RESUMO
The lysosomal cation channel TMEM175 is a Parkinson's disease-related protein and a promising drug target. Unlike whole-cell automated patch-clamp (APC), lysosomal patch-clamp (LPC) facilitates physiological conditions, but is not yet suitable for high-throughput screening (HTS) applications. Here, we apply solid supported membrane-based electrophysiology (SSME), which enables both direct access to lysosomes and high-throughput electrophysiological recordings. In SSME, ion translocation mediated by TMEM175 is stimulated using a concentration gradient at a resting potential of 0 mV. The concentration-dependent K+ response exhibited an I/c curve with two distinct slopes, indicating the existence of two conducting states. We measured H+ fluxes with a permeability ratio of PH/PK = 48,500, which matches literature findings from patch-clamp studies, validating the SSME approach. Additionally, TMEM175 displayed a high pH dependence. Decreasing cytosolic pH inhibited both K+ and H+ conductivity of TMEM175. Conversely, lysosomal pH and pH gradients did not have major effects on TMEM175. Finally, we developed HTS assays for drug screening and evaluated tool compounds (4-AP, Zn as inhibitors; DCPIB, arachidonic acid, SC-79 as enhancers) using SSME and APC. Additionally, we recorded EC50 data for eight blinded TMEM175 enhancers and compared the results across all three assay technologies, including LPC, discussing their advantages and disadvantages.
Assuntos
Eletrofisiologia Cardíaca , Ensaios de Triagem em Larga Escala , Potenciais da Membrana , Cátions , LisossomosRESUMO
Historically, pharmacological therapies have used mechanisms such as γ-aminobutyric acid A (GABAA) receptor potentiation to drive sleep through broad suppression of central nervous system activity. With the discovery of orexin signaling loss as the etiology underlying narcolepsy, a disorder associated with hypersomnolence, orexin antagonism emerged as an alternative approach to attenuate orexin-induced wakefulness more selectively. Dual orexin receptor antagonists (DORAs) block the activity of orexin 1 and 2 receptors to both reduce the threshold to transition into sleep and attenuate orexin-mediated arousal. Among DORAs evaluated clinically, suvorexant has pharmacokinetic properties engineered for a plasma half-life appropriate for rapid sleep onset and maintenance at low to moderate doses. Unlike GABAA receptor modulators, DORAs promote both non-rapid eye movement (NREM) and REM sleep, do not disrupt sleep stage-specific quantitative electroencephalogram spectral profiles, and allow somnolence indistinct from normal sleep. The preservation of cognitive performance and the ability to arouse to salient stimuli after DORA administration suggest further advantages over historical therapies.
Assuntos
Azepinas/uso terapêutico , Descoberta de Drogas/métodos , Antagonistas dos Receptores de Orexina/uso terapêutico , Receptores de Orexina/fisiologia , Medicamentos Indutores do Sono/uso terapêutico , Distúrbios do Início e da Manutenção do Sono/tratamento farmacológico , Triazóis/uso terapêutico , Animais , Azepinas/química , Azepinas/farmacologia , Descoberta de Drogas/tendências , Humanos , Antagonistas dos Receptores de Orexina/química , Antagonistas dos Receptores de Orexina/farmacologia , Receptores de Orexina/química , Estrutura Secundária de Proteína , Medicamentos Indutores do Sono/química , Medicamentos Indutores do Sono/farmacologia , Distúrbios do Início e da Manutenção do Sono/metabolismo , Triazóis/química , Triazóis/farmacologiaRESUMO
Chronic insomnia is defined as a persistent difficulty with sleep initiation maintenance or non-restorative sleep. The therapeutic standard of care for this condition is treatment with gamma-aminobutyric acid (GABA)A receptor modulators, which promote sleep but are associated with a panoply of side effects, including cognitive and memory impairment. Dual orexin receptor antagonists (DORAs) have recently emerged as an alternative therapeutic approach that acts via a distinct and more selective wake-attenuating mechanism with the potential to be associated with milder side effects. Given their distinct mechanism of action, the current work tested the hypothesis that DORAs and GABAA receptor modulators differentially regulate neurochemical pathways associated with differences in sleep architecture and cognitive performance induced by these pharmacological mechanisms. Our findings showed that DORA-22 suppresses the release of the wake neurotransmitter histamine in the lateral hypothalamus, prefrontal cortex, and hippocampus with no significant alterations in acetylcholine levels. In contrast, eszopiclone, commonly used as a GABAA modulator, inhibited acetylcholine secretion across brain regions with variable effects on histamine release depending on the extent of wakefulness induction. In normal waking rats, eszopiclone only transiently suppressed histamine secretion, whereas this suppression was more obvious under caffeine-induced wakefulness. Compared with the GABAA modulator eszopiclone, DORA-22 elicits a neurotransmitter profile consistent with wake reduction that does not impinge on neurotransmitter levels associated with cognition and rapid eye movement sleep.
Assuntos
Acetilcolina/metabolismo , Hipocampo/efeitos dos fármacos , Histamina/metabolismo , Região Hipotalâmica Lateral/efeitos dos fármacos , Antagonistas dos Receptores de Orexina/farmacologia , Piperidinas/farmacologia , Córtex Pré-Frontal/efeitos dos fármacos , Triazóis/farmacologia , Animais , Hipocampo/metabolismo , Masculino , Córtex Pré-Frontal/metabolismo , Ratos , Sono/efeitos dos fármacos , Sono/fisiologia , Distúrbios do Início e da Manutenção do Sono/fisiopatologia , Vigília/efeitos dos fármacos , Ácido gama-Aminobutírico/farmacologiaRESUMO
In an ongoing effort to explore the use of orexin receptor antagonists for the treatment of insomnia, dual orexin receptor antagonists (DORAs) were structurally modified, resulting in compounds selective for the OX2R subtype and culminating in the discovery of 23, a highly potent, OX2R-selective molecule that exhibited a promising in vivo profile. Further structural modification led to an unexpected restoration of OX1R antagonism. Herein, these changes are discussed and a rationale for selectivity based on computational modeling is proposed.
Assuntos
Antagonistas dos Receptores de Orexina/farmacologia , Orexinas/antagonistas & inibidores , Animais , Eletroencefalografia , Eletromiografia , Estrutura Molecular , Antagonistas dos Receptores de Orexina/química , RatosRESUMO
Neuronal inclusions of hyperphosphorylated and aggregated tau protein are a pathological hallmark of several neurodegenerative tauopathies, including Alzheimer's disease (AD). The hypothesis of tau transmission in AD has emerged from histopathological studies of the spatial and temporal progression of tau pathology in postmortem patient brains. Increasing evidence in cellular and animal models supports the phenomenon of intercellular spreading of tau. However, the molecular and cellular mechanisms of pathogenic tau transmission remain unknown. The studies described herein investigate tau pathology propagation using human neurons derived from induced pluripotent stem cells. Neurons were seeded with full-length human tau monomers and oligomers and chronic effects on neuronal viability and function were examined over time. Tau oligomer-treated neurons exhibited an increase in aggregated and phosphorylated pathological tau. These effects were associated with neurite retraction, loss of synapses, aberrant calcium homeostasis, and imbalanced neurotransmitter release. In contrast, tau monomer treatment did not produce any measureable changes. This work supports the hypothesis that tau oligomers are toxic species that can drive the spread of tau pathology and neurodegeneration. SIGNIFICANCE STATEMENT: Several independent studies have implicated tau protein as central to Alzheimer's disease progression and cell-to-cell pathology propagation. In this study, we investigated the ability of different tau species to propagate pathology in human neurons derived from induced pluripotent stem cells, which to date has not been shown. We demonstrated that tau oligomers, but not monomers, induce accumulation of pathological, hyperphosphorylated tau. This effect was accompanied with neurite degeneration, loss of synapses, aberrant calcium homeostasis, imbalanced neurotransmitter release, and ultimately with neuronal death. This study bridges various tau pathological phenotypes into a single and relevant induced pluripotent stem cell neuronal model of human disease that can be applied to the discovery of the mechanisms of tau-induced neurodegeneration.
Assuntos
Células-Tronco Pluripotentes Induzidas/fisiologia , Degeneração Neural/metabolismo , Degeneração Neural/patologia , Neurônios/metabolismo , Proteínas tau/metabolismo , Proteínas tau/toxicidade , Análise de Variância , Cálcio/metabolismo , Sobrevivência Celular , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Humanos , Masculino , Microfluídica , Microscopia de Força Atômica , Neurotransmissores/metabolismo , Fosforilação , Transporte Proteico/fisiologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Proteínas tau/químicaRESUMO
Orexin signaling, known to modulate arousal and vigilance, is also involved in nociception as orexin neurons project to regions of the brain and spinal cord involved in pain processing, and the administration of orexin peptides can alter pain response in a wide range of preclinical models. Pharmacological treatment with the potent, selective and structurally distinct dual orexin receptor antagonists (ORAs) DORA-12 and DORA-2 significantly reduced pain responses during both phases I and II of the mouse formalin pain model and significantly reversed hyperalgesia in the rat complete Freund's adjuvant pain model, respectively. Significant antinociceptive effects of DORA-12 in the formalin model were also observed in orexin 1 receptor (OX1R) knockout mice, but not orexin 2 receptor (OX2R) or OX1R/OX2R double knockout mice. Mechanical hypersensitivity was significantly reduced with a series of structurally distinct, potent and highly selective ORAs (DORA-2, DORA-12 and DORA-22) in the rat spinal nerve ligation (SNL) injury model of neuropathic pain. Selective pharmacological targeting of OX2R with 2-SORA-7 also reduced pain responses in acute inflammatory (complete Freund's adjuvant) and neuropathic (SNL) rat pain models. Performance on the rotarod test of psychomotor performance and baseline thermal sensitivity were not affected in OX1R/OX2R knockout mice or ORA-treated mice, indicating that the observed pain-reducing effects were not due to sedation or motor deficits. These findings indicate that ORAs have pain-reducing effects across a number of acute and chronic neuropathic preclinical mouse and rat pain models. Further studies on the potential pain-relieving effects of orexin receptor antagonism are warranted.
Assuntos
Analgésicos/farmacologia , Antagonistas dos Receptores de Orexina/farmacologia , Animais , Modelos Animais de Doenças , Hiperalgesia/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neuralgia/fisiopatologia , Ratos , Ratos Sprague-DawleyRESUMO
While a correlation between blockade of the orexin 2 receptor (OX2R) with either a dual orexin receptor antagonist (DORA) or a selective orexin 2 receptor antagonist (2-SORA) and a decrease of wakefulness is well established, less is known about selective blockade of the orexin 1 receptor (OX1R). Therefore, a highly selective orexin 1 antagonist (1-SORA) with suitable properties to allow in vivo interrogation of OX1R specific pharmacology in preclinical species remains an attractive target. Herein, we describe the discovery of an optimized 1-SORA series in the piperidine ether class. Notably, a 4,4-difluoropiperidine core coupled with a 2-quinoline ether linkage provides OX1R selective compounds. The combination with an azabenzimidazole or imidazopyridine amide substituent leads to analogs 47 and 51 with >625-fold functional selectivity for OX1R over OX2R in rat. Compounds 47 and 51 possess clean off-target profiles and the required pharmacokinetic and physical properties to be useful as 1-SORA tool compounds.
Assuntos
Antagonistas dos Receptores de Orexina/química , Antagonistas dos Receptores de Orexina/farmacologia , Receptores de Orexina/metabolismo , Piperidinas/química , Piperidinas/farmacologia , Animais , Descoberta de Drogas , Humanos , Piperidinas/farmacocinética , Ratos , Ratos Transgênicos , Relação Estrutura-AtividadeRESUMO
Familial Parkinson's disease cases have recently been associated with the leucine rich repeat kinase 2 (LRRK2) gene. It has been hypothesized that inhibition of the LRRK2 protein may have the potential to alter disease pathogenesis. A dihydrobenzothiophene series of potent, selective, orally bioavailable LRRK2 inhibitors were identified from a high-throughput screen of the internal Merck sample collection. Initial SAR studies around the core established the series as a tractable small molecule lead series of LRRK2 inhibitors for potential treatment of Parkinson's disease. It was also found that incorporation of a lactam into the core drastically improved the CNS and DMPK properties of these small molecules.
Assuntos
Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Tiofenos/farmacologia , Administração Oral , Disponibilidade Biológica , Relação Dose-Resposta a Droga , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Simulação de Acoplamento Molecular , Estrutura Molecular , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/química , Relação Estrutura-Atividade , Tiofenos/síntese química , Tiofenos/químicaRESUMO
Herein, we present the identification of a novel class of pyrazolopyrimidine phosphodiesterase 10A (PDE10A) inhibitors. Beginning with a lead molecule (1) identified through a fragment-based drug discovery (FBDD) effort, lead optimization was enabled by rational design, X-ray crystallography, metabolic and off-target profiling, and fragment scaffold-hopping. We highlight the discovery of PyP-1, a potent, highly selective, and orally bioavailable pyrazolopyrimidine inhibitor of PDE10A. PyP-1 exhibits sub-nanomolar potency (PDE10A Ki=0.23nM), excellent pharmacokinetic (PK) and physicochemical properties, and a clean off-target profile. It displays dose-dependent efficacy in numerous pharmacodynamic (PD) assays that measure potential for anti-psychotic activity and cognitive improvement. PyP-1 also has a clean preclinical profile with respect to cataleptic potential in rats, prolactin secretion, and weight gain, common adverse events associated with currently marketed therapeutics. Further, PyP-1 displays in vivo preclinical target engagement as measured by PET enzyme occupancy in concert with [(11)C]MK-8193, a novel PDE10A PET tracer.
Assuntos
Descoberta de Drogas , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Inibidores de Fosfodiesterase/farmacologia , Diester Fosfórico Hidrolases/metabolismo , Esquizofrenia/tratamento farmacológico , Animais , Cristalografia por Raios X , Cães , Relação Dose-Resposta a Droga , Compostos Heterocíclicos de 4 ou mais Anéis/síntese química , Compostos Heterocíclicos de 4 ou mais Anéis/química , Humanos , Macaca mulatta , Modelos Moleculares , Estrutura Molecular , Inibidores de Fosfodiesterase/síntese química , Inibidores de Fosfodiesterase/química , Ratos , Ratos Wistar , Esquizofrenia/enzimologia , Relação Estrutura-AtividadeRESUMO
A hallmark of Alzheimer's disease (AD) brain is the amyloid ß (Aß) plaque, which is comprised of Aß peptides. Multiple lines of evidence suggest that Aß oligomers are more toxic than other peptide forms. We sought to develop a robust assay to quantify oligomers from CSF. Antibody 19.3 was compared in one-site and competitive ELISAs for oligomer binding specificity. A two-site ELISA for oligomers was developed using 19.3 coupled to a sensitive, bead-based fluorescent platform able to detect single photons of emitted light. The two-site ELISA was >2500× selective for Aß oligomers over Aß monomers with a limit of detection â¼ 0.09 pg/ml in human CSF. The lower limit of reliable quantification of the assay was 0.18 pg/ml and the antibody pairs recognized Aß multimers comprised of either synthetic standards, or endogenous oligomers isolated from confirmed human AD and healthy control brain. Using the assay, a significant 3- to 5-fold increase in Aß oligomers in human AD CSF compared with comparably aged controls was demonstrated. The increase was seen in three separate human cohorts, totaling 63 AD and 54 controls. CSF oligomers ranged between 0.1 and 10 pg/ml. Aß oligomer levels did not strongly associate with age or gender, but had an inverse correlation with MMSE score. The C statistic for the Aß oligomer ROC curve was 0.86, with 80% sensitivity and 88% specificity to detect AD, suggesting reasonable discriminatory power for the AD state and the potential for utility as a diagnostic marker.
Assuntos
Envelhecimento/líquido cefalorraquidiano , Doença de Alzheimer/líquido cefalorraquidiano , Doença de Alzheimer/diagnóstico , Peptídeos beta-Amiloides/líquido cefalorraquidiano , Adulto , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/psicologia , Peptídeos beta-Amiloides/imunologia , Anticorpos/imunologia , Especificidade de Anticorpos , Biomarcadores/líquido cefalorraquidiano , Ensaio de Imunoadsorção Enzimática , Reações Falso-Positivas , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Testes Neuropsicológicos , Fragmentos de Peptídeos/líquido cefalorraquidiano , Curva ROC , Reprodutibilidade dos Testes , Espalhamento de RadiaçãoRESUMO
BACKGROUND: The neuropeptides orexin A and B play a role in reward and feeding and are critical for arousal. However, it was not initially appreciated that most prepro-orexin synthesizing neurons are almost exclusively concentrated in the perifornical hypothalamus, which when stimulated elicits panic-associated behavior and cardiovascular responses in rodents and self-reported "panic attacks" and "fear of dying" in humans. More recent studies support a role for the orexin system in coordinating an integrative stress response. For instance, orexin neurons are highly reactive to anxiogenic stimuli, are hyperactive in anxiety pathology, and have strong projections to anxiety and panic-associated circuitry. Although the two cognate orexin receptors are colocalized in many brain regions, the orexin 2 receptor (OX2R) most robustly maps to the histaminergic wake-promoting region, while the orexin 1 receptor (OX1R) distribution is more exclusive and dense in anxiety and panic circuitry regions, such as the locus ceruleus. Overall, this suggests that OX1Rs play a critical role in mobilizing anxiety and panic responses. METHODS: Here, we used a CO2 -panic provocation model to screen a dual OX1/2R antagonist (DORA-12) to globally inhibit orexin activity, then a highly selective OX1R antagonist (SORA1, Compound 56) or OX2R antagonist (SORA2, JnJ10397049) to assess OX1R and OX2R involvement. RESULTS: All compounds except the SORA2 attenuated CO2 -induced anxiety-like behaviors, and all but the SORA2 and DORA attenuated CO2 -induced cardiovascular responses. CONCLUSIONS: SORA1s may represent a novel method of treating anxiety disorders, with no apparent sedative effects that were present with a benzodiazepine.
Assuntos
Sistema Nervoso Autônomo/fisiopatologia , Encéfalo/metabolismo , Neurônios , Receptores de Orexina/metabolismo , Transtorno de Pânico/metabolismo , Transtorno de Pânico/psicologia , Ansiedade/metabolismo , Ansiedade/fisiopatologia , Ansiedade/psicologia , Nível de Alerta , Dióxido de Carbono , Sistema Cardiovascular/efeitos dos fármacos , Humanos , Neurônios/metabolismo , Pânico , Transtorno de Pânico/induzido quimicamente , Transtorno de Pânico/fisiopatologia , RecompensaRESUMO
Highly selective orexin receptor antagonists (SORAs) of the orexin 2 receptor (OX2R) have become attractive targets both as potential therapeutics for insomnia as well as biological tools to help further elucidate the underlying pharmacology of the orexin signaling pathway. Herein, we describe the discovery of a novel piperidine ether 2-SORA class identified by systematic lead optimization beginning with filorexant, a dual orexin receptor antagonist (DORA) that recently completed Phase 2 clinical trials. Changes to the ether linkage and pendant heterocycle of filorexant were found to impart significant selectivity for OX2R, culminating in lead compound PE-6. PE-6 displays sub-nanomolar binding affinity and functional potency on OX2R while maintaining >1600-fold binding selectivity and >200-fold functional selectivity versus the orexin 1 receptor (OX1R). PE-6 bears a clean off-target profile, a good overall preclinical pharmacokinetic (PK) profile, and reduces wakefulness with increased NREM and REM sleep when evaluated in vivo in a rat sleep study. Importantly, subtle structural changes to the piperidine ether class impart dramatic changes in receptor selectivity. To this end, our laboratories have identified multiple piperidine ether 2-SORAs, 1-SORAs, and DORAs, providing access to a number of important biological tool compounds from a single structural class.
Assuntos
Éteres/química , Antagonistas dos Receptores de Orexina , Piperidinas/química , Pirimidinas/química , Animais , Cães , Avaliação Pré-Clínica de Medicamentos , Éteres/síntese química , Éteres/farmacocinética , Meia-Vida , Humanos , Receptores de Orexina/metabolismo , Piperidinas/metabolismo , Ligação Proteica , Pirimidinas/metabolismo , Ratos , Sono/efeitos dos fármacos , Relação Estrutura-AtividadeRESUMO
Dual orexin receptor antagonists (DORAs), or orexin 1 (OX1) and orexin 2 (OX2) receptor antagonists, have demonstrated clinical utility for the treatment of insomnia. Medicinal chemistry efforts focused on the reduction of bioactivation potential of diazepane amide 1 through the modification of the Western heterocycle resulted in the discovery of suvorexant, a DORA recently approved by the FDA for the treatment of insomnia. A second strategy towards reducing bioactivation risk is presented herein through the exploration of monocyclic quinazoline isosteres, namely substituted pyrimidines. These studies afforded potent DORAs with significantly reduced bioactivation risk and efficacy in rodent sleep models. Surprisingly, side products from the chemistry used to produce these DORAs yielded isomeric pyrimidine-containing diazepane amides possessing selective OX2R antagonist (2-SORA) profiles. Additional exploration of these isomeric pyrimidines uncovered potent 2-SORA diazepane amides with sleep efficacy in mouse EEG studies.
Assuntos
Descoberta de Drogas , Antagonistas dos Receptores de Orexina/farmacologia , Receptores de Orexina/metabolismo , Pirimidinas/farmacologia , Quinazolinas/farmacologia , Distúrbios do Início e da Manutenção do Sono/tratamento farmacológico , Animais , Modelos Animais de Doenças , Cães , Relação Dose-Resposta a Droga , Humanos , Camundongos , Microssomos Hepáticos/efeitos dos fármacos , Modelos Moleculares , Estrutura Molecular , Antagonistas dos Receptores de Orexina/síntese química , Antagonistas dos Receptores de Orexina/química , Pirimidinas/síntese química , Pirimidinas/química , Quinazolinas/síntese química , Quinazolinas/química , Ratos , Relação Estrutura-AtividadeRESUMO
Antagonism of orexin receptors has shown clinical efficacy as a novel paradigm for the treatment of insomnia and related disorders. Herein, molecules related to the dual orexin receptor antagonist filorexant were transformed into compounds that were selective for the OX2R subtype. Judicious selection of the substituents on the pyridine ring and benzamide groups led to 6b; which was highly potent, OX2R selective, and exhibited excellent development properties.
Assuntos
Antagonistas dos Receptores de Orexina/química , Receptores de Orexina/química , Piperidinas/química , Triazóis/química , Animais , Cães , Meia-Vida , Camundongos , Antagonistas dos Receptores de Orexina/farmacocinética , Antagonistas dos Receptores de Orexina/uso terapêutico , Receptores de Orexina/metabolismo , Piperidinas/farmacocinética , Piperidinas/uso terapêutico , Ligação Proteica , Pirimidinas/química , Ratos , Distúrbios do Início e da Manutenção do Sono/tratamento farmacológico , Distúrbios do Início e da Manutenção do Sono/veterinária , Relação Estrutura-Atividade , Triazóis/farmacocinética , Triazóis/uso terapêuticoRESUMO
Phosphodiesterase 10A (PDE10A) inhibition has recently been identified as a potential mechanism to treat multiple symptoms that manifest in schizophrenia. In order to facilitate preclinical development and support key proof-of-concept clinical trials of novel PDE10A inhibitors, it is critical to discover positron emission tomography (PET) tracers that enable plasma concentration/PDE10A occupancy relationships to be established across species with structurally diverse PDE10A inhibitors. In this Letter, we describe how a high-throughput screening hit was optimized to provide [(11)C]MK-8193 (8j), a PET tracer that supports the determination of plasma concentration/PDE10A occupancy relationships for structurally diverse series of PDE10A inhibitors in both rat and rhesus monkey.
Assuntos
Descoberta de Drogas , Compostos Heterocíclicos com 2 Anéis/química , Inibidores de Fosfodiesterase/metabolismo , Inibidores de Fosfodiesterase/farmacologia , Diester Fosfórico Hidrolases/metabolismo , Tomografia por Emissão de Pósitrons , Animais , Encéfalo/metabolismo , Radioisótopos de Carbono , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Compostos Heterocíclicos com 2 Anéis/síntese química , Macaca mulatta , Modelos Moleculares , Estrutura Molecular , Inibidores de Fosfodiesterase/síntese química , Inibidores de Fosfodiesterase/química , Diester Fosfórico Hidrolases/sangue , Ratos , Relação Estrutura-AtividadeRESUMO
Orexin signaling is essential for normal regulation of arousal and behavioral state control and represents an attractive target for therapeutics combating insomnia. Alternatively termed hypocretins, these neuropeptides were named to reflect sequence similarity to incretins and their potential to promote feeding. Current nomenclature reflects these molecular and biochemical discovery approaches in which HCRT, HCRTR1, and HCRTR2 genes encode prepro-orexin, the orexin 1 receptor (OX(1)) and the orexin 2 receptor (OX(2))-gene names designated by the Human Genome Organization and receptor names designated by the International Union of Basic and Clinical Pharmacology. Orexinergic neurons are most active during wakefulness and fall silent during inactive periods, a prolonged disruption in signaling most profoundly resulting in hypersomnia and narcolepsy. Hcrtr2 mutations underlie the etiology of canine narcolepsy, deficiencies in orexin-producing neurons are observed in the human disorder, and ablation of mouse orexin neurons or the Hcrt gene results in a narcolepsy-cataplexy phenotype. The development of orexin receptor antagonists and genetic models targeting components of the orexin pathway have elucidated the OX(2) receptor-specific role in histamine-mediated arousal and the contribution of both receptors in brainstem pathways involved in vigilance state gating. Orexin receptor antagonists of varying specificity uncovered additional roles beyond sleep and feeding that include addiction, depression, anxiety, and potential influences on peripheral physiology. Combined genetic and pharmacological approaches indicate that orexin signaling may represent a confluence of sleep, feeding, and reward pathways. Selective orexin receptor antagonism takes advantage of these properties toward the development of novel insomnia therapeutics.
Assuntos
Hipnóticos e Sedativos/farmacologia , Neurotransmissores/farmacologia , Receptores Acoplados a Proteínas G/classificação , Receptores de Neuropeptídeos/classificação , Animais , Nível de Alerta/efeitos dos fármacos , Nível de Alerta/genética , Ensaios Clínicos como Assunto , Evolução Molecular , Humanos , Hipnóticos e Sedativos/química , Hipnóticos e Sedativos/uso terapêutico , Agências Internacionais , Ligantes , Modelos Moleculares , Estrutura Molecular , Narcolepsia/tratamento farmacológico , Narcolepsia/genética , Neurotransmissores/química , Neurotransmissores/uso terapêutico , Receptores de Orexina , Conformação Proteica , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Receptores Acoplados a Proteínas G/genética , Receptores de Neuropeptídeos/agonistas , Receptores de Neuropeptídeos/antagonistas & inibidores , Receptores de Neuropeptídeos/genética , Distúrbios do Início e da Manutenção do Sono/tratamento farmacológico , Distúrbios do Início e da Manutenção do Sono/genética , Terminologia como AssuntoRESUMO
Cholinergic neurons in the basal forebrain and the brainstem form extensive projections to a number of thalamic nuclei. Activation of cholinergic afferents during distinct behavioral states can regulate neuronal firing, transmitter release at glutamatergic and GABAergic synapses, and synchrony in thalamic networks, thereby controlling the flow of sensory information. These effects are thought to be mediated by slow and persistent increases in extracellular ACh levels, resulting in the modulation of populations of thalamic neurons over large temporal and spatial scales. However, the synaptic mechanisms underlying cholinergic signaling in the thalamus are not well understood. Here, we demonstrate highly reliable cholinergic transmission in the mouse thalamic reticular nucleus (TRN), a brain structure essential for sensory processing, arousal, and attention. We find that ACh release evoked by low-frequency stimulation leads to biphasic excitatory-inhibitory (E-I) postsynaptic responses, mediated by the activation of postsynaptic α4ß2 nicotinic ACh receptors (nAChRs) and M2 muscarinic ACh receptors (mAChRs), respectively. In addition, ACh can bind to mAChRs expressed near cholinergic release sites, resulting in autoinhibition of release. We show that the activation of postsynaptic nAChRs by transmitter release from only a small number of individual axons is sufficient to trigger action potentials in TRN neurons. Furthermore, short trains of cholinergic synaptic inputs can powerfully entrain ongoing TRN neuronal activity. Our study demonstrates fast and precise synaptic E-I signaling mediated by ACh, suggesting novel computational mechanisms for the cholinergic control of neuronal activity in thalamic circuits.
Assuntos
Acetilcolina/metabolismo , Potenciais de Ação/fisiologia , Núcleos Intralaminares do Tálamo/fisiologia , Neurônios/fisiologia , Transmissão Sináptica/fisiologia , Potenciais de Ação/efeitos dos fármacos , Animais , Inibidores da Colinesterase/farmacologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/fisiologia , Feminino , Núcleos Intralaminares do Tálamo/efeitos dos fármacos , Masculino , Camundongos , Neurônios/efeitos dos fármacos , Fisostigmina/farmacologia , Receptores Colinérgicos/metabolismo , Sinapses/efeitos dos fármacos , Sinapses/fisiologia , Transmissão Sináptica/efeitos dos fármacosRESUMO
The axon initial segment (AIS) is a specialized neuronal subcompartment located at the beginning of the axon that is crucially involved in both the generation of action potentials and the regulation of neuronal polarity. We recently showed that prolonged neuronal depolarization produces a distal shift of the entire AIS structure away from the cell body, a change associated with a decrease in neuronal excitability. Here, we used dissociated rat hippocampal cultures, with a major focus on the dentate granule cell (DGC) population, to explore the signaling pathways underlying activity-dependent relocation of the AIS. First, a pharmacological screen of voltage-gated calcium channels (VGCCs) showed that AIS relocation is triggered by activation of L-type Cav1 VGCCs with negligible contribution from any other VGCC subtypes. Additional pharmacological analysis revealed that downstream signaling events are mediated by the calcium-sensitive phosphatase calcineurin; inhibition of calcineurin with either FK506 or cyclosporin A totally abolished both depolarization- and optogenetically-induced activity-dependent AIS relocation. Furthermore, calcineurin activation is sufficient for AIS plasticity, because expression of a constitutively active form of the phosphatase resulted in relocation of the AIS of DGCs without a depolarizing stimulus. Finally, we assessed the role of calcineurin in other forms of depolarization-induced plasticity. Neither membrane resistance changes nor spine density changes were affected by FK506 treatment, suggesting that calcineurin acts via a separate pathway to modulate AIS plasticity. Together, these results emphasize calcineurin as a vital player in the regulation of intrinsic plasticity as governed by the AIS.
Assuntos
Axônios/metabolismo , Calcineurina/metabolismo , Transdução de Sinais/fisiologia , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Animais , Benzamidas/farmacologia , Calcineurina/genética , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Células Cultivadas , Channelrhodopsins , Espinhas Dendríticas/metabolismo , Embrião de Mamíferos , Antagonistas de Aminoácidos Excitatórios/farmacologia , Hipocampo/citologia , Proteínas de Homeodomínio/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Fatores de Transcrição NFATC/genética , Fatores de Transcrição NFATC/metabolismo , Neurônios/citologia , Estimulação Luminosa , Piperidinas/farmacologia , Ratos , Ratos Wistar , Transdução de Sinais/genética , Transfecção , Proteínas Supressoras de Tumor/metabolismoRESUMO
Slow waves represent one of the prominent EEG signatures of non-rapid eye movement (non-REM) sleep and are thought to play an important role in the cellular and network plasticity that occurs during this behavioral state. These slow waves of natural sleep are currently considered to be exclusively generated by intrinsic and synaptic mechanisms within neocortical territories, although a role for the thalamus in this key physiological rhythm has been suggested but never demonstrated. Combining neuronal ensemble recordings, microdialysis, and optogenetics, here we show that the block of the thalamic output to the neocortex markedly (up to 50%) decreases the frequency of slow waves recorded during non-REM sleep in freely moving, naturally sleeping-waking rats. A smaller volume of thalamic inactivation than during sleep is required for observing similar effects on EEG slow waves recorded during anesthesia, a condition in which both bursts and single action potentials of thalamocortical neurons are almost exclusively dependent on T-type calcium channels. Thalamic inactivation more strongly reduces spindles than slow waves during both anesthesia and natural sleep. Moreover, selective excitation of thalamocortical neurons strongly entrains EEG slow waves in a narrow frequency band (0.75-1.5 Hz) only when thalamic T-type calcium channels are functionally active. These results demonstrate that the thalamus finely tunes the frequency of slow waves during non-REM sleep and anesthesia, and thus provide the first conclusive evidence that a dynamic interplay of the neocortical and thalamic oscillators of slow waves is required for the full expression of this key physiological EEG rhythm.
Assuntos
Potenciais de Ação/fisiologia , Neurônios/fisiologia , Sono/fisiologia , Tálamo/fisiologia , Animais , Canais de Cálcio Tipo T/metabolismo , Córtex Cerebral/fisiologia , Eletroencefalografia , Masculino , Ratos , Ratos WistarRESUMO
T-type calcium channels (T/Ca(v)3-channels) are implicated in various physiologic and pathophysiologic processes such as epilepsy, sleep disorders, hypertension, and cancer. T-channels are the target of endogenous signaling lipids including the endocannabinoid anandamide, the ω3-fatty acids, and the lipoamino-acids. However, the precise molecular mechanism by which these molecules inhibit T-current is unknown. In this study, we provided a detailed electrophysiologic and pharmacologic analysis indicating that the effects of the major N-acyl derivatives on the Ca(v)3.3 current share many similarities with those of TTA-A2 [(R)-2-(4-cyclopropylphenyl)-N-(1-(5-(2,2,2-trifluoroethoxy)pyridin-2-yl)ethyl)acetamide], a synthetic T-channel inhibitor. Using radioactive binding assays with the TTA-A2 derivative [(3)H]TTA-A1 [(R)-2-(4-(tert-butyl)phenyl)-N-(1-(5-methoxypyridin-2-yl)ethyl)acetamide], we demonstrated that polyunsaturated lipids, which inhibit the Ca(v)3.3 current, as NAGly (N-arachidonoyl glycine), NASer (N-arachidonoyl-l-serine), anandamide, NADA (N-arachidonoyl dopamine), NATau (N-arachidonoyl taurine), and NA-5HT (N-arachidonoyl serotonin), all displaced [(3)H]TTA-A1 binding to membranes prepared from cells expressing Ca(v)3.3, with Ki in a micromolar or submicromolar range. In contrast, lipids with a saturated alkyl chain, as N-arachidoyl glycine and N-arachidoyl ethanolamine, which did not inhibit the Ca(v)3.3 current, had no effect on [(3)H]TTA-A1 binding. Accordingly, bio-active lipids occluded TTA-A2 effect on Ca(v)3.3 current. In addition, TTA-Q4 [(S)-4-(6-chloro-4-cyclopropyl-3-(2,2-difluoroethyl)-2-oxo-1,2,3,4-tetrahydroquinazolin-4-yl)benzonitrile], a positive allosteric modulator of [(3)H]TTA-A1 binding and TTA-A2 functional inhibition, acted in a synergistic manner to increase lipid-induced inhibition of the Ca(v)3.3 current. Overall, our results demonstrate a common molecular mechanism for the synthetic T-channel inhibitors and the endogenous lipids, and indicate that TTA-A2 and TTA-Q4 could be important pharmacologic tools to dissect the involvement of T-current in the physiologic effects of endogenous lipids.