Structural basis of GABA reuptake inhibition.

γ-Aminobutyric acid (GABA) transporter 1 (GAT1)1 regulates neuronal excitation of the central nervous system by clearing the synaptic cleft of the inhibitory neurotransmitter GABA upon its release from synaptic vesicles. Elevating the levels of GABA in the synaptic cleft, by inhibiting GABA reuptake transporters, is an established strategy to treat neurological disorders, such as epilepsy2. Here we determined the cryo-electron microscopy structure of full-length, wild-type human GAT1 in complex with its clinically used inhibitor tiagabine3, with an ordered part of only 60 kDa. Our structure reveals that tiagabine locks GAT1 in the inward-open conformation, by blocking the intracellular gate of the GABA release pathway, and thus suppresses neurotransmitter uptake. Our results provide insights into the mixed-type inhibition of GAT1 by tiagabine, which is an important anticonvulsant medication. Its pharmacodynamic profile, confirmed by our experimental data, suggests initial binding of tiagabine to the substrate-binding site in the outward-open conformation, whereas our structure presents the drug stalling the transporter in the inward-open conformation, consistent with a two-step mechanism of inhibition4. The presented structure of GAT1 gives crucial insights into the biology and pharmacology of this important neurotransmitter transporter and provides blueprints for the rational design of neuromodulators, as well as moving the boundaries of what is considered possible in single-particle cryo-electron microscopy of challenging membrane proteins.

Development of tricyclic N-benzyl-4-hydroxybutanamide derivatives as inhibitors of GABA transporters mGAT1-4 with anticonvulsant, antinociceptive, and antidepressant activity.

γ-Aminobutyric acid (GABA) neurotransmission has a significant impact on the proper functioning of the central nervous system. Numerous studies have indicated that inhibitors of the GABA transporters mGAT1-4 offer a promising strategy for the treatment of several neurological disorders, including epilepsy, neuropathic pain, and depression. Following our previous results, herein, we report the synthesis, biological evaluation, and structure-activity relationship studies supported by molecular docking and molecular dynamics of a new series of N-benzyl-4-hydroxybutanamide derivatives regarding their inhibitory potency toward mGAT1-4. This study allowed us to identify compound 23a (N-benzyl-4-hydroxybutanamide bearing a dibenzocycloheptatriene moiety), a nonselective GAT inhibitor with a slight preference toward mGAT4 (pIC50 = 5.02 ± 0.11), and compound 24e (4-hydroxy-N-[(4-methylphenyl)-methyl]butanamide bearing a dibenzocycloheptadiene moiety) with relatively high inhibitory activity toward mGAT2 (pIC50 = 5.34 ± 0.09). In a set of in vivo experiments, compound 24e successively showed predominant anticonvulsant activity and antinociception in the formalin model of tonic pain. In contrast, compound 23a showed significant antidepressant-like properties in mice. These results were consistent with the available literature data, which indicates that, apart from seizure control, GABAergic neurotransmission is also involved in the pathophysiology of several psychiatric diseases, however alternative mechanisms underlying this action cannot be excluded. Finally, it is worth noting that the selected compounds showed unimpaired locomotor skills that have been indicated to give reliable results in behavioral assays.

N-Substituted Nipecotic Acids as (S)-SNAP-5114 Analogues with Modified Lipophilic Domains.

Potential mGAT4 inhibitors derived from the lead substance (S)-SNAP-5114 have been synthesized and characterized for their inhibitory potency. Variations from the parent compound included the substitution of one of its aromatic 4-methoxy and 4-methoxyphenyl groups, respectively, with a more polar moiety, including a carboxylic acid, alcohol, nitrile, carboxamide, sulfonamide, aldehyde or ketone function, or amino acid partial structures. Furthermore, it was investigated how the substitution of more than one of the aromatic 4-methoxy groups affects the potency and selectivity of the resulting compounds. Among the synthesized test substances (S)-1-{2-[(4-formylphenyl)bis(4-methoxyphenyl)-methoxy]ethyl}piperidine-3-carboxylic acid, that features a carbaldehyde function in place of one of the aromatic 4-methoxy moieties of (S)-SNAP-5114, was found to have a pIC50 value of 5.89±0.07, hence constituting a slightly more potent mGAT4 inhibitor than the parent substance while showing comparable subtype selectivity.

Synthesis and biological evaluation of novel N-substituted nipecotic acid derivatives with a cis-alkene spacer as GABA uptake inhibitors.

To discover new, potent, and selective inhibitors for the murine gamma-aminobutyric acid transporter 4 (mGAT4), the structure-activity relationship (SAR) study of a new cis-alkene analog family based on DDPM-1457 [(S)-2], which previously showed promising inhibitory potency at and subtype selectivity for mGAT4, was conducted. To uncover the importance of the differences between the trans- and the cis-alkene moiety in the spacer, the present publication describes the synthesis of the new compounds via catalytic hydrogenation with Lindlar's catalyst. The biological results collected by the SAR study revealed that analog rac-7j characterized by a four-instead of a three-carbon atom spacer with a cis double bond applying to the majority of the studied compounds displays a surprisingly high potency at mGAT1 (pIC50 = 6.00 ±â€¯0.04) and at the same time a reasonable potency at mGAT4 (pIC50 = 4.82).

MS-Based Screening of 5-Substituted Nipecotic Acid Derived Hydrazone Libraries as Ligands of the GABA Transporter†1.

A screening of compound libraries based on nipecotic acid derivatives with lipophilic residues attached to the scarcely explored 5-position of the core structure was used for the search of new inhibitors of the γ-aminobutyric acid (GABA) transporter 1 (mGAT1). The generated compound libraries, which were based on hydrazone chemistry commonly used in dynamic combinatorial chemistry but rendered pseudostatic, were screened for their binding affinities toward mGAT1 by means of MS Binding Assays. With nipecotic acid derived hydrazone rac-16 h [rac-(3R,5S)-{5-[(E)-2-{[5-(2-phenylethynyl)thiophen-2-yl]methylidene}hydrazin-1-yl]piperidine-3-carboxylic acid}-sodium chloride (1/2)], one hit was found and evaluated displaying sub-micromolar potency (pKi =6.62±0.04) and a noncompetitive interaction mode at mGAT1. By bearing a 5-(2-phenylethynyl)thiophen-2-yl residue attached to the 5-position of nipecotic acid via a three-atom spacer, compound rac-16 h contains a structural moiety so far unprecedented for these kinds of bioactive molecules, and complements novel 5-substituted nipecotic acid derived ligands of mGAT1 revealed in a recently published screening campaign. This new class of ligands, with an inhibition mode distinct from that of benchmark mGAT1 inhibitors, could serve as research tools for investigations of mGAT1-mediated GABA transport.

Studies on the Activity of Selected Highly Lipophilic Compounds toward hGAT1 Inhibition. Part II.

In this paper, we describe the latest results involving molecular modeling and pharmacodynamic studies of the selected highly lipophilic compounds acting by human GABA transporter 1 (hGAT1) inhibition. The chemical interaction of 17 GABA analogues with a model of hGAT1 is described using the molecular docking method. The biological role of GAT1 is related to the regulation of GABA level in the central nervous system and GAT1 inhibition plays an important role in the control of seizure threshold. To confirm that GAT1 can be also a molecular target for drugs used to treat other neurological and psychiatric diseases (e.g., pain and anxiety), in the in vivo part of this study, potential antinociceptive and anxiolytic-like properties of tiagabine, a selective GAT1 inhibitor, are described.

Generation and screening of pseudostatic hydrazone libraries derived from 5-substituted nipecotic acid derivatives at the GABA transporter mGAT4.

The γ-aminobutyric acid (GABA) transporter mGAT4 represents a promising drug target for the treatment of epilepsy and other neurological disorders; however, the lack of highly potent and selective inhibitors for mGAT4 still retards its pharmacological elucidation. Herein, the generation and screening of pseudostatic combinatorial hydrazone libraries at the murine GABA transporter mGAT4 for the search of novel GABA uptake inhibitors is described. The hydrazone libraries contained more than 1100 compounds derived from nipecotic acid derivatives substituted at the 5-position instead, as common, at the 1-position of the core structure. Two hits were found and evaluated, which display potencies in the lower micromolar range at mGAT4 and its human equivalent hGAT3. These compounds possess a lipophilic moiety derived from a biphenyl residue attached to the 5-position of the hydrophilic nipecotic acid moiety via a three-atom spacer. Thus, the novel structures with potencies close to that of the bench mark mGAT4 inhibitor (S)-SNAP-5114 add new insights into the structure-activity relationship of mGAT4 inhibitors and could provide a promising starting point for the development of new mGAT4 inhibitors with even higher potencies.

Identification of Pyrrolidine-3-acetic Acid Derived Oximes as Potent Inhibitors of γ-Aminobutyric Acid Transporter†1 through Library Screening with MS Binding Assays.

In this study, pyrrolidine-3-acetic acid derived oxime libraries were applied to the concept of library screening by MS Binding Assays, as a powerful technique to reveal new potent murine γ-aminobutyric acid transporter subtype (mGAT1) inhibitors. Library generation was accomplished by condensation of an excess of pyrrolidine-3-acetic acid bearing a hydroxylamine unit with various libraries, each composed of eight different aldehydes. The oxime libraries have been screened by means of competitive MS Binding Assays and, as a consequence, the most active libraries were further investigated through deconvolution experiments to identify single oximes responsible for the observed activity on the target mGAT1. All identified hits were finally resynthesized to characterize them with respect to their binding affinities, and a set of new potent inhibitors with the pyrrolidine-3-acetic acid motif were found, of which the most potent oxime, possessing a 2',4'-dichlorobiphenyl residue, displayed a binding affinity in the low nanomolar range (pKi =7.87±0.01).

Synthesis and biological evaluation of novel N-substituted nipecotic acid derivatives with a trans-alkene spacer as potent GABA uptake inhibitors.

Our study presents the synthesis and structure-activity relationship (SAR) of novel N-substituted nipecotic acid derivatives closely related to DDPM-1457 [(S)-2a], a chemically stable analog of (S)-SNAP-5114 (1), in the pursuit of finding new and potent mGAT4 selective inhibitors. Iminium ion chemistry served as key step for the preparation of the desired, new N-substituted nipecotic acid derivatives containing a variety of different heterocycles attached to the nipecotic acid moiety via a trans-alkene spacer. The target compounds were characterized with regard to their potency at and subtype selectivity for the GABA transporters mGAT1-mGAT4.

Docking and pharmacodynamic studies on hGAT1 inhibition activity in the presence of selected neuronal and astrocytic inhibitors. Part I.

Inhibition of 4-aminobutanoic acid (GABA) uptake is a strategy for enhancing GABA transmission. The utility of this approach is demonstrated by the successful development of such agents for the treatment of epilepsy and pain. Existing reports on acute brain slice preparations indicate the intersecting of complementary channels and receptors sets between astrocytes and neurons cells. Thorough analysis of astroglial cells by means of molecular and functional studies demonstrated their active modulatory role in intercellular communication. The chemical interactions between sixteen GABA analogues and isoform of hGAT1 is outlined in the light of molecular docking results. In the in vivo part antinociceptive properties of racemic nipecotic acid, its R and S enantiomers and isonipecotic acid, each administered intraperitoneally at 3 fixed doses (10, 30 and 100 mg/kg), were assessed in a thermally-induced acute pain model i.e. the mouse hot plate test. Docking analyses provided complex binding energies, specific h-bond components, and h-bond properties, such as energies, distances and angles. In vivo tests revealed statistically significant antinociceptive properties of isonipecotic acid (10 and 30 mg/kg), R-nipecotic acid (30 and 100 mg/kg) and S-nipecotic acid (100 mg/kg) in mice. The docking data endorse the hypothesis of correlation between the strength of their chemical interactions with hGAT1 and analgesic action of studied compounds.

Design and synthesis of cyclopropane-based conformationally restricted GABA analogues as selective inhibitors for betaine/GABA transporter 1.

We previously designed and synthesized a series of cyclopropane-based conformationally restricted analogues of γ-aminobutyric acid (GABA). The study demonstrated that the critical conformation of the analogues that selectively active to betaine/GABA transporter 1 (BGT1) subtype is the trans-syn-form, in which the amino and carboxyl groups are in trans-configuration and the cyclopropane ring and the carboxyl group are in syn-arrangement. In this study, we designed and synthesized cyclopropane-based GABA analogues, which were conformationally restricted in the trans-syn-form by cyclopropylic strain based on the stereochemistry of the carbon adjacent to cyclopropane. Their conformation was confirmed as the syn-form by calculations and NMR studies, and their pharmacological evaluation clarified that compounds 11a and 11d had the BGT1 selectivity, although their inhibitory effects were insufficient.

Design, Synthesis, Evaluation and Computational Studies of Nipecotic Acid-Acetonaphthone Hybrids as Potential Antiepileptic Agents.

BACKGROUND: Nipecotic acid is considered to be one of the most potent inhibitors of neuronal and glial γ-aminobutyric acid (GABA) uptake in vitro. However, nipecotic acid does not readily cross the blood-brain barrier (BBB) following peripheral administration, owing to its hydrophilic nature. OBJECTIVE: A series of substituted acetonaphthones tethered nipecotic acid derivatives were designed and synthesized with an aim to improve the lipophilicity and the blood-brain barrier (BBB) permeation. METHODS: Synthesized compounds were tested in mice models of PTZ, pilocarpine, and DMCM induced epilepsy, in vivo. The rota-rod test was performed to determine the acute neurotoxicity of the potential leads (4a, 4b, and 4i). These potential hybrids were also evaluated for their ability to cross the BBB by an in vitro parallel artificial membrane permeability BBB assay (PAMPA-BBB). The leads were subjected to in silico molecular docking and dynamics studies on homology modelled protein of human GABA (γ-amino butyric acid) transporter 1 (GAT1) and prediction of their pharmacokinetic properties. RESULT: Amongst the synthesized derivatives, compounds 3a, 3b, 3i, 4a, 4b, and 4i exhibited increased latency of seizures against subcutaneous pentylenetetrazole (scPTZ) induced seizures in mice. Derivatives 4a, 4b, 4i were more effective compared to nipecotic acid ester counterparts 3a, 3b and 3i placing the importance of the presence of free carboxyl group in the centre. The findings revealed that 4i was comparatively more permeable (Pe= 8.89) across BBB than the standard tiagabine (Pe= 7.86). In silico studies proved the consensual interactions of compound 4i with the active binding pocket. CONCLUSION: Some nipecotic acid-acetonaphthone hybrids with considerable anti-epileptic activity, drug like properties and the ability to permeate the BBB have been successfully synthesized.

Structure-Activity Relationship, Pharmacological Characterization, and Molecular Modeling of Noncompetitive Inhibitors of the Betaine/γ-Aminobutyric Acid Transporter 1 (BGT1).

N-(1-Benzyl-4-piperidinyl)-2,4-dichlorobenzamide 5 (BPDBA) is a noncompetitive inhibitor of the betaine/GABA transporter 1 (BGT1). We here report the synthesis and structure-activity relationship of 71 analogues. We identify 26m as a more soluble 2,4-Cl substituted 3-pyridine analogue with retained BGT1 activity and an improved off-target profile compared to 5. We performed radioligand-based uptake studies at chimeric constructs between BGT1 and GAT3, experiments with site-directed mutated transporters, and computational docking in a BGT1 homology model based on the newly determined X-ray crystal structure of the human serotonin transporter (hSERT). On the basis of these experiments, we propose a binding mode involving residues within TM10 in an allosteric site in BGT1 that corresponds to the allosteric binding pocket revealed by the hSERT crystal structure. Our study provides first insights into a proposed allosteric binding pocket in BGT1, which accommodates the binding site for a series of novel noncompetitive inhibitors.

Delineation of the Role of Astroglial GABA Transporters in Seizure Control.

Studies of GABA transport in neurons and astrocytes have provided evidence that termination of GABA as neurotransmitter is brought about primarily by active transport into the presynaptic, GABAergic nerve endings. There is, however, a considerable transport capacity in the astrocytes surrounding the synaptic terminals, a transport which may limit the availability of transmitter GABA leading to a higher probability of seizure activity governed by the balance of excitatory and inhibitory neurotransmission. Based on this it was hypothesized that selective inhibition of astrocytic GABA transport might prevent such seizure activity. A series of GABA analogs of restricted conformation were synthesized and in a number of collaborative investigations between Prof. Steve White at the University of Utah and medicinal chemists and pharmacologists at the School of Pharmacy and the University of Copenhagen, Denmark, GABA analogs with exactly this pharmacological property were identified. The most important analogs identified were N-methyl-exo-THPO (N-methyl-3-hydroxy-4-amino-4,5,6,7-tetrahydro-1,2-benzisoxazole) and its lipophilic analog EF-1502 ((RS)-4-[N-[1,1-bis(3-methyl-2-thienyl)but-1-en-4-yl]-N-methylamino]-4,5,6,7-tetrahydrobenzo[d]isoxazol-3-ol) both of which turned out to be potent anticonvulsants in animal models of epilepsy.

Development of Highly Potent GAT1 Inhibitors: Synthesis of Nipecotic Acid Derivatives with N-Arylalkynyl Substituents.

A new scaffold of highly potent and mGAT1-selective inhibitors has been developed. Compounds in this class are characterized by an alkyne-type spacer connecting nipecotic acid with an aromatic moiety. Preliminary evaluations made it apparent that a nipecotic acid derivative with an N-butynyl linker and a terminal 2-biphenyl residue exhibiting a binding affinity (pKi ) of 7.61±0.03 to mGAT1 and uptake inhibition (pIC50 ) of 7.00±0.06 selective for mGAT1 could serve as a hit compound. Docking calculations for compounds based on this structure in an hGAT1 homology modeling study indicated binding affinities similar to or even higher than that of the well-known mGAT1 inhibitor tiagabine. Synthesis of the designed compounds was readily carried out by two consecutive cross-coupling reactions, giving flexible access to variously substituted biphenyl subunits. With an appropriate substitution pattern of the biphenyl moiety, the binding affinity of enantiopure (R)-nipecotic acid derivatives to mGAT1 increased to pKi =8.33±0.01, and the uptake inhibitory potency up to pIC50 =7.72±0.02.

Development of MLR and SVM Aided QSAR Models to Identify Common SAR of GABA Uptake Herbal Inhibitors used in the Treatment of Schizophrenia.

BACKGROUND: Alterations in GABAnergic system are implicated in the pathophysiology of schizophrenia. Available antipsychotics that target GABA receptor form a desirable therapeutic strategy in the treatment regimen of schizophrenia, unfortunately, suffer serious setback due to their prolonged side effects. The present investigation focuses on developing QSAR models from the biological activity of herbal compounds and their derivatives that promise to be alternative candidates to GABA uptake inhibitors. METHODS: Three sets of compounds were undertaken in the study to develop QSAR models. The first set consisted of nine compounds which included Magnolol, Honokiol and other GABA acting established compounds. The second set consisted of 16 derivatives of N-diarylalkenylpiperidinecarboxylic acid. The third QSAR dataset was made up of thirty two compounds which were Magnolol and Honokiol derivatives. Multiple linear regressions (MLR) and support vector machine (SVM) supervised quantitative structure-activity relationship (QSAR) models were developed to predict the biological activity of these three sets. The purpose of taking three QSAR sets of diverse chemical structures but identical in their GABA targeting and pharmacological action was to identify common chemical structure features responsible for structure-activity relationship (SAR). RESULTS: Linear and non-linear QSAR models confirmed that the three sets shared common structural descriptors derived from WHIM (Weighted Holistic Invariant Molecular descriptors), 3D-MoRSE and Eigenvalue classes. CONCLUSION: It was concluded that properties like electro negativity and polarizability play a crucial role in controlling the activity of herbal compounds against GABA receptor.

Synthesis and biological evaluation of a series of N-alkylated imidazole alkanoic acids as mGAT3 selective GABA uptake inhibitors.

In this paper, we report the synthesis and biological evaluation of a series of 1,5- and 1,4- substituted derivatives of 1H-imidazol-4-ylacetic acid, a series of 1,2-substituted 3-(1H-imidazol-2-yl)propanoic acid and an N-substituted (2E)-3-(1H-imidazol-2-yl)prop-2-enoic acid as new mGAT3 inhibitors. The lipophilic moieties attached to the N-atom of the parent structures were delineated from the 2-[9-(4-methoxyphenyl)-9H-fluoren-9-yl]oxyethyl residue, known from a prototypic mGAT3 inhibitor. For the structure-activity-relationship studies, the spacer between the N-atom of the imidazole ring and the 2-[9-(4-methoxyphenyl)-9H-fluoren-9-yl] moiety was varied in length from three to six atoms, and in nature being either a pure saturated or unsaturated alkyl chain or an alkyl chain containing up to two ether functions. The compounds were characterized for inhibitory potencies at mouse GABA transporter proteins mGAT1-mGAT4. Among the 1,2-substituted compounds, the N-alkylated (2E)-3-(1H-imidazol-2-yl)prop-2-enoic acid 12e containing a C5O spacer exhibits a pIC50 value of 5.13 ± 0.04 at mGAT3, but is devoid of significant selectivity for this GABA transporter. However, the inhibitory potency displayed by 12e at mGAT3 nominally surpasses that of SNAP-5294 reported as the most potent inhibitor of mGAT3 so far.

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.

Structure activity relationship of selective GABA uptake inhibitors.

A series of ß-amino acids with lipophilic diaromatic side chain was synthesized and characterized pharmacologically on mouse γ-amino butyric acid (GABA) transporter subtypes mGAT1-4 in order to investigate structure activity relationships (SAR) for mGAT2 (corresponding to hBGT-1). Variation in the lipophilic diaromatic side chain was probed to understand the role of the side chain for activity. This yielded several selective compounds of which the best (1R,2S)-5a was more than 10 fold selective towards other subtypes, although potency was moderate. A docking study was performed to investigate possible binding modes of the compounds in mGAT2 suggesting a binding mode similar to that proposed for Tiagabine in hGAT1. Specific interactions between the transporter and the amino acid part of the ligands may account for a reverted preference towards mGAT2 over mGAT1.