Involvement of opioidergic and GABAergic systems in the anti-nociceptive activity of the methanolic extract of Cuscuta Epithymum Murr. in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Cuscuta epithymum Murr. (CE) is a parasitic plant used as a traditional medicine to treat various diseases such as muscle and joint pains and headache different parts of the world, Europe in the north, Asia in the east. AIM OF THE STUDY: In this study, we aimed to investigate the anti-nociceptive effect of the methanolic extract of the aerial parts of CE and its probable mechanism(s) in mice. MATERIALS AND METHODS: The anti-nociceptive activity of different doses of CE methanolic extract (2.5, 5, 10, 25, 50 and 100 mg/kg, i.p.) was assessed using tail flick, formalin and writhing tests. Morphine (5 mg/kg, s.c.) was used as positive control drug. The possible mechanisms were evaluated by using naloxone (4 mg/kg, i.p.), ondansetron (4 mg/kg, i.p.), picrotoxin (0.6 mg/kg, i.p.) and MK-801 (0.03 mg/kg, i.p.). RESULTS: GC-MS analysis indicated that one of the main components of CE extract was terpenoid compounds. The CE extract (25-100 mg/kg), like morphine, reduced tail flick latency and nociceptive response in both phases of the formalin test. We also observed that the extract significantly decreased the number of abdominal contractions dose-dependently from 5 to 100 mg/kg. The results of tail flick and the first phase of formalin test proved that unlike ondansetron and MK-801, naloxone and picotroxin were able to reverse the anti-nociceptive effect of CE extract. CONCLUSION: Our observations showed the anti-nociceptive potential of the CE extract, which may be mediated by opioidergic and GABAergic systems.

Molecular Dynamic Simulations to Probe Stereoselectivity of Tiagabine Binding with Human GAT1.

The human gamma aminobutyric acid transporter subtype 1 (hGAT1) located in the nerve terminals is known to catalyze the neuronal function by the electrogenic reuptake of γ-aminobutyric acid (GABA) with the co-transport of Na+ and Cl- ions. In the past, there has been a major research drive focused on the dysfunction of hGAT1 in several neurological disorders. Thus, hGAT1 of the GABAergic system has been well established as an attractive target for such diseased conditions. Till date, there are various reports about stereo selectivity of -COOH group of tiagabine, a Food and Drug Administration (FDA)-approved hGAT1-selective antiepileptic drug. However, the effect of the stereochemistry of the protonated -NH group of tiagabine has never been scrutinized. Therefore, in this study, tiagabine has been used to explore the binding hypothesis of different enantiomers of tiagabine. In addition, the impact of axial and equatorial configuration of the-COOH group attached at the meta position of the piperidine ring of tiagabine enantiomers was also investigated. Further, the stability of the finally selected four hGAT1-tiagabine enantiomers namely entries 3, 4, 6, and 9 was evaluated through 100 ns molecular dynamics (MD) simulations for the selection of the best probable tiagabine enantiomer. The results indicate that the protonated -NH group in the R-conformation and the -COOH group of Tiagabine in the equatorial configuration of entry 4 provide maximum strength in terms of interaction within the hGAT1 binding pocket to prevent the change in hGAT1 conformational state, i.e., from open-to-out to open-to-in as compared to other selected tiagabine enantiomers 3, 6, and 9.

Design, Synthesis and Biological Evaluation of Benzo[D]Thiazole with Dimethylpyrazol Derivatives.

A series of new benzothiazole derivatives containing dimethylpyrazole were synthesized and evaluated for their anticonvulsant activity, neurotoxicity and cytotoxicity by using the maximal electroshock (MES), rotarod neurotoxicity (TOX) and MTT colorimetric assay. Among the compounds studied, four compounds (6a, 6b, 6g and 6m) showed better anticonvulsant than the others at 300 mg/kg and they also showed anticonvulsant activity at the dose of 100 mg/kg. All the synthetic compounds showed lower neurotoxicity and little cytotoxicity, so that the compounds, which with better activities, also had higher protective index. In particular, the compound 6g, 2-(3,5-dimethyl-1H-pyrazol-1-yl)-6-((2-fluorobenzyl)oxy)benzo[d]thiazole showed better activity with an ED50 value of 160.4 mg/kg and higher protective index (PI) values of 2.74 in the MES test than the standard drugs sodium valproate, which used as positive controls in this study. After that the compound 6g demonstrated antagonistic activity against seizures induced by pentylenetetrazol, which proved 6g maybe exert activity through effecting GABAergic neurotransmission.
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Nectandra grandiflora essential oil and its isolated sesquiterpenoids minimize anxiety-related behaviors in mice through GABAergic mechanisms.

Nectandra grandiflora Ness (Lauraceae) essential oil (EO) main constituent, the sesquiterpenoid (+)-dehydrofukinone (DHF), has sedative and anticonvulsant effects through GABAergic mechanisms. Other DHF-related sesquiterpenoids have been identified in the EO, such as, dehydrofukinone epoxide (DFX), eremophil-11-en-10-ol (ERM) and selin-11-en-4-α-ol (SEL). However, the neuronal effects of these compounds in mammals remain unknown. Therefore, the aim of this study was to evaluate the anxiolytic potential of the N. grandiflora EO and the isolated compounds in in mice. For this purpose, mice were administered orally with vehicle, 10, 30 or 100 mg/kg EO, DHF, DFX, ERM or SEL or 1 mg/kg diazepam. Locomotion and ethological parameters in the open field (OF) and elevated plus maze (EPM) were recorded. We also examined the effect of DFX, ERM and SEL on the membrane potential and calcium influx in synaptosomes, and the presence of the compounds in the cortical tissue using gas chromatography. EOs and isolated compounds reduced anxiety-related parameters in the EPM (open arms time and entries, end activity, head dipping) and OF (center time and entries, total rearing, unprotected rearing, sniffing, grooming) without alter ambulation or induce sedation. Flumazenil (2 mg/kg, i.p.) altered the anxiolytic-like effect of all treatments and vanished the DFX, ERM and SEL-induced changes in membrane potential. However, FMZ did not blocked the DFX-, ERM- and SEL-induced inhibition of calcium influx. Therefore, our results suggest that N. grandiflora EO and isolated compounds induced anxiolytic-like effect in mice due to positive modulation of GABAa receptors and/or inhibition of neuronal calcium influx.

In Silico Methods for the Discovery of Orthosteric GABAB Receptor Compounds.

The GABAB receptor (GABAB-R) is a heterodimeric class C G protein-coupled receptor comprised of the GABAB1a/b and GABAB2 subunits. The endogenous orthosteric agonist γ-amino-butyric acid (GABA) binds within the extracellular Venus flytrap (VFT) domain of the GABAB1a/b subunit. The receptor is associated with numerous neurological and neuropsychiatric disorders including learning and memory deficits, depression and anxiety, addiction and epilepsy, and is an interesting target for new drug development. Ligand- and structure-based virtual screening (VS) are used to identify hits in preclinical drug discovery. In the present study, we have evaluated classical ligand-based in silico methods, fingerprinting and pharmacophore mapping and structure-based in silico methods, structure-based pharmacophores, docking and scoring, and linear interaction approximation (LIA) for their aptitude to identify orthosteric GABAB-R compounds. Our results show that the limited number of active compounds and their high structural similarity complicate the use of ligand-based methods. However, by combining ligand-based methods with different structure-based methods active compounds were identified in front of DUDE-E decoys and the number of false positives was reduced, indicating that novel orthosteric GABAB-R compounds may be identified by a combination of ligand-based and structure-based in silico methods.

A conformationally restricted GABA analogue based on octahydro-1H-cyclopenta[b]pyridine scaffold.

An approach to rel-(4aS,6R,7aR)-octahydro-1H-cyclopenta[b]pyridine-6-carboxylic acid-a bicyclic conformationally restricted γ-aminobutyric acid (GABA) analogue was developed. The eight-step sequence relied on the reaction of 2,3-bis(chloromethyl)pyridine and a C1-binucleophile and the catalytic reduction of the pyridine ring as the key steps and allowed for the preparation of the title compound in 9.0% overall yield. Assessment of the octahydro-1H-cyclopenta[b]pyridine scaffold geometry showed that this template can be considered truly three-dimensional.

[Depakine: 25 years in Russia]. / Depakin: 25 let v Rossii.

The article summarizes domestic and international studies on the development and clinical investigation of valproates including multiple studies of a research team directed by the author. Valproate targets are considered in biological and clinical aspects. A spectrum of action and advantages of the brand drug (depakine) compared to generics and other antiepileptic drugs are discussed. A number of recommendations for practitioners about using valproates are proposed.

HPLC-Based Activity Profiling for GABAA Receptor Modulators in Searsia pyroides Using a Larval Zebrafish Locomotor Assay.

A dichloromethane extract from leaves of Searsia pyroides potentiated gamma aminobutyric acid-induced chloride currents by 171.8 ± 54% when tested at 100 µg/mL in Xenopus oocytes transiently expressing gamma aminobutyric acid type A receptors composed of α1ß2γ2s subunits. In zebrafish larvae, the extract significantly lowered pentylenetetrazol-provoked locomotion when tested at 4 µg/mL. Active compounds of the extract were tracked with the aid of HPLC-based activity profiling utilizing a previously validated zebrafish larval locomotor activity assay. From two active HPLC fractions, compounds 1 - 3 were isolated. Structurally related compounds 4 - 6 were purified from a later eluting inactive HPLC fraction. With the aid of 1H and 13C NMR and high-resolution mass spectrometry, compounds 1 - 6 were identified as analogues of anacardic acid. Compounds 1 - 3 led to a concentration-dependent decrease of pentylenetetrazol-provoked locomotion in the zebrafish larvae model, while 4 - 6 were inactive. Compounds 1 - 3 enhanced gamma aminobutyric acid-induced chloride currents in Xenopus oocytes in a concentration-dependent manner, while 4 - 6 only showed marginal enhancements of gamma aminobutyric acid-induced chloride currents. Compounds 2, 3, and 5 have not been reported previously.

Contrasting actions of a convulsant barbiturate and its anticonvulsant enantiomer on the α1 ß3 γ2L GABAA receptor account for their in vivo effects.

KEY POINTS: Most barbiturates are anaesthetics but unexpectedly a few are convulsants whose mechanism of action is poorly understood. We synthesized and characterized a novel pair of chiral barbiturates that are capable of photolabelling their binding sites on GABAA receptors. In mice the S-enantiomer is a convulsant, but the R-enantiomer is an anticonvulsant. The convulsant S-enantiomer binds solely at an inhibitory site. It is both an open state inhibitor and a resting state inhibitor. Its action is pH independent, suggesting the pyrimidine ring plays little part in binding. The inhibitory site is not enantioselective because the R-enantiomer inhibits with equal affinity. In contrast, only the anticonvulsant R-enantiomer binds to the enhancing site on open channels, causing them to stay open longer. The enhancing site is enantioselective. The in vivo actions of the convulsant S-enantiomer are accounted for by its interactions with GABAA receptors. ABSTRACT: Most barbiturates are anaesthetics but a few unexpectedly are convulsants. We recently located the anaesthetic sites on GABAA receptors (GABAA Rs) by photolabelling with an anaesthetic barbiturate. To apply the same strategy to locate the convulsant sites requires the creation and mechanistic characterization of a suitable agent. We synthesized enantiomers of a novel, photoactivable barbiturate, 1-methyl-5-propyly-5-(m-trifluoromethyldiazirinyl) phenyl barbituric acid (mTFD-MPPB). In mice, S-mTFD-MPPB acted as a convulsant, whereas R-mTFD-MPPB acted as an anticonvulsant. Using patch clamp electrophysiology and fast solution exchange on recombinant human α1 ß3 γ2L GABAA Rs expressed in HEK cells, we found that S-mTFD-MPPB inhibited GABA-induced currents, whereas R-mTFD-MPPB enhanced them. S-mTFD-MPPB caused inhibition by binding to either of two inhibitory sites on open channels with bimolecular kinetics. It also inhibited closed, resting state receptors at similar concentrations, decreasing the channel opening rate and shifting the GABA concentration-response curve to the right. R-mTFD-MPPB, like most anaesthetics, enhanced receptor gating by rapidly binding to allosteric sites on open channels, initiating a rate-limiting conformation change to stabilized open channel states. These states had slower closing rates, thus shifting the GABA concentration-response curve to the left. Under conditions when most GABAA Rs were open, an inhibitory action of R-mTFD-MPPB was revealed that had a similar IC50 to that of S-mTFD-MPPB. Thus, the inhibitory sites are not enantioselective, and the convulsant action of S-mTFD-MPPB results from its negligible affinity for the enhancing, anaesthetic sites. Interactions with these two classes of barbiturate binding sites on GABAA Rs underlie the enantiomers' different pharmacological activities in mice.

Positive modulation of synaptic and extrasynaptic GABAA receptors by an antagonist of the high affinity benzodiazepine binding site.

GABAA receptors are the major inhibitory neurotransmitter receptors in the brain and are the target for many clinically important drugs such as the benzodiazepines. Benzodiazepines act at the high-affinity binding site at the α+/γ- subunit interface. Previously, an additional low affinity binding site for diazepam located in the transmembrane (TM) domain has been described. The compound SJM-3 was recently identified in a prospective screening of ligands for the benzodiazepine binding site and investigated for its site of action. We determined the binding properties of SJM-3 at GABAA receptors recombinantly expressed in HEK-cells using radioactive ligand binding assays. Impact on function was assessed in Xenopus laevis oocytes with electrophysiological experiments using the two-electrode voltage clamp method. SJM-3 was shown to act as an antagonist at the α+/γ- site. At the same time it strongly potentiated GABA currents via the binding site for diazepam in the transmembrane domain. Mutation of a residue in M2 of the α subunit strongly reduced receptor modulation by SJM-3 and a homologous mutation in the ß subunit abolished potentiation. SJM-3 acts as a more efficient modulator than diazepam at the site in the trans-membrane domain. In contrast to low concentrations of benzodiazepines, SJM-3 modulates both synaptic and extrasynaptic receptors. A detailed exploration of the membrane site may provide the basis for the design and identification of subtype-selective modulatory drugs.

Amino acid derivatives as bitter taste receptor (T2R) blockers.

In humans, the 25 bitter taste receptors (T2Rs) are activated by hundreds of structurally diverse bitter compounds. However, only five antagonists or bitter blockers are known. In this study, using molecular modeling guided site-directed mutagenesis, we elucidated the ligand-binding pocket of T2R4. We found seven amino acids located in the extracellular side of transmembrane 3 (TM3), TM4, extracellular loop 2 (ECL2), and ECL3 to be involved in T2R4 binding to its agonist quinine. ECL2 residues Asn-173 and Thr-174 are essential for quinine binding. Guided by a molecular model of T2R4, a number of amino acid derivatives were screened for their ability to bind to T2R4. These predictions were tested by calcium imaging assays that led to identification of γ-aminobutryic acid (GABA) and Nα,Nα-bis(carboxymethyl)-L-lysine (BCML) as competitive inhibitors of quinine-activated T2R4 with an IC50 of 3.2 ± 0.3 µM and 59 ± 18 nM, respectively. Interestingly, pharmacological characterization using a constitutively active mutant of T2R4 reveals that GABA acts as an antagonist, whereas BCML acts as an inverse agonist on T2R4. Site-directed mutagenesis confirms that the two novel bitter blockers share the same orthosteric site as the agonist quinine. The signature residues Ala-90 and Lys-270 play important roles in interacting with BCML and GABA, respectively. This is the first report to characterize a T2R endogenous antagonist and an inverse agonist. The novel bitter blockers will facilitate physiological studies focused on understanding the roles of T2Rs in extraoral tissues.

Multisite organic-inorganic hybrid catalysts for the direct sustainable synthesis of GABAergic drugs.

Multisite organic-inorganic hybrid catalysts have been prepared and applied in a new general, practical, and sustainable synthetic procedure toward industrially relevant GABA derivatives. The domino sequence is composed of seven chemical transformations which are performed in two one-pot reactions. The method produces both enantiomeric forms of the product in high enantiopurity as well as the racemate in good yields after a single column purification step. This protocol highlights major process intensification, catalyst recyclability, and low waste generation.

Gomisin N isolated from Schisandra chinensis augments pentobarbital-induced sleep behaviors through the modification of the serotonergic and GABAergic system.

The fruits of Schisandra chinensis have been used for the treatment of insomnia in oriental countries for more than thousands of years. However, the pharmacological properties and the mechanism of sedative and hypnotic effects have not yet been studied. Gomisin N is one of the major bioactive constituents from the fruits of Schisandra chinensis, and in this paper we reported a detailed study on the effects and mechanisms of Gomisin N on its sedative and hypnotic activity for the first time. These results implied that Gomisin N possessed weak sedative effects on locomotion activity in normal mice, and produced a dose-dependent(5-45 mg/kg, i.p.) increase in sleep duration in pentobarbital-treated mice, thus, itself did not induce sleep at higher dose which was used in this experiment (45 mg/kg, i.p.). It also can reverse the rodent models of insomnia induced by p-chlorophenylalanine (PCPA) and caffeine, which could exhibit a synergistic effect with 5-hydroxytryptophan (5-HTP) as well; furthermore, the hypnotic effects of Gomisin N were inhibited by flumazenil (a specific GABAA-BZD receptor antagonist). Altogether, these results indicated that Gomisin N produced beneficial sedative and hypnotic bioactivity, which might be mediated by the modification of the serotonergic and GABAergic system.

11-trifluoromethyl-phenyldiazirinyl neurosteroid analogues: potent general anesthetics and photolabeling reagents for GABAA receptors.

RATIONALE: While neurosteroids are well-described positive allosteric modulators of gamma-aminobutyric acid type A (GABAA) receptors, the binding sites that mediate these actions have not been definitively identified. OBJECTIVES: This study was conducted to synthesize neurosteroid analogue photolabeling reagents that closely mimic the biological effects of endogenous neurosteroids and have photochemical properties that will facilitate their use as tools for identifying the binding sites for neurosteroids on GABAA receptors. RESULTS: Two neurosteroid analogues containing a trifluromethyl-phenyldiazirine group linked to the steroid C11 position were synthesized. These reagents, CW12 and CW14, are analogues of allopregnanolone (5α-reduced steroid) and pregnanolone (5ß-reduced steroid), respectively. Both reagents were shown to have favorable photochemical properties with efficient insertion into the C-H bonds of cyclohexane. They also effectively replicated the actions of allopregnanolone and pregnanolone on GABAA receptor functions: they potentiated GABA-induced currents in Xenopus laevis oocytes transfected with α1ß2γ2L subunits, modulated [(35)S]t-butylbicyclophosphorothionate binding in rat brain membranes, and were effective anesthetics in Xenopus tadpoles. Studies using [(3)H]CW12 and [(3)H]CW14 showed that these reagents covalently label GABAA receptors in both rat brain membranes and in a transformed human embryonal kidney (TSA) cells expressing either α1 and ß2 subunits or ß3 subunits of the GABAA receptor. Photolabeling of rat brain GABAA receptors was shown to be both concentration-dependent and stereospecific. CONCLUSIONS: CW12 and CW14 have the appropriate photochemical and pharmacological properties for use as photolabeling reagents to identify specific neurosteroid-binding sites on GABAA receptors.

RuBiGABA-2: a hydrophilic caged GABA with long wavelength sensitivity.

We have devised a new caged GABA based on ruthenium bipyridyl coordination chemistry. This phototrigger delivers GABA upon irradiation with wavelengths up to 532 nm undergoing heterolytic photocleavage, in a clean and very fast (a few nanoseconds) photoreaction. With an absorptivity coefficient ε(MAX) = 5300 M(-1) cm(-1) at 447 nm and a quantum efficiency φ ~ 0.09, RuBiGABA-2 is among the most active caged-GABAs, especially at long wavelengths. This highly hydrophilic caged GABA can be synthesized in a simple one-pot reaction. The synthesis, chemical characterization and photochemical properties are presented. Finally, the usefulness of this caged compound is demonstrated by photodelivering free GABA on leech motoneurons.

Effects of propofol and other GABAergic phenols on membrane molecular organization.

GABA(A) receptor is the main inhibitory receptor of the central nervous system. The phenols propofol and thymol have been shown to act on this receptor. GABA(A) is an intrinsic protein, the activity of which may be affected by physical changes in the membrane. Taking into account the lipophilicity of phenols, their interaction with the membrane and a consequent non-specific receptor modulation cannot be discarded. By using Langmuir films, we analyze the comparative effects on the molecular properties of the membrane exerted by propofol, thymol and other related compounds, the activities of which on the GABA(A) are under investigation in our laboratory. All the compounds were able to expand phospholipid films, by their incorporation into the monolayer being favored by less-packed structures. Nonetheless, they were able to be incorporated at lateral pressures above the equilibrium pressure estimated for a natural membrane. Epifluorescence images revealed their presence between phospholipid molecules, probably at the head-group region. Hence, all results indicated that the phenols studied were clearly able to interact with membranes, suggesting that their anesthetic activity could be the combined result of their interaction with specific receptor proteins and with their surrounding lipid molecules modulating the supramolecular organization of the receptor environment.

Phenazepam: the drug that came in from the cold.

In the past few years there has been concern in Western Europe and in the US about the rise in abuse of phenazepam, a benzodiazepine that was originally developed in the USSR in the mid- to late 1970s.(1-4) Although phenazepam is one of the most widely prescribed benzodiazepines in Russia and other commonwealth of independent state (CIS) countries, it has not been licensed elsewhere in the world. Due to very limited licensed geographical distribution, there is very little peer-reviewed literature that is not written in Russian. In this article, we review the current state of what is currently known about phenazepam. This information on phenazepam and how it can be detected in biological specimens should assist the forensic community in identifying phenazepam in routine toxicology screening and interpreting any phenazepam concentrations that are obtained.

Comparative study of contents of several bioactive components in fruiting bodies and mycelia of culinary-medicinal mushrooms.

Mushrooms have been consumed for thousands of years, and several bioactive components were found therein, including lovastatin, γ-aminobutyric acid (GABA) and ergothioneine. The study reported herein was to analyze these three bioactive components in 15 fruiting bodies and 9 mycelia of 19 species of mushrooms from genera Agaricus, Agrocybe, Auricularia, Boletus, Ganoderma, Hypsizygus, Inonotus, Lentinus, Morchella, Pleurotus, Tremella, Termitomyces, and Volvariella. The results show that Hypsizygus marmoreus contained the highest amount of lovastatin (628.05 mg/kg) in fruiting bodies and Morchella esculenta contained the highest amount (1438.42 mg/ kg) in mycelia. Agaricus brasiliensis contained the highest amount of GABA (1844.85 mg/kg) in fruiting bodies, and mycelia of Boletus edulis, Pleurotus citrinopileatus, and Termitomyces albuminosus contained extraordinarily higher amounts (1274.03, 1631.67, and 2560.00 mg/kg, respectively). Volvariella volvacea contained the highest amount of ergothioneine (537.27 mg/kg) in fruiting bodies and mycelia; Boletus edulis, Pleurotus ferulae, and P. salmoneostramineus contained relatively higher amount of ergothioneine too (258.03, 250.23, and 222.08 mg/kg, respectively). However, none of these components was detected in fruiting bodies of Inonotus obliquus. In conclusion, these three bioactive components were commonly found in most mushrooms, and these results might be related to their beneficial effects.

Identification of GABA A receptor modulators in Kadsura longipedunculata and assignment of absolute configurations by quantum-chemical ECD calculations.

A petroleum ether extract of Kadsura longipedunculata enhanced the GABA-induced chloride current (I(GABA)) by 122.5±0.3% (n=2) when tested at 100 µg/ml in Xenopuslaevis oocytes expressing GABA A receptors (α(1)ß(2)γ(2S) subtype) in two-microelectrode voltage clamp measurements. Thirteen compounds were subsequently identified by HPLC-based activity profiling as responsible for GABA A receptor activity and purified in preparative scale. 6-Cinnamoyl-6,7-dihydro-7-myrceneol and 5,6-dihydrocuparenic acid were thereby isolated for the first time. The determination of the absolute stereochemistry of these compounds was achieved by comparison of experimental and calculated ECD spectra. All but one of the 13 isolated compounds from K. longipedunculata potentiated I(GABA) through GABA A receptors composed of α(1)ß(2)γ(2S) subunits in a concentration-dependent manner. Potencies ranged from 12.8±3.1 to 135.6±85.7 µM, and efficiencies ranged from 129.7±36.8% to 885.8±291.2%. The phytochemical profiles of petroleum ether extracts of Kadsura japonica fruits (114.1±2.6% potentiation of I(GABA) at 100 µg/ml, n=2), and Schisandra chinensis fruits (inactive at 100 µg/ml) were compared by HPLC-PDA-ESIMS with that of K. longipedunculata.