Coronaridine congeners potentiate GABAA receptors and induce sedative activity in mice in a benzodiazepine-insensitive manner.

To determine whether (+)-catharanthine induces sedative- or anxiolytic/anxiogenic-like activity in male mice, proper animal paradigms were used. The results showed that (+)-catharanthine induces sedative-like activity in the 63-72 mg/Kg dose range in a flumazenil-insensitive manner, but neither this effect nor anxiolytic/anxiogenic-like activity was observed at lower doses. To determine the underlying molecular mechanism of the sedative-like activity, electrophysiological and radioligand binding experiments were performed with (+)-catharanthine and (±)-18-methoxycoronaridine [(±)-18-MC] on GABAA (GABAARs) and glycine receptors (GlyRs). Coronaridine congeners both activated and potentiated a variety of human (h) GABAARs, except hρ1. (+)-Catharanthine-induced potentiation followed this receptor selectivity (EC50's in µM): hα1ß2 (4.6 ± 0.8) > hα2ß2γ2 (12.6 ± 3.8) ~ hα1ß2γ2 (14.4 ± 4.6) indicating that both α1 and α2 are equally important, whereas γ2 is not necessary. (+)-Catharanthine was >2-fold more potent and efficient than (±)-18-MC at hα1ß2γ2. (+)-Catharanthine also potentiated, whereas (±)-18-MC inhibited, hα1 GlyRs with very low potency. Additional [3H]-flunitrazepam competition binding experiments using rat cerebellum membranes clearly demonstrated that these ligands do not bind to the benzodiazepine site. This is supported by the observed activity at hα1ß2 (lacking the BDZ site) and similar effects between α1- and α2-containing GABAARs. Our study shows, for the first time, that (+)-catharanthine induced sedative-like effects in mice, and coronaridine congeners potentiated human α1ß2γ2, α1ß2, and hα2ß2γ2, but not ρ1, GABAARs, both in a benzodiazepine-insensitive fashion, whereas only (+)-catharanthine slightly potentiated GlyRs.

Postsynaptic plasticity of GABAergic synapses.

The flexibility of neuronal networks is believed to rely mainly on the plasticity of excitatory synapses. However, like their excitatory counterparts, inhibitory synapses also undergo several forms of synaptic plasticity. This review examines recent advances in the understanding of the molecular mechanisms leading to postsynaptic GABAergic plasticity. Specifically, modulation of GABAA receptor (GABAAR) number at postsynaptic sites plays a key role, with the interaction of GABAARs with the scaffold protein gephyrin and other postsynaptic scaffold/regulatory proteins having particular importance. Our understanding of these molecular interactions are progressing, based on recent insights into the processes of GABAAR lateral diffusion, gephyrin dynamics, and gephyrin nanoscale organization. This article is part of the special issue entitled 'Mobility and trafficking of neuronal membrane proteins'.

Steady-state activation of the high-affinity isoform of the α4ß2δ GABAA receptor.

Activation of GABAA receptors consisting of α4, ß2 (or ß3), and δ subunits is a major contributor to tonic inhibition in several brain regions. The goal of this study was to analyze the function of the α4ß2δ receptor in the presence of GABA and other endogenous and clinical activators and modulators under steady-state conditions. We show that the receptor has a high constitutive open probability (~0.1), but is only weakly activated by GABA that has a maximal peak open probability (POpen,peak) of 0.4, taurine (maximal POpen,peak = 0.4), or the endogenous steroid allopregnanolone (maximal POpen,peak = 0.2). The intravenous anesthetic propofol is a full agonist (maximal POpen,peak = 0.99). Analysis of currents using a cyclic three-state Resting-Active-Desensitized model indicates that the maximal steady-state open probability of the α4ß2δ receptor is ~0.45. Steady-state open probability in the presence of combinations of GABA, taurine, propofol, allopregnanolone and/or the inhibitory steroid pregnenolone sulfate closely matched predicted open probability calculated assuming energetic additivity. The results suggest that the receptor is active in the presence of physiological concentrations of GABA and taurine, but, surprisingly, that receptor activity is only weakly potentiated by propofol.

[Association between Elimination Half-life of Benzodiazepines and Falls in the Elderly: A Meta-analysis of Observational Studies].

Benzodiazepine receptor agonists (BZDRAs) have been associated with an increased risk of falls in the elderly. However, the association between the elimination half-life (t1/2) of BZDRAs and the difference between benzodiazepines (BZDs) and non-benzodiazepines (Z-drugs) has not been clarified. By conducting a meta-analysis of observational studies, we compared the risk of falls with respect to 1) short-acting BZDRAs (t1/2<12 h) vs. long-acting BZDRAs (t1/2≥12 h) and 2) BZDs vs. Z-drugs in elderly patients. Data were retrieved from MEDLINE, the Cochrane Library, and Igaku Chuo Zasshi. In total, 13 observational studies from 12 articles were included in our study (short-acting BZDRAs, n=12; long-acting BZDRAs, n=9; BZDs, n=13; Z-drugs, n=7). The risk of falls was significantly increased by the use of short-acting BZDRAs [Odds ratio (OR) (95% Confidence interval (CI)): 2.00 (1.46-2.73)], long-acting BZDRAs [OR (95%CI): 2.16 (1.61-2.89)], BZDs [OR (95%CI): 1.67 (1.31-2.13)], and Z-drugs [OR (95%CI): 2.42 (1.35-4.34)] compared to the risk in BZDRAs non-users. The increased risk of falls in elderly patients was similar in each group and unrelated to t1/2. This study suggested that all BZDRAs including Z-drugs should be avoided in elderly patients.

In vitro and in vivo GABAA Receptor Interaction of the Propanidid Metabolite 4-(2-[Diethylamino]-2-Oxoethoxy)-3-Methoxy-Benzeneacetic Acid.

BACKGROUND: Propanidid is a γ-aminobutyric acid type A (GABAA) receptor agonist general anesthetic and its primary metabolite is 4-(2-[diethylamino]-2-oxoethoxy)-3-methoxy-benzeneacetic acid (DOMBA). Despite having a high water solubility at physiologic pH that might predict low-affinity GABAA receptor interactions, DOMBA is reported to have no effect on GABAA receptor currents, possibly because the DOMBA concentrations studied were simply insufficient to modulate GABAA receptors. Our objectives were to measure the propanidid and DOMBA concentration responses on -GABAA receptors and to measure the behavioral responses of DOMBA in mice at concentrations that affect GABAA receptor currents in vitro. METHODS: GABAA receptors were expressed in oocytes using clones for the human GABAA α1, ß2 and γ2s subunits. The effects of DOMBA (0.2-10 mmol/L) and propanidid (0.001-1 mmol/L) on oocyte GABAA currents were studied using standard 2-electrode voltage clamp techniques. Based on in vitro results, 6 mice received -DOMBA 32 mg intraperitoneal and were observed for occurrence of neurologic effects and DOMBA plasma concentration was measured by liquid chromatography tandem mass spectrometry. RESULTS: DOMBA both directly activates GABAA receptors and antagonizes its GABA-mediated opening in a concentration-dependent manner at concentrations between 5-10 and 0.5-10 mmol/L respectively. In vivo, DOMBA produced rapid onset sedation at plasma concentrations that correlate with direct GABAA receptor activation. CONCLUSION: DOMBA modulation of GABAA receptors is associated with sedation in mice. Metabolites of propanidid analogues currently in development may similarly modulate GABAA, and impaired elimination of these metabolites could produce clinically relevant neurophysiologic effects.

A mutational and molecular dynamics study of the cys-loop GABA receptor Hco-UNC-49 from Haemonchus contortus: Agonist recognition in the nematode GABA receptor family.

The UNC-49 receptor is a unique nematode γ-aminobutyric acid (GABA)-gated chloride channel that may prove to be a novel target for the development of nematocides. Here we have characterized various charged amino acid residues in and near the agonist binding site of the UNC-49 receptor from the parasitic nematode Haemonchus contorts. Utilizing the Caenorhabditis elegans GluCl crystal structure as a template, a model was generated and various charged residues [D83 (loop D), E131 (loop A), H137 (pre-loop E), R159 (Loop E), E185 (Loop B) and R241 (Loop C)] were investigated based on their location and conservation. These residues may contribute to structure, function, and molecular interactions with agonists. It was found that all residues chosen were important for receptor function to varying degrees. Results of the mutational analysis and molecular simulations suggest that R159 may be interacting with D83 by an ionic interaction that may be crucial for general GABA receptor function. We have used the results from this study as well as knowledge of residues involved in GABA receptor binding to identify sequence patterns that may assist in understanding the function of lesser known GABA receptor subunits from parasitic nematodes.

Probing the molecular basis for affinity/potency- and efficacy-based subtype-selectivity exhibited by benzodiazepine-site modulators at GABAA receptors.

The extracellular α(+)/γ2(-) interface in the α1,2,3,5ßγ2 GABAA receptor harbours the allosteric binding site targeted by benzodiazepines and newer generations of subtype-selective modulators. We have probed the molecular determinants for the affinity/potency-based α1-preference exhibited by the hypnotic zolpidem (Ambien®, Stilnox®) and the efficacy-based α3-over-α1 selectivity displayed by the analgesic NS11394. Binding affinities and functional properties of the modulators were characterized at wild-type, concatenated, mutant and chimeric α1,3ß2γ2S receptors expressed in tsA201 cells and Xenopus oocytes by [3H]flumazenil binding and two-electrode voltage clamp electrophysiology. Substitution of Gly201 in α1 with the corresponding Glu in α3 completely eliminated the α1-over-α3 preference exhibited by zolpidem. In contrast, the reverse α3-E225G mutation did not yield corresponding increases in the binding affinity or modulatory potency of zolpidem at α3ß2γ2S, and two additional molecular elements in the extracellular domain of the α-subunit were found also to contribute to its α1-preference. Interestingly, the α1-Gly201/α3-Glu225 residue was also a key determinant of the efficacy-based α3-over-α1 selectivity exhibited by NS11394, and a pronounced correlation existed between the side-chain bulkiness of this residue and the modulatory efficacy of NS11394 at the receptor. The subtype-selectivity determinants identified for zolpidem and NS11394 were found also to apply in different degrees to the α1-preferring modulator indiplon and the α3-over-α1 selective modulator L-838,417, respectively. In conclusion, the molecular origins of subtype-selectivity exhibited by benzodiazepine-site modulators at the α1,2,3,5ßγ2 GABAA receptor seem more complex than previously appreciated, and the importance of the α1-Gly201/α3-Glu225 residue for both potency- and efficacy-based subtype-selective modulation through this site is likely to be rooted in different molecular mechanisms.

Identification of chemically diverse GABAA agonists as potential anti-epileptic agents using structure-guided virtual screening, ADMET, quantum mechanics and clinical validation through off-target analysis.

Development of resistance against existing anti-epileptic drugs has alarmed the scientific innovators to find novel potential chemical starting points for the treatment of epilepsy and GABAA inhibition is a promising drug target strategy against epilepsy. The crystal structure of a subtype-selective ß3-homopentameric ligand-gated ion channel of GABAA receptor has been used for the first time for screening the Asinex library for discovery of GABAA agonists as potential anti-epileptic agents. Co-crystallized ligand established the involvement of part of the ß7-ß8 loop (Glu155 and Tyr157) and ß9-ß10 loop (Phe200 and Tyr205) residues as the crucial amino acids in effective binding, an essential feature, being hydrogen bond or ionic interaction with Glu155 residue. Top ranked hits were further subjected to binding energy estimation, ADMET analysis and ligand efficiency matric calculations as consecutive filters. About 19 compounds qualifying all parameters possessed interaction of one positively charged group with Glu155 with good CNS drug-like properties. Simulation studies were performed on the apo protein, its complex with co-crystallized ligand and the best hit qualifying all screening parameters. The best hit was also analyzed using Quantum mechanical studies, off-target analysis and hit modification. The off-target analysis emphasized that these agents did not have any other predicted side-effects.

Path to the Desensitized State of Ligand-Gated Ion Channels: Why Are Inhibitory and Excitatory Receptors Different?

The family of pentameric ligand-gated ion channels (pLGICs) includes both inhibitory and excitatory receptors. Electrophysiological methods have explored the time-dependent ion currents induced by their neurotransmitter agonists. Kinetic modeling requires a minimum of three conformational states: resting, active, and desensitized. However, current traces of inhibitory and excitatory pLGICs differ substantially. Reproducing their basic features requires different state connectivity: whether the desensitized state is accessed from the resting or active state. It is surprising that a property as fundamental as state connectivity would differ within the same family. So, we explore the possibility that the connectivity is the same, but corresponding states differ in function: Analogous states on the free energy landscape have similar structure, but differ in ion conductivity, free energies, and agonist binding affinities. This hypothesis is tested using a kinetic model in which agonist and anesthetics modulate the receptor free energy landscape by adsorbing to the membrane in which the receptor is embedded. It was previously shown that even with only three states, the complex behavior observed for GABAAR is reproduced, including its response to anesthetics. It is demonstrated here that this hypothesis accounts for an important difference between inhibitory and excitatory receptors: their opposite responses to inhalation anesthetics.

Discovery of the first low-shift positive allosteric modulators for the muscarinic M1 receptor.

Positive modulation of the muscarinic M1-receptor has for a long time attracted scientists and drug developers for the potential treatment of Alzheimer's disease or Schizophrenia. The precognitive potential of M1 activation has however not been clinically demonstrated as a result of side effects associated both with agonists and positive allosteric modulators (PAM's) of the M1-receptor. To avoid excessive activation of the M1-receptor we have designed a new screening format and developed the first low-shift positive allosteric modulators for the M1 receptor. Low-shift PAM's offer the potential of "use-dependent" attenuation of transmitter-signaling while avoiding pseudo-agonistic behavior in vivo as a common limitation of the so far described high-shift PAM's. With these novel M1-PAM's, the M1 receptor is potentially the first GPCR for which both, high- and low shift PAM's have become available.

Arginine derivatives of dicarboxylic acids from the parotid gland secretions of common toad Bufo bufo-New agonists of ionotropic γ-aminobutyric acid receptors.

Compounds activating γ-aminobutyric acid type A receptor were isolated from the toad Bufo bufo venom as a result of chromatographic separation. Analysis of the structure of these compounds by mass spectrometry and nuclear magnetic resonance showed that they are arginine derivatives of dicarboxylic acids and represent suberylarginine, pimeloylarginine, and adipoylarginine.

Ester to amide substitution improves selectivity, efficacy and kinetic behavior of a benzodiazepine positive modulator of GABAA receptors containing the α5 subunit.

We have synthesized and characterized MP-III-022 ((R)-8-ethynyl-6-(2-fluorophenyl)-N,4-dimethyl-4H-benzo[f]imidazo[1,5-a][1,4]diazepine-3-carboxamide) in vitro and in vivo as a binding- and efficacy-selective positive allosteric modulator of GABAA receptors containing the α5 subunit (α5GABAARs). By approximation of the electrophysiological responses which the estimated free rat brain concentrations can induce, we demonstrated that convenient systemic administration of MP-III-022 in the dose range 1-10mg/kg may result in a selective potentiation, over a wide range from mild to moderate to strong, of α5ßγ2 GABAA receptors. For eliciting a comparable range of potentiation, the widely studied parent ligand SH-053-2'F-R-CH3 containing an ester moiety needs to be administered over a much wider dose range (10-200mg/kg), but at the price of activating non-α5 GABAARs as well as the desired α5GABAARs at the highest dose. At the dose of 10mg/kg, which elicits a strong positive modulation of α5GABAARs, MP-III-022 caused mild, but significant muscle relaxation, while at doses 1-10mg/kg was devoid of ataxia, sedation or an influence on the anxiety level, characteristic for non-selective benzodiazepines. As an amide compound with improved stability and kinetic properties, MP-III-022 may represent an optimized tool to study the influence of α5GABAARs on the neuronal pathways related to CNS disorders such as schizophrenia, Alzheimer's disease, Down syndrome or autism.

Hippocampal-dependent memory in the plus-maze discriminative avoidance task: The role of spatial cues and CA1 activity.

The plus-maze discriminative avoidance task (PMDAT) has been used to investigate interactions between aversive memory and an anxiety-like response in rodents. Suitable performance in this task depends on the activity of the basolateral amygdala, similar to other aversive-based memory tasks. However, the role of spatial cues and hippocampal-dependent learning in the performance of PMDAT remains unknown. Here, we investigated the role of proximal and distal cues in the retrieval of this task. Animals tested under misplaced proximal cues had diminished performance, and animals tested under both misplaced proximal cues and absent distal cues could not discriminate the aversive arm. We also assessed the role of the dorsal hippocampus (CA1) in this aversive memory task. Temporary bilateral inactivation of dorsal CA1 was conducted with muscimol (0.05 µg, 0.1 µg, and 0.2 µg) prior to the training session. While the acquisition of the task was not altered, muscimol impaired the performance in the test session and reduced the anxiety-like response in the training session. We also performed a spreading analysis of a fluorophore-conjugated muscimol to confirm selective inhibition of CA1. In conclusion, both distal and proximal cues are required to retrieve the task, with the latter being more relevant to spatial orientation. Dorsal CA1 activity is also required for aversive memory formation in this task, and interfered with the anxiety-like response as well. Importantly, both effects were detected by different parameters in the same paradigm, endorsing the previous findings of independent assessment of aversive memory and anxiety-like behavior in the PMDAT. Taken together, these findings suggest that the PMDAT probably requires an integration of multiple systems for memory formation, resembling an episodic-like memory rather than a pure conditioning behavior. Furthermore, the concomitant and independent assessment of emotionality and memory in rodents is relevant to elucidate how these memory systems interact during aversive memory formation. Thus, the PMDAT can be useful for studying hippocampal-dependent memory when it involves emotional content.

The α1, α2, α3, and γ2 subunits of GABAA receptors show characteristic spatial and temporal expression patterns in rhombencephalic structures during normal human brain development.

γ-Aminobutyric acid (GABA) is the most abundant inhibitory neurotransmitter in adult mammalian brain, mediating its actions chiefly via a pentameric chloride ion channel, the GABAA receptor. Nineteen different subunits (α1-6, ß1-3, γ1-3, δ, ε, π, θ, ρ1-3) can give rise to multiple receptor subtypes that are the site of action of many clinically important drugs. In the developing brain, however, GABAA receptors mediate excitatory actions due to an increased chloride concentration within neurons and seem to control cell proliferation, migration, differentiation, synapse maturation, and cell death. Little is known about the distribution of single subunits in the human brain. Here we describe developmental changes in the immunohistochemical distribution of four subunits (α1, α2, α3, and γ2) in the human rhombencephalon. The γ2 was the most abundant subunit in all rhombencephalic structures during development and in adults, whereas α subunits showed a structure- and age-characteristic distribution. The α1 was expressed prenatally in the molecular and Purkinje cell layer, but only postnatally in the granule cell layer and the dentate nucleus. Expression was completely absent in the inferior olivary nucleus. The α2 gradually increased during development, showing some layer specificity in the cerebellar cortex. The α3-immunoreactivity in the cerebellar cortex was relatively weak, but it was abundantly observed in different cell populations in the subcortical cerebellar structures. Structure- and age-characteristic colocalization between subunits during development suggests differences in GABAA receptor composition. Interestingly, subunit expression in several instances differed between human and rodent brain, underlining the importance of immunohistochemical studies in humans.

Investigating the Discriminatory Power of BCS-Biowaiver in Vitro Methodology to Detect Bioavailability Differences between Immediate Release Products Containing a Class I Drug.

The purpose of this work is to investigate the discriminatory power of the Biopharmaceutics Classification System (BCS)-biowaiver in vitro methodology, i.e., to investigate if a BCS-biowaiver approach would have detected the Cmax differences observed between two zolpidem tablets and to identify the cause of the in vivo difference. Several dissolution conditions were tested with three zolpidem formulations: the reference (Stilnox), a bioequivalent formulation (BE), and a nonbioequivalent formulation (N-BE). Zolpidem is highly soluble at pH 1.2, 4.5, and 6.8. Its permeability in Caco-2 cells is higher than that of metoprolol and its transport mechanism is passive diffusion. None of the excipients (alone or in combination) showed any effect on permeability. All formulations dissolved more than 85% in 15 min in the paddle apparatus at 50 rpm in all dissolution media. However, at 30 rpm the nonbioequivalent formulation exhibited a slower dissolution rate. A slower gastric emptying rate was also observed in rats for the nonbioequivalent formulation. A slower disintegration and dissolution or a delay in gastric emptying might explain the Cmax infra-bioavailability for a highly permeable drug with short half-life. The BCS-biowaiver approach would have declared bioequivalence, although the in vivo study was not conclusive but detected a 14% mean difference in Cmax that precluded the bioequivalence demonstration. Nonetheless, these findings suggest that a slower dissolution rate is more discriminatory and that rotation speeds higher than 50 rpm should not be used in BCS-biowaivers, even if a coning effect occurs.

A molecular characterization of the agonist binding site of a nematode cys-loop GABA receptor.

BACKGROUND AND PURPOSE: Cys-loop GABA receptors represent important targets for human chemotherapeutics and insecticides and are potential targets for novel anthelmintics (nematicides). However, compared with insect and mammalian receptors, little is known regarding the pharmacological characteristics of nematode Cys-loop GABA receptors. Here we have investigated the agonist binding site of the Cys-loop GABA receptor UNC-49 (Hco-UNC-49) from the parasitic nematode Haemonchus contortus. EXPERIMENTAL APPROACH: We used two-electrode voltage-clamp electrophysiology to measure channel activation by classical GABA receptor agonists on Hco-UNC-49 expressed in Xenopus laevis oocytes, along with site-directed mutagenesis and in silico homology modelling. KEY RESULTS: The sulphonated molecules P4S and taurine had no effect on Hco-UNC-49. Other classical Cys-loop GABAA receptor agonists tested on the Hco-UNC-49B/C heteromeric channel had a rank order efficacy of GABA > trans-4-aminocrotonic acid > isoguvacine > imidazole-4-acetic acid (IMA) > (R)-(-)-4-amino-3-hydroxybutyric acid [R(-)-GABOB] > (S)-(+)-4-amino-3-hydroxybutyric acid [S(+)-GABOB] > guanidinoacetic acid > isonipecotic acid > 5-aminovaleric acid (DAVA) (partial agonist) > ß-alanine (partial agonist). In silico ligand docking revealed some variation in binding between agonists. Mutagenesis of a key serine residue in binding loop C to threonine had minimal effects on GABA and IMA but significantly increased the maximal response to DAVA and decreased twofold the EC50 for R(-)- and S(+)-GABOB. CONCLUSIONS AND IMPLICATIONS: The pharmacological profile of Hco-UNC-49 differed from that of vertebrate Cys-loop GABA receptors and insect resistance to dieldrin receptors, suggesting differences in the agonist binding pocket. These findings could be exploited to develop new drugs that specifically target GABA receptors of parasitic nematodes.

Different Extracts of Zingiber officinale Decrease Enterococcus faecalis Infection in Galleria mellonella

Dried, fresh and glycolic extracts of Zingiber officinale were obtained to evaluate the action against G. mellonella survival assay against Enterococcus faecalis infection. Eighty larvae were divided into: 1) E. faecalis suspension (control); 2) E. faecalis + fresh extract of Z. officinale (FEO); 3) E. faecalis + dried extract of Z. officinale (DEO); 4) E. faecalis + glycolic extract of Z. officinale (GEO); 5) Phosphate buffered saline (PBS). For control group, a 5 μL inoculum of standardized suspension (107 cells/mL) of E. faecalis (ATCC 29212) was injected into the last left proleg of each larva. For the treatment groups, after E. faecalis inoculation, the extracts were also injected, but into the last right proleg. The larvae were stored at 37 °C and the number of dead larvae was recorded daily for 168 h (7 days) to analyze the survival curve. The larvae were considered dead when they did not show any movement after touching. E. faecalis infection led to the death of 85% of the larvae after 168 h. Notwithstanding, in treatment groups with association of extracts, there was an increase in the survival rates of 50% (GEO), 61% (FEO) and 66% (DEO) of the larvae. In all treatment groups, the larvae exhibited a survival increase with statistically significant difference in relation to control group (p=0.0029). There were no statistically significant differences among treatment groups with different extracts (p=0.3859). It may be concluded that the tested extracts showed antimicrobial activity against E. faecalis infection by increasing the survival of Galleria mellonella larvae.

Extratos seco, fresco e glicólico de Zingiber officinale foram obtidos para avaliar suas ações por meio de ensaio de sobrevivência em G. mellonella contra infecção por Enterococcus faecalis. Oitenta larvas foram divididas em: 1) Suspensão de E. faecalis (controle); 2) E. faecalis + extrato fresco de Z. officinale (FEO); 3) E. faecalis + extrato seco de Z. officinale (DEO); 4) E. faecalis + extrato glicólico de Z. officinale (GEO); 5) Solução tampão fosfato salina (PBS). Para o grupo de controle, 5 µL de inóculo de suspensão padronizada (107 células/mL) de E. faecalis (ATCC 29212) foi injetado na última proleg esquerda de cada lagarta. Para os grupos com tratamento, após a injeção de E. faecalis, os extratos foram injetados na última proleg direita. Após as injeções, as lagartas foram armazenadas a 37 °C e o número de animais mortos foi registrado diariamente em 168 h (7 dias) para analisar a curva de sobrevivência. As lagartas foram consideradas mortas quando elas não mostraram qualquer movimento após o toque. A infecção por E. faecalis levou à morte de 85% das lagartas após 168 h. Não obstante, nos grupos de tratamento com associação dos extratos, houve um aumento nas taxas de sobrevivência de 50% (GEO), 61% (FEO) e 66% (DEO) das lagartas. Em todos os grupos com tratamento, as lagartas apresentaram um aumento na sobrevivência, com diferença estatisticamente significativa em relação ao grupo controle (p=0,0029). Não houve diferença estatisticamente significativa entre os tratamentos com os diferentes extratos (p=0,3859). Pode concluir-se que os extratos testados mostraram atividade antimicrobiana contra a infecção por E. faecalis, aumentando a sobrevivência das lagartas de G. mellonella.

Functional characterization of the 1,5-benzodiazepine clobazam and its major active metabolite N-desmethylclobazam at human GABA(A) receptors expressed in Xenopus laevis oocytes.

The 1,5-benzodiazepine clobazam is indicated for the adjunctive treatment of seizures associated with Lennox-Gastaut syndrome in patients 2 years of age or older in the United States, and for treatment of anxiety and various forms of epilepsy elsewhere. Clobazam has been reported to exhibit different in vivo adverse effects and addiction liability profile than the classic 1,4-benzodiazepines. In this study, it was investigated whether the in vitro pharmacological properties of clobazam and its major active metabolite N-desmethylclobazam could explain some of these clinical differences. The functional properties of the two 1,5-benzodiazepines were characterized at the human γ-aminobutyric acid type A receptor (GABA(A)R) subtypes α1ß2γ(2S), α2ß2γ(2S), α3ß2γ(2S), α5ß2γ(2S) and α6ß2δ expressed in Xenopus laevis oocytes by use of two-electrode voltage-clamp electrophysiology and compared to those exhibited by the 1,4-benzodiazepine clonazepam. All three compounds potentiated GABA EC20-evoked responses through the α(1,2,3,5)ß2γ(2S) GABA(A)Rs in a reversible and concentration-dependent manner, with each displaying similar EC50 values at the four subtypes. Furthermore, the degrees of potentiation of the GABA EC20 currents through the four receptors mediated by saturating modulator concentrations did not differ substantially for any of the three benzodiazepines. The three compounds were substantially less potent (200-3900 fold) as positive allosteric modulators at the α6ß2δ GABA(A)R than at the α(1,2,3,5)ß2γ(2S) receptors. Interestingly, however, clobazam and especially N-desmethylclobazam were highly efficacious potentiators of α6ß2δ receptor signaling. Although this activity component is unlikely to contribute to the in vivo effects of clobazam/N-desmethylclobazam, the 1,5-benzodiazepine could constitute an interesting lead for novel modulators targeting this low-affinity binding site in GABAARs. In conclusion, the non-selective modulation exerted by clobazam, N-desmethylclobazam and clonazepam at the α1ß2γ(2S), α2ß2γ(2S), α3ß2γ(2S) and α5ß2γ(2S) GABA(A)Rs indicate that the observed clinical differences between clobazam and 1,4-benzodiazepines are likely to arise from factors other than their respective pharmacological properties at the GABA(A)Rs as investigated here.

Effect of synthetic steroids on GABAA receptor binding in rat brain.

Neuroactive steroids, like allopregnanolone (A) and pregnanolone (P), bind to specifics sites on the GABAA receptor complex and modulate receptor function. They are capable to inhibit or stimulate the binding of GABAA receptor-specific ligands, like t-butyl-bicyclophosphorothionate, flunitrazepam and muscimol. We have previously characterized a set of oxygen-bridged synthetic steroids (SS) analogs to A or P using synaptosomes. Considering that the subunit composition of the GABAA receptor throughout the central nervous system affects the magnitude of the modulation of the GABAA receptor by NAS, we evaluated the action of two selected SS, in brain sections containing the cerebral cortex (CC) and hippocampus (HC) using quantitative receptor autoradiography. Both SS affected the binding of the three ligands in a similar way to A and P, with some differences on certain CC layers according to the ligand used. One of the SS, the 3α-hydroxy-6,19-epoxypregn-4-ene-20-one (compound 5), behaved similarly to the natural neuroactive steroids. However, significant differences with compound 5 were observed on the HC CA2 region, making it steroid suitable for a specific action. Those differences may be related to structural conformation of the SS and the subunits' composition present on the receptor complex.

5-(Piperidin-4-yl)-3-hydroxypyrazole: A novel scaffold for probing the orthosteric γ-aminobutyric acid type A receptor binding site.

A series of bioisosteric N1- and N2 -substituted 5-(piperidin-4-yl)-3-hydroxypyrazole analogues of the partial GABAA R agonists 4-PIOL and 4-PHP have been designed, synthesized, and characterized pharmacologically. The unsubstituted 3-hydroxypyrazole analogue of 4-PIOL (2 a; IC50 ∼300 µM) is a weak antagonist at the α1 ß2 γ2 GABAA R, whereas substituting the N1- or N2- position with alkyl or aryl substituents resulted in antagonists with binding affinities in the high nanomolar to low micromolar range at native rat GABAA Rs. Docking studies using a α1 ß2 γ2 GABAA R homology model along with the obtained SAR indicate that the N1 -substituted analogues of 4-PIOL and 4-PHP, 2 a-k, and previously reported 3-substituted 4-PHP analogues share a common binding mode to the orthosteric binding site in the receptor. Interestingly, the core scaffold of the N2 -substituted analogues of 4-PIOL and 4-PHP, 3 b-k, are suggested to flip 180° thereby adapting to the binding pocket and addressing a cavity situated above the core scaffold.