RESUMO
BACKGROUND: The gold-standard treatment for Parkinson's disease is L-DOPA, which in the long term often leads to levodopa-induced dyskinesia. Serotonergic neurons are partially responsible for this, by converting L-DOPA into dopamine leading to its uncontrolled release as a "false neurotransmitter". The stimulation of 5-HT1A receptors can reduce involuntary movements but this mechanism is poorly understood. OBJECTIVE: This study aimed to investigate the functionality of 5-HT1A receptors using positron emission tomography in hemiparkinsonian rats with or without dyskinesia induced by 3-weeks daily treatment with L-DOPA. Imaging sessions were performed "off" L-DOPA. METHODS: Each rat underwent a positron emission tomography scan with [18F]F13640, a 5-HT1AR agonist which labels receptors in a high affinity state for agonists, or with [18F]MPPF, a 5-HT1AR antagonist which labels all the receptors. RESULTS: There were decreases of [18F]MPPF binding in hemiparkinsonian rats in cortical areas. In dyskinetic animals, changes were slighter but also found in other regions. In hemiparkinsonian rats, [18F]F13640 uptake was decreased bilaterally in the globus pallidus and thalamus. On the non-lesioned side, binding was increased in the insula, the hippocampus and the amygdala. In dyskinetic animals, [18F]F13640 binding was strongly increased in cortical and limbic areas, especially in the non-lesioned side. CONCLUSION: These data suggest that agonist and antagonist 5-HT1A receptor-binding sites are differently modified in Parkinson's disease and levodopa-induced dyskinesia. In particular, these observations suggest a substantial involvement of the functional state of 5-HT1AR in levodopa-induced dyskinesia and emphasize the need to characterize this state using agonist radiotracers in physiological and pathological conditions.
Assuntos
Discinesia Induzida por Medicamentos , Doença de Parkinson , Receptor 5-HT1A de Serotonina , Agonistas do Receptor 5-HT1 de Serotonina , Antagonistas do Receptor 5-HT1 de Serotonina , Animais , Antiparkinsonianos/toxicidade , Modelos Animais de Doenças , Discinesia Induzida por Medicamentos/metabolismo , Levodopa/toxicidade , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/metabolismo , Ratos , Receptor 5-HT1A de Serotonina/metabolismo , Agonistas do Receptor 5-HT1 de Serotonina/metabolismo , Antagonistas do Receptor 5-HT1 de Serotonina/metabolismoRESUMO
A library of novel anilide and benzylamide derivatives of ω-(4-(2-methoxyphenyl)piperazin-1-yl)alkanoic acids as combined 5-HT1A/5-HT7 receptor ligands and phosphodiesterase PDE4B/PDE7A inhibitors was designed using a structure-based drug design approach. The in vitro studies of 33 newly synthesized compounds (7-39) allowed us to identify 22 as the most promising multifunctional 5-HT1A/5-HT7 receptor antagonist (5-HT1AKi = 8 nM, Kb = 0.04 nM; 5-HT7Ki = 451 nM, Kb = 460 nM) with PDE4B/PDE7A inhibitory activity (PDE4B IC50 = 80.4 µM; PDE7A IC50 = 151.3 µM). Compound 22 exerted a very good ability to passively penetrate through biological membranes and a high metabolic stability in vitro. Moreover, the pharmacological evaluation of 22 showed its procognitive and antidepressant properties in rat behavioral tests. Compound 22 at a dose of 3 mg/kg (i.p.) significantly reversed MK-801-induced episodic memory deficits in the novel object recognition test, while at a dose of 10 mg/kg (i.p.) reduced the immobility time of animals (by about 34%) in the forced swimming test. The antidepressant-like effect produced by compound 22 was stronger than that of escitalopram used as a reference drug. This study opens a new perspective in the search for efficacious drugs for the treatment of cognitive and depressive disorders.
Assuntos
Anilidas/farmacologia , Fármacos do Sistema Nervoso Central/farmacologia , Inibidores da Fosfodiesterase 4/farmacologia , Piperazinas/farmacologia , Antagonistas do Receptor 5-HT1 de Serotonina/farmacologia , Anilidas/síntese química , Anilidas/metabolismo , Animais , Células CHO , Fármacos do Sistema Nervoso Central/síntese química , Fármacos do Sistema Nervoso Central/metabolismo , Cricetulus , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 7/antagonistas & inibidores , Nucleotídeo Cíclico Fosfodiesterase do Tipo 7/metabolismo , Células HEK293 , Humanos , Masculino , Microssomos Hepáticos/metabolismo , Simulação de Acoplamento Molecular , Estrutura Molecular , Teste de Campo Aberto/efeitos dos fármacos , Inibidores da Fosfodiesterase 4/síntese química , Inibidores da Fosfodiesterase 4/metabolismo , Piperazinas/síntese química , Piperazinas/metabolismo , Ligação Proteica , Ratos Wistar , Receptor 5-HT1A de Serotonina/metabolismo , Receptores de Serotonina/metabolismo , Antagonistas do Receptor 5-HT1 de Serotonina/síntese química , Antagonistas do Receptor 5-HT1 de Serotonina/metabolismo , Células Sf9 , Relação Estrutura-AtividadeRESUMO
G protein-coupled receptors (GPCRs) constitute the largest superfamily of membrane proteins in higher eukaryotes, and facilitate information transfer from the extracellular environment to the cellular interior upon activation by ligands. Their role in diverse signaling processes makes them an attractive choice as drug targets. GPCRs are coupled to heterotrimeric G-proteins which represent an important interface through which signal transduction occurs across the plasma membrane upon activation by ligands. To obtain further insight into the molecular details of interaction of G-proteins with GPCRs, in this work, we explored the selectivity of binding of specific agonists and antagonists to the serotonin1A receptor under conditions of progressive G-protein inactivation. The serotonin1A receptor is an important neurotransmitter receptor belonging to the GPCR family and is a popular drug target. By use of a number of agents to inactivate G-proteins, we show here that the serotonin1A receptor displays differential discrimination between agonist and antagonist binding. Our results show a reduction in binding sites of the receptor upon treatment with G-protein inactivating agents. In addition, G-protein coupling efficiency was enhanced when G-proteins were inactivated using urea and alkaline pH. We envision that our results could be useful in achieving multiple signaling states of the receptor by fine tuning the conditions of G-protein inactivation and in structural biology of GPCRs bound to specific ligands.
Assuntos
Descoberta de Drogas , Receptor 5-HT1A de Serotonina/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Agonistas do Receptor 5-HT1 de Serotonina/metabolismo , Antagonistas do Receptor 5-HT1 de Serotonina/metabolismo , Sítios de Ligação , Proteínas de Ligação ao GTP/metabolismo , Concentração de Íons de Hidrogênio , Ligação Proteica , Ureia/farmacologiaRESUMO
In addition to its anticonvulsant effect, low frequency stimulation (LFS) improves learning and memory in kindled animals. In the present study, the role of 5-HT1A receptors in mediating LFS' improving effect on spatial learning and memory was investigated in amygdala-kindled rats. Amygdala kindling was conducted in a semi-rapid kindling stimulations (12 stimulations per day) in male Wistar rats. LFS (4 trains of 0.1 ms pulse duration at 1 Hz, 200 pulses, 50-150 µA, at 5 min intervals) was applied after termination of kindling stimulations. NAD-299 (a selective 5-HT1A receptor antagonist; 2.5 and 5 µg/µl) was microinjected into the hippocampal CA1 before applying LFS. The Morris water maze, and novel object recognition tests were conducted after the last kindling stimulation. Hippocampal samples were also prepared, and 5-HT1A receptor gene expression levels were assessed using quantitative RT-PCR. In kindled animals, LFS reduced impairments in spatial learning and memory in the Morris water maze and novel object recognition tests. Microinjection of NAD doses of 5 µg/µl reduced the effects of LFS on learning and memory. The gene expression level of 5-HT1A receptors increased significantly in the hippocampus of amygdala-kindled rats. However, LFS applied after kindling stimulations inhibited this effect. It seems that activation of 5-HT1A receptors in the CA1 field is necessary for LFS' improving effects on spatial learning and memory in kindled animals; although surprisingly, LFS application prevented the elevation in gene expression of 5-HT1A receptors in kindled animals.
Assuntos
Transtornos da Memória/metabolismo , Receptor 5-HT1A de Serotonina/metabolismo , Antagonistas do Receptor 5-HT1 de Serotonina/metabolismo , Tonsila do Cerebelo/metabolismo , Animais , Anticonvulsivantes/farmacologia , Região CA1 Hipocampal/metabolismo , Modelos Animais de Doenças , Estimulação Elétrica , Terapia por Estimulação Elétrica/métodos , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Excitação Neurológica/efeitos dos fármacos , Masculino , Memória , Ratos , Ratos Wistar , Convulsões/metabolismo , Aprendizagem EspacialRESUMO
BACKGROUND: Most previous studies have focused on the effects of social defeat in male juvenile individuals. Whether chronic social defeat stress in adulthood affects female emotion and the underlying mechanisms remains unclear. METHODS: Using highly aggressive adult female mandarin voles (Microtus mandarinus), the present study aimed to determine the effects of chronic social defeat stress on anxiety- and depression-like behaviors in adult female rodents and investigate the neurobiological mechanisms underlying these effects. RESULTS: Exposure of adult female voles to social defeat stress for 14 days reduced the time spent in the central area of the open field test and in the open arms of the elevated plus maze and lengthened the immobility time in the tail suspension and forced swimming tests, indicating increased anxiety- and depression-like behaviors. Meanwhile, defeated voles exhibited increased neural activity in the prelimbic cortex of the medial prefrontal cortex. Furthermore, chronic social defeat stress reduced serotonin projections and levels of serotonin 1A receptors in the medial prefrontal cortex-prelimbic cortex. Intra-prelimbic cortex microinjections of the serotonin 1A receptor agonist 8-OH-DPAT reversed the alterations in emotional behaviors, whereas injections of the serotonin 1A receptor antagonist WAY-100635 into the prelimbic cortex of control voles increased the levels of anxiety- and depression-like behaviors. CONCLUSIONS: Taken together, our results demonstrated that chronic social defeat stress increased anxiety- and depression-like behaviors in adult female voles, and these effects were mediated by the action of serotonin on the serotonin 1A receptors in the prelimbic cortex. The serotonin system may be a promising target to treat emotional disorders induced by chronic social defeat stress.
Assuntos
Córtex Pré-Frontal/metabolismo , Receptor 5-HT1A de Serotonina/metabolismo , Agonistas do Receptor 5-HT1 de Serotonina/metabolismo , Antagonistas do Receptor 5-HT1 de Serotonina/metabolismo , Comportamento Social , Estresse Psicológico/metabolismo , Animais , Arvicolinae , Doença Crônica , Feminino , Córtex Pré-Frontal/efeitos dos fármacos , Distribuição Aleatória , Serotonina/metabolismo , Agonistas do Receptor 5-HT1 de Serotonina/farmacologia , Antagonistas do Receptor 5-HT1 de Serotonina/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Estresse Psicológico/psicologiaRESUMO
In previous study, a series of benzamides was identified as potent antipsychotic agents. As a continuation of the program to discover novel antipsychotics, herein we reported the evaluation of a series of pyridinecarboxamide derivatives. The most promising compound 7h not only held good activities on dopamine D2, serotonin 5-HT1A and 5-HT2A receptors, but also exhibited low potency for α1A, H1 and 5-HT2C receptors, indicating a low propensity of side effects like orthostatic hypotension and weight gain. Furthermore, 7h exhibited more potent antipsychotic-like effect than aripiprazole in behavioral studies. The preliminary results were promising enough for further research around this scaffold.
Assuntos
Antipsicóticos/farmacologia , Ácidos Picolínicos/farmacologia , Animais , Antipsicóticos/síntese química , Antipsicóticos/química , Antipsicóticos/metabolismo , Aripiprazol/farmacologia , Antagonistas dos Receptores de Dopamina D2/síntese química , Antagonistas dos Receptores de Dopamina D2/química , Antagonistas dos Receptores de Dopamina D2/metabolismo , Antagonistas dos Receptores de Dopamina D2/farmacologia , Humanos , Masculino , Camundongos Endogâmicos ICR , Microssomos Hepáticos/metabolismo , Estrutura Molecular , Ácidos Picolínicos/síntese química , Ácidos Picolínicos/química , Ácidos Picolínicos/metabolismo , Risperidona/farmacologia , Antagonistas do Receptor 5-HT1 de Serotonina/síntese química , Antagonistas do Receptor 5-HT1 de Serotonina/química , Antagonistas do Receptor 5-HT1 de Serotonina/metabolismo , Antagonistas do Receptor 5-HT1 de Serotonina/farmacologia , Antagonistas do Receptor 5-HT2 de Serotonina/síntese química , Antagonistas do Receptor 5-HT2 de Serotonina/química , Antagonistas do Receptor 5-HT2 de Serotonina/metabolismo , Antagonistas do Receptor 5-HT2 de Serotonina/farmacologia , Relação Estrutura-AtividadeRESUMO
In the present study, we used a simultaneous PET-MR experimental design to investigate the effects of functionally different compounds (agonist, partial agonist, and antagonist) on 5-HT1B receptor (5-HT1BR) occupancy and the associated hemodynamic responses. In anesthetized male nonhuman primates (n = 3), we used positron emission tomography (PET) imaging with the radioligand [11C]AZ10419369 administered as a bolus followed by constant infusion to measure changes in 5-HT1BR occupancy. Simultaneously, we measured changes in cerebral blood volume (CBV) as a proxy of drug effects on neuronal activity. The 5-HT1BR partial agonist AZ10419369 elicited a dose-dependent biphasic hemodynamic response that was related to the 5-HT1BR occupancy. The magnitude of the response was spatially overlapping with high cerebral 5-HT1BR densities. High doses of AZ10419369 exerted an extracranial tissue vasoconstriction that was comparable to the less blood-brain barrier-permeable 5-HT1BR agonist sumatriptan. By contrast, injection of the antagonist GR127935 did not elicit significant hemodynamic responses, even at a 5-HT1BR cerebral occupancy similar to the one obtained with a high dose of AZ10419369. Given the knowledge we have of the 5-HT1BR and its function and distribution in the brain, the hemodynamic response informs us about the functionality of the given drug: changes in CBV are only produced when the receptor is stimulated by the partial agonist AZ10419369 and not by the antagonist GR127935, consistent with low basal occupancy by endogenous serotonin.SIGNIFICANCE STATEMENT We here show that combined simultaneous positron emission tomography and magnetic resonance imaging uniquely enables the assessment of CNS active compounds. We conducted a series of pharmacological interventions to interrogate 5-HT1B receptor binding and function and determined blood-brain barrier passage of drugs and demonstrate target involvement. Importantly, we show how the spatial and temporal effects on brain hemodynamics provide information about pharmacologically driven downstream CNS drug effects; the brain hemodynamic response shows characteristic dose-related effects that differ depending on agonistic or antagonistic drug characteristics and on local 5-HT1B receptor density. The technique lends itself to a comprehensive in vivo investigation and understanding of drugs' effects in the brain.
Assuntos
Encéfalo/metabolismo , Agonismo Parcial de Drogas , Imageamento por Ressonância Magnética/métodos , Tomografia por Emissão de Pósitrons/métodos , Agonistas do Receptor 5-HT1 de Serotonina/metabolismo , Antagonistas do Receptor 5-HT1 de Serotonina/metabolismo , Animais , Encéfalo/diagnóstico por imagem , Encéfalo/efeitos dos fármacos , Relação Dose-Resposta a Droga , Macaca mulatta , Masculino , Receptor 5-HT1B de Serotonina/metabolismo , Agonistas do Receptor 5-HT1 de Serotonina/farmacologia , Antagonistas do Receptor 5-HT1 de Serotonina/farmacologiaRESUMO
Positron emission tomography (PET) is widely applied in central nervous system (CNS) drug development for assessment of target engagement in vivo. As the majority of PET investigations have addressed drug interaction at a single binding site, findings of multitarget engagement have been less frequently reported and have often been inconsistent with results obtained in vitro. AZD3676 [N,N-dimethyl-7-(4-(2-(pyridin-2-yl)ethyl)piperazin-1-yl) benzofuran-2-carboxamide] is a novel combined serotonin (5-hydroxytryptamine) 5-HT1A and 5-HT1B receptor antagonist that was developed for the treatment of cognitive impairment in Alzheimer's disease. Here, we evaluated the properties of AZD3676 as a CNS drug by combining in vitro and ex vivo radioligand binding techniques, behavioral pharmacology in rodents, and PET imaging in nonhuman primates. Target engagement in the nonhuman primate brain was assessed in PET studies by determination of drug-induced occupancy using receptor-selective radioligands. AZD3676 showed preclinical properties consistent with CNS drug potential, including nanomolar receptor affinity and efficacy in rodent models of learning and memory. In PET studies of the monkey brain, AZD3676 inhibited radioligand binding in a dose-dependent manner with similar affinity at both receptors. The equally high affinity at 5-HT1A and 5-HT1B receptors as determined in vivo was not predicted from corresponding estimates obtained in vitro, suggesting more than 10-fold selectivity for 5-HT1A versus 5-HT1B receptors. These findings support the further integrated use of PET for confirmation of multitarget occupancy of CNS drugs. Importantly, earlier introduction of PET studies in nonhuman primates may reduce future development costs and the requirement for animal experiments in preclinical CNS drug development programs.
Assuntos
Benzofuranos/metabolismo , Benzofuranos/farmacologia , Piperazinas/metabolismo , Piperazinas/farmacologia , Tomografia por Emissão de Pósitrons , Psicotrópicos/metabolismo , Psicotrópicos/farmacologia , Quinazolinas/metabolismo , Quinazolinas/farmacologia , Animais , Proteínas Sanguíneas/metabolismo , Encéfalo/diagnóstico por imagem , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/fisiologia , Humanos , Macaca fascicularis , Memória/efeitos dos fármacos , Camundongos , Receptor 5-HT1A de Serotonina/metabolismo , Receptor 5-HT1B de Serotonina/metabolismo , Antagonistas do Receptor 5-HT1 de Serotonina/metabolismo , Antagonistas do Receptor 5-HT1 de Serotonina/farmacologiaRESUMO
G-protein-coupled receptors (GPCRs) are involved in a wide range of physiological processes, and they have attracted considerable attention as important targets for developing new medicines. A central and largely unresolved question in drug discovery, which is especially relevant to GPCRs, concerns ligand selectivity: Why do certain molecules act as activators (agonists) whereas others, with nearly identical structures, act as blockers (antagonists) of GPCRs? To address this question, we employed all-atom, long-timescale molecular dynamics simulations to investigate how two diastereomers (epimers) of dihydrofuroaporphine bind to the serotonin 5-HT1A receptor and exert opposite effects. By using molecular interaction fingerprints, we discovered that the agonist could mobilize nearby amino acid residues to act as molecular switches for the formation of a continuous water channel. In contrast, the antagonist epimer remained firmly stabilized in the binding pocket.
Assuntos
Receptor 5-HT1A de Serotonina/metabolismo , Aporfinas/química , Aporfinas/metabolismo , Sítios de Ligação , Ligantes , Simulação de Dinâmica Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Receptor 5-HT1A de Serotonina/química , Agonistas do Receptor 5-HT1 de Serotonina/química , Agonistas do Receptor 5-HT1 de Serotonina/metabolismo , Antagonistas do Receptor 5-HT1 de Serotonina/química , Antagonistas do Receptor 5-HT1 de Serotonina/metabolismo , Estereoisomerismo , Água/química , Água/metabolismoRESUMO
PET imaging studies using 5-HT1A receptor radiotracers show a decreased density of this receptor in hippocampi of patients with Alzheimer's disease (AD) at advanced stages. However, current 5-HT1A receptor radiopharmaceuticals used in neuroimaging are antagonists, thought to bind to 5-HT1A receptors in different functional states (i.e., both the one which displays high affinity for agonists and is thought to mediate receptor activation, as well as the state which has low affinity for agonists). Comparing the PET imaging obtained using an agonist radiotracer, which binds selectively to functional receptors, with the PET imaging obtained using an antagonist radiotracer would therefore provide original information on 5-HT1A receptor impairment during AD. Quantitative autoradiography using [(18)F]F13640 and [(18)F]MPPF, a 5-HT1A agonist and antagonist, respectively, was measured in hippocampi of patients with AD (n = 25, at different Braak stages) and control subjects (n = 9). The neuronal density was measured in the same tissues by NeuN immunohistochemistry. The specific binding of both radiotracers was determined by addition of WAY-100635, a selective 5-HT1A receptor antagonist. The autoradiography distribution of both 5-HT1A PET radiotracers varied across hippocampus regions. The highest binding density was in the pyramidal layer of CA1. Incubation with Gpp(NH)p, a non-hydrolysable analogue of GTP, reduced significantly [(18)F]F13640 binding in hippocampal regions, confirming its preferential interaction with G-coupled receptors, and slightly increased [(18)F]MPPF binding. In the CA1 subfield, [(18)F]F13640 binding was significantly decreased at Braak stages I/II (-19%), Braak stages III/IV (-23%), and Braak stages V/VI (-36%) versus control. In contrast, [(18)F]MPPF binding was statistically reduced only at the most advanced Braak stages V/VI compared to control (-33%). Since [(18)F]F13640 and [(18)F]MPPF can be used in vivo in humans, this neuropharmacological paradigm supports testing the concept of functional imaging using agonist radiopharmaceuticals in future clinical studies.
Assuntos
Doença de Alzheimer/metabolismo , Tomografia por Emissão de Pósitrons , Compostos Radiofarmacêuticos/metabolismo , Receptor 5-HT1A de Serotonina/metabolismo , Agonistas do Receptor 5-HT1 de Serotonina/metabolismo , Antagonistas do Receptor 5-HT1 de Serotonina/metabolismo , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/patologia , Autopsia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Piperidinas/metabolismo , Ligação Proteica/fisiologia , Piridinas/metabolismoRESUMO
The present study aimed at evaluation of prophylactic efficacy and possible mechanisms of asiaticoside (AS) based standardized extract of Centella asiatica (L.) Urban leaves (INDCA) in animal models of migraine. The effects of oral and intranasal (i.n.) pretreatment of INDCA (acute and 7-days subacute) were evaluated against nitroglycerine (NTG, 10 mg·kg(-1), i.p.) and bradykinin (BK, 10 µg, intra-arterial) induced hyperalgesia in rats. Tail flick latencies (from 0 to 240 min) post-NTG treatment and the number of vocalizations post-BK treatment were recorded as a measure of hyperalgesia. Separate groups of rats for negative (Normal) and positive (sumatriptan, 42 mg·kg(-1), s.c.) controls were included. The interaction of INDCA with selective 5-HT1A, 5-HT1B, and 5-HT1D receptor antagonists (NAN-190, Isamoltane hemifumarate, and BRL-15572 respectively) against NTG-induced hyperalgesia was also evaluated. Acute and sub-acute pre-treatment of INDCA [10 and 30 mg·kg(-1) (oral) and 100 µg/rat (i.n.) showed significant anti-nociception activity, and reversal of the NTG-induced hyperalgesia and brain 5-HT concentration decline. Oral pre-treatment with INDCA (30 mg·kg(-1), 7 d) showed significant reduction in the number of vocalization. The anti-nociceptive effects of INDCA were blocked by 5-HT1A and 5-HT1B but not 5-HT1D receptor antagonists. In conclusion, INDCA demonstrated promising anti-nociceptive effects in animal models of migraine, probably through 5-HT1A/1B medicated action.
Assuntos
Transtornos de Enxaqueca/prevenção & controle , Profilaxia Pré-Exposição , Receptores 5-HT1 de Serotonina/efeitos dos fármacos , Triterpenos/farmacologia , Administração Intranasal , Administração Oral , Animais , Bradicinina , Centella , Feminino , Hiperalgesia/induzido quimicamente , Hiperalgesia/prevenção & controle , Masculino , Transtornos de Enxaqueca/induzido quimicamente , Modelos Animais , Nitroglicerina , Nociceptividade/efeitos dos fármacos , Extratos Vegetais , Folhas de Planta/química , Ratos , Ratos Wistar , Tempo de Reação , Antagonistas do Receptor 5-HT1 de Serotonina/metabolismo , Cauda/fisiologia , Triterpenos/administração & dosagemRESUMO
The monoamine neurotransmitter serotonin (5-HT) regulates a wide spectrum of human physiology through the 5-HT receptor family. One such receptor, the 5-HT1A receptor (HTR1A), is the most widely studied subtype and represents a significant molecular target in medicinal and therapeutic fields. Yeast-based fluorescent reporter systems have proven to be especially useful for GPCR assays, since detection using a fluorescent reporter considerably simplifies measurement procedures. However, previously reported systems using enhanced green fluorescent protein (EGFP) as the reporter in yeast still showed low signal-to-noise (S/N) ratios, making EGFP difficult to apply as an easily accessible tool. Therefore, we constructed a refined yeast-based GPCR biosensor employing a high-sensitivity strain that incorporated both a Gα-engineered receptor and a fluorescent reporter (ZsGreen). As we report here, the refined yeast-based fluorescent biosensor was applied successfully to antagonist characterization and analysis of site-directed mutants of the HTR1A receptor. Pindolol, a known antagonist of HTR1A, specifically inhibited agonist-induced signaling, demonstrating the ease of evaluating inhibition effects using our reporter strain. Characterization of site-specific receptor mutants confirmed the role of specific targeted residues, including the highly conserved DRY motif, in the activation of HTR1A. Thus, our refined yeast biosensor strain, which incorporates a ZsGreen reporter and an engineered Gα receptor, is expected to serve as a simple and practical sensing tool for evaluating the ligand candidates and defining residues important to the function of human GPCRs. Biotechnol.
Assuntos
Mutagênese Sítio-Dirigida/métodos , Receptor 5-HT1A de Serotonina/química , Saccharomyces cerevisiae/metabolismo , Antagonistas do Receptor 5-HT1 de Serotonina/metabolismo , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Plasmídeos , Receptor 5-HT1A de Serotonina/genética , Receptor 5-HT1A de Serotonina/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Transdução de SinaisRESUMO
To obtain potential antidepressants and/or antipsychotics, a series of new long-chain arylpiperazine derivatives of 8-alkoxy-purine-2,6-dione (10-24) and dihydro[1,3]oxazolo[2,3-f]purinedione (30-34) were synthesized and their serotonin (5-HT1A , 5-HT2A , 5-HT6 , 5-HT7 ) and dopamine (D2 ) receptor affinities were determined. The study allowed the identification of some potent 5-HT1A /5-HT7 /D2 ligands with moderate affinity for 5-HT2A sites. The binding mode of representative compounds from both chemical classes (11 and 31) in the site of 5-HT1A receptor was analyzed in computational studies. In functional in vitro studies, the selected compounds 15 and 16 showed antagonistic properties for the evaluated receptors. 8-Methoxy-7-{4-[4-(2-methoxyphenyl)-piperazin-1-yl]-butyl}-1,3-dimethyl-purine-2,6-dione (15) showed a lack of activity in terms and under the conditions of the forced swim, four plate and amphetamine-induced hyperactivity tests in mice, probably as a result of its high first pass effect in the liver.
Assuntos
Antidepressivos/farmacologia , Antipsicóticos/farmacologia , Antagonistas dos Receptores de Dopamina D2/farmacologia , Purinas/farmacologia , Receptor 5-HT1A de Serotonina/metabolismo , Receptores de Dopamina D2/metabolismo , Antagonistas do Receptor 5-HT1 de Serotonina/farmacologia , Antagonistas do Receptor 5-HT2 de Serotonina/farmacologia , Animais , Antidepressivos/síntese química , Antidepressivos/metabolismo , Antipsicóticos/síntese química , Antipsicóticos/metabolismo , Comportamento Animal/efeitos dos fármacos , Biotransformação , Antagonistas dos Receptores de Dopamina D2/síntese química , Antagonistas dos Receptores de Dopamina D2/metabolismo , Desenho de Fármacos , Ligantes , Fígado/metabolismo , Masculino , Camundongos , Estrutura Molecular , Atividade Motora/efeitos dos fármacos , Ligação Proteica , Purinas/síntese química , Purinas/metabolismo , Receptor 5-HT1A de Serotonina/efeitos dos fármacos , Receptor 5-HT2A de Serotonina/efeitos dos fármacos , Receptor 5-HT2A de Serotonina/metabolismo , Receptores de Dopamina D2/química , Receptores de Dopamina D2/efeitos dos fármacos , Receptores de Serotonina/efeitos dos fármacos , Receptores de Serotonina/metabolismo , Antagonistas do Receptor 5-HT1 de Serotonina/síntese química , Antagonistas do Receptor 5-HT1 de Serotonina/metabolismo , Antagonistas do Receptor 5-HT2 de Serotonina/síntese química , Antagonistas do Receptor 5-HT2 de Serotonina/metabolismo , Relação Estrutura-Atividade , NataçãoRESUMO
In our previous paper, we have reported that some 8-alkoxy-1,3-dimethyl-1H-purine-2,6(3H,7H)-dione derivatives possessed high affinity and displayed agonistic activity for the serotonin 5-HT1A receptor. In order to examine the influence of the substituent in the position 8 of the purine moiety on the affinity for the serotonin 5-HT1A , 5-HT2A , and 5-HT7 receptors, a series of 7-arylpiperazynylalkyl and 7-tetrahydroisoquinolinylalkyl (THIQ) derivatives of 8-amino-1,3-dimethyl-1H-purine-2,6(3H,7H)-dione were synthesized. All the final compounds were investigated in in vitro competition binding experiments for serotonin 5-HT1A , 5-HT2A , and 5-HT7 receptors. The structure-affinity relationships for this group of compounds were discussed. For selected compounds, functional assays for the 5-HT1A receptor were carried out. The results of the assays indicated that these groups of derivatives possessed antagonistic activity for this receptor.
Assuntos
Purinas/metabolismo , Receptor 5-HT1A de Serotonina/metabolismo , Antagonistas do Receptor 5-HT1 de Serotonina/metabolismo , Animais , Ligação Competitiva , Desenho de Fármacos , Ligantes , Estrutura Molecular , Ligação Proteica , Purinas/síntese química , Purinas/farmacologia , Ensaio Radioligante , Ratos , Receptor 5-HT1A de Serotonina/efeitos dos fármacos , Receptor 5-HT2A de Serotonina/efeitos dos fármacos , Receptor 5-HT2A de Serotonina/metabolismo , Receptores de Serotonina/efeitos dos fármacos , Receptores de Serotonina/metabolismo , Antagonistas do Receptor 5-HT1 de Serotonina/síntese química , Antagonistas do Receptor 5-HT1 de Serotonina/farmacologia , Relação Estrutura-AtividadeRESUMO
A predictive quantitative structure - activity relationships model of arylpiperazines as high-affinity 5-HT(1A) receptor ligands was developed using CORAL software (http://www.insilico.eu/CORAL). Simplified molecular input-line entry system (SMILES) was used as representation of the molecular structure of the arylpiperazines. The balance of correlations was used in the Monte Carlo optimization aimed to build up optimal descriptors for one-variable models. The robustness of this model has been tested in four random splits into the sub-training, calibration, and test set. The obtained results reveal good predictive potential of the applied approach: correlation coefficients (r²) for the test sets of the four random splits are 0.9459, 0.9249, 0.9473 and 0.9362.
Assuntos
Antidepressivos/química , Modelos Moleculares , Piperazinas/química , Receptor 5-HT1A de Serotonina/química , Antagonistas do Receptor 5-HT1 de Serotonina/química , Antidepressivos/metabolismo , Antidepressivos/farmacologia , Inteligência Artificial , Calibragem , Humanos , Cinética , Ligantes , Conformação Molecular , Método de Monte Carlo , Piperazinas/metabolismo , Piperazinas/farmacologia , Relação Quantitativa Estrutura-Atividade , Receptor 5-HT1A de Serotonina/metabolismo , Antagonistas do Receptor 5-HT1 de Serotonina/metabolismo , Antagonistas do Receptor 5-HT1 de Serotonina/farmacologia , SoftwareRESUMO
The metabolism of elzasonan has been examined in vitro using hepatic microsomes from human and recombinant heterologously expressed P450 enzymes (rCYP). Metabolism occurs primarily via oxidative N-demethylation to form M4 and oxidation reactions to form elzasonan N-oxide (M5) and 5-hydroxyelzasonan metabolite (M3). Additionally, elzasonan was shown to be metabolized to the novel cyclized indole metabolite (M6) which undergoes subsequent oxidation to form the iminium ion metabolite (M3a). The rCYP data was normalized relative to the levels of each CYP form in native human liver microsomes to better assess the contribution of each rCYP in the metabolism of elzasonan. Results demonstrated the involvement of CYP3A4 in the pathways leading to M3a, M3, M5 and M6 and CYP2C8 in the formation of M4. Kinetic constants for the formation of M3 were determined and correlation and inhibition studies suggested that CYP3A4 is primarily responsible for the formation of M3 and CYP2C19 plays a very minor role in its formation. Cytochrome b5 has shown to be an essential component in P450 3A4 catalyzed 5-hydroxyelzasonan formation and provides insights on the disconnect between human liver microsomes data and that of rCYP. Furthermore, rCYP3A4 containing b5 are useful models for predicting the rates for liver microsomes P450-dependent drug oxidations and should be utilized routinely.
Assuntos
Morfolinas/metabolismo , Piperazinas/metabolismo , Receptor 5-HT1B de Serotonina/metabolismo , Antagonistas do Receptor 5-HT1 de Serotonina/metabolismo , Isótopos de Carbono , Cromatografia Líquida de Alta Pressão , Inibidores das Enzimas do Citocromo P-450 , Sistema Enzimático do Citocromo P-450/metabolismo , Inibidores Enzimáticos/farmacologia , Humanos , Isoenzimas/metabolismo , Cinética , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/enzimologia , Morfolinas/química , Oxirredução/efeitos dos fármacos , Piperazinas/química , Proteínas Recombinantes/metabolismo , Antagonistas do Receptor 5-HT1 de Serotonina/químicaRESUMO
[Carbonyl-(11)C]WAY-100635 is a potent and effective antagonist for the 5-HT(1A) receptor subtype. We aimed to assess the status of [carbonyl-(11)C]WAY-100635 and its main radio-metabolites, [carbonyl-(11)C]desmethyl-WAY-100635 and [carbonyl-(11)C]cyclohexanecarboxylic acid, on the basis of an improved radio-HPLC method. Common methods were characterized by preparative HPLC columns with long runtimes and/or high flow rates. Considering the short half-life of C-11, we developed a more rapid and solvent saving HPLC assay, allowing a fast, efficient and reliable quantification of these major metabolites.
Assuntos
Piperazinas/metabolismo , Piridinas/metabolismo , Receptor 5-HT1A de Serotonina/metabolismo , Cromatografia Líquida de Alta Pressão/métodos , Humanos , Piperazinas/sangue , Piperazinas/isolamento & purificação , Piridinas/sangue , Piridinas/isolamento & purificação , Antagonistas do Receptor 5-HT1 de Serotonina/metabolismoRESUMO
Serotonin (5-HT) 1A receptors exist in high and low affinity states. Agonist ligands bind preferentially to the high affinity state receptors, providing a more functionally relevant measure than antagonist binding. We now report comparison of 5-HT(1A) binding in vivo using both [¹¹C]CUMI-101 (agonist) and [¹¹C]WAY100635 (antagonist) in nonhuman primates. PET studies show that both tracers bind to known 5-HT(1A) receptor (5-HT(1A)R)-rich regions of baboon brain. The binding (BP(F)) of [¹¹C]CUMI-101 was lower on an average of 55% across the regions of interest (ROIs) compared to [¹¹C]WAY100635. This ratio is consistent with the in vitro binding data of agonist and antagonist 5-HT(1A)R ligands previously reported.
Assuntos
Encéfalo/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Receptor 5-HT1A de Serotonina/metabolismo , Agonistas do Receptor 5-HT1 de Serotonina/metabolismo , Antagonistas do Receptor 5-HT1 de Serotonina/metabolismo , Animais , Mapeamento Encefálico , Radioisótopos de Carbono , Humanos , Cinética , Ligantes , Papio , Piperazinas/metabolismo , Tomografia por Emissão de Pósitrons , Piridinas/metabolismo , Triazinas/metabolismoRESUMO
RATIONALE: The serotonin 5-HT(1B) receptor is a potential target for the pharmacologic treatment of depression. Positron emission tomography (PET) determination of 5-HT(1B) receptor occupancy with drug candidates targeting this receptor in non-human primate and human subjects may facilitate translation of research from animal models and guide dose selection for clinical studies. AZD3783 is a recently developed, orally bioavailable 5-HT(1B) receptor antagonist with potential antidepressant properties. OBJECTIVES: To determine the relationship between plasma concentration of AZD3783 and occupancy at primate brain 5-HT(1B) receptors using PET and the radioligand [(11)C]AZ10419369. METHODS: PET studies with [(11)C]AZ10419369 were performed in three non-human primates at baseline and after intravenous injection of AZD3783. Subsequently, PET measurements were undertaken in six human subjects at baseline and after administration of different single oral doses of AZD3783 (1-40 mg). RESULTS: After administration in non-human primates and human subjects, AZD3783 reduced regional [(11)C]AZ10419369 binding in a dose-dependent and saturable manner. The AZD3783 plasma concentration required for 50% receptor occupancy (K (i,plasma)) for monkeys was 25 and 27 nmol/L in occipital cortex and striatum, respectively. Corresponding estimates for human occipital cortex and ventral striatum were 24 and 18 nmol/L, respectively. CONCLUSIONS: The potential antidepressant AZD3783 binds in a saturable manner to brain 5-HT(1B) receptors with a similar in vivo affinity for human and monkey receptors. [(11)C]AZ10419369 can be successfully used to determine occupancy at brain 5-HT(1B) receptors in vivo and constitutes a useful tool for dose selection in clinical studies with 5-HT(1B) receptor compounds.
Assuntos
Benzopiranos/metabolismo , Morfolinas/metabolismo , Tomografia por Emissão de Pósitrons/métodos , Receptor 5-HT1B de Serotonina/efeitos dos fármacos , Antagonistas do Receptor 5-HT1 de Serotonina/metabolismo , Administração Oral , Adulto , Animais , Benzopiranos/administração & dosagem , Encéfalo/metabolismo , Relação Dose-Resposta a Droga , Humanos , Macaca fascicularis , Masculino , Morfolinas/administração & dosagem , Piperazinas , Ligação Proteica , Compostos Radiofarmacêuticos , Receptor 5-HT1B de Serotonina/metabolismo , Especificidade da Espécie , Adulto JovemRESUMO
The metabolism, pharmacokinetics, and excretion of a potent and selective 5-hydroxytryptamine(1B) receptor antagonist elzasonan have been studied in six healthy male human subjects after oral administration of a single 10-mg dose of [(14)C]elzasonan. Total recovery of the administered dose was 79% with approximately 58 and 21% of the administered radioactive dose excreted in feces and urine, respectively. The average t(1/2) for elzasonan was 31.5 h. Elzasonan was extensively metabolized, and excreta and plasma were analyzed using mass spectrometry and NMR spectroscopy to elucidate the structures of metabolites. The major component of drug-related material in the excreta was in the feces and was identified as 5-hydroxyelzasonan (M3), which accounted for approximately 34% of the administered dose. The major human circulating metabolite was identified as the novel cyclized indole metabolite (M6) and accounted for â¼65% of the total radioactivity. A mechanism for the formation of M6 is proposed. Furthermore, metabolism-dependent covalent binding of drug-related material was observed upon incubation of [(14)C]elzasonan with liver microsomes, and data suggest that an indole iminium ion is involved. Overall, the major metabolic pathways of elzasonan were due to aromatic hydroxylation(s) of the benzylidene moiety, N-oxidation at the piperazine ring, N-demethylation, indirect glucuronidation, and oxidation, ring closure, and subsequent rearrangement to form M6.