RESUMEN
Cannabinoid receptors (CBRs) play an important role in a variety of physiological functions and have been considered drug targets for obesity and psychiatric disorders. In particular, the CB1R is highly expressed in brain regions crucial to learning and memory processes, and several lines of evidence indicate that pharmacological blockade of this receptor could have therapeutic applications in the treatment of cognitive disorders. In this study, we investigated whether MK-7128 (0.1, 0.3, and 1 mg/kg, orally), a novel and selective CB1R inverse agonist, could improve learning and memory deficits induced by scopolamine (1 mg/kg, subcutaneously) in mice. The investigators also assessed CB1R occupancy in the brain to ensure target engagement of MK-7128, and showed that MK-7128 significantly improved both Y-maze spontaneous alternation and object habituation performance in scopolamine-treated mice and inhibits the binding of radioiodinated AM251 in murine cortex and hippocampus. These data indicate that MK-7128 improves cognitive performance in a model of cholinergic hypofunction and suggest that efficacy is achieved at relatively low levels of CB1R occupancy in the brain. Our results extend earlier findings suggesting a role of CB1Rs in the modulation of memory processes and a potential therapeutic application for CB1R inverse agonists in cognitive disorders.
Asunto(s)
Azetidinas/farmacología , Agonismo Inverso de Drogas , Trastornos de la Memoria/tratamiento farmacológico , Oxadiazoles/farmacología , Receptor Cannabinoide CB1/agonistas , Animales , Azetidinas/administración & dosificación , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/metabolismo , Trastornos del Conocimiento/tratamiento farmacológico , Trastornos del Conocimiento/fisiopatología , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Masculino , Aprendizaje por Laberinto/efectos de los fármacos , Trastornos de la Memoria/fisiopatología , Ratones , Ratones Endogámicos C57BL , Oxadiazoles/administración & dosificación , Piperidinas/metabolismo , Unión Proteica , Pirazoles/metabolismo , EscopolaminaRESUMEN
Administration of Neuropeptide S (NPS) has been shown to produce arousal, that is, independent of novelty and to induce wakefulness by suppressing all stages of sleep, as demonstrated by EEG recordings in rat. Medicinal chemistry efforts have identified a quinolinone class of potent NPSR antagonists that readily cross the blood-brain barrier. We detail here optimization efforts resulting in the identification of a potent NPSR antagonist which dose-dependently and specifically inhibited (125)I-NPS binding in the CNS when administered to rats.
Asunto(s)
Receptores de Neuropéptido/antagonistas & inhibidores , Amidas/síntesis química , Amidas/química , Amidas/farmacología , Animales , Barrera Hematoencefálica/metabolismo , Sistema Nervioso Central/metabolismo , Humanos , Radioisótopos de Yodo/química , Unión Proteica , Quinolonas/síntesis química , Quinolonas/química , Quinolonas/farmacología , Ratas , Receptores de Neuropéptido/metabolismo , Relación Estructura-ActividadRESUMEN
INTRODUCTION: Poly ADP-ribose polymerase (PARP) maintains genomic integrity by repairing DNA strand breaks, however over-activation of PARP following neural tissue injury is hypothesized to cause neuronal death. Therefore, PARP inhibitors have potential for limiting neural injury under certain conditions. A reliable method for assessing PARP activity in brain is critical for development of novel inhibitors with CNS activity. We developed the PARP In Situ Activity (PISA) assay to provide a direct, quantitative assessment of CNS PARP activity in vitro or in vivo. METHODS: The assay utilized brain sections from rats with striatal kainic acid (KA) lesions and 3H- or biotinylated NAD+ as the substrate to assess PARP activity. Following optimization of the assay, it was used to assess in vitro and in vivo efficacy of known and novel PARP inhibitors. The assay also was used to assess PARP activity in male and female gonad-intact and ovariectomized rats. RESULTS: Using 3H-NAD+ as the substrate, PARP activity was greater (p<0.01) in tissue from KA-lesioned vs. non-lesioned rats. Using biotinylated NAD+ it was revealed that PARP activity was present ipsilateral to the KA injection site, and labeling was blocked by incubation with excess unlabeled NAD+ or PARP inhibitors. The PARP inhibitor, 3-aminobenzamide and several novel inhibitors reduced (p<0.01) polymerase activity in vitro. Furthermore, the inhibitor MRLSD303 reduced (p<0.001) PARP activity in vivo in both male and female rats. Finally, administration of the novel PARP inhibitor MRLIT115 dose-dependently reduced (p<0.001) polymerase activity in vivo. DISCUSSION: The PISA assay provides a direct, quantitative method for assessing PARP activity in vitro and provides critical information on factors underlying in vivo efficacy of chemical inhibitors including brain penetration and target engagement. These findings support use of the PISA assay as a screening tool for testing efficacy of PARP inhibitors in brain.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Poli(ADP-Ribosa) Polimerasas/metabolismo , Animales , Benzamidas/farmacología , Encéfalo/efectos de los fármacos , Encéfalo/enzimología , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Activación Enzimática/fisiología , Femenino , Masculino , Modelos Animales , Ratas , Ratas Sprague-DawleyRESUMEN
Glutamate is a major neurotransmitter in the central nervous system, and abnormal glutamate neurotransmission has been implicated in many neurological disorders, including schizophrenia, Alzheimer's disease, Parkinson's disease, addiction, anxiety, depression, epilepsy, and pain. Metabotropic glutamate receptors (mGluRs) activate intracellular signaling cascades in a G protein-dependent manner, which offer the opportunity for developing drugs that regulate glutamate neurotransmission in a functionally selective manner. In the present study, we further characterize the human mGluR2 (hmGluR2) potentiator binding site by showing that the substitution of the three amino acids found to be required for hmGluR2 potentiation, specifically Ser(688), Gly(689), and Asn(735), with the homologous hmGluR3 amino acids, inactivates the positive allosteric modulator activity of several structurally unique mGluR2 potentiators. Based on the characterization of the hmGluR2 potentiator binding site, we developed a novel scintillation proximity assay that was able to discriminate between compounds that were hmGluR2-specific potentiators, and those that were active on both hmGluR2 and hmGluR3. In addition, we substituted Ser(688), Gly(689), and Asn(735) into hmGluR3 and created an active hmGluR2 allosteric modulation site on the hmGluR3 receptor.
Asunto(s)
Sitio Alostérico/genética , Aminoácidos/metabolismo , Receptores de Glutamato Metabotrópico/química , Receptores de Glutamato Metabotrópico/genética , Regulación Alostérica/genética , Secuencia de Aminoácidos , Sustitución de Aminoácidos/genética , Aminoácidos/química , Aminoácidos/genética , Animales , Línea Celular , Células Cultivadas , Humanos , Masculino , Datos de Secuencia Molecular , Mutación Puntual , Unión Proteica/genética , Ratas , Ratas Sprague-DawleyRESUMEN
We have identified and synthesized a series of biphenyl-carboxylic acid indanones as allosteric potentiators of the metabotropic glutamate receptor 2. Structure-activity relationship studies directed toward improving the potency and the brain to plasma ratio of the initial lead led to the discovery of 5 and 23 (EC50=111 and 5 nM, respectively).
Asunto(s)
Compuestos de Bifenilo/síntesis química , Indanos/síntesis química , Receptores de Glutamato Metabotrópico/agonistas , Regulación Alostérica , Animales , Compuestos de Bifenilo/metabolismo , Compuestos de Bifenilo/farmacocinética , Química Encefálica , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Humanos , Indanos/metabolismo , Indanos/farmacocinética , Ratas , Receptores de Glutamato Metabotrópico/metabolismo , Esquizofrenia/tratamiento farmacológico , Relación Estructura-Actividad , Distribución TisularRESUMEN
Metabotropic glutamate receptor 2 (mGluR2) has been implicated in a variety of CNS disorders, including schizophrenia. Disclosed herein is the development of a new series of allosteric potentiators of mGluR2. Structure-activity relationship studies in conjunction with pharmacokinetic data led to the discovery of indole 5, which is active in an animal model for schizophrenia.
Asunto(s)
Acetofenonas/farmacología , Modelos Animales de Enfermedad , Receptores de Glutamato Metabotrópico/metabolismo , Esquizofrenia/tratamiento farmacológico , Esquizofrenia/metabolismo , Acetofenonas/química , Acetofenonas/farmacocinética , Regulación Alostérica/efectos de los fármacos , Animales , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Reactivos de Enlaces Cruzados/química , Humanos , Ketamina/farmacología , Estructura Molecular , Ratas , Esquizofrenia/inducido químicamente , Relación Estructura-ActividadRESUMEN
We have identified and synthesized a brain penetrant propanoic acid as an allosteric potentiator of the metabotropic glutamate receptor 2. Structure-activity relationship studies directed toward improving the potency, level of potentiation and brain penetration led to the discovery of 8 (EC50=1200 nM, 77% potentiation, 119% brain/plasma in rat, 20 mpk i.p., brain level of 5700 nM).
Asunto(s)
Encéfalo/fisiología , Butanos/síntesis química , Butanos/farmacología , Propionatos/síntesis química , Propionatos/farmacología , Receptores de Glutamato Metabotrópico/fisiología , Regulación Alostérica , Animales , Encéfalo/efectos de los fármacos , Butanos/farmacocinética , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Cinética , Conformación Molecular , Éteres Fenílicos , Propionatos/farmacocinética , Ratas , Receptores de Glutamato Metabotrópico/efectos de los fármacos , Relación Estructura-ActividadRESUMEN
We have identified and synthesized a series of phenyl-tetrazolyl and 4-thiopyridyl indanones as allosteric potentiators of the metabotropic glutamate receptor 2. Structure activity relationship studies directed toward improving the potency and level of potentiation, as well as PK properties, led to the discovery of 28 (EC50=186 nM), which displayed activity in a rodent model for schizophrenia.
Asunto(s)
Indanos/farmacología , Receptores de Glutamato Metabotrópico/agonistas , Regulación Alostérica , Animales , Encéfalo/metabolismo , Modelos Animales de Enfermedad , Indanos/farmacocinética , Modelos Químicos , Estructura Molecular , Unión Proteica , Ratas , Esquizofrenia/tratamiento farmacológico , Relación Estructura-ActividadRESUMEN
We have identified and synthesized a series of 4-thiopyridyl acetophenones as positive allosteric potentiators of the metabotropic glutamate receptor 2. Structure-activity relationship studies directed toward replacement of the tetrazole in the initial lead led to the discovery of 16 (EC(50)=340 nM), which showed improved brain penetration over the initial lead.
Asunto(s)
Acetofenonas/metabolismo , Piridinas/química , Piridinas/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Acetofenonas/química , Regulación Alostérica/fisiología , Animales , Encéfalo/metabolismo , Línea Celular , Humanos , Unión Proteica/fisiología , Ratas , Receptores de Glutamato Metabotrópico/agonistas , Relación Estructura-Actividad , TetrazolesRESUMEN
We have identified and synthesized a series of aryl-tetrazoyl acetophenones as positive allosteric potentiators of the metabotropic glutamate receptor 2. Structure activity relationship studies directed toward improving the potency and level of potentiation led to the discovery of 22 (EC(50)=93nM, 128% potentiation).
Asunto(s)
Acetofenonas/síntesis química , Receptores de Glutamato Metabotrópico/agonistas , Tetrazoles/síntesis química , Acetofenonas/química , Acetofenonas/farmacología , Regulación Alostérica , Estereoisomerismo , Relación Estructura-Actividad , Tetrazoles/química , Tetrazoles/farmacologíaRESUMEN
Pyrimidine methyl anilines as potent and selective mGlu2 potentiators are described. Findings from the structure-activity-relationship investigations are discussed.
Asunto(s)
Compuestos de Anilina/síntesis química , Pirimidinas/síntesis química , Receptores de Glutamato Metabotrópico/agonistas , Compuestos de Anilina/química , Compuestos de Anilina/farmacología , Pirimidinas/química , Pirimidinas/farmacología , Ensayo de Unión Radioligante , Relación Estructura-Actividad , Sulfonamidas/síntesis química , Sulfonamidas/química , Sulfonamidas/farmacologíaRESUMEN
Herein we disclose the discovery of a new class of positive allosteric potentiators of the metabotropic glutamate receptor 2 (mGlu2), phenyl-tetrazolyl acetophenones, e.g. 1-(2-hydroxy-3-propyl-4-[4-[4-(2H-tetrazol-5-yl)phenoxy]butoxy]phenyl) ethanone (4). These potentiators were shown to have no effect in the absence of glutamate as well as no effect at mGlu3 or the other mGlu receptors. The compounds were also evaluated in rodent models with potential relevance for schizophrenia, and 4 was shown to have activity in the inhibition of ketamine-induced norepinephrine release and ketamine-induced hyperactivity. This represents the first example of the efficacy of mGlu2 receptor potentiators in these models.
Asunto(s)
Acetofenonas/síntesis química , Acetofenonas/farmacología , Regulación Alostérica , Receptores de Glutamato Metabotrópico/agonistas , Animales , Conducta/efectos de los fármacos , Encéfalo/metabolismo , Relación Dosis-Respuesta a Droga , Ácido Glutámico/farmacología , Hipercinesia/tratamiento farmacológico , Norepinefrina/metabolismo , Ratas , Esquizofrenia/tratamiento farmacológico , Relación Estructura-ActividadRESUMEN
In the present study, we describe the characterization of a positive allosteric modulator at metabotropic glutamate subtype 2 receptors (mGluR2). N-(4-(2-Methoxyphenoxy)-phenyl-N-(2,2,2-trifluoroethylsulfonyl)-pyrid-3-ylmethylamine (LY487379) is a selective positive allosteric modulator at human mGluR2 and is without activity at human mGluR3. Furthermore, LY487379 has no intrinsic agonist or antagonist activity at hmGluR2, as determined by functional guanosine 5'(gamma-[35S]thio)triphosphate ([35S]GTPgammaS) binding, single-cell Ca2+ imaging, and electrophysiological studies. However, LY487379 markedly potentiated glutamate-stimulated [35S]GTPgammaS binding in a concentration-dependent manner at hmGluR2, shifting the glutamate dose-response curve leftward by 3-fold and increasing the maximum levels of [35S]GTPgammaS stimulation. This effect of LY487479 was also observed to a greater extent on the concentration-response curves to selective hmGluR2/3 agonists. In radioligand binding studies to rat cortical membranes, LY487379 increased the affinity of the radiolabeled agonist, [3H]DCG-IV, without affecting the binding affinity of the radiolabeled antagonist, [3H]LY341495. In rat hippocampal slices, coapplication of LY487379 potentiated synaptically evoked mGluR2 responses. Finally, to elucidate the site of action, we systematically exchanged segments and single amino acids between hmGluR2 and hmGluR3. Substitution of Ser688 and/or Gly689 in transmembrane IV along with Asn735 located in transmembrane segment V, with the homologous amino acids of hmGluR3, completely eliminated LY487379 allosteric modulation of hmGluR2. We propose that this allosteric binding site defines a pocket that is different from the orthosteric site located in the amino terminal domain.