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1.
Eur J Med Chem ; 164: 378-390, 2019 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-30611057

RESUMO

As part of an effort to identify druggable diacylglycerol kinase alpha (DGKα) inhibitors, we used an in-silico approach based on chemical homology with the two commercially available DGKα inhibitors R59022 and R59949. Ritanserin and compound AMB639752 emerged from the screening of 127 compounds, showing an inhibitory activity superior to the two commercial inhibitors, being furthermore specific for the alpha isoform of diacylglycerol kinase. Interestingly, AMB639752 was also devoid of serotoninergic activity. The ability of both ritanserin and AMB639752, by inhibiting DGKα in intact cells, to restore restimulation induced cell death (RICD) in SAP deficient lymphocytes was also tested. Both compounds restored RICD at concentrations lower than the two previously available inhibitors, indicating their potential use for the treatment of X-linked lymphoproliferative disease 1 (XLP-1), a rare genetic disorder in which DGKα activity is deregulated.


Assuntos
Diacilglicerol Quinase/antagonistas & inibidores , Avaliação Pré-Clínica de Medicamentos/métodos , Transtornos Linfoproliferativos/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Morte Celular/efeitos dos fármacos , Simulação por Computador , Humanos , Piperidinas , Pirimidinonas , Quinazolinonas , Ritanserina , Tiazóis
2.
Mol Pharmacol ; 94(5): 1246-1255, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30158316

RESUMO

Ritanserin was tested in the clinic as a serotonin receptor inverse agonist but recently emerged as a novel kinase inhibitor with potential applications in cancer. Here, we discovered that ritanserin induced apoptotic cell death of non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) cells via a serotonin-independent mechanism. We used quantitative chemical proteomics to reveal a ritanserin-dependent kinase network that includes key mediators of lipid [diacylglycerol kinase α, phosphatidylinositol 4-kinase ß] and protein [feline encephalitis virus-related kinase, rapidly accelerated fibrosarcoma (RAF)] signaling, metabolism [eukaryotic elongation factor 2 kinase, eukaryotic translation initiation factor 2-α kinase 4], and DNA damage response [tousled-like kinase 2] to broadly kill lung tumor cell types. Whereas ritanserin exhibited polypharmacology in NSCLC proteomes, this compound showed unexpected specificity for c-RAF in the SCLC subtype, with negligible activity against other kinases mediating mitogen-activated protein kinase signaling. Here we show that ritanserin blocks c-RAF but not B-RAF activation of established oncogenic signaling pathways in live cells, providing evidence in support of c-RAF as a key target mediating its anticancer activity. Given the role of c-RAF activation in RAS-mutated cancers resistant to clinical B-RAF inhibitors, our findings may have implications in overcoming resistance mechanisms associated with c-RAF biology. The unique target landscape combined with acceptable safety profiles in humans provides new opportunities for repositioning ritanserin in cancer.


Assuntos
Apoptose/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma de Células Pequenas/metabolismo , Carcinoma de Células Pequenas/patologia , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Proteômica , Ritanserina/farmacologia , Sequência de Aminoácidos , Linhagem Celular Tumoral , Reposicionamento de Medicamentos , Células HEK293 , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-raf/química , Proteínas Proto-Oncogênicas c-raf/efeitos dos fármacos , Serotonina/metabolismo
3.
J Virol ; 92(8)2018 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-29437972

RESUMO

JC polyomavirus (JCPyV) establishes a lifelong persistence in roughly half the human population worldwide. The cells and tissues that harbor persistent virus in vivo are not known, but renal tubules and other urogenital epithelial cells are likely candidates as virus is shed in the urine of healthy individuals. In an immunosuppressed host, JCPyV can become reactivated and cause progressive multifocal leukoencephalopathy (PML), a fatal demyelinating disease of the central nervous system. Recent observations indicate that JCPyV may productively interact with cells in the choroid plexus and leptomeninges. To further study JCPyV infection in these cells, primary human choroid plexus epithelial cells and meningeal cells were challenged with virus, and their susceptibility to infection was compared to the human glial cell line, SVG-A. We found that JCPyV productively infects both choroid plexus epithelial cells and meningeal cells in vitro Competition with the soluble receptor fragment LSTc reduced virus infection in these cells. Treatment of cells with neuraminidase also inhibited both viral infection and binding. Treatment with the serotonin receptor antagonist, ritanserin, reduced infection in SVG-A and meningeal cells. We also compared the ability of wild-type and sialic acid-binding mutant pseudoviruses to transduce these cells. Wild-type pseudovirus readily transduced all three cell types, but pseudoviruses harboring mutations in the sialic acid-binding pocket of the virus failed to transduce the cells. These data establish a novel role for choroid plexus and meninges in harboring virus that likely contributes not only to meningoencephalopathies but also to PML.IMPORTANCE JCPyV infects greater than half the human population worldwide and causes central nervous system disease in patients with weakened immune systems. Several recent reports have found JCPyV in the choroid plexus and leptomeninges of patients with encephalitis. Due to their role in forming the blood-cerebrospinal fluid barrier, the choroid plexus and leptomeninges are also poised to play roles in virus invasion of brain parenchyma, where infection of macroglial cells leads to the development of progressive multifocal leukoencephalopathy, a severely debilitating and often fatal infection. In this paper we show for the first time that primary choroid plexus epithelial cells and meningeal cells are infected by JCPyV, lending support to the association of JCPyV with meningoencephalopathies. These data also suggest that JCPyV could use these cells as reservoirs for the subsequent invasion of brain parenchyma.


Assuntos
Plexo Corióideo , Células Epiteliais , Vírus JC/metabolismo , Leucoencefalopatia Multifocal Progressiva , Meninges , Ritanserina/farmacologia , Linhagem Celular , Plexo Corióideo/metabolismo , Plexo Corióideo/patologia , Plexo Corióideo/virologia , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Células Epiteliais/virologia , Humanos , Leucoencefalopatia Multifocal Progressiva/tratamento farmacológico , Leucoencefalopatia Multifocal Progressiva/metabolismo , Leucoencefalopatia Multifocal Progressiva/patologia , Leucoencefalopatia Multifocal Progressiva/virologia , Meninges/metabolismo , Meninges/patologia , Meninges/virologia
4.
Cell ; 172(4): 719-730.e14, 2018 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-29398112

RESUMO

Drugs frequently require interactions with multiple targets-via a process known as polypharmacology-to achieve their therapeutic actions. Currently, drugs targeting several serotonin receptors, including the 5-HT2C receptor, are useful for treating obesity, drug abuse, and schizophrenia. The competing challenges of developing selective 5-HT2C receptor ligands or creating drugs with a defined polypharmacological profile, especially aimed at G protein-coupled receptors (GPCRs), remain extremely difficult. Here, we solved two structures of the 5-HT2C receptor in complex with the highly promiscuous agonist ergotamine and the 5-HT2A-C receptor-selective inverse agonist ritanserin at resolutions of 3.0 Å and 2.7 Å, respectively. We analyzed their respective binding poses to provide mechanistic insights into their receptor recognition and opposing pharmacological actions. This study investigates the structural basis of polypharmacology at canonical GPCRs and illustrates how understanding characteristic patterns of ligand-receptor interaction and activation may ultimately facilitate drug design at multiple GPCRs.


Assuntos
Ergotamina/química , Receptor 5-HT2C de Serotonina/química , Ritanserina/química , Agonistas do Receptor 5-HT2 de Serotonina/química , Antagonistas do Receptor 5-HT2 de Serotonina/química , Células HEK293 , Humanos , Obesidade/tratamento farmacológico , Obesidade/metabolismo , Domínios Proteicos , Receptor 5-HT2C de Serotonina/metabolismo , Esquizofrenia/tratamento farmacológico , Esquizofrenia/metabolismo , Relação Estrutura-Atividade , Transtornos Relacionados ao Uso de Substâncias/tratamento farmacológico , Transtornos Relacionados ao Uso de Substâncias/metabolismo
6.
Biochemistry ; 57(2): 231-236, 2018 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-29155586

RESUMO

Diacylglycerol kinases (DGKs) regulate lipid metabolism and cell signaling through ATP-dependent phosphorylation of diacylglycerol to biosynthesize phosphatidic acid. Selective chemical probes for studying DGKs are currently lacking and are needed to annotate isoform-specific functions of these elusive lipid kinases. Previously, we explored fragment-based approaches to discover a core fragment of DGK-α (DGKα) inhibitors responsible for selective binding to the DGKα active site. Here, we utilize quantitative chemical proteomics to deconstruct widely used DGKα inhibitors to identify structural regions mediating off-target activity. We tested the activity of a fragment (RLM001) derived from a nucleotide-like region found in the DGKα inhibitors R59022 and ritanserin and discovered that RLM001 mimics ATP in its ability to broadly compete at ATP-binding sites of DGKα as well as >60 native ATP-binding proteins (kinases and ATPases) detected in cell proteomes. Equipotent inhibition of activity-based probe labeling by RLM001 supports a contiguous ligand-binding site composed of C1, DAGKc, and DAGKa domains in the DGKα active site. Given the lack of available crystal structures of DGKs, our studies highlight the utility of chemical proteomics in revealing active-site features of lipid kinases to enable development of inhibitors with enhanced selectivity against the human proteome.


Assuntos
Diacilglicerol Quinase/antagonistas & inibidores , Inibidores Enzimáticos/química , Proteômica/métodos , Ritanserina/análogos & derivados , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Diacilglicerol Quinase/metabolismo , Relação Dose-Resposta a Droga , Desenho de Drogas , Estrutura Molecular , Proteínas Recombinantes/metabolismo , Ritanserina/química , Ritanserina/metabolismo , Ritanserina/farmacologia , Relação Estrutura-Atividade
7.
Neuro Oncol ; 20(2): 192-202, 2018 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-29048560

RESUMO

Background: The mesenchymal phenotype in glioblastoma (GBM) and other cancers drives aggressiveness and treatment resistance, leading to therapeutic failure and recurrence of disease. Currently, there is no successful treatment option available against the mesenchymal phenotype. Methods: We classified patient-derived GBM stem cell lines into 3 subtypes: proneural, mesenchymal, and other/classical. Each subtype's response to the inhibition of diacylglycerol kinase alpha (DGKα) was compared both in vitro and in vivo. RhoA activation, liposome binding, immunoblot, and kinase assays were utilized to elucidate the novel link between DGKα and geranylgeranyltransferase I (GGTase I). Results: Here we show that inhibition of DGKα with a small-molecule inhibitor, ritanserin, or RNA interference preferentially targets the mesenchymal subtype of GBM. We show that the mesenchymal phenotype creates the sensitivity to DGKα inhibition; shifting GBM cells from the proneural to the mesenchymal subtype increases ritanserin activity, with similar effects in epithelial-mesenchymal transition models of lung and pancreatic carcinoma. This enhanced sensitivity of mesenchymal cancer cells to ritanserin is through inhibition of GGTase I and downstream mediators previously associated with the mesenchymal cancer phenotype, including RhoA and nuclear factor-kappaB. DGKα inhibition is synergistic with both radiation and imatinib, a drug preferentially affecting proneural GBM. Conclusions: Our findings demonstrate that a DGKα-GGTase I pathway can be targeted to combat the treatment-resistant mesenchymal cancer phenotype. Combining therapies with greater activity against each GBM subtype may represent a viable therapeutic option against GBM.


Assuntos
Neoplasias Encefálicas/tratamento farmacológico , Diacilglicerol Quinase/antagonistas & inibidores , Glioblastoma/patologia , Ritanserina/farmacologia , Animais , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Diacilglicerol Quinase/genética , Feminino , Humanos , Camundongos Endogâmicos BALB C , NF-kappa B/metabolismo , Transdução de Sinais/efeitos dos fármacos
8.
Neuroreport ; 29(1): 54-58, 2018 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-29135714

RESUMO

It has been suggested that serotonin (5-HT) may be implicated in functional recovery after stroke; however, the underlying molecular mechanisms remain unknown. Here, the role of 5-HT was verified using ritanserin, a potent 5-HT2A receptor antagonist, and protein expression and modification were analyzed to further understand the association between paralysis recovery and molecular mechanisms in the brain. Experimental cerebral cortex infarctions were induced by photothrombosis in rats. Voluntary exercise was initiated 2 days after surgery. Motor performance was then measured using the rotarod test. Differences in protein expression and phosphorylation in the perilesional cortex were analyzed using western blot. In behavioral evaluations, performance in the rotarod test was significantly increased by exercise. However, there was a significantly lower value in time until falling after combined exercise and ritanserin administration compared with that of exercise alone. Protein expression analysis revealed that phosphorylation of protein kinase C (PKC) α, PKCε, and growth-associated protein 43 (GAP43) was significantly upregulated by exercise. These effects were attenuated by ritanserin administration. These data suggest that 5-HT may be related to the underlying mechanisms of exercise-dependent paralysis recovery, that is, exercise-dependent plasticity through the phosphorylation of PKC and GAP43.


Assuntos
Infarto Cerebral/complicações , Infarto Cerebral/tratamento farmacológico , Recuperação de Função Fisiológica/efeitos dos fármacos , Ritanserina/uso terapêutico , Antagonistas da Serotonina/uso terapêutico , Animais , Infarto Cerebral/reabilitação , Modelos Animais de Doenças , Proteína GAP-43/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Locomoção/efeitos dos fármacos , Masculino , Atividade Motora/efeitos dos fármacos , Condicionamento Físico Animal/métodos , Proteína Quinase C/metabolismo , Ratos , Ratos Sprague-Dawley
9.
J Headache Pain ; 18(1): 104, 2017 Oct 11.
Artigo em Inglês | MEDLINE | ID: mdl-29022279

RESUMO

BACKGROUND: Dihydroergotamine (DHE) is an acute antimigraine agent that displays affinity for dopamine D2-like receptors, serotonin 5-HT1/2 receptors and α1/α2-adrenoceptors. Since activation of vascular α1/α2-adrenoceptors results in systemic vasopressor responses, the purpose of this study was to investigate the specific role of α1- and α2-adrenoceptors mediating DHE-induced vasopressor responses using several antagonists for these receptors. METHODS: For this purpose, 135 male Wistar rats were pithed and divided into 35 control and 100 pretreated i.v. with ritanserin (100 µg/kg; to exclude the 5-HT2 receptor-mediated systemic vasoconstriction). Then, the vasopressor responses to i.v. DHE (1-3100 µg/kg, given cumulatively) were determined after i.v. administration of some α1/α2-adrenoceptor antagonists. RESULTS: In control animals (without ritanserin pretreatment), the vasopressor responses to DHE were: (i) unaffected after prazosin (α1; 30 µg/kg); (ii) slightly, but significantly, blocked after rauwolscine (α2; 300 µg/kg); and (iii) markedly blocked after prazosin (30 µg/kg) plus rauwolscine (300 µg/kg). In contrast, after pretreatment with ritanserin, the vasopressor responses to DHE were: (i) attenuated after prazosin (α1; 10 and 30 µg/kg) or rauwolscine (α2; 100 and 300 µg/kg); (ii) markedly blocked after prazosin (30 µg/kg) plus rauwolscine (300 µg/kg); (iii) attenuated after 5-methylurapidil (α1A; 30-100 µg/kg), L-765,314 (α1B; 100 µg/kg), BMY 7378 (α1D; 30-100 µg/kg), BRL44408 (α2A; 100-300 µg/kg), imiloxan (α2B; 1000-3000 µg/kg) or JP-1302 (α2C; 1000 µg/kg); and (iv) unaffected after the corresponding vehicles (1 ml/kg). CONCLUSION: These results suggest that the DHE-induced vasopressor responses in ritanserin-pretreated pithed rats are mediated by α1- (probably α1A, α1B and α1D) and α2- (probably α2A, α2B and α2C) adrenoceptors. These findings could shed light on the pharmacological profile of the vascular side effects (i.e. systemic vasoconstriction) produced by DHE and may lead to the development of more selective antimigraine drugs devoid vascular side effects.


Assuntos
Di-Hidroergotamina/farmacologia , Receptores Adrenérgicos alfa 1/fisiologia , Receptores Adrenérgicos alfa 2/fisiologia , Ritanserina/farmacologia , Vasoconstrição/fisiologia , Vasoconstritores/farmacologia , Animais , Pressão Sanguínea/efeitos dos fármacos , Pressão Sanguínea/fisiologia , Relação Dose-Resposta a Droga , Masculino , Ratos , Ratos Wistar , Antagonistas da Serotonina/farmacologia , Vasoconstrição/efeitos dos fármacos
10.
Cell Chem Biol ; 24(7): 870-880.e5, 2017 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-28712745

RESUMO

Diacylglycerol kinases (DGKs) are integral components of signal transduction cascades that regulate cell biology through ATP-dependent phosphorylation of the lipid messenger diacylglycerol. Methods for direct evaluation of DGK activity in native biological systems are lacking and needed to study isoform-specific functions of these multidomain lipid kinases. Here, we utilize ATP acyl phosphate activity-based probes and quantitative mass spectrometry to define, for the first time, ATP and small-molecule binding motifs of representative members from all five DGK subtypes. We use chemical proteomics to discover an unusual binding mode for the DGKα inhibitor, ritanserin, including interactions at the atypical C1 domain distinct from the ATP binding region. Unexpectedly, deconstruction of ritanserin yielded a fragment compound that blocks DGKα activity through a conserved binding mode and enhanced selectivity against the kinome. Collectively, our studies illustrate the power of chemical proteomics to profile protein-small molecule interactions of lipid kinases for fragment-based lead discovery.


Assuntos
Diacilglicerol Quinase/metabolismo , Ligantes , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Sítios de Ligação , Cromatografia Líquida de Alta Pressão , Diacilglicerol Quinase/química , Diacilglicerol Quinase/genética , Células HEK293 , Humanos , Marcação por Isótopo , Ketanserina/química , Ketanserina/metabolismo , Peptídeos/análise , Ligação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteoma/análise , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Ritanserina/química , Ritanserina/metabolismo , Espectrometria de Massas em Tandem
11.
J Pharmacol Sci ; 134(2): 131-138, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28647281

RESUMO

We previously demonstrated that nicotine elicited kinetic tremor by elevating the neural activity of the inferior olive via α7 nicotinic acetylcholine (nACh) receptors. Since α7 nACh receptors reportedly facilitate synaptic monoamine release, we explored the role of 5-HT receptors in induction and/or modulation of nicotine tremor. Treatment of mice with nicotine induced kinetic tremor that normally appeared during movement. The 5-HT1A agonist, 8-hydroxydipropylaminotetraline (8-OH-DPAT), significantly enhanced nicotine-induced tremor and the action of 8-OH-DPAT was antagonized by WAY-100135 (5-HT1A antagonist). In addition, the cerebral 5-HT depletion by repeated treatment with p-chlorophenylalanine did not reduce, but rather potentiated the facilitatory effects of 8-OH-DPAT. In contrast, the 5-HT2 agonist, 2,5-dimethoxy-4-iodoamphetamine (DOI), significantly attenuated nicotine tremor, which was antagonized by ritanserin (5-HT2 antagonist). The 5-HT3 agonist SR-57227 did not affect nicotine-induced tremor. Furthermore, when testing the direct actions of 5-HT antagonists, nicotine tremor was inhibited by WAY-100135, but was unaffected by ritanserin, ondansetron (5-HT3 antagonist) or SB-258585 (5-HT6 antagonist). These results suggest that postsynaptic 5-HT1A receptors are involved in induction of nicotine tremor mediated by α7 nACh receptors. In addition, 5-HT2 receptors have an inhibitory modulatory role in induction of nicotine tremor.


Assuntos
Nicotina/toxicidade , Receptores de Serotonina/metabolismo , Tremor/metabolismo , 8-Hidroxi-2-(di-n-propilamino)tetralina/farmacologia , 8-Hidroxi-2-(di-n-propilamino)tetralina/uso terapêutico , Anfetaminas/farmacologia , Animais , Fenclonina/farmacologia , Humanos , Masculino , Camundongos , Ondansetron/farmacologia , Piperazinas/farmacologia , Receptor 5-HT1A de Serotonina/metabolismo , Ritanserina/farmacologia , Antagonistas da Serotonina/farmacologia , Agonistas do Receptor de Serotonina/farmacologia , Agonistas do Receptor de Serotonina/uso terapêutico , Tremor/induzido quimicamente , Tremor/tratamento farmacológico
12.
Behav Brain Res ; 325(Pt A): 72-78, 2017 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-28202410

RESUMO

Dysfunctions of the serotonergic system have been suggested to be important in the neurobiology of schizophrenia. Patients with schizophrenia exhibit deficits in an operational measure of sensorimotor gating: prepulse inhibition (PPI) of startle. PPI is the normal reduction in the startle response caused by a low intensity non-startling stimulus (prepulse) which is presented shortly before the startle stimulus (pulse). The hallucinogen 2,5-dimethoxy-4-iodoamphetamine (DOI), a 5-hydroxytryptamine(HT)2 receptor agonist disrupted PPI in rats. The inferior colliculus (IC) is a critical nucleus of the auditory pathway mediating acoustic PPI. The activation of the IC by the acoustic prepulse reduces startle magnitude. The present study investigated the role of serotonergic transmission in the IC on the expression of acoustic PPI. For that we investigated whether 5-HT2A receptor activation or blockade would affect this response. Unilateral microinjection of DOI (10µg/0.3µl) into the IC disrupted PPI, while microinjection of the 5-HT2A receptor antagonist ritanserin (4µg/0.3µl), into this structure did not alter PPI. We also examined the ability of the atypical antipsychotic clozapine (5.0mg/kg; I.P.) to reverse the disruption of PPI produced by unilateral microinjections of DOI into the IC of rats. Pretreatment with clozapine blocked DOI-induced disruption of PPI. Altogether, these results suggest that serotonin-mediated mechanisms of the IC are involved in the expression of PPI in rodents and that this response is sensitive to atypical antipsychotic clozapine.


Assuntos
Anfetaminas/administração & dosagem , Antipsicóticos/administração & dosagem , Clozapina/administração & dosagem , Colículos Inferiores/efeitos dos fármacos , Colículos Inferiores/fisiologia , Inibição Pré-Pulso/efeitos dos fármacos , Animais , Masculino , Microinjeções , Ratos Wistar , Receptor 5-HT2A de Serotonina/fisiologia , Ritanserina/administração & dosagem , Agonistas do Receptor 5-HT2 de Serotonina/administração & dosagem , Antagonistas do Receptor 5-HT2 de Serotonina/administração & dosagem
13.
Biochem Pharmacol ; 123: 29-39, 2017 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-27974147

RESUMO

Diacylglycerol kinase alpha (DGKα) catalyzes the conversion of diacylglycerol (DAG) to phosphatidic acid (PA). Recently, DGKα was identified as a therapeutic target in various cancers, as well as in immunotherapy. Application of small-molecule DGK inhibitors, R59022 and R59949, induces cancer cell death in vitro and in vivo. The pharmacokinetics of these compounds in mice, however, are poor. Thus, there is a need to discover additional DGK inhibitors not only to validate these enzymes as targets in oncology, but also to achieve a better understanding of their biology. In the present study, we investigate the activity of ritanserin, a compound structurally similar to R59022, against DGKα. Ritanserin, originally characterized as a serotonin (5-HT) receptor (5-HTR) antagonist, underwent clinical trials as a potential medicine for the treatment of schizophrenia and substance dependence. We document herein that ritanserin attenuates DGKα kinase activity while increasing the enzyme's affinity for ATP in vitro. In addition, R59022 and ritanserin function as DGKα inhibitors in cultured cells and activate protein kinase C (PKC). While recognizing that ritanserin attenuates DGK activity, we also find that R59022 and R59949 are 5-HTR antagonists. In conclusion, ritanserin, R59022 and R59949 are combined pharmacological inhibitors of DGKα and 5-HTRs in vitro.


Assuntos
Diacilglicerol Quinase/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Pirimidinonas/farmacologia , Ritanserina/farmacologia , Tiazóis/farmacologia , Trifosfato de Adenosina/metabolismo , Diacilglicerol Quinase/metabolismo , Eletroforese em Gel de Poliacrilamida , Células HEK293 , Células HeLa , Humanos , Cinética
14.
Behav Brain Res ; 316: 294-304, 2017 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-27616344

RESUMO

The electrical stimulation of the dorsolateral columns of the periaquedutal grey matter (dlPAG) or deep layers of the superior colliculus (dlSC) evokes defensive behaviours followed by an antinociceptive response. Monoaminergic brainstem reticular nuclei are suggested to comprise the endogenous pain modulatory system. The aim of the present work was to investigate the role played by 5-HT2 subfamily of serotonergic receptors of the nucleus raphe magnus (NRM) and the gigantocellularis/paragigantocellularis pars α reticular nuclei (Gi/PGiα) in the elaboration of instinctive fear-induced antinociception elicited by electrical stimulation of dlPAG or of dlSC. The nociceptive thresholds were measured by the tail-flick test in Wistar rats. The 5-HT2A/2C-serotonergic receptors antagonist ritanserin was microinjected at different concentrations (0.05, 0.5 and 5.0µg/0.2µL) either in Gi/PGiα or in NRM. The blockade of 5-HT2 receptors in both Gi/PGiα and NRM decreased the innate fear-induced antinociception elicited by electrical stimulation of the dlSC or the dlPAG. These findings indicate that serotonin is involved in the hypo-algesia induced by unconditioned fear-induced behavioural responses and the 5-HT2A/2C-serotonergic receptor subfamily in neurons situated in the Gi/PGiα complex and NRM are critically recruited in pain modulation during the panic-like emotional behaviour.


Assuntos
Medo/fisiologia , Núcleo Magno da Rafe/metabolismo , Substância Cinzenta Periaquedutal/fisiologia , Núcleos da Rafe/metabolismo , Receptor 5-HT2A de Serotonina/metabolismo , Receptor 5-HT2C de Serotonina/metabolismo , Colículos Superiores/fisiologia , Animais , Condicionamento Clássico , Estimulação Elétrica , Masculino , Vias Neurais/fisiologia , Dor/patologia , Dor/fisiopatologia , Medição da Dor , Limiar da Dor/fisiologia , Ratos , Ratos Wistar , Ritanserina/farmacologia , Antagonistas da Serotonina/farmacologia
15.
Behav Brain Res ; 314: 181-9, 2016 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-27506653

RESUMO

Little is known about the pharmacological effects of amphetamine derivatives. In the present study, the effect on social preference and anxiety-like behavior of 2,5-dimetoxy-4-bromo-amphetamine hydrobromide (DOB) and para-methoxyamphetamine (PMA), in comparison with 3,4 methylenedioxymethamphetamine (MDMA) was investigated in zebrafish, an emerging model to study emotional behavior in an inexpensive and quick manner. DOB (0.05-2mg/kg), PMA (0.0005-2mg/kg) or MDMA (0.25-20mg/kg), given i.m. to adult zebrafish, progressively increased the time spent in the proximity of nacre fish picture in a social preference test. However, high doses were ineffective. Similarly, in the novel tank diving and light-dark tests the compounds elicited a progressive anxiolytic effect in terms of time spent in the upper half of the tank and in the light compartment, respectively. All the above effects were interpolated by symmetrical parabolas. The 5-HT2A/C antagonist ritanserin (0.025-2.5mg/kg) in association with the maximal effective dose of MDMA, DOB and PMA blocked both the social and anxiolytic effect. Taken together these findings demonstrate for the first time the prosocial and anxiolytic properties of DOB and PMA and focus on the mechanisms of their action through the serotonergic-like system suggesting a potential clinical application.


Assuntos
Ansiolíticos/farmacologia , Comportamento Animal/efeitos dos fármacos , N-Metil-3,4-Metilenodioxianfetamina/farmacologia , Ritanserina/farmacologia , Animais , Ansiedade/tratamento farmacológico , Ansiedade/psicologia , Alucinógenos/farmacologia , Atividade Motora/efeitos dos fármacos , Peixe-Zebra
16.
Psychopharmacology (Berl) ; 233(15-16): 3031-9, 2016 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-27318987

RESUMO

RATIONALE: The synthetic phenethylamines are recreational drugs known to produce psychostimulant effects. However, their abuse potential has not been widely studied. OBJECTIVES: Here, we investigated the rewarding and the hallucinatory effects of 2,5-dimetoxy-4-bromo-amphetamine hydrobromide (DOB) and para-methoxyamphetamine (PMA) in comparison with the classical 3,4-methylenedioxymethamphetamine (MDMA). In addition, the role of serotonin 5-HT2-like receptor on the abovementioned effects was evaluated. METHODS: Zebrafish were intramuscularly (i.m.) treated with a wide range of doses of DOB (0.1-20 mg/kg), PMA (0.0005-2 mg/kg), or MDMA (0.5-160 mg/kg). Animals were submitted to a conditioned place preference (CPP) task, to investigation of the rewarding properties, and to the evaluation of hallucinatory behavior in terms of appearance of a trance-like behavior. The serotonin 5-HT2 subtype receptor antagonist ritanserin (0.025-2.5 mg/kg) in association with the maximal effective dose of MDMA, DOB, and PMA was given i.m., and the effect on CPP or hallucinatory behavior was evaluated. RESULTS: MDMA and its derivatives exhibited CPP in a biphasic fashion, being PMA the most potent. This effect was accompanied, for DOB (2 mg/kg) and PMA (0.1 mg/kg), by a trance-like hallucinatory behavior. MDMA at a high dose as 160 mg/kg did not induce any hallucinatory behavior. Ritanserin significantly blocked the rewarding and hallucinatory effects suggesting the involvement of serotonin 5HT2 subtype receptor. CONCLUSION: Collectively, these findings demonstrate for the first time that the rewarding properties of DOB and PMA are accompanied by hallucinatory behavior through a serotonergic system and reinforce zebrafish as an emerging experimental model for screening new hallucinogens.


Assuntos
2,5-Dimetoxi-4-Metilanfetamina/análogos & derivados , Anfetaminas/farmacologia , Comportamento Animal/efeitos dos fármacos , Alucinógenos/farmacologia , N-Metil-3,4-Metilenodioxianfetamina/farmacologia , Receptores 5-HT2 de Serotonina/metabolismo , Agonistas do Receptor de Serotonina/farmacologia , 2,5-Dimetoxi-4-Metilanfetamina/farmacologia , Animais , Feminino , Masculino , Recompensa , Ritanserina/farmacologia , Antagonistas do Receptor 5-HT2 de Serotonina/farmacologia , Peixe-Zebra
17.
Brain Res Bull ; 121: 59-67, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26772625

RESUMO

Fast ripples (FR, 250-600 Hz) are field potentials that occur only in those areas capable of generating seizures, such as the hippocampus, and modulation of FR by serotonin has been reported. Therefore, we hypothesized that the receptor antagonists 5HT1A and 5HT2A, B, C will increase FR in rats treated with kainic acid (KA, 0.8 µg/0.5 µl). For this purpose, the intracranial EEG recordings of the hippocampus from animals treated with KA and the serotonin antagonists WAY100135 and ritanserin (dose 0.2mg/Kg, i.p) were analyzed. In addition, morphologic parameters were analyzed after staining samples with cresyl violet, Timm stain, NeuN and GFAP and observing immunofluorescence. The results showed an increase in the number of events of FR (p<0.0001) and duration of each FR event after the administration of WAY100135 (p<0.030). Additionally, there was an increase in the number of events of FR (p<0.0001) and amplitude of FR after ritanserin administration (p<0.014). In relation to changes in unspecified cells, there was a significant decrement in the width of the CA3 pyramidal layer of the hippocampus (p<0.001), and there were no significant changes in reactive glia and fiber sprouting. However, a slight gain of astrocytes marked with GFAP and larger astrocytes with more projections were observed. In conclusion, these results support the modulation of FR by serotonin with participation of the 5HT1A receptor as a possible mediator of the effect. However the exact mechanisms resulting in such effect is not known.


Assuntos
Ondas Encefálicas/efeitos dos fármacos , Agonistas de Aminoácidos Excitatórios/farmacologia , Ácido Caínico/farmacologia , Convulsões/induzido quimicamente , Convulsões/fisiopatologia , Antagonistas da Serotonina/farmacologia , Análise de Variância , Animais , Relação Dose-Resposta a Droga , Eletroencefalografia , Proteína Glial Fibrilar Ácida/metabolismo , Masculino , Fosfopiruvato Hidratase/metabolismo , Piperazinas/farmacologia , Ratos , Ratos Wistar , Ritanserina/farmacologia
18.
Amino Acids ; 48(2): 349-56, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26371055

RESUMO

Glycine receptors (GlyRs) permeable to chloride only mediate tonic inhibition in the cerebral cortex where glycinergic projection is completely absent. The functional modulation of GlyRs was largely studied in subcortical brain regions with glycinergic transmissions, but the function of cortical GlyRs was rarely addressed. Serotonin could broadly modulate many ion channels through activating 5-HT2 receptor, but whether cortical GlyRs are subjected to serotonergic modulation remains unexplored. The present study adopted patch clamp recordings to examine functional regulation of strychnine-sensitive GlyRs currents in cultured cortical neurons by DOI (2,5-Dimethoxy-4-iodoamphetamine), a 5-HT2A/C receptor agonist. DOI caused a concentration-dependent reduction of GlyR currents with unchanged reversal potential. This reduction was blocked by the selective receptor antagonists (ritanserin and risperidone) and G protein inhibitor (GDP-ß-s) demonstrated that the reducing effect of DOI on GlyR current required the activation of 5-HT2A/C receptors. Strychnine-sensitive tonic currents revealed the inhibitory tone mediated by nonsynaptic GlyRs, and DOI similarly reduced the tonic inhibition. The impaired microtube-dependent trafficking or clustering of GlyRs was thought to be involved in that nocodazole as a microtube depolymerizing drug largely blocked the inhibition mediated by 5-HT2A/C receptors. Our results suggested that activation of 5-HT2A/C receptors might suppress cortical tonic inhibition mediated by GlyRs, and the findings would provide important insight into serotonergic modulation of tonic inhibition mediated by GlyRs, and possibly facilitate to develop the therapeutic treatment of neurological diseases such as tinnitus through regulating cortical GlyRs.


Assuntos
Córtex Auditivo/metabolismo , Neurônios/metabolismo , Receptor 5-HT2A de Serotonina/metabolismo , Receptor 5-HT2C de Serotonina/metabolismo , Receptores da Glicina/metabolismo , Anfetaminas/farmacologia , Animais , Células Cultivadas , Proteínas de Ligação ao GTP/metabolismo , Guanosina Difosfato/análogos & derivados , Guanosina Difosfato/farmacologia , Canais Iônicos/metabolismo , Microtúbulos/metabolismo , Nocodazol/farmacologia , Técnicas de Patch-Clamp , Ratos , Ratos Sprague-Dawley , Risperidona/farmacologia , Ritanserina/farmacologia , Agonistas do Receptor 5-HT2 de Serotonina/farmacologia , Antagonistas do Receptor 5-HT2 de Serotonina/farmacologia , Estricnina/farmacologia , Tionucleotídeos/farmacologia , Moduladores de Tubulina/farmacologia
19.
Expert Rev Anti Infect Ther ; 14(2): 177-91, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26655489

RESUMO

Progressive multifocal encephalopathy (PML) caused by JC virus was frequently encountered in AIDS patients before combination antiretroviral therapy (cART). Incidence decreased and the outcome improved with cART. The immune reconstitution with cART is beneficial for HIV-infected patients and is an effective treatment for PML. However, when it is excessive an inflammatory response immune syndrome might occur with deterioration of PML. So far, no specific therapy has proven efficacious in small clinical trials in spite of some optimistic case reports. Combination of drugs targeted at different stages of JC virus life cycle seems to have a better effect. Passive and active immune therapies, immune competence "boosters" appear promising. New future approaches such as gene editing are not far away.


Assuntos
Fármacos Anti-HIV/uso terapêutico , Terapia Antirretroviral de Alta Atividade/métodos , Antivirais/uso terapêutico , Infecções por HIV/tratamento farmacológico , Síndrome Inflamatória da Reconstituição Imune/induzido quimicamente , Leucoencefalopatia Multifocal Progressiva/tratamento farmacológico , Antagonistas da Serotonina/uso terapêutico , Cidofovir , Coinfecção , Citarabina/uso terapêutico , Citosina/análogos & derivados , Citosina/uso terapêutico , Infecções por HIV/complicações , Infecções por HIV/imunologia , Humanos , Síndrome Inflamatória da Reconstituição Imune/imunologia , Leucoencefalopatia Multifocal Progressiva/complicações , Leucoencefalopatia Multifocal Progressiva/imunologia , Mefloquina/uso terapêutico , Mianserina/análogos & derivados , Mianserina/uso terapêutico , Mirtazapina , Organofosfonatos/uso terapêutico , Ritanserina/uso terapêutico
20.
Behav Pharmacol ; 26(7 Spec No): 681-90, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25932719

RESUMO

We have recently found that isolation-reared mice show hyperactivity during an encounter with an intruder. However, it is not known whether encounter-induced hyperactivity may model some aspects of psychiatric disorders. The present study examined the pharmacological profile of encounter-induced hyperactivity in isolation-reared mice. Encounter-induced hyperactivity was reduced by acute administration of various antidepressants including the tricyclic antidepressant desipramine (10 mg/kg), the selective serotonin (5-HT) reuptake inhibitors fluvoxamine (10 mg/kg) and paroxetine (10 mg/kg), the 5-HT/noradrenaline reuptake inhibitors venlafaxine (10 mg/kg) and duloxetine (10 mg/kg), the antipsychotic drug risperidone (0.01 mg/kg), the 5-HT2 antagonist ritanserin (1 mg/kg), and the glucocorticoid receptor antagonist RU-43044 (30 mg/kg). The α2 adrenoceptor agonist clonidine (0.03 mg/kg) and the 5-HT4 receptor agonist BIMU8 (30 mg/kg) also reduced encounter-induced hyperactivity. The effect of desipramine was blocked by the α2 adrenoceptor antagonist idazoxan (0.3 mg/kg). The effect of fluvoxamine was blocked by the 5-HT4 receptor antagonist GR125487 (3 mg/kg), but not the 5-HT1A receptor antagonist WAY100635 (1 mg/kg), the 5-HT3 receptor antagonist azasetron (3 mg/kg), or the 5-HT6 receptor antagonist SB399885 (3 mg/kg). The effect of venlafaxine was blocked by the simultaneous administration of idazoxan (0.3 mg/kg) and GR125487 (3 mg/kg), but not by either compound alone. These findings suggest that encounter-induced hyperactivity in isolation-reared mice is a robust model for testing the pharmacological profile of antidepressants, although the range of antidepressants tested is limited and some non-antidepressants are also effective. The present study also shows a key role of α2 and 5-HT4 receptors in the antidepressant effect in this model.


Assuntos
Atividade Motora/efeitos dos fármacos , Comportamento Social , Isolamento Social/psicologia , Animais , Animais não Endogâmicos , Benzimidazóis/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Desipramina/farmacologia , Cloridrato de Duloxetina/farmacologia , Fluvoxamina/farmacologia , Hidroxicorticosteroides/farmacologia , Idazoxano/farmacologia , Indóis/farmacologia , Masculino , Camundongos , Testes Neuropsicológicos , Oxazinas/farmacologia , Paroxetina/farmacologia , Piperazinas/farmacologia , Psicotrópicos/farmacologia , Piridinas/farmacologia , Risperidona/farmacologia , Ritanserina/farmacologia , Sulfonamidas/farmacologia , Cloridrato de Venlafaxina/farmacologia
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