric acid (GABA) exerts its inhibitory effect by binding to three different classes of receptors: GABAA-, GABAB- and GABAC receptors. GABAA- and GABAC are both ligand gated chloride ion channels, while GABAB are G-protein coupled receptors. GABAA receptors are important therapeutic targets for anxiety disorders, cognitive disorders, epilepsies, mood disorders, schizophrenia and sleep disorders. Due to the commercial interest, many compound classes, such as benzodiazepines, steroids, and barbiturates, are known to allosterically modify the effect of GABA by binding to distinct sites of the GABAA receptors.

In vivo saturation binding of GABA‐A receptor ligands to estimate receptor occupancy using liquid chromatography/tandem mass spectrometry

Typically, the dose-occupancy curves for GABA-A receptor ligands are determined using in vivo binding of [3H]flumazenil. This study describes in vivo binding experiments without the use of tracer ligands. Bound and free fractions were measured directly using a highly sensitive LC/MS/MS detection method after in vivo administration of the GABA-A ligands zolpidem, (RS)-zopiclone, L-838417 and flumazenil, to demonstrate affinity and saturation of the filter-retained, membrane-bound fraction. The in vivo binding of flumazenil and L-838417 both saturated around 200 nM, at a similar level to the specific binding of (S)-zopiclone after doses of the racemic zopiclone, using (R)-zopiclone to estimate non-specific binding. This saturable component represented an estimate of benzodiazepine binding sites available on GABA-A receptors in vivo (200 nM). Dose-occupancy curves were constructed to estimate the dose required to achieve 50% occupancy and matched estimates obtained with tracer methods. In contrast to tracer methods, this method is uniquely suitable to the demonstration of stereoselective binding of the (S)-isomer in vivo after doses of racemic zopiclone. These results demonstrate that the LC/MS/MS measurements of total drug concentrations typically used in early drug development can be adapted to provide information about receptor occupancy in vivo.

Characterisation and partial purification of the GABAB receptor from the rat cerebellum using the novel antagonist [[formula omitted]]CGP 62349

The novel GABAB receptor antagonist [3H]CGP 62349 binds rat cerebellar synaptosomal membranes with high affinity at a single population of sites (Kd=0.9 nM, Bmax=760 fmol/mg protein). Solubilisation with 1% Triton X-100/0.5 M NaCl/10% glycerol resulted in a marked increase in [3H]CGP 62349 binding (Kd=0.5 nM, Bmax=1285 fmol/mg protein). Competition of [3H]CGP 62349 binding with a range of GABAergic ligands yielded the same rank order at membrane-bound and soluble receptors: CGP 54626=CGP 55845≫GABA>(−)-baclofen>CGP 35348=CGP 36742. The GABAA ligand isoguvacine did not displace [3H]CGP 62349 binding. Partial purification of [3H]CGP 62349 binding sites was obtained by sucrose density centrifugation and a predominant protein in the peak binding fraction was recognised by an anti-GABAB receptor antibody and had a molecular weight similar to the recombinant expressed GABABR1a. These results demonstrate that [3H]CGP 62349 provides a useful additional tool for further characterisation of the pharmacology and biochemistry of the native GABAB receptor.

Arginine residue 120 of the human GABAA receptor alpha 1, subunit is essential for GABA binding and chloride ion current gating.

The effect of mutating the conserved amino acid residue arginine 120 to lysine in the GABAA receptor alpha 1 subunit was studied. In electrophysiological experiments, the arginine 120 lysine (R120K) mutation in the alpha 1 subunit, when co-expressed with beta 2 and gamma 2 subunits in Sf-9 insect cells, induces a 180-fold rightward shift of the GABA dose-response curve compared with wild type alpha 1 beta 2 gamma 2s GABAA receptors. The diazepam potentiation of GABA-gated chloride ion currents was not affected. The binding of the GABAA ligands [3H]muscimol and [3H]SR 95531 to alpha 1 (R120K) beta 2 gamma 2s GABAA receptors was abolished but the binding affinity of the benzodiazepine receptor ligand [3H]flunitrazepam was unchanged. These results suggest that the arginine residue 120 in the alpha 1 subtype of the GABAA receptor is essential for GABA binding.

530 Effect of zolpidem and zopiclone on cognitive and attentional function in healthy young volunteers — event-related potential study

The present study has addressed whether acute ethanol administration mediates changes in GABA release and GABAA receptor density in the rat mesolimbic system. In vivo microdialysis was performed in the ventral pallidum and between the ventral tegmental area and substantia nigra of conscious ethanol-naive rats. Extracellular levels of endogenous GABA were stable in both brain regions and not significantly affected following administration of 5 ml of 20% ethanol by gavage, despite clear overt behavioral signs of intoxication. Two hours following ethanol administration, animals were decapitated and the brains processed for autoradiography. Adjacent tissue sections were incubated with [3H]SR95531 or [3H]muscimol and the resulting autoradiograms quantified. Binding of both radioligands was significantly reduced in the striatum of rats treated with ethanol compared to vehicle (15 ± 2% for [3H]SR95531 and 33 ± 6% for [3H]muscimol). In contrast, ethanol had no effect on [3H]SR95531 binding in hippocampus, while that of [3H]muscimol was increased by 100 ± 30%. Ethanol had no effect on the binding of either radioligand in all other areas examined. Therefore, while acute ethanol had no effect on the release of GABA in either the ventral pallidum or ventral tegmental area, changes in the binding density of GABAA ligands were observed in selected brain regions.

Muscimol increases acetylcholine release by directly stimulating adult striatal cholinergic interneurons

Because GabaA ligands increase acetylcholine (ACh) release from adult striatal slices, we hypothesized that activation of GabaA receptors on striatal cholinergic interneurons directly stimulates ACh secretion. Fractional [ 3H]ACh release was recorded during perifusion of acutely dissociated, [ 3H]choline-labeled, adult male rat striata. The GabaA agonist, muscimol, immediately stimulated release maximally ∼300% with EC 50=∼1 μM. This action was enhanced by the allosteric GabaA receptor modulators, diazepam and secobarbital, and inhibited by the GabaA antagonist, bicuculline, by ligands for D2 or muscarinic cholinergic receptors or by low calcium buffer, tetrodotoxin or vesamicol. Membrane depolarization inversely regulated muscimol-stimulated secretion. Release of endogenous and newly synthesized ACh was stimulated in parallel by muscimol without changing choline release. Muscimol pretreatment inhibited release evoked by K + depolarization or by receptor-mediated stimulation with glutamate. Thus, GabaA receptors on adult striatal cholinergic interneurons directly stimulate voltage- and calcium-dependent exocytosis of ACh stored in vesamicol-sensitive synaptic vesicles. The action depends on the state of membrane polarization and apparently depolarizes the membrane in turn. This functional assay demonstrates that excitatory GabaA actions are not limited to neonatal tissues. GabaA-stimulated ACh release may be prevented in situ by normal tonic dopaminergic and muscarinic input to cholinergic neurons.

The Chemistry and Pharmacology of GABAA and GABAs Ligands

Abstract: The inhibitory neurotransmitter y-aminobutyrate (GA8A) elicits its physiological effects through interactions with two distinct classes of cell­ surface receptors in the central nervous system. The GA8AA receptor is a member of the superfamily of ligand-gate8 ion channels and is regulated allosterically at a number of distinct sites. The complex is a heterooligomeric integral membrane protein made up of a, p and y subunits. In addition to the site at which GA8A itself binds, radioligand binding studies have defined at least three other high-affinity binding sites on the receptor complex, the benzodiazepine receptor site, the picrotoxin site (which also recognizes cage-convulsants) and the barbiturate site. There is also evidence for an avermectin 81a site and a steroid site. The ligands which bind at the various sites on this complex are chemically quite diverse and mediate a wide variety of pharmacological actions and have a continuum of intrinsic activity ranging from full agonist (anxiolytic, hypnotic, and anticonvulsant agents) through antagonist to inverse agonists (proconvulsants and anxiogenic agents). The GA8A 8 receptor displays a neuronal distribution and pharmacology distinct from that of the GA8AA receptor. The GA8A 8 receptor is a metabotropic receptor which is associated with an inhibitor GTP-binding protein regulating adenylate cyclase, calcium and potassium channels. 8aclofen and phaclofen are selective agonist and antagonist for GA8A8. In this review we will present the most current thinking regarding the importance of the various receptor subtypes, the syntheses of the various ligands that modulate GA8AA and GA8A 8 receptors and their pharmacology

Zinc Inhibition of t‐[3H]Butylbicycloorthobenzoate Binding to the GABAA Receptor Complex

The effect of Zn2+ on t-[3H]butylbicycloorthobenzoate ([3H]TBOB) binding to the GABAA receptor complex was studied autoradiographically in rat brain. Zn2+ inhibited [3H]TBOB binding in a dose-dependent manner at physiological concentrations. Saturation analysis revealed noncompetitive inhibition in various brain regions. The inhibitory effect of Zn2+ had regional heterogeneity; regions showing the greatest inhibition of [3H]TBOB binding were cortical laminae I-III, most areas of hippocampus, striatum, septum, and cerebellar cortex. Regions with relatively less inhibition of [3H]TBOB binding included cortical laminae V-VI, thalamus, superior colliculus, inferior colliculus, and central gray matter. The effect of Zn2+ and those of other GABAA ligands, such as benzodiazepines, bicuculline, isoguvacine, and picrotoxin, on [3H]TBOB binding seemed to be additive. Ni2+, Cd2+, and Cu2+ also inhibited [3H]TBOB binding with a regional heterogeneity similar to that produced by Zn2+. These results are consistent with Zn2+ acting at the previously detected recognition site on the GABAA receptor complex, distinct from the picrotoxin, GABA, and benzodiazepine sites. The regional heterogeneity of the Zn2+ effect may reflect differential regional distribution of GABAA receptor subtypes among brain regions. Other divalent cations probably act at the Zn2+ binding site.

Studies on the neuronal systems involved in the oestrogen-negative feedback effect on gonadotrophin releasing hormone neurons in the ewe.

Oestrogen can act on the brain to exert negative and positive feedback effects on the secretion of gonadotrophin releasing hormone (GnRH), but this cannot be effected through the GnRH cells themselves because they have no oestrogen receptors. We have used intraventricular injection of agonists and antagonists of various transmitters to determine which systems are involved in the control of GnRH by steroid hormones. We have also developed a model for the injection of drugs directly into the medial pre-optic area of the sheep brain to study the action on the GnRH cell bodies. Opioid systems seem to be involved in the mediation of the feedback effects of progesterone, but not oestrogen. Noradrenaline appears to be involved in the negative feedback regulation of GnRH secretion during the anoestrous period. Thus, under the strong negative influence of oestrogen at this time GnRH/luteinizing hormone (LH) secretion is reduced but can be restored by the preoptic micro-injection of noradrenaline. Gamma-amino-butyric acid (GABA) agonists and antagonists inhibit LH secretion when injected into the medial pre-optic region of the hypothalamus of ovariectomized ewes with or without oestrogen treatment. We have no evidence that GABA is involved in the negative feedback regulation of GnRH, since neither agonists nor antagonists will reverse the effects of steroid feedback. There appears to be a shift in the GABA receptor subtype function with season; in the breeding season only GABAA ligands are effective, whereas during the non-breeding season both GABAA and GABAB type ligands will affect LH secretion. This suggests that the seasonal shift in responsiveness to the negative feedback effects of oestrogen involves a shift in the function of GABA receptor subtypes.