GABA Transporter Subtype Research Articles

Combination of MS Binding Assays and affinity selection mass spectrometry for screening of structurally homogenous libraries as exemplified for a focused oxime library addressing the neuronal GABA transporter 1.

This study presents an efficient screening approach based on combination of mass spectrometry (MS) based binding assays (MS Binding Assays) and affinity selection mass spectrometry (ASMS) customized for screening of structurally homogeneous libraries sharing a common mass spectrometric fragmentation pattern. After reaction of a nipecotic acid derivative possessing a hydroxylamine functionality with aldehydes, the resulting oxime library was screened accordingly toward the GABA transporter subtype 1 (GAT1), a drug target for several neurological disorders. After assessing sublibraries' activities for inhibition of reporter ligand binding, hits in active ones were directly identified. This could be achieved by recording mass transitions for the reporter ligand as well as those predicted for the library components in a single LC-MS/MS run with a triple quadrupole mass spectrometer in the multiple reaction monitoring mode. Identification of hits with a predefined affinity could be reliably accomplished by calculation of IC50-values from specific binding concentrations of library constituents and reporter ligand. Application of this strategy revealed six hits, from which two of them were resynthesized for further biological evaluation. Thereby, the best one displayed a pKi of 7.38 in MS Binding Assays and a pIC50 of 6.82 in [3H]GABA uptake assays for GAT1.

Extensive astrocyte metabolism of γ-aminobutyric acid (GABA) sustains glutamine synthesis in the mammalian cerebral cortex.

Synaptic transmission is closely linked to brain energy and neurotransmitter metabolism. However, the extent of brain metabolism of the inhibitory neurotransmitter γ-aminobutyric acid (GABA), and the relative metabolic contributions of neurons and astrocytes, are yet unknown. The present study was designed to investigate the functional significance of brain GABA metabolism using isolated mouse cerebral cortical slices and slices of neurosurgically resected neocortical human tissue of the temporal lobe. By using dynamic isotope labeling, with [15 N]GABA and [U-13 C]GABA as metabolic substrates, we show that both mouse and human brain slices exhibit a large capacity for GABA metabolism. Both the nitrogen and the carbon backbone of GABA strongly support glutamine synthesis, particularly in the human cerebral cortex, indicative of active astrocytic GABA metabolism. This was further substantiated by pharmacological inhibition of the primary astrocytic GABA transporter subtype 3 (GAT3), by (S)-SNAP-5114 or 1-benzyl-5-chloro-2,3-dihydro-1H-indole-2,3-dione (compound 34), leading to significant reductions in oxidative GABA carbon metabolism. Interestingly, this was not the case when tiagabine was used to specifically inhibit GAT1, which is predominantly found on neurons. Finally, we show that acute GABA exposure does not directly stimulate glycolytic activity nor oxidative metabolism in cultured astrocytes, but can be used as an additional substrate to enhance uncoupled respiration. These results clearly show that GABA is actively metabolized in astrocytes, particularly for the synthesis of glutamine, and challenge the current view that synaptic GABA homeostasis is maintained primarily by presynaptic recycling.

A Library Screening Strategy Combining the Concepts of MS Binding Assays and Affinity Selection Mass Spectrometry

The primary objective of early drug development is to identify hits and leads for a target of interest. To achieve this aim, rapid, and reliable screening techniques for a huge number of compounds are needed. Mass spectrometry based binding assays (MS Binding Assays) represent a well-established technique for library screening based on competitive binding experiments revealing active sublibraries due to reduced binding of a reporter ligand and following hit identification for active libraries by deconvolution in further competitive binding experiments. In the present study, we combined the concepts of MS Binding Assays and affinity selection mass spectrometry (ASMS) to improve the efficiency of the hit identification step. In that case, only a single competitive binding experiment is performed that is in the first step analyzed for reduced binding of the reporter ligand and—only if a sublibrary is active—additionally for specific binding of individual library components. Subsequently, affinities of identified hits as well as activities of reduced sublibraries (i.e., all sublibrary components without hit) are assessed in additional competitive binding experiments. We exemplified this screening concept for the identification of ligands addressing the most widespread GABA transporter subtype in the brain (GAT1) studying in the beginning a library composed of 128 and further on a library of 1,280 well-characterized GAT1 inhibitors, drug substances, and pharmacological tool compounds. Determination of sublibraries' activities was done by quantification of bound NO711 as reporter ligand and hit identification for the active ones achieved in a further LC-ESI-MS/MS run in the multiple reaction monitoring mode enabling detection of all sublibrary components followed by hit verification and investigation of reduced sublibraries in further competitive binding experiments. In this way, we could demonstrate that all GAT1 inhibitors reducing reporter ligand binding below 50% at a concentration of 1 μM are detected reliably without generation of false positive or false negative hits. As the described strategy is apart from its reliability also highly efficient, it can be assumed to become a valuable tool in early drug research, especially for membrane integrated drug targets that are often posing problems in established screening techniques.

Functional characterization of the GABA transporter GAT-1 from the deep-sea mussel Bathymodiolus septemdierum

Mammalian γ-aminobutyric acid (GABA) transporter subtype 1 (GAT-1) is a specific transporter for GABA, an inhibitory neurotransmitter in GABA-ergic neurons. GAT-1 belongs to the GAT group, in which five related transporters, GAT-2, GAT-3, GAT-4, CT1, and TAUT are known in mammals. By contrast, the deep-sea mussel, Bathymodiolus septemdierum has only two GAT group members, BsGAT-1 and BsTAUT, and their function in environmental adaptation is of interest to better understand the physiology of deep-sea organisms. Compared with BsTAUT, the function of BsGAT-1 is unknown. Here, we report the functional characterization of BsGAT-1. Analyses of BsGAT-1 expressed in Xenopus oocytes showed that it could transport GABA in a Na+- and Cl−-dependent manner, with Km and Vmax values of 0.58 μM and 1.97 pmol/oocyte/h, respectively. BsGAT-1 activity was blocked by the GAT-1 selective inhibitors SKF89976A and ACHC. Competition assays indicated that BsGAT-1 has no affinity for taurine and thiotaurine. These characteristics were common with those of mammalian GAT-1, suggesting its conserved function in the nervous system. However, BsGAT-1 showed a certain affinity for hypotaurine, which is involved in sulfide detoxification in hydrothermal vent-specific animals. This result suggests an additional role for BsGAT-1 in sulfide detoxification, which may be specific to the deep-sea mussel. In a tissue distribution analysis, BsGAT-1 mRNA expression was observed in various tissues. The expression in the adductor and byssus retractor muscles, labial palp, and foot, which possibly contain ganglia, suggested a function in the neural system, while BsGAT-1 expression in other tissues might be related to sulfide detoxification.

Novel, highly potent and in vivo active inhibitor of GABA transporter subtype 1 with anticonvulsant, anxiolytic, antidepressant and antinociceptive properties

Background and purposeSince GABAergic dysfunction underlies a variety of neurological and psychiatric disorders, numerous strategies leading to the augmentation of GABAergic neurotransmission have been introduced. One of them is the inhibition of GABA reuptake from the synaptic cleft mediated by four plasma membrane GABA transporters (GAT1-4). GAT1 which is exclusively expressed in the brain is an interesting target for centrally acting drugs. In this research, pharmacological properties of a novel, highly potent and selective inhibitor of GAT1, the guvacine derivative named DDPM-2571, were assessed in vivo. Experimental approachPharmacological effects and pharmacokinetics of intraperitoneally administered DDPM-2571 were assessed in CD-1 mice. Key resultsDDPM-2571 was quickly distributed into the brain and was highly effective in the prevention of chemically-induced seizures (pentylenetetrazole and pilocarpine models) and 6-Hz convulsions. It demonstrated significant anxiolytic-like and antidepressant-like properties. DDPM-2571 had antinociceptive properties, both in the hot plate test and in the second phase of the formalin test. Within the dose range tested, it did not impair animals' motor skills, but it impaired cognition and potentiated scopolamine-induced cognitive deficits in the passive avoidance task. Conclusions and implicationsDue to GAT1 inhibition, DDPM-2571 is effective in mouse models of chemically-induced seizures, anxiety, depression, acute and tonic pain. At biologically active doses, it does not impair animals' motor skills, but it might induce memory deficits. Taken together, DDPM-2571 can be regarded as a promising lead structure in the search for new centrally acting drugs and a potent pharmacological tool to study the biological role of GAT1.

Straightforward and effective synthesis of γ-aminobutyric acid transporter subtype 2-selective acyl-substituted azaspiro[4.5]decanes.

Supply of major metabolites such as γ-aminobutyric acid (GABA), β-alanine and taurine is an essential instrument that shapes signalling, proper cell functioning and survival in the brain and peripheral organs. This background motivates the synthesis of novel classes of compounds regulating their selective transport through various fluid-organ barriers via the low-affinity γ-aminobutyric acid (GABA) transporter subtype 2 (GAT2). Natural and synthetic spirocyclic compounds or therapeutics with a range of structures and biological activity are increasingly recognised in this regard. Based on pre-validated GABA transport activity, straightforward and efficient synthesis method was developed to provide an azaspiro[4.5]decane scaffold, holding a variety of charge, substituent and 3D constrain of spirocyclic amine. Investigation of the azaspiro[4.5]decane scaffold in cell lines expressing the four GABA transporter subtypes led to the discovery of a subclass of a GAT2-selective compounds with acyl-substituted azaspiro[4.5]decane core.

Application of MS Transport Assays to the Four Human γ-Aminobutyric Acid Transporters.

γ-Aminobutyric acid (GABA) transporters (GATs) are promising drug targets for various diseases associated with imbalances in GABAergic neurotransmission. For the development of new drugs or pharmacological tools addressing GATs, screening techniques to identify new inhibitors and to characterize their potency at each GAT subtype are indispensable. By now, the technique by far dominating is based on radiolabeled GABA. We recently described "MS Transport Assays" for hGAT-1 by employing ((2) H6 )GABA as the substrate. In the present study, we applied this approach to all four human GAT subtypes and determined the KM values for GAT-mediated transport of ((2) H6 )GABA at each subtype. Furthermore, a comprehensive set of GAT inhibitors reflecting the whole range of potency and subtype selectivity known so far was evaluated for their potency. The comparison of pIC50 values obtained in conventional [(3) H]GABA uptake assays with those obtained in MS Transport Assays indicated the reliability of the latter. The MS Transport Assays enable a throughput similar to that of conventional radiometric transport assays performed in a 96-well format but avoid the use of radiolabeled substrates.

Identification of the First Highly Subtype-Selective Inhibitor of Human GABA Transporter GAT3

Screening a library of small-molecule compounds using a cell line expressing human GABA transporter 3 (hGAT3) in a [(3)H]GABA uptake assay identified isatin derivatives as a new class of hGAT3 inhibitors. A subsequent structure-activity relationship (SAR) study led to the identification of hGAT3-selective inhibitors (i.e., compounds 20 and 34) that were superior to the reference hGAT3 inhibitor, (S)-SNAP-5114, in terms of potency (low micromolar IC50 values) and selectivity (>30-fold selective for hGAT3 over hGAT1/hGAT2/hBGT1). Further pharmacological characterization of compound 20 (5-(thiophen-2-yl)indoline-2,3-dione) revealed a noncompetitive mode of inhibition at hGAT3. This suggests that this compound class, which has no structural resemblance to GABA, has a binding site different from the substrate, GABA. This was supported by a molecular modeling study that suggested a unique binding site that matched the observed selectivity, inhibition kinetics, and SAR of the compound series. These compounds are the most potent GAT3 inhibitors reported to date that provide selectivity for GAT3 over other GABA transporter subtypes.

Anticonvulsant active inhibitor of GABA transporter subtype 1, tiagabine, with activity in mouse models of anxiety, pain and depression

BackgroundTiagabine, a selective inhibitor of GABA transporter subtype 1 is used as an add-on therapy of partial seizures in humans but its mechanism of action suggests other potential medical indications for this drug. In this research we assess its pharmacological activity in several screening models of seizures, pain, anxiety and depression in mice. MethodsFor pharmacological tests tiagabine was administered intraperitoneally 60min before the assay. Behavioral tests were performed using models of chemically and electrically induced seizures, thermal acute pain and formalin-induced tonic pain. Anxiolytic-like properties were evaluated using the four plate test and the elevated plus maze test. Antidepressant-like activity was assessed in the forced swim test. In addition, to exclude false positive results in these assays, the influence of tiagabine on animals’ locomotor activity and motor coordination was investigated, too. ResultsTiagabine demonstrated anticonvulsant properties in chemically induced seizures (pentylenetetrazole and pilocarpine seizures). At the dose of 100mg/kg it also elevated the seizure threshold for electrically induced seizures by 31.6% (p<0.01), but it had no activity in the maximal electroshock seizure test. Tiagabine showed anxiolytic-like and antidepressant-like effects. Although it apparently reduced animals’ nociceptive responses in pain tests, these activities rather resulted from its sedative and motor-impairing properties demonstrated in the locomotor activity and the rotarod tests, respectively. ConclusionsThe results obtained in the present study suggest that tiagabine, apart its anticonvulsant effect, has anxiolytic-like, sedative and antidepressant-like properties. In view of this, it can be potentially used in the treatment of anxiety and mood disorders.

MS transport assays for γ-aminobutyric acid transporters--an efficient alternative for radiometric assays.

Transport assays for neurotransmitters based on radiolabeled substrates are widely spread and often indispensable in basic research and the drug development process, although the use of radioisotopes is inherently coupled to issues concerning radioactive waste and safety precautions. To overcome these disadvantages, we developed mass spectrometry (MS)-based transport assays for γ-aminobutyric acid (GABA), which is the major inhibitory neurotransmitter in the central nervous system (CNS). These "MS Transport Assays" provide all capabilities of [(3)H]GABA transport assays and therefore represent the first substitute for the latter. The performance of our approach is demonstrated for GAT1, the most important GABA transporter (GAT) subtype. As GABA is endogenously present in COS-7 cells employed as hGAT1 expression system, ((2)H6)GABA was used as a substrate to differentiate transported from endogenous GABA. To record transported ((2)H6)GABA, a highly sensitive, short, robust, and reliable HILIC-ESI-MS/MS quantification method using ((2)H2)GABA as an internal standard was developed and validated according to the Center for Drug Evaluation and Research (CDER) guidelines. Based on this LC-MS quantification, a setup to characterize hGAT1 mediated ((2)H6)GABA transport in a 96-well format was established, that enables automated processing and avoids any sample preparation. The K(m) value for ((2)H6)GABA determined for hGAT1 is in excellent agreement with results obtained from [(3)H]GABA uptake assays. In addition, the established assay format enables efficient determination of the inhibitory potency of GAT1 inhibitors, is capable of identifying those inhibitors transported as substrates, and furthermore allows characterization of efflux. The approach described here combines the strengths of LC-MS/MS with the high efficiency of transport assays based on radiolabeled substrates and is applicable to all GABA transporter subtypes.

Conformationally Restricted GABA with Bicyclo[3.1.0]hexane Backbone as the First Highly Selective BGT-1 Inhibitor.

On the basis of the three-dimensional diversity-oriented conformational restriction strategy using key chiral cyclopropane units, we previously identified 3 ((2S,3R)-4-amino-3,4-methanobutyric acid) with a chiral trans-cyclopropane structure as a γ-aminobutyric acid (GABA) transporter inhibitor selective for GABA transporter (GAT) subtypes GAT-3 and BGT-1 (betaine/GABA transporter-1). Further conformational restriction of 3 with the rigid bicyclo[3.1.0]hexane backbone led to the successful development of the first highly potent and selective BGT-1 inhibitor 4 (IC50 = 0.59 μM). The bioactive conformation of 3 for BGT-1 was also identified.

Pharmacological identification of a guanidine-containing β-alanine analogue with low micromolar potency and selectivity for the betaine/GABA transporter 1 (BGT1).

The γ-aminobutyric acid (GABA) transporters (GATs) are key membrane transporter proteins involved in the termination of GABAergic signaling at synapses in the mammalian brain and proposed drug targets in neurological disorders such as epilepsy. To date, four different GAT subtypes have been identified: GAT1, GAT2, GAT3 and the betaine/GABA transporter 1 (BGT1). Owing to the lack of potent and subtype selective inhibitors of the non-GAT1 GABA transporters, the physiological role and therapeutic potential of these transporters remain to be fully understood. Based on bioisosteric replacement of the amino group in β-alanine or GABA, a series of compounds was generated, and their pharmacological activity assessed at human GAT subtypes. Using a cell-based [(3)H]GABA uptake assay, several selective inhibitors at human BGT1 were identified. The guanidine-containing compound 9 (2-amino-1,4,5,6-tetrahydropyrimidine-5-carboxylic acid hydrochloride) displayed more than 250 times greater potency than the parent compound β-alanine at BGT1 and is thus the most potent inhibitor reported to date for this subtype (IC50 value of 2.5 µM). In addition, compound 9 displayed about 400, 16 and 40 times lower inhibitory potency at GAT1, GAT2 and GAT3, respectively. Compound 9 was shown to be a substrate for BGT1 and to have an overall similar pharmacological profile at the mouse orthologue. Compound 9 constitutes an interesting pharmacological tool for specifically investigating the cellular pharmacology of BGT1 and is the first small-molecule substrate identified with such a high selectivity for BGT1 over the three other GAT subtypes.

The GABA Synapse as a Target for Antiepileptic Drugs: A Historical Overview Focused on GABA Transporters

It is clear that normal neuronal function relies on a tight balance between excitatory and inhibitory neurotransmission. Inhibitory signaling through the GABAergic system can be tightly regulated at the level of GABA uptake via GABA transporters (GAT). As such, selectively modulating the GABA uptake process through pharmacological agents has been an area of active investigation over several decades. These studies have demonstrated that inhibition of astroglial, but not neuronal, GATs may be preferred for anticonvulsant action. To date, four distinct GAT subtypes have been identified and efforts to selectively target these transporters have led to the proliferation of pharmacological agents aimed at augmenting extrasynaptic GABA levels. These pharmacological tools have provided novel and informative insight into the role of GABA and GABAergic signaling in the brain, but have also provided critical information concerning the regulation of CNS disorders associated with an imbalance in inhibitory tone, such as epilepsy. One such compound with notable inhibitory effects at GATs, tiagabine, has demonstrated clinical anticonvulsant efficacy, and is, to date, the only approved GAT inhibitor for clinical use. Thus, efforts to identify and develop GAT subtype-specific compounds continue to be an area of active investigation for the management of epilepsy and other CNS disorders. Herein, the historical efforts to elucidate the role of GABA in the synapse, as well as the role of GAT inhibitors as anticonvulsants, are described.

Antiallodynic Action of 1-(3-(9H-Carbazol-9-yl)-1-propyl)-4-(2-methyoxyphenyl)-4-piperidinol (NNC05-2090), a Betaine/GABA Transporter Inhibitor

The GABAergic system in the spinal cord has been shown to participate in neuropathic pain in various animal models. GABA transporters (GATs) play a role in controlling the synaptic clearance of GABA; however, their role in neuropathic pain remains unclear. In the present study, we compared the betaine/GABA transporter (BGT-1) with other GAT subtypes to determine its participation in neuropathic pain using a mouse model of sciatic nerve ligation. 1-(3-(9H-Carbazol-9-yl)-1-propyl)-4-(2-methyoxyphenyl)-4-piperidinol (NNC05-2090), an inhibitor that displays moderate selectivity for BGT-1, had an antiallodynic action on model mice treated through both intrathecally and intravenous administration routes. On the other hand, SKF89976A, a selective GAT-1 inhibitor, had a weak antiallodynic action, and (S)-SNAP5114, an inhibitor that displays selectivity for GAT-3, had no antiallodynic action. Systemic analysis of these compounds on GABA uptake in CHO cells stably expressing BGT-1 revealed that NNC05-2090 not only inhibited BGT-1, but also serotonin, noradrenaline, and dopamine transporters, using a substrate uptake assay in CHO cells stably expressing each transporter, with IC50: 5.29, 7.91, and 4.08 μM, respectively. These values were similar to the IC50 value at BGT-1 (10.6 μM). These results suggest that the antiallodynic action of NNC05-2090 is due to the inhibition of both BGT-1 and monoamine transporters.

Essential Roles of GABA Transporter-1 in Controlling Rapid Eye Movement Sleep and in Increased Slow Wave Activity after Sleep Deprivation

GABA is the major inhibitory neurotransmitter in the mammalian central nervous system that has been strongly implicated in the regulation of sleep. GABA transporter subtype 1 (GAT1) constructs high affinity reuptake sites for GABA and regulates GABAergic transmission in the brain. However, the role of GAT1 in sleep-wake regulation remains elusive. In the current study, we characterized the spontaneous sleep-wake cycle and responses to sleep deprivation in GAT1 knock-out (KO) mice. GAT1 KO mice exhibited dominant theta-activity and a remarkable reduction of EEG power in low frequencies across all vigilance stages. Under baseline conditions, spontaneous rapid eye movement (REM) sleep of KO mice was elevated both during the light and dark periods, and non-REM (NREM) sleep was reduced during the light period only. KO mice also showed more state transitions from NREM to REM sleep and from REM sleep to wakefulness, as well as more number of REM and NREM sleep bouts than WT mice. During the dark period, KO mice exhibited more REM sleep bouts only. Six hours of sleep deprivation induced rebound increases in NREM and REM sleep in both genotypes. However, slow wave activity, the intensity component of NREM sleep was briefly elevated in WT mice but remained completely unchanged in KO mice, compared with their respective baselines. These results indicate that GAT1 plays a critical role in the regulation of REM sleep and homeostasis of NREM sleep.

The Lignan (−)-Hinokinin Displays Modulatory Effects on Human Monoamine and GABA Transporter Activities

The neurotransmitter transporters of the SLC6 family play critical roles in the regulation of neurotransmission and are the primary targets of therapeutic agents used to treat clinical disorders involving compromised neurotransmitter signaling. The dopamine and norepinephrine transporters have been implicated in clinical disorders such as attention deficit hyperactivity disorder (ADHD) and substance abuse. The GABA transporters (GATs) serve as a target for anxiolytic, antidepressant, and antiepileptic therapies. In this work, the interaction with neurotransmitter transporters was characterized for a derivative of the lignan (-)-cubebin (1), namely, (-)-hinokinin (2). Using in vitro pharmacological assays, 2 selectively inhibited the human dopamine and norepinephrine transporters, in a noncompetitive manner possibly mediated by binding to a novel site within the transporters, and displayed low affinity for the serotonin transporter. Compound 2 also specifically inhibited the GAT-1 GABA transporter subtype. Compound 2 is not a substrate of the carriers as it had no effect on the efflux of either of the neurotransmitters investigated. This compound is inactive toward glutamate and glycine transporters. These results suggest that 2 may serve as a tool to develop new therapeutic drugs for ADHD and anxiety that target the DAT, NET, and GAT-1 transporters.

GABA transporter-1 inhibitor NO-711 alters the EEG power spectra and enhances non-rapid eye movement sleep during the active phase in mice

GABA transporter subtype 1 (GAT1) constructs high affinity reuptake sites for GABA in the CNS and regulates GABAergic transmission. Compounds that inhibit GAT1 are targets often used for the treatment of epilepsy; however sedation has been reported as a side effect of these agents, indicating potential sedative and/or hypnotic uses for these compounds. In the current study, we observed the sleep behaviors of mice treated with NO-711, a selective GAT1 inhibitor, in order to elucidate the role of GAT1 in sleep–wake regulation during the active phase. The data revealed that NO-711 at a high dose of 10mg/kg caused a marked enhancement of EEG activity in the frequency ranges of 3–25Hz during wakefulness as well as rapid eye movement (REM) sleep. During the non-REM (NREM) sleep, NO-711 (10mg/kg) elevated EEG activity in the frequency ranges of 1.5–6.75Hz. Similar changes were found in mice treated with a low dose of 3mg/kg. NO-711 administered i.p. at a dose of 1, 3 or 10mg/kg significantly shortened the sleep latency of NREM sleep, increased the amount of NREM sleep and the number of NREM sleep episodes. NO-711 did not affect the sleep latency and the amount of REM sleep. NO-711 dose-dependently increased c-Fos expression in sleep-promoting nucleus of the ventrolateral preoptic area and median preoptic area. However, c-Fos expression was decreased in the wake-promoting nuclei, tuberomammillary nucleus and lateral hypothalamus. These results indicate that NO-711 can increase NREM sleep in mice.

Evaluation of anxiolytic-like, anticonvulsant, antidepressant-like and antinociceptive properties of new 2-substituted 4-hydroxybutanamides with affinity for GABA transporters in mice

PurposeThe inhibition of plasma membrane GABA transporters (GATs) is responsible for anxiolytic-like, anticonvulsant, antinociceptive and antidepressant-like effects in mice. It also influences animals' motor coordination and their sensitivity to ethanol. The aim of this study was to assess the pharmacological activity of two novel 2-substituted 4-hydroxybutanamides (BM 130 and BM 131) in some screening models. An attempt has been made to establish the relationship between the inhibition of GAT subtype and the observed in vivo activity. MethodsThe affinity for GAT subtypes was evaluated by means of [3H]GABA uptake assay. It indicated that BM 130 inhibited GAT1 and GAT2, whereas BM 131 inhibited GAT1 and GAT3. In mice anxiolytic-like, antidepressant-like, anticonvulsant and antinociceptive properties of the test compounds were assessed. Their influence on motor coordination, locomotor activity and the ability to potentiate effects of subnarcotic doses of ethanol was also tested. ResultsBoth compounds administered intraperitoneally exerted a significant anxiolytic-like effect in the four plate test with ED50 values 3.4 and 7.9mg/kg, respectively. At 30mg/kg they reduced duration of immobility in the forced swim test for 33% and 19%, respectively. They had no effect on electroconvulsive threshold or pain reactivity in the hot plate assay but they were antinociceptive in the acetic acid-induced writhing test (ED50 values were 12.7 and 18.6mg/kg, respectively) and in both phases of the formalin test (ED50 values in the first phase were 10.2 and 2.1mg/kg for BM 130 and BM 131, respectively). No motor adverse effects were observed in mice pretreated with the test compounds in the rotarod or chimney tests but BM 131 caused a transient but statistically significant decrease of animals' locomotor activity. ConclusionsIn mice BM 130 and BM 131 have anxiolytic-like, antidepressant-like and antinociceptive properties which can be attributed to their affinity for not only mGAT1 but also mGAT2–4.

Cyclopropane-based conformational restriction of GABA by a stereochemical diversity-oriented strategy: Identification of an efficient lead for potent inhibitors of GABA transports

A series of cyclopropane-based conformationally restricted γ-aminobutyric acid (GABA) analogs with stereochemical diversity, that is, the trans- and cis-2,3-methano analogs Ia and Ib and their enantiomers ent-Ia and ent-Ib, and also the trans- and cis-3,4-methano analogs IIa and IIb and their enantiomers ent-IIa and ent-Iib, were synthesized from the chiral cyclopropane units Type-a and Type-b that we developed. These analogs were systematically evaluated with four GABA transporter (GAT) subtypes. The trans-3,4-methano analog IIa had inhibitory effects on GAT3 (IC50=23.9μM) and betaine-GABA transporter1 (5.48μM), indicating its potential as an effective lead compound for the development of potent GAT inhibitors due to its hydrophilic and low molecular weight properties and excellent ligand efficiency.

Discovery of a subtype selective inhibitor of the human betaine/GABA transporter 1 (BGT-1) with a non-competitive pharmacological profile

The γ-aminobutyric acid (GABA) transporters (GATs) are essential regulators of the activity in the GABAergic system through their continuous uptake of the neurotransmitter from the synaptic cleft and extrasynaptic space. Four GAT subtypes have been identified to date, each displaying different pharmacological properties and expression patterns. The present study focus on the human betaine/GABA transporter 1 (BGT-1), which has recently emerged as a new target for treatment of epilepsy. However, the lack of selective inhibitors of this transporter has impaired the exploration of this potential considerably. With the objective of identifying novel compounds displaying selectivity for BGT-1, we performed a screening of a small compound library at cells expressing BGT-1 using a [3H]GABA uptake assay. The screening resulted in the identification of the compound N-(1-benzyl-4-piperidinyl)-2,4-dichlorobenzamide (BPDBA), a selective inhibitor of the human BGT-1 transporter with a non-competitive profile exhibiting no significant inhibitory activity at the other three human GAT subtypes. The selectivity profile of the compound was subsequently confirmed at cells expressing the four mouse GAT subtypes. Thus, BPDBA constitutes a potential useful pharmacological tool compound for future explorations of the function of the BGT-1 subtype.