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1.
Talanta ; 278: 126500, 2024 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-38991407

RESUMEN

Impaired expression of GABA transporters (GATs) is closely related to the pathogenesis of among others Parkinson's disease and epilepsy. As such, lipophilic nipecotic acid analogs have been extensively studied as GAT1-addressing drugs and radioligands but suffer from limited brain uptake due to the zwitterionic properties of the nipecotic acid moiety. Bioisosteric replacement of the carboxylic acid group is a promising strategy to improve the brain uptake, though it requires knowledge on the binding of these isosteres to GAT1. To screen nipecotic acid isosteres for their affinity to GAT1 in a time- and cost-effective manner, this research aims to develop a molecular imprinted polymer (MIP) that mimics the natural binding site of GAT1 and can act as an alternative screening tool to the current radiometric and mass spectrometry cellular-based assays. To this end, a nipecotic acid MIP was created using the electropolymerization of ortho-phenylenediamine (oPD) by cyclic voltammetry (CV). The optimization of the generated receptor layer was achieved by varying the scan rate (50-250 mV/s) and number of CV cycles (5-12), yielding an optimized MIP with an average imprinting factor of 2.6, a linear range of 1-1000 nm, and a theoretical LOD of 0.05 nm, as analyzed by electrical impedance spectroscopy (EIS). Selectivity studies facilitated the investigation of major binding interactions between the MIP and the substrate, building an experimental model that compares characteristics of various analogs. Results from this model indicate that the substrate carboxylic acid group plays a more important role in binding than an amine group, after comparing the binding of cyclohexanecarboxylic acid (average IF of 1.7) and piperidine (average IF of 0.46). The research culminates in a discussion regarding the feasibility of the in vitro model, comparing the synthetic system against the biological performance of GAT1. Thus, evaluating if it is possible to generate a synthetic GAT1 mimic, and if so, provide directions for follow-up research.


Asunto(s)
Polímeros Impresos Molecularmente , Ácidos Nipecóticos , Polímeros Impresos Molecularmente/química , Ácidos Nipecóticos/química , Ácidos Nipecóticos/metabolismo , Humanos , Proteínas Transportadoras de GABA en la Membrana Plasmática/metabolismo , Proteínas Transportadoras de GABA en la Membrana Plasmática/química , Impresión Molecular
2.
Biochem Pharmacol ; 180: 114156, 2020 10.
Artículo en Inglés | MEDLINE | ID: mdl-32682759

RESUMEN

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 ß-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.


Asunto(s)
Complemento C5a/antagonistas & inhibidores , Complemento C5a/metabolismo , Receptor de Anafilatoxina C5a/antagonistas & inhibidores , Receptor de Anafilatoxina C5a/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Compuestos de Anilina/metabolismo , Compuestos de Anilina/farmacología , Animales , Células CHO , Complemento C5a/farmacología , Cricetinae , Cricetulus , Relación Dosis-Respuesta a Droga , Células HEK293 , Humanos , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ácidos Nipecóticos/metabolismo , Ácidos Nipecóticos/farmacología , Péptidos Cíclicos/metabolismo , Péptidos Cíclicos/farmacología , Unión Proteica/efectos de los fármacos , Unión Proteica/fisiología
3.
Glia ; 68(3): 646-655, 2020 03.
Artículo en Inglés | MEDLINE | ID: mdl-31692106

RESUMEN

Microglial cells are the immune cells of the brain that, by sensing the microenvironment, permit a correct brain development and function. They communicate with other glial cells and with neurons, releasing and responding to a number of molecules that exert effects on surrounding cells. Among these, neurotransmitters and, in particular, gamma-aminobutyric acid (GABA) has recently gained interest in this context. We demonstrated the expression of GABA transporter 1 (GAT-1) in microglial cells both in soma and cell processes. We show that microglial cell treatment with 1,2,5,6-tetrahydro-1-[2-[[(diphenylmethylene)amino]oxy]ethyl]-3-pyridinecarboxylic acid hydrochloride (NNC-711), a potent and selective GAT-1 inhibitor, significantly reduced Na+ -dependent GABA uptake. On the other hand, GABA uptake was significantly increased by cell treatment with (S)-1-[2-[tris(4-methoxyphenyl)methoxy]ethyl]-3-piperidinecarboxylic acid (SNAP-5114), a GAT-2/3 inhibitor, and this effect was completely blocked by the botulinum toxin BoNT/C1, that specifically cleaves and inactives syntaxin 1A (STX1A). Overall, these findings show that microglial cells express GAT-1 and indicate that STX1A plays an important role in the regulation of GAT-1-dependent GABA uptake in microglia.


Asunto(s)
Corteza Cerebral/metabolismo , Proteínas Transportadoras de GABA en la Membrana Plasmática/metabolismo , Microglía/metabolismo , Sintaxina 1/metabolismo , Animales , Neuronas/metabolismo , Ácidos Nipecóticos/metabolismo , Transmisión Sináptica/fisiología , Ácido gamma-Aminobutírico/metabolismo
4.
Bioorg Med Chem ; 26(12): 3668-3687, 2018 07 23.
Artículo en Inglés | MEDLINE | ID: mdl-29886082

RESUMEN

In this study, we present the synthesis and structure-activity relationships (SAR) of novel N-substituted nipecotic acid derivatives closely related to (S)-SNAP-5114 (2) in the pursuit of finding new and potent mGAT4 selective inhibitors. By the use of iminium ion chemistry, a series of new N-substituted nipecotic acid derivatives containing a variety of heterocycles, and an alkyne spacer were synthesized. Biological evaluation of the prepared compounds showed, how the inhibitory potency and subtype selectivity for the murine GABA transporters (mGATs) were influenced by the performed modifications.


Asunto(s)
Alquinos/química , Proteínas Transportadoras de GABA en la Membrana Plasmática/química , Proteínas Transportadoras de GABA en la Membrana Plasmática/metabolismo , Inhibidores de Recaptación de GABA/síntesis química , Ácidos Nipecóticos/química , Animales , Inhibidores de Recaptación de GABA/metabolismo , Células HEK293 , Humanos , Ratones , Ácidos Nipecóticos/metabolismo , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/metabolismo , Relación Estructura-Actividad , Ácido gamma-Aminobutírico/metabolismo
5.
Neurochem Res ; 43(2): 306-315, 2018 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-29127598

RESUMEN

Inhibitory signaling in the ventral tegmental area (VTA) is involved in the mechanism of action for many drugs of abuse. Although drugs of abuse have been shown to alter extracellular γ-aminobutyric acid (GABA) concentration in the VTA, knowledge on how uptake mechanisms are regulated in vivo is limited. Quantitative (no-net-flux) microdialysis is commonly used to examine the extracellular concentration and clearance of monoamine neurotransmitters, however it is unclear whether this method is sensitive to changes in clearance for amino acid neurotransmitters such as GABA. The purpose of this study was to determine whether changes in GABA uptake are reflected by in vivo extraction fraction within the VTA. Using quantitative (no-net-flux) microdialysis adapted for transient conditions, we examined the effects of local perfusion with the GABA uptake inhibitor, nipecotic acid, in the VTA of Long Evans rats. Basal extracellular GABA concentration and in vivo extraction fraction were 44.4 ± 1.9 nM (x-intercepts from 4 baseline regressions using a total of 24 rats) and 0.19 ± 0.01 (slopes from 4 baseline regressions using a total of 24 rats), respectively. Nipecotic acid (50 µM) significantly increased extracellular GABA concentration to 170 ± 4 nM and reduced in vivo extraction fraction to 0.112 ± 0.003. Extraction fraction returned to baseline following removal of nipecotic acid from the perfusate. Conventional microdialysis substantially underestimated the increase of extracellular GABA concentration due to nipecotic acid perfusion compared with that obtained from the quantitative analysis. Together, these results show that inhibiting GABA uptake mechanisms within the VTA alters in vivo extraction fraction measured using microdialysis and that in vivo extraction fraction may be an indirect measure of GABA clearance.


Asunto(s)
Microdiálisis , Neuronas/metabolismo , Área Tegmental Ventral/metabolismo , Ácido gamma-Aminobutírico/metabolismo , Aminoácidos/metabolismo , Animales , Dopamina/metabolismo , Espacio Extracelular/metabolismo , Inhibidores de Recaptación de GABA/metabolismo , Microdiálisis/métodos , Ácidos Nipecóticos/metabolismo , Núcleo Accumbens/efectos de los fármacos , Ratas Long-Evans
6.
ChemMedChem ; 12(5): 362-371, 2017 03 07.
Artículo en Inglés | MEDLINE | ID: mdl-28125164

RESUMEN

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.


Asunto(s)
Proteínas Transportadoras de GABA en la Membrana Plasmática/química , Inhibidores de Recaptación de GABA/química , Ácidos Nipecóticos/química , Sitios de Unión , Proteínas Transportadoras de GABA en la Membrana Plasmática/metabolismo , Inhibidores de Recaptación de GABA/síntesis química , Inhibidores de Recaptación de GABA/metabolismo , Células HEK293 , Humanos , Concentración 50 Inhibidora , Simulación del Acoplamiento Molecular , Ácidos Nipecóticos/síntesis química , Ácidos Nipecóticos/metabolismo , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/metabolismo , Estructura Terciaria de Proteína , Relación Estructura-Actividad
7.
Eur J Pharm Sci ; 96: 362-372, 2017 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-27721044

RESUMEN

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.


Asunto(s)
Anticonvulsivantes/metabolismo , Anticonvulsivantes/farmacología , Proteínas Transportadoras de GABA en la Membrana Plasmática/metabolismo , Simulación del Acoplamiento Molecular/métodos , Neuralgia/metabolismo , Dimensión del Dolor/efectos de los fármacos , Animales , Anticonvulsivantes/uso terapéutico , Relación Dosis-Respuesta a Droga , Proteínas Transportadoras de GABA en la Membrana Plasmática/química , Humanos , Masculino , Ratones , Neuralgia/tratamiento farmacológico , Ácidos Nipecóticos/metabolismo , Ácidos Nipecóticos/farmacología , Ácidos Nipecóticos/uso terapéutico , Dimensión del Dolor/métodos , Unión Proteica/fisiología , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Estructura Secundaria de Proteína , Tiagabina
8.
ChemMedChem ; 11(5): 509-18, 2016 Mar 04.
Artículo en Inglés | MEDLINE | ID: mdl-26804464

RESUMEN

Well-known inhibitors of the γ-aminobutyric acid (GABA) transporter GAT1 share a common scaffold of a small cyclic amino acid linked by an alkyl chain to a moiety with two aromatic rings. Tiagabine, the only FDA-approved GAT1 inhibitor, is a typical example. Some small amino acids such as (R)-nipecotic acid are medium-to-strong binders of GAT1, but similar compounds, such as proline, are very weak binders. When substituted with 4,4-diphenylbut-3-en-1-yl (DPB) or 4,4-bis(3-methylthiophen-2-yl)but-3-en-1-yl (BTB) groups, the resulting compounds have similar pKi and pIC50 values, even though the pure amino acids have very different values. To investigate if small amino acids and their substituted counterparts share a similar binding mode, we synthesized butyl-, DPB-, and BTB-substituted derivatives of small amino acids. Supported by the results of docking studies, we propose different binding modes not only for unsubstituted und substituted, but also for strong- and weak-binding amino acids. These data lead to the conclusion that following a fragment-based approach, not pure but N-butyl-substituted amino acids should be used as starting points, giving a better estimate of the activity when a BTB or DPB substituent is added.


Asunto(s)
Proteínas Transportadoras de GABA en la Membrana Plasmática/metabolismo , Simulación del Acoplamiento Molecular , Ácidos Nipecóticos/metabolismo , Unión Proteica
9.
J Med Chem ; 58(5): 2149-58, 2015 Mar 12.
Artículo en Inglés | MEDLINE | ID: mdl-25679268

RESUMEN

Elevating GABA levels in the synaptic cleft by inhibiting its reuptake carrier GAT1 is an established approach for the treatment of CNS disorders like epilepsy. With the increasing availability of crystal structures of transmembrane transporters, structure-based approaches to elucidate the molecular basis of ligand-transporter interaction also become feasible. Experimental data guided docking of derivatives of the GAT1 inhibitor tiagabine into a protein homology model of GAT1 allowed derivation of a common binding mode for this class of inhibitors that is able to account for the distinct structure-activity relationship pattern of the data set. Translating essential binding features into a pharmacophore model followed by in silico screening of the DrugBank identified liothyronine as a drug potentially exerting a similar effect on GAT1. Experimental testing further confirmed the GAT1 inhibiting properties of this thyroid hormone.


Asunto(s)
Agonistas del GABA/metabolismo , Proteínas Transportadoras de GABA en la Membrana Plasmática/química , Proteínas Transportadoras de GABA en la Membrana Plasmática/metabolismo , Simulación del Acoplamiento Molecular , Ácidos Nipecóticos/metabolismo , Triyodotironina/farmacología , Ácido gamma-Aminobutírico/metabolismo , Simulación por Computador , Agonistas del GABA/química , Células HEK293 , Ensayos Analíticos de Alto Rendimiento , Humanos , Modelos Moleculares , Estructura Molecular , Ácidos Nipecóticos/química , Relación Estructura-Actividad , Tiagabina , Triyodotironina/química
10.
Chem Biol ; 21(12): 1648-59, 2014 Dec 18.
Artículo en Inglés | MEDLINE | ID: mdl-25500222

RESUMEN

Inducible Hsp70 (Hsp70i) is overexpressed in a wide spectrum of human tumors, and its expression correlates with metastasis, poor outcomes, and resistance to chemotherapy in patients. Identification of small-molecule inhibitors selective for Hsp70i could provide new therapeutic tools for cancer treatment. In this work, we used fluorescence-linked enzyme chemoproteomic strategy (FLECS) to identify HS-72, an allosteric inhibitor selective for Hsp70i. HS-72 displays the hallmarks of Hsp70 inhibition in cells, promoting substrate protein degradation and growth inhibition. Importantly, HS-72 is selective for Hsp70i over the closely related constitutively active Hsc70. Studies with purified protein show HS-72 acts as an allosteric inhibitor, reducing ATP affinity. In vivo HS-72 is well-tolerated, showing bioavailability and efficacy, inhibiting tumor growth and promoting survival in a HER2+ model of breast cancer. The HS-72 scaffold is amenable to resynthesis and iteration, suggesting an ideal starting point for a new generation of anticancer therapeutics targeting Hsp70i.


Asunto(s)
Bencimidazoles/química , Bencimidazoles/farmacología , Proteínas HSP70 de Choque Térmico/antagonistas & inhibidores , Proteínas HSP70 de Choque Térmico/metabolismo , Ácidos Nipecóticos/química , Ácidos Nipecóticos/farmacología , Piperidinas/química , Piperidinas/farmacología , Regulación Alostérica/efectos de los fármacos , Animales , Bencimidazoles/metabolismo , Bencimidazoles/farmacocinética , Disponibilidad Biológica , Caspasas/metabolismo , Proliferación Celular/efectos de los fármacos , Evaluación Preclínica de Medicamentos , Activación Enzimática/efectos de los fármacos , Células HEK293 , Proteínas HSP70 de Choque Térmico/química , Humanos , Ratones , Modelos Moleculares , Ácidos Nipecóticos/metabolismo , Ácidos Nipecóticos/farmacocinética , Permeabilidad , Piperidinas/metabolismo , Piperidinas/farmacocinética , Agregado de Proteínas/efectos de los fármacos , Estructura Terciaria de Proteína , Ensayos Antitumor por Modelo de Xenoinjerto
11.
Biol Pharm Bull ; 37(5): 817-25, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-24790004

RESUMEN

Taurine transporter (TauT/SLC6A6) is an "honorary" γ-aminobutyric acid (GABA) transporter because of its low affinity for GABA. The sequence analysis of TauT implied the role of Gly57, Phe58, Leu306 and Glu406 in the substrate recognition of TauT, and amino acid-substitutions were performed. Immunocytochemistry supported no marked effect of mutations on the expression of TauT. TauT-expressing oocytes showed a reduction in [(3)H]taurine uptake by G57E, F58I, L306Q and E406C, and change in [(3)H]GABA uptake by G57E and E406C, suggesting their significant roles in the function of TauT. G57E lost the activity of [(3)H]taurine and [(3)H]GABA uptake, suggesting that Gly57 is involved in the determination of substrate pocket volume and in the interaction with substrates. E406C exhibited a decrease and an increase in the affinity for taurine and GABA, respectively, suggesting the involvement of Glu406 in the substrate specificity of TauT. The inhibition study supported the role of Glu406 in the substrate specificity since [(3)H]taurine and [(3)H]GABA uptake by E406C was less sensitive to taurine and ß-alanine, and more sensitive to GABA and nipecotic acid than was the case with wild type of TauT. F58I had an increased affinity for GABA, suggesting the involvement of Phe58 in the substrate accessibility. The kinetic parameters showed the decreased and increased affinities of L306Q for taurine and GABA, respectively, supporting that substrate recognition of TauT is conformationally regulated by the branched-side chain of Leu306. In conclusion, the present results suggest that these residues play important roles in the transport function and substrate specificity of TauT.


Asunto(s)
Aminoácidos/metabolismo , Transporte Biológico/genética , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Proteínas de Transporte de Membrana/química , Proteínas de Transporte de Membrana/metabolismo , Taurina/metabolismo , Ácido gamma-Aminobutírico/metabolismo , Animales , Glicoproteínas de Membrana/genética , Proteínas de Transporte de Membrana/genética , Mutagénesis Sitio-Dirigida , Ácidos Nipecóticos/metabolismo , Oocitos/metabolismo , Especificidad por Sustrato/genética , Tritio , Xenopus laevis , beta-Alanina/metabolismo
12.
Biomed Chromatogr ; 27(5): 641-54, 2013 May.
Artículo en Inglés | MEDLINE | ID: mdl-23225341

RESUMEN

Binding assays for the γ-aminobutyric acid (GABA) transporter GAT3 can be assumed to significantly facilitate screening for respective inhibitors. As appropriate labeled ligands for this promising drug target are not available so far, we started efforts to set up mass spectrometry-based binding assays (MS binding assays), for which labeled markers are not required. Therefore, we developed a sensitive and rapid LC-ESI-MS/MS quantification method for DDPM-1007 {(RS)-1-[4,4,4-Tris(4-methoxyphenyl)but-2-en-1-yl]piperidine-3-carboxylic acid}, one of the most potent GAT3 inhibitors yet known, as a potential GAT3 marker. Using a 50 × 2 mm C(8) column in combination with a mobile phase composed of 10 mM ammonium bicarbonate buffer pH 8.0 and acetonitrile (60:40, v/v) at a flow rate of 450 µL/min DDPM-1007 could be analyzed in the positive multiple reaction monitoring mode [(m/z) 502.5 → 265.4] within a chromatographic cycle time of 3 min. Deuterated DDPM-1007 [((2)H(9))DDPM-1007] was synthesized and employed as internal standard. This way DDPM-1007 could be quantified in a range from 100 pM to 10 nM in the matrix resulting from respective binding experiments without any sample preparation. The established quantification method met the requirements of the FDA guidance for bioanalytical method validation concerning linearity and intra- and inter-batch accuracy. Based on this LC-ESI-MS/MS quantification preliminary MS binding assays employing membrane preparations obtained from a stably GAT3 expressing HEK293 cell line and DDPM-1007 as nonlabeled GAT3 marker could be performed. In these experiments specific binding of DDPM-1007 at GAT3 could be unambiguously detected. Additionally, the established LC-MS method provides a suitable analytical tool for further pharmacokinetic characterization of DDPM-1007, as exemplified for its logD determination.


Asunto(s)
Biomarcadores/análisis , Cromatografía Líquida de Alta Presión/métodos , Proteínas Transportadoras de GABA en la Membrana Plasmática/análisis , Espectrometría de Masa por Ionización de Electrospray/métodos , Biomarcadores/metabolismo , Proteínas Transportadoras de GABA en la Membrana Plasmática/metabolismo , Células HEK293 , Humanos , Modelos Lineales , Ácidos Nipecóticos/análisis , Ácidos Nipecóticos/química , Ácidos Nipecóticos/metabolismo , Unión Proteica , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Espectrometría de Masa por Ionización de Electrospray/normas , Simportadores/antagonistas & inhibidores , Espectrometría de Masas en Tándem/métodos , Espectrometría de Masas en Tándem/normas
13.
Future Med Chem ; 3(2): 163-75, 2011 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-21428811

RESUMEN

In 1950, γ-aminobutyric acid (GABA) was discovered in the brain and in 1967 it was recognized as an inhibitory neurotransmitter. The discovery of the benzodiazepines Librium® (launched in 1960) and Valium® by Sternbach initiated huge research activities resulting in 50 marketed drugs. In 1975, Haefely found that GABA is involved in the actions of benzodiazepines. The baclofen-sensitive, bicuculline-insensitive GABA(B) receptor was discovered by Bowery in 1980, and the baclofen-insensitive, bicuculline-insensitive GABA(C) receptor by Johnston in 1984. Barnard & Seeburg reported the cloning of the GABA(A) receptor in 1987, Cutting the GABA(C) receptor in 1991 and Bettler the GABA(B1a) and GABA(B1b) receptors in 1997. Six groups cloned the GABA(B2) receptor in 1998/1999 showing that the GABA(B) receptor functions as a heterodimer with GABA(B1b)/GABA(B2) mediating postsynaptic inhibition and GABA(B1a)/GABA(B2) mediating presynaptic inhibition. Möhler and McKernan dissected the pharmacology of the benzodiazepine-receptor subtypes. Antagonists and positive allosteric modulators of GABA(B) receptors were discovered in 1987 and 2001, respectively. GABA transporter inhibitor, tiagabine, was launched in 1996, a GABA aminotransferase inhibitor, vigabatrin, in 1998 and a glutamic acid decarboxylase activator, pregabalin, in 2004. Most recently, brain-penetrating GABA(C)-receptor antagonists were reported in 2009.


Asunto(s)
Ácido gamma-Aminobutírico/metabolismo , 4-Aminobutirato Transaminasa/metabolismo , Animales , Benzodiazepinas/química , Benzodiazepinas/metabolismo , Bestrofinas , Canales de Cloruro/metabolismo , Proteínas del Ojo/metabolismo , Agonistas del GABA/química , Agonistas del GABA/metabolismo , Antagonistas del GABA/química , Antagonistas del GABA/metabolismo , Proteínas Transportadoras de GABA en la Membrana Plasmática/química , Proteínas Transportadoras de GABA en la Membrana Plasmática/metabolismo , Glutamato Descarboxilasa/metabolismo , Humanos , Estructura Molecular , Ácidos Nipecóticos/química , Ácidos Nipecóticos/metabolismo , Receptores de GABA-A/metabolismo , Receptores de GABA-B/metabolismo , Sinapsis/metabolismo , Sinapsis/ultraestructura , Tiagabina , Ácido gamma-Aminobutírico/química
14.
J Med Chem ; 53(6): 2354-63, 2010 Mar 25.
Artículo en Inglés | MEDLINE | ID: mdl-20170117

RESUMEN

Here we studied the capacity of N-MePhe-(N-MePhe)(3)-CONH(2), Cha-(N-MePhe)(3)-CONH(2), and 2Nal-(N-MePhe)(3)-CONH(2) to carry various drugs (cargos) in in vitro blood-brain barrier (BBB) models in order to determine the versatility of these peptides as BBB-shuttles for drug delivery to the brain. Using SPPS, the peptides were coupled to GABA, Nip, and ALA to examine their passive BBB permeation by means of PAMPA and their lipophilicity by IAMC. Unaided, these nonpermeating drugs alone did not cross the PAMPA barrier and the BBB passively; however, the peptides tested as potential BBB shuttles transferred them by passive transfer through the PAMPA phospholipid. The permeability of peptides that showed the highest permeability in PAMPA, and Ac-N-MePhe-(N-MePhe)(3)-CONH(2) as the parent peptide was also examined in bovine brain microvessel endothelial cells (BBMECs). These peptide-based BBB shuttles open up the possibility to overcome the formidable obstacle of the BBB, thereby achieving drug delivery to the brain.


Asunto(s)
Barrera Hematoencefálica/metabolismo , Portadores de Fármacos/metabolismo , Oligopéptidos/metabolismo , Fenilalanina/análogos & derivados , Ácido Aminolevulínico/química , Ácido Aminolevulínico/metabolismo , Animales , Animales Recién Nacidos , Astrocitos/citología , Astrocitos/metabolismo , Transporte Biológico , Vasos Sanguíneos/citología , Encéfalo/irrigación sanguínea , Bovinos , Permeabilidad de la Membrana Celular , Células Cultivadas , Cromatografía Líquida de Alta Presión , Técnicas de Cocultivo , Portadores de Fármacos/química , Portadores de Fármacos/farmacocinética , Células Endoteliales/citología , Células Endoteliales/metabolismo , Espectrometría de Masas , Estructura Molecular , Ácidos Nipecóticos/química , Ácidos Nipecóticos/metabolismo , Oligopéptidos/química , Oligopéptidos/farmacocinética , Fenilalanina/química , Ratas , Ácido gamma-Aminobutírico/química , Ácido gamma-Aminobutírico/metabolismo
15.
J Chromatogr B Analyt Technol Biomed Life Sci ; 878(17-18): 1356-64, 2010 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-20036623

RESUMEN

The present study describes the use of short columns to speed up LC-MS quantification in MS binding assays. The concept of MS binding assays follows closely the principle of traditional radioligand binding but uses MS for the quantification of bound marker thus eliminating the need for a radiolabelled ligand. The general strategy of increasing the throughput of this type of binding assay by the use of short columns is exemplified for NO 711 binding addressing GAT1, the most prevalent GABA transporter in the CNS. Employing short RP-18 columns with the dimension of 20 mm x 2 mm and 10 mm x 2 mm at flow rates up to 1000 microL/min in an isocratic mode retention times of 8-9s and chromatographic cycle times of 18s could be achieved. Based on the internal standard [(2)H(10)]NO 711 fast chromatography methods were developed for four different columns that enabled quantification of NO 711 in a range from 50 pM up to 5 nM directly out of reconstituted matrix samples without further sample preparation. A validation of the established methods with respect to linearity, intra- and inter-batch accuracy and precision showed that the requirements according to the FDA guideline for bioanalytical methods are met. Furthermore the established short column methods were applied to the quantification of NO 711 in saturation experiments. The results obtained (i.e., K(d)- and B(max)-values) were almost identical as compared to those determined employing standard column dimension (55 mm x 2 mm).


Asunto(s)
Cromatografía Liquida/métodos , Proteínas Transportadoras de GABA en la Membrana Plasmática/metabolismo , Ensayo de Unión Radioligante/métodos , Espectrometría de Masas en Tándem/métodos , Animales , Cromatografía Liquida/instrumentación , Deuterio/química , Ratones , Ácidos Nipecóticos/química , Ácidos Nipecóticos/metabolismo , Oximas/química , Oximas/metabolismo , Reproducibilidad de los Resultados , Sensibilidad y Especificidad
16.
Neurochem Res ; 33(8): 1610-7, 2008 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-18437566

RESUMEN

Mouse cerebral cortical mini-slices were used in a superfusion system to monitor depolarization-induced (55 mM K(+)) release of preloaded [2,3-(3)H]GABA and to investigate the biosynthesis of glutamate, GABA and aspartate during physiological and depolarizing (55 mM K(+)) conditions from either [1,6-(13)C]glucose or [U-(13)C]glutamine. Depolarization-induced GABA release could be reduced (50%) by the GABA transport inhibitor tiagabine (25 microM) or by replacing Ca(2+) with Co(2+). In the presence of both tiagabine and Co(2+) (1 mM), release was abolished completely. The release observed in the presence of 25 microM tiagabine thus represents vesicular release. Superfusion in the presence of [1,6-(13)C]glucose led to considerable labeling in the three amino acids, the labeling in glutamate and aspartate being increased after depolarization. This condition had no effect on GABA labeling. For all three amino acids, the distribution of label in the different carbon atoms revealed on increased tricarboxylic acid (TCA) activity during depolarization. When [U-(13)C]glutamine was used as substrate, labeling in glutamate was higher than that in GABA and aspartate and the fraction of glutamate and aspartate being synthesized by participation of the TCA cycle was increased by depolarization, an effect not seen for GABA. However, GABA synthesis reflected TCA cycle involvement to a much higher extent than for glutamate and aspartate. The results show that this preparation of brain tissue with intact cellular networks is well suited to study metabolism and release of neurotransmitter amino acids under conditions mimicking neural activity.


Asunto(s)
Isótopos de Carbono/química , Corteza Cerebral/metabolismo , Glucosa , Glutamina , Ácido gamma-Aminobutírico/metabolismo , Aminoácidos/metabolismo , Animales , Cobalto/metabolismo , Agonistas del GABA/metabolismo , Glucosa/análogos & derivados , Glucosa/metabolismo , Glutamina/química , Glutamina/metabolismo , Homeostasis , Ratones , Ácidos Nipecóticos/metabolismo , Tiagabina
17.
Anal Bioanal Chem ; 391(1): 309-16, 2008 May.
Artículo en Inglés | MEDLINE | ID: mdl-18345533

RESUMEN

Following a recently developed concept of MS binding assays based on the quantification of a native marker by LC-MS a procedure to study binding of a low-affinity marker in kinetic, saturation, and competition experiments was established. Separation of bound and unbound marker-the most crucial step of the assay-could be effectively achieved by filtration in a 96-well-format. MS binding assays according to this procedure allowed the reliable characterization of NO 711 binding to mGAT1 in presence of physiological NaCl concentrations. Comparing the results obtained in the present study with those from experiments using 1 mol L(-1) NaCl in the incubation milieu reveals remarkable differences with respect to the marker's affinity and kinetics and to the investigated test compound's potency. [figure: see text]


Asunto(s)
Aciltransferasas/metabolismo , Biomarcadores/análisis , Cromatografía Liquida , Proteínas Transportadoras de GABA en la Membrana Plasmática/análisis , Espectrometría de Masas , Ácidos Nipecóticos/metabolismo , Oximas/metabolismo , Aciltransferasas/química , Unión Competitiva , Bioensayo , Biomarcadores/química , Línea Celular , Cromatografía Liquida/métodos , Antagonistas del GABA/química , Antagonistas del GABA/metabolismo , Proteínas Transportadoras de GABA en la Membrana Plasmática/química , Humanos , Cinética , Ligandos , Hígado/citología , Espectrometría de Masas/métodos , N-Acetilglucosaminiltransferasas , Ácidos Nipecóticos/química , Oximas/química
18.
Aging Clin Exp Res ; 18(3): 257-60, 2006 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-16804373

RESUMEN

BACKGROUND AND AIMS: In spite of the fact that GABA is a significant transmitter, little is known about the GABA system in aging, compared with other transmitter systems. [(3)H]tiagabine is a ligand for GABAergic neurons, which binds with 10-fold higher affinity to the GABA uptake site than [(3)H]nipecotic acid. The aim of this study was to study the binding of [(3)H]tiagabine to the GABA transporter 1, GAT-1, in human frontal cortex and cingulate cortex from individuals of varying ages. METHODS: [(3)H]tiagabine binding experiments were conducted on post-mortem brain tissue from 19 individuals (age range 17-78 years) without known neurological or psychiatric disorders. Binding data vs age and postmortem interval was analysed by Pearson correlation. RESULTS: The density of [(3)H]tiagabine binding to GAT- 1 decreased significantly with increasing age in the frontal cortex, whereas binding affinity was unchanged. No significant alterations in binding parameters were observed in the cingulate cortex. No correlation was found between post-mortem delay and the number of [(3)H]tiagabine binding sites. CONCLUSIONS: According to the present study, presynaptical alterations in the GABA system are correlated with aging in the frontal cortex of the human brain. Further studies involving a broader range of brain regions seem warranted, to confirm the present findings and to enlarge knowledge about the GABA system in aging.


Asunto(s)
Envejecimiento/metabolismo , Lóbulo Frontal/metabolismo , Proteínas Transportadoras de GABA en la Membrana Plasmática/metabolismo , Ácidos Nipecóticos/metabolismo , Terminales Presinápticos/metabolismo , Adulto , Anciano , Sitios de Unión , Femenino , Giro del Cíngulo/metabolismo , Humanos , Técnicas In Vitro , Masculino , Persona de Mediana Edad , Tiagabina , Tritio
19.
Appl Environ Microbiol ; 71(4): 1971-6, 2005 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-15812028

RESUMEN

The natural product rapamycin, produced during fermentation by Streptomyces hygroscopicus, is known for its potent antifungal, immunosuppressive, and anticancer activities. During rapamycin biosynthesis, the amino acid l-pipecolate is incorporated into the rapamycin molecule. We investigated the use of precursor-directed biosynthesis to create new rapamycin analogs by substitution of unusual l-pipecolate analogs in place of the normal amino acid. Our results suggest that the l-pipecolate analog (+/-)-nipecotic acid inhibits the biosynthesis of l-pipecolate, thereby limiting the availability of this molecule for rapamycin biosynthesis. We used (+/-)-nipecotic acid in our precursor-directed biosynthesis studies to reduce l-pipecolate availability and thereby enhance the incorporation of other pipecolate analogs into the rapamycin molecule. We describe here the use of this method for production of two new sulfur-containing rapamycin analogs, 20-thiarapamycin and 15-deoxo-19-sulfoxylrapamycin, and report measurement of their binding to FKBP12.


Asunto(s)
Regulación Bacteriana de la Expresión Génica , Precursores de Proteínas/metabolismo , Sirolimus/análogos & derivados , Sirolimus/metabolismo , Streptomyces/metabolismo , Biotecnología/métodos , Ácidos Nipecóticos/metabolismo , Ácidos Pipecólicos/metabolismo , Streptomyces/genética , Streptomyces/crecimiento & desarrollo , Proteína 1A de Unión a Tacrolimus/metabolismo
20.
BMC Biochem ; 5: 16, 2004 Nov 17.
Artículo en Inglés | MEDLINE | ID: mdl-15548327

RESUMEN

BACKGROUND: In establishing structure-function relationships for membrane transport proteins, the interpretation of phenotypic changes can be problematic, owing to uncertainties in protein expression levels, sub-cellular localization, and protein-folding fidelity. A dual-label competitive transport assay called "Transport Specificity Ratio" (TSR) analysis has been developed that is simple to perform, and circumvents the "expression problem," providing a reliable TSR phenotype (a constant) for comparison to other transporters. RESULTS: Using the Escherichia coli GABA (4-aminobutyrate) permease (GabP) as a model carrier, it is demonstrated that the TSR phenotype is largely independent of assay conditions, exhibiting: (i) indifference to the particular substrate concentrations used, (ii) indifference to extreme changes (40-fold) in transporter expression level, and within broad limits (iii) indifference to assay duration. The theoretical underpinnings of TSR analysis predict all of the above observations, supporting that TSR has (i) applicability in the analysis of membrane transport, and (ii) particular utility in the face of incomplete information on protein expression levels and initial reaction rate intervals (e.g., in high-throughput screening situations). The TSR was used to identify gab permease (GabP) variants that exhibit relative changes in catalytic specificity (kcat/Km) for [14C]GABA (4-aminobutyrate) versus [3H]NA (nipecotic acid). CONCLUSIONS: The TSR phenotype is an easily measured constant that reflects innate molecular properties of the transition state, and provides a reliable index of the difference in catalytic specificity that a carrier exhibits toward a particular pair of substrates. A change in the TSR phenotype, called a Delta(TSR), represents a specificity shift attributable to underlying changes in the intrinsic substrate binding energy (DeltaGb) that translocation catalysts rely upon to decrease activation energy (Delta G(T)(++). TSR analysis is therefore a structure-function tool that enables parsimonious scanning for positions in the protein fold that couple to the transition state, creating stability and thereby serving as functional determinants of catalytic power (efficiency, or specificity).


Asunto(s)
Modelos Moleculares , Transportadores de Anión Orgánico/química , Transportadores de Anión Orgánico/fisiología , Pliegue de Proteína , Relación Estructura-Actividad Cuantitativa , Unión Competitiva/fisiología , Catálisis , Escherichia coli/enzimología , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/fisiología , Proteínas Transportadoras de GABA en la Membrana Plasmática , Regulación Bacteriana de la Expresión Génica/fisiología , Regulación Enzimológica de la Expresión Génica/fisiología , Variación Genética/fisiología , Ácidos Nipecóticos/metabolismo , Transportadores de Anión Orgánico/genética , Transportadores de Anión Orgánico/metabolismo , Fenotipo , Especificidad por Sustrato/fisiología , Ácido gamma-Aminobutírico/metabolismo
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