GLP-1-directed NMDA receptor antagonism for obesity treatment.

The N-methyl-D-aspartate (NMDA) receptor is a glutamate-activated cation channel that is critical to many processes in the brain. Genome-wide association studies suggest that glutamatergic neurotransmission and NMDA receptor-mediated synaptic plasticity are important for body weight homeostasis1. Here we report the engineering and preclinical development of a bimodal molecule that integrates NMDA receptor antagonism with glucagon-like peptide-1 (GLP-1) receptor agonism to effectively reverse obesity, hyperglycaemia and dyslipidaemia in rodent models of metabolic disease. GLP-1-directed delivery of the NMDA receptor antagonist MK-801 affects neuroplasticity in the hypothalamus and brainstem. Importantly, targeting of MK-801 to GLP-1 receptor-expressing brain regions circumvents adverse physiological and behavioural effects associated with MK-801 monotherapy. In summary, our approach demonstrates the feasibility of using peptide-mediated targeting to achieve cell-specific ionotropic receptor modulation and highlights the therapeutic potential of unimolecular mixed GLP-1 receptor agonism and NMDA receptor antagonism for safe and effective obesity treatment.

Muscimol inhibits plasma membrane rupture and ninjurin-1 oligomerization during pyroptosis.

Pyroptosis is a cell death process that causes inflammation and contributes to numerous diseases. Pyroptosis is mediated by caspase-1 family proteases that cleave the pore-forming protein gasdermin D, causing plasma membrane rupture and release of pathogenic cellular contents. We previously identified muscimol as a small molecule that prevents plasma membrane rupture during pyroptosis via an unidentified mechanism. Here, we show that muscimol has reversible activity to prevent cellular lysis without affecting earlier pyroptotic events. Although muscimol is a well-characterized agonist for neuronal GABAA receptors, muscimol protection is not altered by GABAA receptor antagonists or recapitulated by other GABAA agonists, suggesting that muscimol acts via a novel mechanism. We find that muscimol blocks oligomerization of ninjurin-1, which is required for plasma membrane rupture downstream of gasdermin D pore formation. Our structure-activity relationship studies reveal distinct molecular determinants defining inhibition of pyroptotic lysis compared to GABAA binding. In addition, we demonstrate that muscimol reduces lethality during LPS-induced septic shock. Together, these findings demonstrate that ninjurin-1-mediated plasma membrane rupture can be pharmacologically modulated and pave the way toward identification of therapeutic strategies for pathologic conditions associated with pyroptosis.

Pregnenolone sulfate analogues differentially modulate GABAA receptor closed/desensitised states.

BACKGROUND AND PURPOSE: GABAA receptors are regulated by numerous classes of allosteric modulators. However, regulation of receptor macroscopic desensitisation remains largely unexplored and may offer new therapeutic opportunities. Here, we report the emerging potential for modulating desensitisation with analogues of the endogenous inhibitory neurosteroid, pregnenolone sulfate. EXPERIMENTAL APPROACH: New pregnenolone sulfate analogues were synthesised incorporating various heterocyclic substitutions located at the C-21 position on ring D. The pharmacological profiles of these compounds were assessed using electrophysiology and recombinant GABAA receptors together with mutagenesis, molecular dynamics simulations, structural modelling and kinetic simulations. KEY RESULTS: All seven analogues retained a negative allosteric modulatory capability whilst exhibiting diverse potencies. Interestingly, we observed differential effects on GABA current decay by compounds incorporating either a six- (compound 5) or five-membered heterocyclic ring (compound 6) on C-21, which was independent of their potencies as inhibitors. We propose that differences in molecular charges, and the targeted binding of analogues to specific states of the GABAA receptor, are the most likely cause of the distinctive functional profiles. CONCLUSIONS AND IMPLICATIONS: Our findings reveal that heterocyclic addition to inhibitory neurosteroids not only affected their potency and macroscopic efficacy but also affected innate receptor mechanisms that underlie desensitisation. Acute modulation of macroscopic desensitisation will determine the degree and duration of GABA inhibition, which are vital for the integration of neural circuit activity. Discovery of this form of modulation could present an opportunity for next-generation GABAA receptor drug design and development.

Exploring the NCS-382 Scaffold for CaMKIIα Modulation: Synthesis, Biochemical Pharmacology, and Biophysical Characterization of Ph-HTBA as a Novel High-Affinity Brain-Penetrant Stabilizer of the CaMKIIα Hub Domain.

Ca2+/calmodulin-dependent protein kinase II alpha (CaMKIIα) is a brain-relevant kinase and an emerging drug target for ischemic stroke and neurodegenerative disorders. Despite reported CaMKIIα inhibitors, their usefulness is limited by low subtype selectivity and brain permeability. (E)-2-(5-Hydroxy-5,7,8,9-tetrahydro-6H-benzo[7]annulen-6-ylidene)acetic acid (NCS-382) is structurally related to the proposed neuromodulator, γ-hydroxybutyric acid, and is a brain-penetrating high nanomolar-affinity ligand selective for the CaMKIIα hub domain. Herein, we report the first series of NCS-382 analogs displaying improved affinity and preserved brain permeability. Specifically, we present Ph-HTBA (1i) with enhanced mid-nanomolar affinity for the CaMKIIα binding site and a marked hub thermal stabilization effect along with a distinct CaMKIIα Trp403 flip upon binding. Moreover, Ph-HTBA has good cellular permeability and low microsomal clearance and shows brain permeability after systemic administration to mice, signified by a high Kp, uu value (0.85). Altogether, our study highlights Ph-HTBA as a promising candidate for CaMKIIα-associated pharmacological interventions and future clinical development.

The CaMKIIα hub ligand Ph-HTBA promotes neuroprotection after focal ischemic stroke by a distinct molecular interaction.

Ca2+/calmodulin-dependent protein kinase II alpha (CaMKIIα) is a potential target for acute neuroprotection due to its key role in physiological and pathological glutamate signaling. The hub domain organizes the CaMKII holoenzyme into large oligomers, and additional functional effects on holoenzyme activation have lately emerged. We recently reported that compounds related to the proposed neuromodulator γ-hydroxybutyrate (GHB) selectively bind to the CaMKIIα hub domain and increase hub thermal stabilization, which is believed to have functional consequences and to mediate neuroprotection. However, the detailed molecular mechanism is unknown. In this study, we functionally characterize the novel and brain permeable GHB analog (E)-2-(5-hydroxy-2-phenyl-5,7,8,9-tetrahydro-6H-benzo[7]annulen-6-ylidene)acetic acid (Ph-HTBA). Administration of a single dose of Ph-HTBA at a clinically relevant time point (3-6 h after photothrombotic stroke) promotes neuroprotection with a superior effect at low doses compared to the smaller GHB analog 3-hydroxycyclopent-1-enecarboxylic acid (HOCPCA). In contrast to HOCPCA, Ph-HTBA reduces Ca2+-stimulated CaMKIIα Thr286 autophosphorylation in primary cortical neurons and substrate phosphorylation of recombinant CaMKIIα, potentially contributing to its neuroprotective effect. Supported by previous in silico docking studies, we suggest that Ph-HTBA makes distinct molecular interactions with the hub cavity, which may contribute to its differential functional profile and superior neuroprotective effect compared to HOCPCA. Together, this highlights Ph-HTBA as a promising tool to study hub functionality, but also as a good candidate for clinical development.

Discovery and Optimization of 5-Hydroxy-Diclofenac toward a New Class of Ligands with Nanomolar Affinity for the CaMKIIα Hub Domain.

The Ca2+/calmodulin-dependent protein kinase II α (CaMKIIα) is a brain-relevant kinase involved in long-term potentiation and synaptic plasticity. We have recently pinpointed the CaMKIIα hub domain as the long-sought-after high-affinity target of γ-hydroxybutyrate ligands substantiated with a high-resolution cocrystal of 5-hydroxydiclofenac (3). Herein, we employed in silico approaches to rationalize and guide the synthesis and pharmacological characterization of a new series of analogues circumventing chemical stability problems associated with 3. The oxygen-bridged analogue 4d showed mid-nanomolar affinity and notable ligand-induced stabilization effects toward the CaMKIIα hub oligomer. Importantly, 4d displayed superior chemical and metabolic stability over 3 by showing excellent chemical stability in phosphate-buffered saline and high resistance to form reactive intermediates and subsequent sulfur conjugates. Altogether, our study highlights 4d as a new CaMKIIα hub high-affinity ligand with enhanced pharmacokinetic properties, representing a powerful tool compound for allosteric regulation of kinase activity with subtype specificity.

Structure-Activity Studies of 3,9-Diazaspiro[5.5]undecane-Based γ-Aminobutyric Acid Type A Receptor Antagonists with Immunomodulatory Effect.

The 3,9-diazaspiro[5.5]undecane-based compounds 2027 and 018 have previously been reported to be potent competitive γ-aminobutyric acid type A receptor (GABAAR) antagonists showing low cellular membrane permeability. Given the emerging peripheral application of GABAAR ligands, we hypothesize 2027 analogs as promising lead structures for peripheral GABAAR inhibition. We herein report a study on the structural determinants of 2027 in order to suggest a potential binding mode as a basis for rational design. The study identified the importance of the spirocyclic benzamide, compensating for the conventional acidic moiety, for GABAAR ligands. The structurally simplified m-methylphenyl analog 1e displayed binding affinity in the high-nanomolar range (Ki = 180 nM) and was superior to 2027 and 018 regarding selectivity for the extrasynaptic α4ßδ subtype versus the α1- and α2- containing subtypes. Importantly, 1e was shown to efficiently rescue inhibition of T cell proliferation, providing a platform to explore the immunomodulatory potential for this class of compounds.

Molecular Determinants and Pharmacological Analysis for a Class of Competitive Non-transported Bicyclic Inhibitors of the Betaine/GABA Transporter BGT1.

The betaine/GABA transporter 1 (BGT1) is a member of the GABA transporter (GAT) family with still elusive function, largely due to a lack of potent and selective tool compounds. Based on modeling, we here present the design, synthesis and pharmacological evaluation of five novel conformationally restricted cyclic GABA analogs related to the previously reported highly potent and selective BGT1 inhibitor (1S,2S,5R)-5-aminobicyclo[3.1.0]hexane-2-carboxylic acid (bicyclo-GABA). Using [3H]GABA radioligand uptake assays at the four human GATs recombinantly expressed in mammalian cell lines, we identified bicyclo-GABA and its N-methylated analog (2) as the most potent and selective BGT1 inhibitors. Additional pharmacological characterization in a fluorescence-based membrane potential assay showed that bicyclo-GABA and 2 are competitive inhibitors, not substrates, at BGT1, which was validated by a Schild analysis for bicyclo-GABA (pK B value of 6.4). To further elaborate on the selectivity profile both compounds were tested at recombinant α1ß2γ2 GABAA receptors. Whereas bicyclo-GABA showed low micromolar agonistic activity, the N-methylated 2 was completely devoid of activity at GABAA receptors. To further reveal the binding mode of bicyclo-GABA and 2 binding hypotheses of the compounds were obtained from in silico-guided mutagenesis studies followed by pharmacological evaluation at selected BGT1 mutants. This identified the non-conserved BGT1 residues Q299 and E52 as the molecular determinants driving BGT1 activity and selectivity. The binding mode of bicyclo-GABA was further validated by the introduction of activity into the corresponding GAT3 mutant L314Q (38 times potency increase cf. wildtype). Altogether, our data reveal the molecular determinants for the activity of bicyclic GABA analogs, that despite their small size act as competitive inhibitors of BGT1. These compounds may serve as valuable tools to selectively and potently target BGT1 in order to decipher its elusive pharmacological role in the brain and periphery such as the liver and kidneys.

GHB analogs confer neuroprotection through specific interaction with the CaMKIIα hub domain.

Ca2+/calmodulin-dependent protein kinase II alpha subunit (CaMKIIα) is a key neuronal signaling protein and an emerging drug target. The central hub domain regulates the activity of CaMKIIα by organizing the holoenzyme complex into functional oligomers, yet pharmacological modulation of the hub domain has never been demonstrated. Here, using a combination of photoaffinity labeling and chemical proteomics, we show that compounds related to the natural substance γ-hydroxybutyrate (GHB) bind selectively to CaMKIIα. By means of a 2.2-Å x-ray crystal structure of ligand-bound CaMKIIα hub, we reveal the molecular details of the binding site deep within the hub. Furthermore, we show that binding of GHB and related analogs to this site promotes concentration-dependent increases in hub thermal stability believed to alter holoenzyme functionality. Selectively under states of pathological CaMKIIα activation, hub ligands provide a significant and sustained neuroprotection, which is both time and dose dependent. This is demonstrated in neurons exposed to excitotoxicity and in a mouse model of cerebral ischemia with the selective GHB analog, HOCPCA (3-hydroxycyclopent-1-enecarboxylic acid). Together, our results indicate a hitherto unknown mechanism for neuroprotection by a highly specific and unforeseen interaction between the CaMKIIα hub domain and small molecule brain-penetrant GHB analogs. This establishes GHB analogs as powerful tools for investigating CaMKII neuropharmacology in general and as potential therapeutic compounds for cerebral ischemia in particular.

Molecular Determinants Underlying Delta Selective Compound 2 Activity at δ-Containing GABAA Receptors.

Delta selective compound 2 (DS2; 4-chloro-N-[2-(2-thienyl)imidazo[1,2-a]pyridin-3-yl]benzamide) is one of the most widely used tools to study selective actions mediated by δ-subunit-containing GABAA receptors. DS2 was discovered over 10 years ago, but despite great efforts, the precise molecular site of action has remained elusive. Using a combination of computational modeling, site-directed mutagenesis, and cell-based pharmacological assays, we probed three potential binding sites for DS2 and analogs at α 4 ß 1 δ receptors: an α 4 (+) δ (-) interface site in the extracellular domain (ECD), equivalent to the diazepam binding site in αßγ 2 receptors, and two sites in the transmembrane domain (TMD) - one in the α 4 (+) ß 1 (-) and one in the α 4 (-) ß 1 (+) interface, with the α 4 (-) ß 1 (+) site corresponding to the binding site for etomidate and a recently disclosed low-affinity binding site for diazepam. We show that mutations in the ECD site did not abrogate DS2 modulation. However, mutations in the TMD α 4 (+) ß 1 (-) interface, either α 4(S303L) of the α 4 (+) side or ß 1(I289Q) of the ß 1 (-) side, convincingly disrupted the positive allosteric modulation by DS2. This was consistently demonstrated both in an assay measuring membrane potential changes and by whole-cell patch-clamp electrophysiology and rationalized by docking studies. Importantly, general sensitivity to modulators was not compromised in the mutated receptors. This study sheds important light on the long-sought molecular recognition site for DS2, refutes the misconception that the selectivity of DS2 for δ-containing receptors is caused by a direct interaction with the δ-subunit, and instead points toward a functional selectivity of DS2 and its analogs via a surprisingly well conserved binding pocket in the TMD. SIGNIFICANCE STATEMENT: δ-Containing GABAA receptors represent potential drug targets for the treatment of several neurological conditions with aberrant tonic inhibition, yet no drugs are currently in clinical use. With the identification of the molecular determinants responsible for positive modulation by the known compound delta selective compound 2, the ground is laid for design of ligands that selectively target δ-containing GABAA receptor subtypes, for better understanding of tonic inhibition, and ultimately, for rational development of novel drugs.

Exploring the molecular determinants for subtype-selectivity of 2-amino-1,4,5,6-tetrahydropyrimidine-5-carboxylic acid analogs as betaine/GABA transporter 1 (BGT1) substrate-inhibitors.

We have previously identified 2-amino-1,4,5,6-tetrahydropyrimidine-5-carboxylic acid (ATPCA) as the most potent substrate-inhibitor of the betaine/GABA transporter 1 (BGT1) (IC50 2.5 µM) reported to date. Herein, we characterize the binding mode of 20 novel analogs and propose the molecular determinants driving BGT1-selectivity. A series of N1-, exocyclic-N-, and C4-substituted analogs was synthesized and pharmacologically characterized in radioligand-based uptake assays at the four human GABA transporters (hGATs) recombinantly expressed in mammalian cells. Overall, the analogs retained subtype-selectivity for hBGT1, though with lower inhibitory activities (mid to high micromolar IC50 values) compared to ATPCA. Further characterization of five of these BGT1-active analogs in a fluorescence-based FMP assay revealed that the compounds are substrates for hBGT1, suggesting they interact with the orthosteric site of the transporter. In silico-guided mutagenesis experiments showed that the non-conserved residues Q299 and E52 in hBGT1 as well as the conformational flexibility of the compounds potentially contribute to the subtype-selectivity of ATPCA and its analogs. Overall, this study provides new insights into the molecular interactions governing the subtype-selectivity of BGT1 substrate-inhibitors. The findings may guide the rational design of BGT1-selective pharmacological tool compounds for future drug discovery.

Sex-specific alterations in GABA receptor-mediated responses in laterodorsal tegmentum are associated with prenatal exposure to nicotine.

Smoking during pregnancy is associated with deleterious physiological and cognitive effects on the offspring, which are likely due to nicotine-induced alteration in the development of neurotransmitter systems. Prenatal nicotine exposure (PNE) in rodents is associated with changes in behaviors controlled in part by the pontine laterodorsal tegmentum (LDT), and LDT excitatory signaling is altered in a sex and age-dependent manner by PNE. As effects on GABAergic LDT signaling are unknown, we used calcium imaging to evaluate GABAA receptor- (GABAA R as well as GABAA -ρ R) and GABAB receptor (GABAB R)-mediated calcium responses in LDT brain slices from female and male PNE mice in two different age groups. Overall, in older PNE females, changes in calcium induced by stimulation of GABAA R and GABAB R, including GABAA -ρ R were shifted toward calcium rises. In both young and old males, PNE was associated with alterations in calcium mediated by all three receptors; however, the GABAA R was the most affected. These results show for the first time that PNE is associated with alterations in GABAergic transmission in the LDT in a sex- and age-dependent manner, and these data are the first to show PNE-associated alterations in functionality of GABA receptors in any nucleus. PNE-associated alterations in LDT GABAergic transmission within the LDT would be expected to alter output to target regions and could play a role in LDT-implicated, negative behavioral outcomes following gestational exposure to smoking. Accordingly, our data provide further supportive evidence of the importance of eliminating the consumption of nicotine during pregnancy.

Discovery of a new class of orthosteric antagonists with nanomolar potency at extrasynaptic GABAA receptors.

Brain GABAΑ receptors are ionotropic receptors belonging to the class of Cys-loop receptors and are important drug targets for the treatment of anxiety and sleep disorders. By screening a compound library (2,112 compounds) at recombinant human α4ß1δ GABAΑ receptors heterologously expressed in a HEK cell line, we identified a scaffold of spirocyclic compounds with nanomolar antagonist activity at GABAΑ receptors. The initial screening hit 2027 (IC50 of 1.03 µM) was used for analogue search resulting in 018 (IC50 of 0.088 µM). 018 was most potent at α3,4,5-subunit containing receptors, thus showing preference for forebrain-expressed extrasynaptic receptors. Schild analysis of 018 at recombinant human α4ß1δ receptors and displacement of [3H]muscimol binding in rat cortical homogenate independently confirmed a competitive profile. The antagonist profile of 018 was further validated by whole-cell patch-clamp electrophysiology, where kinetic studies revealed a slow dissociation rate and a shallow hill slope was observed. Membrane permeability studies showed that 2027 and 018 do not cross membranes, thus making the compounds less attractive for studying central GABAΑ receptors effects, but conversely more attractive as tool compounds in relation to emerging peripheral GABAΑ receptor-mediated effects of GABA e.g. in the immune system.

Silencing of spontaneous activity at α4ß1/3δ GABAA receptors in hippocampal granule cells reveals different ligand pharmacology.

BACKGROUND AND PURPOSE: The δ-subunit-containing GABAA receptors, α4 ß1 δ and α4 ß3 δ, in dentate gyrus granule cells (DGGCs) are known to exhibit both spontaneous channel openings (i.e. constitutive activity) and agonist-induced current. The functional implications of spontaneous gating are unclear. In this study, we tested the hypothesis that constitutively active α4 ß1/3 δ receptors limit agonist efficacy. EXPERIMENTAL APPROACH: Whole-cell electrophysiological recordings of adult male rat and mouse hippocampal DGGCs were used to characterize known agonists and antagonists at δ-subunit-containing GABAA receptors. To separate constitutive and agonist-induced currents, different recording conditions were employed. KEY RESULTS: Recordings at either 24°C or 34°C, including the PKC autoinhibitory peptide (19-36) intracellularly, removed spontaneous gating by GABAA receptors. In the absence of spontaneous gating, DGGCs responded to the α4 ß1/3 δ orthosteric agonist Thio-THIP with a four-fold increased efficacy relative to recording conditions favouring constitutive activity. Surprisingly, the neutral antagonist gabazine was unable to antagonize the current by Thio-THIP. Furthermore, a current was elicited by gabazine alone only when the constitutive current was silenced (EC50 2.1 µM). The gabazine-induced current was inhibited by picrotoxin, potentiated by DS2, completely absent in δ-/- mice and reduced in ß1 -/- mice, but could not be replicated in human α4 ß1/3 δ receptors expressed heterologously in HEK cells. CONCLUSION AND IMPLICATIONS: Kinase activity infers spontaneous gating in α4 ß1/3 δ receptors in DGGCs. This significantly limits the efficacy of GABAA agonists and has implications in pathologies involving aberrant excitability caused by phosphorylation (e.g. addiction and epilepsy). In such cases, the efficacy of δ-preferring GABAA ligands may be reduced.

Developing New 4-PIOL and 4-PHP Analogues for Photoinactivation of γ-Aminobutyric Acid Type A Receptors.

The critical roles played by GABAA receptors as inhibitory regulators of excitation in the central nervous system has been known for many years. Aberrant GABAA receptor function and trafficking deficits have also been associated with several diseases including anxiety, depression, epilepsy, and insomnia. As a consequence, important drug groups such as the benzodiazepines, barbiturates, and many general anesthetics have become established as modulators of GABAA receptor activity. Nevertheless, there is much we do not understand about the roles and mechanisms of GABAA receptors at neural network and systems levels. It is therefore crucial to develop novel technologies and especially chemical entities that can interrogate GABAA receptor function in the nervous system. Here, we describe the chemistry and characterization of a novel set of 4-PIOL and 4-PHP analogues synthesized with the aim of developing a toolkit of drugs that can photoinactivate GABAA receptors. Most of these new analogues show higher affinities/potencies compared with the respective lead compounds. This is indicative of cavernous areas being present near their binding sites that can be potentially associated with novel receptor interactions. The 4-PHP azide-analogue, 2d, possesses particularly impressive nanomolar affinity/potency and is an effective UV-inducible photoinhibitor of GABAA receptors with considerable potential for photocontrol of GABAA receptor function in situ.

Synthesis and Pharmacological Evaluation of [11C]4-Methoxy-N-[2-(thiophen-2-yl)imidazo[1,2-a]pyridin-3-yl]benzamide as a Brain Penetrant PET Ligand Selective for the δ-Subunit-Containing γ-Aminobutyric Acid Type A Receptors.

The α4/6ßδ-containing GABAA receptors are involved in a number of brain diseases. Despite the potential of a δ-selective imaging agent, no PET radioligand is currently available for in vivo imaging. Here, we report the characterization of DS2OMe (1) as a candidate radiotracer, 11C-labeling, and subsequent evaluation of [11C]DS2OMe in a domestic pig as a PET radioligand for visualization of the δ-containing GABAA receptors.

Designing Poly-agonists for Treatment of Metabolic Diseases: Challenges and Opportunities.

Obesity, type 2 diabetes, and the numerous associated metabolic co-morbidities are growing global threats to public health. Despite recent progress in pharmacotherapies for metabolic diseases, the current treatment options have limited efficacy and provide mostly symptomatic relief with little or no impact on disease reversal. Thus, improved therapies are urgently needed. As a result, the scientific community has increasingly invested in leveraging new pathophysiological insights into more efficacious pharmacotherapies for metabolic complications. A heightened understanding of the large, interindividual variation in responsiveness to certain metabolic medicines combined with advances in engineering multi-agonist candidates are important steps towards this goal. Additionally, the emerging pharmacological concept of peptide-mediated targeting of small molecules for tissue-specific delivery holds promise for more powerful treatment solutions in the future. In this review, we summarize recent advances in medicinal chemistry and molecular pharmacology that have enabled the engineering of several, novel, poly-agonist drug candidates for treatment of metabolic diseases, and we discuss the recent results from clinical trials assessing the efficacy and safety of glucagon-like peptide (GLP)-1/glucagon and GLP-1/GIP co-agonists.

Five-Membered N-Heterocyclic Scaffolds as Novel Amino Bioisosteres at γ-Aminobutyric Acid (GABA) Type A Receptors and GABA Transporters.

Given the heterogeneity within the γ-aminobutyric acid (GABA) receptor and transporter families, a detailed insight into the pharmacology is still relatively sparse. To enable studies of the physiological roles governed by specific receptor and transporter subtypes, a series of GABA analogues comprising five-membered nitrogen- and sulfur-containing heterocycles as amine bioisosteres were synthesized and pharmacologically characterized at native and selected recombinant GABAA receptors and GABA transporters. The dihydrothiazole and imidazoline analogues, 5-7, displayed moderate GAT activities and GABAA receptor binding affinities in the mid-nanomolar range ( Ki, 90-450 nM). Moreover, they exhibited full and equipotent agonist activity compared to GABA at GABAA-αßγ receptors but somewhat lower potency as partial agonists at the GABAA-ρ1 receptor. Stereoselectivity was observed for compounds 4 and 7 for the GABAA-αßγ receptors but not the GABAA-ρ1 receptor. This study illustrates how subtle differences in these novel amino GABA bioisosteres result in diverse pharmacological profiles in terms of selectivity and efficacy.

Structural and molecular aspects of betaine-GABA transporter 1 (BGT1) and its relation to brain function.

É£-aminobutyric-acid (GABA) functions as the principal inhibitory neurotransmitter in the central nervous system. Imbalances in GABAergic neurotransmission are involved in the pathophysiology of various neurological diseases such as epilepsy, Alzheimer's disease and stroke. GABA transporters (GATs) facilitate the termination of GABAergic signaling by transporting GABA together with sodium and chloride from the synaptic cleft into presynaptic neurons and surrounding glial cells. Four different GATs have been identified that all belong to the solute carrier 6 (SLC6) transporter family: GAT1-3 (SLC6A1, SLC6A13, SLC6A11) and betaine/GABA transporter 1 (BGT1, SLC6A12). BGT1 has emerged as an interesting target for treating epilepsy due to animal studies that reported anticonvulsant effects for the GAT1/BGT1 selective inhibitor EF1502 and the BGT1 selective inhibitor RPC-425. However, the precise involvement of BGT1 in epilepsy remains elusive because of its controversial expression levels in the brain and the lack of highly selective and potent tool compounds. This review gathers the current structural and functional knowledge on BGT1 with emphasis on brain relevance, discusses all available compounds, and tries to shed light on the molecular determinants driving BGT1 selectivity. This article is part of the issue entitled 'Special Issue on Neurotransmitter Transporters'.