RESUMO
4-anilino quinazolines have been identified as inhibitors of HCV replication. The target of this class of compounds was proposed to be the viral protein NS5A, although unequivocal proof has never been presented. A 4-anilino quinazoline moiety is often found in kinase inhibitors, leading us to formulate the hypothesis that the anti-HCV activity displayed by these compounds might be due to inhibition of a cellular kinase. Type III phosphatidylinositol 4-kinase α (PI4KIIIα) has recently been identified as a host factor for HCV replication. We therefore evaluated AL-9, a compound prototypical of the 4-anilino quinazoline class, on selected phosphatidylinositol kinases. AL-9 inhibited purified PI4KIIIα and, to a lesser extent, PI4KIIIß. In Huh7.5 cells, PI4KIIIα is responsible for the phosphatidylinositol-4 phosphate (PI4P) pool present in the plasma membrane. Accordingly, we observed a gradual decrease of PI4P in the plasma membrane upon incubation with AL-9, indicating that this agent inhibits PI4KIIIα also in living cells. Conversely, AL-9 did not affect the level of PI4P in the Golgi membrane, suggesting that the PI4KIIIß isoform was not significantly inhibited under our experimental conditions. Incubation of cells expressing HCV proteins with AL-9 induced abnormally large clusters of NS5A, a phenomenon previously observed upon silencing PI4KIIIα by RNA interference. In light of our findings, we propose that the antiviral effect of 4-anilino quinazoline compounds is mediated by the inhibition of PI4KIIIα and the consequent depletion of PI4P required for the HCV membranous web. In addition, we noted that HCV has a profound effect on cellular PI4P distribution, causing significant enrichment of PI4P in the HCV-membranous web and a concomitant depletion of PI4P in the plasma membrane. This observation implies that HCV--by recruiting PI4KIIIα in the RNA replication complex--hijacks PI4P metabolism, ultimately resulting in a markedly altered subcellular distribution of the PI4KIIIα product.
Assuntos
1-Fosfatidilinositol 4-Quinase/metabolismo , Antivirais/farmacologia , Hepacivirus/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Fosfatos de Fosfatidilinositol/metabolismo , 1-Fosfatidilinositol 4-Quinase/antagonistas & inibidores , 1-Fosfatidilinositol 4-Quinase/química , Domínio Catalítico/efeitos dos fármacos , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Inibidores Enzimáticos/farmacologia , Hepacivirus/patogenicidade , Hepatócitos/metabolismo , Hepatócitos/virologia , Interações Hospedeiro-Patógeno , Humanos , Quinazolinas/farmacologia , Proteínas não Estruturais Virais/metabolismo , Replicação ViralRESUMO
OBJECTIVE: Integrin-mediated cell adhesion to type I fibrillar collagen regulates gene and protein expression, whereas little is known of its effect on lipid metabolism. In the present study, we examined the effect of type I fibrillar collagen on cholesterol biosynthesis in human aortic smooth muscle cells (SMCs). METHODS AND RESULTS: SMCs were cultured on either fibrillar or monomer collagen for 48 hours and [(14)C]-acetate incorporation into cholesterol was evaluated. Fibrillar collagen reduced by 72.9+/-2.6% cholesterol biosynthesis without affecting cellular cholesterol levels. Fibrillar collagen also reduced 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA) promoter activity (-72.6+/-7.3%), mRNA (-58.7+/-6.4%), protein levels (-35.5+/-8.5%), and enzyme activity (-37.7+/-2.2%). Intracellular levels of the active form of sterol regulatory element binding proteins (SREBP) 1a was decreased by 60.7+/-21.7% in SMCs cultured on fibrillar collagen, whereas SREBP2 was not significantly affected (+12.1+/-7.1%). The overexpression of the active form of SREBP1a rescued the downregulation of fibrillar collagen on HMG-CoA reductase levels. Blocking antibody to alpha2 integrin partially reversed the downregulation of HMG-CoA reductase mRNA expression. Finally, fibrillar collagen led to an intracellular accumulation of unprenylated Ras. CONCLUSIONS: Our study demonstrated that alpha2 beta 1 integrin interaction with fibrillar collagen affected the expression of HMG-CoA reductase, which led to the inhibition of cholesterol biosynthesis in human SMCs.
Assuntos
Colesterol/biossíntese , Colágenos Fibrilares/fisiologia , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Aorta Torácica/metabolismo , Adesão Celular , Células Cultivadas , Regulação Enzimológica da Expressão Gênica , Humanos , Hidroximetilglutaril-CoA Redutases/genética , Hidroximetilglutaril-CoA Redutases/fisiologia , Integrina alfa2beta1/fisiologia , Músculo Liso Vascular/citologia , Prenilação de Proteína , RNA Mensageiro/análise , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Proteínas ras/metabolismoRESUMO
The expansion of a polyglutamine tract in the ubiquitously expressed huntingtin protein causes Huntington's disease (HD), a dominantly inherited neurodegenerative disease. We show that the activity of the cholesterol biosynthetic pathway is altered in HD. In particular, the transcription of key genes of the cholesterol biosynthetic pathway is severely affected in vivo in brain tissue from HD mice and in human postmortem striatal and cortical tissue; this molecular dysfunction is biologically relevant because cholesterol biosynthesis is reduced in cultured human HD cells, and total cholesterol mass is significantly decreased in the CNS of HD mice and in brain-derived ST14A cells in which the expression of mutant huntingtin has been turned on. The transcription of the genes of the cholesterol biosynthetic pathway is regulated via the activity of sterol regulatory element-binding proteins (SREBPs), and we found an approximately 50% reduction in the amount of the active nuclear form of SREBP in HD cells and mouse brain tissue. As a consequence, mutant huntingtin reduces the transactivation of an SRE-luciferase construct even under conditions of SREBP overexpression or in the presence of an exogenous N-terminal active form of SREBP. Finally, the addition of exogenous cholesterol to striatal neurons expressing mutant huntingtin prevents their death in a dose-dependent manner. We conclude that the cholesterol biosynthetic pathway is impaired in HD cells, mice, and human subjects, and that the search for HD therapies should also consider cholesterol levels as both a potential target and disease biomarker.
Assuntos
Colesterol/biossíntese , Doença de Huntington/metabolismo , Doença de Huntington/patologia , Neurônios/fisiologia , Análise de Variância , Animais , Western Blotting/métodos , Morte Celular/efeitos dos fármacos , Morte Celular/fisiologia , Células Cultivadas , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Relação Dose-Resposta a Droga , Fibroblastos/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Hidroximetilglutaril-CoA Redutases/genética , Hidroximetilglutaril-CoA Redutases/metabolismo , Imuno-Histoquímica/métodos , Camundongos , Neurônios/efeitos dos fármacos , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Transporte Proteico/genética , RNA Mensageiro/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Fatores de Tempo , Transfecção/métodosRESUMO
AIM: This work aimed to investigate the ability of different formulations of bioactive glass (BAG)-S53P4 to interfere with bacterial biofilm produced on prosthetic material by methicillin-resistant Staphylococcus aureus and multi-drug-resistant Pseudomonas aeruginosa. MATERIALS & METHODS: Antibiofilm activity of three formulations of bioglass was assessed at different time points through two different analyses: Crystal Violet and confocal laser scanning microscopy assays. RESULTS: Significant differences in the whole biofilm were observed between BAG-S53P4-treated and control samples, while no marked changes in antibiofilm activity were observed among the tested formulations. Data from colorimetric assay were confirmed by confocal laser scanning microscopy analysis, which evidenced the significant reduction in biomass and a decrease of total cell volume when both S. aureus and P. aeruginosa biofilms were treated with BAG-S53P4. CONCLUSION: BAG-S53P4 can be considered as an excellent adjuvant in the treatment of prosthetic infections related to biofilm.
Assuntos
Biofilmes/efeitos dos fármacos , Vidro/química , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Próteses e Implantes/microbiologia , Pseudomonas aeruginosa/efeitos dos fármacos , Antibacterianos , Aderência Bacteriana/efeitos dos fármacos , Biomassa , Farmacorresistência Bacteriana Múltipla , Humanos , Testes de Sensibilidade Microbiana , Microscopia Confocal , Infecções por Pseudomonas/tratamento farmacológico , Infecções por Pseudomonas/prevenção & controle , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/prevenção & controleRESUMO
Prosthetic joint infections (PJIs) are becoming a growing public health concern in developed countries as more people undergo arthroplasty for bone fixation or joint replacement. Because a wide range of bacterial strains responsible for PJIs can produce biofilms on prosthetic implants and because the biofilm structure confers elevated bacterial resistance to antibiotic therapy, new drugs and therapies are needed to improve the clinical outcome of treatment of PJIs. Antimicrobial photodynamic therapy (APDT), a non-antibiotic broad-spectrum antimicrobial treatment, is also active against multidrug-resistant micro-organisms such as meticillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa. APDT uses a photosensitiser that targets bacterial cells following exposure to visible light. APDT with RLP068/Cl, a novel photosensitiser, was studied by confocal laser scanning microscopy (CLSM) to evaluate the disruption of MRSA and P. aeruginosa biofilms on prosthetic material. Quantitative CLSM studies showed a reduction in biofilm biomass (biofilm disruption) and a decrease in viable cell numbers, as determined by standard plate counting, in the S. aureus and P. aeruginosa biofilms exposed to APDT with the photosensitiser RLP068/Cl. APDT with RLP068/Cl may be a useful approach to the treatment of PJI-associated biofilms.
Assuntos
Antibacterianos/farmacologia , Biofilmes/efeitos dos fármacos , Indóis/farmacologia , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Compostos Organometálicos/farmacologia , Fármacos Fotossensibilizantes/farmacologia , Pseudomonas aeruginosa/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Biofilmes/efeitos da radiação , Contagem de Colônia Microbiana , Meios de Cultura , Violeta Genciana , Humanos , Luz , Staphylococcus aureus Resistente à Meticilina/crescimento & desenvolvimento , Staphylococcus aureus Resistente à Meticilina/efeitos da radiação , Staphylococcus aureus Resistente à Meticilina/ultraestrutura , Viabilidade Microbiana/efeitos dos fármacos , Viabilidade Microbiana/efeitos da radiação , Microscopia Confocal , Próteses e Implantes/microbiologia , Pseudomonas aeruginosa/crescimento & desenvolvimento , Pseudomonas aeruginosa/efeitos da radiação , Pseudomonas aeruginosa/ultraestrutura , TitânioRESUMO
Exogenous expression of pharmacological targets in transformed cell lines has been the traditional platform for high throughput screening of small molecules. However, exogenous expression in these cells is limited by aberrant dosage, or its toxicity, the potential lack of interaction partners, and alterations to physiology due to transformation itself. Instead, primary cells or cells differentiated from precursors are more physiological, but less amenable to exogenous expression of reporter systems. To overcome this challenge, we stably expressed c-Photina, a Ca(2+)-sensitive photoprotein, driven by a ubiquitous promoter in a mouse embryonic stem (mES) cell line. The same embryonic stem cell line was also used to generate a transgenic mouse that expresses c-Photina in most tissues. We show here that these cells and mice provide an efficient source of primary cells, cells differentiated from mES cells, including cardiomyocytes, neurons, astrocytes, macrophages, endothelial cells, pancreatic islet cells, stably and robustly expressing c-Photina, and may be exploited for miniaturized high throughput screening. Moreover, we provide evidence that the transgenic mice may be suitable for ex-vivo bioimaging studies in both cells and tissues.
Assuntos
Cálcio/metabolismo , Células-Tronco Embrionárias/metabolismo , Proteínas Luminescentes/metabolismo , Animais , Diferenciação Celular , Células Cultivadas , Células-Tronco Embrionárias/citologia , Camundongos , Camundongos TransgênicosRESUMO
The use of engineered mouse embryonic stem (mES) cells in high-throughput screening (HTS) can offer new opportunities for studying complex targets in their native environment, increasing the probability of discovering more meaningful hits. The authors have generated and developed a mouse embryonic stem cell line called c-Photina mES stably expressing a Ca(2+)-activated photoprotein as a reporter gene. This reporter cell line retains the ability to differentiate into any cell lineage and can be used for miniaturized screening processes in 384-well microplates. The c-Photina mES cell line is particularly well suited for the study of the pharmacological modulation of target genes that induce Ca(2+) mobilization. The authors differentiated this mES reporter cell line into neuronal cells and screened the LOPAC(1280) library monitoring the agonistic or antagonistic activities of compounds. They also demonstrate the possibility to generate and freeze bulk preparations of cells at an intermediate stage of differentiation and enriched in neural precursor cells, which retain the ability to form fully functional neural networks once thawed. The proposed cell model is of high value for HTS purposes because it offers a more physiological environment to the targets of interest and the possibility of using frozen batches of neural precursor cells.