RESUMO
M4 muscarinic receptors are highly expressed in the striatum and cortex, brain regions that are involved in diseases such as Parkinson's disease, schizophrenia, and dystonia. Despite potential therapeutic advantages of specifically targeting the M4 receptor, it has been historically challenging to develop highly selective ligands, resulting in undesired off-target activity at other members of the muscarinic receptor family. Recently, we have reported first-in-class, potent, and selective M4 receptor antagonists. As an extension of that work, we now report the development and characterization of a radiolabeled M4 receptor antagonist, [3H]VU6013720, with high affinity (pKd of 9.5 ± 0.2 at rat M4, 9.7 at mouse M4, and 10 ± 0.1 at human M4 with atropine to define nonspecific binding) and no significant binding at the other muscarinic subtypes. Binding assays using this radioligand in rodent brain tissues demonstrate loss of specific binding in Chrm4 knockout animals. Dissociation kinetics experiments with various muscarinic ligands show differential effects on the dissociation of [3H]VU6013720 from M4 receptors, suggesting a binding site that is overlapping but may be distinct from the orthosteric site. Overall, these results demonstrate that [3H]VU6013720 is the first highly selective antagonist radioligand for the M4 receptor, representing a useful tool for studying the basic biology of M4 as well for the support of M4 receptor-based drug discovery. SIGNIFICANCE STATEMENT: This manuscript describes the development and characterization of a novel muscarinic (M) acetylcholine subtype 4 receptor antagonist radioligand, [3H]VU6013720. This ligand binds to or overlaps with the acetylcholine binding site, providing a highly selective radioligand for the M4 receptor that can be used to quantify M4 protein expression in vivo and probe the selective interactions of acetylcholine with M4 versus the other members of the muscarinic receptor family.
Assuntos
Acetilcolina , Receptores Muscarínicos , Ratos , Humanos , Camundongos , Animais , Acetilcolina/metabolismo , Receptores Muscarínicos/metabolismo , Receptor Muscarínico M4/metabolismo , Atropina , Ligantes , Colinérgicos , Antagonistas Muscarínicos/farmacologia , Antagonistas Muscarínicos/metabolismo , Receptor Muscarínico M2/metabolismo , Ensaio Radioligante , Receptor Muscarínico M1/metabolismoRESUMO
In this manuscript, we report a series of chiral 6-azaspiro[2.5]octanes and related spirocycles as highly potent and selective antagonists of the muscarinic acetylcholine receptor subtype 4 (mAChR4). Chiral separation and subsequent X-ray crystallographic analysis of early generation analogs revealed the R enantiomer to possess excellent human and rat M4 potency, and further structure-activity relationship (SAR) studies on this chiral scaffold led to the discovery of VU6015241 (compound 19). Compound 19 is characterized by high M4 potency and selectivity across multiple species, excellent aqueous solubility, and moderate brain exposure in rodents after intraperitoneal administration.
Assuntos
Antagonistas Muscarínicos/farmacologia , Receptor Muscarínico M4/antagonistas & inibidores , Relação Dose-Resposta a Droga , Humanos , Estrutura Molecular , Antagonistas Muscarínicos/síntese química , Antagonistas Muscarínicos/química , Receptor Muscarínico M4/metabolismo , Relação Estrutura-AtividadeRESUMO
A high throughput screen (HTS) identified a novel, but weak (EC50 = 6.2 µM, 97% Glu Max) mGlu4 PAM chemotype based on a 1,4-thiazepane core, VU0544412. Reaction development and chemical optimization delivered a potent mGlu4 PAM VU6022296 (EC50 = 32.8 nM, 108% Glu Max) with good CNS penetration (Kp = 0.45, Kp,uu = 0.70) and enantiopreference. Finally, VU6022296 displayed robust, dose-dependent efficacy in reversing Haloperidol-Induced Catalepsy (HIC), a rodent preclinical Parkinson's disease model.
Assuntos
Catalepsia/tratamento farmacológico , Modelos Animais de Doenças , Descoberta de Drogas , Fármacos Neuroprotetores/farmacologia , Doença de Parkinson/tratamento farmacológico , Receptores de Glutamato Metabotrópico/antagonistas & inibidores , Regulação Alostérica/efeitos dos fármacos , Animais , Catalepsia/induzido quimicamente , Relação Dose-Resposta a Droga , Haloperidol , Camundongos , Estrutura Molecular , Fármacos Neuroprotetores/síntese química , Fármacos Neuroprotetores/química , Receptores de Glutamato Metabotrópico/metabolismo , Relação Estrutura-AtividadeRESUMO
This Letter describes the further lead optimization of the CHT inhibitor probe, ML352 (VU0476201), and the development of VU6001221, an improved in vivo tool. A multi-dimensional optimization effort encountered steep SAR, and ultimately, subtle tuning of the electronics of the central phenyl core provided VU6001221, a CHT inhibitor with comparable potency for choline uptake inhibition as ML352, yet improved PK and CNS penetration. Moreover, VU6001221 enabled evaluation, for the first time, of a CHT inhibitor in a standard preclinical rodent cognition model, novel object recognition (NOR). We observed VU6001221 to elicit a dose-responsive increase in NOR, raising the possibility of agonism of synaptic α7 nicotinic ACh receptors by elevated extracellular choline, that if confirmed would represent a novel molecular strategy to enhance cognition.
Assuntos
Benzamidas/farmacologia , Isoxazóis/farmacologia , Proteínas de Membrana Transportadoras/efeitos dos fármacos , Oxazóis/farmacologia , Piperidinas/farmacologia , Animais , Benzamidas/química , Benzamidas/farmacocinética , Relação Dose-Resposta a Droga , Meia-Vida , Concentração Inibidora 50 , Isoxazóis/química , Isoxazóis/farmacocinética , Oxazóis/química , Oxazóis/farmacocinética , Piperidinas/química , Piperidinas/farmacocinética , Ratos , Relação Estrutura-AtividadeRESUMO
Ligands for the serotonin 2B receptor (5-HT2B) have shown potential to treat pulmonary arterial hypertension in preclinical models but cannot be used in humans because of predicted off-target neurological effects. The aim of this study was to develop novel systemically restricted compounds targeting 5-HT2B. Here, we show that mice treated with VU6047534 had decreased RVSP compared with control treatment in both the prevention and intervention studies using Sugen-hypoxia. VU6047534 is a novel 5-HT2B partial agonist that is peripherally restricted and able to both prevent and treat Sugen-hypoxia-induced pulmonary arterial hypertension. We have synthesized and characterized a structurally novel series of 5-HT2B ligands with high potency and selectivity for the 5-HT2B receptor subtype. Next-generation 5-HT2B ligands with similar characteristics, and predicted to be systemically restricted in humans, are currently advancing to investigational new drug-enabling studies.
RESUMO
BACKGROUND: Altered permeability of the blood-brain barrier (BBB) is a feature of numerous neurological conditions including multiple sclerosis, cerebral malaria, viral hemorrhagic fevers and acute hemorrhagic leukoencephalitis. Our laboratory has developed a murine model of CD8 T cell-initiated central nervous system (CNS) vascular permeability in which vascular endothelial growth factor (VEGF) signaling plays a prominent role in BBB disruption. FINDINGS: In this study, we addressed the hypothesis that in vivo blockade of VEGF signal transduction through administration of peptide (ATWLPPR) to inhibit neuropilin-1 (NRP-1) would have a therapeutic effect following induction of CD8 T cell-initiated BBB disruption. We report that inhibition of NRP-1, a co-receptor that enhances VEGFR2 (flk-1) receptor activation, decreases vascular permeability, brain hemorrhage, and mortality in this model of CD8 T cell-initiated BBB disruption. We also examine the expression pattern of VEGFR2 (flk-1) and VEGFR1 (flt-1) mRNA expression during a time course of this condition. We find that viral infection of the brain leads to increased expression of flk-1 mRNA. In addition, flk-1 and flt-1 expression levels decrease in the striatum and hippocampus in later time points following induction of CD8 T cell-mediated BBB disruption. CONCLUSION: This study demonstrates that NRP-1 is a potential therapeutic target in neuro-inflammatory diseases involving BBB disruption and brain hemorrhage. Additionally, the reduction in VEGF receptors subsequent to BBB disruption could be involved in compensatory negative feedback as an attempt to reduce vascular permeability.
Assuntos
Linfócitos T CD8-Positivos/fisiologia , Permeabilidade Capilar/fisiologia , Sistema Nervoso Central/fisiologia , Neuropilina-1/metabolismo , Animais , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Modelos Animais , Neuropilina-1/antagonistas & inibidores , Peptídeos/farmacologia , RNA Mensageiro/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Parkinson's disease (PD) is a debilitating neurodegenerative disorder associated with severe motor impairments caused by the loss of dopaminergic innervation of the striatum. Previous studies have demonstrated that positive allosteric modulators (PAMs) of metabotropic glutamate receptor 4 (mGlu4), including N-phenyl-7-(hydroxyimino) cyclopropa[b]chromen-1a-carboxamide, can produce antiparkinsonian-like effects in preclinical models of PD. However, these early mGlu4 PAMsexhibited unsuitable physiochemical properties for systemic dosing, requiring intracerebroventricular administration and limiting their broader utility as in vivo tools to further understand the role of mGlu4 in the modulation of basal ganglia function relevant to PD. In the present study, we describe the pharmacologic characterization of a systemically active mGlu4 PAM, N-(3-chlorophenyl)picolinamide (VU0364770), in several rodent PD models. VU0364770 showed efficacy alone or when administered in combination with L-DOPA or an adenosine 2A (A2A) receptor antagonist currently in clinical development (preladenant). When administered alone, VU0364770 exhibited efficacy in reversing haloperidol-induced catalepsy, forelimb asymmetry-induced by unilateral 6-hydroxydopamine (6-OHDA) lesions of the median forebrain bundle, and attentional deficits induced by bilateral 6-OHDA nigrostriatal lesions in rats. In addition, VU0364770 enhanced the efficacy of preladenant to reverse haloperidol-induced catalepsy when given in combination. The effects of VU0364770 to reverse forelimb asymmetry were also potentiated when the compound was coadministered with an inactive dose of L-DOPA, suggesting that mGlu4 PAMs may provide L-DOPA-sparing activity. The present findings provide exciting support for the potential role of selective mGlu4 PAMs as a novel approach for the symptomatic treatment of PD and a possible augmentation strategy with either L-DOPA or A2A antagonists.
Assuntos
Antagonistas do Receptor A2 de Adenosina/uso terapêutico , Levodopa/uso terapêutico , Doença de Parkinson/tratamento farmacológico , Ácidos Picolínicos/uso terapêutico , Receptores de Glutamato Metabotrópico/agonistas , Ácido 3,4-Di-Hidroxifenilacético/metabolismo , Antagonistas do Receptor A2 de Adenosina/sangue , Antagonistas do Receptor A2 de Adenosina/metabolismo , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/patologia , Encéfalo/fisiopatologia , Sinalização do Cálcio/efeitos dos fármacos , Catalepsia/induzido quimicamente , Catalepsia/tratamento farmacológico , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/metabolismo , Corpo Estriado/patologia , Corpo Estriado/fisiopatologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Quimioterapia Combinada , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/metabolismo , Ácido Glutâmico/farmacologia , Células HEK293 , Haloperidol/farmacologia , Humanos , Levodopa/metabolismo , Masculino , Monoaminoxidase/metabolismo , Doença dos Neurônios Motores/induzido quimicamente , Doença dos Neurônios Motores/tratamento farmacológico , Doença dos Neurônios Motores/metabolismo , Doença dos Neurônios Motores/patologia , Doença dos Neurônios Motores/fisiopatologia , Oxidopamina/farmacologia , Ácidos Picolínicos/sangue , Ácidos Picolínicos/metabolismo , Ácidos Picolínicos/farmacocinética , Ácidos Picolínicos/farmacologia , Ligação Proteica , Desempenho Psicomotor/efeitos dos fármacos , Pirimidinas/sangue , Pirimidinas/metabolismo , Pirimidinas/uso terapêutico , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Tempo de Reação/efeitos dos fármacos , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Glutamato Metabotrópico/genéticaRESUMO
Dysregulation of the blood-brain barrier (BBB) is a hallmark feature of numerous neurologic disorders as diverse as multiple sclerosis, stroke, epilepsy, viral hemorrhagic fevers, cerebral malaria, and acute hemorrhagic leukoencephalitis. CD8 T cells are one immune cell type that have been implicated in promoting vascular permeability in these conditions. Our laboratory has created a murine model of CD8 T cell-mediated CNS vascular permeability using a variation of the Theiler's murine encephalomyelitis virus system traditionally used to study multiple sclerosis. Previously, we demonstrated that CD8 T cells have the capacity to initiate astrocyte activation, cerebral endothelial cell tight junction protein alterations and CNS vascular permeability through a perforin-dependent process. To address the downstream mechanism by which CD8 T cells promote BBB dysregulation, in this study, we assess the role of vascular endothelial growth factor (VEGF) expression in this model. We demonstrate that neuronal expression of VEGF is significantly upregulated prior to, and coinciding with, CNS vascular permeability. Phosphorylation of fetal liver kinase-1 is significantly increased early in this process indicating activation of this receptor. Specific inhibition of neuropilin-1 significantly reduced CNS vascular permeability and fetal liver kinase-1 activation, and preserved levels of the cerebral endothelial cell tight junction protein occludin. Our data demonstrate that CD8 T cells initiate neuronal expression of VEGF in the CNS under neuroinflammatory conditions, and that VEGF may be a viable therapeutic target in neurologic disease characterized by inflammation-induced BBB disruption.
Assuntos
Linfócitos T CD8-Positivos/fisiologia , Permeabilidade Capilar , Sistema Nervoso Central/irrigação sanguínea , Regulação da Expressão Gênica/imunologia , Inflamação , Fator A de Crescimento do Endotélio Vascular/genética , Animais , Barreira Hematoencefálica/metabolismo , Sistema Nervoso Central/patologia , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/metabolismo , Fosforilação , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
In the course of optimizing an M1 PAM chemotype, introduction of an ether moiety unexpectedly abolished M1 PAM activity while engendering a "molecular switch" to afford a weak, pure mGlu5 PAM. Further optimization was able to deliver a potent (mGlu5 EC50 = 520 nM, 63% Glu Max), centrally penetrant (Kp = 0.83), MPEP-site binding mGlu5 PAM 17a (VU6036486) that reversed amphetamine-induced hyperlocomotion. A pronounced "magic methyl" effect was noted with a regioisomeric methyl congener, leading to a change in pharmacology to afford a potent (mGlu5 IC50 = 110 nM, 3% Glu Min), centrally penetrant (Kp = 0.94), MPEP-site binding NAM 28d (VU6044766) that displayed anxiolytic activity in a mouse marble burying assay. These data further support the growing body of literature concerning the existence of G protein-coupled receptor (GPCR) allosteric privileged structures, and the value and impact of subtle methyl group walks, as well as the highly productive fluorine walk, around allosteric ligand cores to stabilize unique GPCR conformations.
RESUMO
Nonselective antagonists of muscarinic acetylcholine receptors (mAChRs) that broadly inhibit all five mAChR subtypes provide an efficacious treatment for some movement disorders, including Parkinson's disease and dystonia. Despite their efficacy in these and other central nervous system disorders, antimuscarinic therapy has limited utility due to severe adverse effects that often limit their tolerability by patients. Recent advances in understanding the roles that each mAChR subtype plays in disease pathology suggest that highly selective ligands for individual subtypes may underlie the antiparkinsonian and antidystonic efficacy observed with the use of nonselective antimuscarinic therapeutics. Our recent work has indicated that the M4 muscarinic acetylcholine receptor has several important roles in opposing aberrant neurotransmitter release, intracellular signaling pathways, and brain circuits associated with movement disorders. This raises the possibility that selective antagonists of M4 may recapitulate the efficacy of nonselective antimuscarinic therapeutics and may decrease or eliminate the adverse effects associated with these drugs. However, this has not been directly tested due to lack of selective antagonists of M4. Here, we utilize genetic mAChR knockout animals in combination with nonselective mAChR antagonists to confirm that the M4 receptor activation is required for the locomotor-stimulating and antiparkinsonian efficacy in rodent models. We also report the synthesis, discovery, and characterization of the first-in-class selective M4 antagonists VU6013720, VU6021302, and VU6021625 and confirm that these optimized compounds have antiparkinsonian and antidystonic efficacy in pharmacological and genetic models of movement disorders.
RESUMO
Herein, we report the SAR leading to the discovery of VU6028418, a potent M4 mAChR antagonist with high subtype-selectivity and attractive DMPK properties in vitro and in vivo across multiple species. VU6028418 was subsequently evaluated as a preclinical candidate for the treatment of dystonia and other movement disorders. During the characterization of VU6028418, a novel use of deuterium incorporation as a means to modulate CYP inhibition was also discovered.
RESUMO
This Letter describes the chemical optimization of a new series of muscarinic acetylcholine receptor subtype 1 (M1) positive allosteric modulators (PAMs) based on novel tricyclic triazolo- and imidazopyridine lactam cores, devoid of M1 agonism, e.g., no M1 ago-PAM activity, in high expressing recombinant cell lines. While all the new tricyclic congeners afforded excellent rat pharmacokinetic (PK) properties (CLp < 8 mL/min/kg and t1/2 > 5 h), regioisomeric triazolopyridine analogues were uniformly not CNS penetrant ( Kp < 0.05), despite a lack of hydrogen bond donors. However, removal of a single nitrogen atom to afford imidazopyridine derivatives proved to retain the excellent rat PK and provide high CNS penetration ( Kp > 2), despite inclusion of a basic nitrogen. Moreover, 24c was devoid of M1 agonism in high expressing recombinant cell lines and did not induce cholinergic seizures in vivo in mice. Interestingly, all of the new M1 PAMs across the diverse tricyclic heterocyclic cores possessed equivalent CNS MPO scores (>4.5), highlighting the value of both "medicinal chemist's eye" and experimental data, e.g., not sole reliance (or decision bias) on in silico calculated properties, for parameters as complex as CNS penetration.
Assuntos
Descoberta de Drogas/métodos , Imidazóis/farmacologia , Lactamas/farmacologia , Agonistas Muscarínicos/farmacologia , Piridinas/farmacologia , Receptor Muscarínico M1/agonistas , Regulação Alostérica/efeitos dos fármacos , Regulação Alostérica/fisiologia , Animais , Descoberta de Drogas/tendências , Humanos , Imidazóis/química , Lactamas/química , Camundongos , Agonistas Muscarínicos/química , Piridinas/química , Ratos , Receptor Muscarínico M1/fisiologiaRESUMO
Herein, we report the chemical optimization of a new series of M1 positive allosteric modulators (PAMs) based on a novel pyrrolo[2,3-b]pyridine core, developed via scaffold hopping and iterative parallel synthesis. The vast majority of analogs in this series proved to display robust cholinergic seizure activity. However, by removal of the secondary hydroxyl group, VU6007477 resulted with good rat M1 PAM potency (EC50 = 230 nM, 93% ACh max), minimal M1 agonist activity (agonist EC50 > 10 µM), good CNS penetration (rat brain/plasma K p = 0.28, K p,uu = 0.32; mouse K p = 0.16, K p,uu = 0.18), and no cholinergic adverse events (AEs, e.g., seizures). This work demonstrates that within a chemical series prone to robust M1 ago-PAM activity, SAR can result, which affords pure M1 PAMs, devoid of cholinergic toxicity/seizure liability.
RESUMO
Highly selective positive allosteric modulators (PAMs) of the M1 subtype of muscarinic acetylcholine receptor have emerged as an exciting new approach for improving cognitive function in patients suffering from Alzheimer's disease and schizophrenia. However, excessive activation of M1 is known to induce seizure activity and have actions in the prefrontal cortex (PFC) that could impair cognitive function. We now report a series of pharmacological, electrophysiological, and behavioral studies in which we find that recently reported M1 PAMs, PF-06764427 and MK-7622, have robust agonist activity in cell lines and agonist effects in the mouse PFC, and have the potential to overactivate the M1 receptor and disrupt PFC function. In contrast, structurally distinct M1 PAMs (VU0453595 and VU0550164) are devoid of agonist activity in cell lines and maintain activity dependence of M1 activation in the PFC. Consistent with the previously reported effect of PF-06764427, the ago-PAM MK-7622 induces severe behavioral convulsions in mice. In contrast, VU0453595 does not induce behavioral convulsions at doses well above those required for maximal efficacy in enhancing cognitive function. Furthermore, in contrast to the robust efficacy of VU0453595, the ago-PAM MK-7622 failed to improve novel object recognition, a rodent assay of cognitive function. These findings suggest that in vivo cognition-enhancing efficacy of M1 PAMs can be observed with PAMs lacking intrinsic agonist activity and that intrinsic agonist activity of M1 PAMs may contribute to adverse effects and reduced efficacy in improving cognitive function.
Assuntos
Colinérgicos/farmacologia , Nootrópicos/farmacologia , Receptor Muscarínico M1/metabolismo , Regulação Alostérica , Animais , Células CHO , Cricetulus , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/fisiologia , Masculino , Camundongos Endogâmicos C57BL , Córtex Pré-Frontal/efeitos dos fármacos , Córtex Pré-Frontal/fisiologia , Células Piramidais/efeitos dos fármacos , Células Piramidais/fisiologia , Ratos , Ratos Sprague-Dawley , Receptor Muscarínico M1/agonistas , Receptor Muscarínico M1/genética , Reconhecimento Psicológico/efeitos dos fármacos , Técnicas de Cultura de TecidosRESUMO
Selective activation of the M1 subtype of muscarinic acetylcholine receptor, via positive allosteric modulation (PAM), is an exciting strategy to improve cognition in schizophrenia and Alzheimer's disease patients. However, highly potent M1 ago-PAMs, such as MK-7622, PF-06764427, and PF-06827443, can engender excessive activation of M1, leading to agonist actions in the prefrontal cortex (PFC) that impair cognitive function, induce behavioral convulsions, and result in other classic cholinergic adverse events (AEs). Here, we report a fundamentally new and highly selective M1 PAM, VU0486846. VU0486846 possesses only weak agonist activity in M1-expressing cell lines with high receptor reserve and is devoid of agonist actions in the PFC, unlike previously reported ago-PAMs MK-7622, PF-06764427, and PF-06827443. Moreover, VU0486846 shows no interaction with antagonist binding at the orthosteric acetylcholine (ACh) site (e.g., neither bitopic nor displaying negative cooperativity with [3H]-NMS binding at the orthosteric site), no seizure liability at high brain exposures, and no cholinergic AEs. However, as opposed to ago-PAMs, VU0486846 produces robust efficacy in the novel object recognition model of cognitive function. Importantly, we show for the first time that an M1 PAM can reverse the cognitive deficits induced by atypical antipsychotics, such as risperidone. These findings further strengthen the argument that compounds with modest in vitro M1 PAM activity (EC50 > 100 nM) and pure-PAM activity in native tissues display robust procognitive efficacy without AEs mediated by excessive activation of M1. Overall, the combination of compound assessment with recombinant in vitro assays (mindful of receptor reserve), native tissue systems (PFC), and phenotypic screens (behavioral convulsions) is essential to fully understand and evaluate lead compounds and enhance success in clinical development.
Assuntos
Cognição/efeitos dos fármacos , Condicionamento Psicológico/efeitos dos fármacos , Comportamento Exploratório/efeitos dos fármacos , Morfolinas/farmacologia , Córtex Pré-Frontal/efeitos dos fármacos , Pirazóis/farmacologia , Regulação Alostérica , Animais , Antipsicóticos/toxicidade , Células CHO , Disfunção Cognitiva/induzido quimicamente , Disfunção Cognitiva/fisiopatologia , Cricetulus , Medo , Camundongos , Morfolinas/toxicidade , Pirazóis/toxicidade , Ratos , Risperidona/toxicidade , Convulsões/induzido quimicamenteRESUMO
The dorsolateral striatum is critically involved in movement control and motor learning. Striatal function is regulated by a variety of neuromodulators including acetylcholine. Previous studies have shown that cholinergic activation excites striatal principal projection neurons, medium spiny neurons (MSNs), and this action is mediated by muscarinic acetylcholine subtype 1 receptors (M1) through modulating multiple potassium channels. In the present study, we used electrophysiology techniques in conjunction with optogenetic and pharmacological tools to determine the long-term effects of striatal cholinergic activation on MSN intrinsic excitability. A transient increase in acetylcholine release in the striatum by optogenetic stimulation resulted in a long-lasting increase in excitability of MSNs, which was associated with hyperpolarizing shift of action potential threshold and decrease in afterhyperpolarization (AHP) amplitude, leading to an increase in probability of EPSP-action potential coupling. The M1 selective antagonist VU0255035 prevented, while the M1 selective positive allosteric modulator (PAM) VU0453595 potentiated the cholinergic activation-induced persistent increase in MSN intrinsic excitability, suggesting that M1 receptors are critically involved in the induction of this long-lasting response. This M1 receptor-dependent long-lasting change in MSN intrinsic excitability could have significant impact on striatal processing and might provide a novel mechanism underlying cholinergic regulation of the striatum-dependent motor learning and cognitive function. Consistent with this, behavioral studies indicate that potentiation of M1 receptor signaling by VU0453595 enhanced performance of mice in cue-dependent water-based T-maze, a dorsolateral striatum-dependent learning task.
Assuntos
Corpo Estriado/citologia , Potenciais Pós-Sinápticos Excitadores/fisiologia , Neurônios/fisiologia , Receptor Muscarínico M1/metabolismo , Acetilcolina/farmacologia , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/genética , Animais , Channelrhodopsins , Colina O-Acetiltransferase/genética , Colina O-Acetiltransferase/metabolismo , Colinérgicos/farmacologia , Sinais (Psicologia) , Antagonistas de Aminoácidos Excitatórios/farmacologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Aprendizagem/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Atividade Motora/efeitos dos fármacos , Atividade Motora/genética , Neurônios/efeitos dos fármacos , Estimulação LuminosaRESUMO
Selective potentiation of the mGlu5 subtype of metabotropic glutamate (mGlu) receptor using positive allosteric modulators (PAMs) has robust cognition-enhancing effects in rodent models that are relevant for schizophrenia. Until recently, these effects were thought to be due to potentiation of mGlu5-induced modulation of NMDA receptor (NMDAR) currents and NMDAR-dependent synaptic plasticity. However, "biased" mGlu5 PAMs that do not potentiate mGlu5 effects on NMDAR currents show efficacy that is similar to that of prototypical mGlu5 PAMs, suggesting that NMDAR-independent mechanisms must be involved in these actions. We now report that synaptic activation of mGlu5 is required for a form of long-term depression (mLTD) in mouse prefrontal cortex (PFC) that is induced by activation of M1 muscarinic acetylcholine (mAChR) receptors, which was previously thought to be independent of mGlu5 activation. Interestingly, a biased mGlu5 PAM, VU0409551, that does not potentiate mGlu5 modulation of NMDAR currents, potentiated induction of mLTD. Furthermore, coactivation of mGlu5 and M1 receptors increased GABAA-dependent inhibitory tone in the PFC pyramidal neurons, which likely contributes to the observed mLTD. Finally, systemic administration of the biased mGlu5 PAM reversed deficits in mLTD and associated cognitive deficits in a model of cortical disruption caused by repeated phencyclidine exposure that is relevant for schizophrenia and was previously shown to be responsive to selective M1 muscarinic receptor PAMs. These studies provide exciting new insights into a novel mechanism by which mGlu5 PAMs can reverse deficits in PFC function and cognition that is independent of modulation of NMDAR currents.
Assuntos
Colinérgicos/farmacologia , Córtex Pré-Frontal/efeitos dos fármacos , Receptor de Glutamato Metabotrópico 5/metabolismo , Esquizofrenia/tratamento farmacológico , Transmissão Sináptica/efeitos dos fármacos , Animais , Antipsicóticos/farmacologia , Cognição/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Plasticidade Neuronal/efeitos dos fármacos , Fenciclidina/farmacologia , Córtex Pré-Frontal/metabolismo , Receptores de N-Metil-D-Aspartato/efeitos dos fármacos , Receptores de N-Metil-D-Aspartato/metabolismoRESUMO
Activation of ß-adrenergic receptors (ßARs) enhances both the induction of long-term potentiation (LTP) in hippocampal CA1 pyramidal cells and hippocampal-dependent cognitive function. Interestingly, previous studies reveal that coincident activation of group II metabotropic glutamate (mGlu) receptors with ßARs in the hippocampal astrocytes induces a large increase in cyclic-AMP (cAMP) accumulation and release of adenosine. Adenosine then acts on A1 adenosine receptors at neighboring excitatory Schaffer collateral terminals, which could counteract effects of activation of neuronal ßARs on excitatory transmission. On the basis of this, we postulated that activation of the specific mGlu receptor subtype that mediates this response could inhibit ßAR-mediated effects on hippocampal synaptic plasticity and cognitive function. Using novel mGlu receptor subtype-selective allosteric modulators along with knockout mice we now report that the effects of mGlu2/3 agonists on ßAR-mediated increases in cAMP accumulation are exclusively mediated by mGlu3. Furthermore, mGlu3 activation inhibits the ability of the ßAR agonist isoproterenol to enhance hippocampal LTP, and this effect is absent in slices treated with either a glial toxin or an adenosine A1 receptor antagonist. Finally, systemic administration of the mGlu2/3 agonist LY379268 disrupted contextual fear memory in a manner similar to the effect of the ßAR antagonist propranolol, and this effect was reversed by the mGlu3-negative allosteric modulator VU0650786. Taken together, these data suggest that mGlu3 can influence astrocytic signaling and modulate ßAR-mediated effects on hippocampal synaptic plasticity and cognitive function.
Assuntos
AMP Cíclico/metabolismo , Potenciação de Longa Duração/fisiologia , Consolidação da Memória/fisiologia , Receptores Adrenérgicos beta/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Animais , Córtex Cerebral/citologia , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Condicionamento Psicológico/efeitos dos fármacos , Condicionamento Psicológico/fisiologia , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Potenciação de Longa Duração/efeitos dos fármacos , Masculino , Consolidação da Memória/efeitos dos fármacos , Camundongos Endogâmicos ICR , Camundongos Knockout , Neurotransmissores/farmacologia , Ratos Sprague-Dawley , Receptores de Glutamato Metabotrópico/genética , Técnicas de Cultura de TecidosRESUMO
Both historical clinical and recent preclinical data suggest that the M1 muscarinic acetylcholine receptor is an exciting target for the treatment of Alzheimer's disease and the cognitive and negative symptom clusters in schizophrenia; however, early drug discovery efforts targeting the orthosteric binding site have failed to afford selective M1 activation. Efforts then shifted to focus on selective activation of M1 via either allosteric agonists or positive allosteric modulators (PAMs). While M1 PAMs have robust efficacy in rodent models, some chemotypes can induce cholinergic adverse effects (AEs) that could limit their clinical utility. Here, we report studies aimed at understanding the subtle structural and pharmacological nuances that differentiate efficacy from adverse effect liability within an indole-based series of M1 ago-PAMs. Our data demonstrate that closely related M1 PAMs can display striking differences in their in vivo activities, especially their propensities to induce adverse effects. We report the discovery of a novel PAM in this series that is devoid of observable adverse effect liability. Interestingly, the molecular pharmacology profile of this novel PAM is similar to that of a representative M1 PAM that induces severe AEs. For instance, both compounds are potent ago-PAMs that demonstrate significant interaction with the orthosteric site (either bitopic or negative cooperativity). However, there are subtle differences in efficacies of the compounds at potentiating M1 responses, agonist potencies, and abilities to induce receptor internalization. While these differences may contribute to the differential in vivo profiles of these compounds, the in vitro differences are relatively subtle and highlight the complexities of allosteric modulators and the need to focus on in vivo phenotypic screening to identify safe and effective M1 PAMs.
Assuntos
Regulação Alostérica/efeitos dos fármacos , Descoberta de Drogas , Agonistas Muscarínicos/química , Agonistas Muscarínicos/farmacologia , Receptor Muscarínico M1/efeitos dos fármacos , Animais , Humanos , Camundongos , Agonistas Muscarínicos/síntese química , Ratos , Receptor Muscarínico M1/metabolismo , Relação Estrutura-AtividadeRESUMO
Schizophrenia is associated with disruptions in N-methyl-D-aspartate glutamate receptor subtype (NMDAR)-mediated excitatory synaptic signaling. The metabotropic glutamate receptor subtype 5 (mGlu5) is a closely associated signaling partner with NMDARs and regulates NMDAR function in forebrain regions implicated in the pathology of schizophrenia. Efficacy of mGlu5 positive allosteric modulators (PAMs) in animal models of psychosis and cognition was previously attributed to potentiation of NMDAR function. To directly test this hypothesis, we identified VU0409551 as a novel mGlu5 PAM that exhibits distinct stimulus bias and selectively potentiates mGlu5 coupling to Gαq-mediated signaling but not mGlu5 modulation of NMDAR currents or NMDAR-dependent synaptic plasticity in the rat hippocampus. Interestingly, VU0409551 produced robust antipsychotic-like and cognition-enhancing activity in animal models. These data provide surprising new mechanistic insights into the actions of mGlu5 PAMs and suggest that modulation of NMDAR currents is not critical for in vivo efficacy. VIDEO ABSTRACT.