Mary Chebib

The 'ABC' of GABA receptors: a brief review.

1. In the mammalian central nervous system, GABA is the main inhibitory neurotransmitter. GABA is a highly flexible molecule and, thus, can exist in many low‐energy conformations. Conformationally restricted analogues of GABA have been used to help identify three major GABA receptors, termed GABAA, GABAB and GABAC receptors.

Flavonoid modulation of GABAA receptors

There has been a resurgence of interest in synthetic and plant‐derived flavonoids as modulators of γ‐amino butyric acid‐A (GABAA) receptor function influencing inhibition mediated by the major inhibitory neurotransmitter GABA in the brain. Areas of interest include (i) flavonoids that show subtype selectivity in recombinant receptor studies in vitro consistent with their behavioural effects in vivo, (ii) flumazenil‐insensitive modulation of GABAA receptor function by flavonoids, (iii) the ability of some flavonoids to act as second‐order modulators of first‐order modulation by benzodiazepines and (iv) the identification of the different sites of action of flavonoids on GABAA receptor complexes. An emerging area of interest is the activation of GABAA receptors by flavonoids in the absence of GABA. The relatively rigid shape of flavonoids means that they are useful scaffolds for the design of new therapeutic agents. Like steroids, flavonoids have wide‐ranging effects on numerous biological targets. The challenge is to understand the structural determinants of flavonoid effects on particular targets and to develop agents specific for these targets.

Alpha9 nicotinic acetylcholine receptors and the treatment of pain.

Chronic pain is a vexing worldwide problem that causes substantial disability and consumes significant medical resources. Although there are numerous analgesic medications, these work through a small set of molecular mechanisms. Even when these medications are used in combination, substantial amounts of pain often remain. It is therefore highly desirable to develop treatments that work through distinct mechanisms of action. While agonists of nicotinic acetylcholine receptors (nAChRs) have been intensively studied, new data suggest a role for selective antagonists of nAChRs. alpha-Conotoxins are small peptides used offensively by carnivorous marine snails known as Conus. A subset of these peptides known as alpha-conotoxins RgIA and Vc1.1 produces both acute and long lasting analgesia. In addition, these peptides appear to accelerate the recovery of function after nerve injury, possibly through immune mediated mechanisms. Pharmacological analysis indicates that RgIA and Vc1.1 are selective antagonists of alpha9alpha10 nAChRs. A recent study also reported that these alpha9alpha10 antagonists are also potent GABA-B agonists. In the current study, we were unable to detect RgIA or Vc1.1 binding to or action on cloned GABA-B receptors expressed in HEK cells or Xenopus oocytes. We review the background, findings and implications of use of compounds that act on alpha9* nAChRs.(1).

The β-amyloid protein of Alzheimer’s disease binds to membrane lipids but does not bind to the α7 nicotinic acetylcholine receptor

Accumulation of the amyloid protein (Aβ) in the brain is an important step in the pathogenesis of Alzheimer’s disease. However, the mechanism by which Aβ exerts its neurotoxic effect is largely unknown. It has been suggested that the peptide can bind to the α7 nicotinic acetylcholine receptor (α7nAChR). In this study, we examined the binding of Aβ1‐42 to endogenous and recombinantly expressed α7nAChRs. Aβ1‐42 did neither inhibit the specific binding of α7nAChR ligands to rat brain homogenate or slice preparations, nor did it influence the activity of α7nAChRs expressed in Xenopus oocytes. Similarly, Aβ1‐42 did not compete for α‐bungarotoxin‐binding sites on SH‐SY5Y cells stably expressing α7nAChRs. The effect of the Aβ1‐42 on tau phosphorylation was also examined. Although Aβ1‐42 altered tau phosphorylation in α7nAChR‐transfected SH‐SY5Y cells, the effect of the peptide was unrelated to α7nAChR expression or activity. Binding studies using surface plasmon resonance indicated that the majority of the Aβ bound to membrane lipid, rather than to a protein component. Fluorescence anisotropy experiments indicated that Aβ may disrupt membrane lipid structure or fluidity. We conclude that the effects of Aβ are unlikely to be mediated by direct binding to the α7nAChR. Instead, we speculate that Aβ may exert its effects by altering the packing of lipids within the plasma membrane, which could, in turn, influence the function of a variety of receptors and channels on the cell surface.

GABAC Receptors as Drug Targets

GABA(C) receptors are the least studied of the three major classes of GABA receptors. The physiological roles of GABA(C) receptors are still being unravelled and the pharmacology of these receptors is being developed. A range of agents has been described that act on GABA(C) receptors with varying degrees of specificity as agonists, partial agonists, antagonists and allosteric modulators. Pharmacological differences are known to exist between subtypes of cloned GABA(C) receptors that have been cloned from mammalian sources. There is evidence for functional GABA(C) receptors in the retina, spinal cord, superior colliculus, pituitary and gastrointestinal tract. Given the lower abundance and less widespread distribution of GABA(C) receptors in the CNS compared to GABA(A) receptors, GABA(C) receptors may be a more selective drug target than GABA(A) receptors. The major indications for drugs acting on GABA(C) receptors are in the treatment of visual, sleep and cognitive disorders. The most promising leads are THIP, a GABA(C) receptor antagonist in addition to its well known activity as a GABA(A) receptor partial agonist, which is being evaluated for sleep therapy, and CGP36742, an orally active GABA(B) and GABA(C) receptor antagonist, which enhances cognition. Analogues of THIP and CGP36742, such as aza-THIP, that are selective for GABA(C) receptors are being developed. TPMPA and related compounds such as P4MPA, PPA and SEPI are also important leads for the development of systemically active selective GABA(C) receptor antagonists.

Novel, Potent, and Selective GABAC Antagonists Inhibit Myopia Development and Facilitate Learning and Memory

This study reports pharmacological and physiological effects of cis- and trans-(3-aminocyclopentanyl)butylphosphinic acid (cis- and trans-3-ACPBPA). These compounds are conformationally restricted analogs of the orally active GABAB/C receptor antagonist (3-aminopropyl)-n-butylphosphinic acid (CGP36742 or SGS742). cis-[IC50(ρ1) = 5.06 μM and IC50(ρ2) = 11.08 μM; n = 4] and trans-3-ACPMPA [IC50(ρ1) = 72.58 μM and IC50(ρ2) = 189.7 μM; n = 4] seem competitive at GABAC receptors expressed in Xenopus laevis oocytes, having no effect as agonists (1 mM) but exerting weak antagonist (1 mM) effects on human GABAA and GABAB receptors. cis-3-ACPBPA was more potent and selective than the trans-compound, being more than 100 times more potent at GABAC than GABAA or GABAB receptors. cis-3-ACPBPA was further evaluated on dissociated rat retinal bipolar cells and dose-dependently inhibited the native GABAC receptor (IC50 = 47 ± 4.5 μM; n = 6). When applied to the eye as intravitreal injections, cis- and trans-3-ACPBPA prevented experimental myopia development and inhibited the associated vitreous chamber elongation, in a dose-dependent manner in the chick model. Doses only 10 times greater than required to inhibit recombinant GABAC receptors caused the antimyopia effects. Using intraperitoneal administration, cis- (30 mg/kg) and trans-3-ACPBPA (100 mg/kg) enhanced learning and memory in male Wistar rats; compared with vehicle there was a significant reduction in time for rats to find the platform in the Morris water maze task (p < 0.05; n = 10). As the physiological effects of cis- and trans-3-ACPBPA are similar to those reported for CGP36742, the memory and refractive effects of CGP36742 may be due in part to its GABAC activity.

α4βδ GABA(A) receptors are high-affinity targets for γ-hydroxybutyric acid (GHB).

γ-Hydroxybutyric acid (GHB) binding to brain-specific high-affinity sites is well-established and proposed to explain both physiological and pharmacological actions. However, the mechanistic links between these lines of data are unknown. To identify molecular targets for specific GHB high-affinity binding, we undertook photolinking studies combined with proteomic analyses and identified several GABAA receptor subunits as possible candidates. A subsequent functional screening of various recombinant GABAA receptors in Xenopus laevis oocytes using the two-electrode voltage clamp technique showed GHB to be a partial agonist at αβδ- but not αβγ-receptors, proving that the δ-subunit is essential for potency and efficacy. GHB showed preference for α4 over α(1,2,6)-subunits and preferably activated α4β1δ (EC50 = 140 nM) over α4β(2/3)δ (EC50 = 8.41/1.03 mM). Introduction of a mutation, α4F71L, in α4β1(δ)-receptors completely abolished GHB but not GABA function, indicating nonidentical binding sites. Radioligand binding studies using the specific GHB radioligand [3H](E,RS)-(6,7,8,9-tetrahydro-5-hydroxy-5H-benzocyclohept-6-ylidene)acetic acid showed a 39% reduction (P = 0.0056) in the number of binding sites in α4 KO brain tissue compared with WT controls, corroborating the direct involvement of the α4-subunit in high-affinity GHB binding. Our data link specific GHB forebrain binding sites with α4-containing GABAA receptors and postulate a role for extrasynaptic α4δ-containing GABAA receptors in GHB pharmacology and physiology. This finding will aid in elucidating the molecular mechanisms behind the proposed function of GHB as a neurotransmitter and its unique therapeutic effects in narcolepsy and alcoholism.

GABAC RECEPTOR ION CHANNELS

1. The present review gives an overview of studies conducted on GABAC receptors over the past 10 years since the author started at the University of Sydney. It concentrates on the structure–activity relationship profiles of the receptor and how these studies were used to: (i) develop selective GABAC receptor ligands; and (ii) understand the impact of amino acid changes on GABAC receptor pharmacology and function.

Structure and Activity of (2,8)-Dicarba-(3,12)-cystino α-ImI, an α-Conotoxin Containing a Nonreducible Cystine Analogue†

The alpha-conotoxins are potent and selective antagonists of nicotinic acetylcholine receptors (nAChR). Exploitation of these and other peptides in research and clinical settings has been hampered by the lability of the disulfide bridges that are essential for toxin structure and activity. One solution to this problem is replacement of cystine bridges with nonreducible dicarba linkages. We explore this approach by determining the solution structure and functional characteristics of a dicarba analogue of the alpha-conotoxin alpha-ImI, (2,8)-dicarba-(3,12)-cystino alpha-ImI. The structure of the dicarba analogue was similar to that of native alpha-ImI, with differences attributable to the different covalent geometry of the disulfide and dicarba bridges. Dicarba-alpha-ImI maintained inhibitory activity of nAChR comparable to that of native alpha-ImI in two in vitro assays. These findings confirm the potential of the dicarba linkage to improve stability while maintaining alpha-conotoxin function.

Modulation of Ionotropic GABA Receptors by Natural Products of Plant Origin

Publisher Summary This chapter discusses the modulation of ionotropic γ‐aminobutyric acid (GABA) receptors by natural products of plant origin. There is an impressive array of natural products that are known to influence the function of ionotropic receptors for GABA, the major inhibitory neurotransmitter in the brain. The major chemical classes of such natural products are flavonoids, terpenoids, phenols, and polyacetylenic alcohols. The interaction of flavonoids with benzodiazepine modulatory sites on GABAA receptors lead to the great interest in flavonoids as positive modulators of such receptors, many of the interactions between flavonoids and GABAA receptors do not involve classical flumazenil‐sensitive benzodiazepine sites. There are significant synergistic interactions between some of these positive modulators such as between substances isolated from Valeriana officinalis. Thus, the sleep inducing effects of hesperidin are potentiated by 6‐methylapigenin, while the sedating and sleep inducing effects of valerenic acid are potentiated when co‐administered with the flavonoid glycoside linarin. The discovery of second order positive modulators adds new dimension to the concept of the allosteric modulation of GABAA receptors. Second order positive modulators act only in conjunction with a specific first order positive modulator.