How does α1Histidine102 affect the binding of modulators to α1ß2γ2 GABAA receptors? molecular insights from in silico experiments.

The activation of GABAA receptors by the neurotransmitter gamma-aminobutyric acid mediates the rapid inhibition response in the central nervous system of mammals. Many neurological and mental health disorders arise from alterations in the structure or function of these pentameric ion channels. GABAA receptors are targets for numerous drugs, including benzodiazepines, which bind to α1ß2γ2 GABAA receptors with high affinity to a site in the extracellular domain, between subunits α1 and γ2. It has been established experimentally that the binding of these drugs depends on the presence of one particular amino acid in the α1 subunit: histidine 102. However, the specific role it plays in the intermolecular interaction has not been elucidated. In this work, we applied in silico methods to understand whether certain protonation and rotamer states of α1His102 facilitate the binding of modulators. We analysed diazepam binding, a benzodiazepine, and the antagonist flumazenil to the GABAA receptor using molecular dynamics simulations and adaptive biasing force simulations. The binding free energy follows changes in the protonation state for both ligands, and rotameric states of α1His102 were specific for the different compounds, suggesting distinct preferences for positive allosteric modulators and antagonists. Moreover, in the presence of diazepam and favoured by a neutral tautomer, we identified a water molecule that links loops A, B, and C and may be relevant to the modulation mechanism.

Electrical stimulation of the posterior insula induces mechanical analgesia in a rodent model of neuropathic pain by modulating GABAergic signaling and activity in the pain circuitry.

The insula has emerged as a critical target for electrical stimulation since it influences pathological pain states. We investigated the effects of repetitive electrical stimulation of the insular cortex (ESI) on mechanical nociception, and general locomotor activity in rats subjected to chronic constriction injury (CCI) of the sciatic nerve. We also studied neuroplastic changes in central pain areas and the involvement of GABAergic signaling on ESI effects. CCI rats had electrodes implanted in the left agranular posterior insular cortex (pIC), and mechanical sensitivity was evaluated before and after one or five daily consecutive ESIs (15 min each, 60 Hz, 210 µs, 1 V). Five ESIs (repetitive ESI) induced sustained mechanical antinociception from the first to the last behavioral assessment without interfering with locomotor activity. A marked increase in Fos immunoreactivity in pIC and a decrease in the anterior and mid-cingulate cortex, periaqueductal gray and hippocampus were noticed after five ESIs. The intrathecal administration of the GABAA receptor antagonist bicuculline methiodide reversed the stimulation-induced antinociception after five ESIs. ESI increased GAD65 levels in pIC but did not interfere with GABA, glutamate or glycine levels. No changes in GFAP immunoreactivity were found in this work. Altogether, the results indicate the efficacy of repetitive ESI for the treatment of experimental neuropathic pain and suggest a potential influence of pIC in regulating pain pathways partially through modulating GABAergic signaling.

AZD6280, a novel partial γ-aminobutyric acid A receptor modulator, demonstrates a pharmacodynamically selective effect profile in healthy male volunteers.

OBJECTIVE: AZD6280 is a novel γ-aminobutyric acid A receptor modulator with higher in vitro efficacy at the α2,3 subtypes as compared to the α1 and α5 subtypes. This study compared the pharmacodynamic effects of single oral dose AZD6280 10 mg and 40 mg on the central nervous system with 2 mg of lorazepam. METHODS: Sixteen healthy males were enrolled into the double-blind, randomized, 4-way crossover study. Two validated central nervous system test batteries, Neurocart and CogState, were administered to measure drug effects on cognition, neurophysiologic function, and psychomotor and subjective feelings. Statistical analysis was performed using mixed model analysis of variance, with fixed factors of treatment, period, time and treatment by time, and random factors of subject, subject by treatment and subject by time, and the average prevalue as covariate. RESULTS: Most pharmacodynamic parameters were affected by lorazepam. AZD6280 induced dose-dependent smaller-than-lorazepam effects on saccadic peak velocity (SPV) (AZD6280, 10 mg vs. AZD6280, 40 mg vs. lorazepam [deg/s]: -22.6 vs. -50.0 vs. -62.9, P < 0.001), whereas the impacts on adaptive-tracking, body-sway, smooth-pursuit, and the one-card-learning tests were significant but much smaller than lorazepam. Thus, the slopes of regression lines for the ΔLog(Sway)-ΔSPV, ΔTracking-ΔSPV, and ΔSmooth-ΔSPV relations were flatter with AZD6280 than with lorazepam. AZD6280 caused a distinct electroencephalography signature from that of lorazepam. CONCLUSIONS: The SPV responses to AZD6280 suggest potential concentration-related anxiolytic effects, whereas the smaller SPV-normalized effects of AZD6280 on various non-SPV pharmacodynamic parameters suggest a more favorable side effect profile compared to lorazepam. Overall, the pharmacodynamic profile of AZD6280 matches the pharmacological specificity and selectivity of this compound at the α2,3 γ-aminobutyric acid A receptor subtypes.

Surface active benzodiazepine-bromo-alkyl conjugate for potential GABAA-receptor purification.

A conjugable analogue of the benzodiazepine 5-(2-hydroxyphenyl)-7-nitro-benzo[e][1,4]diazepin-2(3H)-one containing a bromide C(12)-aliphatic chain (BDC) at nitrogen N1 was synthesized. One-pot preparation of this benzodiazepine derivative was achieved using microwave irradiation giving 49% yield of the desired product. BDC inhibited FNZ binding to GABA(A)-R with an inhibition binding constant K(i) = 0.89 µM and expanded a model membrane packed up to 35 mN m(-1) when penetrating in it from the aqueous phase. BDC exhibited surface activity, with a collapse pressure π = 9.8 mN m(-1) and minimal molecular area A(min) = 52 Å(2)/molecule at the closest molecular packing, resulted fully and non-ideally mixed with a phospholipid in a monolayer up to a molar fraction x≅ 0.1. A geometrical-thermodynamic analysis along the π-A phase diagram predicted that at low x(BDC) (<0.1) and at all π, including the equilibrium surface pressures of bilayers, dpPC-BDC mixtures dispersed in water were compatible with the formation of planar-like structures. These findings suggest that, in a potential surface grafted BDC, this compound could be stabilize though London-type interactions within a phospholipidic coating layer and/or through halogen bonding with an electron-donor surface via its terminal bromine atom while GABA(A)-R might be recognized through the CNZ moiety.

Heightened aggression after chronic flunitrazepam in male rats: potential links to cortical and caudate-putamen-binding sites.

RATIONALE: Higher doses of benzodiazepines induce sedation. However, in low to moderate doses, benzodiazepines can increase aggressive behavior both after acute and chronic administration. The determinants for increasing aggression after chronic intake of flunitrazepam, a so-called date rape drug, in violence-prone individuals are incompletely understood. OBJECTIVES: The aim of this study is to assess the effects of acute and chronic treatment with flunitrazepam on male aggression in resident rats. We also examined possible changes in binding to benzodiazepine receptors throughout the brain of rats that display aggressive behavior after repeated flunitrazepam treatment using quantitative receptor autoradiography. MATERIALS AND METHODS: The behaviors of the male Wistar resident rats (n = 35) toward a male intruder were recorded for 10 min twice a week. The salient aggressive and non-aggressive elements in the resident rat's behavior were analyzed. Initially, the dose-dependent effects of flunitrazepam (0.01, 0.03, 0.1, 0.18, and 0.3 mg/kg) or vehicle were determined in all rats; subsequently, 0.3 mg/kg per day flunitrazepam was administered for 42 days (n = 15), and a parallel group was treated with vehicle (n = 20). After the chronic treatment, the flunitrazepam (0, 0.01, 0.03, 0.1, 0.18, and 0.3 mg/kg) effects were again assessed. RESULTS: The most significant finding is the escalation of aggression after chronic treatment with flunitrazepam. A previously sedative 0.3 mg/kg dose of flunitrazepam engendered very high levels of attack bites, sideways threats, and aggressive postures (total aggression) after 6 weeks of daily administration. Individual differences emerged, and these were associated with decreased binding to benzodiazepine receptors, mainly in the limbic structures such as the cingulate cortex (cingulate areas 1 and 2) and caudate-putamen (posterior part) of aggressive animals, suggesting that these areas are pivotal in the control of emotional and aggressive behavior. CONCLUSIONS: Chronic flunitrazepam produces changes in receptor binding in discrete areas of the cingulate cortex and caudate-putamen that are proposed to be part of the mechanisms for increased expression of aggressive behavior.

Isolation and identification of 6-methylapigenin, a competitive ligand for the brain GABA(A) receptors, from Valeriana wallichii.

Using the guidance by a competitive assay for the benzodiazepine binding site in the GABA(A) receptor, active compounds were isolated from the rhizomes and roots of Valeriana wallichii DC. The UV, NMR and mass spectral data permitted the identification of 6-methylapigenin. This flavonoid has a Ki = 495 nM for the BDZ-bs and a GABA ratio of 1.6-2.0, which suggests possible agonistic properties. The calculated percentage of 6-methylapigenin in the crude drug is in the range: 0.013% to 0.0013%.

In ovo chronic neurosteroid treatment affects the function and allosteric interactions of GABA(A) receptor modulatory sites.

We investigated the effects of in ovo chronic administration of the endogenous neurosteroid epipregnanolone (5beta-pregnan-3beta-ol-20-one) on the GABA(A) receptor complex present in chick optic lobe synaptic membranes. Chronic epipregnanolone treatment failed to exert any effect on the chick optic lobe total protein content and wet weight at the different doses tested. [3H]Flunitrazepam control binding remained unaltered after neurosteroid exposure, however, the positive allosteric modulation of this ligand by 4 microM allopregnanolone was reduced in a dose-dependent manner by neurosteroid treatment. Embryo exposure to 30 microM epipregnanolone decreased allopregnanolone EC(50) and E(max) values. Analyses of saturation binding isotherms disclosed that such administration had no effect on K(d) and B(max) values for [3H]flunitrazepam and [3H]GABA binding. [3H]GABA binding modulation disclosed an increase in allopregnanolone EC(50) value with a decrease in its E(max) value. Diazepam EC(50) and E(max) values were enhanced, while low affinity sodium pentobarbital EC(50) value was reduced by epipregnanolone treatment. The investigation of the GABA(A) receptor function revealed that administration of this neurosteroid reduces the efficacy of GABA to induce 36Cl(-) influx into microsacs prepared from chick optic lobe. These results indicate that endogenous neurosteroid epipregnanolone chronically administered in ovo produces homologous uncoupling between steroid modulatory sites, and those corresponding to benzodiazepine and GABA receptors. Thus epipregnanolone is able to induce heterologous changes in the allosteric linkage between benzodiazepine and barbiturate modulatory sites, and the GABA receptor site. Taken jointly with results on epipregnanolone enhancing effects on [3H]flunitrazepam and [3H]GABA binding, in the context of its endogenous synthesis, our present findings support this neurosteroid as the endogenous modulator of GABA(A) receptor sites and function during chick optic lobe development.

Prolonged exposure to hypobaric hypoxia transiently reduces GABA(A) receptor number in mice cerebral cortex.

The central nervous system is severely affected by hypoxic conditions, which produce alterations in neural cytoarchitecture and neurotransmission, resulting in a variety of neuropathological conditions such as convulsive states, neurobehavioral impairment and motor CNS alterations. Some of the neuropathologies observed in hypobaric hypoxia, corresponding to high altitude conditions, have been correlated with a loss of balance between excitatory and inhibitory neurotransmission, produced by alterations in glutamatergic and GABAergic receptors. In the present work, we have studied the effect of chronic hypobaric hypoxia (506 hPa, 18 h/day x 21 days) applied to adult male mice on GABA(A) receptors from cerebral cortex, to determine whether hypoxic exposure may irreversibly affect central inhibitory neurotransmission. Saturation curves for [3H]GABA specifically bound to GABA(A) receptors in isolated synaptic membranes showed a 30% decrease in maximal binding capacity after hypoxic exposure (Bmax control, 4.70+/-0.19, hypoxic, 3.33+/-0.10 pmol/mg protein), with no effect on GABA binding sites affinity (Kd control: 159.3+/-13.3 nM, hypoxic: 164.2+/-15.1 nM). Decreased B(max) values were observed up to the 10th post-hypoxic day, returning to control values by the 15th post-hypoxic day. Pharmacological properties of GABA(A) receptor were also affected by hypoxic exposure, with a 45 to 51% increase in the maximal effect by positive allosteric modulators (pentobarbital and 5alpha-pregnan-3alpha-ol-20-one). We conclude that long-term hypoxia produces a significant but reversible reduction on GABA binding to GABA(A) receptor sites in cerebral cortex, which may reflect an adaptive response to this sustained pathophysiological state.

Synthesis and preliminary pharmacological evaluation of coumestans with different patterns of oxygenation.

Five coumestans with different patterns of oxygenation in rings A and D were synthesized from resorcinol and aromatic aldehydes, and screened for their antimyotoxic activity. The most potent compound (2b, IC50 = 1 microM) was selected for study of its pharmacological profile.

Presence of an endogenous factor in avian CNS with agonistic action on benzodiazepine receptors.

In the present paper we describe the presence in avian CNS of an endogenous inhibitor of [3H]flunitrazepam binding. This compound was extracted from a synaptic membrane fraction isolated from chick optic lobe and brain using an exhaustive aqueous washing procedure, then purified by means of solid-phase extraction with C18 cartridges and several HPLC steps until an homogeneous peak was obtained. Its chemical structure was studied by size-exclusion chromatography of the purified material which indicated that it possesses a molecular weight below 1350. Although its inhibitory activity was lost by HCl treatment, its peptidic nature was ruled out by an amino acid and N-terminal sequence analyses. Ultraviolet absorption spectrum showed two main peaks at 230 and 280 nm. The endogenous compound was found to inhibit competitively [3H]flunitrazepam binding to its recognition site without affecting [3H]GABA binding to the same receptor complex. The behavior of the endogenous factor in an "in vitro" GABA "shift" test and GABA-dependent chloride flux experiments were similar to that of benzodiazepine receptor agonists. In conclusion, these results demonstrate the existence in avian CNS of a competitive endogenous inhibitor of benzodiazepine binding with agonistic action on benzodiazepine receptors.

Estimation of the binding affinity constants of soluble ligand-receptor complexes by a rapid filtration technique: [3H]-flunitrazepam-bovine serum albumin as an example.

A method for determining the equilibrium dissociation constant (KA) of a soluble ligand (L) from a soluble receptor (A) in the presence of another solid phase receptor (R) for the same ligand was developed. The total and nonspecific binding of L to R was measured in the presence and in the absence of A. The separation of bound and free L was done by a rapid filtration technique so that only the complex RL, but not AL, was recovered. An apparent dissociation constant (KR,app) was calculated from the saturation curve obtained in the presence of A. The magnitude of KA could be determined from this KR,app and the value of the equilibrium dissociation constant of the complex R-L (KR) calculated from the saturation curve in the absence of A. The equality of the Bmax values obtained in the presence and in the absence of A assured the accuracy in the determination of KA so that the fulfillment of this condition could be used as an internal control. For the correct definition of nonspecific binding, the displacement agent (L1) should be used at concentrations within the range 10(2).KR < L1 < 10. K4. This fact constraints the applicability of the method to systems where KA/KR > 10(3). The highest sensitivity of the method can be attained when 0.33 < [At]/KA < 3. The equilibrium binding constant of [3H]-flunitrazepam to non-delipidized bovine serum albumin determined by the present approach (31 +/- 7 mumol/L) did not differ significantly from the literature.