RESUMEN

22q11.2, 15q13.3, and 1q21.1 microdeletions attract considerable interest by conferring high risk for a range of neuropsychiatric disorders, including schizophrenia and autism. A fundamental open question is whether divergent or convergent neural mechanisms mediate this genetic pleiotropic association with the same behavioral phenotypes. We use a combination of rodent microdeletion models with high-field neuroimaging to perform a comparative whole-brain characterization of functional and structural mechanisms linked to high-risk states. Resting-state functional and structural magnetic resonance imaging data were acquired on mice carrying heterozygous microdeletions in 22q11.2 (N = 12), 15q13.3 (N = 11), and 1q21.1 (N = 11) loci. We performed network-based statistic, graph, and morphometric analyses. The three microdeletions did not share significant systems-level features. Instead, morphometric analyses revealed microcephaly in 1q21.1 and macrocephaly in 15q13.3 deletions, whereas cerebellar volume was specifically reduced in 22q11.2 deletion. In function, 22q11.2 deletion mice showed widespread cortical hypoconnectivity, accompanied by opposing hyperconnectivity in dopaminergic pathways, which was confirmed by graph analysis. 1q21.1 exhibited distinct changes in posterior midbrain morphology and function, especially in periaqueductal gray, whereas 15q13.3 demonstrated alterations in auditory/striatal system. The combination of cortical hypoconnectivity and dopaminergic hyperconnectivity and reduced cerebellum in 22q11.2 deletion mirrors key neurodevelopmental features of schizophrenia, whereas changes in midbrain and auditory/striatal morphology and topology in 1q21.1 and 15q13.3 rather indicate focal processes possibly linked to the emergence of abnormal salience perception and hallucinations. In addition to insights into pathophysiological processes in these microdeletions, our results establish the general point that microdeletions might increase risk for overlapping neuropsychiatric phenotypes through separable neural mechanisms.

Asunto(s)

Variaciones en el Número de Copia de ADN , Esquizofrenia , Animales , Encéfalo/diagnóstico por imagen , Deleción Cromosómica , Ratones , Fenotipo , Esquizofrenia/diagnóstico por imagen , Esquizofrenia/genética

RESUMEN

The multimodal antidepressant vortioxetine is thought to mediate its pharmacological effects via 5-HT1A receptor agonism, 5-HT1B receptor partial agonism, 5-HT1D, 5-HT3, 5-HT7 receptor antagonism and 5-HT transporter inhibition. Here we studied vortioxetine's functional effects across species (canine, mouse, rat, guinea pig and human) in cellular assays with heterologous expression of 5-HT3A receptors (in Xenopus oocytes and HEK-293 cells) and in mouse neuroblastoma N1E-115 cells with endogenous expression of 5-HT3A receptors. Furthermore, we studied the effects of vortioxetine on activity of CA1 Stratum Radiatum interneurons in rat hippocampus slices using current- and voltage-clamping methods. The patched neurons were subsequently filled with biocytin for confirmation of 5-HT3 receptor mRNA expression by in situ hybridization. Whereas, both vortioxetine and the 5-HT3 receptor antagonist ondansetron potently antagonized 5-HT-induced currents in the cellular assays, vortioxetine had a slower off-rate than ondansetron in oocytes expressing 5-HT3A receptors. Furthermore, vortioxetine's but not ondansetron's 5-HT3 receptor antagonistic potency varied considerably across species. Vortioxetine had the highest potency at rat and the lowest potency at guinea pig 5-HT3A receptors. Finally, in 5-HT3 receptor-expressing GABAergic interneurons from the CA1 stratum radiatum, vortioxetine and ondansetron blocked depolarizations induced by superfusion of either 5-HT or the 5-HT3 receptor agonist mCPBG. Taken together, these data add to a growing literature supporting the idea that vortioxetine may inhibit GABAergic neurotransmission in some brain regions via a 5-HT3 receptor antagonism-dependent mechanism and thereby disinhibit pyramidal neurons and enhance glutamatergic signaling.

Asunto(s)

Potenciales de Acción/efectos de los fármacos , Antidepresivos/farmacología , Interneuronas/efectos de los fármacos , Células Piramidales/efectos de los fármacos , Antagonistas del Receptor de Serotonina 5-HT3/farmacología , Vortioxetina/farmacología , Potenciales de Acción/fisiología , Animales , Región CA1 Hipocampal/efectos de los fármacos , Región CA1 Hipocampal/metabolismo , Perros , Ácido Glutámico/metabolismo , Cobayas , Células HEK293 , Humanos , Interneuronas/metabolismo , Ratones , Ondansetrón/farmacología , Oocitos , Células Piramidales/metabolismo , Ratas , Receptores de Serotonina 5-HT3/metabolismo , Serotonina/metabolismo , Técnicas de Cultivo de Tejidos , Xenopus laevis , Ácido gamma-Aminobutírico/metabolismo

RESUMEN

BACKGROUND: The hemizygous 22q11.2 microdeletion is a common copy number variant in humans. The deletion confers high risk for neurodevelopmental disorders, including autism and schizophrenia. Up to 41% of deletion carriers experience psychotic symptoms. METHODS: We present a new mouse model (Df(h22q11)/+) of the deletion syndrome (22q11.2DS) and report on, to our knowledge, the most comprehensive study undertaken to date in 22q11.2DS models. The study was conducted in male mice. RESULTS: We found elevated postpubertal N-methyl-D-aspartate (NMDA) receptor antagonist-induced hyperlocomotion, age-independent prepulse inhibition (PPI) deficits and increased acoustic startle response (ASR). The PPI deficit and increased ASR were resistant to antipsychotic treatment. The PPI deficit was not a consequence of impaired hearing measured by auditory brain stem responses. The Df(h22q11)/+ mice also displayed increased amplitude of loudness-dependent auditory evoked potentials. Prefrontal cortex and dorsal striatal elevations of the dopamine metabolite DOPAC and increased dorsal striatal expression of the AMPA receptor subunit GluR1 was found. The Df(h22q11)/+ mice did not deviate from wild-type mice in a wide range of other behavioural and biochemical assays. LIMITATIONS: The 22q11.2 microdeletion has incomplete penetrance in humans, and the severity of disease depends on the complete genetic makeup in concert with environmental factors. In order to obtain more marked phenotypes reflecting the severe conditions related to 22q11.2DS it is suggested to expose the Df(h22q11)/+ mice to environmental stressors that may unmask latent psychopathology. CONCLUSION: The Df(h22q11)/+ model will be a valuable tool for increasing our understanding of the etiology of schizophrenia and other psychiatric disorders associated with the 22q11DS.

Asunto(s)

Envejecimiento/fisiología , Síndrome de DiGeorge/fisiopatología , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Filtrado Sensorial/fisiología , Ácido 3,4-Dihidroxifenilacético/metabolismo , Envejecimiento/efectos de los fármacos , Animales , Percepción Auditiva/fisiología , Cuerpo Estriado/metabolismo , Modelos Animales de Enfermedad , Potenciales Evocados Auditivos del Tronco Encefálico , Antagonistas de Aminoácidos Excitadores/farmacología , Masculino , Ratones Endogámicos C57BL , Actividad Motora/efectos de los fármacos , Corteza Prefrontal/metabolismo , Receptores AMPA/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Reflejo de Sobresalto/fisiología

RESUMEN

A chromosomal microdeletion at the 22q11.2 locus is associated with extensive cognitive impairments, schizophrenia and other psychopathology in humans. Previous reports indicate that mouse models of the 22q11.2 microdeletion syndrome (22q11.2DS) may model the genetic basis of cognitive deficits relevant for neuropsychiatric disorders such as schizophrenia. To assess the models usefulness for drug discovery, a novel mouse (Df(h22q11)/+) was assessed in an extensive battery of cognitive assays by partners within the NEWMEDS collaboration (Innovative Medicines Initiative Grant Agreement No. 115008). This battery included classic and touchscreen-based paradigms with recognized sensitivity and multiple attempts at reproducing previously published findings in 22q11.2DS mouse models. This work represents one of the most comprehensive reports of cognitive functioning in a transgenic animal model. In accordance with previous reports, there were non-significant trends or marginal impairment in some tasks. However, the Df(h22q11)/+ mouse did not show comprehensive deficits; no robust impairment was observed following more than 17 experiments and 14 behavioral paradigms. Thus - within the current protocols - the 22q11.2DS mouse model fails to mimic the cognitive alterations observed in human 22q11.2 deletion carriers. We suggest that the 22q11.2DS model may induce liability for cognitive dysfunction with additional "hits" being required for phenotypic expression.

Asunto(s)

Cognición , Síndrome de DiGeorge/psicología , Modelos Animales de Enfermedad , Ratones Transgénicos , Animales , Atención , Estudios de Cohortes , Discriminación en Psicología , Descubrimiento de Drogas , Función Ejecutiva , Femenino , Inhibición Psicológica , Aprendizaje , Masculino , Memoria , Ratones Endogámicos C57BL , Pruebas Neuropsicológicas , Fenotipo , Investigación Biomédica Traslacional

RESUMEN

RATIONALE: A microdeletion at locus 15q13.3 is associated with high incidence rates of psychopathology, including schizophrenia. A mouse model of the 15q13.3 microdeletion syndrome has been generated (Df[h15q13]/+) with translational utility for modelling schizophrenia-like pathology. Among other deficits, schizophrenia is characterised by dysfunctions in prefrontal cortical (PFC) inhibitory circuitry and attention. OBJECTIVES: The objective of this study is to assess PFC-dependent functioning in the Df(h15q13)/+ mouse using electrophysiological, pharmacological, and behavioural assays. METHOD: Experiments 1-2 investigated baseline firing and auditory-evoked responses of PFC interneurons and pyramidal neurons. Experiment 3 measured pyramidal firing in response to intra-PFC GABAA receptor antagonism. Experiments 4-6 assessed PFC-dependent attentional functioning through the touchscreen 5-choice serial reaction time task (5-CSRTT). Experiments 7-12 assessed reversal learning, paired-associate learning, extinction learning, progressive ratio, trial-unique non-match to sample, and object recognition. RESULTS: In experiments 1-3, the Df(h15q13)/+ mouse showed reduced baseline firing rate of fast-spiking interneurons and in the ability of the GABAA receptor antagonist gabazine to increase the firing rate of pyramidal neurons. In assays of auditory-evoked responses, PFC interneurons in the Df(h15q13)/+ mouse had reduced detection amplitudes and increased detection latencies, while pyramidal neurons showed increased detection latencies. In experiments 4-6, the Df(h15q13)/+ mouse showed a stimulus duration-dependent decrease in percent accuracy in the 5-CSRTT. The impairment was insensitive to treatment with the partial α7nAChR agonist EVP-6124. The Df(h15q13)/+ mouse showed no cognitive impairments in experiments 7-12. CONCLUSION: The Df(h15q13)/+ mouse has multiple dysfunctions converging on disrupted PFC processing as measured by several independent assays of inhibitory transmission and attentional function.

Asunto(s)

Trastorno por Déficit de Atención con Hiperactividad/genética , Trastorno por Déficit de Atención con Hiperactividad/fisiopatología , Eliminación de Gen , Corteza Prefrontal/fisiopatología , Esquizofrenia/genética , Esquizofrenia/fisiopatología , Psicología del Esquizofrénico , Animales , Trastorno por Déficit de Atención con Hiperactividad/psicología , Conducta Animal/efectos de los fármacos , Deleción Cromosómica , Trastornos de los Cromosomas/genética , Cromosomas Humanos Par 15/genética , Modelos Animales de Enfermedad , Potenciales Evocados Auditivos/efectos de los fármacos , Extinción Psicológica/efectos de los fármacos , Antagonistas del GABA/farmacología , Humanos , Discapacidad Intelectual/genética , Interneuronas/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Células Piramidales/efectos de los fármacos , Piridazinas/farmacología , Tiempo de Reacción/efectos de los fármacos , Receptores de GABA-A/efectos de los fármacos , Aprendizaje Inverso/efectos de los fármacos , Convulsiones/genética

RESUMEN

The metabotropic glutamate (Glu) receptors (mGluRs) play key roles in modulating excitatory neurotransmission in the brain. In all, eight subtypes have been identified and divided into three groups, group I (mGlu1,5), group II (mGlu2,3), and group III (mGlu4,6-8). In this article, we present a L-2,4-syn-substituted Glu analogue, 1d, which displays selective agonist activity at mGlu2 over the remaining mGluR subtypes. A modeling study and redesign of the core scaffold led to the stereoselective synthesis of four new conformationally restricted Glu analogues, 2a-d. Most interestingly, 2a retained a selective agonist activity profile at mGlu2 (EC50 in the micromolar range), whereas 2c/2d were both selective agonists at group III, subtypes mGlu4,6,8. In general, 2d was 20-fold more potent than 2c and potently activated mGlu4,6,8 in the low-mid nanomolar range.

Asunto(s)

Ácido Glutámico/análogos & derivados , Ácido Glutámico/farmacología , Receptores de Glutamato Metabotrópico/agonistas , Animales , Células Cultivadas , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Ácido Glutámico/síntesis química , Ácido Glutámico/química , Células HEK293 , Humanos , Modelos Moleculares , Estructura Molecular , Ratas , Relación Estructura-Actividad

RESUMEN

In a small fraction of patients with schizophrenia or autism, alleles of copy-number variants (CNVs) in their genomes are probably the strongest factors contributing to the pathogenesis of the disease. These CNVs may provide an entry point for investigations into the mechanisms of brain function and dysfunction alike. They are not fully penetrant and offer an opportunity to study their effects separate from that of manifest disease. Here we show in an Icelandic sample that a few of the CNVs clearly alter fecundity (measured as the number of children by age 45). Furthermore, we use various tests of cognitive function to demonstrate that control subjects carrying the CNVs perform at a level that is between that of schizophrenia patients and population controls. The CNVs do not all affect the same cognitive domains, hence the cognitive deficits that drive or accompany the pathogenesis vary from one CNV to another. Controls carrying the chromosome 15q11.2 deletion between breakpoints 1 and 2 (15q11.2(BP1-BP2) deletion) have a history of dyslexia and dyscalculia, even after adjusting for IQ in the analysis, and the CNV only confers modest effects on other cognitive traits. The 15q11.2(BP1-BP2) deletion affects brain structure in a pattern consistent with both that observed during first-episode psychosis in schizophrenia and that of structural correlates in dyslexia.

Asunto(s)

Trastorno Autístico/genética , Cognición/fisiología , Variaciones en el Número de Copia de ADN/genética , Predisposición Genética a la Enfermedad , Esquizofrenia/genética , Adolescente , Adulto , Anciano , Encéfalo/anomalías , Encéfalo/anatomía & histología , Encéfalo/metabolismo , Estudios de Casos y Controles , Deleción Cromosómica , Cromosomas Humanos/genética , Cromosomas Humanos Par 15/genética , Dislexia/genética , Femenino , Fertilidad/genética , Heterocigoto , Humanos , Islandia , Discapacidades para el Aprendizaje/genética , Imagen por Resonancia Magnética , Masculino , Persona de Mediana Edad , Pruebas Neuropsicológicas , Fenotipo , Adulto Joven

RESUMEN

The isoxazol-3-one tautomer of the bicyclic isoxazole, 5,6,7,8-tetrahydro-4H-isoxazolo[4,5-d]azepin-3-ol (THAZ), has previously been shown to be a weak GABA(A) and glycine receptor antagonist. In the present study, the potential in this scaffold has been explored through the synthesis and pharmacological characterization of a series of N- and O-substituted THAZ analogues. The analogues N-Bn-THAZ (3d) and O-Bn-THAZ (4d) were found to be potent agonists of the human 5-HT(2A) and 5-HT(2C) receptors. Judging from an elaborate pharmacological profiling at numerous other CNS targets, the 3d analogue appears to be selective for the two receptors. Administration of 3d substantially improved the cognitive performance of mice in a place recognition Y-maze model, an effect fully reversible by coadministration of the selective 5-HT(2C) antagonist SB242084. In conclusion, as novel bioavailable cognitive enhancers that most likely mediate their effects through 5-HT(2A) and/or 5-HT(2C) receptors, the isoxazoles 3d and 4d constitute interesting leads for further medicinal chemistry development.

Asunto(s)

Azepinas/química , Azepinas/farmacología , Cognición/efectos de los fármacos , Receptor de Serotonina 5-HT2A/efectos de los fármacos , Receptor de Serotonina 5-HT2C/efectos de los fármacos , Agonistas de Receptores de Serotonina/química , Agonistas de Receptores de Serotonina/farmacología , Azepinas/síntesis química , Disponibilidad Biológica , Diseño de Fármacos , Células HEK293 , Humanos

RESUMEN

The present study aimed at investigating the theory that free (unbound) active site concentrations are the best predictors of target binding of compounds blocking the serotonin transporter (Sert) in the central nervous system (CNS). Thirteen serotonin reuptake inhibitors were evaluated for their Sert-binding affinities in vitro and in vivo in rats together with their unbound fractions in plasma and brain. Cortical Sert occupancy was used in vivo to acquire EC50-estimates from total plasma, free plasma, whole brain, and free brain concentrations after acute drug administration. The in vitro-in vivo Sert occupancy analyses showed that the best correlation was achieved when unbound brain concentrations were employed. Unbound brain concentrations also provided a better correlation when compared with unbound plasma concentrations, which could be related to lack of equilibrium between plasma and brain at time of measurements or involvement of active brain efflux processes. In addition, brain-free fractions were shown to be directly correlated to the lipophilicity of the compounds. These data emphasize the use and impact of applying free fraction data in assessment of pharmacological in vitro-in vivo correlations and demonstrates its use to validate in vivo Sert occupancy as pharmacodynamic marker for serotonin reuptake inhibitors in rats.

Asunto(s)

Sistema Nervioso Central/efectos de los fármacos , Sistema Nervioso Central/metabolismo , Inhibidores Selectivos de la Recaptación de Serotonina/metabolismo , Inhibidores Selectivos de la Recaptación de Serotonina/farmacología , Proteínas de Transporte de Serotonina en la Membrana Plasmática/metabolismo , Animales , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Línea Celular , Perros , Relación Dosis-Respuesta a Droga , Humanos , Concentración de Iones de Hidrógeno/efectos de los fármacos , Concentración 50 Inhibidora , Masculino , Ratas , Ratas Sprague-Dawley , Estándares de Referencia , Inhibidores Selectivos de la Recaptación de Serotonina/sangre , Inhibidores Selectivos de la Recaptación de Serotonina/química , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/metabolismo

RESUMEN

Intrahippocampal injections of aggregated amyloid-beta (Abeta)1-42 in rats result in memory impairment and in reduction of hippocampal 5-HT2A receptor levels. In order to investigate how changes in 5-HT2A levels and functionality relate to the progressive accumulation of Abeta protein, we studied 5-HT2A receptor regulation in double transgenic AbetaPPswe/PS1dE9 mice which display excess production of Abeta and age-dependent increase in amyloid plaques. Three different age-groups, 4-month-old, 8- month-old, and 11-month-old were included in the study. [3H]-MDL100907, [3H]-escitalopram, and [11C]-PIB autoradiography was performed for measuring 5-HT2A receptor, serotonin transporter (SERT), and Abeta plaque levels in medial prefrontal cortex (mPFC), prefrontal cortex (PFC), frontoparietal cortex (FPC), dorsal and ventral hippocampus, and somatosensory cortex. To investigate 5-HT2A receptor functionality, animals were treated with the 5-HT2A receptor agonist DOI and head-twitch response (HTR) subsequently recorded. Expression level of the immediate early gene c-fos was measured by in situ hybridization. We found that the age-related increase in Abeta plaque burden was accompanied by a significant decrease in 5-HT2A receptor binding in mPFC in the 11-month-old group. The changes in 5-HT2A receptor binding correlated negatively with [11C]-PIB binding and were not accompanied by decreases in SERT binding. Correspondingly, 11-month-old transgenic mice showed diminished DOI-induced HTR and reduced increase in expression of c-fos mRNA in mPFC and FPC. These observations point towards a direct association between Abeta accumulation and changes in 5-HT2A receptor expression that is independent of upstream changes in the serotonergic system.

Asunto(s)

Precursor de Proteína beta-Amiloide/biosíntesis , Precursor de Proteína beta-Amiloide/genética , Placa Amiloide/patología , Receptor de Serotonina 5-HT2A/genética , Receptor de Serotonina 5-HT2A/fisiología , Envejecimiento/fisiología , Anfetaminas/farmacología , Compuestos de Anilina/farmacocinética , Animales , Autorradiografía , Citalopram/farmacocinética , Interpretación Estadística de Datos , Fluorobencenos/farmacocinética , Genes fos/genética , Movimientos de la Cabeza/efectos de los fármacos , Humanos , Hibridación in Situ , Indicadores y Reactivos , Masculino , Ratones , Ratones Transgénicos , Piperidinas/farmacocinética , Antagonistas de la Serotonina/farmacocinética , Agonistas de Receptores de Serotonina/farmacología , Inhibidores Selectivos de la Recaptación de Serotonina/farmacocinética , Tiazoles/farmacocinética

RESUMEN

The discovery of the first class of subtype-selective inhibitors of the human excitatory amino acid transporter subtype 1 (EAAT1) and its rat orthologue GLAST is reported. An opening structure-activity relationship of 25 analogues is presented that addresses the influence of substitutions at the 4- and 7-positions of the parental skeleton 2-amino-5-oxo-5,6,7,8-tetrahydro-4H-chromene-3-carbonitrile. The most potent analogue 1o displays high nanomolar inhibitory activity at EAAT1 and a >400-fold selectivity over EAAT2 and EAAT3, making it a highly valuable pharmacological tool.

Asunto(s)

Benzopiranos/farmacología , Transportador 1 de Aminoácidos Excitadores/antagonistas & inhibidores , Nitrilos/farmacología , Animales , Benzopiranos/química , Descubrimiento de Drogas , Humanos , Nitrilos/química , Ratas , Relación Estructura-Actividad

RESUMEN

In the mammalian central nervous system (CNS), the action of sodium dependent excitatory amino acid transporters (EAATs) is responsible for termination of glutamatergic neurotransmission by reuptake of ( S) -glutamate (Glu) from the synaptic cleft. Five EAAT subtypes have been identified, of which EAAT1-4 are present in the CNS, while EAAT5 is localized exclusively in the retina. In this study, we have used an enantioselective chemo-enzymatic strategy to synthesize 10 new Glu analogues 2a- k ( 2d is exempt) with different functionalities in the 4 R-position and characterized their pharmacological properties at the human EAAT1-3. In particular, one compound, 2k, displayed a significant preference as inhibitor of the EAAT2 subtype over EAAT1,3. The compound also displayed very low affinities toward ionotropic and metabotropic Glu receptors, making it the most selective EAAT2 inhibitor described so far.

Asunto(s)

Antagonistas de Aminoácidos Excitadores/síntesis química , Antagonistas de Aminoácidos Excitadores/farmacología , Proteínas de Transporte de Glutamato en la Membrana Plasmática/antagonistas & inhibidores , Glutaratos/síntesis química , Glutaratos/farmacología , Línea Celular , Transportador 2 de Aminoácidos Excitadores , Humanos , Espectroscopía de Resonancia Magnética , Potenciales de la Membrana/efectos de los fármacos , Espectrometría de Masa por Ionización de Electrospray , Estereoisomerismo

RESUMEN

( S)-Glutamic acid (Glu) is the major excitatory neurotransmitter in the central nervous system (CNS) activating the plethora of ionotropic Glu receptors (iGluRs) and metabotropic Glu receptors (mGluRs). In this paper, we present a chemo-enzymatic strategy for the enantioselective synthesis of five new Glu analogues 2a- f ( 2d is exempt) holding a functionalized substituent in the 4-position. Nine Glu analogues 2a- j are characterized pharmacologically at native 2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA), kainic acid (KA), and N-methyl- d-aspartic acid (NMDA) receptors in rat synaptosomes as well as in binding assays at cloned rat iGluR5-7 subtypes. A detailed in silico study address as to why 2h is a high-affinity ligand at iGluR5-7 ( K i = 3.81, 123, 57.3 nM, respectively), while 2e is only a high affinity ligand at iGluR5 ( K i = 42.8 nM). Furthermore, a small series of commercially available iGluR ligands are characterized in iGluR5-7 binding.

Asunto(s)

Ácido Glutámico/análogos & derivados , Receptores de Glutamato/efectos de los fármacos , Aminación , Animales , Ácido Glutámico/biosíntesis , Ácido Glutámico/síntesis química , Ácido Glutámico/farmacología , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Ratas , Espectrofotometría Infrarroja , Relación Estructura-Actividad , Sinaptosomas/efectos de los fármacos

RESUMEN

A series of 4-aryl-5-(4-piperidyl)-3-isoxazolol GABAA antagonists have been synthesized and pharmacologically characterized. The meta-phenyl-substituted compounds 9k and 9m and the para-phenoxy-substituted compound 9l all display high affinities (Ki=10-70 nM) and antagonist potencies in the low nanomolar range (Ki=9-10 nM). These potencies are significantly higher than those of previously reported 4-PIOL antagonists and considerably higher than that of the standard GABAA antagonist SR 95531.

Asunto(s)

Antagonistas de Receptores de GABA-A , Isoxazoles/síntesis química , Piperidinas/síntesis química , Piridazinas/síntesis química , Animales , Femenino , Humanos , Técnicas In Vitro , Isoxazoles/química , Isoxazoles/farmacología , Modelos Moleculares , Conformación Molecular , Oocitos/efectos de los fármacos , Oocitos/fisiología , Técnicas de Placa-Clamp , Piperidinas/química , Piperidinas/farmacología , Piridazinas/química , Piridazinas/farmacología , Relación Estructura-Actividad , Xenopus

RESUMEN

Two 3-(5-tetrazolylmethoxy) analogues, 1a and 1b, of (RS)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA), a selective AMPA receptor agonist, and (RS)-2-amino-3-(5-tert-butyl-3-hydroxy-4-isoxazolyl)propionic acid (ATPA), a GluR5-preferring agonist, were synthesized. Compounds 1a and 1b were pharmacologically characterized in receptor binding assays, and electrophysiologically on homomeric AMPA receptors (GluR1-4), homomeric (GluR5 and GluR6) and heteromeric (GluR6/KA2) kainic acid receptors, using two-electrode voltage-clamped Xenopus laevis oocytes expressing these receptors. Both analogues proved to be antagonists at all AMPA receptor subtypes, showing potencies (Kb=38-161 microM) similar to that of the AMPA receptor antagonist (RS)-2-amino-3-[3-(carboxymethoxy)-5-methyl-4-isoxazolyl]propionic acid (AMOA) (Kb=43-76 microM). Furthermore, the AMOA analogue, 1a, blocked two kainic acid receptor subtypes (GluR5 and GluR6/KA2), showing sevenfold preference for GluR6/KA2 (Kb=19 microM). Unlike the iGluR antagonist (S)-2-amino-3-[5-tert-butyl-3-(phosphonomethoxy)-4-isoxazolyl]propionic acid [(S)-ATPO], the corresponding tetrazolyl analogue, 1b, lacks kainic acid receptor effects. On the basis of docking to a crystal structure of the isolated extracellular ligand-binding core of the AMPA receptor subunit GluR2 and a homology model of the kainic acid receptor subunit GluR5, we were able to rationalize the observed structure-activity relationships.

Asunto(s)

Antagonistas de Aminoácidos Excitadores/farmacología , Receptores de Glutamato/efectos de los fármacos , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/farmacología , Animales , Línea Celular , Simulación por Computador , Electrofisiología , Agonistas de Aminoácidos Excitadores/síntesis química , Agonistas de Aminoácidos Excitadores/química , Agonistas de Aminoácidos Excitadores/farmacología , Antagonistas de Aminoácidos Excitadores/síntesis química , Antagonistas de Aminoácidos Excitadores/química , Isoxazoles/química , Modelos Moleculares , Oocitos/efectos de los fármacos , Oocitos/metabolismo , Técnicas de Placa-Clamp , Propionatos/química , Ratas , Receptores de Glutamato/genética , Receptores de Glutamato/metabolismo , Xenopus laevis , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/análogos & derivados , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/síntesis química

RESUMEN

We have previously described a series of competitive GABA(A) antagonists derived from the low-efficacy partial agonist 5-(4-piperidyl)-3-isoxazolol (4-PIOL, 4). The 2-naphthylmethyl analogue, 4-(2-naphthylmethyl)-5-(4-piperidyl)-3-isoxazolol (5), provided affinity for the GABA(A) receptor site higher than that of the standard GABA(A) receptor antagonist, SR 95531 (3). Molecular modeling studies of these compounds exposed a cavity at the receptor recognition site capable of accommodating aromatic groups of substantial size in the 4-position in the 3-isoxazolol ring. Here we present a series of analogues of 5, with various substituents in different positions in the naphthyl ring system (6a-k), and compounds with aromatic substituents directly attached to the 4-position of the 3-isoxazolol ring (7l-n). The compounds have been pharmacologically characterized using receptor-binding assays and electrophysiological whole-cell patch-clamp techniques. All of the tested compounds show affinity for the GABA(A) receptor site. While the 5-, 7-, and 8-bromo analogues, 6b-d, showed receptor affinities (K(i) = 45, 109, and 80 nM, respectively) comparable with that of 5 (K(i) = 49 nM), the 1-bromo analogue, 6a, provided the highest receptor affinity of the series (K(i) = 10 nM). Introduction of a series of different substituents in the 1-position in the 2-naphthyl ring system led to compounds, 6e-k, with retained high affinity for the GABA(A) receptor (K(i) = 16-250 nM). Introduction of a phenyl ring directly into the 4-position on the 3-isoxazolol ring gave a 41-fold increase in affinity relative to that of 4-PIOL. In whole-cell patch-clamp recordings from cultured cerebral cortical neurons, all of the tested compounds were able to inhibit the effect of the specific GABA(A) agonist isoguvacine, 6a showing antagonist potency (IC(50) = 42 nM) markedly higher than that of 3 (IC(50) = 240 nM). Molecular modeling studies, based on the compounds described, emphasized the importance of the distal ring in 5 for receptor affinity and the considerable dimensions of the proposed receptor cavity. Furthermore, the phenyl rings in 7l and in 6k were shown to represent highly favorable positions for an aromatic ring in previously unexplored receptor regions in terms of a pharmacophore model.

Asunto(s)

Antagonistas de Receptores de GABA-A , Isoxazoles/síntesis química , Animales , Sitios de Unión , Células Cultivadas , Corteza Cerebral/citología , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/fisiología , Isoxazoles/química , Isoxazoles/farmacología , Ligandos , Masculino , Ratones , Modelos Moleculares , Naftalenos/síntesis química , Naftalenos/química , Naftalenos/farmacología , Neuronas/efectos de los fármacos , Neuronas/fisiología , Técnicas de Placa-Clamp , Piperidinas/síntesis química , Piperidinas/química , Piperidinas/farmacología , Ensayo de Unión Radioligante , Ratas , Ratas Sprague-Dawley , Relación Estructura-Actividad

RESUMEN

The two diastereomeric pairs of acidic amino acids 5-(2-amino-2-carboxyethyl)-4,5-dihydroisoxazole-3-carboxylic acid (8A/8B) and 4-(2-amino-2-carboxyethyl)-5,5-dimethyl-4,5-dihydroisoxazole-3-carboxylic acid (10A/10B) were prepared via a strategy based on a 1,3-dipolar cycloaddition. The four amino acids were tested at ionotropic and metabotropic glutamate receptors. None of the compounds was active, neither as agonists nor as antagonists, at 1 mM on metabotropic receptors (mGluR1, -2, -4, and -5 expressed in CHO cell lines). Conversely, the pair of stereoisomers 8A/8B showed a remarkable affinity, antagonist potency, and selectivity for NMDA receptors, when tested on ionotropic glutamate receptors. The affinity of 8A proved to be 5 times higher than that of diastereomer 8B (K(i) values 0.21 and 0.96 microM, respectively). Furthermore, compounds 8A and 8B exhibited a noteworthy anticonvulsant activity in in vivo tests on DBA/2 mice. Derivative 10A was inactive at all ionotropic glutamate receptors, whereas its stereoisomer 10B displayed a seizable binding to both NMDA and AMPA receptors.

Asunto(s)

Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Animales , Diseño de Fármacos , Masculino , Ratones , Ratones Endogámicos DBA , Conformación Molecular , Ratas , Relación Estructura-Actividad

RESUMEN

We have previously used homologation of (S)-glutamic acid (Glu) and Glu analogs as an approach to the design of selective ligands for different subtypes of Glu receptors. (RS)-2-Amino-3-(3-carboxy-5-methyl-4-isoxazolyl)propionic acid (ACPA), which is an isoxazole homolog of Glu, is a very potent agonist at the (RS)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA) subgroup of Glu receptors and a moderately potent ligand for the kainic acid (KA) subgroup of Glu receptors. The enantiomers of ACPA were previously obtained by chiral HPLC resolution. Prompted by pharmacological interest in ACPA, we have now prepared the (S)- and (R)-enantiomers of ACPA by stereocontrolled syntheses using (1R,2R,5R)- and (1S,2S,5S)-2-hydroxy-3-pinanone, respectively, as chiral auxiliaries. Furthermore, the 5-ethyl analog of ACPA, Ethyl-ACPA, was synthesized, and (S)- and (R)-Ethyl-ACPA were also prepared using this method. The absolute configurations of (S)- and (R)-ACPA were established by X-ray crystallographic analysis of a protected (1S,2S,5S)-2-hydroxy-3-pinanone imine derivative of (R)-ACPA. The absolute stereochemistry of (S)- and (R)-Ethyl-ACPA was assigned on the basis of a comparison of their properties with those of the enantiomers of ACPA, employing elution order on chiral HPLC columns, as well as circular dichroism (CD) spectroscopy in combination with time-dependent density functional theory. The structural and electronic basis for the Cotton effect observed for such analogs is examined. The lower homolog of ACPA, (RS)-2-amino-2-(3-carboxy-5-methyl-4-isoxazolyl)acetic acid (1), which is a Glu analog, was also synthesized. Affinities and neuroexcitatory effects were determined using rat brain membranes and cortical wedges, respectively, at native AMPA, KA, and N-methyl-D-aspartic acid (NMDA) receptors. The molecular pharmacology of (S)- and (R)-ACPA and (S)- and (R)-Ethyl-ACPA was evaluated at homomeric cloned subtypes of AMPA receptors (iGluR1o,3o,4o) and of KA receptors (iGluR5,6), expressed in Xenopus laevis oocytes. The cloned receptors mGluR1alpha, mGluR2, and mGluR4a, expressed in CHO cell lines, were used to study the effects of the five compounds at metabotropic Glu receptors. In accordance with ligand-receptor complexes known from X-ray crystallography, the conformationally restricted Glu analog 1 was inactive at all Glu receptors studied, and the R-forms of ACPA and Ethyl-ACPA were very weak or inactive at these receptors. At AMPA receptor subtypes, (S)-ACPA and (S)-Ethyl-ACPA showed equally potent agonist effects at iGluR1o and iGluR3o, whereas (S)-Ethyl-ACPA was 6-fold more potent than (S)-ACPA at iGluR4o. (S)-ACPA and (S)-Ethyl-ACPA were approximately an order of magnitude less potent at iGluR5 than at AMPA receptor subtypes, and neither compound showed detectable effects at iGluR6. The binding mode of (S)-Ethyl-ACPA at iGluR2 was examined by docking to the (S)-ACPA-iGluR2 complex.

Asunto(s)

Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/síntesis química , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/farmacología , Animales , Células CHO , Cromatografía Líquida de Alta Presión , Dicroismo Circular , Cricetinae , Cristalografía por Rayos X , Electrofisiología , Ácido Glutámico/metabolismo , Enlace de Hidrógeno , Concentración 50 Inhibidora , Conformación Molecular , Óvulo , Ratas , Receptores de Glutamato/genética , Receptores de Glutamato/metabolismo , Sistemas de Mensajero Secundario/efectos de los fármacos , Estereoisomerismo , Xenopus , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/análogos & derivados , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/química

RESUMEN

In this study, we have determined and compared the pharmacological profiles of ibotenic acid and its isothiazole analogue thioibotenic acid at native rat ionotropic glutamate (iGlu) receptors and at recombinant rat metabotropic glutamate (mGlu) receptors expressed in mammalian cell lines. Thioibotenic acid has a distinct pharmacological profile at group III mGlu receptors compared with the closely structurally related ibotenic acid; the former is a potent (low microm) agonist, whereas the latter is inactive. By comparing the conformational energy profiles of ibotenic and thioibotenic acid with the conformations preferred by the ligands upon docking to mGlu1 and models of the other mGlu subtypes, we propose that unlike other subtypes, group III mGlu receptor binding sites require a ligand conformation at an energy level which is prohibitively expensive for ibotenic acid, but not for thioibotenic acid. These studies demonstrate how subtle differences in chemical structures can result in profound differences in pharmacological activity.

Asunto(s)

Agonistas de Aminoácidos Excitadores/farmacología , Ácido Iboténico/farmacología , Receptores de Glutamato Metabotrópico/agonistas , Tiazoles/farmacología , Animales , Células CHO , Cricetinae , Cricetulus , Agonistas de Aminoácidos Excitadores/química , Ácido Iboténico/química , Técnicas In Vitro , Ligandos , Masculino , Ratones , Modelos Moleculares , Conformación Molecular , Ensayo de Unión Radioligante , Ratas , Receptores de Glutamato Metabotrópico/clasificación , Receptores de Glutamato Metabotrópico/fisiología , Tiazoles/química

RESUMEN

Bicyclic acidic amino acids (+/-)-6 and (+/-)-7, which are conformationally constrained homologues of glutamic acid, were prepared via a strategy based on a 1,3-dipolar cycloaddition. The new amino acids were tested toward ionotropic and metabotropic glutamate receptor subtypes; both of them behaved as antagonists at mGluR1,5 and as agonists at mGluR2. Furthermore, whereas (+/-)-6 was inactive at all ionotropic glutamate receptors, (+/-)-7 displayed a quite potent antagonism at the NMDA receptors. In the in vivo tests on DBA/2 mice, the compounds displayed an anticonvulsant activity. The interesting pharmacological profile of (+/-)-7 qualifies it as a lead of novel neuroprotective agents.

Asunto(s)

Aminoácidos Acídicos/síntesis química , Aminoácidos Dicarboxílicos/síntesis química , Anticonvulsivantes/síntesis química , Ácidos Dicarboxílicos/síntesis química , Compuestos Heterocíclicos con 2 Anillos/síntesis química , Isoxazoles/síntesis química , Aminoácidos Acídicos/química , Aminoácidos Acídicos/farmacología , Aminoácidos Dicarboxílicos/química , Aminoácidos Dicarboxílicos/farmacología , Animales , Anticonvulsivantes/química , Anticonvulsivantes/farmacología , Células CHO , Corteza Cerebral/metabolismo , Cricetinae , Cristalografía por Rayos X , Ácidos Dicarboxílicos/química , Ácidos Dicarboxílicos/farmacología , Agonistas de Aminoácidos Excitadores/síntesis química , Agonistas de Aminoácidos Excitadores/química , Agonistas de Aminoácidos Excitadores/farmacología , Antagonistas de Aminoácidos Excitadores/síntesis química , Antagonistas de Aminoácidos Excitadores/química , Antagonistas de Aminoácidos Excitadores/farmacología , Compuestos Heterocíclicos con 2 Anillos/química , Compuestos Heterocíclicos con 2 Anillos/farmacología , Técnicas In Vitro , Isoxazoles/química , Isoxazoles/farmacología , Masculino , Ratones , Ratones Endogámicos DBA , Conformación Molecular , Ratas , Receptor del Glutamato Metabotropico 5 , Receptores de Glutamato Metabotrópico/agonistas , Receptores de Glutamato Metabotrópico/antagonistas & inhibidores , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Estereoisomerismo