GABAA Receptor Channel Pharmacology

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GABAA Receptor Channel Pharmacology Graham A.R. Johnston* The Adrien Albert Laboratory of Medicinal Chemistry, Department of Pharmacology, The University of Sydney, NSW 2006, Australia Abstract: GABA A receptor channels are ubiquitous in the mammalian central nervous system mediating fast inhibitory neurotransmission by becoming permeant to chloride ions in response to GABA. The emphasis of this review is on the rich chemical diversity of ligands that influence GABAA receptor function. Such diversity provides many avenues for the design and development of new chemical entities acting on GABAA receptors. There is also a significant diversity of GABAA receptor subtypes composed of different protein subunits. The discovery of subtype specific agents is a major challenge in the continuing development of GABAA receptor pharmacology. Leads for the discovery of new chemical entities that influence GABAA receptors come from using recombinant GABAA receptors of known subunit composition as has been elegantly demonstrated by the refining of benzodiazepine actions with α1 subunit preferring agents showing sedative properties but not anxiolytic properties. The most recent advances in the therapeutic use of agents acting on GABAA receptors concern the promotion of sound sleep. Many herbal medicines are used to promote sleep and many of their active ingredients include flavonoids and terpenoids known to modulate GABAA receptor function.

INTRODUCTION GABA (γ-aminobutyric acid), the major inhibitory neurotransmitter in the brain, is essential for the overall balance between neuronal excitation and inhibition that is vital to normal brain function. Too much inhibition, or too little excitation, can lead to coma, depression, low blood pressure, sedation or sleep. Too much excitation, or too little inhibition, can result in a range of conditions including convulsions, anxiety, high blood pressure, restlessness and insomnia. Either imbalance in the extreme can result in death. The exact symptoms depend on what regions of the brain are involved and exactly what nerve cells are out of balance. Restoration of the balance between excitation and inhibition is a major aim of therapies that target GABAmediated neuronal inhibition. GABA produces neuronal inhibition by acting on an amazing diversity of membrane-bound receptors. These receptors can be divided into two major types: ionotropic receptors that are ligand-gated ion channels (GABAA and GABAC receptors), and metabotropic receptors that are Gprotein coupled receptors (GABAB receptors) that act via second messengers [1, 2]. The ionotropic GABA receptors belong to the nicotinicoid superfamily of ligand-gated ion channels as described by Le Novere and Changeux [3] that includes nicotinic acetylcholine, strychnine-sensitive glycine and 5HT3 receptors. The family of ionotropic GABA receptors is divided into two subfamilies, GABAA and GABAC receptors, on the basis of their ability to form endogenous heteromeric and homomeric receptors respectively, and differences in their physiological and pharmacological properties [1], although GABAC receptors are sometimes classified as subtypes of GABAA receptors *Address correspondence to this author at the The Adrien Albert Laboratory of Medicinal Chemistry, Department of Pharmacology, The University of Sydney, D06, Sydney, NSW 2006, Australia; Tel: 61 2 9351 6117; Fax: 61 2 9351 2891; E-mail: [email protected] 1381-6128/05 $50.00+.00

[4]. There is also diversity in GABAB metabotropic GABA receptors that are heteromeric dimers [5]. There is evidence for the existence of functional GABA A, GABAB and GABAC receptors, as well as strychnine-sensitive glycine receptors, on a single population of retinal ganglion cells [6]. Like other members of the nicotinicoid superfamily of ligand-gated ion channels, ionotropic GABA receptors are considered to consist of 5 protein subunits arranged around a central pore that constitutes the actual ion channel [1]. Each subunit has a large extracellular N-terminal domain which incorporates part of the agonist/antagonist binding site, followed by three membrane spanning domains (M1-3), an intracellular loop of variable length and a fourth membrane spanning domain (M4), with the C-terminal end being extracellular. Each subunit arranges itself such that the second membrane-spanning domain (M2) forms the wall of the channel pore and the overall charge of the domain determines whether the channel conducts anions or cations. Both GABAA and GABAC receptors are GABA-gated chloride ion channels causing inhibition of neuronal firing, with GABAA receptors being heteromeric, i.e. made up of different subunits (e.g. α1, β2 and γ2 subunits) and GABAC receptors being homomeric (e.g. made up exclusively of either ρ1, ρ2 or ρ3 subunits; in addition ‘pseudoheteromeric’ GABAC receptors made up of ρ1 and ρ2 subunits have been described). The cytoplasmic loop, between the third and fourth transmembrane domains (M3 and M4), is believed to be the target for protein kinases, required for subcellular targeting and membrane clustering of the receptor. There are 16 different subunits comprising the GABAA receptor family: α1-6, β1-3, γ1-3, δ, ε, π and θ [7]. In addition, there are splice variants of many of these subunits. If all of these subunits could co-assemble to form functional pentameric receptors the total number of GABAA receptors would be huge. Even if the combinations were restricted to those containing two α, two β and one other subunit, then more than 2000 different GABAA receptors could exist [8]. © 2005 Bentham Science Publishers Ltd.

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In fact, studies of native GABAA receptors suggest that there may be less than 20 widely occurring GABAA receptor subtype combinations, with the major combinations being α1β2/3γ2, α3β3γ2 and α2β3γ2 [7, 9]. This review is directed at some recent highlights, together with a re-evaluation of some older data, relevant to GABAA receptors as therapeutic targets with an emphasis on the chemical diversity of ligands that influence GABAA receptor function. Such diversity provides many avenues for the design and development of new chemical entities acting on GABAA receptors. There are many reviews on aspects GABAA receptors including methodological approaches to the study of GABAA receptors [10, 11], GABAA receptor subtypes [7, 12-15], neurosteroid modulation [16, 17], drug interactions [18], specific agonists and partial agonists [19], receptor recycling and regulation [20], novel modulators [21], medicinal chemistry [1, 22], and analysis of GABAA receptors through mouse genetics [23]. GABAC receptors as therapeutic targets have been the subject of a recent review [24]. THERAPEUTIC USES OF AGENTS ACTING ON GABAA RECEPTORS Agents acting on GABAA receptors have widespread therapeutic use as anaesthetics, anticonvulsants, anxiolytics and sedative-hypnotics. In the main, these agents act to increase GABA-mediated synaptic inhibition either by directly activating GABAA receptors or, more usually, by enhancing the action of GABA on GABAA receptors. This latter action is known as positive modulation [8] and is considered to involve agents acting on allosteric sites on GABAA receptors remote from the GABA recognition sites (orthosteric sites). Such allosteric sites are regarded as good targets for the development of subtype specific drugs since there is generally greater diversity between receptor subtypes in amino acid sequence at allosteric sites than at orthosteric sites [25]. Agents that reduce the action of GABA on GABAA receptors are known as negative allosteric modulators (once known as ‘inverse agonists’); they have the opposite actions to those of the classical benzodiazepines. Agents that block the actions of both positive and negative allosteric modulators are known as neutralising allosteric modulators, e.g. the classical benzodiazepine ‘antagonist’ flumazenil [8]. Benzodiazepines and barbiturates are examples of widely used therapeutic agents that act as positive allosteric modulators at GABAA receptors. A rich chemical diversity of agents acting GABAA receptors is known [1]. The discovery of subtype specific GABAA receptor agents is a major challenge in the continuing development of therapeutic agents acting on specific wild type and mutant GABAA receptors. DISORDERS INVOLVING GABAA RECEPTORS Not surprisingly, GABA as the major inhibitory neurotransmitter is involved, directly or indirectly, in many disorders of brain function. The major disorders for which GABAA receptors represent important therapeutic targets include anxiety disorders, cognitive disorders, epilepsies, moods disorders, schizophrenia and sleep disorders.

Graham A.R. Johnston

Heritable mutations are known to occur across the nicotinicoid superfamily of ligand-gated ion channels including GABAA receptors [26]. Angelman syndrome, a neurodevelopmental disorder characterised by severe mental retardation, epilepsy and delayed motor development has been associated with deletions of GABAA receptor β3 subunits [27]. GABAA receptor β3 knockout mice have epilepsy and a phenotype with marked similarities to Angelman syndrome [28, 29]. GABA systems have been implicated in the pathogenesis of anxiety, depression and insomnia. These symptoms are part of the core and comorbid psychiatric disturbances in post-traumatic stress disorder (PTSD). In a study of PTSD patients, heterozygosity of β3 GABAA receptor subunits was associated with higher levels of anxiety, insomnia, social dysfunction and depression than found in homozygosity [30]. There is increasing evidence for GABA abnormalities in mood disorders [31] and in alcohol dependence [32, 33]. Some of the neurological symptoms of guanidinoacetate methyltransferase deficiency may be due to the partial agonist action of increased levels of guanidinoacetate on GABAA receptors [34]. The use of herbal medicines to treat depression may be associated with actions on GABAA receptors [35]. GABAA receptor ligands have a potential role in the treatment of schizophrenia [36, 37], acute ischaemic stroke [38], tinnitus [39] and impaired cognition [40]. Epilepsies Studies on the genetics of human epilepsies show that epilepsy syndromes that have monogenic inheritance are associated with mutations that encode subunits of voltage gated and ligand gated ion channels [41]. Heritable mutations in GABAA receptor subunits are strongly implicated in idiopathic generalised epilepsies [42]. Mutations in γ2 GABAA receptor subunits have been described in two families with generalised epilepsy syndromes [43, 44]. One mutation associated with febrile seizures and generalised epilepsy is in the extracellular loop connecting the TM2 and TM3 domains. Studies using recombinant receptors in frog oocytes revealed that this mutant showed smaller GABAactivated currents than did receptors lacking this mutation [43]. The other mutation was in the distal part of the N terminus thought to make up part of the benzodiazepine binding pocket and was associated with childhood absence epilepsy and febrile seizures. Studies in frog oocytes showed that this mutation had diminished sensitivity to positive allosteric modulation by benzodiazepines [44]. These findings have been questioned recently as a result of patch clamp studies using a mammalian expression system (HEK cells) that showed that the TM2-TM3 loop γ2 GABAA receptor mutation resulted in faster deactivation rates, while the N terminus mutant reduced current amplitude without altering benzodiazepine sensitivity [45]. Mutations in intracellular regions of γ2 GABAA receptor subunits between TM1 and TM2 and between TM3 and TM4 have also been associated with epilepsies. It appears that all of these mutations in γ2 GABAA receptor subunits may result in diminished synaptic inhibition mediated by GABAA receptors and epilepsy by diverse mechanisms [46]. In addition to

GABAA Receptor Channel Pharmacology

these mutations, a γ2 GABAA receptor subunit splice site mutation has been associated with childhood absence epilepsy and febrile convulsions, but it is not known how this influences GABAA receptor properties [47]. An association analysis has shown that polymorphism in the γ2 GABAA receptor gene is a susceptibility factor for febrile seizures [48]. A mutation in the TM3 region of the α1 GABAA receptor subunit that influences both the efficacy and affinity of GABA has been associated with juvenile myoclonic epilepsy [49]. Studies on recombinant receptors containing this TM3 α1 mutation show that it results in reduced channel open time with no change in single channel conductance [50]. Although heritable epilepsies represent only a small fraction of epilepsies, the observed mutations associated with heritable epilepsies provide clues as to possible problems with GABAA receptors implicated in other epilepsies. They also provide targets for pharmacogenomic therapies using drugs acting on specific mutant GABAA receptors.

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different pharmacological actions of benzodiazepines result from interactions with different GABAA receptor subtypes was pivotal to these studies [14]. O N

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The treatment of insomnia is regarded as a developing market for agents acting on GABAA receptors. Drugs currently used to treat insomnia include zolpidem (Ambien), zaleplon (Sonata) and zopiclone (Imovane). These drugs, Fig. (1), show some selectivity for α1 subunit containing GABAA receptors, acting as positive allosteric modulators. The structurally related indiplon, Fig. (1), which is in phase III clinical trials for the treatment of insomnia, acts in a similarly selective manner [54, 55]. Also in phase III clinical trials is gaboxadol (THIP), a directly acting GABAA receptor partial agonist, discussed in Section 5, Fig. (3), that interacts with a GABAA receptor population that is insensitive to benzodiazepines, zolpidem, zaleplon, zolpidem and indiplon [56]. Many herbal preparations are used to promote sleep. For example, chamomile tea contains the flavonoid apigenin (see Section 7.1) which has been shown to enhance the positive allosteric modulating effects of benzodiazepines on GABAA receptors [24] and Valerian contains a variety of agents (see Section 7.2) that act on GABAA receptors [57]. GABAA RECEPTOR SUBUNITS The importance of drug interactions with receptor subtypes made up of specific protein subunits has been highlighted by a number of recent findings involving genetically modified mice and ligands that show some selectivity for particular receptor subtypes. The finding that

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Sleep Disorders GABA systems are known to play an important role in sleep and positive allosteric modulators of GABAA receptors are widely used to promote restful sleep [51]. Two observations indicate the importance of β3 GABA A receptor subunits in sleep. Oleamide, an endogenous sleep promoting fatty acid, is inactive in β3 GABAA receptor subunit knockout mice [52]. A mutation in β3 GABAA receptor subunits has been described in a patient with chronic insomnia. Functional characterisation of this mutant showed a slower rate of desensitisation compared with normal GABAA receptors [53].

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Indiplon

Fig. (1). Structures of sedative/hypnotic substances that interact preferentially with α1 subunit containing GABAA receptors.

It must be remembered, however, that it is not only the nature of the protein subunits that influence the diversity of function of GABAA receptor subtypes in vivo. Major contributors to this diversity are: presynaptic factors including release probability and number of release sites; factors that determine synaptic GABA transients in the cleft, including diffusion and the actions of GABA transporters; and postsynaptic factors, including GABA receptor subtypes, their location and number, their modulation by endogenous and exogenous factors, and their interactions with postsynaptic-anchoring proteins [58]. The transport, clustering and turnover of GABA A receptors are known to be influenced by a variety of proteins}. [20]. The phosphorylation state of the subunits is very important with GABAA receptors as with many other ligand-gated ion channels [59]. For example, the sensitivity of GABAA receptors to ethanol is dependent on receptor phosphorylation; mice lacking protein kinase Cε show increased sensitivity to ethanol while those lacking protein kinase Cγ show decreased sensitivity [59]. Furthermore, there are many endogenous ligands that influence GABAA receptor function in vivo including metal ions such zinc [60], steroids [17], and chemicals derived from our diet such as flavonoids [57]. Influence of α1 and α2 Subunits – Sedative and Anxiolytic Actions of Benzodiazepines Benzodiazepines are considered to act on GABAA receptors at a binding pocket at the interface between the γ2 subunit and α subunits that contain a conserved histidine residue in the benzodiazepine binding domain on the

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Graham A.R. Johnston

extracellular N-terminus (α1, α2, α3 and α5 subunits). Mutation of this histidine to an arginine results in GABAA receptors that are insensitive to benzodiazepines in vitro [61]. GABAA receptors containing α4 or α6 subunits are relatively insensitive to benzodiazepines.

α1 subtypes of GABAA receptors are being developed, e.g. 3-heteroaryl-2-pyridones [67]. Recently a series of 3-phenyl6-(2-pyridyl)methoxy-1, 2, 4-triazolo[3, 4-a]phthalazines have been developed with Compound 62, Fig. (2), showing binding selectivity for α2, α3 and α5 over α1 subunits and acting as an anxiolytic in the rat elevated plus maze [68]. It showed a good pharmacokinetic profile making it a useful tool to explore the effect of a GABAA α2/α3 selective agonist in vivo. The sedative effects of the α1-selective agent zolpidem are diminished in the α1 knock-in mouse, consistent with sedation being mediated via α1-containing GABAA receptors [69].

Knock-in point mutations in the genes that code for α1 or α2 subunits produced mice that had different responses to benzodiazepines. Mice with the mutant α1 subunits showed the normal anxiolytic responses to benzodiazepines but not the sedative effects [62, 63]. The reverse was true for mice with the mutant α2 subunits showing sedative but not anxiolytic effects in responses to benzodiazepines [64]. While there is general agreement on the importance of the α1 GABAA receptor subunit in the sedative actions of benzodiazepines, there has been some doubt on the relative contributions of the α2 and α3 subunits to the anxiolytic action due to confounding effects on locomotor activity that influence the assessment of anxiety [65]. Furthermore, in profiling the influence of a range of agents on benzodiazepine binding differences in functional activity need to be taken into account [66]. Ultimately, validation of GABAA receptor subtype-selective drugs needs to be carried out using genetically modified mice [12]. Drugs selective for α2-containing GABAA receptors (found in about 15% of diazepam-sensitive GABA A receptors) would be expected to be anxiolytics with greatly reduced sedative effects compared to the non-selective benzodiazepines currently in clinical use [14]. The non-sedative anxiolytic L-838, 417, Fig. (2), is a neutralising modulator at α1 containing GABAA receptors but is a positive modulator at α2, α3 and α5 containing GABAA receptors [63]; this agent thus offers a double pronged approach to anxiolysis without sedation by enhancing the action of GABA at α2, α3 and α5 containing GABAA receptors while diminishing the action of endogenous benzodiazepines on α1 containing GABAA receptors. New chemical entities with functional selectivity for α2 over

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Importance of α5 Subunits in Spatial Memory GABAA receptor α5 subunits account for less than 5% of GABAA receptors in the brain. They are localised mainly to the hippocampus where they may play a key role in cognitive processes by controlling a component of synaptic transmission in the CA1 [72]. Mice lacking the α5 gene show improved performance in the Morris water maze model of spatial learning, whereas the performance in nonhippocampal-dependent learning and in anxiety tasks were unaltered in comparison with wild-type controls [73]. Novel selective α5 negative allosteric modulators, e.g. 6, 6 - dimethyl - 3 - (2 - hydroxyethyl) thio - 1 - (thiazol - 2 - yl) - 6, 7 dihydro-2-benzothiophen-4(5H)-one (Compound 43, Fig. (2)) have been developed that enhance spatial learning but lack the convulsant or proconvulsant activity associated with non-selective GABAA receptor negative allosteric modulators [74].

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Quinolone antibiotics have long been known to interact with receptors for GABA and other neurotransmitters [70]. Modification of norfloxacin has yielded molecules such as Compound 4, Fig. (2), that positively modulates GABAA receptors with α2 subunit selectivity and is a non-sedating anxiolytic [71].

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Fig. (2). Structures of substances showing selectivity for GABAA receptors containing specific subunits.

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GABAA Receptor Channel Pharmacology

Furosemide and α6-Subunits The loop diuretic furosemide, Fig (2), has been described as ‘the most receptor-subtype specific’ allosteric modulator acting on GABAA receptors [75]. Furosemide acts as a negative allosteric modulator of α6 subunit containing GABAA receptors. Such GABAA receptors are largely restricted to the cerebellum and show low sensitivity to the classic GABAA antagonist bicuculline and to positive modulation by diazepam, but can be influenced by other benzodiazepine receptor ligands [76, 77]. Furosemide exhibits approximately 100-fold selectivity for α6 containing receptors over α1 containing receptors. It also acts on α4 containing receptors. Mutation of a threonine to a isoleucine in the TM1 region of α1 subunits increases furosemide sensitivity by 20-fold [78]. Structure-activity studies show that the diuretic properties of agents related to furosemide are distinct from the α6 GABAA receptor negative modulation [79]. The positive allosteric modulator (+)ROD188 shows selectivity for α6 GABAA receptors [80]. The GABAC receptor agonist cis-4-aminocrotonic acid also acts as a bicuculline-sensitive agonist at recombinant α6β2γ2S GABAA receptors that are insensitive to the GABAC receptor antagonist TPMPA [81]. Niflumic acid, a nonsteroidal anti-inflammatory drug, acts as an antagonist at recombinant α6β2 receptors in a manner similar to that of furosemide, but also acts as a positive modulator at α1β2γ2 receptors [82]. Gene knockout of the α6 subunit in mice resulted in an associated inhibition of δ subunit expression without influence on exploratory activity in the open field or learning in a horizontal wire task [83]. Other studies showed the lack of effect of α6 knockout on responses to ethanol, pentobarbital and general anaesthetics [84]. In a rotating rod test, however, α6 knockout mice were significantly more impaired by diazepam than were wild-type mice [85]. This diazepam-induced ataxia in α6 knockout mice could be reversed by flumazenil, indicating the involvement of the remaining α1β2/3γ2 GABAA receptors on the cerebellar granule cells. This led to the conclusion that α6 subunitdependent actions in the cerebellar cortex could be compensated by other receptor subtypes; but, if not for the α6 subunit, patients on benzodiazepine medication would suffer considerably from ataxic side-effects [85]. There is electrophysiological evidence for the coexistence of α1 and α6 subunits in a single functional GABAA receptor [86], for furosemide-sensitive and furosemide-insensitive GABAmediated effects on cerebellar granule cells [87] and for a tonic diazepam-sensitive GABA-mediated inhibition on cultured rodent cerebellar granule cells [88]. Allelic variants in α6 GABAA subunits are associated with abdominal obesity and cortisol secretion [89]. In a study of 100 patients of the effects of midazolam, a point mutation (Pro385Ser) in the α6 GABAA receptor subunit did not affect baseline sedation, anxiety or memory, but significantly attenuated the anxiolytic affect of low-dose midazolam [90]. Anaesthesia and Sedation Involving β2 and β3 Subunits The intravenous general anaesthetic etomidate provides another example of distinct actions involving different GABAA receptor subtypes, in this case involving β2 and β3

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subunits. Using genetically modified mice with etomidateinsensitive β2 subunits, it was shown that in wild-type mice etomidate produces sedation via the β2 subunit and anaesthesia via the β3 subunit [91]. Furthermore, the recovery of function in the genetically modified mice was considerably improved after etomidate anaesthesia suggesting that β3 selective agents could be used as anaesthetics with significantly improved recovery profile [91]. The β2 subunit has been shown to mediate the hypothermic effect of etomidate [92]. Loreclezole and mefenamic acid show similar selectivity to etomidate with respect to β2 and β3 subunits [93]. Salicylidene salicylhydrazide, Fig. (2), has been shown to be a selective inhibitor of GABAA receptors that contain β1 subunits and thus may be a useful agent with which to study β subunit selectivity [94]. Splice Variants of γ2 Subunits and Sedation The γ2 GABA A receptor subunit is generally considered to be vital to the classical actions of benzodiazepines on GABAA receptors. Alternate splicing results in two splice variants, a short (γ2S) and a long (γ2L) variant. Mice lacking the γ2L variant are more sensitive to the sedative effects of midazolam and zolpidem, while responses to etomidate and barbiturates are unchanged [95]. It is suggested that the lack of the γ2L variant may shift the state of α1-containing GABAA receptors from a negative allosteric modulator preferring conformation towards a positive allosteric modulator preferring conformation. Ethanol and δ Subunits The importance of the δ GABAA receptor subunit has been highlighted by the discovery that ethanol at low concentrations known to affect humans enhances the action of GABA on α4β3δ and α6β3δ receptor subtypes [96]. Reproducible ethanol enhancement of GABA responses occurred at 3 mM, i.e. concentrations that are reached with moderate ethanol consumption producing blood-ethanol levels well below the legal level for driving in most countries. Ethanol has been long known to influence the functioning of a variety of receptors usually at concentrations in excess of 50 mM. This had been true for recombinant GABAA receptors [97] until the studies on δ subunit containing receptors. The δ subunits appear to associate almost exclusively with α4 and α6 subunits forming functional receptors that are 50 fold more sensitive to GABA and desensitise more slowly than receptor subtypes that do not contain δ subunits [83, 98]. The δ subunit protein is expressed in brain regions expressing α4 (high in thalamus, dentate gyrus, striatum and outer cortical layers and low in hippocampus) and α6 subunit proteins (cerebellum) and appears to be associated with extrasynaptic rather than synaptic GABA A receptors [99]. Knocking out the δ subunit gene in mice reduces their sensitivity to neurosteroids [100] and increases their susceptibility to seizures [101]. Knocking out the α6 subunit gene in mice does not alter their susceptibility to ethanol [84]. GABAA RECEPTOR AGONISTS Muscimol and THIP, Fig. (3), are widely used as selective GABA A receptor agonists [1]. However, they have

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potent actions on GABAC receptors which mean that interpretation of studies with these agents should be treated with some caution. No “selective” GABAA receptor agonist is known that does not have significant action on either GABAB and/or GABA C receptors. Muscimol, a conformationally restricted analogue of GABA in which a hydroxyisoxazole moiety replaces the carboxyl group of GABA [102], is more potent at GABAC receptors than at GABAA receptors [103]. THIP and Ionotropic GABA Receptors THIP (Gaboxadol, 4, 5, 6, 7-tetrahydroisoxazolo(5, 4-c) pyridin-3-ol), Fig. (3), is a conformationally restricted analogue of muscimol [102]. It is a potent GABAA receptor partial agonist of high efficacy [104] that has proved to be a moderately potent GABAC receptor antagonist [103]. Unlike GABA, both muscimol and THIP pass the blood-brain barrier on systemic administration [105]. Muscimol is psychoactive, while THIP is a potent analgesic. Side effects of THIP (including sedation, dizziness, and blurred vision) meant that it had too low a therapeutic index to be therapeutically useful as an analgesic [106, 107]. There is renewed interest in THIP with respect to sleep therapy [19] as it produces slow wave sleep and reduces spindling activity in non rapid eye movement sleep in humans [108]. It does appear that receptors other than classical benzodiazepine-sensitive, bicuculline-sensitive GABAA receptors are involved in the effects of THIP on pain perception and sleep. THIP-induced analgesia is not sensitive to bicuculline indicating that GABAA receptors are not involved [109]. The GABAC receptor antagonist action of THIP may contribute to its analgesic action [24]. The analgesic action of THIP in rats is blocked by subconvulsant doses of picrotoxinin [110], a known GABAC receptor antagonist. Benzodiazepine-sensitive GABAA receptors do not appear to be involved in the effects of THIP on sleep patterns [108]. The GABAC receptor antagonist TPMPA has been used to probe the involvement of GABAC receptors in sleep-waking behaviour [111]. The binding of THIP to rat brain membranes, unlike that of GABA and muscimol, is not stimulated by diazepam [112]. THIP was devoid of the anticonvulsant and antiepileptogenic effects shown by diazepam and alphaxalone in pentamethylenetetrazolekindled mice [113]. Clinical studies with THIP have indicated that sleep quality improving effects are obtained at plasma concentrations of the order of 1 µM [108]. THIP shows considerable variation in potency on recombinant receptors: THIP acts on α1β3γ2S recombinant GABAA receptors expressed in oocytes as a partial agonist (EC50 350µM) and more potently and as a full agonist on α5β3γ3 (EC50 40 µM) and α5β3γ3 (EC50 29 µM) recombinant receptors [114]. On α4β3γ2 recombinant receptors THIP acts as a partial agonist (EC50 102 µM) and on α4β3δ as a ‘superagonist’ (EC50 6 µM) [115]. On recombinant GABAC receptors THIP acts as an antagonist (Kb 32 µM for ρ1 [103] and 10 µM for ρ3 receptors [116]. On this basis, α4β3δ GABAA and ρ3 GABAC receptors are the most likely GABA

Graham A.R. Johnston

receptors to respond to clinically relevant 1 µM plasma concentrations of THIP. Studies on the interactions between THIP, benzodiazepines and ethanol in the rat cortical wedge preparation provide evidence for THIP acting on benzodiazepineinsensitive GABAA receptors in intact tissue possibly containing α4 subunits [56]. Rotarod studies on the effects of THIP on motor performance in rats showed a lack of crosstolerance with benzodiazepines [117]. In neither study did ethanol show a potentiation of the effects of THIP [56, 117]. This is interesting in view of the recent findings discussed in section 4.5. of the sensitivity of α4β3δ GABA A receptors to ethanol [96] and may suggest that this receptor subtype is not involved in some of the actions of THIP. Studies on the effects of THIP in δ-subunit knockout mice may help sort out the role of α4β3δ GABAA receptors in THIP-induced analgesia and sleep. The rat cortical wedge preparation has yielded evidence of the importance of GABAA receptor ligands acting at extrasynaptic receptors [118]. In this preparation THIP acted as a full agonist with an EC50 of 8 µM [118]. In contrast, an unusually low activity of THIP has been reported on GABA receptors on isolated rat dorsal roots, a tissue that certainly does not contain any synapses [119]. THIP was 20 times weaker than GABA in depolarising these dorsal roots but at least 20 times more potent than GABA is depressing overall spontaneous synaptic activity in the rat hemisected spinal cord as recorded in the ventral roots. As in the rat cortical wedge preparation, the actions of THIP in the hemisected spinal cord may involve extrasynaptic GABA receptors, but the results in isolated dorsal roots clearly show that not all extrasynaptic GABA receptors show high sensitivity to activation by THIP. Interestingly, extrasynaptic GABAA receptor channels in hippocampal slices are known to be modulated by diazepam [120]. OH H2N O

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GABAA Receptor Channel Pharmacology

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4-PIOL as a Low Efficacy Partial GABAA Receptor Agonist

became apparent that a wide range of natural and synthetic steroids have anaesthetic actions.

Partial agonists offer certain advantages over full agonists [19]. Full agonists may induce receptor desensitisation that can lead to tolerance and subsequent withdrawal symptoms. The non-fused THIP analogue 4-PIOL, Fig. (3) (5-(4-piperidyl)isoxazol-3-ol), is a low efficacy GABAA receptor partial agonist that exhibits a predominantly antagonist profile [19]. Its activity varies with different recombinant GABAA receptor subtypes and in the rat cortical wedge preparation, 4-PIOL behaves as a high efficacy partial agonist [118]. Patch clamp studies on hippocampal neurones show that 4-PIOL is a non-desensitising partial agonist whose action can be potentiated by benzodiazepines and barbiturates [121]. Studies on analogues of 4-PIOL substituted in the 4-position of the isoxazole ring yielded GABAA receptor antagonists of increased potency, e.g. 4naphthyl-methyl-4-PIOL, Fig. (3), with a linear correlation between the lipophilicity of the 4-subsitutent and antagonist activity, providing evidence for a hydrophobic binding pocket at the GABA recognition site [122].

The synthetic steroid anaesthetic alphaxalone, Fig. (4), was the first steroid shown to act as a positive modulator of GABAA receptors [128]. This was followed by the discovery that steroid hormone metabolites, e.g. 3α-hydroxy-5αpregnan-20-one, Fig, (4), that occur in the brain are ‘barbiturate-like modulators’ of the GABAA receptor [129]. This led to the concept that neurosteroids can directly modulate GABAA receptors on the cell surface rather than acting on receptors in the nucleus regulating gene expression. Steroids produced outside the brain are also important modulators of GABAA receptors. For example, THDOC, Fig (4), 3α, 21-dihydroxy-5α-pregnan-20-one, is a ‘neuroactive steroid’ because the sole source of this steroid appears to be the adrenals. Nonetheless, THDOC is found in the brain where its concentration is increased during stress [130]. 3α-Hydroxy-5α-pregnan-20-one and THDOC are among the most potent known steroid modulators of GABA A receptors. O

O

“Superagonists” at GABAA Receptors Studies on a series of GABA amides revealed substances that could act as partial, full or superagonists as assessed by the stimulation of chloride influx into mouse brain synaptoneurosomes in a bicuculline- and picrotoxinin-sensitive manner [123]. Compound 5b (N, N’-1, 4-butanediylbis [4-aminobutanamide]), Fig. (3), produced a maximum response that was 150% that of GABA and was thus descried as a “superagonist”. It showed similar affinity to THIP, a partial agonist with a maximum response 65% that of GABA. The apparent ‘superagonist’ action of compound 5b could mean that GABA is in fact a partial agonist in these experiments. While the exact nature of this “superagonist” action remains to be determined in functional assays using recombinant GABAA receptors of known subunit composition, the concept of ‘superagonists’ opens up another possible approach to therapeutic agents acting at GABAA receptors. THIP has been shown to display superagonist behaviour at α4β3δ receptors with a maximum response 160% that of GABA [124].

OH

HO

THDOC O

O O

HO

HO

H Alphaxalone

H Ganaxolone

O HO

The CNS depressant action of steroids has been known since 1927 when it was shown that injection of a colloidal suspension of cholesterol into cats caused deep anaesthesia [125]. Subsequently, cholesterol was found to potentiate the anaesthetic actions of pentobarbitone [126], but it was not until the extensive investigations of Seyle [127] that it

H

5α-Pregnan-3α-ol-20-one

STEROIDS THAT INFLUENCE GABAA RECEPTORS A variety of steroids are known to influence GABAA receptors via non-genomic actions that are rapid in onset and offset. These neuroactive steroids include neurosteroids (i.e. steroids that are synthesised in the brain), sex steroids that originate in the gonads, and corticosteroids that are made in the adrenal cortex, together with a range of synthetic steroids and steroid analogues [16]. Most interest is centred on neuroactive steroids that act as potent positive allosteric modulators and have anxiolytic, anticonvulsant, analgesic, anaesthetic and sedative actions [17].

HO

H

OH OH OH

O

O Cortisol

Nandrolone

Fig. (4). Steroids that act on GABAA receptor function.

Studies using 3α-hydroxy-5α-pregnan-20-one indicate that its positive modulatory actions on GABAA receptors are only modestly influenced by the α-, β- or γ-subunits, [131]. The inclusion of either an ε or δ subunit however dramatically alters the response with the ε subunit reducing and the δ subunit augmenting the efficacy of modulation by 3αhydroxy-5α-pregnan-20-one [131]. The enhanced efficacy of δ subunit containing GABAA receptors has also been reported for THDOC [132]. As noted above, knocking out

1874 Current Pharmaceutical Design, 2005, Vol. 11, No. 15

the δ subunit gene in mice reduces their sensitivity to neurosteroids [100]. These findings clearly distinguish steroid positive modulation of GABAA receptors from flumazenilsensitive positive modulation by benzodiazepines but do suggest some similarities with the modulation induced by volatile anaesthetics and ethanol. Experiments with alphaxalone on chimeric GABAA receptors indicate that the site of action for steroids is not the same as that for volatile anaesthetics and ethanol [133]. Studies comparing the positive modulator effects on α6β3γ2L GABA A receptors of 4 structurally distinct general anaesthetics – propofol, pentobarbitone, etomidate and 3α-hydroxy-5α-pregnan-20one – showed that the action of all but 3α-hydroxy-5αpregnan-20-one depended critically on a single amino acid in TM2 [134]. A novel neuroactive steroid, 6-aza-3α-hydroxy-5βpregnan-20-one, has been used to photoaffinity label rat brain membranes [135]. It labelled a protein identified as voltage-dependant anion channel-1 (VDAC-1) that coimmunoprecipitated with the β2 and β3 subunits of the GABAA receptor, suggesting that neuroactive steroids may modulate GABA A receptor function by binding to VDAC-1 as an accessory protein [135]. Further studies using VDAC-1 deficient mice have suggested that VDAC-1 is unlikely to be involved in steroid modulation of GABAA receptors [136]. In addition to alphaxalone, there are a number of synthetic steroids that are known to modulate GABAA receptor function. Anabolic steroids such as nandrolone, Fig. (4), and stanazolol induce region- and subunit-specific rapid modulation of GABAA receptor-mediated currents in the rat forebrain [137]. The antiepileptic agent, ganaxolone, Fig. (4), belongs to a novel class of neuroactive steroids called epalons which specifically modulate GABAA receptors in the central nervous system (CNS). Chemically related to progesterone but devoid of any hormonal activity, the epalons have potent antiepileptic, anxiolytic, sedative and hypnotic activities in animals [138]. Ganaxolone has demonstrated outstanding efficacy and better tolerability in children with intractable infantile spasms [139]. It has, however, been reported to exacerbate absence seizures in animal models [140]. Cortisol, Fig. (4), is a potent bidirectional modulator of the action of GABA on GABAA receptors in the guinea-pig ileum enhancing at low (1-10 pM) concentrations and inhibiting at higher (10-1000 nM) concentrations [141]. Cortisone is a potent non-competitive inhibitor of these GABAA receptors acting at concentrations as low as 1 pM [142]. These corticosteroids are thus the most potent agents modulating GABAA receptors. The actions of cortisol may be restricted to particular GABAA receptor subtypes since cortisol has little effect on GABAA responses in the rat cuneate nucleus [143]. Biphasic effects of corticosteroids have been described on TBPS binding to rat brain membranes, low (nM) concentrations enhancing binding and higher (µM) concentrations inhibiting, the effect of nM concentrations indicative of an antagonist action as observed at these concentrations on GABA responses in the guineapig ileum [144]. Cortisol (10 µM) has been shown to rapidly increase the spontaneous firing frequency of neurones in rat paraventricular nucleus and to inhibit whole cell potassium

Graham A.R. Johnston

currents, suggesting the cortisol may act indirectly via inactivating potassium channels [145]. High affinity binding sites (nM) for corticosterone have been described on brain membranes [146], and corticosterone is known to influence the expression and activity of GABAA receptors in the hippocampus [147]. Given the risk of memory decline in patients on corticosteroids [148], further investigations of the effects of these steroids on GABAA receptors seem warranted. NATURAL PRODUCTS AND GABAA RECEPTORS With increasing community acceptance of herbal medicines and functional foods there is increasing interest in natural products that may influence brain function. There is a view that natural substances are inherently safer than unnatural substances, i.e. synthetic chemicals. This view is mistaken as many of the most toxic chemicals are in fact natural products and the majority of therapeutically beneficial drugs are synthetic. It is the molecular structure and dose that determine the effects of substances on human health, not whether they are of natural or synthetic origin [149]. There is now an impressive array of natural products in addition to steroids that are known to influence GABAA receptor function including substances found in beverages such as tea, red wine and whiskey, and in herbal preparations including Ginkgo biloba and Ginseng [57]. Natural substances represent a rich diversity in chemical structures that can lead to the development of new therapeutic agents. Flavonoids and GABAA Receptors Flavonoids are found in all plants in high abundance and exhibit a considerable chemical diversity with more than 5,000 different flavonoids having been described. Fruits, vegetables, and beverages such as tea and red wine are major sources of flavonoids our diet [150]. It has been estimated that the average daily intake of flavonoids is 1-2 g [151]. Many flavonoids are polyphenolic and are thus strongly antioxidant [152]. They have a wide variety of biological activities and are being studied intensively as anticancer agents [153]. Flavonoids have a range of activities on GABAA receptors [154]. Flavonoids were first linked to GABAA receptors when three isoflavans isolated from bovine urine were shown to inhibit diazepam binding to brain membranes [155]. The most potent compound was 3’, 7-dihydroxyisoflavan, Fig. (5), with an IC50 of 45 µM. Further studies searching for diazepam-like substances using benzodiazepine binding assays led to the discovery that the biflavonoid amentoflavone, Fig. (5), sometimes known as biapigenin, was capable of displacing benzodiazepine binding to rat brain membranes with a nM affinity comparable to that of diazepam [156]. These investigations used amentoflavone isolated from Karmelitter Geist, an alcoholic tincture of various plants used to treat anxiety and epilepsy. However it was concluded that amentoflavone cannot be responsible for any pharmacological effects of the plant extract as amentoflavone did not influence flunitrazepam binding in the brain in vivo following i.v. administration to mice [156]. It was suggested that amentoflavone was either rapidly metabolised or did not cross the blood brain barrier, but a

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recent study does indicate that amentoflavone does cross the blood brain barrier [157]. Amentoflavone occurs in a variety of herbal preparations including St John’s wort [158] and Ginkgo biloba [159]. A comprehensive battery of in vitro binding assays has shown that amentoflavone influences a variety of G-protein coupled receptors for serotonin, dopamine and opioids at nM concentrations while having no effect on the binding of muscimol to GABAA receptors [160]. Using recombinant α1β2γ2L GABAA receptors expressed in oocytes, amentoflavone has been shown recently to be a relatively weak (4 µM) negative allosteric modulator of GABA action acting independently of classical flumazenil-sensitive benzodiazepine modulatory sites [159]. These studies on amentoflavone illustrate the difficulties of studying flavonoid actions – the variety of effects, the lack of selectivity, the need for functional assays and the mismatch between in vitro and in vivo findings.

used for classical benzodiazepines’ and it was devoid of anticonvulsant effects [161]. These finding are in contrast to a later study in rats where apigenin was shown to reduce the latency of onset of picrotoxin-induced convulsions and to reduce locomotor activity but was devoid of anxiolytic or muscle relaxant activities [162]. This later study showed that apigenin could reduce GABA-activated chloride currents in cultured cerebellar granule cells, an action that could be blocked by flumazenil and thus likely to involve classical benzodiazepine allosteric sites on GABAA receptors. The inhibitory action of apigenin on locomotor behaviour, however, could not be blocked by flumazenil and thus could not ‘be ascribed to an interaction with GABAA-benzodiazepine receptors, but to other neurotransmitter systems’ [162]. Another study from the same group reported that apigenin exerted sedative effects on locomotor activity in rats in a flumazenil-insensitive manner, whereas chrysin, a structurally related flavonoid lacking the 4’-hydroxy substituent of apigenin, showed a clear flumazenil-sensitive anxiolytic effect in addition to the flumazenil-insensitive sedation [163]. The apparent discrepancy between the behavioural effects of apigenin on mice [161] and rats [162] may be due to mice having higher baseline levels of anxiety.

Apigenin, Fig. (5), a component of Matricicaria recutita flowers (chamomile), has been characterised as a centrallyacting benzodiazepine ligand with anxiolytic effects [161]. Infusions of chamomile flowers are widely used as a tea to promote sleep. Apigenin competitively inhibited (Ki 4 µM) the binding of flunitrazepam to brain membranes without influencing the binding of muscimol to GABAA receptors. Apigenin was described as having ‘a clear anxiolytic effect in mice in the elevated plus maze without evidencing sedation or muscle relaxation effects at doses similar to those HO

Recent studies on recombinant receptors in oocytes have shown that µM apigenin inhibited the activation of α1β1γ2S GABAA receptors in a flumazenil-insensitive manner and had a similar effect on ρ1 GABAC receptors [164]. Other

O

HO

O

OH OH 3',7-Dihydroxyisoflavan

O

OH

Genistein OH

OH O HO

O

O HO

OH

OH

O Amentoflavone R2

HO

O

O R1

R1 OH

O

R1=H, R2=OH Apigenin R1=MeO, R2=OH Hispidulin R1=MeO, R2=H Oroxylin A R1=Me, R2=OH 6-Methylapigenin

Fig. (5). Flavonoids that act on GABAA receptors.

NO 2

Q O

R=NO2 3',6-Dinitroflavone R=Cl 6-Chloro-3'-nitroflavone

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studies on recombinant α1β2γ2L GABA A receptors describe an inhibitory effect of apigenin on GABA responses and, in addition, describe an enhancement of the diazepam-induced positive allosteric modulation of GABA responses by apigenin [57, 165]. Such a second order modulation by apigenin of benzodiazepine modulation of the activation by GABA of GABAA receptors may indicate that apigenin needs to work through an endogenous benzodiazepine system to influence behaviour in a flumazenil-sensitive manner. Overall, it seems that the effects of apigenin on GABAA receptors are complex and involve both flumazenilsensitive and flumazenil–insensitive components, and that other receptors could be involved in the behavioural effects of apigenin. Genistein, Fig. (5), the isoflavone equivalent of apigenin, is a phytoestrogen with a wide variety of pharmacological effects on animal cells [166]. It is widely used as a tyrosine kinase inhibitor but its action as a negative modulator of the action of GABA on recombinant GABAA receptors is the result of a direct action on the receptors and is independent of tyrosine kinase [167, 168]. Hispidulin, Fig. (5), 4’, 5, 7-trihydroxy-6-methoxyflavone, i.e. the 6-methoxy derivative of apigenin), was isolated together with apigenin from Salvia officinalis (Sage) recently using a benzodiazepine binding assay-guided fractionation [169]. Hispidulin was some 30 times more potent that apigenin in displacing flumazenil binding. Preparations of sage have been used in herbal medicine to assist memory [170, 171] and an extract of Salvia lavandulaefolia (Spanish sage) has been shown to enhance memory in healthy young volunteers [172]. Hispidulin has been shown to act as a positive allosteric modulator of α1, 3, 5, 6β2γ2S GABAA receptor subtypes showing little subtype selectivity being a little more potent at α1, 2, 5β2γ2S subtypes than at α3, 6β2γ2S subtypes [173]. The positive modulatory action of 10 µM hispidulin at α1β2γ2S receptors was reduced from 47% to 17% by flumazenil, indicating that sites other than classical flumazenil-sensitive benzodiazepine sites were involved in the action of hispidulin. As hispidulin did not influence the action of GABA on α1β2 GABAA receptors, hispidulin does not interact with low affinity flumazenilinsensitive benzodiazepine sites [174] in contrast to other flavonoids such as 6-methylflavone [175]. Of significance is the ability of hispidulin to act as a positive modulator at α6β2γ2L GABAA receptors unlike diazepam; 10 µM hispidulin enhanced the action of GABA at these receptors by 65%, this action being reduced by 1 µM flumazenil to 37% [173]. Hispidulin was shown to have an anticonvulsant action in seizure prone Mongolian gerbils and to pass the blood brain barrier [173]. Flavonoids structurally related to hispidulin, and that influence benzodiazepine binding, have been isolated from Scutellaria baicalensis, an important herb in traditional Chinese medicine [176]. Oroxylin A, Fig. (5), 5, 7-dihydroxy-6-methoxyflavone, i.e. hispidulin lacking the 4’-hydroxy group), inhibits flunitrazepam binding at 1 µM and on oral administration as a neutralising allosteric modulator blocking the anxiolytic, myorelaxant and motor incoordination effects, but not the sedative and anticonvulsant effects elicited by diazepam [177]. 6-Methylapigenin, Fig. (5), 4’,5,7-dihydroxy-6-methylflavone) isolated from Valeriana wallichii, a known sedative herb, influences benzodiazepine binding at 0.5 µM in manner suggesting it

Graham A.R. Johnston

may be a positive modulator of GABAA receptors [178]. Thus, flavones substituted in the 6-position with a methoxy or methyl substituent have interesting effects on GABAA receptor function and may contribute to the properties of some herbal preparations. Natural and synthetic 2’-hydroxysubstituted flavones are also of interest [179]. Several flavonoid glycosides including goodyerin [180], linarin and hesperidin [181] are also being studied as sedative and anticonvulsant agents likely to interact with GABAA receptors. Using a combinatorial chemistry approach, a range of relatively simple flavones have been synthesised and evaluated initially for activity in a benzodiazepine binding assay [182]. This approach led to some very interesting compounds that were further evaluated in a variety of pharmacological tests as GABA A receptor ligands [154]. The most active anxiolytic flavone was 3’, 6-dinitroflavone, Fig. (5), which was 30 times more potent than diazepam. It was orally active and had minimal sedative action at anxiolytic doses. In contrast, 6-chloro-3’-nitroflavone, Fig. (5), had no anxiolytic properties and abolished the anxiolytic, anticonvulsant and amnesic effects of diazepam [154]. There have been extensive structure-activity studies aimed as developing models of flavonoid pharmacophores for their interaction with GABAA receptors [183-187]. A problem common to most of these studies is that the activity data is based on ligand binding studies to what is now known to be a mixture of benzodiazepine binding sites. Given our increased knowledge of the diversity of benzodiazepine and flavonoid actions on cloned receptors of defined subunit composition, future structure-activity studies need to be based on data from functional studies on GABA receptors of known subunit composition [175]. Terpenoids and GABAA Receptors Terpenoids are widespread in plants, especially in what are known as essential oils that can be extracted from plants and have a wide range of uses from perfume constituents to paint thinners. Terpenoids are oxygenated products formally derived from C5 isoprene units and are classified by the number of C5 units in their structure. Thus monoterpenoids have 2xC5 units, sesquiterpenoids 3xC5 units, diterpenoids 4xC5 units and triterpenoids 6xC5 units. The most widely used terpenoid in studies on GABAA receptors is the sesquiterpenoid lactone picrotoxinin, Fig. (6), a noncompetitive antagonist at GABAA receptors [1]. A number of other terpenoids, however, are of interest for their actions on GABAA receptors. Bilobalide, Fig. (6), a sesquiterpenoid lactone from Ginkgo biloba that bears some structural similarities to picrotoxinin, including a lipophilic side chain and a hydrophilic cage, is also a non-competitive antagonist at GABAA receptors [188]. Both bilobalide and picrotoxinin appear to act at sites in the chloride channel of GABAA receptors and are thus negative allosteric modulators. The cognition-enhancing effects of Ginkgo extracts may be partly mediated by bilobalide acting to enhance hippocampal pyramidal neuronal excitability [189]. While picrotoxinin is a convulsant, bilobalide is an anticonvulsant [189, 190]. As with the α5 subunit preferring negative allosteric modulator

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O O

O

H

HO

OH O

O

O

H

O O

O Picortoxinin

O O

OH

H

Bilobalide

O

O HO

α-Thujone

O

Thymoquinone

Thymol COOH

HO

HO

O

H (+)-Borneol

Valerenic acid

lsocurcumenol

Fig. (6). Terpenoids that act on GABAA receptors.

mentioned in Section 4.2, the lack of convulsant action in an agent that reduces GABA action may be important for enhancement of cognition. The lack of convulsant action of bilobalide may result from subunit selectivity but this has yet to be established. The structurally-related ginkgolides, especially ginkgolide B, also act as negative modulators at GABAA receptors [191]. They also inhibit strychninesensitive glycine receptors and platelet activating factor [191, 192]. Bilobalide and the ginkgolides reduce barbiturate-induced sleeping time in mice, an effect perhaps relevant to the clinically observed ‘vigilance-enhancing’ and antidepressant-like actions of Ginkgo extracts [193]. The monoterpenoid α-thujone, Fig. (6), is a psychoactive component of absinthe, a liqueur popular in France in the 19th and early 20th centuries. It is found in extracts of woodworm and some other herbal medicines and beverages since ancient Egyptian times [194]. α-Thujone is a convulsant that acts as a negative allosteric modulator of GABAA receptors [195]. It also acts as an antagonist of 5HT3 receptors by influencing agonist-induced desensitisation [194]. The structurally-related substance thymol, Fig. (6), a constituent of thyme essential oil, is a flumazenil-insensitive positive allosteric modulator of GABAA receptors [196]. At higher concentrations, thymol had a direct action on GABAA receptors similar to that of the anaesthetic propofol and other phenols [197]. The anticonvulsant effects of thymoquinone, the major constituent of Nigella sativa seeds, may be due to positive modulation of GABAA receptors [198].

(+)-Borneol, Fig. (6), a monoterpenoid found in many essential oils, is a flumazenil-insensitive positive allosteric modulator of recombinant GABAA receptors of low affinity but very high efficacy producing 12 fold enhancement of the action of 10 µM GABA at a concentration of 450 µM [57, 199]. (+)-Borneol is found in high concentrations in extracts of Valerian officinalis that are widely used to reduce the latency of sleep onset, the depth of sleep and the perception of well-being. Extracts of Valerian are known to contain a large number of constituents including flavonoids and terpenoids, many of which are considered to be active at GABAA receptors. The sesquiterpenoid valerenic acid, Fig. (6), has a direct partial agonist action on GABAA receptors [200]. Isocurcumenol, Fig. (6), a sesquiterpenoid from Cyperus rotundus, was found to inhibit [H-3]Ro15-1788 binding and enhance [H-3]flunitrazepam binding in the presence of GABA in a manner consistent with it acting as a positive allosteric modulator [201]. Ginsenosides, triterpenoid glycosides that are the major active constituents of Panax ginseng, are known to negatively modulate nicotinic and NMDA receptor activity. Of a series of ginsenosides, ginsenoside Rc was the most potent (EC50 53 µM) in enhancing the action of GABA on recombinant α1β1γ2S GABAA receptors expressed in oocytes [202]. Sage and GABAA Receptors Sage has been used widely to treat memory deficits and extracts of Salvia lavandulaefolia (Spanish Sage) have been

1878 Current Pharmaceutical Design, 2005, Vol. 11, No. 15

Graham A.R. Johnston

shown to enhance memory in healthy young volunteers [172]. In addition to the flavonoids apigenin, hispidulin and linarin (see section 7.1.), a number of terpenoids have been extracted from varieties of Salvia (sage) that influence benzodiazepine binding [169]. The diterpenoid lactone galdosol, Fig. (7) from the common sage Saliva officinalis, inhibited flumazenil binding at 0.8 µM [169]. The diterpenoid quinone miltirone, Fig. (7), from the Chinese medicinal herb Salvia miltriorrhiza, inhibited flunitrazepam binding at 0.3 µM and was orally active in animal models as a tranquilliser without muscle relaxant properties [203]. Structure-activity studies on miltirone led to the development of a synthetic compound that was much more potent than miltirone on flunitrazepam binding (IC50 0.05 µM) [204].

receptors in addition to the flavonoids, terpenoids and ethanol discussed in previous sections. GABA itself occurs widely in plants being involved in pH regulation, nitrogen storage, plant development and defence [207]. High levels of GABA in plants extracts may be a confusing factor in evaluating the effects of such extracts on GABAA receptors in binding or functional assays, but such GABA will not normally influence GABAA receptors in the brain on ingestion due to the blood brain barrier.

may

Tea and coffee contain a range of chemicals in addition to GABA that have been shown to influence recombinant bovine α1β1 GABA A receptors. Extracts of green, oolong or black tea contained catechins, especially (-)-epicatechin gallate and (-)-epigallocatechin gallate, that inhibited GABA responses and alcohols, such as leaf alcohol and linalool, Fig. (8), that enhanced GABA responses at concentrations of 1 mM [208]. Coffee extracts contained theophylline, which inhibited GABA responses in a non-competitive mechanism (Ki 0.55 mM), and theobromine, which inhibited in a competitive manner (Ki 3.8 mM), while a number of compounds including 1-octen-3-ol and sotolone, Fig. (8), enhanced GABA responses [209]. When 1-octen-3-ol (100 mg/kg) was orally administered to mice prior to intraperitoneal administration of pentobarbitone, the sleeping time of mice induced by pentobarbital increased significantly [209]. Sotolone is a key component in the “nutty” and “spicy-like” aroma of oxidative aged port wine [210]. Many components in the fragrance of whiskey, in particular ethyl 3-phenylpropanoate, Fig. (8), strongly enhanced GABAA responses [211]. When applied to mice through respiration, ethyl 3-phenylpropanoate delayed the onset of convulsions induced by pentylenetetrazole. The extract of other alcoholic drinks such as wine, sake, brandy, and shochu also potentiated GABA responses to varying degrees [211]. Although these fragrant components are present in alcoholic drinks at low concentrations (extremely small quantities compared with ethanol), they may also modulate the mood or consciousness through the potentiation of GABAA responses after absorption into the brain because these hydrophobic fragrant compounds are easily absorbed into the brain through the blood-brain barrier and are several thousands times as potent as ethanol in the potentiation of GABAA receptor-mediated responses [211]. The aging of whiskey results in enhanced potency of the fragrance in potentiating GABAA responses and in prolonging pentobarbitone-induced sleeping time in mice [212]. As all of the above tests on chemicals from tea, coffee and whiskey were carried out on recombinant bovine α1β1 GABAA receptors, the observed effects are independent of classical benzodiazepine-sensitive sites. Several perfume constituents have been shown to act as positive modulators of GABAA receptors including the terpenoids eugenol, citronellol and hinokitol, Fig. (8) [213]. The tea flavonoid (-)-epigallocatechin gallate has been shown to be some 10 times more potent than apigenin as a second order modulator of the positive modulation by diazepam of α1β2γ2L human recombinant GABAA receptors expressed in oocytes [165].

There are many chemicals in food and beverages, together with other chemicals in our environment, that are known to be capable of influencing the function of GABAA

Simple disaccharides are able to enhance TBOB binding to GABAA receptors [214]. Lactose (EC50 1.5 µM) was 100600 fold more potent than maltose or sucrose. Lactose did not influence flunitrazepam binding and its effects on TBOB

The structurally-related diterpenoids carnosic acid and carnosol, Fig. (7), extracted from Salvia officinalis, while not influencing diazepam or muscimol binding, did inhibit TBPS binding [205]. This suggests that, like flavonoids, diterpenoids can influence GABAA receptors in a manner independent of classical benzodiazepine sites and could be missed in benzodiazepine binding assays. The structures of galdosol, carnosic acid and carnosol, Fig. (7), contain the oisopropylphenolic moiety that is present in thymol, Fig. (6), and the anaesthetic agent propofol. O O

OH HO

O O Miltirone

Galdosol

OH HO

OH HO

HO2 C O O Carnosic acid

Garnosol

Fig. (7). Diterpenoids from Salvia that influence GABAA receptors.

Sage also contains α-thujone, Fig. (6), a known GABAA receptor antagonist as noted above, which may influence the GABA enhancing effects of hispidulin, galdosol, miltirone, carnosic acid, carnosol and related compounds in sage extracts. The levels of α-thujone in individual sage plants are known to vary considerably [206]. Dietary and Environmental Influence GABAA Receptors

Chemicals

that

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Current Pharmaceutical Design, 2005, Vol. 11, No. 15

OH

OH

HO

1879

HO Leaf alcohol

Citronellol

OMe

Linalool

1-Octen-3-ol

O

O

O

HO

EtO

O OH

Eugenol

Ethyl 3-phenylpropanoate

Hinekitol

HO Sotolone

Fig. (8). Volatile substances in beverages and perfumes that influence GABAA receptor.

binding could be blocked by GABA. Regional differences in the potency of lactose enhancement of TBOB binding suggest that the effect might be GABAA receptor subtype selective [214]. As noted in section 6, cholesterol has been long known to produce deep anaesthesia in cats following injection of a colloidal suspension [125] and to potentiate pentobarbitoneinduced anaesthesia [126]. Dietary cholesterol and agents that alter cholesterol levels may influence GABAA receptor function in the brain. Alterations in membrane cholesterol in dissociated hippocampal neurones alters GABAA receptor properties [215]. Cholesterol enrichment increased the positive modulatory effects of the nonsteroidal agents propofol, flunitrazepam and pentobarbitone but reduced the positive modulatory effects of the steroids pregnanolone and alphaxalone. Depletion of membrane cholesterol increased the effects of pregnanolone and alphaxalone without influencing the effects of the nonsteroidal modulators. Increases in dietary cholesterol in rats has been shown to depress brain waves as measured by EEG [216], an effect that may be attributable to changes in GABAA receptor function. There has been speculation about an association between brain cholesterol and Alzheimer’s disease and the suggestion that cholesterol-lowering strategies influence the progression of this disease [217]. There is some evidence that statins can reduce cholesterol turnover in the brain thus enabling statins to reduce the incidence of Alzheimer’s disease [218]. Such treatments might influence GABAA receptor function through alteration of cholesterol levels in the brain. Inhaled drugs of abuse such as the solvents toluene, 1, 1, 1-trichloroethane and trichlorethylene act as positive modulators of GABAA receptors, acting in a manner suggesting that their sites of action may overlap with those of ethanol and volatile anaesthetics [219]. Another study, however, found toluene to be an antagonist of the activation of GABAA receptors [220]. CONCLUSIONS When I reviewed GABAA receptor pharmacology in 1996 [8] the then literature prompted a conclusion that there appeared to be at least 11 distinct sites on GABAA receptors for interactions with specific ligands. The likely sites were:

(1) agonist/partial agonist/competitive antagonist recognition sites; (2) picrotoxinin sites; (3) sedative-hypnotic barbiturate sites; (4) neuroactive steroid sites; (5) benzodiazepine sites; (6) ethanol sites; (7) sites for inhalation anaesthetics; (8) sites for furosemide associated with α6 subunits; (9) sites for Zn2+; (10) sites for a variety of divalent cations, such as Ca2+, Sr2+, Ba2+, Cd2+, Mn 2+, and Mg2+; and (11) sites for La3+. I noted that it was likely that there were subtypes of neuroactive steroid sites and that there were certainly subtypes of benzodiazepine sites. In addition, I noted that there were possibly sites associated with (a) phospholipids interacting with GABAA receptor protein subunits, (b) cyclic nucleotide protein kinase activity involved phosphorylation of the intracellular loop of some GABAA receptor protein subunits, and (c) the interaction of GABAA receptors and microtubules that may anchor receptor clusters at postsynaptic membranes. The situation has become even more complex since 1996. Thanks to the use of genetically modified mice we now know the importance of the different types of GABAA receptor subunits for the actions of particular agents – e.g. α1 and α2 subunits for the sedative and anxiolytic actions of benzodiazepines respectively, α5 subunits for agents influencing spatial memory, and δ subunits for the potent action of ethanol. Thanks to the use of recombinant receptor technology, expressing GABAA receptors of known subunit composition, we now have detailed knowledge of the actions of the increasingly chemically diverse range of natural products on GABAA receptor function. We know of flavonoids that influence GABAA receptor function as positive and negative allosteric modulators, some of these actions of flavonoids being sensitive to the classical benzodiazepine antagonist flumazenil and other actions being insensitive. It is a similar story with terpenoids. Thus it is likely that there are at least two distinct sites, flumazenilsensitive and flumazenil-insensitive, on GABAA receptors for flavonoids and terpenoids of which the flumazenilsensitive sites may overlap with classical benzodiazepine sites. It appears likely that the various proposed sites on GABAA receptors overlap and interact making it difficult to put a meaningful figure on the number of distinct sites though my 1996 figure of 11 now seems very conservative. We await detailed three-dimensional structural information

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on the various subtypes of GABAA receptors in order to work out exactly where all of these chemically diverse ligands interact. Such information is starting to emerge through homology modelling based on a 4Å resolution structure of a nicotinic receptor [221], e.g. the structure of the proposed propofol binding site involving the M2 and M3 regions of GABAA receptors [222]. The diversity of sites on GABAA receptors represent targets for the further development of specific agents acting on particular GABAA receptor subtypes. The structures of the various ligands described in this and other reviews serve as leads for the discovery of new chemical entities for the treatment of disorders involving specific GABAA receptors.

Graham A.R. Johnston [17] [18] [19] [20] [21] [22] [23]

ACKNOWLEDGEMENTS The author is grateful to his many colleagues who have contributed to his studies on GABAA receptors including Robin Allan, Erica Campbell, Mary Chebib, Rujee Duke, Renee Granger, Belinda Hall, Jane Hanrahan, Shelley Huang, Povl Krogsgaard-Larsen, Ken Mewett, Hue Tran and Paul Whiting, and to the Australian National Health and Medical Research Council and Polychip Pharmaceuticals for financial support.

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