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1.
Methods Mol Biol ; 1350: 95-116, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26820855

RESUMO

BacMams are modified baculoviruses that contain mammalian expression cassettes for gene delivery and expression in mammalian cells. BacMams have become an integral part of the recombinant mammalian gene expression toolbox in research labs worldwide. Construction of transfer vectors is straightforward using basic molecular biology protocols. Virus generation is based on common methods used with the baculovirus insect cell expression system. BacMam transduction of mammalian cells requires minimal modifications to familiar cell culture methods. This chapter highlights the BacMam transfer vector pHTBV.


Assuntos
Baculoviridae/genética , Engenharia Genética/métodos , Animais , Baculoviridae/isolamento & purificação , Células CHO , Clonagem Molecular , Cricetinae , Cricetulus , Enzimas de Restrição do DNA/genética , DNA Complementar/genética , Expressão Gênica , Vetores Genéticos/genética , Células HEK293 , Humanos , Técnicas de Amplificação de Ácido Nucleico , Células Sf9 , Spodoptera , Transformação Genética
2.
PLoS One ; 10(3): e0119738, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25780921

RESUMO

Endoplasmic reticulum stress plays a critical role to restore the homeostasis of protein production in eukaryotic cells. This vital process is hence involved in many types of diseases including COPD. PERK, one branch in the ER stress signaling pathways, has been reported to activate NRF2 signaling pathway, a known protective response to COPD. Based on this scientific rationale, we aimed to identify PERK activators as a mechanism to achieve NRF2 activation. In this report, we describe a phenotypic screening assay to identify PERK activators. This assay measures phosphorylation of GFP-tagged eIF2α upon PERK activation via a cell-based LanthaScreen technology. To obtain a robust assay with sufficient signal to background and low variation, multiple parameters were optimized including GFP-tagged eIF2α BacMam concentration, cell density and serum concentration. The assay was validated by a tool compound, Thapsigargin, which induces phosphorylation of eIF2α. In our assay, this compound showed maximal signal window of approximately 2.5-fold with a pEC50 of 8.0, consistent with literature reports. To identify novel PERK activators through phosphorylation of eIF2α, a focused set of 8,400 compounds was screened in this assay at 10 µM. A number of hits were identified and validated. The molecular mechanisms for several selected hits were further characterized in terms of PERK activation and effects on PERK downstream components. Specificity of these compounds in activating PERK was demonstrated with a PERK specific inhibitor and in PERK knockout mouse embryonic fibroblast (MEF) cells. In addition, these hits showed NRF2-dependent anti-oxidant gene induction. In summary, our phenotypic screening assay is demonstrated to be able to identify PERK specific activators. The identified PERK activators could potentially be used as chemical probes to further investigate this pathway as well as the link between PERK activation and NRF2 pathway activation.


Assuntos
Estresse do Retículo Endoplasmático , Ensaios de Triagem em Larga Escala/métodos , Fator 2 Relacionado a NF-E2/metabolismo , eIF-2 Quinase/fisiologia , Animais , Células Cultivadas , Fator de Iniciação 2 em Eucariotos/análise , Fator de Iniciação 2 em Eucariotos/metabolismo , Proteínas de Fluorescência Verde/análise , Homeostase , Camundongos , Fenótipo , Fosforilação , Biossíntese de Proteínas , Transdução de Sinais , Bibliotecas de Moléculas Pequenas , Tapsigargina/química , eIF-2 Quinase/antagonistas & inibidores , eIF-2 Quinase/metabolismo
3.
J Biomol Screen ; 18(10): 1212-22, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24163393

RESUMO

Sickle cell anemia (SCA) is a genetic disorder of the ß-globin gene. SCA results in chronic ischemia with pain and tissue injury. The extent of SCA symptoms can be ameliorated by treatment with drugs, which result in increasing the levels of γ-globin in patient red blood cells. Hydroxyurea (HU) is a Food and Drug Administration-approved drug for SCA, but it has dose-limiting toxicity, and patients exhibit highly variable treatment responses. To identify compounds that may lead to the development of better and safer medicines, we have established a method using primary human bone marrow day 7 erythroid progenitor cells (EPCs) to screen for compounds that induce γ-globin production. First, human marrow CD34(+) cells were cultured and expanded for 7 days and characterized for the expression of erythroid differentiation markers (CD71, CD36, and CD235a). Second, fresh or cryopreserved EPCs were treated with compounds for 3 days in 384-well plates followed by γ-globin quantification by an enzyme-linked immunosorbent assay (ELISA), which was validated using HU and decitabine. From the 7408 compounds screened, we identified at least one new compound with confirmed γ-globin-inducing activity. Hits are undergoing analysis in secondary assays. In this article, we describe the method of generating fit-for-purpose EPCs; the development, optimization, and validation of the ELISA and secondary assays for γ-globin detection; and screening results.


Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Células Precursoras Eritroides/metabolismo , Ativação Transcricional/efeitos dos fármacos , gama-Globinas/genética , Anemia Falciforme/tratamento farmacológico , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Ácido Butírico/farmacologia , Sobrevivência Celular , Metilases de Modificação do DNA/antagonistas & inibidores , Metilases de Modificação do DNA/metabolismo , Decitabina , Ensaio de Imunoadsorção Enzimática , Epigênese Genética/efeitos dos fármacos , Células Precursoras Eritroides/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Humanos , Cultura Primária de Células , gama-Globinas/metabolismo
4.
J Biomol Screen ; 17(7): 900-11, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22644265

RESUMO

In this study, the development of an image-based high-content screening (HCS) binding assay for the seven-transmembrane (7TM) receptor Smoothened (Smo) is described. Using BacMam-based gene delivery of Smo, BODIPY-cyclopamine as a fluorescent probe, and a confocal imaging system, a robust 384-well assay that could be used for high-throughput compound profiling activities was developed. The statistically robust HCS binding assay was developed through optimization of multiple parameters, including cell transduction conditions, Smo expression levels, the image analysis algorithm, and staining procedures. Evaluation of structurally diverse compounds, including functional Smo activators, inhibitors, and related analogs, demonstrated good compound potency correlations between high-content imaging binding, membrane fluorescence polarization binding, and gene reporter assays. Statistical analysis of data from a screening test set of compounds at a single 10-µM concentration suggested that the high-content imaging Smo binding assay is amenable for use in hit identification. The 384-well HCS assay was rapidly developed and met statistical assay performance targets, thus demonstrating its utility as a fluorescent whole-cell binding assay suitable for compound screening and profiling.


Assuntos
Benzamidas/metabolismo , Benzimidazóis/metabolismo , Cicloexilaminas/metabolismo , Avaliação Pré-Clínica de Medicamentos/métodos , Ensaios de Triagem em Larga Escala/métodos , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Receptores Acoplados a Proteínas G/metabolismo , Tiofenos/metabolismo , Algoritmos , Baculoviridae/genética , Benzamidas/química , Benzamidas/farmacologia , Benzimidazóis/química , Benzimidazóis/farmacologia , Linhagem Celular , Cicloexilaminas/química , Cicloexilaminas/farmacologia , Corantes Fluorescentes , Genes Reporter , Células HEK293 , Humanos , Morfolinas/química , Morfolinas/metabolismo , Morfolinas/farmacologia , Piperazinas/química , Piperazinas/metabolismo , Piperazinas/farmacologia , Ligação Proteica , Purinas/química , Purinas/metabolismo , Purinas/farmacologia , Pirazóis/química , Pirazóis/metabolismo , Pirazóis/farmacologia , Receptor Smoothened , Tiofenos/química , Tiofenos/farmacologia
5.
Assay Drug Dev Technol ; 10(6): 514-24, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22574653

RESUMO

Nuclear-factor-E2-related transcription factor 2 (Nrf2) regulates a large panel of Phase II genes and plays an important role in cell survival. Nrf2 activation has been shown as preventing cigarette smoke-induced alveolar enlargement in mice. Therefore, activation of the Nrf2 protein by small-molecule activators represents an attractive therapeutic strategy that is used for chronic obstructive pulmonary disease. In this article, we describe a cell-based luciferase enzyme fragment complementation assay that identifies Nrf2 activators. This assay is based on the interaction of Nrf2 with its nuclear partner MafK or runt-related transcription factor 2 (RunX2) and is dependent on the reconstitution of a "split" luciferase. Firefly luciferase is split into two fragments, which are genetically fused to Nrf2 and MafK or RunX2, respectively. BacMam technology was used to deliver the fusion constructs into cells for expression of the tagged proteins. When the BacMam-transduced cells were treated with Nrf2 activators, the Nrf2 protein was stabilized and translocated into the nucleus where it interacted with MafK or RunX2. The interaction of Nrf2 and MafK or RunX2 brought together the two luciferase fragments that form an active luciferase. The assay was developed in a 384-well format and was optimized by titrating the BacMam concentration, transduction time, cell density, and fetal bovine serum concentration. It was further validated with known Nrf2 activators. Our data show that this assay is robust, sensitive, and amenable to high throughput screening of a large compound collection for the identification of novel Nrf2 activators.


Assuntos
Teste de Complementação Genética/métodos , Ensaios de Triagem em Larga Escala/métodos , Luciferases/metabolismo , Fator 2 Relacionado a NF-E2/efeitos dos fármacos , Algoritmos , Automação , Contagem de Células , Clonagem Molecular , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Meios de Cultura , Citomegalovirus/genética , Interpretação Estatística de Dados , Dimetil Sulfóxido/farmacologia , Vetores Genéticos , Células HEK293 , Humanos , Fator 2 Relacionado a NF-E2/agonistas , Reação em Cadeia da Polimerase em Tempo Real , Bibliotecas de Moléculas Pequenas , Transdução Genética
6.
J Biomol Screen ; 17(1): 99-107, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22086723

RESUMO

Histone proteins are subject to several modifications, including phosphorylation, acetylation, methylation, sumoylation, and ubiquitination. These posttranslational modifications play critical roles in chromatin structure and gene transcription. Because of their involvement in the progression of a variety of diseases, histone modifications are attracting increased attention. We report herein a high-throughput DELFIA assay to quantify H3K27me3 in the prostate cancer cell line, PC3. Using a high binding MaxiSorp plate, we were able to eliminate the need for the capture antibody. We also developed an effective method, a combination of "freeze-thaw" and 0.2 N HCl, to extract histone proteins in PC3 cells cultured in a 384-well plate. To compensate for cell viability change, we normalized H3K27me3 signal to the total amount of H3 in each sample well. As a result, we show that the assay has a good dynamic range with a robust assay window. Using a methlytransferase inhibitor, DZNep, we show that the change of H3K27me3 signal is target specific. This method simplifies the logistics in screening and profiling and reduces the cost per well to an acceptable level for high-throughput screening. The findings presented here should be applicable to other assays involving binding and extraction of histone proteins.


Assuntos
Imunofluorescência , Ensaios de Triagem em Larga Escala , Histonas/isolamento & purificação , Histonas/metabolismo , Lisina/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Ensaios de Triagem em Larga Escala/economia , Histona Metiltransferases , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Histonas/análise , Humanos , Elementos da Série dos Lantanídeos/química , Masculino , Metilação , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia
7.
J Biomol Screen ; 16(1): 53-64, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-20938045

RESUMO

A 2-step kinase assay was developed and used in a high-throughput screen (HTS) of more than 1 million compounds in an effort to identify c-Abl tyrosine kinase activators. This assay employed a 2-step phosphorylation reaction: in the first step, purified recombinant c-Abl was activated by incubating with compound in the presence of adenosine triphosphate (ATP). In the second step, the TAMRA-labeled IMAP Abltide substrate was added to allow phosphorylation of the substrate to occur. The assay was calibrated such that inactive c-Abl protein was activated by ATP alone to a degree that it not only demonstrated a measurable c-Abl activity but also maintained a robust assay window for screening. The screen resulted in 8624 primary hits with >30% response. Further analysis showed that 1024 had EC(50) <10 µM with a max % response of >50%. These hits were structurally and chemically diverse with possibly different mechanisms for activating c-Abl. In addition, selective hits were shown to be cell permeable and were able to induce c-Abl activation as determined by In-Cell Western (ICW) analysis of HEK-MSRII cells transduced with BacMam virus expressing full-length c-Abl.


Assuntos
Ativadores de Enzimas/farmacologia , Ensaios de Triagem em Larga Escala/métodos , Proteínas Proto-Oncogênicas c-abl/agonistas , Proteínas Proto-Oncogênicas c-abl/metabolismo , Trifosfato de Adenosina/metabolismo , Baculoviridae/genética , Bioensaio , Descoberta de Drogas , Vetores Genéticos/genética , Células HEK293 , Humanos , Fosforilação , Bibliotecas de Moléculas Pequenas/farmacologia , Transfecção
8.
Protein Expr Purif ; 75(1): 1-14, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-20692346

RESUMO

Multi-protein complexes are involved in essentially all cellular processes. A protein's function is defined by a combination of its own properties, its interacting partners, and the stoichiometry of each. Depending on binding partners, a transcription factor can function as an activator in one instance and a repressor in another. The study of protein function or malfunction is best performed in the relevant context. While many protein complexes can be reconstituted from individual component proteins after being produced individually, many others require co-expression of their native partners in the host cells for proper folding, stability, and activity. Protein co-expression has led to the production of a variety of biological active complexes in sufficient quantities for biochemical, biophysical, structural studies, and high throughput screens. This article summarizes examples of such cases and discusses critical considerations in selecting co-expression partners, and strategies to achieve successful production of protein complexes.


Assuntos
Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Animais , Expressão Gênica , Humanos , Complexos Multiproteicos/isolamento & purificação , Mapeamento de Interação de Proteínas , Proteínas/genética , Proteínas/isolamento & purificação , Proteínas/metabolismo
9.
Curr Gene Ther ; 10(3): 168-73, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20392201

RESUMO

Modern drug discovery programs utilize a wide variety of technologies to aid in identification of potential drug targets, and progress them through the often long and winding path of finding novel drug-like molecules. Recombinant cell-based assays are an important tool in the drug discovery process for investigating the biological mechanisms of potential drug targets and conducting screening campaigns in the hunt for biologically active molecules. Historically, stable cell lines expressing the target protein(s) of interest have been used for these assays. Although such cell lines can be useful, their development can be laborious and the resulting cell line affords little experimental flexibility. Transient gene expression approaches provide an alternative to the often tedious task of developing and maintaining numerous stable cell lines. Recently the unique properties of modified baculoviruses, containing mammalian expression cassettes and referred to as BacMam viruses, have been exploited to facilitate rapid and reproducible transient cell-based assay development. This review will focus on the many features of BacMam virus gene delivery that make it a powerful system for cell-based assay development and screening.


Assuntos
Baculoviridae/genética , Técnicas de Transferência de Genes , Terapia Genética/métodos , Animais , Linhagem Celular , Expressão Gênica , Genes , Mamíferos/genética
10.
J Biomol Screen ; 15(4): 388-97, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20228280

RESUMO

Superoxide affects many normal and pathogenic cellular processes, and the detection of superoxide produced by cells is therefore of interest for potential therapeutic applications. To develop a high-throughput cell-based assay for the detection of extracellular superoxide production that could be run in a 384-well or 1536-well format, 2 luminescent reagents, Lucigenin and Diogenes, and one fluorescent reagent, Oxyburst Green BSA, were tested. HL-60 cells, which had been differentiated to a neutrophil-like phenotype with DMSO and frozen in large batches, were used in assays. All 3 superoxide detection reagents performed well statistically in terms of IC(50) reproducibility and met a desired Z' value requirement of >0.4. When tested against a 1408-compound test set at 5 or 10 microM compound concentration, a higher hit rate was obtained with the 2 luminescent reagents compared with that obtained with the fluorescent Oxyburst Green BSA reagent. The Oxyburst Green BSA assay was ultimately chosen for compound profiling and high-throughput screening activities. This 1536 superoxide detection assay using cryopreserved differentiated HL-60 cells represents a shifting paradigm toward the utilization of more therapeutically relevant cells in early drug development activities.


Assuntos
Ensaios de Triagem em Larga Escala/métodos , Superóxidos/metabolismo , Citocromos c/metabolismo , Células HL-60 , Humanos , Cinética , Oniocompostos/farmacologia , Acetato de Tetradecanoilforbol/farmacologia , Fatores de Tempo
11.
Mol Biotechnol ; 45(3): 207-17, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20339956

RESUMO

Epoxyeicosatrienoic acids (EETs) play important protective functions in cardiovascular and renal systems. Under physiological conditions, EETs are quickly converted by the soluble epoxide hydrolase (sEH) to diols which do not have the beneficiary roles. Inhibition of sEH with small molecules to increase the concentration of EETs therefore provides an attractive therapeutic strategy for cardiovascular diseases. We describe here the development of a high throughput cell-based assay to measure sEH activity and screen small molecular compounds as sEH inhibitors. This assay is based on the technology of fluorescence polarization (FP), utilizing a Cy3B labeled 14,15-DHET ligand and a rabbit anti-14,15-DHET antibody. With the optimized assay, we measured the cellular sEH activity of several cell lines expressing endogenous sEH as well as sEH BacMam transduced HEK-293 cells. The inhibitory effect of several known sEH inhibitors was evaluated in sEH BacMam transduced HEK-293 cells. Our data show that there is good agreement of pIC(50) values obtained between the FP format and a commercially available ELISA kit. To our knowledge, this is the first report of a high throughput cell-based assay for screening sEH inhibitors.


Assuntos
Ácido 8,11,14-Eicosatrienoico/análise , Epóxido Hidrolases/química , Ensaios de Triagem em Larga Escala/métodos , Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Ácido 8,11,14-Eicosatrienoico/química , Ácido 8,11,14-Eicosatrienoico/metabolismo , Animais , Carbocianinas/química , Carbocianinas/metabolismo , Linhagem Celular , Ensaio de Imunoadsorção Enzimática , Epóxido Hidrolases/antagonistas & inibidores , Epóxido Hidrolases/metabolismo , Imunoensaio de Fluorescência por Polarização , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Cabras , Humanos , Imunoglobulina G/metabolismo , Concentração Inibidora 50 , Ligação Proteica , Coelhos , Reprodutibilidade dos Testes
12.
Methods Mol Biol ; 552: 199-211, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19513651

RESUMO

BacMam viruses are modified baculoviruses that contain mammalian expression cassettes for viral gene delivery and transient expression in mammalian cells. They are easily, inexpensively, and rapidly generated and provide a versatile solution for G protein-coupled receptor (GPCR) cell-based assay development. Using BacMam technology, target gene expression levels are easily controlled and simultaneous delivery of multiple genes is possible, for example, coexpression of a receptor and a G protein or a reporter gene. BacMam viruses are compatible with the GPCR cell-based assay formats typically used in high-throughput screening and provide an unparalleled level of experimental flexibility that is simply not possible when using stable recombinant cell lines.


Assuntos
Baculoviridae/genética , Neoplasias Ósseas/metabolismo , Técnicas de Transferência de Genes , Osteossarcoma/metabolismo , Receptores Acoplados a Proteínas G/genética , Neoplasias Ósseas/genética , Vetores Genéticos , Humanos , Osteossarcoma/genética , Receptores Acoplados a Proteínas G/metabolismo , Transdução Genética , Células Tumorais Cultivadas
13.
Protein Expr Purif ; 65(2): 251-60, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19297698

RESUMO

We describe here two strategies to produce biologically active chemokines with authentic N-terminal amino acid residues. The first involves producing the target chemokine with an N-terminal 6xHis-SUMO tag in Escherichia coli as inclusion bodies. The fusion protein is solubilized and purified with Ni-NTA-agarose in denaturing reagents. This is further followed by tag removal and refolding in a redox refolding buffer. The second approach involves expressing the target chemokine with an N-terminal 6xHis-Trx-SUMO tag in an engineered E. coli strain that facilitates formation of disulfide bonds in the cytoplasm. Following purification of the fusion protein via Ni-NTA and tag removal, the target chemokine is refolded without redox buffer and purified by reverse phase chromatography. Using the procedures, we have produced more than 15 biologically active chemokines, with a yield of up to 15 mg/L.


Assuntos
Quimiocinas/biossíntese , Quimiocinas/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/metabolismo , Quimiocinas/isolamento & purificação , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Oxirredução , Reação em Cadeia da Polimerase , Engenharia de Proteínas , Dobramento de Proteína , Proteínas Recombinantes de Fusão/isolamento & purificação
14.
Expert Opin Drug Discov ; 4(3): 243-56, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23489124

RESUMO

BACKGROUND: Adenovirus, retrovirus and lentivirus-based vectors, originally engineered and optimized for in vivo and ex vivo gene therapy, have become increasingly useful for viral-mediated gene delivery to support in vitro cell-based assays. Viral vectors underpin functional genomics screening of cDNA, shRNA and aptamer libraries, are used for a variety of target validation studies and importantly, for high-throughput cell-based drug discovery and compound profiling assays. The baculovirus/insect cell expression system had gained prevalence as a tool for recombinant protein production when it was observed that recombinant baculovirus vectors too could serve as efficient gene delivery vehicles for a wide range of mammalian cells. Although the use of baculovirus vectors in vivo has lagged behind retroviral, adenoviral and lentiviral vectors, they have gained prominence for development of in vitro cell-based assays due to the ease of generation, broad host range and excellent biosafety profile. There is an increasing emphasis on cell-based assays in high-throughput automated drug discovery laboratories and a variety of commercially available viral-vectors can be used for supporting these assays. OBJECTIVE: We compare and contrast the current viral-mediated gene delivery vector systems and highlight their suitability for cell-based drug discovery assays. CONCLUSION: Viral-mediated gene delivery is increasingly being used in support of genome scale target validation studies and cell-based assay development for specific drug target genes such as ion channels, G protein-coupled receptors and intracellular enzymes. The choice of a delivery system over another for a particular application is largely dictated by the cell types and cell lines in use, virus cellular tropism, assay throughput, safety requirements and ease/cost of reagent generation.

15.
Mol Biotechnol ; 39(2): 127-34, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18327553

RESUMO

Cortisol is an important glucocorticoid in humans that regulates many physiological processes. Human 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) converts cortisone to cortisol in vivo and has emerged as an appealing therapeutic target for treating metabolic diseases. Here, we report a sensitive and robust high-throughput (HT) cell-based assay for screening 11beta-HSD1 inhibitors. This assay utilizes a HEK293 cell line transduced by a BacMam virus expressing human 11beta-HSD1. The enzyme activity in the cells was measured by quantifying cortisol levels released into the cell culture supernatant via a competitive homogenous time-resolved fluorescence (HTRF) method. We show that 11beta-HSD1 activity in supernatant of BacMam-transduced HEK293 cells increases with 11beta-HSD1 BacMam virus load in a dose-dependent manner, and is comparable to the enzyme activity detected in differentiated mouse adipocytes. In addition, we show that co-expression of hexose-6-phosphate dehydrogenase (H6PDH) is not required for the enzyme to function effectively as an oxo-reductase. This assay has been developed in low-volume 384-well format and it is sensitive, robust, and amenable to HT screening.


Assuntos
Fluorimunoensaio/métodos , Rim/enzimologia , Transdução Genética/métodos , 11-beta-Hidroxiesteroide Desidrogenase Tipo 1/antagonistas & inibidores , 11-beta-Hidroxiesteroide Desidrogenase Tipo 1/metabolismo , Células 3T3-L1 , Adipócitos/enzimologia , Animais , Desidrogenases de Carboidrato/metabolismo , Sobrevivência Celular , Cortisona/metabolismo , Meios de Cultura/análise , Humanos , Hidrocortisona/metabolismo , Camundongos
16.
Methods Mol Biol ; 388: 95-114, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17951767

RESUMO

BacMams are modified baculoviruses that contain mammalian expression cassettes for gene delivery and expression in mammalian cells. The BacMam system combines the advantages of viral transient expression, ease in generation, and a wide cell tropism. It enables rapid, facile, and flexible gene over-expression experiments to be performed in a variety of mammalian cell lines. Conversion of baculovirus vectors to BacMam vectors involves replacement of the viral specific expression cassette with a mammalian expression cassette or the addition of a mammalian expression cassette. Viruses are produced using standard methods in a few weeks. Mammalian cells transduced with the BacMam viruses have been routinely used as substitutes for stable cell lines.


Assuntos
Baculoviridae/genética , Expressão Gênica , Vetores Genéticos/genética , Nucleopoliedrovírus/genética , Animais , Linhagem Celular , DNA Viral/genética , Citometria de Fluxo , Humanos , Luciferases/genética , Luciferases/metabolismo , Mariposas/virologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transfecção/métodos
17.
Drug Discov Today ; 12(9-10): 396-403, 2007 May.
Artigo em Inglês | MEDLINE | ID: mdl-17467576

RESUMO

Membrane protein targets constitute a key segment of drug discovery portfolios and significant effort has gone into increasing the speed and efficiency of pursuing these targets. However, issues still exist in routine gene expression and stable cell-based assay development for membrane proteins, which are often multimeric or toxic to host cells. To enhance cell-based assay capabilities, modified baculovirus (BacMam virus) gene delivery technology has been successfully applied to the transient expression of target proteins in mammalian cells. Here, we review the development, full implementation and benefits of this platform-based gene expression technology in support of SAR and HTS assays across GlaxoSmithKline.


Assuntos
Baculoviridae/genética , Desenho de Fármacos , Transfecção/métodos , Animais , Indústria Farmacêutica/métodos , Expressão Gênica , Vetores Genéticos/genética , Humanos , Modelos Biológicos , Tecnologia Farmacêutica/métodos
18.
Expert Opin Drug Discov ; 2(12): 1669-81, 2007 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23488908

RESUMO

The recombinant baculovirus/insect cell system was firmly established as a leading method for recombinant protein production when a new potential use for these viruses was revealed in 1995. It was reported that engineered recombinant baculoviruses could deliver functional expression cassettes to mammalian cell types; a system which has come to be known as BacMam gene delivery. In the field of high-throughput screening the failure of many common transient gene delivery methods in reproducibility and cell survival has caused investigators to routinely apply stable cell lines in support of cell-based assays. The ease of use, versatility, safety and economics of the BacMam system makes transient gene delivery a viable option in the high-throughput screening setting and in most instances circumvents many of the limitations of stable cell lines. Although a few pharmaceutical companies have embraced the technology, its use is poised to become more widespread with increased familiarity and the emergence of enabling products based on the BacMam system.

20.
Br J Pharmacol ; 148(2): 173-90, 2006 May.
Artigo em Inglês | MEDLINE | ID: mdl-16547525

RESUMO

Several peptidic urotensin-II (UT) receptor antagonists exert 'paradoxical' agonist activity in recombinant cell- and tissue-based bioassay systems, likely the result of differential urotensin-II receptor (UT receptor) signal transduction/coupling efficiency between assays. The present study has examined this phenomenon in mammalian arteries and recombinant UT-HEK (human embryonic kidney) cells.BacMam-mediated recombinant UT receptor upregulation in HEK cells augmented agonist activity for all four peptidic UT ligands studied. The nominal rank order of relative intrinsic efficacy was U-II>urantide ([Pen(5)-DTrp(7)-Orn(8)]hU-II(4-11))>SB-710411 (Cpa-c[DCys-Pal-DTrp-Lys-Val-Cys]-Cpa-amide)>>GSK248451 (Cin-c[DCys-Pal-DTrp-Orn-Val-Cys]-His-amide) (the relative coupling efficiency of recombinant HEK cells was cat>human>>rat UT receptor). The present study further demonstrated that the use of high signal transduction/coupling efficiency isolated blood vessel assays (primate>cat arteries) is required in order to characterize UT receptor antagonism thoroughly. This cannot be attained simply by using the rat isolated aorta, an artery with low signal transduction/coupling efficiency in which low-efficacy agonists appear to function as antagonists. In contrast to the 'low-efficacy agonists' urantide and SB-710411, GSK248451 functioned as a potent UT receptor antagonist in all native isolated tissues studied (UT receptor selectivity was confirmed in the rat aorta). Further, GSK248451 exhibited an extremely low level of relative intrinsic activity in recombinant HEK cells (4-5-fold less than seen with urantide). Since GSK248451 (1 mg kg(-1), i.v.) blocked the systemic pressor actions of exogenous U-II in the anaesthetized cat, it represents a suitable peptidic tool antagonist for delineating the role of U-II in the aetiology of mammalian cardiometabolic diseases.


Assuntos
Fragmentos de Peptídeos/farmacologia , Peptídeos Cíclicos/farmacologia , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Urotensinas/farmacologia , Animais , Artérias/efeitos dos fármacos , Artérias/fisiologia , Ligação Competitiva/efeitos dos fármacos , Pressão Sanguínea/efeitos dos fármacos , Cálcio/metabolismo , Gatos , Linhagem Celular , Relação Dose-Resposta a Droga , Haplorrinos , Humanos , Técnicas In Vitro , Masculino , Fragmentos de Peptídeos/metabolismo , Peptídeos Cíclicos/química , Ensaio Radioligante , Ratos , Ratos Sprague-Dawley , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/metabolismo , Urotensinas/metabolismo , Vasoconstrição/efeitos dos fármacos , Vasoconstritores/farmacologia
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