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1.
Eur J Med Chem ; 57: 149-61, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23047231

RESUMO

Newly synthesized sulfonilamidothiopyrimidone derivatives and a subset of 14 sulfonilamidothiopyrimidones and thiopyrimidones selected by an MTT assays cell viability guided selection from an in house collection were evaluated to determine the inhibitory effect on the PGE(2) formation in human peripheral blood lymphocytes (PBLs) using commercial ELISA. The newly synthesized sulfonilamidothiopyrimidone derivatives showed interesting pharmacological activities. Preliminary in vitro assays showed that compounds 2-5 are endowed with very high activity. Compound 2 was the most active as hCOX-2 inhibitor. The observed effects were not due to an inhibition of cell proliferation as proved by the BrdU assay. Western blot of COX-2 confirmed the inhibition on the PGE(2) secretion. Further evidence on the inhibitory potential and selectivity as COX-2 inhibitors of the selected compounds came from the in vitro screening. In order to better rationalize the action and the binding mode of these compounds, docking studies were carried out. These studies were in agreement with the biological data. Compounds 2-5 were able to fit into the active site of COX-2 with highest scores and interaction energies. Furthermore, compound 2, which showed an inhibition of around 50% on PGE(2) production, was the best scored in all the docking calculations carried out.


Assuntos
Inibidores de Ciclo-Oxigenase 2/síntese química , Ciclo-Oxigenase 2/metabolismo , Leucócitos Mononucleares/efeitos dos fármacos , Pirimidinonas/síntese química , Sulfonamidas/síntese química , Tiadiazóis/síntese química , Western Blotting , Bromodesoxiuridina , Domínio Catalítico , Morte Celular/efeitos dos fármacos , Inibidores de Ciclo-Oxigenase 2/farmacologia , Dinoprostona/antagonistas & inibidores , Dinoprostona/biossíntese , Humanos , Interleucina-2/farmacologia , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/metabolismo , Simulação de Acoplamento Molecular , Pirimidinonas/farmacologia , Relação Estrutura-Atividade , Sulfonamidas/farmacologia , Termodinâmica , Tiadiazóis/farmacologia
2.
PLoS One ; 6(1): e16100, 2011 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-21298033

RESUMO

There is increasing evidence that soluble factors in inflammatory central nervous system diseases not only regulate the inflammatory process but also directly influence electrophysiological membrane properties of neurons and astrocytes. In this context, the cytokine TNF-α (tumor necrosis factor-α) has complex injury promoting, as well as protective, effects on neuronal viability. Up-regulated TNF-α expression has also been found in various neurodegenerative diseases such as cerebral malaria, AIDS dementia, Alzheimer's disease, multiple sclerosis, and stroke, suggesting a potential pathogenic role of TNF-α in these diseases as well. We used the neuroblastoma cells SK-N-MC. Transcriptional activity was measured using luciferase reporter gene assays by using lipofectin. We performed cotransfection experiments of NFAT (nuclear factor of activated T cells) promoter constructed with a dominant negative version of NFAT (dn-NFAT). Cell death was performed by MTT (3-(4,5-dimethylthiazol-2-yl)5,5-diphenyltetrazolium bromide) and TUNEL assays. NFAT translocation was confirmed by Western blot. Involvement of NFAT in cell death was assessed by using VIVIT. P53, Fas-L, caspase-3, and caspase-9 expressions were carried out by Western blot. The mechanisms involved in TNF-α-induced cell death were assessed by using microarray analysis. TNF-α causes neuronal cell death in the absence of glia. TNF-α treatment results in nuclear translocation of NFAT through activation of calcineurin in a Ca(2+) independent manner. We demonstrated the involvement of FasL/Fas, cytochrome c, and caspase-9 but the lack of caspase-3 activation. NB cell death was absolutely reverted in the presence of VIVIT, and partially diminished by anti-Fas treatment. These data demonstrate that TNF-α promotes FasL expression through NFAT activation in neuroblastoma cells and this event leads to increased apoptosis through independent caspase-3 activation.


Assuntos
Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Fatores de Transcrição NFATC/metabolismo , Neuroblastoma/patologia , Fator de Necrose Tumoral alfa/farmacologia , Transporte Ativo do Núcleo Celular , Calcineurina/metabolismo , Linhagem Celular Tumoral , Proteína Ligante Fas/genética , Humanos , Análise em Microsséries , Transcrição Gênica
3.
Rev. neurol. (Ed. impr.) ; 52(2): 101-111, 16 ene., 2011. tab, ilus
Artigo em Espanhol | IBECS | ID: ibc-86969

RESUMO

La neuroinflamación constituye un proceso clave en la neuropatogénesis del virus del sida como consecuencia de la activación aberrante de receptores de quimiocinas (CXCR4, CX3CR1 y CCR5), ya que la liberación de citocinas proinflamatorias por las células infectadas amplifica la neurotoxicidad microglial y genera lipoperóxidos y especies reactivas de oxígeno que, en última instancia, dañan la neurona. Por otro lado, la neurotoxina Tat induce alteraciones dendríticas por interacción con el receptor LRP (receptor de lipoproteínas de baja densidad) e induce una excesiva estimulación de los receptores de N-metil D-aspartato. Además, la interacción aberrante de la glucoproteína gp120 con el receptor CXCR4 induce apoptosis dependiente de caspasa 3 (también libera ceramida) y activa las proteínas apoptóticas p53 y retinoblastoma como mecanismos neurotóxicos asociados a la disfunción neural en el virus de la inmunodeficiencia humana 1 (VIH-1). Asimismo, la gliosis/activación microglial y la liberación de factores virales por los monocitos infectados, y el incremento de determinadas quimiocinas en el líquido cefalorraquídeo (MCP-1 y fractalcina, entre otras), contribuyen a la neuropatogénesis del VIH-1. Por otro lado, se han detectado depósitos de alfa-sinucleína y de beta-amiloide en cerebros post mortem de seropositivos de edad avanzada. Además, se han descrito varios marcadores sistémicos relacionados con los efectos degenerativos del virus y de sus neurotoxinas en el sistema nervioso central, tales como osteopontina, CD163 y fractalcina, entre otros. Por último, se han realizado ensayos clínicos basados en estrategias protectoras relacionadas con la inhibición de proteínas apoptóticas (inhibidores de GSK-3 beta), con inhibidores de la activación microglial (minociclina), antioxidantes (selegilina) o factores tróficos (IGF-1, hormona del crecimiento o eritropoyetina), que muestran efectos beneficiosos como tratamientos complementarios a la terapia antirretroviral (AU)


Neuroinflammation is a key process in the neuropathogenesis of AIDS virus since as a result of the aberrant activation of the chemokine receptors (CXCR4, CX3CR1 and CR5) produces proinflammatory cytokine release by infected cells, increases microglial neurotoxicity and generates lipoperoxides and reactive oxygen species (ROS) that eventually damage the neuron. Moreover, the neurotoxin Tat produces dendritic loss by interacting with the low-density lipoprotein receptor (LRP) and also overstimulates N-methyl D-aspartate receptors (NMDA). Furthermore, the aberrant interaction of glycoprotein gp120 with the CXCR4 chemokine receptor causes caspase-3-dependent apoptosis (ceramide is also released) activating apoptotic proteins (p53 and retinoblastoma), which are part of the neurotoxic mechanisms associated to neuronal dysfunction in neuroAIDS. Similarly, gliosis/microglial activation and the release of neurotoxic factors by infected monocytes with elevated amounts of certain chemokines in the cerebrospinal fluid (MCP-1 and fractalkine, among others) contribute to the neuropathogenesis of HIV-1. Alpha-synuclein and beta amyloid deposits have also been detected in post mortem brains of seropositives patients. In addition, there are studies have detected several systemic markers related with the degenerative effects of the virus and its neurotoxins on the central nervous system; such as osteopontin, CD163 and fractalkine, among others. Lastly, clinical trials have been conducted using protective strategies related that attempt to inhibit apoptotic proteins (GSK-3 beta), microglial activation inhibitors (minocycline), antioxidants (selegiline) or trophic factors (IGF-1, growth hormone or erythropoietin). These trials have shown that their treatments are beneficial and complementary to treat complications of HIV/AIDS (AU)


Assuntos
Humanos , Complexo AIDS Demência/tratamento farmacológico , Infecções por HIV/complicações , Proteína gp120 do Envelope de HIV/efeitos adversos , Quimiocinas CX3C , Antirretrovirais/uso terapêutico , Microglia
4.
PLoS One ; 5(12): e15360, 2010 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-21170329

RESUMO

BACKGROUND: Acetaminophen (AAP) is widely prescribed for treatment of mild pain and fever in western countries. It is generally considered a safe drug and the most frequently reported adverse effect associated with acetaminophen is hepatotoxicity, which generally occurs after acute overdose. During AAP overdose, encephalopathy might develop and contribute to morbidity and mortality. Our hypothesis is that AAP causes direct neuronal toxicity contributing to the general AAP toxicity syndrome. METHODOLOGY/PRINCIPAL FINDINGS: We report that AAP causes direct toxicity on rat cortical neurons both in vitro and in vivo as measured by LDH release. We have found that AAP causes concentration-dependent neuronal death in vitro at concentrations (1 and 2 mM) that are reached in human plasma during AAP overdose, and that are also reached in the cerebrospinal fluid of rats for 3 hours following i.p injection of AAP doses (250 and 500 mg/kg) that are below those required to induce acute hepatic failure in rats. AAP also increases both neuronal cytochrome P450 isoform CYP2E1 enzymatic activity and protein levels as determined by Western blot, leading to neuronal death through mitochondrial-mediated mechanisms that involve cytochrome c release and caspase 3 activation. In addition, in vivo experiments show that i.p. AAP (250 and 500 mg/kg) injection induces neuronal death in the rat cortex as measured by TUNEL, validating the in vitro data. CONCLUSIONS/SIGNIFICANCE: The data presented here establish, for the first time, a direct neurotoxic action by AAP both in vivo and in vitro in rats at doses below those required to produce hepatotoxicity and suggest that this neurotoxicity might be involved in the general toxic syndrome observed during patient APP overdose and, possibly, also when AAP doses in the upper dosing schedule are used, especially if other risk factors (moderate drinking, fasting, nutritional impairment) are present.


Assuntos
Acetaminofen/farmacologia , Apoptose , Neurônios/citologia , Animais , Antioxidantes/metabolismo , Antipiréticos/farmacologia , Benzimidazóis/farmacologia , Citocromo P-450 CYP2E1/metabolismo , Fragmentação do DNA , Radicais Livres , L-Lactato Desidrogenase/metabolismo , Fígado/efeitos dos fármacos , Neurônios/metabolismo , Dor , Isoformas de Proteínas , Ratos
5.
J Alzheimers Dis ; 22(2): 493-505, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20847443

RESUMO

Both amyloid-ß peptide 1-42 (Aß1-42) formation and cyclooxygenase-2 (COX-2) have been involved in the pathogenesis of Alzheimer's disease (AD), a devastating neurological disorder. However, the relationship between Aß1-42 and COX-2 is unclear. We found that the addition of Aß1-42 to astrocytoma cultures induced COX-2 mRNA and protein and PGE2 synthesis in primary human astrocytes and in human astrocytoma cell lines. Moreover, Aß1-42 induced COX-2 promoter transcription. Deletion of nuclear factor-κB (NF-κB) sites of the promoter diminished Aß1-42-COX-2 dependent transcription. In agreement with this, Aß1-42 induced transcription of NF-κB reporter gene. In contrast, Aß1-42 neither did not induce NFAT not AP-1 factors activation suggesting that both NFAT and AP-1 was not necessary to control COX-2 transcription induced by Aß1-42. Over expression of NF-κB inhibitory subunit, IκB, completely abrogated Aß1-42-induced COX-2 activity in U-87 cells, whereas the opposite effect was shown when p65/rel A NF-κB was over expressed. In addition, Aß1-42 induced p65/rel A subunit translocation to the nucleus and binding to the distal site of the COX-2 promoter. The importance of NF-κB in COX-2 induction and PGE2 synthesis by Aß1-42 was corroborated by using the pharmacological inhibitor of the NF-κB pathway, PDTC. In addition, Aß1-42 treated astrocytoma supernatants were toxic for neuroblastoma cells, an effect which was blocked by PDTC. Summing up, our results indicate that Aß1-42 was able to induce COX-2 and PGE2 synthesis in astrocytic cells through a NF-κB dependent mechanism. This may have implicated in our understanding of AD pathology.


Assuntos
Peptídeos beta-Amiloides/farmacologia , Astrócitos/efeitos dos fármacos , Ciclo-Oxigenase 2/metabolismo , Dinoprostona/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , NF-kappa B/metabolismo , Fragmentos de Peptídeos/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Ciclo-Oxigenase 2/genética , Dinoprostona/genética , Relação Dose-Resposta a Droga , Interações Medicamentosas , Inibidores Enzimáticos/farmacologia , Humanos , Luciferases/genética , Nitrobenzenos/farmacologia , Prolina/análogos & derivados , Prolina/farmacologia , RNA Mensageiro/metabolismo , Sulfonamidas/farmacologia , Tiocarbamatos/farmacologia , Transfecção/métodos
6.
J Neurochem ; 113(3): 772-83, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20180883

RESUMO

Neurodegenerative and neuroinflammatory disorders are commonly associated with local chemokine release. In other way, emerging data indicate that the prostaglandin E2 (PGE(2)), one of the major prostaglandins produced in the brain, play a central role in several pathological diseases. In this study, we investigated the relationship between CXCL12, cyclooxygenase (COX)-2 and PGE(2) in human brain cells. CXCL12 induced COX-2 and secretion of PGE(2) in a dose-dependent manner in human astrocytes. This induction was abolished by treatment with pertussis toxin and AMD3100, confirming the role of CXCR4 signaling. The nuclear factor-kappaB involvement was confirmed by using pyrrolidine dithiocarbamate, and with transient transfection assays. Over-expression of inhibitory proteins of nuclear factor-kappaB abrogated COX-2 induction, and CXCL12 induced p65/relA translocation. Culture supernatants from CXCL12-treated astrocytes reduced viability of neuroblastoma cells, and COX inhibitors abrogated this toxicity. Therefore, the relationship between chemokines and PGs could differentially influence the pathogenic network responsible for neurodegeneration.


Assuntos
Astrócitos/enzimologia , Quimiocina CXCL12/metabolismo , Ciclo-Oxigenase 2/biossíntese , Regulação Enzimológica da Expressão Gênica/fisiologia , NF-kappa B/metabolismo , Neurônios/patologia , Astrócitos/efeitos dos fármacos , Western Blotting , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Quimiocina CXCL12/biossíntese , Quimiocina CXCL12/efeitos dos fármacos , Quimiocina CXCL12/genética , Ciclo-Oxigenase 2/genética , Dinoprostona/biossíntese , Indução Enzimática/efeitos dos fármacos , Indução Enzimática/fisiologia , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Luciferases/metabolismo , NF-kappa B/genética , Neurônios/efeitos dos fármacos , Plasmídeos/genética , RNA Mensageiro/análise , RNA Mensageiro/biossíntese , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Transfecção
7.
An. R. Acad. Farm ; 74(4): 1-19, oct.-dic. 2008.
Artigo em Espanhol | IBECS | ID: ibc-135204

RESUMO

La infección cerebral por el VIH puede resultar en una profunda afectación del conocimiento, comportamiento y capacidades motoras, todo ello conocido como complejo demencia sida (CDS) en adultos y encefalopatía en niños (EP). Aunque la introducción de la TARGA ha prolongado y mejorado la vida de los individuos infectados, parece claro que la terapia antirretroviral no proporcional una protección completa frente al daño neurológico observado en esta enfermedad. La demencia-VIH es un complejo fenómeno que aparece como resultado de varios mecanismos causados por múltiples mediadores utilizando diferentes vías de señalización intracelular. Ya que el VIH no parece infectar masivamente a las neuronas, el daño neuronal es probablemente inducido por factores solubles entre los que se incluyen proteínas virales (gp120 y Tat principalmente), citocinas proinflamatorias, quimiocinas y prostaglandinas liberadas por macrófagos cerebrales infectados y células de la microglía activadas y/o infectadas. Estos factores solubles son además los responsables de la activación de células no infectadas aumentando y perpetuando de este modo el daño cerebral. Es pues necesario que la terapia esté dirigida no solo a limitar la infección de la microglía sino también la producción de estos mediadores inflamatorios y la activación de los astrocitos, para poder disminuir el daño neuronal observado en esta enfermedad (AU)


Brain HIV-1-infection may result in a syndrome of profound cognitive, behavioural and motor impairment known as AIDS dementia complex (ADC) in adults and HIV-related encephalopathy in children. Although the introduction of HAART has prolonged and improved the lives of infected individuals, it is clear that HAART does not provide complete protection against neurological damage in HIV/AIDS. HIV- 1associated dementia is a complex phenomenon, which could be the result of several mechanisms caused by those players using different intracellular signalling pathways. Since HIV-1 does not easily infect neuronal cells, neuronal damage is likely to be induced by soluble factors including viral proteins, proinflammatory cytokines, chemokines or prostaglandins released by HIV-1-infected macrophages or microglial cells. These soluble factors are also responsible for activation of uninfected cells and, thus, for spreading and perpetuation of brain damage. Thus, it is necessary an antiretroviral therapy that not only inhibit microglia infection, but also diminish proinflammatory factors and astrocytic activation to reduce neuronal damage (AU)


Assuntos
Humanos , Masculino , Feminino , Inflamação/complicações , Inflamação/diagnóstico , HIV/genética , HIV/metabolismo , Complexo AIDS Demência/diagnóstico , Complexo AIDS Demência/metabolismo , Citocinas/administração & dosagem , Citocinas/análise , Quimiocinas/análise , Inflamação/metabolismo , HIV/crescimento & desenvolvimento , HIV/isolamento & purificação , Complexo AIDS Demência/genética , Complexo AIDS Demência/transmissão , Complexo AIDS Demência/virologia , Citocinas/deficiência , Citocinas/provisão & distribuição , Quimiocinas/uso terapêutico
8.
Virology ; 380(1): 144-51, 2008 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-18752821

RESUMO

HIV-2 invades CNS and causes neurological disease as well as HIV-1 does. Induction of COX-2 in CNS of HIV-1 infected people has been proposed as a cause of cognitive impairment, so we tested whether HIV-2 may cause damage by a similar mechanism. COX-2 mRNA and protein expression were induced in human astrocytes upon interaction with HIV-2, being this induction abrogated by CXCR4 antagonists. HIV-2 induced COX-2 promoter transcription and deletion of the two NF-kappaB binding sites of the promoter abrogated this induction. Neither AP-1 nor NFAT seem to be involved in COX-2 transcriptional activation. Overexpression of IkappaBalpha completely abrogated COX-2 induction, and transfection of p65/relA NF-kappaB induced COX-2 transcription. Interestingly, HIV-2 activated NF-kappaB by inducing p65/relA transactivating activity through Ser536 phosphorylation. Moreover, the astrocyte activation induced by HIV-2 was abrogated by different COX inhibitors. In summary, HIV-2 induces COX-2 in human astrocytes depending on CXCR4 coreceptor.


Assuntos
Astrócitos/virologia , Ciclo-Oxigenase 2/metabolismo , Inibidores de Ciclo-Oxigenase/farmacologia , HIV-2/metabolismo , NF-kappa B/metabolismo , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Linhagem Celular , Ciclo-Oxigenase 2/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , HIV-2/genética , Humanos , NF-kappa B/genética , Transcrição Gênica
9.
J Immunol ; 180(1): 530-40, 2008 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-18097055

RESUMO

Both the HIV-1 protein Tat and cyclooxygenase-2 (COX-2) have been involved in the neuropathogenesis associated with HIV-1 infection. However, the relationship among them has not been addressed. Here, we found that extracellular Tat was able to induce COX-2 mRNA and protein expression and PGE2 synthesis in astrocytoma cell lines and primary human astrocytes. Moreover, Tat induced COX-2 promoter transcription. Deletion of NF-kappaB sites of the promoter did not diminish Tat-dependent transcription. Interestingly, Tat did not induce NF-kappaB activity, suggesting that NF-kappaB was not necessary to control COX-2 transcription induced by Tat. In contrast, deletion or mutation of the NFAT and/or AP-1 site abrogated COX-2 induction by Tat. Moreover, Tat induced transcription of NFAT- and AP-1-dependent reporter genes. Transfection of a dominant negative c-Jun mutant protein, TAM-67, or of a dominant negative version of NFAT, efficiently blocked the induction of COX-2 promoter by Tat, confirming the requirement of both transcription factors. Moreover, Tat induced NFAT translocation to the nucleus and binding to the distal site of the COX-2 promoter. The importance of NFAT and AP-1 in COX-2 induction and PGE2 synthesis by Tat was corroborated by using pharmacological inhibitors of the NFAlphaTau, ERK, and JNK pathways. In summary, our results indicate that HIV-1 Tat was able to induce COX-2 and PGE2 synthesis in astrocytic cells through an NFAT/AP-1-dependent mechanism.


Assuntos
Astrócitos/virologia , Ciclo-Oxigenase 2/metabolismo , Dinoprostona/metabolismo , HIV-1/metabolismo , Fatores de Transcrição NFATC/metabolismo , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Linhagem Celular , Núcleo Celular/metabolismo , Ciclo-Oxigenase 2/genética , Regulação da Expressão Gênica , Humanos , NF-kappa B/metabolismo , Fatores de Transcrição NFATC/antagonistas & inibidores , Neuroglia/metabolismo , Neuroglia/virologia , Regiões Promotoras Genéticas/efeitos dos fármacos , Linfócitos T/imunologia , Fator de Transcrição AP-1/antagonistas & inibidores , Fator de Transcrição AP-1/metabolismo , Transcrição Gênica/efeitos dos fármacos , Regulação para Cima , Produtos do Gene tat do Vírus da Imunodeficiência Humana/farmacologia
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