Glycine Receptor Drug Discovery.

Postsynaptic glycine receptor (GlyR) chloride channels mediate inhibitory neurotransmission in the spinal cord and brain stem, although presynaptic and extrasynaptic GlyRs are expressed more widely throughout the brain. In humans, GlyRs are assembled as homo- or heteromeric pentamers of α1-3 and ß subunits. GlyR malfunctions have been linked to a range of neurological disorders including hyperekplexia, temporal lobe epilepsy, autism, breathing disorders, and chronic inflammatory pain. Although it is possible that GlyRs may eventually be clinically targeted for a variety of neurological disorders, most research to date has focused on developing GlyR-targeted treatments for chronic pain. Inflammatory pain sensitization is caused by inflammatory mediators downregulating the magnitude of α3 GlyR-mediated inhibitory postsynaptic currents in spinal nociceptive neurons. Consistent with this paradigm, it is now well established that the selective enhancement of α3 GlyR current magnitude is effective in alleviating inflammatory pain. In this review, we briefly describe the physiological roles and pharmacological properties of GlyRs. We then outline the methods commonly used to discover new GlyR-active compounds and review recent progress, in our laboratory and elsewhere, in developing GlyR-targeted analgesics. We conclude that the eventual development of an α3 GlyR-targeted analgesic is an eminently feasible goal. However, in selecting or designing new therapeutic leads, we caution against the automatic exclusion of compounds with potentiating effects on α1 GlyRs. Also, as GlyRs are strongly potentiated by Zn2+ at nanomolar concentrations, we also caution against the identification of false positives caused by contaminating Zn2+ in otherwise pure compound samples.

Xenopus borealis as an alternative source of oocytes for biophysical and pharmacological studies of neuronal ion channels.

For the past 30 years, oocytes from Xenopus laevis have been extensively used to express and characterise ion channels in an easily controlled environment. Here we report the first use of oocytes from the closely related species Xenopus borealis as an alternative expression system for neuronal ion channels. Using the two-electrode voltage-clamp technique, we show that a wide variety of voltage- and ligand-gated ion channels have the same channel properties and pharmacological profiles when expressed in either X. laevis or X. borealis oocytes. Potential advantages of the X. borealis oocytes include a smaller endogenous chloride current and the ability to produce more intense fluorescence signals when studied with voltage-clamp fluorometry. Scanning electron microscopy revealed a difference in vitelline membrane structure between the two species, which may be related to the discrepancy in fluorescence signals observed. We demonstrate that X. borealis oocytes are a viable heterologous system for expression of neuronal ion channels with some potential advantages over X. laevis oocytes for certain applications.

Investigating ion channel conformational changes using voltage clamp fluorometry.

Ion channels are membrane proteins whose functions are governed by conformational changes. The widespread distribution of ion channels, coupled with their involvement in most physiological and pathological processes and their importance as therapeutic targets, renders the elucidation of these conformational mechanisms highly compelling from a drug discovery perspective. Thanks to recent advances in structural biology techniques, we now have high-resolution static molecular structures for members of the major ion channel families. However, major questions remain to be resolved about the conformational states that ion channels adopt during activation, drug modulation and desensitization. Patch-clamp electrophysiology has long been used to define ion channel conformational states based on functional criteria. It achieves this by monitoring conformational changes at the channel gate and cannot detect conformational changes occurring in regions distant from the gate. Voltage clamp fluorometry involves labelling cysteines introduced into domains of interest with environmentally sensitive fluorophores and inferring structural rearrangements from voltage or ligand-induced fluorescence changes. Ion channel currents are monitored simultaneously to verify the conformational status. By defining real time conformational changes in domains distant from the gate, this technique provides unexpected new insights into ion channel structure and function. This review aims to summarise the methodology and highlight recent innovative applications of this powerful technique. This article is part of the Special Issue entitled 'Fluorescent Tools in Neuropharmacology'.

A novel anticonvulsant mechanism via inhibition of complement receptor C5ar1 in murine epilepsy models.

The role of complement system-mediated inflammation is of key interest in seizure and epilepsy pathophysiology, but its therapeutic potential has not yet been explored. We observed that the pro-inflammatory C5a receptor, C5ar1, is upregulated in two mouse models after status epilepticus; the pilocarpine model and the intrahippocampal kainate model. The C5ar1 antagonist, PMX53, was used to assess potential anticonvulsant actions of blocking this receptor pathway. PMX53 was found to be anticonvulsant in several acute models (6Hz and corneal kindling) and one chronic seizure model (intrahippocampal kainate model). The effects in the 6Hz model were not found in C5ar1-deficient mice, or with an inactive PMX53 analogue suggesting that the anticonvulsant effect of PMX53 is C5ar1-specific. In the pilocarpine model, inhibition or absence of C5ar1 during status epilepticus lessened seizure power and protected hippocampal neurons from degeneration as well as halved SE-associated mortality. C5ar1-deficiency during pilocarpine-induced status epilepticus also was accompanied by attenuation of TNFα upregulation by microglia, suggesting that C5ar1 activation results in TNFα release contributing to disease. Patch clamp studies showed that C5a-induced microglial K(+) outward currents were also inhibited with PMX53 providing a potential mechanism to explain acute anticonvulsant effects. In conclusion, our data indicate that C5ar1 activation plays a role in seizure initiation and severity, as well as neuronal degeneration following status epilepticus. The widespread anticonvulsant activity of PMX53 suggests that C5ar1 represents a novel target for improved anti-epileptic drug development which may be beneficial for pharmaco-resistant patients.

Phosphorylation mediated structural and functional changes in pentameric ligand-gated ion channels: implications for drug discovery.

Pentameric ligand-gated ion channels (pLGICs) mediate numerous physiological processes, including fast neurotransmission in the brain. They are targeted by a large number of clinically-important drugs and disruptions to their function are associated with many neurological disorders. The phosphorylation of pLGICs can result in a wide range of functional consequences. Indeed, many neurological disorders result from pLGIC phosphorylation. For example, chronic pain is caused by the protein kinase A-mediated phosphorylation of α3 glycine receptors and nicotine addiction is mediated by the phosphorylation of α4- or α7-containing nicotinic receptors. A recent study demonstrated that phosphorylation can induce a global conformational change in a pLGIC that propagates to the neurotransmitter-binding site. Here we present evidence that phosphorylation-induced global conformational changes may be a universal phenomenon in pLGICs. This raises the possibility of designing drugs to specifically treat disease-modified pLGICs. This review summarizes some of the opportunities available in this area.

Evaluation of in vitro antioxidant and in vivo analgesic potential of Terminalia paniculata aqueous bark extract.

The Terminalia genus includes plants that are used in a variety of food, nutritional products, and traditional medicines. Aqueous bark extract of Terminalia paniculata (TPW) was screened for its antioxidant and analgesic potential. The major polyphenols were identified by high-performance liquid chromatography. In vitro antioxidant potential of TPW was investigated by 1,1-diphenyl-2-picryl hydrazyl (DPPH), 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS(2-)) radical assay, nitric oxide (NO) scavenging, superoxide scavenging (O(2-)), Fe(2+) chelating (O-phenanthroline), and ferric reducing/antioxidant power (FRAP) assay. We evaluated the effects of TPW on cell viability, lipopolysaccharide (LPS)-stimulated reactive oxygen species (ROS), nitrite, and cytokines (interleukin [IL] 6 and tumor necrosis factor alpha [TNF-α]) in RAW 264.7 murine macrophages. Evaluation of analgesic activity of TPW was performed using acetic acid-induced writhing and hot plate test in mice. Phytochemical analysis showed the presence of four polyphenols, namely, gallic acid, ellagic acid, rutin, and quercetin. TPW showed maximum superoxide, ABTS(2-), NO, DPPH inhibition, and Fe(2+-)chelating property at 400 µg/mL, respectively. FRAP value was 4.5±0.25 µg Fe(II)/g. TPW, per se, did not affect RAW 264.7 cell viability. In LPS-induced RAW 264.7 cells, TPW attenuated the elevation in ROS, nitrite, IL-6, and TNF-α levels. TPW (100-400 mg/kg, orally) significantly reduced the number of writhes in a dose-dependent manner compared with the control. Similarly, TPW (400 mg/kg, orally) evoked a significant increase in the maximum percentage effect in the hot plate test. The study suggests the efficacy of aqueous bark extract of T. paniculata as a potential antioxidant and analgesic agent.

Phosphorylation of α3 glycine receptors induces a conformational change in the glycine-binding site.

Inflammatory pain sensitization is initiated by prostaglandin-induced phosphorylation of α3 glycine receptors (GlyRs) that are specifically located in inhibitory synapses on spinal pain sensory neurons. Phosphorylation reduces the magnitude of glycinergic synaptic currents, thereby disinhibiting nociceptive neurons. Although α1 and α3 subunits are both expressed on spinal nociceptive neurons, α3 is a more promising therapeutic target as its sparse expression elsewhere implies a reduced risk of side-effects. Here we compared glycine-mediated conformational changes in α1 and α3 GlyRs to identify structural differences that might be exploited in designing α3-specific analgesics. Using voltage-clamp fluorometry, we show that glycine-mediated conformational changes in the extracellular M2-M3 domain were significantly different between the two GlyR isoforms. Using a chimeric approach, we found that structural variations in the intracellular M3-M4 domain were responsible for this difference. This prompted us to test the hypothesis that phosphorylation of S346 in α3 GlyR might also induce extracellular conformation changes. We show using both voltage-clamp fluorometry and pharmacology that Ser346 phosphorylation elicits structural changes in the α3 glycine-binding site. These results provide the first direct evidence for phosphorylation-mediated extracellular conformational changes in pentameric ligand-gated ion channels, and thus suggest new loci for investigating how phosphorylation modulates structure and function in this receptor family. More importantly, by demonstrating that phosphorylation alters α3 GlyR glycine-binding site structure, they raise the possibility of developing analgesics that selectively target inflammation-modulated GlyRs.

Hypoglycaemic effects of alcoholic root extract of Borassus flabellifer (Linn.) in normal and diabetic rats.

The objective of the study was to investigate the alcoholic (ALEBF) extract of B. flabellifer for their hypoglycaemic effects in normal and diabetic rats. Diabetes was induced in rats by single dose administration of alloxan (120 mg/kg, i.p.) or by injecting dexamethasone (10 mg/kg, i.p.) for 10 days. In normal rats, ALEBF (100, 200 and 400 mg/kg) had significantly decreased the blood glucose level in a dose dependent manner after repeated administration for 7 days. In alloxan induced diabetic rats, extract (ALEBF) had decreased blood sugar level and improved glucose tolerance in alloxan induced diabetic rats at the end of 1st, 2nd , 3rd and 4th week after test extract treatment. However, the insulin levels of extract treated group did not significantly change after 28 days treatment with the extract. It did not alter the insulin levels. In alloxan model, repeated dose administration of ALEBF had showed significant increase in body weight, prevention of elimination of sugar in urine and reduced the mortality rate induced by alloxan. In dexamethasone induced insulin resistance diabetic rats, repeated administration of ALEBF inhibited the increase in blood glucose level, improved glucose tolerance and reduced the insulin levels as compared dexamethasone induced diabetic rats.

Toxicological evaluation of Terminalia paniculata bark extract and its protective effect against CCl4-induced liver injury in rodents.

BACKGROUND: Based on the reported antioxidant and anti-inflammatory potential of Terminalia paniculata, the bark aqueous extract (TPW) was investigated against liver damage. METHODS: Intrinsic cytotoxicity was tested on normal human liver (Chang) cell lines, followed by acute and sub-chronic toxicity studies in mice. TPW was then evaluated against CCl4-induced liver toxicity in rats. Liver enzymes (AST, ALT, and ALP) and antioxidant markers were assessed. The effect of TPW on isolated hepatic cells, post-CCl4 administration, was assessed by isolated mitochondrial membrane staining. The actions of TPW on apoptotic pathway in CCl4-treated Chang cells were also elucidated. RESULTS: TPW was found to be safe at all doses tested in both in vitro and in vivo toxicity studies. TPW (400 mg/kg, p.o.) significantly (*p <0.05) improved liver enzyme activity as compared to CCl4. Also, it improved antioxidant status (GSH, GST, MDA and total thiol) and preserved hepatic cell architecture. TPW pre-treatment significantly attenuated the levels of phospho-p53, p53, cleaved caspase-3, phospho-Bad, Bad and cleaved PARP in CCl4-treated Chang cells, improving the viability considerably. CONCLUSION: The findings support a protective role for Terminalia paniculata in pathologies involving oxidative stress.

Fluorescence-based high-throughput functional profiling of ligand-gated ion channels at the level of single cells.

Ion channels are involved in many physiological processes and are attractive targets for therapeutic intervention. Their functional properties vary according to their subunit composition, which in turn varies in a developmental and tissue-specific manner and as a consequence of pathophysiological events. Understanding this diversity requires functional analysis of ion channel properties in large numbers of individual cells. Functional characterisation of ligand-gated channels involves quantitating agonist and drug dose-response relationships using electrophysiological or fluorescence-based techniques. Electrophysiology is limited by low throughput and high-throughput fluorescence-based functional evaluation generally does not enable the characterization of the functional properties of each individual cell. Here we describe a fluorescence-based assay that characterizes functional channel properties at single cell resolution in high throughput mode. It is based on progressive receptor activation and iterative fluorescence imaging and delivers >100 dose-responses in a single well of a 384-well plate, using α1-3 homomeric and αß heteromeric glycine receptor (GlyR) chloride channels as a model system. We applied this assay with transiently transfected HEK293 cells co-expressing halide-sensitive yellow fluorescent protein and different GlyR subunit combinations. Glycine EC50 values of different GlyR isoforms were highly correlated with published electrophysiological data and confirm previously reported pharmacological profiles for the GlyR inhibitors, picrotoxin, strychnine and lindane. We show that inter and intra well variability is low and that clustering of functional phenotypes permits identification of drugs with subunit-specific pharmacological profiles. As this method dramatically improves the efficiency with which ion channel populations can be characterized in the context of cellular heterogeneity, it should facilitate systems-level analysis of ion channel properties in health and disease and the discovery of therapeutics to reverse pathological alterations.

The relative orientation of the TM3 and TM4 domains varies between α1 and α3 glycine receptors.

Glycine receptors (GlyRs) are anion-conducting members of the pentameric ligand-gated ion channel family. We previously showed that the dramatic difference in glycine efficacies of α1 and α3 GlyRs is largely attributable to their nonconserved TM4 domains. Because mutation of individual nonconserved TM4 residues had little effect, we concluded that the efficacy difference was a distributed effect of all nonconserved TM4 residues. We therefore hypothesized that the TM4 domains of α1 and α3 GlyRs differ in structure, membrane orientation, and/or molecular dynamic properties. Here we employed voltage-clamp fluorometry to test whether their TM4 domains interact differently with their respective TM3 domains. We found a rhodamine fluorophore covalently attached to a homologous TM4 residue in each receptor interacts differentially with a conserved TM3 residue. We conclude that the α1 and α3 GlyR TM4 domains are orientated differently relative to their TM3 domains. This may underlie their differential ability to influence glycine efficacy.

Isolation of anxiolytic principle from ethanolic root extract of Cardiospermum halicacabum.

Cardiospermum halicacabum roots have been used traditionally for the treatment of epilepsy and anxiety disorders. The purpose of this study was to characterize the putative phytoconstituents present in the ethanolic root extract having anxiolytic activity using an elevated plus-maze (EPM) and light dark transition model. Control mice were orally treated with an equal volume of vehicle (4% gum acacia), and positive control mice were treated with diazepam (1mg/kg). In the EPM test, out of pool of 19 master fractions (MF) only MF-14, 16 and 17 significantly (30mg/kg, p<0.05, p<0.01 and p<0.001) increased the number of entries in the open arm. MF-14, 16 and 17 (10, 20 and 30mg/kg) had also increased the time spent by mice in illuminated part of the box significantly (p<0.05, p<0.01 and p<0.001), as compared to control. However, significant changes (p<0.05, p<0.01 and p<0.001) were recorded in other parameters, e.g., rearing, time spent in the closed arm and dark zone in both the models. These results suggested that C. halicacabum root is an effective anxiolytic agent. The phytoconstituent responsible for the observed central effects was isolated from MF-14 and identified as well-known compound, Cardiospermin, a cyanogenic glucoside.

Anti-inflammatory activity of Terminalia paniculata bark extract against acute and chronic inflammation in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Terminalia paniculata Roxb. (Family-Combretaceae) is a wild tree commonly used in traditional ayurvedic medicine for the treatment of inflammation of parotid glands and in menstrual disorders. AIM OF THE STUDY: To explore the folk use of Terminalia paniculata on pharmacological grounds to evaluate the scientific basis of anti-inflammatory activity. MATERIALS AND METHODS: The anti-inflammatory activity of Terminalia paniculata was studied against carrageenan-induced hind paw edema, air pouch inflammation and complete Freund's adjuvant (CFA)-induced arthritis in rats. The aqueous extract of Terminalia paniculata bark (TPW) was administered at the concentrations of 100, 200 and 400mg/kg body weight. RESULTS: TPW showed significant (p<0.05) anti-inflammatory activity by reducing the edema volume in carrageenan-induced paw edema in rats. Further, TPW (400mg/kg) also reduced the carrageenan-induced leukocyte migration (50.92 ± 5.71%) and myeloperoxidase activity (49.31 ± 5.24%) in air pouch exudates. TPW (200mg/kg) exhibits anti-rheumatic and analgesic activities by improving the altered haematological milieu (ESR, CRP, RF, WBC, RBC and Hb) and also by inhibiting the flexion scores and radiographic changes in CFA-induced arthritis. This extract also had significant (p<0.05) effects on the occurrence of secondary lesions compared to CFA control. CONCLUSIONS: Terminalia paniculata bark may be a potential preventive or therapeutic candidate for the treatment of chronic inflammation and arthritis.

Hypoglycaemic and anti-diabetic activity of stem bark extracts Erythrina indica in normal and alloxan-induced diabetic rats.

The objective of the study is to investigate the alcoholic (AlcE) and aqueous (AqsE) extracts of stem bark of Erythrina indica (Papilionaceae) for hypoglycaemic effects in normal and diabetic rats. Diabetes was induced in rats by a single dose administration of alloxan (120 mg/kg, i.p.) or by injecting dexamethasone (10 mg/kg, i.p.) for 10 days. In normal rats, AlcE and AqsE had significantly decreased the blood glucose level (BGL) in a dose dependent manner after repeated administration for 7 days. In alloxan-induced diabetic rats, both the extracts decreased blood sugar levels with significant improvement in glucose tolerance and body weight at the end of 1st, 2nd and 3rd week after test extract treatment. In case of dexamethasone induced insulin resistant diabetic rats, repeated administration of AlcE and AqsE inhibited the increase in blood glucose level and improved glucose tolerance induced by dexamethasone as compared to dexamethasone induced diabetic rats. These results suggest that both extracts possess hypoglycaemic activity in normal as well as in diabetic rats. Among AlcE and AqsE, AqsE possesses better hypoglycaemic activity than AlcE in all the models. Preliminary phytochemical investigations revealed that alcoholic extracts contain carbohydrates, alkaloids, flavonoids, saponins, phytosterols, phenolics and tannins. Aqueous extract contains carbohydrates, alkaloids, flavonoids, glycosides, phytosterols and triterpenoids. These phytoconstituents may be responsible for the hypoglycaemic activity of the plant.