RESUMO
Virus infection of host cells is sensed by innate pattern recognition receptors (PRRs) and induces production of type I interferons (IFNs) and other inflammatory cytokines. These cytokines orchestrate the elimination of the viruses but are occasionally detrimental to the hosts. The outcomes and pathogenesis of viral infection are largely determined by the specific interaction between the viruses and their host cells. Therefore, compounds that either inhibit viral infection or modulate virus-induced cytokine response should be considered as candidates for managing virus infection. The aim of the study was to identify compounds in both categories, using a single cell-based assay. Our screening platform is a HEK293 cell-based reporter assay where the expression of a firefly luciferase is under the control of a human IFN-ß promoter. We have demonstrated that infection of the reporter cell line with a panel of RNA viruses activated the reporter gene expression that correlates quantitatively with the levels of virus replication and progeny virus production, and could be inhibited in a dose-dependent manner by known antiviral compound or inhibitors of PRR signal transduction pathways. Using Dengue virus as an example, a pilot screening of a small molecule library consisting of 26,900 compounds proved the concept that the IFN-ß promoter reporter assay can serve as a convenient high throughput screening platform for simultaneous discovery of antiviral and innate immune response modulating compounds. A representative antiviral compound from the pilot screening, 1-(6-ethoxybenzo[d]thiazol-2-yl)-3-(3-methoxyphenyl) urea, was demonstrated to specifically inhibit several viruses belonging to the family of flaviviridae.
Assuntos
Antivirais/isolamento & purificação , Fusão Gênica Artificial , Avaliação Pré-Clínica de Medicamentos/métodos , Genes Reporter , Fatores Imunológicos/isolamento & purificação , Interferon beta/biossíntese , Vírus de RNA/efeitos dos fármacos , Linhagem Celular , Ensaios de Triagem em Larga Escala , Humanos , Interferon beta/genética , Luciferases de Vaga-Lume/análise , Luciferases de Vaga-Lume/genética , Regiões Promotoras GenéticasRESUMO
Proteolytical cleavage of the picornaviral polyprotein is essential for viral replication. Therefore, viral proteases are attractive targets for anti-viral therapy. Most assays available for testing proteolytical activity of proteases are performed in vitro, using heterologously expressed proteases and peptide substrates. To deal with the disadvantages associated with in vitro assays, we modified a cell-based protease assay for picornavirus proteases. The assay is based on the induction of expression of a firefly luciferase reporter by a chimeric transcription factor in which the viral protease and cleavage sites are inserted between the GAL4 binding domain and the VP16 activation domain. Firefly luciferase expression is dependent on cleavage of the transcription factor by the viral protease. This biosafe assay enables testing the effect of compounds on protease activity in cells while circumventing the need for infection. We designed the assay for 3C proteases (3C(pro)) of various enteroviruses as well as of viruses of several other picornavirus genera, and show that the assay is amenable for use in a high-throughput setting. Furthermore, we show that the spectrum of activity of 3C(pro) inhibitor AG7088 (rupintrivir) not only encompasses enterovirus 3C(pro) but also 3C(pro) of foot-and-mouth disease virus (FMDV), an aphthovirus. In contrary, AG7404 (compound 1), an analogue of AG7088, had no effect on FMDV 3C(pro) activity, for which we provide a structural explanation.
Assuntos
Antivirais/isolamento & purificação , Antivirais/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Picornaviridae/efeitos dos fármacos , Picornaviridae/enzimologia , Proteínas Virais/antagonistas & inibidores , Proteases Virais 3C , Animais , Linhagem Celular , Cisteína Endopeptidases , Genes Reporter , Humanos , Luciferases de Vaga-Lume/análise , Luciferases de Vaga-Lume/genética , Inibidores de Proteases/isolamento & purificação , Inibidores de Proteases/farmacologiaRESUMO
We validated a new method, based on luciferine/luciferase bioluminescence, for drug screening on promastigotes of different Leishmania species. Results obtained with this new, rapid, reproducible, and reliable method are in good accordance with results obtained by the conventional MTT assay. This bioluminescence assay has a lower detection limit.
Assuntos
Antiprotozoários/isolamento & purificação , Antiprotozoários/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Leishmania/efeitos dos fármacos , Medições Luminescentes/métodos , Luciferina de Vaga-Lumes/metabolismo , Luciferases de Vaga-Lume/análise , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
OBJECTIVES: In vivo experimentation is costly and time-consuming, and presents a major bottleneck in anti-tuberculosis drug development. Conventional methods rely on the enumeration of bacterial colonies, and it can take up to 4 weeks for Mycobacterium tuberculosis to grow on agar plates. Light produced by recombinant bacteria expressing luciferase enzymes can be used as a marker of bacterial load, and disease progression can be easily followed non-invasively in live animals by using the appropriate imaging equipment. The objective of this work was to develop a bioluminescence-based mouse model of tuberculosis to assess antibiotic efficacy against M. tuberculosis in vivo. METHODS: We used an M. tuberculosis strain carrying a red-shifted derivative of the firefly luciferase gene (FFlucRT) to infect mice, and monitored disease progression in living animals by bioluminescence imaging before and after treatment with the frontline anti-tuberculosis drug isoniazid. The resulting images were analysed and the bioluminescence was correlated with bacterial counts. RESULTS: Using bioluminescence imaging we detected as few as 1.7â×â10(3) and 7.5â×â10(4) reporter bacteria ex vivo and in vivo, respectively, in the lungs of mice. A good correlation was found between bioluminescence and bacterial load in both cases. Furthermore, a marked reduction in luminescence was observed in living mice given isoniazid treatment. CONCLUSIONS: We have shown that an improved bioluminescent strain of M. tuberculosis can be visualized by non-invasive imaging in live mice during an acute, progressive infection and that this technique can be used to rapidly visualize and quantify the effect of antibiotic treatment. We believe that the model presented here will be of great benefit in early drug discovery as an easy and rapid way to identify active compounds in vivo.
Assuntos
Antituberculosos/administração & dosagem , Luciferases de Vaga-Lume/análise , Mycobacterium tuberculosis/efeitos dos fármacos , Tuberculose/microbiologia , Animais , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos/métodos , Feminino , Genes Reporter , Luciferases de Vaga-Lume/genética , Medições Luminescentes , Camundongos , Dados de Sequência Molecular , Mycobacterium tuberculosis/genética , Análise de Sequência de DNA , Tuberculose/tratamento farmacológico , Imagem Corporal TotalRESUMO
The cyclic adenosine monophosphate response element-binding protein (CREB) is a transcription factor that contributes to memory formation. The transcriptional activity of CREB is induced by its phosphorylation at Ser-133 and subsequent interaction with the CREB-binding protein (CBP)/p300. We designed and optimized firefly split luciferase probe proteins that detect the interaction of the kinase-inducible domain (KID) of CREB and the KIX domain of CBP/p300. The increase in the light intensity of the probe proteins results from the phosphorylation of the responsible serine corresponding to Ser-133 of CREB. Because these proteins have a high signal-to-noise ratio and are nontoxic, it has become possible for the first time to carry out long-term measurement of KID-KIX interaction in living cells. Furthermore, we examined the usefulness of the probe proteins for future high-throughput cell-based drug screening and found several herbal extracts that activated CREB.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Luciferases de Vaga-Lume/metabolismo , Substâncias Luminescentes/metabolismo , Mapeamento de Interação de Proteínas/métodos , Fatores de Transcrição de p300-CBP/metabolismo , Sítios de Ligação , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/química , Células HEK293 , Humanos , Luciferases de Vaga-Lume/análise , Substâncias Luminescentes/análise , Medições Luminescentes/métodos , Fosforilação , Extratos Vegetais/farmacologia , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Fatores de Transcrição de p300-CBP/químicaRESUMO
Intracellular protein aggregation is a common pathologic feature in neurodegenerative diseases such as Huntington' disease, amyotrophic lateral sclerosis and Parkinson' disease. Although progress towards understanding protein aggregation in vitro has been made, little of this knowledge has translated to patient therapy. Moreover, mechanisms controlling aggregate formation and catabolism in cellulo remain poorly understood. One limitation is the lack of tools to quantitatively monitor protein aggregation and disaggregation. Here, we developed a protein-aggregation reporter that uses huntingtin exon 1 containing 72 glutamines fused to the N-terminal end of firefly luciferase (httQ72-Luc). httQ72-Luc fails to aggregate unless seeded by a non-luciferase-containing polyglutamine (polyQ) protein such as Q80-cfp. Upon co-aggregation, httQ72-luc becomes insoluble and loses its enzymatic activity. Using httQ72-Luc with Q80(CFP/YFP) as seeds, we screened the Johns Hopkins Clinical Compound Library and identified leflunomide, a dihydroorotate dehydrogenase inhibitor with immunosuppressive and anti-psoriatic activities, as a novel drug that prevents polyQ aggregation. Leflunomide and its active metabolite teriflunomide inhibited protein aggregation independently of their known role in pyrimidine biosynthesis, since neither uridine treatment nor other pyrimidine biosynthesis inhibitors affected polyQ aggregation. Inducible cell line and cycloheximide-chase experiments indicate that these drugs prevent incorporation of expanded polyQ into an aggregate. This study demonstrates the usefulness of luciferase-based protein aggregate reporters for high-throughput screening applications. As current trials are under-way for teriflunomide in the treatment of multiple sclerosis, we propose that this drug be considered a possible therapeutic agent for polyQ diseases.
Assuntos
Crotonatos/farmacologia , Isoxazóis/farmacologia , Peptídeos/química , Toluidinas/farmacologia , Sequência de Aminoácidos , Linhagem Celular , Avaliação Pré-Clínica de Medicamentos/métodos , Células HEK293 , Humanos , Hidroxibutiratos , Leflunomida , Luciferases de Vaga-Lume/análise , Luciferases de Vaga-Lume/genética , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Nitrilas , Pirimidinas/biossíntese , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/químicaRESUMO
Identification of active compounds in high-throughput screening (HTS) contexts can be substantially improved by applying classical experimental design and statistical inference principles to all phases of HTS studies. The authors present both experimental and simulated data to illustrate how true-positive rates can be maximized without increasing false-positive rates by the following analytical process. First, the use of robust data preprocessing methods reduces unwanted variation by removing row, column, and plate biases. Second, replicate measurements allow estimation of the magnitude of the remaining random error and the use of formal statistical models to benchmark putative hits relative to what is expected by chance. Receiver Operating Characteristic (ROC) analyses revealed superior power for data preprocessed by a trimmed-mean polish method combined with the RVM t-test, particularly for small- to moderate-sized biological hits.