Personalized medicine: Vinpocetine to reverse effects of GABRB3 mutation.

OBJECTIVE: To screen a library of potential therapeutic compounds for a woman with Lennox-Gastaut syndrome due to a Y302C GABRB3 (c.905A>G) mutation. METHODS: We compared the electrophysiological properties of cells with wild-type or the pathogenic GABRB3 mutation. RESULTS: Among 1320 compounds, multiple candidates enhanced GABRB3 channel conductance in cell models. Vinpocetine, an alkaloid derived from the periwinkle plant with anti-inflammatory properties and the ability to modulate sodium and channel channels, was the lead candidate based on efficacy and safety profile. Vinpocetine was administered as a dietary supplement over 6 months, reaching a dosage of 20 mg three times per day, and resulted in a sustained, dose-dependent reduction in spike-wave discharge frequency on electroencephalograms. Improved language and behavior were reported by family, and improvements in global impression of change surveys were observed by therapists blinded to intervention. SIGNIFICANCE: Vinpocetine has potential efficacy in treating patients with this mutation and possibly other GABRB3 mutations or other forms of epilepsy. Additional studies on pharmacokinetics, potential drug interactions, and safety are needed.

Functional and structural interaction of (-)-reboxetine with the human α4ß2 nicotinic acetylcholine receptor.

The interaction of the selective norepinephrine reuptake inhibitor (-)-reboxetine with the human α4ß2 nicotinic acetylcholine receptor (nAChR) in different conformational states was studied by several functional and structural approaches. Patch-clamp and Ca(2+)-influx results indicate that (-)-reboxetine does not activate hα4ß2 nAChRs via interaction with the orthosteric sites, but inhibits agonist-induced hα4ß2 activation by a noncompetitive mechanism. Consistently, the results from the electrophysiology-based functional approach suggest that (-)-reboxetine may act via open channel block; therefore, it is capable of producing a use-dependent type of inhibition of the hα4ß2 nAChR function. We tested whether (-)-reboxetine binds to the luminal [(3)H]imipramine site. The results indicate that, although (-)-reboxetine binds with low affinity to this site, it discriminates between the resting and desensitized hα4ß2 nAChR ion channels. Patch-clamp results also indicate that (-)-reboxetine progressively inhibits the hα4ß2 nAChR with two-fold higher potency at the end of one-second application of agonist, compared with the peak current. The molecular docking studies show that (-)-reboxetine blocks the ion channel at the level of the imipramine locus, between M2 rings 6' and 14'. In addition, we found a (-)-reboxetine conformer that docks in the helix bundle of the α4 subunit, near the middle region. According to molecular dynamics simulations, (-)-reboxetine binding is stable for both sites, albeit less stable than imipramine. The interaction of these drugs with the helix bundle might alter allostericaly the functionality of the channel. In conclusion, the clinical action of (-)-reboxetine may be produced (at least partially) by its inhibitory action on hα4ß2 nAChRs.

Pharmacological properties and predicted binding mode of arylmethylene quinuclidine-like derivatives at the α3ß4 nicotinic acetylcholine receptor (nAChR).

We have carried out a pharmacological evaluation of arylmethylene quinuclidine derivatives interactions with human α3ß4 nAChRs subtype, using cell-based receptor binding, calcium-influx, electrophysiological patch-clamp assays and molecular modeling techniques. We have found that the compounds bind competitively to the α3ß4 receptor with micromolar affinities and some of the compounds behave as non-competitive antagonists (compounds 1, 2 and 3), displaying submicromolar IC(50) values. These evidences suggest a mixed mode of action for these compounds, having interactions at the orthosteric site and more pronounced interactions at an allosteric site to block agonist effects. One of the compounds, 1-benzyl-3-(diphenylmethylene)-1-azoniabicyclo[2.2.2]octane chloride (compound 3), exhibited poorly reversible use-dependent block of α3ß4 channels. We also found that removal of a phenyl group from compound 1 confers a partial agonism to the derived analog (compound 6). Introducing a hydrogen-bond acceptor into the 3-benzylidene quinuclidine derivative (compound 7) increases agonism potency at the α3ß4 receptor subtype. Docking into the orthosteric binding site of a α3ß4 protein structure derived by comparative modeling accurately predicted the experimentally-observed trend in binding affinity. Results supported the notion that binding requires a hydrogen bond formation between the ligand basic nitrogen and the backbone carbonyl oxygen atom of the conserved Trp-149.

Analysis of the Potential Range of Anticlea sibirica L. (Kunth) and Its Changes under Moderate Climate Change in the 21st Century.

The study shows the analysis of the current potential range and the modeling of its changes in the hemiboreal species Anticlea sibirica. The models show the habitat suitability for A. sibirica under moderate climatic changes (RCP4.5) in the middle and second half of the 21st century. For modeling, we used MaxEnt software with the predictors being climate variables from CHELSA Bioclim and a digital elevation model. The modeling has shown that climate change can be favorable for the spread of A. sibirica to the northeastern part of its range by expanding highly suitable habitats in mountainous landscapes along the coast of the Sea of Okhotsk. In the rest of the range, the total area of suitable habitats will decrease. In areas with extremely deteriorating growing conditions, the species will persist in low-competition habitats such as rocky outcrops, riverbanks, and screes. The predicted change in the distribution of A. sibirica indicates a possible strong transformation of the vegetation cover in Siberia and the Urals, even under moderate climate change.

Prediction of habitat suitability for Patrinia sibirica Juss. in the Southern Urals.

The paper presents the results of predictions of the habitat persistence for rare relict of the Pleistocene floristic complex Patrinia sibirica (L.) Juss. in the Southern Urals under various forecasted climate change scenarios. Climate variables from CHELSA BIOCLIM, elevation data (GMTED2010) and coarse fragment content in the top level of soil were used as predictors for modeling in the MaxEnt software. The impact of climate change on P. sibirica habitats under the RCP4.5 and RCP8.5 scenarios calculated from an ensemble of four general circulation models has been analyzed. The modeling has shown that the changes in the habitat suitability depend on the altitude. Deterioration of the habitats could be attributed to a temperature increase in mountain forest locations, and to a precipitation of driest quarter increase in mountain forest-steppe locations. In both cases, this leads to the expansion of forest and shrub vegetation. Monitoring of the habitat persistence of P. sibirica and other relict species of the Pleistocene floristic complex can play a major role in predictions, as their massive decline would constitute that climatic changes exceed the ranges of their fluctuations in the Holocene.

Differential pharmacologies of mecamylamine enantiomers: positive allosteric modulation and noncompetitive inhibition.

(+/-)-Mecamylamine is a racemic mixture of a widely used brain-permeant noncompetitive inhibitor of muscle-type and neuronal nicotinic receptors (NNRs). The present studies evaluated whether the stereoisomers of this drug show different profiles for inhibition of the high-sensitivity (HS) and low-sensitivity (LS) isoforms of the human alpha4beta2 NNR subtype expressed in subclonal human epithelial 1 cells. We found that at low concentrations (micromolar range), TC-5214 [S-(+)-mecamylamine] was more effective than TC-5213 [R-(-)-mecamylamine] in inhibiting the LS alpha4beta2 NNRs. In addition, we demonstrated that TC-5214 potentiated and TC-5213 inhibited agonist-induced activation of HS alpha4beta2 NNRs. The stereoselectivity of mecamylamine enantiomers at HS and LS alpha4beta2 receptors demonstrates that TC-5214 is the preferred stereoisomer for selective activation of HS, whereas it is more effective in suppressing LS receptor function. This feature could be relevant to therapeutic applications where such a selective mechanism of action is required.

Inhibitory autophosphorylation of CaMKII controls PSD association, plasticity, and learning.

To investigate the function of the alpha calcium-calmodulin-dependent kinase II (alphaCaMKII) inhibitory autophosphorylation at threonines 305 and/or 306, we generated knockin mice that express alphaCaMKII that cannot undergo inhibitory phosphorylation. In addition, we generated mice that express the inhibited form of alphaCaMKII, which resembles the persistently phosphorylated kinase at these sites. Our data demonstrate that blocking inhibitory phosphorylation increases CaMKII in the postsynaptic density (PSD), lowers the threshold for hippocampal long-term potentiation (LTP), and results in hippocampal-dependent learning that seems more rigid and less fine-tuned. Mimicking inhibitory phosphorylation dramatically decreased the association of CaMKII with the PSD and blocked both LTP and learning. These data demonstrate that inhibitory phosphorylation has a critical role in plasticity and learning.

Increased neuronal excitability, synaptic plasticity, and learning in aged Kvbeta1.1 knockout mice.

BACKGROUND: Advancing age is typically accompanied by deficits in learning and memory. These deficits occur independently of overt pathology and are often considered to be a part of "normal aging." At the neuronal level, normal aging is known to be associated with numerous cellular and molecular changes, which include a pronounced decrease in neuronal excitability and an altered induction in the threshold for synaptic plasticity. Because both of these mechanisms (neuronal excitability and synaptic plasticity) have been implicated as putative cellular substrates for learning and memory, it is reasonable to propose that age-related changes in these mechanisms may contribute to the general cognitive decline that occurs during aging. RESULTS: To further investigate the relationship between aging, learning and memory, neuronal excitability, and synaptic plasticity, we have carried out experiments with aged mice that lack the auxiliary potassium channel subunit Kvbeta1.1. In aged mice, the deletion of the auxiliary potassium channel subunit Kvbeta1.1 resulted in increased neuronal excitability, as measured by a decrease in the post-burst afterhyperpolarization. In addition, long-term potentiation (LTP) was more readily induced in aged Kvbeta1.1 knockout mice. Finally, the aged Kvbeta1.1 mutants outperformed age-matched controls in the hidden-platform version of the Morris water maze. Interestingly, the enhancements in excitability and learning were both sensitive to genetic background: The enhanced learning was only observed in a genetic background in which the mutants exhibited increased neuronal excitability. CONCLUSIONS: Neuronal excitability is an important determinant of both synaptic plasticity and learning in aged subjects.

High-Throughput Patch Clamp Screening in Human α6-Containing Nicotinic Acetylcholine Receptors.

Nicotine, the addictive component of tobacco products, is an agonist at nicotinic acetylcholine receptors (nAChRs) in the brain. The subtypes of nAChR are defined by their α- and ß-subunit composition. The α6ß2ß3 nAChR subtype is expressed in terminals of dopaminergic neurons that project to the nucleus accumbens and striatum and modulate dopamine release in brain regions involved in nicotine addiction. Although subtype-dependent selectivity of nicotine is well documented, subtype-selective profiles of other tobacco product constituents are largely unknown and could be essential for understanding the addiction-related neurological effects of tobacco products. We describe the development and validation of a recombinant cell line expressing human α6/3ß2ß3V273S nAChR for screening and profiling assays in an automated patch clamp platform (IonWorks Barracuda). The cell line was pharmacologically characterized by subtype-selective and nonselective reference agonists, pore blockers, and competitive antagonists. Agonist and antagonist effects detected by the automated patch clamp approach were comparable to those obtained by conventional electrophysiological assays. A pilot screen of a library of Food and Drug Administration-approved drugs identified compounds, previously not known to modulate nAChRs, which selectively inhibited the α6/3ß2ß3V273S subtype. These assays provide new tools for screening and subtype-selective profiling of compounds that act at α6ß2ß3 nicotinic receptors.

Electrophysiology-Based Assays to Detect Subtype-Selective Modulation of Human Nicotinic Acetylcholine Receptors.

The Family Smoking Prevention and Tobacco Control Act of 2009 (Public Law 111-31) gave the US Food and Drug Administration (FDA) the responsibility for regulating tobacco products. Nicotine is the primary addictive component of tobacco and its effects can be modulated by additional ingredients in manufactured products. Nicotine acts by mimicking the neurotransmitter acetylcholine on neuronal nicotinic acetylcholine receptors (nAChRs), which function as ion channels in cholinergic modulation of neurotransmission. Subtypes within the family of neuronal nAChRs are defined by their α- and ß-subunit composition. The subtype-selective profiles of tobacco constituents are largely unknown, but could be essential for understanding the physiological effects of tobacco products. In this report, we report the development and validation of electrophysiology-based high-throughput screens (e-HTS) for human nicotinic subtypes, α3ß4, α3ß4α5, α4ß2, and α7 stably expressed in Chinese Hamster Ovary cells. Assessment of agonist sensitivity and acute desensitization gave results comparable to those obtained by conventional manual patch clamp electrophysiology assays. The potency of reference antagonists for inhibition of the receptor channels and selectivity of positive allosteric modulators also were very similar between e-HTS and conventional manual patch voltage clamp data. Further validation was obtained in pilot screening of a library of FDA-approved drugs that identified α7 subtype-selective positive allosteric modulation by novel compounds. These assays provide new tools for profiling of nicotinic receptor selectivity.

Slice XVIvo™: a novel electrophysiology system with the capability for 16 independent brain slice recordings.

Here we validate the design and use of a novel, customized electrophysiology system (Slice XVIvo™) that is capable of recording from 16 independent brain slices. The system consists of 16 independent recording chambers in which individual electrodes can be manually manipulated and fixed in order to stimulate and record extracellular responses from 16 brain slices simultaneously. Responses from each brain slice are elicited with individual stimulus isolator units and recorded through separate channels, thus allowing for independent control and analysis of the evoked extracellular activity from each slice. The system was designed to fit on a standard anti-vibration table, thus the Slice XVIvo™ system occupies considerably less space than other currently available multi-slice recording systems. We have demonstrated the utility of the system to obtain stable, extracellular responses from the CA1 region of the hippocampus, as well as induce long-term potentiation. Additionally, we show the utility of the Slice XVIvo™ system to significantly improved throughput for testing compounds in an oxygen and glucose deprivation assay. Overall, we have designed, created and validated a considerably cost- and space-efficient electrophysiology system that greatly improves throughput while minimizing the number of animals used in experiments.

Validation of a high-throughput, automated electrophysiology platform for the screening of nicotinic agonists and antagonists.

High-throughput compound screening using electrophysiology-based assays represents an important tool for biomedical research and drug discovery programs. The recent development and availability of devices capable of performing high-throughput electrophysiology-based screening have brought the need to validate these tools by producing data that are consistent with results obtained with conventional electrophysiological methods. In this study, we compared the response properties of hα3ß4 and hα4ß2 nicotinic receptors to their endogenous ligand acetylcholine (ACh) using three separate electrophysiology platforms: Dynaflow (low-throughput, manual system), PatchXpress 7000A (medium-throughput automated platform), and IonWorks Barracuda (high-throughput automated platform). We found that despite the differences in methodological approaches between these technologies, the EC(50) values from the ACh dose-response curves were consistent between all three platforms. In addition, we have validated the IonWorks Barracuda for both competitive and uncompetitive inhibition assays by using the competitive nicotinic antagonist dihydro-beta-erythroidin (DHßE) and uncompetitive nicotinic antagonist mecamylamine. Furthermore, we have demonstrated the utility of a custom-written algorithm for generating dose-response curves from multiple extrapolated current metrics that allows for discriminating between competitive and uncompetitive inhibition while maintaining high-throughput capacity. This study provides validation of the consistency of results using low-, medium-, and high-throughput electrophysiology platforms and supports their use for screening nicotinic compounds.

A method for bidirectional solution exchange--"liquid bullet" applications of acetylcholine to α7 nicotinic receptors.

Fast solution exchange techniques have revolutionized the study of synaptic transmission and promise to remain an important neuroscience research tool. Here we provide evidence for the hypothesis that using continuous, rapid transitions through an agonist solution can significantly increase the exchange rate around a cell by reducing the diffusion boundary at the membrane. This novel approach of rapid solution exchange during whole-cell recordings--described as a "liquid bullet" (LB) application--takes advantage of a bidirectional solution flow around the cell, allowing for a full solution exchange within a range of several milliseconds. An exchange rate (10-90% rise time) of about 2 ms could be achieved during both agonist application and washout. We recorded whole-cell currents from cells expressing the rapidly desensitizing α7 neuronal nicotinic receptor (NNR) subtype that exhibited very fast rise times of around 4-5 ms. We further demonstrated the advantages of a LB application over conventional methods by the ability of this method to elicit concentration-dependent responses for rapidly desensitizing compounds that were not measurable with conventional agonist applications. In addition, we illustrate the utility of this approach for frequency-based assays through fast, repeated agonist applications at frequencies of 1 Hz and 30 Hz. This approach could therefore be useful for the study of rapid agonist-receptor interactions that closely mimic the physiological conditions in the synaptic cleft during bursts of neuronal activity.

Discovery of (2S,3R)-N-[2-(pyridin-3-ylmethyl)-1-azabicyclo[2.2.2]oct-3-yl]benzo[b]furan-2-carboxamide (TC-5619), a selective α7 nicotinic acetylcholine receptor agonist, for the treatment of cognitive disorders.

(2S,3R)-N-[2-(Pyridin-3-ylmethyl)-1-azabicyclo[2.2.2]oct-3-yl]benzo[b]furan-2-carboxamide (7a, TC-5619), a novel selective agonist of the α7 neuronal nicotinic acetylcholine receptor, has been identified as a promising drug candidate for the treatment of cognitive impairment associated with neurological disorders. 7a demonstrated more than a thousand-fold separation between the affinities for the α7 and α4ß2 receptor subtypes and had no detectable effects on muscle or ganglionic nicotinic receptor subtypes, indicating a marked selectivity for the central nervous system over the peripheral nervous system. Results obtained from homology modeling and docking explain the observed selectivity. 7a had positive effects across cognitive, positive, and negative symptoms of schizophrenia in animal models and was additive or synergistic with the antipsychotic clozapine. Compound 7a, as an augmentation therapy to the standard treatment with antipsychotics, demonstrated encouraging results on measures of negative symptoms and cognitive dysfunction in schizophrenia and was well tolerated in a phase II clinical proof of concept trial in patients with schizophrenia.

Structure-activity studies of 7-heteroaryl-3-azabicyclo[3.3.1]non-6-enes: a novel class of highly potent nicotinic receptor ligands.

The potential for nicotinic ligands with affinity for the α4ß2 or α7 subtypes to treat such diverse diseases as nicotine addiction, neuropathic pain, and neurodegenerative and cognitive disorders has been exhibited clinically for several compounds while preclinical activity in relevant in vivo models has been demonstrated for many more. For several therapeutic programs, we sought nicotinic ligands with various combinations of affinity and function across both subtypes, with an emphasis on dual α4ß2-α7 ligands, to explore the possibility of synergistic effects. We report here the structure-activity relationships (SAR) for a novel series of 7-heteroaryl-3-azabicyclo[3.3.1]non-6-enes and characterize many of the analogues for activity at multiple nicotinic subtypes.

Structural differences determine the relative selectivity of nicotinic compounds for native alpha 4 beta 2*-, alpha 6 beta 2*-, alpha 3 beta 4*- and alpha 7-nicotine acetylcholine receptors.

Mammalian brain expresses multiple nicotinic acetylcholine receptor (nAChR) subtypes that differ in subunit composition, sites of expression and pharmacological and functional properties. Among known subtypes of receptors, alpha 4 beta 2* and alpha 6 beta 2*-nAChR have the highest affinity for nicotine (where * indicates possibility of other subunits). The alpha 4 beta 2*-nAChRs are widely distributed, while alpha 6 beta 2*-nAChR are restricted to a few regions. Both subtypes modulate release of dopamine from the dopaminergic neurons of the mesoaccumbens pathway thought to be essential for reward and addiction. alpha 4 beta 2*-nAChR also modulate GABA release in these areas. Identification of selective compounds would facilitate study of nAChR subtypes. An improved understanding of the role of nAChR subtypes may help in developing more effective smoking cessation aids with fewer side effects than current therapeutics. We have screened a series of nicotinic compounds that vary in the distance between the pyridine and the cationic center, in steric bulk, and in flexibility of the molecule. These compounds were screened using membrane binding and synaptosomal function assays, or recordings from GH4C1 cells expressing h alpha 7, to determine affinity, potency and efficacy at four subtypes of nAChRs found in brain, alpha 4 beta 2*, alpha 6 beta 2*, alpha 7 and alpha 3 beta 4*. In addition, physiological assays in gain-of-function mutant mice were used to assess in vivo activity at alpha 4 beta 2* and alpha 6 beta 2*-nAChRs. This approach has identified several compounds with agonist or partial agonist activity that display improved selectivity for alpha 6 beta 2*-nAChR.

Development and optimization of a high-throughput electrophysiology assay for neuronal alpha4beta2 nicotinic receptors.

Historically, the identification of alpha4beta2 nicotinic acetylcholine receptor ligands has been based on high-throughput radioligand binding, rubidium efflux assays and Ca++ flux assays using a fluorometric imaging plate reader (FLIPR). Among other approaches, low-throughput electrophysiological assays in Xenopus oocytes and two channel application "liquid filament" systems for mammalian cells have been commonly used. More recent technical innovations that have been introduced into the field of electrophysiology allow for automated simultaneous multi-channel operation. Here we report the development and optimization of a high-throughput electrophysiological assay for identifying functionally active alpha4beta2 nicotinic receptor ligands using such a system. Characterization of the test system yielded results comparable to those obtained by other investigators using conventional electrophysiological assays. For example, the concentration-response relationships obtained for alpha4beta2 receptor activation by acetylcholine and nicotine were best described by biphasic Hill equations, and the inhibition of alpha4beta2 receptor currents by the nicotinic antagonist dihydro-beta-erythroidine was consistent with previously published results. Functional up-regulation of alpha4beta2 receptors by prolonged exposure to nicotine or lower temperature was also confirmed. Using this methodology we were able to characterize the activation of alpha4beta2 receptors by multiple compounds in a mammalian cell expression system, exemplifying its utility for rapid identification of novel nicotinic ligands within a screening cascade. Our results demonstrate the utility of this electrophysiological tool for the discovery of alpha4beta2 nicotinic acetylcholine receptor ligands with potential applications in numerous clinical indications.