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The pharmacology of amino acid transporters in the SLC6 family is poorly developed compared to that of the neurotransmitter transporters. To identify new inhibitors of the proline transporter SIT1 (SLC6A20), its expression in Xenopus laevis oocytes was optimized. Trafficking of SIT1 was augmented by co-expression of angiotensin-converting enzyme 2 (ACE2) in oocytes but there was no strict requirement for co-expression of ACE2. A pharmacophore-guided screen identified tiagabine as a potent non-competitive inhibitor of SIT1. To understand its binding mode, we determined the cryo-electron microscopy (cryo-EM) structure of ACE2-SIT1 bound with tiagabine. The inhibitor binds close to the orthosteric proline binding site, but due to its size extends into the cytosolic vestibule. This causes the transporter to adopt an inward-open conformation, in which the intracellular gate is blocked. This study provides the first structural insight into inhibition of SIT1 and generates tools for a better understanding of the ACE2-SIT1 complex. These findings may have significance for SARS-CoV-2 binding to its receptor ACE2 in human lung alveolar cells where SIT1 and ACE2 are functionally expressed., Competing Interests: Conflict of interest The authors declare that they have no conflict of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Avacopan is a small-molecule complement 5a receptor (CD88) antagonist recently approved by the United States Food and Drug Administration as an adjunct therapy in combination with immunosuppressants and corticosteroids for treatment of ANCA-vasculitis. The selective ability of avacopan to inhibit the C5a receptor blocks neutrophil chemoattraction, activation, and adhesion while maintaining other beneficial complement pathways. Therefore, avacopan's unique selective property provides a breakthrough treatment for ANCA- vasculitis given that current therapies of corticosteroid treatment often lead to a decreased quality of life and a possible relapse. Clinical trials prove that avacopan is an excellent adjunctive treatment option, although it is not approved for the primary treatment of ANCA-vasculitis at this time. Initial clinical trials show substantial promise for avacopan, but additional studies with a longer duration will be needed to test for its durability and safety., Competing Interests: Disclosure: The authors declare no conflict of interest., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

Recent experimental data have revealed the complement, and more specifically the C5a compound, plays a major role in the pathophysiology of ANCA associated vasculitis (AAV). The development of avacopan, an oral inhibitor of C5a receptor, has allowed to test the blockade of this immunological pathway, initially in a murine animal model of the disease, followed by a preliminary, phase 2 therapeutic trial in human disease, with promising results. An international phase 3 trial published in 2021 demonstrated that avacopan can be used instead of corticosteroids for the induction therapy of AAV, in association with an immunosuppressive drug such as cyclophosphamide or rituximab. The adjunction of this new-generation immunosuppressive drug does not increase the infectious risk, but seems to amplify the improvement of renal function during the initial treatment of renal vasculitis. These results have led to the recent registration of avacopan, opening new therapeutic options in AAV. © 2022 Published by Elsevier Masson SAS on behalf of Société francophone de néphrologie, dialyse et transplantation., (Copyright © 2022 Société francophone de néphrologie, dialyse et transplantation. Publié par Elsevier Masson SAS. Tous droits réservés.)

Gamma-Aminobutyric Acid (GABA) inhibitory neurotransmitter departs an energetic role in brain signalling system. Levels of GABA in the brain influence human behaviour, diminishes in the degree of GABA can cause seizures, depression, Parkinson's. To put it plainly, it plays a basic part in the significant issues of mind. It is exceptionally important to cure the issues linked to GABA. Writing overview proposed that nipecotic acid is an intense GABA reuptake inhibitor. This scaffold is likewise present in one of the promoted anticonvulsant drugs 'Tiagabine'. Tiagabine is only drug in the market which works through this mechanism however the medication is regulated with one more prescription for the synergistic impact. Nipecotic acid has several disadvantages such as it can't cross the blood-brain barrier because of its hydrophilic and zwitterionic nature. To avoid this problem nipecotic acid scaffold hybrids with the different aromatic groups can enhance the physical (lipophilicity) as well as biological properties of the resultant compound. So, there is a dire requirement for compounds that work through this mechanism. Several medicinal chemists and researchers are already working in this field and developed outstanding newer molecules. This review article compiles these developed new hybrids along with design strategies, structure-activity relationship, and biological activity as well as in silico studies. This review also demonstrates the synthesis of nipecotic acid and the core mechanism through which nipecotic acid acts as a GABA reuptake inhibitor., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Masson SAS. All rights reserved.)

Avacopan (TAVNEOS™) is a complement 5a receptor (C5aR) antagonist developed by ChemoCentryx for the treatment of autoimmune diseases including anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis. The therapeutic effects of avacopan are attributed to the inhibition of C5aR activity on neutrophils, however, the exact mechanism of therapeutic efficacy in patients with ANCA-associated vasculitis has not been established. In September 2021, avacopan received its first approval in Japan for the treatment of microscopic polyangiitis (MPA) and granulomatosis with polyangiitis (GPA), the two most common forms of ANCA-associated vasculitis, where it is being commercialized by Kissei Pharmaceutical through a partnership with Vifor Pharma. In October 2021, avacopan was approved in the USA as an adjunctive treatment in adults for severe active ANCA-associated vasculitis (specifically MPA and GPA) in combination with standard therapy including glucocorticoids (avacopan does not eliminate glucocorticoid use). Avacopan has received a positive opinion in the EU, and is also undergoing regulatory review in Switzerland and Canada. Avacopan is being investigated for the treatment of complement component 3 glomerulopathy, hidradenitis suppurativa, lupus nephritis and IgA nephropathy. This article summarizes the milestones in the development of avacopan leading to these first approvals in Japan and the USA., (© 2021. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Complement activation is key to anti-microbial defenses by directly acting on microbes and indirectly by triggering cellular immune responses. Complement activation may also contribute to the pathogenesis of numerous inflammatory and immunological diseases. Consequently, intense research focuses on developing therapeutics that block pathology-causing complement activation while preserving anti-microbial complement activities. However, the pace of research is slowed down significantly by the limitations of current tools for evaluating complement-targeting therapeutics. Moreover, the effects of potential therapeutic agents on innate immune cells, like neutrophils, are not fully understood. Here, we employ microfluidic assays and measure chemotaxis, phagocytosis, and swarming changes in human neutrophils ex vivo in response to various complement-targeting agents. We show that whereas complement factor 5 (C5) cleavage inhibitor eculizumab blocks all neutrophil anti-microbial functions, newer compounds like the C5 cleavage inhibitor RA101295 and C5a receptor antagonist avacopan inhibit chemotaxis and swarming while preserving neutrophil phagocytosis. These results highlight the utility of microfluidic neutrophil assays in evaluating potential complement-targeting therapeutics., Competing Interests: Authors, DDV, SR, YT, AR and CES were employees of Ra Pharma at the time when this work was performed. DDV, SR, YT, CES and AR were shareholders of Ra Pharma (now part of UCB Pharma). The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Muldur, Vadysirisack, Ragunathan, Tang, Ricardo, Sayegh and Irimia.)

As the most abundant inhibitory neurotransmitter in the mammalian brain, γ-aminobutyric acid (GABA) plays a crucial role in shaping the frequency and amplitude of oscillations, which suggests a role for GABA in shaping the topography of functional connectivity and activity. This study explored the effects of pharmacologically blocking the reuptake of GABA (increasing local concentrations) using the GABA transporter 1 (GAT1) blocker, tiagabine (15 mg). In a placebo-controlled crossover design, we collected resting magnetoencephalography (MEG) recordings from 15 healthy individuals prior to, and at 1-, 3- and 5- hours post, administration of tiagabine and placebo. We quantified whole brain activity and functional connectivity in discrete frequency bands. Drug-by-session (2 × 4) analysis of variance in connectivity revealed interaction and main effects. Post-hoc permutation testing of each post-drug recording vs. respective pre-drug baseline revealed consistent reductions of a bilateral occipital network spanning theta, alpha and beta frequencies, across 1- 3- and 5- hour recordings following tiagabine only. The same analysis applied to activity revealed significant increases across frontal regions, coupled with reductions in posterior regions, across delta, theta, alpha and beta frequencies. Crucially, the spatial distribution of tiagabine-induced changes overlap with group-averaged maps of the distribution of GABA A receptors, from flumazenil (FMZ-V T ) PET, demonstrating a link between GABA availability, GABA A receptor distribution, and low-frequency network oscillations. Our results indicate that the relationship between PET receptor distributions and MEG effects warrants further exploration, since elucidating the nature of this relationship may uncover electrophysiologically-derived maps of oscillatory activity as sensitive, time-resolved, and targeted receptor-mapping tools for pharmacological imaging., Competing Interests: Declaration of Competing Interest All authors declare no conflict of interest., (Copyright © 2021. Published by Elsevier B.V.)

Nipecotic acid is considered to be one of the most potent inhibitors of neuronal and glial-aminobutyric acid (GABA) uptake in vitro. Due to its hydrophilic nature, nipecotic acid does not readily cross the blood-brain barrier (BBB). Large neutral amino acids (LAT1)-knotted nipecotic acid prodrug was designed and synthesized with the aim to enhance the BBB permeation by the use of carrier-mediated transport. The synthesized prodrug was tested in animal models of Pentylenetetrazole (PTZ)-induced convulsions in mice. Further pain studies were carried out followed by neurotoxicity estimation by writhing and rota-rod test respectively. HPLC data suggests that the synthesized prodrug has improved penetration through BBB. Nipecotic acid-L-serine ester prodrug with considerable anti-epileptic activity, and the ability to permeate the BBB has been successfully synthesized. Graphical Abstract., Competing Interests: The authors declare no conflict of interest, financial or otherwise, (Thieme. All rights reserved.)

Pathogenic mutations in the copper transporter ATP7B have been hypothesized to affect its protein interaction landscape contributing to loss of function and, thereby, to hepatic copper toxicosis in Wilson disease. Although targeting mutant interactomes was proposed as a therapeutic strategy, druggable interactors for rescue of ATP7B mutants remain elusive. Using proteomics, we found that the frequent H1069Q substitution promotes ATP7B interaction with HSP70, thus accelerating endoplasmic reticulum (ER) degradation of the mutant protein and consequent copper accumulation in hepatic cells. This prompted us to use an HSP70 inhibitor as bait in a bioinformatics search for structurally similar Food and Drug Administration-approved drugs. Among the hits, domperidone emerged as an effective corrector that recovered trafficking and function of ATP7B-H1069Q by impairing its exposure to the HSP70 proteostatic network. Our findings suggest that HSP70-mediated degradation can be safely targeted with domperidone to rescue ER-retained ATP7B mutants and, hence, to counter the onset of Wilson disease., Competing Interests: The authors declare no competing interest.

The hyperactivity of the lateral habenula (LHb) is closely associated with depression. At present, it is unknown how GABA transporter (GAT) in the LHb affects depressive-like behaviors, particularly in Parkinson's disease (PD)-related depression. In this study, unilateral 6-hydroxydopamine lesions of the substantia nigra pars compacta (SNc) in rats induced depressive-like behaviors and led to hyperactivity of LHb neurons compared to sham-lesioned rats. Intra-LHb injection of GAT-1 inhibitor NO-711 produced antidepressant-like responses, decreased firing rate of LHb neurons, and increased levels of LHb extracellular GABA in sham-lesioned and the lesioned rats. Further, the dose producing behavioral effects in the lesioned rats was lower than that of sham-lesioned rats. In the lesioned rats, the duration of inhibitory effect on the firing rate and increased levels of the GABA induced by NO-711 was longer than those in sham-lesioned rats, respectively. Intra-LHb injection of GAT-3 inhibitor SNAP-5114 improved depressive-like behaviors and decreased firing rate of LHb neurons in the lesioned rats, but not in sham-lesioned rats. SNAP-5114 increased LHb GABA levels in the lesioned rats, whereas did not alter that in sham-lesioned rats. These changes were involved in the down-regulated expression of LHb GAT-1 and GAT-3 after lesioning the SNc. These findings suggest that GAT-1 plays a major role in transporting LHb GABA under physiological conditions, and depletion of dopamine increases the transport capacity of GAT-3 in the LHb. Further, the study provides evidence that GAT-1 and GAT-3 in the LHb are involved in the regulation of PD-related depression., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The complement fragment C5a is a core effector of complement activation. C5a, acting through its major receptor C5aR1, exerts powerful pro-inflammatory and immunomodulatory functions. Dysregulation of the C5a-C5aR1 axis has been implicated in numerous immune disorders, and the therapeutic inhibition of this axis is therefore imperative for the treatment of these diseases. A myriad of small-molecule C5aR1 inhibitors have been developed and independently characterised over the past two decades, however the pharmacological properties of these compounds has been difficult to directly compare due to the wide discrepancies in the model, read-out, ligand dose and instrumentation implemented across individual studies. Here, we performed a systematic characterisation of the most commonly reported and clinically advanced small-molecule C5aR1 inhibitors (peptidic: PMX53, PMX205 and JPE1375; non-peptide: W545011, NDT9513727, DF2593A and CCX168). Through signalling assays measuring C5aR1-mediated cAMP and ERK1/2 signalling, and β-arrestin 2 recruitment, this study highlighted the signalling-pathway dependence of the rank order of potencies of the C5aR1 inhibitors. Functional experiments performed in primary human macrophages demonstrated the high insurmountable antagonistic potencies for the peptidic inhibitors as compared to the non-peptide compounds. Finally, wash-out studies provided novel insights into the duration of inhibition of the C5aR1 inhibitors, and confirmed the long-lasting antagonistic properties of PMX53 and CCX168. Overall, this study revealed the potent and prolonged antagonistic activities of selected peptidic C5aR1 inhibitors and the unique pharmacological profile of CCX168, which thus represent ideal candidates to fulfil diverse C5aR1 research and clinical therapeutic needs., (Copyright © 2020 Elsevier Inc. All rights reserved.)

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 pIC 50 value of 5.89±0.07, hence constituting a slightly more potent mGAT4 inhibitor than the parent substance while showing comparable subtype selectivity., (© 2020 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

Activation of hepatic stellate cells (HSCs), characterized by development of a robust actin cytoskeleton and expression of abundant extracellular matrix (ECM) proteins, such as type 1 collagen (COL.1), is a central cellular and molecular event in liver fibrosis. It has been demonstrated that HSCs express both myocardin and myocardin-related transcription factor-A (MRTF-A). However, the biological effects of myocardin and MRTF-A on HSC activation and liver fibrosis, as well as the molecular mechanism under the process, remain unclear. Here, we report that myocardin and MRTF-A's expression and nuclear accumulation are prominently increased during the HSC activation process, accompanied by robust activation of actin cytoskeleton dynamics. Targeting myocardin and MRTF-A binding and function with a novel small molecule, CCG-203971, led to dose-dependent inhibition of HSC actin cytoskeleton dynamics and abrogated multiple functional features of HSC activation (i.e., HSC contraction, migration and proliferation) and decreased COL.1 expression in vitro and liver fibrosis in vivo. Mechanistically, blocking the myocardin and MRTF-A nuclear translocation pathway with CCG-203971 directly inhibited myocardin/MRTF-A-mediated serum response factor (SRF), and Smad2/3 activation in the COL.1α2 promoter and indirectly abrogated actin cytoskeleton-dependent regulation of Smad2/3 and Erk1/2 phosphorylation and their nuclear accumulation. Finally, there was no effect of CCG-203971 on markers of inflammation, suggesting a direct effect of the compound on HSCs and liver fibrosis. These data reveal that myocardin and MRTF-A are two important cotranscriptional factors in HSCs and represent entirely novel therapeutic pathways that might be targeted to treat liver fibrosis. NEW & NOTEWORTHY Myocardin and myocardin-related transcription factor-A (MRTF-A) are upregulated in activated hepatic stellate cells (HSCs) in vitro and in vivo, closely associated with robustly increased actin cytoskeleton remodeling. Targeting myocardin and MRTF-A by CCG-203971 leads to actin cytoskeleton-dependent inhibition of HSC activation, reduced cell contractility, impeded cell migration and proliferation, and decreased COL.1 expression in vitro and in vivo. Dual expression of myocardin and MRTF-A in HSCs may represent novel therapeutic targets in liver fibrosis.

A role for the alternative complement pathway has emerged in the understanding of ANCA vasculitis pathogenesis. Current therapies of ANCA vasculitis are limited by partial efficacy and toxicity and many patients pursue a relapsing course. Improved therapies are needed. Inhibition of the alternative complement pathway component C5a is attractive due to its role in neutrophil activation and migration, and engagement of other inflammatory and thrombotic mechanisms. Two inhibitors of C5a are in clinical development for ANCA vasculitis: avacopan, an oral C5a receptor inhibitor has demonstrated efficacy, safety and steroid sparing in two Phase II trials; and IFX-1, a monoclonal antibody to C5a which is entering Phase II development. Complement inhibition has the potential to contribute to remission induction protocols achieving a higher quality of remission as well as replacing steroids. Confirmation of safety, especially infective risk, and the potential to replace steroids depends on further studies and a role in relapse prevention needs to be explored., (Copyright © 2019 Société francophone de néphrologie, dialyse et transplantation. Published by Elsevier Masson SAS. All rights reserved.)

Background: The lack of anti-fibrotic agents targeting intestinal fibrosis is a large unmet need in inflammatory bowel diseases, including Crohn's disease and ulcerative colitis. Previous studies have found that perinatal tissue (umbilical cord, UC; placenta, PL)-derived mesenchymal stem cells (MSCs) reduce fibrosis in several organs. However, their effects on human intestinal fibrosis are poorly understood. This study investigated the anti-fibrogenic properties and mechanisms of MSCs derived from UC and PL (UC/PL-MSCs) on human primary intestinal myofibroblasts (HIMFs)., Methods: The HIMFs were treated with TGF-β1 and co-cultured with UC/PL-MSCs. We used a small molecular inhibitor CCG-100602 to examine whether serum response factor (SRF) and its transcriptional cofactor myocardin-related transcription factor A (MRTF-A) are involved in TGF-β1-induced fibrogenic activation in HIMFs. The anti-fibrogenic mechanism of UC/PL-MSCs on HIMFs was analyzed by detecting the expression of RhoA, MRTF-A, and SRF in HIMFs., Results: UC/PL-MSCs reduced TGF-β1-induced procollagen1A1, fibronectin, and α-smooth muscle actin expression in HIMFs. This anti-fibrogenic effect was more apparent in the UC-MSCs. TGF-β1 stimulation increased the expressions of RhoA, MRTF-A, and SRF in the HIMFs. TGF-β1 induced the synthesis of procollagen1A1, fibronectin, and α-smooth muscle actin through a MRTF-A/SRF-dependent mechanism. Co-culture with the UC/PL-MSCs downregulated fibrogenesis by inhibition of RhoA, MRTF-A, and SRF expression., Conclusions: UC/PL-MSCs suppress TGF-β1-induced fibrogenic activation in HIMFs by blocking the Rho/MRTF/SRF pathway and could be considered as a novel candidate for stem cell-based therapy of intestinal fibrosis.

A new class of nipecotic acid and guvacine derivatives has been synthesized and characterized for their inhibitory potency at mGAT1-4 and binding affinity for mGAT1. Compounds of the described class are defined by a four-carbon-atom allenyl spacer connecting the nitrogen atom of the nipecotic acid or guvacine head with an aromatic residue. Among the compounds investigated, the mixture of nipecotic acid derivatives rac-{(R a )-1-[4-([1,1':2',1''-terphenyl]-2-yl)buta-2,3-dien-1-yl](3R)-piperidine-3-carboxylic acid} and rac-{(S a )-1-[4-([1,1':2',1''-terphenyl]-2-yl)buta-2,3-dien-1-yl](3R)-piperidine-3-carboxylic acid} (21 p), possessing an o-terphenyl residue, was identified as highly selective and the most potent mGAT1 inhibitor in this study. For the (R)-nipecotic acid derived form of 21 p, the inhibitory potency in [ 3 H]GABA uptake assays was determined as pIC 50 =6.78±0.08, and the binding affinity in MS Binding Assays as pK i =7.10±0.12. The synthesis of the designed compounds was carried out by a two-step procedure, generating the allene moiety via allenylation of terminal alkynes which allows broad variation of the terminal phenyl and biphenyl subunit., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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 (pIC 50  = 6.00 ± 0.04) and at the same time a reasonable potency at mGAT4 (pIC 50  = 4.82)., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Mancozeb (MZ), an organic-metal fungicide used predominantly on vegetables and fruits, has been linked to neurodegeneration and behavioral disruptions in a variety of organisms, including humans. Both γ-aminobutyric acid and dopamine neurons appear to be more vulnerable to MZ exposure than other neuronal populations. Based on these observations, we hypothesized that MZ may be differentially transported into these cells through their presynaptic neurotransmitter transporters. To test this, we pretreated Caenorhabditis elegans with transporter antagonists followed by exposure to various concentrations of MZ. Potential neuroprotection was monitored via green fluorescence associated with various neuron populations in transgenic worm strains. Neurodegeneration associated with subacute MZ treatment (30 min) was not altered by transporter antagonist pretreatment. On the other hand, pretreatment with a dopamine transporter antagonist (GBR12909) appeared to protect dopaminergic neurons from chronic (24 h) MZ treatment. These results are consistent with other reports that dopamine transporter levels or activity may modulate toxicity for neurotoxicants., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The C5a receptor (C5aR) is a G-protein-coupled receptor (GPCR) that can induce strong inflammatory response to the anaphylatoxin C5a. Targeting C5aR has emerged as a novel anti-inflammatory therapeutic method. However, developing potent C5aR antagonists as drugs has proven difficult. Here, we report two crystal structures of human C5aR in ternary complexes with the peptide antagonist PMX53 and a non-peptide antagonist, either avacopan or NDT9513727. The structures, together with other biophysical, computational docking and cell-based signaling data, reveal the orthosteric action of PMX53 and its effect of stabilizing the C5aR structure, as well as the allosteric action of chemically diverse non-peptide C5aR antagonists with different binding poses. Structural comparison analysis suggests the presence of similar allosteric sites in other GPCRs. We also discuss critical structural features of C5aR in activation, including a novel conformation of helix 8. On the basis of our results, we suggest novel strategies for developing C5aR-targeting drugs.

Introduction: ANCA-associated vasculitis (AAV) is a rare but potentially life-threatening disease. Currently used induction treatment (cyclophosphamide or rituximab with high-dose corticosteroids) has significantly improved outcome of AAV, but is associated with high toxicity. Alternative complement pathway activation was shown to play a role in the pathogenesis of AAV, thus providing rationale for the use of avacopan, a selective inhibitor of C5a receptor, in the treatment of AAV. Areas covered: Pharmacokinetic and pharmocodynamic properties of avacopan, clinical efficacy and safety and tolerability of avacopan in so far performed clinical trials in patients with AAV are reviewed and discussed. Expert opinion: Avacopan was shown to have at least similar efficacy compared to high dose corticosteroids in patients with active AAV with renal involvement, while there were no major safety issues reported. Replacement of corticosteroids should decrease the corticosteroid-related toxicity and improve long-term outcome of patients with AAV even though this still needs to be confirmed in a larger trial. Data on long-term outcome of avacopan-treated patients are currently lacking and will be eagerly awaited. In the future, avacopan could replace corticosteroids not only in the induction phase, but also in the maintenance treatment of AAV.

Mechanisms underlying functional recovery after stroke are little known, and effective drug intervention during the delayed stage is desirable. One potential drug target, the protein-protein interaction between neuronal nitric oxide synthase (nNOS) and postsynaptic density protein 95 (PSD-95), is critical to acute ischaemic damage and neurogenesis. We show that nNOS-PSD-95 dissociation induced by microinjection of a recombinant fusion protein, Tat-nNOS-N 1-133 , or systemic administration of a small-molecule, ZL006, from day 4 to day 10 after photothrombotic ischaemia in mice reduced excessive tonic inhibition in the peri-infarct cortex and ameliorated motor functional outcome. We also demonstrated improved neuroplasticity including increased dendrite spine density and synaptogenesis after reducing excessive tonic inhibition by nNOS-PSD-95 dissociation. Levels of gamma-aminobutyric acid (GABA) and GABA transporter-3/4 (GAT-3/4) are increased in the reactive astrocytes in the peri-infarct cortex. The GAT-3/4-selective antagonist SNAP-5114 reduced tonic inhibition and promoted function recovery, suggesting that increased tonic inhibition in the peri-infarct cortex was due to GABA release from reversed GAT-3/4 in reactive astrocytes. Treatments with Tat-nNOS-N 1-133 or ZL006 after ischaemia inhibited astrocyte activation and GABA production, prevented the reversal of GAT-3/4, and consequently decreased excessive tonic inhibition and ameliorated functional outcome. The underlying molecular mechanisms were associated with epigenetic inhibition of glutamic acid decarboxylase 67 and monoamine oxidase B expression through reduced NO production. The nNOS-PSD-95 interaction is thus a potential target for functional restoration after stroke and ZL006, a small molecule inhibitor of this interaction, is a promising pharmacological lead compound. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd., (Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

GABA is a principal neurotransmitter in the hypothalamic suprachiasmatic nucleus (SCN) that contributes to intercellular communication between individual circadian oscillators within the SCN network and the stability and precision of the circadian rhythms. GABA transporters (GAT) regulate the extracellular GABA concentration and modulate GABA A receptor (GABA A R)-mediated currents. GABA transport inhibitors were applied to study how GABA A R-mediated currents depend on the expression and function of GAT. Nipecotic acid inhibits GABA transport and induced an inward tonic current in concentration-dependent manner during whole cell patch-clamp recordings from SCN neurons. Application of either the selective GABA transporter 1 (GAT1) inhibitors NNC-711 or SKF-89976A, or the GABA transporter 3 (GAT3) inhibitor SNAP-5114, produced only small changes of the baseline current. Coapplication of GAT1 and GAT3 inhibitors induced a significant GABA A R-mediated tonic current that was blocked by gabazine. GAT inhibitors decreased the amplitude and decay time constant and increased the rise time of spontaneous GABA A R-mediated postsynaptic currents. However, inhibition of GAT did not alter the expression of either GAT1 or GAT3 in the hypothalamus. Thus GAT1 and GAT3 functionally complement each other to regulate the extracellular GABA concentration and GABA A R-mediated synaptic and tonic currents in the SCN. Coapplication of SKF-89976A and SNAP-5114 (50 µM each) significantly reduced the circadian period of Per1 expression in the SCN by 1.4 h. Our studies demonstrate that GAT are important regulators of GABA A R-mediated currents and the circadian clock in the SCN. NEW & NOTEWORTHY In the suprachiasmatic nucleus (SCN), the GABA transporters GAT1 and GAT3 are expressed in astrocytes. Inhibition of these GABA transporters increased a tonic GABA current and reduced the circadian period of Per1 expression in SCN neurons. GAT1 and GAT3 showed functional cooperativity: inhibition of one GAT increased the activity but not the expression of the other. Our data demonstrate that GABA transporters are important regulators of GABA A receptor-mediated currents and the circadian clock., (Copyright © 2017 the American Physiological Society.)

The ability to quantify synaptic function at the level of cortical microcircuits from non-invasive data would be enormously useful in the study of neuronal processing in humans and the pathophysiology that attends many neuropsychiatric disorders. Here, we provide proof of principle that one can estimate inter-and intra-laminar interactions among specific neuronal populations using induced gamma responses in the visual cortex of human subjects - using dynamic causal modelling based upon the canonical microcircuit (CMC; a simplistic model of a cortical column). Using variability in induced (spectral) responses over a large cohort of normal subjects, we find that the predominant determinants of gamma responses rest on recurrent and intrinsic connections between superficial pyramidal cells and inhibitory interneurons. Furthermore, variations in beta responses were mediated by inter-subject differences in the intrinsic connections between deep pyramidal cells and inhibitory interneurons. Interestingly, we also show that increasing the self-inhibition of superficial pyramidal cells suppresses the amplitude of gamma activity, while increasing its peak frequency. This systematic and nonlinear relationship was only disclosed by modelling the causes of induced responses. Crucially, we were able to validate this form of neurophysiological phenotyping by showing a selective effect of the GABA re-uptake inhibitor tiagabine on the rate constants of inhibitory interneurons. Remarkably, we were able to recover the pharmacodynamics of this effect over the course of several hours on a per subject basis. These findings speak to the possibility of measuring population specific synaptic function - and its response to pharmacological intervention - to provide subject-specific biomarkers of mesoscopic neuronal processes using non-invasive data. Finally, our results demonstrate that, using the CMC as a proxy, the synaptic mechanisms that underlie the gain control of neuronal message passing within and between different levels of cortical hierarchies may now be amenable to quantitative study using non-invasive (MEG) procedures., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Accumulating evidence indicates that cannabinoid CB1 receptor ligands play a pivotal role in seizures, not only in preclinical studies on animals, but also in clinical settings. This study was aimed at characterizing the influence of arachidonyl-2'-chloroethylamide (ACEA-a selective cannabinoid CB1 receptor agonist) co-administered with phenylmethylsulfonyl fluoride (PMSF) on the anticonvulsant potency of various antiepileptic drugs (clobazam, lacosamide, levetiracetam, phenobarbital, tiagabine and valproate) in the 6-Hz corneal stimulation model. Psychomotor seizures in male albino Swiss mice were evoked by a current (32 mA, 6 Hz, 3 s stimulus duration) delivered via corneal electrodes. Potential adverse effects produced by the antiepileptic drugs in combination with ACEA+PMSF were assessed using the chimney test (motor performance), passive avoidance task (remembering and acquisition of learning), and grip-strength test (muscular strength). Brain concentrations of antiepileptic drugs were measured by HPLC to exclude any pharmacokinetic contribution to the observed effect. ACEA (5 mg/kg, i.p.) + PMSF (30 mg/kg, i.p.) significantly potentiated the anticonvulsant potency of levetiracetam (P<0.05), but not that of clobazam, lacosamide, phenobarbital, tiagabine or valproate in the 6-Hz corneal stimulation model. Moreover, ACEA+PMSF did not significantly affect total brain concentrations of levetiracetam in mice. No behavioral side effects were observed in animals receiving combinations of the studied antiepileptic drugs with ACEA+PMSF. In conclusion, the combined administration of ACEA+PMSF with levetiracetam is associated with beneficial anticonvulsant pharmacodynamic interaction in the 6-Hz corneal stimulation model. The selective activation of cannabinoid CB1 receptor-mediated neurotransmission in the brain may enhance levetiracetam-related suppression of seizures in epilepsy patients, contributing to the efficacious treatment of epilepsy in future.

(1) Background: Temporal lobe epilepsy (TLE) is the most common type of epilepsy in adults. It is also the one with the highest percentage of drug-resistance to the current available anti-epileptic drugs (AED). Additionaly, most antiepileptic drugs are only able to control seizures in epileptogenesis, but do not decrease the hippocampal neurodegenerative process. TLE patients have a reduced population of interneuronal cells, which express Parvalbumin (PV) proteins. This reduction is directly linked to seizure frequency and severity in the chronic period of epilepsy. There is therefore a need to seek new therapies with a disease-modifying profile, and with efficient antiepileptic and neuroprotective properties. Parawixin2, a compound isolated from the venom of the spider Parawixia bistriata , has been shown to inhibit GABA transporters (GAT) and to have acute anticonvulsant effects in rats. (2) Methods: In this work, we studied the effects of Parawixin2 and Tiagabine (an FDA- approved GAT inhibitor), and compared these effects in a TLE model. Rats were subjected to lithium-pilocarpine TLE model and the main features were evaluated over a chronic period including: (a) spontaneous recurrent seizures (SRS), (b) neuronal loss, and (c) PV cell density in different regions of the hippocampus (CA1, CA3, DG and Hilus). (3) Results: Parawixin2 treatment reduced SRS frequency whereas Tiagabine did not. We also found a significant reduction in neuronal loss in CA3 and in the hilus regions of the hippocampus, in animals treated with Parawixin2. Noteworthy, Parawixin2 significantly reversed PV cell loss observed particularly in DG layers. (4) Conclusions: Parawixin2 exerts a promising neuroprotective and anti-epileptic effect and has potential as a novel agent in drug design., Competing Interests: The authors declare no conflict of interest.

The series of experiments herein evaluated prototype drugs representing different mechanisms of antiseizure, antinociceptive or antidepressant action in a battery of preclinical pain models in adult male CF#1 mice (formalin, writhing, and tail flick) and Sprague Dawley rats partial sciatic nerve ligation (PSNL). In the formalin assay, phenytoin (PHT, 6 mg/kg), sodium valproate (VPA, 300 mg/kg), amitriptyline (AMI, 7.5 and 15 mg/kg), gabapentin (GBP, 30 and 70 mg/kg), tiagabine (TGB, 5 and 15 mg/kg), and acetominophen (APAP, 250 and 500 mg/kg) reduced both phases of the formalin response to ≤ 25% of vehicle-treated mice. In the acetic acid induced writhing assay, VPA (300 mg/kg), ethosuximide (ETX, 300 mg/kg), morphine (MOR, 5 & 10 mg/kg), GBP (10, 30, and 60 mg/kg), TGB (15 mg/kg), levetiracetam (LEV, 300 mg/kg), felbamate (FBM, 80 mg/kg) and APAP (250 mg/kg) reduced writhing to ≤ 25% of vehicle-treated mice. In the tail flick test, MOR (1.25-5 mg/kg), AMI (15 mg/kg) and TGB (5 mg/kg) demonstrated significant antinociceptive effects. Finally, carbamazepine (CBZ, 20 and 50 mg/kg), VPA, MOR (2 and 4 mg/kg), AMI (12 mg/kg), TPM (100 mg/kg), lamotrigine (LTG, 40 mg/kg), GBP (60 mg/kg), TGB (15 mg/kg), FBM (35 mg/kg), and APAP (250 mg/kg) were effective in the PSNL model. Thus, TGB was the only prototype compound with significant analgesic effects in each of the four models, while AMI, GBP, APAP, and MOR each improved three of the four pain phenotypes. This study highlights the importance evaluating novel targets in a variety of pain phenotypes.

Stimulated by the results of a recent paper on the effects of tiagabine, a selective inhibitor of the main GABA transporter GAT-1, on oligodendrogenesis, we verified the possibility that GAT-1 may be expressed in oligodendrocytes using immunocytochemical methods and functional assays. Light microscopic analysis of the subcortical white matter of all animals revealed the presence of numerous GAT-1+ cells of different size (from 3 to 29 µm) and morphology. An electron microscope analysis revealed that, besides fibrous astrocytes and interstitial neurons, GAT-1 immunoreactivity was present in immature and mature oligodendrocytes. Co-localization studies between GAT-1 and markers specific for oligodendrocytes (NG2 and RIP) showed that about 12% of GAT-1 positive cells in the white matter were immature oligodendrocytes, while about 15% were mature oligodendrocytes. In vitro functional assays showed that oligodendrocytes exhibit tiagabine-sensitive Na + -dependent GABA uptake. Although relationships between GABA and oligodendrocytes have been known for many years, this is the first demonstration that GAT-1 is expressed in oligodendrocytes. The present results on the one hand definitely closes the era of "neuronal" and "glial" GABA transporters, on the other they suggest that oligodendrocytes may contribute to pathophysiology of the several diseases in which GAT-1 have been implicated to date. GLIA 2017;65:514-522., (© 2017 Wiley Periodicals, Inc.)

Introduction: Obstructive sleep apnea (OSA) severity is markedly reduced during slow-wave sleep (SWS) even in patients with a severe disease. The reason for this improvement is uncertain but likely relates to non-anatomical factors (i.e. reduced arousability, chemosensitivity, and increased dilator muscle activity). The anticonvulsant tiagabine produces a dose-dependent increase in SWS in subjects without OSA. This study aimed to test the hypothesis that tiagabine would reduce OSA severity by raising the overall arousal threshold during sleep., Aims and Methods: After a baseline physiology night to assess patients' OSA phenotypic traits, a placebo-controlled, double-blind, crossover trial of tiagabine 12 mg administered before sleep was performed in 14 OSA patients. Under each condition, we assessed the effects on sleep and OSA severity using standard clinical polysomnography., Results: Tiagabine increased slow-wave activity (SWA) of the electroencephalogram (1-4 Hz) compared to placebo (1.8 [0.4] vs. 2.0 [0.5] LogμV2, p = .04) but did not reduce OSA severity (apnea-hypopnea index [AHI] 41.5 [20.3] vs. 39.1 [16.5], p > .5). SWS duration (25 [20] vs. 26 [43] mins, p > .5) and arousal threshold (-26.5 [5.0] vs. -27.6 [5.1] cmH2O, p = .26) were also unchanged between nights., Conclusions: Tiagabine modified sleep microstructure (increase in SWA) but did not change the duration of SWS, OSA severity, or arousal threshold in this group of OSA patients. Based on these findings, tiagabine should not be considered as a therapeutic option for OSA treatment., (© Sleep Research Society 2016. Published by Oxford University Press on behalf of the Sleep Research Society. All rights reserved. For permissions, please e-mail journals.permissions@oup.com.)

Background/aims: Our previous studies demonstrated that intrinsic aortic smooth muscle cell (VSMC) stiffening plays a pivotal role in aortic stiffening in aging and hypertension. However, the underlying molecular mechanisms remain largely unknown. We here hypothesized that Rho kinase (ROCK) acts as a novel mediator that regulates intrinsic VSMC mechanical properties through the serum response factor (SRF) /myocardin pathway and consequently regulates aortic stiffness and blood pressure in hypertension., Methods: Four-month old male spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats were studied. Aortic stiffness was measured by echography. Intrinsic mechanical properties of VSMCs were measured by atomic force microscopy (AFM) in vitro., Results: Compared to WKY rats, SHR showed a significant increase in aortic stiffness and blood pressure, which is accompanied by a remarkable cell stiffening and ROCK activation in thoracic aortic (TA) VSMCs. Theses alterations in SHR were abolished by Y-27632, a specific inhibitor of ROCK. Additionally, boosted filamentous/globular actin ratio was detected in TA VSMCs from SHR versus WKY rats, resulting in an up-regulation of SRF and myocardin expression and its downstream stiffness-associated genes including α-smooth muscle actin, SM22, smoothelin and myosin heavy chain 11. Reciprocally, these alterations in SHR TA VSMCs were also suppressed by Y-27632. Furthermore, a specific inhibitor of SRF/myocardin, CCG-100602, showed a similar effect to Y-27632 in SHR in both TA VSMCs stiffness in vitro and aorta wall stiffness in vivo., Conclusion: ROCK is a novel mediator modulating aortic VSMC stiffness through SRF/myocardin signaling which offers a therapeutic target to reduce aortic stiffening in hypertension., (© 2017 The Author(s). Published by S. Karger AG, Basel.)

The chemical interaction of nine antiepileptic drugs (tiagabine, gabapentin, pregabalin, lamotrigine, zonisamide, valproic acid, valpromide, vigabatrin, progabide) and two endogenous metabolites (4-aminobutanoic acid, 4-hydroxybutanoic acid) with a model of human GABA transporter 1 (hGAT1) is described using the molecular docking method. To establish the role of hGAT1 in chronic pain, tiagabine, a selective hGAT1 inhibitor, was assessed in the in vivo experiments for its antiallodynic properties in two mouse models of neuropathic pain. Docking analyses performed in this study provided the complex binding energies, specific hydrogen bond components, and hydrogen bond properties such as energies, distances and angles. The data of the docking studies strongly support the assumption that the antiepileptic and analgesic actions of the studied drugs can be at least in part related to the strength of their chemical interactions with hGAT1. In vivo experiments with tiagabine confirmed the involvement of hGAT1 in the regulation of the mechanical nociceptive threshold in neuropathic pain., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Inactivation of GABA-mediated neurotransmission is achieved by high-affinity transporters located at both GABAergic neurons and the surrounding astrocytes. Early studies of the pharmacological properties of neuronal and glial GABA transporters suggested that different types of transporters might be expressed in the two cell types, and such a scenario was confirmed by the cloning of four distinctly different GABA transporters from a number of different species. These GABA-transport entities have been extensively characterized using a large number of GABA analogues of restricted conformation, and several of these compounds have been shown to exhibit pronounced anticonvulsant activity in a variety of animal seizure models. As proof of concept of the validity of this drug development approach, one GABA-transport inhibitor, tiagabine, has been developed as a clinically active antiepileptic drug. This review provides a detailed account of efforts to design new subtype-selective GABA-transport inhibitors aiming at identifying novel antiepileptic drug candidates.

Melanoma is the most dangerous form of skin cancer with the majority of deaths arising from metastatic disease. Evidence implicates Rho-activated gene transcription in melanoma metastasis mediated by the nuclear localization of the transcriptional coactivator, myocardin-related transcription factor (MRTF). Here, we highlight a role for Rho and MRTF signaling and its reversal by pharmacologic inhibition using in vitro and in vivo models of human melanoma growth and metastasis. Using two cellular models of melanoma, we clearly show that one cell type, SK-Mel-147, is highly metastatic, has high RhoC expression, and MRTF nuclear localization and activity. Conversely, SK-Mel-19 melanoma cells have low RhoC expression, and decreased levels of MRTF-regulated genes. To probe the dependence of melanoma aggressiveness to MRTF transcription, we use a previously developed small-molecule inhibitor, CCG-203971, which at low micromolar concentrations blocks nuclear localization and activity of MRTF-A. In SK-Mel-147 cells, CCG-203971 inhibits cellular migration and invasion, and decreases MRTF target gene expression. In addition, CCG-203971-mediated inhibition of the Rho/MRTF pathway significantly reduces cell growth and clonogenicity and causes G 1 cell-cycle arrest. In an experimental model of melanoma lung metastasis, the RhoC-overexpressing melanoma cells (SK-Mel-147) exhibited pronounced lung colonization compared with the low RhoC-expressing SK-Mel-19. Furthermore, pharmacologic inhibition of the MRTF pathway reduced both the number and size of lung metastasis resulting in a marked reduction of total lung tumor burden. These data link Rho and MRTF-mediated signaling with aggressive phenotypes and support targeting the MRTF transcriptional pathway as a novel approach to melanoma therapeutics. Mol Cancer Ther; 16(1); 193-204. ©2016 AACR., (©2016 American Association for Cancer Research.)

The frequency of visual gamma oscillations is determined by both the neuronal excitation-inhibition balance and the time constants of GABAergic processes. The gamma peak frequency has been linked to sensory processing, cognitive function, cortical structure, and may have a genetic contribution. To disentangle the intricate relationship among these factors, accurate and reliable estimates of peak frequency are required. Here, a bootstrapping approach that provides estimates of peak frequency reliability, thereby increasing the robustness of the inferences made on this parameter was developed. The method using both simulated data and real data from two previous pharmacological MEG studies of visual gamma with alcohol and tiagabine was validated. In particular, the study by Muthukumaraswamy et al. [] (Neuropsychopharmacology 38(6):1105-1112), in which GABAergic enhancement by tiagabine had previously demonstrated a null effect on visual gamma oscillations, contrasting with strong evidence from both animal models and very recent human studies was re-evaluated. After improved peak frequency estimation and additional exclusion of unreliably measured data, it was found that the GABA reuptake inhibitor tiagabine did produce, as predicted, a marked decrease in visual gamma oscillation frequency. This result demonstrates the potential impact of objective approaches to data quality control, and provides additional translational evidence for the mechanisms of GABAergic transmission generating gamma oscillations in humans. Hum Brain Mapp 37:3882-3896, 2016. © 2016 Wiley Periodicals, Inc., (© 2016 The Authors Human Brain Mapping Published by Wiley Periodicals, Inc.)

The complement 5a receptor has been an attractive therapeutic target for many autoimmune and inflammatory disorders. However, development of a selective and potent C5aR antagonist has been challenging. Here we describe the characterization of CCX168 (avacopan), an orally administered selective and potent C5aR inhibitor. CCX168 blocked the C5a binding, C5a-mediated migration, calcium mobilization, and CD11b upregulation in U937 cells as well as in freshly isolated human neutrophils. CCX168 retains high potency when present in human blood. A transgenic human C5aR knock-in mouse model allowed comparison of the in vitro and in vivo efficacy of the molecule. CCX168 effectively blocked migration in in vitro and ex vivo chemotaxis assays, and it blocked the C5a-mediated neutrophil vascular endothelial margination. CCX168 was effective in migration and neutrophil margination assays in cynomolgus monkeys. This thorough in vitro and preclinical characterization enabled progression of CCX168 into the clinic and testing of its safety, tolerability, pharmacokinetic, and pharmacodynamic profiles in a Phase 1 clinical trial in 48 healthy volunteers. CCX168 was shown to be well tolerated across a broad dose range (1 to 100 mg) and it showed dose-dependent pharmacokinetics. An oral dose of 30 mg CCX168 given twice daily blocked the C5a-induced upregulation of CD11b in circulating neutrophils by 94% or greater throughout the entire day, demonstrating essentially complete target coverage. This dose regimen is being tested in clinical trials in patients with anti-neutrophil cytoplasmic antibody-associated vasculitis. Trial Registration ISRCTN registry with trial ID ISRCTN13564773., Competing Interests: The work was funded by ChemoCentryx, Inc. The ChemoCentryx, Inc. website notes that CCX168 is under development by this company and have market exclusivity. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

In this work we investigated the effect of nanomolar concentrations of nitric oxide on the release of gamma-aminobutyric acid (GABA) from rat brain nerve terminals using a radioisotope method with [3H]GABA and a spectrofluorimetric method with Ca2+-sensitive probe Fluo-4 AM. It was shown that in the presen­ce of dithiothreitol (DTT), nitric oxide donor SNAP at concentration, in which it produces NO in the nanomolar range, caused Ca2+-independent [3H]GABA release from nerve terminals. The applications of 4-aminopyridine (4-AP) and nipecotic acid (NA), as the inducers of GABA release from vesicular and cytoplasmic pools, showed that the maximum of SNAP/+DTT-induced [3H]GABA release was registered at 10th min of incubation and coincided in time with significant increase (almost double) in NA-induced [3H]GABA release. At this time point, 4-AP-induced release of [3H]GABA was drastically reduced. At the 15th min of incubation of nerve terminals with SNAP/+DTT, the opposite picture was observed: the decrease in NA- and increase in 4-AP-induced [3H]GABA release. Thus, nitric oxide in the form of S-nitrosothiols at nanomolar concentrations causes Ca2+-independent GABA leakage from synaptic vesicles into cytosol with subsequent release from nerve terminals. The reuptake of the neurotransmitter and its re-accumulation in synaptic vesicles occur later.

An estimated three billion people are at risk of Dengue virus (DENV) infection worldwide and there are currently no approved therapeutic interventions for DENV infection. Due to the relatively small size of the DENV genome, DENV is reliant on host factors throughout the viral life cycle. The inducible form of Heat Shock Protein 70 (Hsp70i) has been implicated as a host factor in DENV pathogenesis, however the complete role remains to be elucidated. Here we further illustrate the importance of Hsp70i in dengue virus pathogenesis and describe the antiviral activity of the allosteric small molecule inhibitor that is selective for Hsp70i, called HS-72. In monocytes, Hsp70i is expressed at low levels preceding DENV infection, but Hsp70i expression is induced upon DENV infection. Targeting Hsp70i with HS-72, results in a dose dependent reduction in DENV infected monocytes, while cell viability was maintained. HS-72 works to reduce DENV infection by inhibiting the entry stage of the viral life cycle, through disrupting the association of Hsp70i with the DENV receptor complex. This work highlights Hsp70i as an antiviral target and HS-72 as a potential anti-DENV therapeutic agent., (Copyright © 2016 Elsevier B.V. All rights reserved.)

In this study, we disclose the design and synthesis of novel 4-susbtituted nipecotic acid derivatives as inhibitors of the GABA transporter mGAT1. Based on molecular modeling studies the compounds are assumed to adopt a binding pose similar to that of the potent mGAT1 inhibitor nipecotic acid. As substitution in 4-position should not cause an energetically unfavorable orientation of nipecotic acid as it is the case for N-substituted derivatives this is expected to lead to highly potent binders. For the synthesis of novel 4-substituted nipecotic acid derivatives a linear synthetic strategy was employed. As a key step, palladium catalyzed cross coupling reactions were used to attach the required biaryl moieties to the ω-position of the alkenyl- or alkynyl spacers of varying length in the 4-position of the nipecotic acid scaffold. The resulting amino acids were characterized with respect to their binding affinities and inhibitory potencies at mGAT1. Though the biological activities found were generally insignificant to poor, two compounds, one of which possesses a reasonable binding affinity for mGAT1, rac-57, the other a notable inhibitory potency at mGAT4, rac-84, both displaying a slight subtype selectivity for the individual transporters, could be identified., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

A new series of potent and selective mGAT1 inhibitors has been identified, featuring a nipecotic acid residue and an N-butenyl linker with a 2-biphenyl residue at the ω-position. Docking, combined with MD calculations, revealed a binding mode for the new compounds similar to that of tiagabine, the only mGAT1 inhibitor currently approved as antiepileptic drug. For the synthesis, a Suzuki-Miyaura cross-coupling reaction was used as a key step by which variously substituted biaryl subunits were assembled. Biological evaluation revealed several compounds that possess binding affinities and inhibitory potencies toward mGAT1, together with subtype selectivities against mGAT2-mGAT4 that were similar to or even higher than those for tiagabine. A derivative carrying the 2',4'-dichloro-2-biphenyl moiety attached to N-but-3-enylnipecotic acid at the terminal position of the linker chain was found to be the most potent binder, with the racemic form of the compound displaying a binding affinity of 8.05±0.13 (pKi ), while the R enantiomer exhibited an affinity value of 8.33±0.06 (pKi )., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

γ-Aminobutyric acid (GABA), the principal brain inhibitory neurotransmitter, modulates inflammatory and neurodegenerative disease. Here, we tested the hypothesis that central GABAergic neurotransmission mediates the detrimental inflammatory, hemodynamic, and cardiac autonomic actions of endotoxemia. The effects of drugs that block GABA receptors or interfere with GABA uptake or degradation on blood pressure (BP), heart rate (HR), and HR variability (HRV) responses elicited by i.v. lipopolysaccharide (LPS) were assessed in conscious rats. The hypotensive effect of LPS (10 mg/kg) was blunted after intracisternal (i.c.) administration of bicuculline (GABAA receptor antagonist) or saclofen (GABAB receptor antagonist). By contrast, the concomitant LPS-evoked tachycardia and decreases in time domain and frequency domain indices of HRV (measures of cardiac autonomic control) were abolished upon treatment with bicuculline but not saclofen. Increases in serum tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) caused by LPS disappeared in the presence of bicuculline or saclofen, whereas LPS-evoked increases in serum nitric oxide metabolites (NOx) were counteracted by bicuculline only. None of the endotoxemia effects was altered in rats treated with i.c. tiagabine (GABA reuptake inhibitor) or vigabatrin (GABA transaminase inhibitor). These data suggest a major role for central GABAA receptors in the inflammatory and cardiovascular effects of endotoxemia.

The aim of the present study was to investigate if basic GABA-mimetics interact with the taurine transporter (TauT, Slc6a6), and to find a suitable cell based model that is robust towards extracellular changes in osmolality during uptake studies. Taurine uptake was measured in human Caco-2 cells, porcine LLC-PK1 cells, and rat SKPT cells using radiolabelled taurine. Hyperosmotic conditions were obtained by incubation with raffinose (final osmolality of 500mOsm) for 24h prior to the uptake experiments. Expression of the taurine transporter, TauT, was investigated at the mRNA level by real-time PCR. Uptake of the GABA-mimetics gaboxadol and vigabatrin was investigated in SKPT cells, and quantified by liquid scintillation or HPLC-MS/MS analysis, respectively. The uptake rate of [(3)H]-taurine was Na(+) and Cl(-) and concentration dependent with taurine with an apparent Vmax of 6.3±1.6pmolcm(-2)min(-1) and a Km of 24.9±15.0μM. β-alanine, nipecotic acid, gaboxadol, GABA, vigabatrin, δ-ALA and guvacine inhibited the taurine uptake rate in a concentration dependent manner. The order of affinity for TauT was β-alanine>GABA>nipecotic acid>guvacine>δ-ALA>vigabatrin>gaboxadol with IC50-values of 0.04, 1.07, 2.02, 4.19, 4.94, 31.4 and 39.9mM, respectively. In conclusion, GABA mimetics inhibited taurine uptake in hyperosmotic rat renal SKPT cells. SKPT cells, which seem to be a useful model for investigating taurine transport in the short-term presence of high concentrations of osmolytes. Furthermore, analogues of β-alanine appear to have higher affinities for TauT than GABA-analogues., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Background: GABAergic neurotransmission is involved in long-term potentiation, a neurophysiological basis for learning and memory. On the other hand, GABA-enhancing drugs may impair memory and learning in humans and animals. The present study aims at investigating the effect of GAT1 inhibitor tiagabine on memory and learning., Methods: Albino Swiss (CD-1) and C57BL/6J mice were used in the passive avoidance (PA), Morris water maze (MWM) and radial arm water maze (RAWM) tasks. Scopolamine (1mg/kg ip) was applied to induce cognitive deficits., Results: In the retention trial of PA scopolamine reduced step-through latency as compared to vehicle-treated mice, and pretreatment with tiagabine did not have any influence on this effect. In MWM the results obtained for vehicle-treated mice, scopolamine-treated group and combined scopolamine+tiagabine-treated mice revealed variable learning abilities in these groups. Tiagabine did not impair learning in the acquisition trial. In RAWM on day 1 scopolamine-treated group made nearly two-fold more errors than vehicle-treated mice and mice that received combined scopolamine and tiagabine. Learning abilities in the latter group were similar to those of vehicle-treated mice in the corresponding trial block on day 1, except for the last trial block, during which tiagabine+scopolamine-injected mice made more errors than control mice and the scopolamine-treated group. In all groups a complete reversal of memory deficits was observed in the last trial block of day 2., Conclusions: The lack of negative influence of tiagabine on cognitive functions in animals with scopolamine-induced memory impairments may be relevant for patients treated with this drug., (Copyright © 2015 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.)

Chronic morphine exposure produces morphine tolerance. One of the mechanisms of morphine tolerance involves γ-aminobutric acid (GABA), whose level is regulated by GABA transporter 1 (GAT-1). The aim of this study was to investigate the expression of GAT-1 in the spinal cord during morphine treatment. Morphine was administrated to rats via drinking water for 21 days. On day 21, a single dose of morphine (10mg/kg) was injected, followed by the administration of 5% formalin after 30 min. Expression of GAT-1 in the lumbar spinal cord during morphine treatment was analyzed by Western blotting and immunohistochemistry assay. In another set of experiments, a morphine-tolerant group was treated with a GAT-1 inhibitor, ethyl nipecotate (60 mg/kg), 5 min prior to the formalin test. To assess a possible analgesic effect of the GAT-1 inhibitor, a non-tolerant group was injected only with ethyl nipecotate 5 min prior to the formalin test. Our results indicated that a chronic consumption of morphine led to morphine tolerance. Morphine tolerance was also concomitant with GAT-1 up-regulation in the lumbar spinal cord. The GAT-1 inhibitor ethyl nipecotate improved the antinociceptive effect of morphine in the morphine-tolerant group. Ethyl nipecotate also had an antinociceptive effect on the non-tolerant group. Thus, our data suggest that GAT-1 overexpression in the spinal cord plays an important role in morphine tolerance., (Copyright © 2015. Published by Elsevier B.V.)

Microglial activation and inflammation are associated with progressive neuronal apoptosis in neurodegenerative disorders such as Parkinson's disease (PD). γ-Aminobutyric acid (GABA), the major inhibitory neurotransmitter in the central nervous system, has recently been shown to play an inhibitory role in the immune system. Tiagabine, a piperidine derivative, enhances GABAergic transmission by inhibiting GABA transporter 1 (GAT 1). In the present study, we found that tiagabine pretreatment attenuated microglial activation, provided partial protection to the nigrostriatal axis and improved motor deficits in a methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD. The protective function of tiagabine was abolished in GAT 1 knockout mice that were challenged with MPTP. In an alternative PD model, induced by intranigral infusion of lipopolysaccharide (LPS), microglial suppression and subsequent neuroprotective effects of tiagabine were demonstrated. Furthermore, the LPS-induced inflammatory activation of BV-2 microglial cells and the toxicity of conditioned medium toward SH-SY5Y cells were inhibited by pretreatment with GABAergic drugs. The attenuation of the nuclear translocation of nuclear factor κB (NF-κB) and the inhibition of the generation of inflammatory mediators were the underlying mechanisms. Our results suggest that tiagabine acts as a brake for nigrostriatal microglial activation and that it might be a novel therapeutic approach for PD.

Key Points: Throughout life, fear learning is indispensable for survival and neural plasticity in the lateral amygdala underlies this learning and storage of fear memories. During development, properties of fear learning continue to change into adulthood, but currently little is known about changes in amygdala circuits that enable these behavioural transitions. In recordings from neurons in lateral amygdala brain slices from infant up to adult mice, we show that spontaneous and evoked excitatory and inhibitory synaptic transmissions mature into adolescence. At this time, increased inhibitory activity and signalling has the ability to restrict the function of excitation by presynaptic modulation, and may thus enable precise stimulus associations to limit fear generalization from adolescence onward. Our results provide a basis for addressing plasticity mechanisms that underlie altered fear behaviour in young animals., Abstract: Convergent evidence suggests that plasticity in the lateral amygdala (LA) participates in acquisition and storage of fear memory. Sensory inputs from thalamic and cortical areas activate principal neurons and local GABAergic interneurons, which provide feed-forward inhibition that tightly controls LA activity and plasticity via pre- and postsynaptic GABAA and GABAB receptors. GABAergic control is also critical during fear expression, generalization and extinction in adult animals. During rodent development, properties of fear and extinction learning continue to change into early adulthood. Currently, few studies have assessed physiological changes in amygdala circuits that may enable these behavioural transitions. To obtain first insights, we investigated changes in spontaneous and sensory input-evoked inhibition onto LA principal neurons and then focused on GABAB receptor-mediated modulation of excitatory sensory inputs in infant, juvenile, adolescent and young adult mice. We found that spontaneous and sensory-evoked inhibition increased during development. Physiological changes were accompanied by changes in dendritic morphology. While GABAB heteroreceptors were functionally expressed on sensory afferents already early in development, they could only be physiologically recruited by sensory-evoked GABA release to mediate heterosynaptic inhibition from adolescence onward. Furthermore, we found GABAB -mediated tonic inhibition of sensory inputs by ambient GABA that also emerged in adolescence. The observed increase in GABAergic drive may be a substrate for providing modulatory GABA. Our data suggest that GABAB -mediated tonic and evoked presynaptic inhibition can suppress sensory input-driven excitation in the LA to enable precise stimulus associations and limit generalization of conditioned fear from adolescence onward., (© 2015 The Authors. The Journal of Physiology © 2015 The Physiological Society.)

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.

Background: Neuropathic pain (NPP) is likely the result of repetitive high-frequency bursts of peripheral afferent activity leading to long-lasting changes in synaptic plasticity in the spinal dorsal horn. Drugs that promote γ-aminobutyric acid (GABA) activity in the dorsal horn provide partial relief of neuropathic symptoms. The authors examined how in vivo silencing of the GABA receptor type A (GABAA) α2 gene in dorsal root ganglia (DRG) controls NPP., Methods: After crush injury to the right sciatic nerve of female rats, the α2 GABAA antisense and mismatch oligodeoxynucleotides or NO-711 (a GABA uptake inhibitor) were applied to the L5 DRG. In vivo behavioral assessment of nociception was conducted before the injury and ensuing 10 days (n = 4 to 10). In vitro quantification of α2 GABAA protein and electrophysiological studies of GABAA currents were performed on acutely dissociated L5 DRG neurons at relevant time points (n = 6 to 14)., Results: NPP postcrush injury of a sciatic nerve in adult female rats coincides with significant down-regulation of the α2 subunit expression in the ipsilateral DRG (approximately 30%). Selective down-regulation of α2 expression in DRGs significantly worsens mechanical (2.55 ± 0.75 to 5.16 ± 1.16) and thermal (7.97 ± 0.96 to 5.51 ± 0.75) hypersensitivity in crush-injured animals and causes development of significant mechanical (2.33 ± 0.40 to 5.00 ± 0.33) and thermal (10.80 ± 0.29 to 7.34 ± 0.81) hypersensitivity in sham animals (data shown as mean ± SD). Conversely, up-regulation of endogenous GABA via blockade of its uptake in DRG alleviates NPP., Conclusion: The GABAA receptor in the DRG plays an important role in pathophysiology of NPP caused by sciatic nerve injury and represents promising target for novel pain therapies.

γ-Aminobutyric acid (GABA) clearance from the extracellular space after release from neurons involves reuptake into terminals and astrocytes through GABA transporters (GATs). The relative flows through these two pathways for GABA released from neurons remains unclear. This study determines the effect of tiagabine, a selective inhibitor of neuronal GAT-1, on the rates of glutamate (Glu) and GABA metabolism and GABA resynthesis via the GABA-glutamine (Gln) cycle. Halothane-anesthetized rats were administered tiagabine (30 mg/kg, i.p.) and 45 min later received an intravenous infusion of either [1,6-(13)C2]glucose (in vivo) or [2-(13)C]acetate (ex vivo). Nontreated rats served as controls. Metabolites and (13)C enrichments were measured with (1)H-[(13)C]-nuclear magnetic resonance spectroscopy and referenced to their corresponding endpoint values measured in extracts from in situ frozen brain. Metabolic flux estimates of GABAergic and glutamatergic neurons were determined by fitting a metabolic model to the (13)C turnover data measured in vivo during [1,6-(13)C2]glucose infusion. Tiagabine-treated rats were indistinguishable (P > 0.05) from controls in tissue amino acid levels and in (13)C enrichments from [2-(13)C]acetate. Tiagabine reduced average rates of glucose oxidation and neurotransmitter cycling in both glutamatergic neurons (↓18%, CMR(glc(ox)Glu): control, 0.27 ± 0.05 vs. tiagabine, 0.22 ± 0.04 µmol/g/min; ↓11%, V(cyc(Glu-Gln)): control 0.23 ± 0.05 vs. tiagabine 0.21 ± 0.04 µmol/g/min and GABAergic neurons (↓18-25%, CMR(glc(ox)GABA): control 0.09 ± 0.02 vs. tiagabine 0.07 ± 0.03 µmol/g/min; V(cyc(GABA-Gln)): control 0.08 ± 0.02 vs. tiagabine 0.07 ± 0.03 µmol/g/min), but the changes in glutamatergic and GABAergic fluxes were not significant (P > 0.10). The results suggest that any reduction in GABA metabolism by tiagabine might be an indirect response to reduced glutamatergic drive rather than direct compensatory effects., (© 2015 Wiley Periodicals, Inc.)