Investigating the role of loop c hydrophilic residue 'T244' in the binding site of ρ1 GABAC receptors via site mutation and partial agonism

The loop C hydrophilic residue, threonine 244 lines the orthosteric binding site of ρ1 GABAC receptors was studied by point mutation into serine, alanine and cysteine, and tested with GABA, some representative partial agonists and antagonists. Thr244 has a hydroxyl group essential for GABA activity that is constrained by the threonine methyl group, orienting it toward the binding site. Significant decreases in activation effects of the studied ligands at ρ1 T244S mutant receptors, suggests a critical role for this residue. Results of aliphatic and heteroaromatic partial agonists demonstrate different pharmacological effects at ρ1 T244S mutant receptors when co-applied with GABA EC50 responses. ρ1 T244A and ρ1 T244C mutant receptors have minimal sensitivity to GABA at high mM concentrations, whereas, the ρ1 WT partial agonists, β-alanine and MTSEA demonstrate more efficacy and potency, respectively, than GABA at these mutant receptors. This study explores the role of Thr244 in the binding of agonists as an initial step during channel gating by moving loop C towards the ligand

Zolpidem is a potent stoichiometry-selective modulator of α1β3 GABAA receptors : evidence of a novel benzodiazepine site in the α1-α1 interface

Zolpidem is not a typical GABAA receptor hypnotic. Unlike benzodiazepines, zolpidem modulates tonic GABA currents in the rat dorsal motor nucleus of the vagus, exhibits residual effects in mice lacking the benzodiazepine binding site, and improves speech, cognitive and motor function in human patients with severe brain injury. The receptor by which zolpidem mediates these effects is not known. In this study we evaluated binary α1β3 GABAA receptors in either the 3α1:2β3 or 2α1:3β3 subunit stoichiometry, which differ by the existence of either an α1-α1 interface, or a β3-β3 interface, respectively. Both receptor stoichiometries are readily expressed in Xenopus oocytes, distinguished from each other by using GABA, zolpidem, diazepam and Zn2+. At the 3α1:2β3 receptor, clinically relevant concentrations of zolpidem enhanced GABA in a flumazenil-sensitive manner. The efficacy of diazepam was significantly lower compared to zolpidem. No modulation by either zolpidem or diazepam was detected at the 2α1:3β3 receptor, indicating that the binding site for zolpidem is at the α1-α1 interface, a site mimicking the classical α1-γ2 benzodiazepine site. Activating α1β3 (3α1:2β3) receptors may, in part, mediate the physiological effects of zolpidem observed under distinct physiological and clinical conditions, constituting a potentially attractive drug target

Cannabigerolic acid, a major biosynthetic precursor molecule in cannabis, exhibits divergent effects on seizures in mouse models of epilepsy

Background and Purpose: Cannabis has been used to treat epilepsy for millennia, with such use validated by regulatory approval of cannabidiol (CBD) for Dravet syndrome. Unregulated artisanal cannabis-based products used to treat children with intractable epilepsies often contain relatively low doses of CBD but are enriched in other phytocannabinoids. This raises the possibility that other cannabis constituents might have anticonvulsant properties. Experimental Approach: We used the Scn1a+/− mouse model of Dravet syndrome to investigate the cannabis plant for phytocannabinoids with anticonvulsant effects against hyperthermia-induced seizures. The most promising, cannabigerolic acid (CBGA), was further examined against spontaneous seizures and survival in Scn1a+/− mice and in electroshock seizure models. Pharmacological effects of CBGA were surveyed across multiple drug targets. Key Results: The initial screen identified three phytocannabinoids with novel anticonvulsant properties: CBGA, cannabidivarinic acid (CBDVA) and cannabigerovarinic acid (CBGVA). CBGA was most potent and potentiated the anticonvulsant effects of clobazam against hyperthermia-induced and spontaneous seizures, and was anticonvulsant in the MES threshold test. However, CBGA was proconvulsant in the 6-Hz threshold test and a high dose increased spontaneous seizure frequency in Scn1a+/− mice. CBGA was found to interact with numerous epilepsy-relevant targets including GPR55, TRPV1 channels and GABAA receptors. Conclusion and Implications: These results suggest that CBGA, CBDVA and CBGVA may contribute to the effects of cannabis-based products in childhood epilepsy. Although these phytocannabinoids have anticonvulsant potential and could be lead compounds for drug development programmes, several liabilities would need to be overcome before CBD is superseded by another in this class

Ligand-gated ion channels in genetic disorders and the question of efficacy

Whole-genome sequencing has unearthed a substantial number of individual variants in ion channels associated with genetic disorders. Ligand-gated ion channels, including glycine, γ-aminobutyric acid type A and nicotinic acetylcholine receptors, have long been known to harbour genetic variants associated with hyperekplexia and different forms of epilepsy. In some of these cases, missense variants enhance or impair the intrinsic ability of the receptor to convert ligand binding to channel opening, or the efficacy of receptor activation. We review the current understanding of how ligand-gated ion channels are activated and the properties that define the efficacy of an agonist, and how these properties can be altered by disease-causing variants. Additionally, we consider the mechanisms defining drug modulation of receptors and consider how this may differ in genetic variants. This fundamental knowledge is likely to be essential in understanding how effective treatments will be for patients with genetic variants in ligand-gated ion channels

Venom-derived modulators of epilepsy-related ion channels

Epilepsy is characterised by spontaneous recurrent seizures that are caused by an imbalance between neuronal excitability and inhibition. Since ion channels play fundamental roles in the generation and propagation of action potentials as well as neurotransmitter release at a subset of excitatory and inhibitory synapses, their dysfunction has been linked to a wide variety of epilepsies. Indeed, these unique proteins are the major biological targets for antiepileptic drugs. Selective targeting of a specific ion channel subtype remains challenging for small molecules, due to the high level of homology among members of the same channel family. As a consequence, there is a growing trend to target ion channels with biologics. Venoms are the best known natural source of ion channel modulators, and venom peptides are increasingly recognised as potential therapeutics due to their high selectivity and potency gained through millions of years of evolutionary selection pressure. Here we describe the major ion channel families involved in the pathogenesis of various types of epilepsy, including voltage-gated Na+, K+, Ca2+ channels, Cys-loop receptors, ionotropic glutamate receptors and P2X receptors, and currently available venom-derived peptides that target these channel proteins. Although only a small number of venom peptides have successfully progressed to the clinic, there is reason to be optimistic about their development as antiepileptic drugs, notwithstanding the challenges associated with development of any class of peptide drug

High and low GABA sensitivity α4β2δ GABAA receptors are expressed in Xenopus laevis oocytes with divergent stoichiometries

GABAA receptors that contain the a4 and d subunits are thought to be located extrasynaptically, mediating tonic currents elicited by low concentrations of GABA. These a4bd receptors are modulated by neurosteroids and certain anesthetics, identifying them as important drug targets in research. However, pharmacological studies on these receptors have often yielded variable results, possibly due to the expression of receptors in different stoichiometries or arrangements. In this study, we injected different ratios of a4, b2 and d cRNA into Xenopus oocytes and measured the sensitivity to GABA and DS2 activation of the resulting receptor populations. By creating a matrix of RNA injection ratios from stock RNA concentrations, we were able to compare the changes in pharmacology between injection ratios where the ratio of only one subunit was altered. We identified two distinct populations of receptors, the first with an EC50 value of approximately 100 nM to GABA, a low Hill slope of approximately 0.3 and substantial direct activation by DS2. The second population had an EC50 value of approximately 1 lM to GABA, a steeper Hill slope of 1 and little direct activation, but substantial potentiation, by DS2. The second population was formed with high a4 ratios and low b2 ratios, but altering the ratio of d subunit injected had little effect. We propose that receptors with high sensitivity to GABA and direct activation by DS2 are the result of a greater number of b2 subunits being incorporated into the receptor

The de novo GABRA4 p.Thr300Ile variant found in a patient with early-onset intractable epilepsy and neurodevelopmental abnormalities displays gain-of-function traits

Overall, our observations complement the study by Vogel et al.1 and highlight an important role for gain-of-function extrasynaptic receptors in the etiology of epilepsy and neurodevelopmental disorders. The challenge now is to accumulate enough variants in the GABRA4 and GARBD genes to robustly define the clinical phenotype that is associated with distinct functional changes in each gene

A pharmacological characterization of GABA, THIP and DS2 at binary α4β3 and β38 receptors : GABA activates β38 receptors via the β3(+)8(-) interface

There is growing evidence that GABA (γ-aminobutyric acid) can activate GABAA receptors (GABAARs) in the absence of an α subunit. In this study, we compared the pharmacology of homomeric and binary α4, β3 or δ subunits with ternary α4β3δ to identify subunit interfaces that contribute to the pharmacology of GABA, THIP, and DS2, and the antagonists, Zn2þ, gabazine and bicuculline. β3δ receptors form functional GABA-gated channels when expressed in Xenopus oocytes with a pharmacology that differs to homomeric β3, binary α4β3 and ternary α4β3δ receptors. GABA had similar potency at α4β3 and β3δ receptors (25 mM and 26 mM, respectively) but differed at α4β3δ receptors where GABA exhibited a biphasic concentration-response (EC50 (1)¼12.6 nM; EC50 (2)¼6.3 μM). THIP activated β3δ receptors (EC50¼456 μM) but was a more potent activator of α4β3 (EC50¼27 μM) and α4β3δ receptors (EC50 (1)¼ 27.5 nM; EC50 (2)¼29.5 μΜ), indicating that the α4 subunit significantly contribute to its potency. The δpreferring modulator, DS2 had marginal or no effect at β3δ and α4β3 receptors, indicating a role for both the α4 and δ subunits for its potency. Gabazine inhibited GABA-elicited currents at β3δ receptors whereas bicuculline activated these receptors. Mutational analysis verified that GABA binds to the β3(þ) δ() interface formed by the β3 and δ subunits. In conclusion, evaluating agents against binary GABAARs such as β3δ and α4β3 receptors enables identification of interfaces that may contribute to the pharmacology of the more complex ternary α4β3δ receptors

A pharmacological assessment of agonists and modulators at α4β2γ2 and α4β28 GABAA receptors : the challenge in comparing apples with oranges

Extrasynaptically located γ-aminobutyric acid (GABA) receptors type A are often characterized by the presence of a δ subunit in the receptor complex. δ-Containing receptors respond to low ambient concentrations of GABA, or respond to spillover of GABA from the synapse, and give rise to tonic inhibitory currents. In certain brain regions, e.g. thalamocortical neurons, tonic inhibition is estimated to represent the majority of total GABA-mediated inhibition, which has raised substantial interest in extrasynaptic receptors as potential drug targets. Thalamocortical neurons typically express 4β2/3δ receptors, however, these have proven difficult to study in recombinant in vitro expression systems due to the inherently low current levels elicited in response to GABA. In this study, we sought to characterize a range of agonists and positive allosteric modulators at α4β2δ and α4β2γ2 receptors. All tested agonists (GABA, THIP, muscimol, and taurine) displayed between 8 and 22 fold increase in potency at the 4β2δ receptor. In contrast, modulatory potencies of steroids (allopregnanolone, THDOC and alfaxalone), anesthetics (etomidate, pentobarbital) and Delta-Selective agents 1 and 2 (DS1 and DS2) were similar at α4β2δ and α4β2γ2 receptors. When evaluating modulatory efficacies,the neurosteroids and anesthetics displayed highest efficacy at α4β2γ2 receptors whereas DS1 and in particular DS2 had highest efficacy at α4β2δ receptors. Overall, several key messages emerged: (i) none of the tested compounds displayed significant selectivity and a great need for identifying new δ-selective compounds remains; (ii) α4β2δ and α4β2γ2receptors have such divergent intrinsic activation properties that valid comparisons of modulator efficacies are at best challenging

Coadministered cannabidiol and clobazam : preclinical evidence for both pharmacodynamic and pharmacokinetic interactions

Objective: Cannabidiol (CBD) has been approved by the US Food and Drug Administration (FDA) to treat intractable childhood epilepsies, such as Dravet syn-drome and Lennox‐Gastaut syndrome. However, the intrinsic anticonvulsant activity of CBD has been questioned due to a pharmacokinetic interaction between CBD and a first‐line medication, clobazam. This recognized interaction has led to speculation that the anticonvulsant efficacy of CBD may simply reflect CBD augmenting clobazam exposure. The present study aimed to address the nature of the interaction between CBD and clobazam. Methods: We examined whether CBD inhibits human CYP3A4 and CYP2C19 mediated metabolism of clobazam and N‐desmethylclobazam (N‐CLB), respectively, and performed studies assessing the effects of CBD on brain and plasma pharma-cokinetics of clobazam in mice. We then used the Scn1a+/− mouse model of Dravet syndrome to examine how CBD and clobazam interact. We compared anticonvulsant effects of CBD‐clobazam combination therapy to monotherapy against thermally‐in-duced seizures, spontaneous seizures and mortality in Scn1a+/− mice. In addition, we used Xenopus oocytes expressing γ‐aminobutyric acid (GABA)A receptors to investigate the activity of GABAA receptors when treated with CBD and clobazam together. Results: CBD potently inhibited CYP3A4 mediated metabolism of clobazam and CYP2C19 mediated metabolism of N‐CLB. Combination CBD‐clobazam treatment resulted in greater anticonvulsant efficacy in Scn1a+/− mice, but only when an anticonvulsant dose of CBD was used. It is important to note that a sub‐anticonvulsant dose of CBD did not promote greater anticonvulsant effects despite increasing plasma clobazam concentrations. In addition, we delineated a novel pharmacodynamic mechanism where CBD and clobazam together enhanced inhibitory GABAA receptor activation