Publications by authors named "Robert S Ames"

23 Publications

  • Page 1 of 1

Gene Expression in Mammalian Cells Using BacMam, a Modified Baculovirus System.

Methods Mol Biol 2016 ;1350:95-116

Platform Technology and Science, GlaxoSmithKline, 1250 S. Collegeville Road, Collegeville, PA, 19426, USA.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/978-1-4939-3043-2_5DOI Listing
October 2016

Identification and characterization of PERK activators by phenotypic screening and their effects on NRF2 activation.

PLoS One 2015 17;10(3):e0119738. Epub 2015 Mar 17.

Department of Biological Sciences, Platform Technology and Science, GlaxoSmithKline, Collegeville, Pennsylvania, United States of America.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0119738PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4363567PMC
February 2016

Development of phenotypic screening assays for γ-globin induction using primary human bone marrow day 7 erythroid progenitor cells.

J Biomol Screen 2013 Dec 25;18(10):1212-22. Epub 2013 Oct 25.

1Molecular Discovery Research, GlaxoSmithKline, Collegeville, PA, USA.

Sickle cell anemia (SCA) is a genetic disorder of the β-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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1177/1087057113499776DOI Listing
December 2013

The development of a high-content screening binding assay for the smoothened receptor.

J Biomol Screen 2012 Aug 29;17(7):900-11. Epub 2012 May 29.

Department of Biological Reagents and Assay Development, Molecular Discovery Research, GlaxoSmithKline, Collegeville, PA 19426-0989, USA.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1177/1087057112447872DOI Listing
August 2012

Development of a cell-based high throughput luciferase enzyme fragment complementation assay to identify nuclear-factor-e2-related transcription factor 2 activators.

Assay Drug Dev Technol 2012 Dec 10;10(6):514-24. Epub 2012 May 10.

Department of Biological Reagents and Assay Development, Platform Technology and Science, GlaxoSmithKline Pharmaceuticals, Collegeville, Pennsylvania 19426, USA.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1089/adt.2011.436DOI Listing
December 2012

A cell-based high-throughput screening assay to measure cellular histone h3 lys27 trimethylation with a modified dissociation-enhanced lanthanide fluorescent immunoassay.

J Biomol Screen 2012 Jan 15;17(1):99-107. Epub 2011 Nov 15.

Biological Reagents and Assay Development, Platform Technology and Science, GlaxoSmithKline, Collegeville, PA 19426, USA.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1177/1087057111422378DOI Listing
January 2012

Assay development and high-throughput screening of small molecular c-Abl kinase activators.

J Biomol Screen 2011 Jan 11;16(1):53-64. Epub 2010 Oct 11.

Biological Reagents and Assay Development, GlaxoSmithKline Pharmaceuticals, Collegeville, PA, USA.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1177/1087057110384133DOI Listing
January 2011

Production of protein complexes via co-expression.

Protein Expr Purif 2011 Jan 6;75(1):1-14. Epub 2010 Aug 6.

Biological Reagents & Assay Development, Platform Technology & Science, GlaxoSmithKline R&D, 1250 South Collegeville Road, Collegeville, PA 19426, USA.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.pep.2010.07.015DOI Listing
January 2011

Baculovirus gene delivery: a flexible assay development tool.

Curr Gene Ther 2010 Jun;10(3):168-73

GlaxoSmithKline, 5 Moore Drive, Research Triangle Park, North Carolina 27709, USA.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2174/156652310791321224DOI Listing
June 2010

Development of a high throughput cell-based assay for soluble epoxide hydrolase using BacMam technology.

Mol Biotechnol 2010 Jul;45(3):207-17

Biological Reagents and Assay Development, Molecular Discovery Research, GlaxoSmithKline, 1250 S. Collegeville Road, Collegeville, PA 19426, USA.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s12033-010-9271-8DOI Listing
July 2010

Development of a high-throughput cell-based assay for superoxide production in HL-60 cells.

J Biomol Screen 2010 Apr 12;15(4):388-97. Epub 2010 Mar 12.

Department of Screening and Compound Profiling, GlaxoSmithKline R&D, Collegeville, Pennsylvania 19426, USA.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1177/1087057109359687DOI Listing
April 2010

BacMam: versatile gene delivery technology for GPCR assays.

Methods Mol Biol 2009 ;552:199-211

Molecular Discovery Research, Biological Reagents and Assay Development, GlaxoSmithKline R&D, Collegeville, PA, USA.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/978-1-60327-317-6_14DOI Listing
July 2009

Optimized procedures for producing biologically active chemokines.

Protein Expr Purif 2009 Jun;65(2):251-60

GlaxoSmithKline, Biological Reagents & Assay Development, Mail Code: UE0548, 709 Swedeland Road, King of Prussia, PA 19406, USA.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.pep.2009.01.017DOI Listing
June 2009

Viral-mediated gene delivery for cell-based assays in drug discovery.

Expert Opin Drug Discov 2009 Mar;4(3):243-56

Molecular Discovery Research, GlaxoSmithKline R&D, UE0433, 709 Swedeland Road, King of Prussia, PA 19406, USA +1 610 270 7602 ; +1 610 270 7359 ;

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1517/17460440902751599DOI Listing
March 2009

Development of a high-throughput cell-based assay for 11beta-hydroxysteroid dehydrogenase type 1 using BacMam technology.

Mol Biotechnol 2008 Jun;39(2):127-34

Biological Reagents and Assay Development, GlaxoSmithKline, 1250 S. Collegeville Road, Collegeville, PA 19426, USA.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s12033-008-9050-yDOI Listing
June 2008

BacMam technology and its application to drug discovery.

Expert Opin Drug Discov 2007 Dec;2(12):1669-81

Molecular Discovery Research, GlaxoSmithKline R&D, UE0433, 709 Swedeland Road, King of Prussia, PA 19406, USA +1 610 270 7602 ; +1 610 270 7359 ;

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1517/17460441.2.12.1669DOI Listing
December 2007

Gene expression in mammalian cells using BacMam, a modified baculovirus system.

Methods Mol Biol 2007 ;388:95-114

Department of Gene Expression and Protein Biochemistry, GlaxoSmithKline, King of Prussia, PA, USA.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/978-1-59745-457-5_5DOI Listing
November 2007

Implementation of BacMam virus gene delivery technology in a drug discovery setting.

Drug Discov Today 2007 May 23;12(9-10):396-403. Epub 2007 Mar 23.

Molecular Discovery Research, GlaxoSmithKline, 5 Moore Drive, Research Triangle Park, NC 27709, USA.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.drudis.2007.02.017DOI Listing
May 2007

Baculoviruses and mammalian cell-based assays for drug screening.

Adv Virus Res 2006 ;68:255-86

Department of Gene Expression and Protein Biochemistry GlaxoSmithKline Discovery Research, Research Triangle Park, North Carolina 27709, USA.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/S0065-3527(06)68007-XDOI Listing
November 2006

The peptidic urotensin-II receptor ligand GSK248451 possesses less intrinsic activity than the low-efficacy partial agonists SB-710411 and urantide in native mammalian tissues and recombinant cell systems.

Br J Pharmacol 2006 May;148(2):173-90

Department of Vascular Biology and Thrombosis, GlaxoSmithKline, 709 Swedeland Road, King of Prussia, PA 19406, USA.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/sj.bjp.0706716DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1617064PMC
May 2006

Structure-function studies of the C3a-receptor: C-terminal serine and threonine residues which influence receptor internalization and signaling.

Eur J Immunol 2003 Apr;33(4):920-7

Department of Medical Microbiology, Medical School Hannover, Hannover, Germany.

The anaphylatoxic peptide C3a is a pro-inflammatory mediator generated during complement activation, whose specific G protein coupled receptor is expressed on granulocytes, monocytes, mast cells, activated lymphocytes, and in the nervous tissue. We have generated RBL-2H3 cell clones stably expressing mutants of the human C3a-receptor (C3aR) with combined alanine (Ala) substitutions of ten C-terminal serine (Ser) or threonine (Thr) residues, which may represent putative phosphorylation sites to characterize their role in ligand-induced C3aR internalization and signaling. Ser475/479 and Thr480/481 as well as Ser449 seemed not to be involved in ligand-induced receptor internalization. Either directly or by a conformational change they even "inhibit" C3aR internalization. In contrast, mutants with Ala substitutions at Ser465/470 and Thr463/466 were poorly internalized, and Thr463 seemed to be the most important C-terminal Thr or Ser residue directly effecting receptor internalization. However, it is likely that other C3aR regions additionally participate in this negative feed-back mechanism since even mutants with multiple Ala substitutions still internalized to a limited degree. Interestingly, in a mutant with a single exchange of Ser449 to Ala, the signal transduction assessed by a Ca(2+) assay and [(35)S]GTP gamma S-binding on HEK cells transiently co-transfected with G-alpha 16 or G-alpha O, respectively, was severely impaired, indicating that this residue of C3aR is involved in G protein coupling.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/eji.200323293DOI Listing
April 2003

Progressive epitope-blocked panning of a phage library for isolation of human RSV antibodies.

J Immunol Methods 2002 May;263(1-2):123-32

Department of Molecular Biology, GlaxoSmithKline Pharmaceuticals, 709 Swedeland Road, P.O. Box 1539, King of Prussia, PA 19406, USA.

Epitope-blocked panning is an approach to mining antigen-specific diversity from phage display antibody libraries. Previously, we developed and used this method to recover a neutralizing antibody to respiratory syncytial virus (RSV) by blocking a dominant response to a nonneutralizing epitope on a recombinant derivative of the viral F antigen. We have extended this approach to the blocking of multiple epitopes simultaneously, which led to the recovery of new antibodies of different specificity, including one new neutralizing activity. A phage display Fab library was selected on recombinant F antigen in the presence of three representative antibodies recovered in the unblocked and subsequent single-blocked panning procedures. Restriction endonuclease fingerprinting of 13 F+ clones revealed seven unique Fabs. DNA sequence analysis of five of these clones revealed five new light chains in combination with different heavy chains, three of which were very similar or identical to Fabs previously isolated from this library. The blocking antibodies did not compete with the new Fabs, demonstrating effective masking of their binding sites in the panning procedure. Conversely, these Fabs did show variable inhibition of two of the blocking antibodies suggesting a close proximity or interdependence of their epitopes. One of the antibodies did inhibit virus infection, albeit with modest potency. These results demonstrate that epitope-blocked panning is a self-progressing approach to retrieving diverse antibodies from phage libraries.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/s0022-1759(02)00032-7DOI Listing
May 2002

Expression of the complement C3a and C5a receptors after permanent focal ischemia: An alternative interpretation.

Glia 2002 Apr;38(2):169-73

Department of Microbiology, University of Alabama, Birmingham, 35294, USA.

The receptors for the complement anaphylatoxins C3a and C5a are expressed by glial cells and neurons in normal and inflamed brain. Previous studies demonstrated modest elevations in mRNA expression of these receptors in a model of focal cerebral ischemia. Using a similar model system for both mice and rats, we report markedly different patterns of anaphylatoxin receptor mRNA expression in cerebral ischemia. C5a receptor expression was dramatically elevated within 3 h after middle cerebral artery occlusion, while C3aR expression was reduced to 25% of control animals. By 24 h post-occlusion, expression of both receptors was higher than at any other time point examined. This increased expression at late time points after occlusion is most likely the result of massive infiltration of leukocytes expressing the receptors. We also observed increased receptor mRNA expression in sham-operated animals, indicating that the procedures used for arterial occlusion affects mechanisms regulating receptor expression. This latter result highlights the importance of including this important control group in ischemic model systems for proper interpretation of changes in gene expression.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/glia.10069DOI Listing
April 2002