RESUMEN
We report the synthesis and in vitro activity of a series of novel substituted N-{3-[(1,1-dioxido-1,2-benzothiazol-3-yl)(phenyl)amino]propyl}benzamide analogs. These analogs showed potent inhibitory activity against Kv1.3. Several demonstrated similar potency to the known Kv1.3 inhibitor PAP-1 when tested under the IonWorks patch clamp assay conditions. Two compounds 13i and 13rr were advanced further as potential tool compounds for in vivo validation studies.
Asunto(s)
Benzamidas/química , Benzamidas/farmacología , Benzotiazoles/química , Canal de Potasio Kv1.3/antagonistas & inhibidores , Amidas , Animales , Benzotiazoles/farmacología , Línea Celular , Humanos , Proteínas Asociadas a Pancreatitis , Técnicas de Placa-Clamp , Ratas , Relación Estructura-ActividadRESUMEN
We report the synthesis and in vitro activity of a series of novel N-{3-[(1,1-dioxido-1,2-benzothiazol-3-yl)(phenyl)amino]propyl}benzamide analogs. These analogs showed potent inhibitory activity against Kv1.3. Several compounds, including compound 8b, showed similar potency to the known Kv1.3 inhibitor PAP-1 when tested under the IonWorks patch clamp assay conditions.
Asunto(s)
Benzamidas/química , Benzotiazoles/química , Canal de Potasio Kv1.3/antagonistas & inhibidores , Amidas , Animales , Benzamidas/farmacología , Benzotiazoles/farmacología , Línea Celular , Humanos , Proteínas Asociadas a Pancreatitis , Técnicas de Placa-Clamp , Relación Estructura-Actividad , Sulfonamidas , Urea/análogos & derivadosRESUMEN
Today, many thousands of recombinant proteins, ranging from cytosolic enzymes to membrane-bound proteins, have been successfully produced in baculovirus-infected insect cells. Yet, in addition to its value in producing recombinant proteins in insect cells and larvae, this viral vector system continues to evolve in new and unexpected ways. This is exemplified by the development of engineered insect cell lines to mimic mammalian cell glycosylation of expressed proteins, baculovirus display strategies and the application of the virus as a mammalian-cell gene delivery vector. Novel vector design and cell engineering approaches will serve to further enhance the value of baculovirus technology.
Asunto(s)
Baculoviridae/genética , Biotecnología/métodos , Mejoramiento Genético/métodos , Vectores Genéticos/genética , Ingeniería de Proteínas/métodos , Proteínas Recombinantes/biosíntesis , Transfección/métodos , Animales , Biotecnología/tendencias , Humanos , Insectos , Mamíferos , Ingeniería de Proteínas/tendenciasRESUMEN
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.
Asunto(s)
Baculoviridae/genética , Diseño de Fármacos , Transfección/métodos , Animales , Industria Farmacéutica/métodos , Expresión Génica , Vectores Genéticos/genética , Humanos , Modelos Biológicos , Tecnología Farmacéutica/métodosRESUMEN
The authors describe the use of modified baculoviruses containing mammalian expression cassettes (BacMam technology) in steroid nuclear receptor reporter assays designed for screening and profiling agonist and antagonist compounds. Baculo-viruses were constructed that express full-length human genes for mineralocorticoid receptor (MR), glucocorticoid receptor (GR), progesterone receptor A (PR-A), and progesterone receptor B (PR-B) from the cytomegalovirus immediate early promoter. A virus carrying the mouse mammary tumor virus-firefly luciferase (MMTV-Luc) cassette was generated to provide a suitable reporter construct. Feasibility studies with BacMam-MR in single-dose tests of 1000 compounds showed high correlation to the standard transfection-based assay results. Likewise, in dose-response experiments, BacMam-based assays for GR and PR-B produced potency and efficacy values similar to transfection assay results. At various receptor/reporter ratios, the BacMam assays showed good flexibility, demonstrating consistent signal-to-background (S/B) ratios and compound potencies. Increasing transduction time from 24 to 48 h provided no benefit, actually reducing overall assay performance as measured by S/B and Z' values. The BacMam technology was applied in studies of isoforms PR-A and PR-B, which showed similar responses to a series of agonists. Taken together, the results demonstrate the utility of steroid nuclear receptor BacMam constructs for compound screening procedures with high reproducibility, reduced turnaround time, and lower cost.
Asunto(s)
Baculoviridae/genética , ADN Recombinante/genética , Receptores de Progesterona/análisis , Receptores de Progesterona/genética , Animales , Línea Celular , Línea Celular Tumoral , Chlorocebus aethiops , Humanos , Isoformas de Proteínas/análisis , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptores de Progesterona/metabolismo , Transducción Genética , TransfecciónRESUMEN
The nuclear receptor (NR) superfamily represents a major class of drug targets for the pharmaceutical industry. Strategies for the development of novel, more selective and safer compounds aimed at these receptors are now emerging. Reporter assays have been used routinely for the identification and characterisation of NR ligands. As the NR drug development process evolves, the increase in screening demand in terms of both capacity and complexity has necessitated the development of novel assay formats with increased throughput and flexibility. BacMam technology, a modified baculovirus system for over-expressing genes of interest in mammalian cells has helped answer this requirement. BacMam has many advantages over traditional gene delivery systems including high transduction efficiencies, broad cell host range, speed, cost and ease of generation and use. As outlined in this review, the technology has shown itself to be robust and efficient in various NR assay formats including transactivation (ER alpha/beta, MR, PR and PXR) and transrepression (GR-NFkappaB). In addition, the flexibility of this system will allow greater multiplexing of receptor, reporter, and cell host combinations as NR assays become more complex in order to relate better to relevant cellular and biological systems.
Asunto(s)
Baculoviridae/genética , Receptores Citoplasmáticos y Nucleares/genética , Tecnología Farmacéutica/métodos , Transfección/métodos , Animales , Regulación de la Expresión Génica/genética , Vectores Genéticos/genética , Humanos , Receptores Citoplasmáticos y Nucleares/antagonistas & inhibidores , Receptores de Esteroides/antagonistas & inhibidores , Receptores de Esteroides/genéticaRESUMEN
The baculovirus expression system has been used extensively for the expression of recombinant proteins in insect cells. Recently, recombinant baculovirus vectors engineered to contain mammalian cell-active promoter elements, have been used successfully for transient and stable gene delivery in a broad spectrum of primary and established mammalian cells. The application of modified baculoviruses for in vivo gene delivery has also been demonstrated. In contrast to other commonly used viral vectors, baculoviruses have the unique property of replicating in insect cells while being incapable of initiating a replication cycle and producing infectious virus in mammalian cells. The viruses can be readily manipulated, accommodate large insertions of foreign DNA, initiate little to no microscopically observable cytopathic effect in mammalian cells and have a good biosafety profile. These attributes will undoubtedly lead to the increased application and continued development of this system for efficient gene delivery into mammalian cells. Who said you can't teach an old dog new tricks?
Asunto(s)
Baculoviridae/genética , ADN Recombinante/genética , ADN Viral/genética , Mamíferos/genética , Mamíferos/virología , Animales , Regulación Viral de la Expresión Génica/genética , Técnicas de Transferencia de Gen , Genes Virales , Vectores Genéticos/genética , Humanos , Proteínas Estructurales Virales/genéticaRESUMEN
The initial event by which M-tropic HIV strains gain access to cells is via interaction of the viral envelope protein gp120 with the host cell CCR5 coreceptor and CD4. Inhibition of this event reduces viral fusion and entry into cells in vitro. The authors have employed BacMam baculovirus-mediated gene transduction to develop a cell/cell fusion assay that mimics the HIV viral/cell fusion process and allows high-throughput quantification of this fusion event. The assay design uses human osteosarcoma (HOS) cells stably transfected with cDNAs expressing CCR5, CD4, and long terminal repeat (LTR)-luciferase as the recipient host cell. An HEK-293 cell line transduced with BacMam viral constructs to express the viral proteins gp120, gp41, tat, and rev represents the virus. Interaction of gp120 with CCR5/CD4 results in the fusion of the 2 cells and transfer of tat to the HOS cell cytosol; tat, in turn, binds to the LTR region on the luciferase reporter and activates transcription, resulting in an increase in cellular luciferase activity. In conclusion, the cell/cell fusion assay developed has been demonstrated to be a robust and reproducible high-throughput surrogate assay that can be used to assess the effects of compounds on gp120/CCR5/CD4-mediated viral fusion into host cells.
Asunto(s)
Baculoviridae/genética , Antígenos CD4/metabolismo , Fusión Celular , Proteína gp120 de Envoltorio del VIH/metabolismo , VIH , Piperidinas , Receptores CCR5/metabolismo , Amidas/farmacología , Ácido Butírico/farmacología , Antagonistas de los Receptores CCR5 , Línea Celular , Línea Celular Tumoral , Óxidos N-Cíclicos/farmacología , Dimetilsulfóxido/farmacología , Productos del Gen env/metabolismo , Productos del Gen rev/genética , Productos del Gen rev/metabolismo , Productos del Gen tat/genética , Productos del Gen tat/metabolismo , Duplicado del Terminal Largo de VIH/genética , Humanos , Oximas , Plásmidos , Piridinas/farmacología , Compuestos de Amonio Cuaternario/farmacología , Transducción Genética , Transfección , Productos del Gen rev del Virus de la Inmunodeficiencia Humana , Productos del Gen tat del Virus de la Inmunodeficiencia HumanaRESUMEN
We report that modified baculoviruses, termed BacMam viruses, can efficiently deliver multiple genes into mammalian cells to generate a heterologous transcription factor/reporter gene system. Using human estrogen receptor (ER) as a model nuclear receptor, we demonstrate how this approach can be successfully applied to assay development in Saos-2 human osteosarcoma cells. BacMam viruses containing full-length cDNAs were constructed for both human ER subtypes, ERalpha and ERbeta, and a third BacMam virus containing an ER-responsive reporter gene cassette. Using these viruses, we found that BacMam-ER expression/reporter constructs could be used to profile the effects of the agonist 17beta-estradiol and the partial agonist raloxifene in human Saos-2 cells. A comparison of assay data obtained with the BacMam-based system with that using standard DNA transfections demonstrates that the two systems are functionally equivalent, giving comparable EC(50) and IC(50) values for estrogen and estrogen plus raloxifene treatments, respectively. Our results indicate that BacMam-mediated gene transfer offers a novel and efficient method for delivery of nuclear receptors and associated genes for mammalian cell-based assay development.
Asunto(s)
Baculoviridae/genética , Neoplasias Óseas/genética , Osteosarcoma/genética , Receptores de Estrógenos/genética , Proteínas Recombinantes de Fusión/fisiología , Transfección/métodos , Animales , Baculoviridae/metabolismo , Neoplasias Óseas/metabolismo , Línea Celular Tumoral , Vectores Genéticos , Humanos , Osteosarcoma/metabolismo , Receptores de Estrógenos/fisiología , Proteínas Recombinantes de Fusión/síntesis química , Proteínas Recombinantes de Fusión/genética , Transducción Genética , Xenopus laevisRESUMEN
A relatively recent advance in the use of recombinant baculoviruses is their use for delivery of genes and genetic elements into mammalian cells. Baculovirus vectors retrofitted with mammalian gene promoters have been shown to efficiently deliver and express genes in a broad assortment of cell types. These baculovirus transductions are simple to perform, reproducible, and demonstrate no overt cell toxicity. Baculovirus-mediated gene delivery is particularly useful for repetitive or moderately high-throughput procedures such as cell-based assays, or for situations where transfection procedures are inadequate.
Asunto(s)
Baculoviridae/genética , Técnicas de Transferencia de Gen , Animales , Línea Celular , Humanos , Plásmidos , beta-Galactosidasa/genéticaRESUMEN
The development of cell-based assays for cellular receptors, ion channels, and transporters requires the delivery and expression of transgenes. Viral-mediated gene delivery is a particularly attractive approach for this purpose because of its efficiency and potential to deliver genes to a wide variety of cell types. Recombinant baculoviruses, long used to deliver genes to insect cells for overexpression, also effectively transfer genes to mammalian cells. The only required modification to the virus for this purpose is the addition of transgene expression cassettes controlled by mammalian cell-active promoters. These so-called BacMam viruses are useful for developing mammalian cell-based assays for investigating the function of recombinant proteins and for assessing the action of pharmacological modulators of their function. The use of such viruses is gaining popularity because of the ease of optimizing assay conditions, the ability to deliver multiple gene products, and of their flexibility in terms of host cells and levels of transgene expression. BacMam-mediated assays may be used for studying a wide variety of target proteins and assay technologies. Described in this unit is an example of BacMam-mediated gene delivery to configure a cell-based assay for pharmacological assessment of a G protein-coupled receptor. A protocol is also provided describing the use of a GFP-expressing BacMam to assess the susceptibility of new cell lines to transduction by the virus.
Asunto(s)
Baculoviridae , Proteínas de Unión al GTP/metabolismo , Técnicas de Transferencia de Gen , Vectores Genéticos/genética , Receptores Acoplados a Proteínas G/genética , Animales , Calcio/metabolismo , Línea Celular , Humanos , Indicadores y Reactivos , Mamíferos , Soluciones , Transducción Genética/métodosRESUMEN
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.
Asunto(s)
Baculoviridae/genética , Técnicas de Transferencia de Gen , Terapia Genética/métodos , Animales , Línea Celular , Expresión Génica , Genes , Mamíferos/genéticaRESUMEN
Functional expression of recombinant proteins has become a routine, but critical tool in modern molecular biology. Since their introduction, the use of baculovirus vectors to produce proteins for purification has become one of the most widely-used viral gene delivery systems as expression levels obtained are difficult to match with any other eukaryotic expression system. Extensive engineering to simplify and accelerate the process of recombinant virus construction has made this system accessible to virtually any modern biological laboratory. The utility of baculoviruses has been broadened with the discovery that appropriately modified virus can mediate gene expression in a wide variety of mammalian cell lines, and thus can function as a flexible cell-based assay development tool. The wide range of applications and potential for commercialization of products leads to consideration of a number of aspects of the system.
Asunto(s)
Baculoviridae/genética , Vectores Genéticos , Ingeniería de Proteínas/métodos , Animales , Línea Celular , ADN Viral/metabolismo , Regulación Viral de la Expresión Génica , Técnicas de Transferencia de Gen , Insectos/genética , Mamíferos , Transfección/métodosRESUMEN
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.
RESUMEN
The expression of recombinant proteins following transduction of CHO cells with recombinant baculoviruses containing a mammalian expression cassette with the CMV-promoter is enhanced by the addition of trichostatin A (TSA), a specific histone deacetylase inhibitor. To further investigate the effect of TSA treatment on protein production following BacMam transduction, viruses containing various viral promoters (SV40, CMV, and RSV) and one cellular promoter (human ubiquitin C) were compared with regard to expression level of a gfp-luciferase fusion protein following transduction of CHO, COS-1, and HEK293 cells. The overall effect on expression appears to be cell specific, indicating that different mechanisms are active within different cell lines. Further, COS cells transfected with naked viral DNA, plasmids, and baculovirus particles were compared in regard to TSA treatment. The increase in reporter gene expression observed following BacMam transduction and TSA treatment were greater than those for transfection of either naked viral DNA or plasmid DNA.
Asunto(s)
Baculoviridae/genética , Genes Bacterianos , Ácidos Hidroxámicos/farmacología , Regiones Promotoras Genéticas , Animales , Baculoviridae/efectos de los fármacos , Células CHO , Células COS , Cricetinae , Cricetulus , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
A variety of transfection approaches have been used to deliver plasmids encoding ion channel genes into cells. We have used the baculovirus transduction system, BacMam, to demonstrate transient expression of multi-subunit KATP channels in CHO-K1 and HEK-293 EBNA cells using sulfonylurea receptor 1 (SUR), SUR2A, SUR2B, and KIR 6.2 genes. [3H]-glyburide binding, patch clamp, and DiBAC4(3) measurements of membrane potential changes were used to monitor channel expression. BacMam delivery of each SUR isoform with KIR6.2 was demonstrated based on its pharmacological profiles. Expression levels of SUR1 and KIR6.2 were titrated by varying the viral concentration or time of virus addition, with functional activity measured in as little as 4-6 hours posttransduction. Further increases in BacMam virus induced sufficient KATP expression to dominate membrane potential without pharmacological opening of the channel. Independently altering treatment with virus containing either the SUR1 or KIR6.2 gene revealed interactions among subunits during formation of functional channels in the plasma membrane. This study demonstrates the utility and versatility of BacMam as a valuable gene delivery tool for the study of ion channel function.
Asunto(s)
Transportadoras de Casetes de Unión a ATP , Canales de Potasio de Rectificación Interna/genética , Canales de Potasio de Rectificación Interna/metabolismo , Canales de Potasio/genética , Canales de Potasio/metabolismo , Receptores de Droga/genética , Receptores de Droga/metabolismo , Animales , Baculoviridae/genética , Células CHO , Línea Celular , Cricetinae , Expresión Génica , Gliburida/metabolismo , Humanos , Potenciales de la Membrana , Plásmidos/genética , Canales de Potasio/química , Canales de Potasio de Rectificación Interna/química , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Droga/química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Receptores de Sulfonilureas , Transducción GenéticaRESUMEN
Previously we demonstrated the rapid generation of affinity matured monoclonal antibody (MAb) producing cell lines following gene gun delivery of DNA using a mammalian expression vector (pAlpha/hFc), which enables the expression of human Fc-chimera proteins in vivo. Here we compare the pAlpha/hFc vector to modified vectors that replace human IgG(1) with either a Glutathione-S-Transferase (GST) fusion protein or a mouse IgG(2c) (mFc) fusion protein. We report that in vivo expression of a GST-chimera results in the rapid generation of affinity matured MAbs, comparable with antibodies raised using the pAlpha/hFc vector, that were reactive with annexin V. The mFc vector failed to induce early antigen-specific B-cell responses suitable for MAb development.