Long-term immune cell profiling in stroke patients with or without infections.

PURPOSE: Infections are frequent complications in acute ischemic stroke and may be caused by an altered immune response influencing brain damage. We compared long-term immune responses in stroke patients with or without infections during the recovery period by performing a long-term profiling of clinically relevant inflammatory parameters from stroke onset until day 49. MATERIALS AND METHODS: Thirty-four stroke patients were retrospectively included and divided into two groups depending on infection status. Group 1 had no infections (N = 17) and group 2 had post-admission infection (N = 17). The patients were evaluated carefully for infections and evolution of the peripheral inflammatory response. Neutrophils, monocytes, lymphocytes, total leukocytes and C-reactive protein were evaluated in relation to the occurrence and development of infections. In both patient groups, an acute boost in neutrophils and monocytes were observed whereas the opposite was true for lymphocytes. RESULTS: In Group 1, neutrophils and monocytes approached normal levels after 20-30 days, but remained elevated in Group 2. We found an increase in neutrophils (p = 0.01) and leukocytes (p < 0.01) as well as C-reactive protein (p < 0.01) among infected patients. Lymphocytes remained depressed in Group 2, while Group 1 slowly approached baseline levels. In both groups, CRP levels initially increased with a slow return to baseline levels. From day 0 to 49 after stroke, uninfected patients generally experienced a decline in leukocytes, neutrophils and monocytes (all p < 0.05), while no similar changes happened among infected patients. CONCLUSIONS: Our study provides an overview of general immune cell kinetics after stroke related to infection status. Immune cell numbers were severely disturbed for weeks after the insult, independent of infection status, although infected patients achieved the highest cell counts of neutrophils, leukocytes and for C-reactive protein. The sustained depression of lymphocytes, especially and paradoxically among infected patients, warrants future studies into the mechanisms behind this, with potential for future therapies aimed at restoring normal immunity and thereby improving patient outcome.

Delineation of the functional properties exhibited by the Zinc-Activated Channel (ZAC) and its high-frequency Thr128Ala variant (rs2257020) in Xenopus oocytes.

The signalling characteristics of the Zinc-Activated Channel (ZAC), a member of the Cys-loop receptor (CLR) superfamily, are presently poorly elucidated. The ZACN polymorphism c.454G>A encoding for the Thr128Ala variation in ZAC is found in extremely high allele frequencies across ethnicities. In this, the first study of ZAC in Xenopus oocytes by TEVC electrophysiology, ZACThr128 and ZACAla128 exhibited largely comparable pharmacological and signalling characteristics, but interestingly the Zn2+- and H+-evoked current amplitudes in ZACAla128-oocytes were dramatically smaller than those in ZACThr128-oocytes. While the variation thus appeared to impact cell surface expression and/or channel properties of ZAC, the similar expression properties exhibited by ZACThr128 and ZACAla128 in transfected mammalian cells indicated that their distinct functionalities could arise from the latter. In co-expression experiments, wild-type and variant ZAC subunits assembled efficiently into "heteromeric" complexes in HEK293 cells, while the concomitant presence of ZACAla128 in ZACThr128:ZACAla128-oocytes did not exert a dominant negative effect on agonist-evoked current amplitudes compared to those in ZACThr128-oocytes. Finally, the structural determinants of the functional importance of the 1-hydroxyethyl side-chain of Thr128 appeared to be subtle, as agonist-evoked current amplitudes in ZACSer128-, ZACVal128- and ZACIle128-oocytes also were substantially lower than those in ZACThr128-oocytes. In conclusion, the functional properties exhibited by ZAC in this work substantiate the notion of it being an atypical CLR. While the impact of the Thr128Ala variation on ZAC functionality in oocytes is striking, it remains to be investigated whether and to which extent this translates into an in vivo setting and thus could constitute a source of inter-individual variation in ZAC physiology.

An in silico kinetic model of 8-oxo-7,8-dihydro-2-deoxyguanosine and 8-oxo-7,8-dihydroguanosine metabolism from intracellular formation to urinary excretion.

Oxidatively generated DNA damage is of paramount importance in a wide range of physiological and pathophysiological processes. Urinary 8-oxo-7,8-dihydro-2-deoxyguanosine (8-oxodG) is often used as an outcome marker in studies on the role of oxidatively generated DNA damage, but its exact relation to intracellular damage levels and variations in DNA repair have been unclear. Using a new approach of quantitative kinetic modeling inspired by pharmacokinetics, we find evidence that in steady state - i.e. when systemic consequences of given change in damage or cellular removal rates have stabilized - the urinary excretion of 8-oxodG is closely correlated to rates of damage and intracellular 8-oxodG levels, but independent of the rate of cellular removal. Steady state was calculated to occur within approximately 12 h. A similar pattern was observed in a model of the corresponding RNA marker 8-oxo-7,8-dihydroguanosine (8-oxoGuo), but with steady-state occurring slower (up to 5 d). These data have significant implications for the planning of studies and interpretation of data involving urinary 8-oxodG/8-oxoGuo excretion as outcome.HighlightsThe kinetics of 8-oxodG/8-oxoGuo formation, removal and excretion were simulated in silico.The model was based on existing data on 8-oxodG/8-oxoGuo levels and removal/excretion rates.Intracellular 8-oxodG/8-oxoGuo was closely correlated with urinary excretion in steady state.Changes in removal rates did not influence urinary excretion of 8-oxodG/8-oxoGuo.

Impaired GABAergic inhibition in the prefrontal cortex of early postnatal phencyclidine (PCP)-treated rats.

A compromised γ-aminobutyric acid (GABA)ergic system is hypothesized to be part of the underlying pathophysiology of schizophrenia. N-methyl-D-aspartate (NMDA) receptor hypofunction during neurodevelopment is proposed to disrupt maturation of interneurons causing an impaired GABAergic transmission in adulthood. The present study examines prefrontal GABAergic transmission in adult rats administered with the NMDA receptor channel blocker, phencyclidine (PCP), for 3 days during the second postnatal week. Whole-cell patch-clamp recordings from pyramidal cells in PCP-treated rats showed a 22% reduction in the frequency of miniature inhibitory postsynaptic currents in layer II/III, but not in layer V pyramidal neurons of the prefrontal cortex. Furthermore, early postnatal PCP treatment caused insensitivity toward effects of the GABA transporter 1 (GAT-1) inhibitor, 1,2,5,6-tetrahydro-1-[2-[[(diphenyl-methylene)amino]oxy]ethyl]-3-pyridinecarboxylic acid, and also diminished currents passed by δ-subunit-containing GABAA receptors in layer II/III pyramidal neurons. The observed impairments in GABAergic function are compatible with the alteration of GABAergic markers as well as cognitive dysfunction observed in early postnatal PCP-treated rats and support the hypothesis that PCP administration during neurodevelopment affects the functionality of interneurons in later life.

The GABAA antagonist DPP-4-PIOL selectively antagonises tonic over phasic GABAergic currents in dentate gyrus granule cells.

GABAA receptors mediate two different types of inhibitory currents: phasic inhibitory currents when rapid and brief presynaptic GABA release activates postsynaptic GABAA receptors and tonic inhibitory currents generated by low extrasynaptic GABA levels, persistently activating extrasynaptic GABAA receptors. The two inhibitory current types are mediated by different subpopulations of GABAA receptors with diverse pharmacological profiles. Selective antagonism of tonic currents is of special interest as excessive tonic inhibition post-stroke has severe pathological consequences. Here we demonstrate that phasic and tonic GABAA receptor currents can be selectively inhibited by the antagonists SR 95531 and the 4-PIOL derivative, 4-(3,3-diphenylpropyl)-5-(4-piperidyl)-3-isoxazolol hydrobromide (DPP-4-PIOL), respectively. In dentate gyrus granule cells, SR 95531 was found approximately 4 times as potent inhibiting phasic currents compared to tonic currents (IC50 values: 101 vs. 427 nM). Conversely, DPP-4-PIOL was estimated to be more than 20 times as potent inhibiting tonic current compared to phasic current (IC50 values: 0.87 vs. 21.3 nM). Consequently, we were able to impose a pronounced reduction in tonic GABA mediated current (>70 %) by concentrations of DPP-4-PIOL, at which no significant effect on the phasic current was seen. Our findings demonstrate that selective inhibition of GABA mediated tonic current is possible, when targeting a subpopulation of GABAA receptors located extrasynaptically using the antagonist, DPP-4-PIOL.

Leukocyte infiltration in experimental stroke.

Stroke is one of the leading causes of death worldwide. At present, the only available treatment is thrombolysis, which should be initiated no later than 4.5 hours after onset of symptoms. Several studies have shown that an attenuation of the inflammatory response in relation to stroke could widen the therapeutic window. However, the immune system has important functions following infarction, such as removal of dead cells and the subsequent astrocytosis as well as prevention of post-ischemic infection. Hence, detailed knowledge concerning the temporal profile of leukocyte infiltration is necessary in order to develop new and effective treatments.The purpose of this review is to determine the temporal profile of leukocyte (neutrophil granulocytes, macrophages and T-cells) infiltration following experimental stroke. We found that the number of neutrophil granulocytes peaks between day 1 and 3 after experimental stroke, with short occlusion times (30 and 60 minutes of middle cerebral artery occlusion (MCAO)) leading to a later peak in response (P <0.001). Macrophages/microglia were found to peak later than day 3 and stay in the infarcted area for longer time periods, whereas duration of occlusion had no influence on the temporal infiltration (P = 0.475). Studies on T-cell infiltration are few; however, a tendency towards infiltration peak at later time points (from day 4 onwards) was seen.This review provides a framework for the instigation of post-stroke anti-inflammatory treatment, which could prove beneficial and widen the therapeutic window compared to current treatment options.

Structure-Activity Studies of 3,9-Diazaspiro[5.5]undecane-Based γ-Aminobutyric Acid Type A Receptor Antagonists with Immunomodulatory Effect.

The 3,9-diazaspiro[5.5]undecane-based compounds 2027 and 018 have previously been reported to be potent competitive γ-aminobutyric acid type A receptor (GABAAR) antagonists showing low cellular membrane permeability. Given the emerging peripheral application of GABAAR ligands, we hypothesize 2027 analogs as promising lead structures for peripheral GABAAR inhibition. We herein report a study on the structural determinants of 2027 in order to suggest a potential binding mode as a basis for rational design. The study identified the importance of the spirocyclic benzamide, compensating for the conventional acidic moiety, for GABAAR ligands. The structurally simplified m-methylphenyl analog 1e displayed binding affinity in the high-nanomolar range (Ki = 180 nM) and was superior to 2027 and 018 regarding selectivity for the extrasynaptic α4ßδ subtype versus the α1- and α2- containing subtypes. Importantly, 1e was shown to efficiently rescue inhibition of T cell proliferation, providing a platform to explore the immunomodulatory potential for this class of compounds.

Discovery of a new class of orthosteric antagonists with nanomolar potency at extrasynaptic GABAA receptors.

Brain GABAΑ receptors are ionotropic receptors belonging to the class of Cys-loop receptors and are important drug targets for the treatment of anxiety and sleep disorders. By screening a compound library (2,112 compounds) at recombinant human α4ß1δ GABAΑ receptors heterologously expressed in a HEK cell line, we identified a scaffold of spirocyclic compounds with nanomolar antagonist activity at GABAΑ receptors. The initial screening hit 2027 (IC50 of 1.03 µM) was used for analogue search resulting in 018 (IC50 of 0.088 µM). 018 was most potent at α3,4,5-subunit containing receptors, thus showing preference for forebrain-expressed extrasynaptic receptors. Schild analysis of 018 at recombinant human α4ß1δ receptors and displacement of [3H]muscimol binding in rat cortical homogenate independently confirmed a competitive profile. The antagonist profile of 018 was further validated by whole-cell patch-clamp electrophysiology, where kinetic studies revealed a slow dissociation rate and a shallow hill slope was observed. Membrane permeability studies showed that 2027 and 018 do not cross membranes, thus making the compounds less attractive for studying central GABAΑ receptors effects, but conversely more attractive as tool compounds in relation to emerging peripheral GABAΑ receptor-mediated effects of GABA e.g. in the immune system.

Increasing cellular lifespan with a flow system in organotypic culture of the Laterodorsal Tegmentum (LDT).

Organotypic brain culture is an experimental tool widely used in neuroscience studies. One major drawback of this technique is reduced neuronal survival across time, which is likely exacerbated by the loss of blood flow. We have designed a novel, tube flow system, which is easily incorporated into the commonly-used, standard semi-permeable membrane culture methodology which has significantly enhanced neuronal survival in a brain stem nucleus involved in control of motivated and arousal states: the laterodorsal tegmental nucleus (LDT). Our automated system provides nutrients and removes waste in a comparatively aseptic environment, while preserving temperature, and oxygen levels. Using immunohistochemistry and electrophysiology, our system was found superior to standard techniques in preserving tissue quality and survival of LDT cells for up to 2 weeks. In summary, we provide evidence for the first time that the LDT can be preserved in organotypic slice culture, and further, our technical improvements of adding a flow system, which likely enhanced perfusion to the slice, were associated with enhanced neuronal survival. Our perfusion system is expected to facilitate organotypic experiments focused on chronic stimulations and multielectrode recordings in the LDT, as well as enhance neuronal survival in slice cultures originating from other brain regions.

The sodium channel activator Lu AE98134 normalizes the altered firing properties of fast spiking interneurons in Dlx5/6+/- mice.

Mental disorders such as schizophrenia are associated with impaired firing properties of fast spiking inhibitory interneurons (FSINs) causing reduced task-evoked gamma-oscillation in prefrontal cortex. The voltage-gated sodium channel NaV1.1 is highly expressed in PV-positive interneurons, but only at low levels in principal cells. Positive modulators of Nav1.1 channels are for this reason considered potential candidates for the treatment of cognitive disorders. Here we examined the effect of the novel positive modulator of voltage-gated sodium channels Lu AE98134. We found that Lu AE98134 facilitated the sodium current mediated by NaV1.1 expressed in HEK cells by shifting its activation to more negative values, decreasing its inactivation kinetics and promoting a persistent inward current. In a slice preparation from the brain of adult mice, Lu AE98134 promoted the excitability of fast spiking interneurons by decreasing the threshold for action potentials. We then tested if Lu AE98134 could normalize the altered firing properties of FSINs in Dlx5/6+/- mutant mice. FSINs of this model for schizophrenia are characterized by broader action potentials and higher spike threshold. We found that in the presence of Lu AE98134, the firing frequency was increased while the spike duration and the threshold were decreased. Compounds with similar mode of action appear as promising candidates for restoring cognitive deficits present in schizophrenia.

Differences in kinetics of structurally related competitive GABA(A) receptor antagonists.

Previously, 4-alkyl and 4-aryl substituted analogues of the low-efficacy partial GABA(A) receptor agonist 5-(4-piperidyl)-3-isothiazole (4-PIOL) have been identified as competitive GABA(A) receptor antagonists. These structurally related competitive antagonists show marked differences in their kinetic properties. The kinetics of 20 4-alkyl and 4-aryl substituted analogues of 4-PIOL, two 4-arylalkyl substituted 3-isothiazolol analogues and the classical GABA(A) receptor antagonist SR95531 was studied in cultured cerebral cortical neurons using whole-cell patch-clamp techniques. The kinetics of the antagonists was studied indirectly by measuring the changes in the response of the full GABA(A) receptor agonist isoguvacine (IGU) induced by concurrent application of an antagonist. When added, the majority of the antagonists did not affect the rate of deactivation of the IGU-induced responses. When removed, however, the majority of the antagonists slowed the reactivation phase of IGU implying that the dissociation of the antagonist from the GABA(A) receptor is the rate-limiting step. Surprisingly, the functional off-rates of the antagonists seemed to correlate better with the lipophilicity of the compounds than with the affinity and potency. This suggests that the dissociation of the tested antagonists from the GABA(A) receptor is restricted by lipophilic interactions, perhaps with the aromatic amino acids surrounding the GABA binding site.

Probing the pharmacophore of ginkgolides as glycine receptor antagonists.

Ginkgolides are antagonists of the inhibitory ligand-gated ion channels for the neurotransmitters glycine and gamma-aminobutyric acid (GABA). In this study the ginkgolide structure was modified in order to investigate the minimum structural requirements for glycine receptor antagonism. The five native ginkgolides and a series of 29 ginkgolide derivatives were characterized at the three glycine receptor subtypes alpha1, alpha1beta, and alpha2, which revealed that only minor changes in the ginkgolide skeleton were allowed for maintaining glycine receptor antagonism. A pharmacophore model was generated and applied in a virtual screening of a compound database (300000 compounds), resulting in the identification of 31 hits. Twenty-seven of these hits were screened for biological activity, but none displayed antagonist activity at the glycine receptors. This strongly suggests the importance of other pharmacophore components in the binding of ginkgolides to glycine receptors, and we propose that the structural rigidity of the ginkgolide molecule may be crucial for its glycine receptor activity.

5-Substituted imidazole-4-acetic acid analogues: synthesis, modeling, and pharmacological characterization of a series of novel gamma-aminobutyric acid(C) receptor agonists.

A series of ring-substituted analogues of imidazole-4-acetic acid (IAA, 4), a partial agonist at both GABAA and GABAC receptors (GABA = gamma-aminobutyric acid), have been synthesized. The synthesized compounds 8a-l have been evaluated as ligands for the alpha1beta2gamma2S GABAA receptors and the rho1 GABAC receptors using the FLIPR membrane potential (FMP) assay and by electrophysiology techniques. None of the tested compounds displayed activity at the GABAA receptors at concentrations up to 1000 microM. However, the 5-Me, 5-Ph, 5-p-Me-Ph, and 5-p-F-Ph IAA analogues, 8a,c,f,g, displayed full agonist activities at the rho1 receptors in the FMP assay (EC50 in the range 22-420 microM). Ligand-protein docking identified the Thr129 in the alpha1 subunit and the corresponding Ser168 residue in rho1 as determinants of the selectivity displayed by the 5-substituted IAA analogues. The fact that GABA, 4, and 8a displayed decreased agonist potencies at a rho1Ser168Thr mutant compared to the WT rho1 receptor strongly supported this hypothesis. However, in contrast to GABA and 4, which exhibited increased agonist potencies at a alpha1(Thr129Ser)beta2gamma2 mutant compared to WT GABAA receptor, the data obtained for 8a at the WT and mutant receptors were nonconclusive.

Anticonvulsive evaluation of THIP in the murine pentylenetetrazole kindling model: lack of anticonvulsive effect of THIP despite functional δ-subunit-containing GABAA receptors in dentate gyrus granule cells.

THIP (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol) is a GABAA receptor agonist with varying potencies and efficacies at γ-subunit-containing receptors. More importantly, THIP acts as a selective superagonist at δ-subunit-containing receptors (δ-GABAA Rs) at clinically relevant concentrations. Evaluation of THIP as a potential anticonvulsant has given contradictory results in different animal models and for this reason, we reevaluated the anticonvulsive properties of THIP in the murine pentylenetetrazole (PTZ) kindling model. As loss of δ-GABAA R in the dentate gyrus has been associated with several animal models of epilepsy, we first investigated the presence of functional δ-GABAA receptors. Both immunohistochemistry and Western blot data demonstrated that δ-GABAA R expression is not only present in the dentate gyrus, but also the expression level was enhanced in the early phase after PTZ kindling. Whole-cell patch-clamp studies in acute hippocampal brain slices revealed that THIP was indeed able to induce a tonic inhibition in dentate gyrus granule cells. However, THIP induced a tonic current of similar magnitude in the PTZ-kindled mice compared to saline-treated animals despite the observed upregulation of δ-GABAA Rs. Even in the demonstrated presence of functional δ-GABAA Rs, THIP (0.5-4 mg/kg) showed no anticonvulsive effect in the PTZ kindling model using a comprehensive in vivo evaluation of the anticonvulsive properties.

Hydroxy-1,2,5-oxadiazolyl moiety as bioisoster of the carboxy function. Synthesis, ionization constants, and pharmacological characterization of gamma-aminobutyric acid (GABA) related compounds.

Three 4-substituted 1,2,5-oxadiazol-3-ols containing aminoalkyl substituents (analogues and homologues of gamma-aminobutyric acid (GABA)) were synthesized to investigate the hydroxy-1,2,5-oxadiazolyl moiety as a bioisoster for a carboxyl group at GABA receptors. The pK(a) values of the target compounds were close to those of GABA. At GABA(A) receptors of cultured cerebral cortical neurons, weak agonist and partial agonist profiles were identified, demonstrating the 4-hydroxy-1,2,5-oxadiazol-3-yl unit to be a nonclassical carboxyl group bioisoster.

Delineation of the functional properties and the mechanism of action of TMPPAA, an allosteric agonist and positive allosteric modulator of 5-HT3 receptors.

We have previously identified a novel class of 5-hydroxytryptamine type 3 receptor (5-HT3R) agonists sharing little structural similarity with orthosteric 5-HT3R ligands (Jørgensen et al., 2011). In the present study we have elucidated the functional characteristics and the mechanism of action of one of these compounds, trans-3-(4-methoxyphenyl)-N-(pentan-3-yl)acrylamide (TMPPAA). In electrophysiological recordings TMPPAA was found to be a highly-efficacious partial agonist equipotent with 5-HT at the 5-HT3A receptor (5-HT3AR) expressed in COS-7 cells and somewhat less potent at the receptor expressed in Xenopus oocytes. The desensitization kinetics of TMPPAA-evoked currents were very different from those mediated by 5-HT. Moreover, repeated TMPPAA applications resulted in progressive current run-down and persistent non-responsiveness of the receptor to TMPPAA, but not to 5-HT. In addition to its direct activation, TMPPAA potentiated 5-HT-mediated 5-HT3AR signalling, and the allosteric link between the two binding sites was corroborated by the analogous ability of 5-HT to potentiate TMPPAA-evoked responses. The agonism and potentiation exerted by TMPPAA at a chimeric α7-nACh/5-HT3A receptor suggested that the ligand acts through the transmembrane domain of 5-HT3AR, a notion further substantiated by its functional properties at chimeric and mutant human/murine 5-HT3ARs. A residue in the transmembrane helix 4 of 5-HT3A was identified as an important molecular determinant for the different agonist potencies exhibited by TMPPAA at human and murine 5-HT3ARs. In conclusion, TMPPAA is a novel allosteric agonist and positive allosteric modulator of 5-HT3Rs, and its aberrant signalling characteristics compared to 5-HT at the 5-HT3AR underline the potential in Cys-loop receptor modulation and activation through allosteric sites.

Astrocytic GABA transporter activity modulates excitatory neurotransmission.

Astrocytes are ideally placed to detect and respond to network activity. They express ionotropic and metabotropic receptors, and can release gliotransmitters. Astrocytes also express transporters that regulate the extracellular concentration of neurotransmitters. Here we report a previously unrecognized role for the astrocytic GABA transporter, GAT-3. GAT-3 activity results in a rise in astrocytic Na+ concentrations and a consequent increase in astrocytic Ca2+ through Na+/Ca2+ exchange. This leads to the release of ATP/adenosine by astrocytes, which then diffusely inhibits neuronal glutamate release via activation of presynaptic adenosine receptors. Through this mechanism, increases in astrocytic GAT-3 activity due to GABA released from interneurons contribute to 'diffuse' heterosynaptic depression. This provides a mechanism for homeostatic regulation of excitatory transmission in the hippocampus.

Potent 4-aryl- or 4-arylalkyl-substituted 3-isoxazolol GABA(A) antagonists: synthesis, pharmacology, and molecular modeling.

We have previously described a series of competitive GABA(A) antagonists derived from the low-efficacy partial agonist 5-(4-piperidyl)-3-isoxazolol (4-PIOL, 4). The 2-naphthylmethyl analogue, 4-(2-naphthylmethyl)-5-(4-piperidyl)-3-isoxazolol (5), provided affinity for the GABA(A) receptor site higher than that of the standard GABA(A) receptor antagonist, SR 95531 (3). Molecular modeling studies of these compounds exposed a cavity at the receptor recognition site capable of accommodating aromatic groups of substantial size in the 4-position in the 3-isoxazolol ring. Here we present a series of analogues of 5, with various substituents in different positions in the naphthyl ring system (6a-k), and compounds with aromatic substituents directly attached to the 4-position of the 3-isoxazolol ring (7l-n). The compounds have been pharmacologically characterized using receptor-binding assays and electrophysiological whole-cell patch-clamp techniques. All of the tested compounds show affinity for the GABA(A) receptor site. While the 5-, 7-, and 8-bromo analogues, 6b-d, showed receptor affinities (K(i) = 45, 109, and 80 nM, respectively) comparable with that of 5 (K(i) = 49 nM), the 1-bromo analogue, 6a, provided the highest receptor affinity of the series (K(i) = 10 nM). Introduction of a series of different substituents in the 1-position in the 2-naphthyl ring system led to compounds, 6e-k, with retained high affinity for the GABA(A) receptor (K(i) = 16-250 nM). Introduction of a phenyl ring directly into the 4-position on the 3-isoxazolol ring gave a 41-fold increase in affinity relative to that of 4-PIOL. In whole-cell patch-clamp recordings from cultured cerebral cortical neurons, all of the tested compounds were able to inhibit the effect of the specific GABA(A) agonist isoguvacine, 6a showing antagonist potency (IC(50) = 42 nM) markedly higher than that of 3 (IC(50) = 240 nM). Molecular modeling studies, based on the compounds described, emphasized the importance of the distal ring in 5 for receptor affinity and the considerable dimensions of the proposed receptor cavity. Furthermore, the phenyl rings in 7l and in 6k were shown to represent highly favorable positions for an aromatic ring in previously unexplored receptor regions in terms of a pharmacophore model.

GABAA receptor partial agonists and antagonists: structure, binding mode, and pharmacology.

A high degree of structural heterogeneity of the GABAA receptors (GABAARs) has been revealed and is reflected in multiple receptor subtypes. The subunit composition of GABAAR subtypes is believed to determine their localization relative to the synapses and adapt their functional properties to the local temporal pattern of GABA impact, enabling phasic or tonic inhibition. Specific GABAAR antagonists are essential tools for physiological and pharmacological elucidation of the different type of GABAAR inhibition. However, distinct selectivity among the receptor subtypes (populations) has been shown for only a few orthosteric ligands. Still, these examples show that it is indeed possible to obtain orthosteric subtype selectivity and they serve as models for further development in the orthosteric GABAAR ligand area. This review presents the very few existing structural classes of orthosteric GABAAR antagonists and describes the development of potent antagonists from partial agonists originally derived from the potent GABAAR agonist muscimol. In this process, several heterocyclic aromatic systems have been used in combination with structural models in order to map the orthosteric binding site and to reveal structural details to be used for obtaining potency and subtype selectivity. The challenges connected to functional characterization of orthosteric GABAAR partial agonists and antagonists, especially with regard to GABAAR stoichiometry and alternative binding sites are discussed. GABAAR antagonists have been essential in defining the tonic current but both remaining issues concerning the GABAARs involved and the therapeutic possibilities of modulating tonic inhibition underline the need for GABAAR antagonists with improved selectivity.

GABA(A) receptor ligands and their therapeutic potentials.

The GABA(A) receptor system is implicated in a number of neurological diseases, making GABA(A) receptor ligands interesting as potential therapeutic agents. Only a few different classes of structures are currently known as ligands for the GABA recognition site on the GABA(A) receptor complex, reflecting the very strict structural requirements for GABA(A) receptor recognition and activation. Within the series of compounds showing agonist activity at the GABA(A) receptor site that have been developed, most of the ligands are structurally derived from the GABA(A) agonists muscimol, THIP or isoguvacine. Using recombinant GABA(A) receptors, functional selectivity has been shown for a number of compounds such as the GABA(A)agonists imidazole-4-acetic acid and THIP, showing highly subunit-dependent potency and maximal response. In the light of the interest in partial GABA(A) receptor agonists as potential therapeutics, structure-activity studies of a number of analogues of 4-PIOL, a low-efficacy partial GABA(A) agonist, have been performed. In this connection, a series of GABA(A) ligands has been developed showing pharmacological profiles from moderately potent low-efficacy partial GABA(A) agonist activity to potent and selective antagonist effect. Only little information about direct acting GABA(A) receptor agonists in clinical studies is available. Results from clinical studies on the effect of the GABA(A) agonist THIP on human sleep pattern shows that the functional consequences of a direct acting agonist are different from those seen after administration of GABA(A) receptor modulators.