GABAB receptor agonist baclofen promotes central nervous system remyelination.

Promoting remyelination is considered as a potential neurorepair strategy to prevent/limit the development of permanent neurological disability in patients with multiple sclerosis (MS). To this end, a number of clinical trials are investigating the potential of existing drugs to enhance oligodendrocyte progenitor cell (OPC) differentiation, a process that fails in chronic MS lesions. We previously reported that oligodendroglia express GABAB receptors (GABAB Rs) both in vitro and in vivo, and that GABAB R-mediated signaling enhances OPC differentiation and myelin protein expression in vitro. Our goal here was to evaluate the pro-remyelinating potential of GABAB R agonist baclofen (Bac), a clinically approved drug to treat spasticity in patients with MS. We first demonstrated that Bac increases myelin protein production in lysolecithin (LPC)-treated cerebellar slices. Importantly, Bac administration to adult mice following induction of demyelination by LPC injection in the spinal cord resulted in enhanced OPC differentiation and remyelination. Thus, our results suggest that Bac repurposing should be considered as a potential therapeutic strategy to stimulate remyelination in patients with MS.

Structure optimization of positive allosteric modulators of GABAB receptors led to the unexpected discovery of antagonists/potential negative allosteric modulators.

Positive allosteric modulators (PAMs) of GABAB receptor represent an interesting alternative to receptor agonists such as baclofen, as they act on the receptor in a more physiological way and thus are devoid of the side effects typically exerted by the agonists. Based on our interest in the identification of new GABAB receptor PAMs, we followed a merging approach to design new chemotypes starting from selected active compounds, such as GS39783, rac-BHFF, and BHF177, and we ended up with the synthesis of four different classes of compounds. The new compounds were tested alone or in the presence of 10 µM GABA using [35S]GTPγS binding assay to assess their functionality at the receptor. Unexpectedly, a number of them significantly inhibited GABA-stimulated GTPγS binding thus revealing a functional switch with respect to the prototype molecules. Further studies on selected compounds will clarify if they act as negative modulators of the receptor or, instead, as antagonists at the orthosteric binding site.

Structural basis of the activation of a metabotropic GABA receptor.

Metabotropic γ-aminobutyric acid receptors (GABAB) are involved in the modulation of synaptic responses in the central nervous system and have been implicated in neuropsychological conditions that range from addiction to psychosis1. GABAB belongs to class C of the G-protein-coupled receptors, and its functional entity comprises an obligate heterodimer that is composed of the GB1 and GB2 subunits2. Each subunit possesses an extracellular Venus flytrap domain, which is connected to a canonical seven-transmembrane domain. Here we present four cryo-electron microscopy structures of the human full-length GB1-GB2 heterodimer: one structure of its inactive apo state, two intermediate agonist-bound forms and an active form in which the heterodimer is bound to an agonist and a positive allosteric modulator. The structures reveal substantial differences, which shed light on the complex motions that underlie the unique activation mechanism of GABAB. Our results show that agonist binding leads to the closure of the Venus flytrap domain of GB1, triggering a series of transitions, first rearranging and bringing the two transmembrane domains into close contact along transmembrane helix 6 and ultimately inducing conformational rearrangements in the GB2 transmembrane domain via a lever-like mechanism to initiate downstream signalling. This active state is stabilized by a positive allosteric modulator binding at the transmembrane dimerization interface.

Exploring the Binding Mechanism of GABAB Receptor Agonists and Antagonists through in Silico Simulations.

GABAB is a G protein-coupled receptor that functions as a constitutive heterodimer composed of the GABAB1a/b and GABAB2 subunits. It mediates slow and prolonged inhibitory neurotransmission in the nervous system, representing an attractive target for the treatment of various disorders. However, the molecular mechanism of the GABAB receptor is not thoroughly understood. Therefore, a better description of the binding of existing agonists and antagonists to this receptor is crucial to improve our knowledge about G protein-coupled receptor structure as well as for helping the development of new potent and more selective therapeutic agents. In this work, we used the recent X-ray cocrystallization data of agonists (GABA and baclofen) and antagonists (2-hydroxysaclofen, SCH50911, and CGP54626) bound to the GABAB orthosteric site together with quantum biochemistry and the molecular fractionation with conjugate caps (MFCC) scheme to describe the individual contribution of each amino acid residue involved in the GABAB-ligand interaction, pointing out differences and similarities among the compounds. Our quantum biochemical computational results show that the total binding energy of the ligands to the GABAB ligand pocket, with radius varying from 2.0 to 9.0 Å, is well-correlated with the experimental binding affinity. In addition, we found that the binding site is very similar for agonists or antagonists, showing small differences in the importance of the most significant amino acids. Finally, we predict the energetic relevance of the regions of the five ligands as well as the influence of each protein lobe on GABAB-ligand binding. These results provide important new information on the binding mechanism of the GABAB receptor and should facilitate the development of new chemicals targeting this receptor.

FRET-Based Sensors Unravel Activation and Allosteric Modulation of the GABAB Receptor.

The main inhibitory neurotransmitter, γ-aminobutyric acid (GABA), modulates many synapses by activating the G protein-coupled receptor GABAB, which is a target for various therapeutic applications. It is an obligatory heterodimer made of GB1 and GB2 that can be regulated by positive allosteric modulators (PAMs). The molecular mechanism of activation of the GABAB receptor remains poorly understood. Here, we have developed FRET-based conformational GABAB sensors compatible with high-throughput screening. We identified conformational changes occurring within the extracellular and transmembrane domains upon receptor activation, which are smaller than those observed in the related metabotropic glutamate receptors. These sensors also allow discrimination between agonists of different efficacies and between PAMs that have different modes of action, which has not always been possible using conventional functional assays. Our study brings important new information on the activation mechanism of the GABAB receptor and should facilitate the screening and identification of new chemicals targeting this receptor.

Liquid chromatography high resolution mass spectrometry for the determination of baclofen and its metabolites in plasma: Application to therapeutic drug monitoring.

Baclofen is used to manage alcohol dependence. This study describes a simple method using liquid chromatography coupled to high-resolution mass spectrometry (LC-HR-MS) developed in plasma samples. This method was optimized to allow quantification of baclofen and determination of metabolic ratio of its metabolites, an oxidative deaminated metabolite of baclofen (M1) and its glucuronide form (M2). The LC-HR-MS method on Exactive® apparatus is a newly developed method with all the advantages of high resolution in full-scan mode for the quantification of baclofen and detection of its metabolites in plasma. The present assay provides a protein precipitation method starting with 100 µL plasma giving a wide polynomial dynamic range (R2 > 0.999) between 10 and 2000 ng/mL and a lower limit of quantitation of 3 ng/mL for baclofen. Intra- and inter-day precisions were <8.1% and accuracies were between 91.2 and 103.3% for baclofen. No matrix effect was observed. The assay was successfully applied to 36 patients following baclofen administration. Plasma concentrations of baclofen were determined between 12.2 and 1399.9 ng/mL and metabolic ratios were estimated between 0.4 and 81.8% for M1 metabolite and on the order of 0.3% for M2 in two samples.

Baclofen and other GABAB receptor agents are allosteric modulators of the CXCL12 chemokine receptor CXCR4.

CXCR4, a receptor for the chemokine CXCL12 (stromal-cell derived factor-1α), is a G-protein-coupled receptor (GPCR), expressed in the immune and CNS and integrally involved in various neurological disorders. The GABAB receptor is also a GPCR that mediates metabotropic action of the inhibitory neurotransmitter GABA and is located on neurons and immune cells as well. Using diverse approaches, we report novel interaction between GABAB receptor agents and CXCR4 and demonstrate allosteric binding of these agents to CXCR4. First, both GABAB antagonists and agonists block CXCL12-elicited chemotaxis in human breast cancer cells. Second, a GABAB antagonist blocks the potentiation by CXCL12 of high-threshold Ca(2+) channels in rat neurons. Third, electrophysiology in Xenopus oocytes and human embryonic kidney cell line 293 cells in which we coexpressed rat CXCR4 and the G-protein inward rectifier K(+) (GIRK) channel showed that GABAB antagonist and agonist modified CXCL12-evoked activation of GIRK channels. To investigate whether GABAB ligands bind to CXCR4, we expressed this receptor in heterologous systems lacking GABAB receptors and performed competition binding experiments. Our fluorescent resonance energy transfer experiments suggest that GABAB ligands do not bind CXCR4 at the CXCL12 binding pocket suggesting allosteric modulation, in accordance with our electrophysiology experiments. Finally, using backscattering interferometry and lipoparticles containing only the CXCR4 receptor, we quantified the binding affinity for the GABAB ligands, confirming a direct interaction with the CXCR4 receptor. The effect of GABAergic agents on CXCR4 suggests new therapeutic potentials for neurological and immune diseases.

Amorphous solid dispersion successfully improved oral exposure of ADX71943 in support of toxicology studies.

ADX71943 is a potent and selective GABA(b) receptor positive allosteric modulator (PAM) which exhibits poor aqueous solubility at all physiologically relevant pHs. The aim of this study was to identify an adequate formulation to improve the solubility of ADX71943 to achieve a sufficiently high plasma exposure after oral administration to support the toxicology program. Considering the overall physicochemical properties and the low solubility of ADX71943 in a variety of solvents, solid dispersion, and particle size reduction have been successfully chosen as potential strategies to improve its oral bioavailability. Both technologies have proven useful in improving the in vitro dissolution profile and as a result of the solubility enhancement, higher bioavailability was obtained in vivo. As the solid dispersion gave better bioavailability (30-fold compared with the neat active pharmaceutical ingredient (API)), this formulation was selected for the toxicology study. Changing the crystalline form of ADX71943 into amorphous state by preparing a solid dispersion has greatly improved its oral bioavailability and has allowed achieving the required plasma concentration needed in toxicology studies.

Decreased GABAA and GABAB receptor functional activity in cannabinoid CB1 receptor knockout mice.

The interaction between brain GABAergic and endocannabinoid systems was evaluated by examining the quantitative and functional status of GABAergic receptors in cannabinoid CB(1) receptor knockout (CB(1)(-/-)) mice. To this aim, GABA(A) ([(3)H]-Muscimol binding assay), GABA(B) (baclofen-stimulated [(35)S]-GTPγS binding assay), GABA(A)α(1), GABA(A)α(2) and GABA(A)γ(2) receptors gene expression (real-time reverse transcriptase polymerase chain reaction [PCR]) were carried out in CB(1)(-/-) and wild-type mice (CB(1)(+/+)). [(3)H]-Muscimol binding assays revealed significant reduction in the density of GABA(A) receptors in CA2 (30%) and DG (28%) of the hippocampus, thalamus (40%), cingulate (28%) and motor cortex (35%) of CB(1)(-/-) mice. Functional activity of metabotropic GABA(B) receptors was measured by evaluating the ability of GABA(B) agonist baclofen to stimulate [(35)S]-GTPγS binding. The results showed significant reduced [(35)S]-GTPγS binding in CA1 (61%), CA3 (51%) and DG (60%) of CB(1)(-/-) mice compared with CB(1)(+/+) mice. Real-time reverse transcriptase PCR was carried out for evaluating gene expression of α(1), α(2) and γ(2) subunits of GABA(A) receptor in the amygdala. The results showed significant reduced GABA(A)α(1) (50%) and GABA(A)α(2) (40%) receptor subunits gene expression in the amygdala of CB(1)(-/-) mice. No difference was observed in GABA(A)γ(2) receptor subunit gene expression. This study provides strong evidence of the involvement of CB(1) receptors in the control of GABAergic responses mediated by GABA(A) and GABA(B) receptors, and suggests a possible role of the endocannabinoid system in the regulation of anxiety-related disorders.