Publications by authors named "Paul S Carter"

10 Publications

  • Page 1 of 1

Rapid Lentiviral Vector Producer Cell Line Generation Using a Single DNA Construct.

Mol Ther Methods Clin Dev 2020 Dec 14;19:47-57. Epub 2020 Aug 14.

GlaxoSmithKline Medicines Research Centre, Gunnels Wood Road, Stevenage SG1 2NY, UK.

Stable suspension producer cell lines for the production of vesicular stomatitis virus envelope glycoprotein (VSVg)-pseudotyped lentiviral vectors represent an attractive alternative to current widely used production methods based on transient transfection of adherent 293T cells with multiple plasmids. We report here a method to rapidly generate such producer cell lines from 293T cells by stable transfection of a single DNA construct encoding all lentiviral vector components. The resulting suspension cell lines yield titers as high as can be achieved with transient transfection, can be readily scaled up in single-use stirred-tank bioreactors, and are genetically and functionally stable in extended cell culture. By removing the requirement for efficient transient transfection during upstream processing of lentiviral vectors and switching to an inherently scalable suspension cell culture format, we believe that this approach will result in significantly higher batch yields than are possible with current manufacturing processes and enable better patient access to medicines based on lentiviral vectors.
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http://dx.doi.org/10.1016/j.omtm.2020.08.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7501408PMC
December 2020

Structurally Diverse Mitochondrial Branched Chain Aminotransferase (BCATm) Leads with Varying Binding Modes Identified by Fragment Screening.

J Med Chem 2016 Mar 16;59(6):2452-67. Epub 2016 Mar 16.

Medicines Research Centre, GlaxoSmithKline R&D , Gunnels Wood Road, Stevenage, Hertfordshire, SG1 2NY, U.K.

Inhibitors of mitochondrial branched chain aminotransferase (BCATm), identified using fragment screening, are described. This was carried out using a combination of STD-NMR, thermal melt (Tm), and biochemical assays to identify compounds that bound to BCATm, which were subsequently progressed to X-ray crystallography, where a number of exemplars showed significant diversity in their binding modes. The hits identified were supplemented by searching and screening of additional analogues, which enabled the gathering of further X-ray data where the original hits had not produced liganded structures. The fragment hits were optimized using structure-based design, with some transfer of information between series, which enabled the identification of ligand efficient lead molecules with micromolar levels of inhibition, cellular activity, and good solubility.
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http://dx.doi.org/10.1021/acs.jmedchem.5b01607DOI Listing
March 2016

The Discovery of in Vivo Active Mitochondrial Branched-Chain Aminotransferase (BCATm) Inhibitors by Hybridizing Fragment and HTS Hits.

J Med Chem 2015 Sep 2;58(18):7140-63. Epub 2015 Jul 2.

GlaxoSmithKline R&D, Medicines Research Centre , Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, U.K.

The hybridization of hits, identified by complementary fragment and high throughput screens, enabled the discovery of the first series of potent inhibitors of mitochondrial branched-chain aminotransferase (BCATm) based on a 2-benzylamino-pyrazolo[1,5-a]pyrimidinone-3-carbonitrile template. Structure-guided growth enabled rapid optimization of potency with maintenance of ligand efficiency, while the focus on physicochemical properties delivered compounds with excellent pharmacokinetic exposure that enabled a proof of concept experiment in mice. Oral administration of 2-((4-chloro-2,6-difluorobenzyl)amino)-7-oxo-5-propyl-4,7-dihydropyrazolo[1,5-a]pyrimidine-3-carbonitrile 61 significantly raised the circulating levels of the branched-chain amino acids leucine, isoleucine, and valine in this acute study.
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http://dx.doi.org/10.1021/acs.jmedchem.5b00313DOI Listing
September 2015

Catalytic in vivo protein knockdown by small-molecule PROTACs.

Nat Chem Biol 2015 Aug 10;11(8):611-7. Epub 2015 Jun 10.

1] Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut, USA. [2] Department of Chemistry, Yale University, New Haven, Connecticut, USA. [3] Department of Pharmacology, Yale University, New Haven, Connecticut, USA.

The current predominant therapeutic paradigm is based on maximizing drug-receptor occupancy to achieve clinical benefit. This strategy, however, generally requires excessive drug concentrations to ensure sufficient occupancy, often leading to adverse side effects. Here, we describe major improvements to the proteolysis targeting chimeras (PROTACs) method, a chemical knockdown strategy in which a heterobifunctional molecule recruits a specific protein target to an E3 ubiquitin ligase, resulting in the target's ubiquitination and degradation. These compounds behave catalytically in their ability to induce the ubiquitination of super-stoichiometric quantities of proteins, providing efficacy that is not limited by equilibrium occupancy. We present two PROTACs that are capable of specifically reducing protein levels by >90% at nanomolar concentrations. In addition, mouse studies indicate that they provide broad tissue distribution and knockdown of the targeted protein in tumor xenografts. Together, these data demonstrate a protein knockdown system combining many of the favorable properties of small-molecule agents with the potent protein knockdown of RNAi and CRISPR.
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http://dx.doi.org/10.1038/nchembio.1858DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4629852PMC
August 2015

Discovery of GSK143, a highly potent, selective and orally efficacious spleen tyrosine kinase inhibitor.

Bioorg Med Chem Lett 2011 Oct 12;21(20):6188-94. Epub 2011 Aug 12.

GlaxoSmithKline R&D, Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire, UK.

The lead optimisation of the diaminopyrimidine carboxamide series of spleen tyrosine kinase inhibitors is described. The medicinal chemistry strategy was focused on optimising the human whole blood activity whilst achieving a sufficient margin over liability kinases and hERG activity. GSK143 is a potent and highly selective SYK inhibitor showing good efficacy in the rat Arthus model.
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http://dx.doi.org/10.1016/j.bmcl.2011.07.082DOI Listing
October 2011

Kinetic assay for characterization of spleen tyrosine kinase activity and inhibition with recombinant kinase and crude cell lysates.

Anal Biochem 2009 Jan 15;384(1):56-67. Epub 2008 Aug 15.

Center for Signal Transduction Research, Cellular Analysis/BioDiscovery Division, Invitrogen, Hopkinton, MA 01748, USA.

Spleen tyrosine kinase (Syk) is involved in the activation of cells implicated in allergic or autoimmune diseases and certain cancers. Therefore, Syk inhibitors may prove to be effective in treating diseases where Syk activity or expression is increased or deregulated. We developed a continuous and direct (noncoupled) fluorescence intensity assay for measuring Syk activity using purified recombinant enzyme or crude lysates generated from anti-immunoglobulin M (IgM) antibody-treated RAMOS cells. The assay is based on the chelation-enhanced fluorophore 8-hydroxy-5-(N,N-dimethylsulfonamido)-2-methylquinoline (referred to as Sox), which has been incorporated into a peptide substrate selected for robust detection of Syk activity. This homogeneous assay is simple to use, provides considerably more information, and has been adapted to a 384-well, low-volume microtiter plate format that can be used for the high-throughput identification and kinetic characterization of Syk inhibitors. The assay can be performed with a wide range of adenosine triphosphate (ATP) concentrations and, therefore, can be used to analyze ATP-competitive and ATP-noncompetitive/allosteric kinase inhibitors. Measurement of Syk activity in RAMOS crude cell lysates or immunoprecipitation (IP) capture formats may serve as a physiologically more relevant enzyme source. These Sox-based continuous and homogeneous assays provide a valuable set of tools for studying Syk signaling and for defining inhibitors that may be more effective in controlling disease.
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http://dx.doi.org/10.1016/j.ab.2008.07.040DOI Listing
January 2009

Necrotising fasciitis: a new management algorithm based on clinical classification.

Int Wound J 2004 Sep;1(3):189-98

Department of Plastic Surgery, Radcliffe Infirmary, Oxford, UK.

Necrotising fasciitis is a rare infection of the subcutaneous tissues. If untreated, it is invariably fatal, and thus a high index of suspicion for the diagnosis is required. The disease's manifestation can range from a fulminant presentation to a subtle and insidious development. The priority in every case is to proceed to radical surgical debridement. On review of the literature and based on our clinical experience, we propose a new classification based on clinical presentation and suggest an algorithm to facilitate the management of this devastating condition. Increasing awareness should be given to the management of the large wounds resulting from the surgical debridement of necrotising fasciitis.
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http://dx.doi.org/10.1111/j.1742-4801.2004.00054.xDOI Listing
September 2004

The structure of MSK1 reveals a novel autoinhibitory conformation for a dual kinase protein.

Structure 2004 Jun;12(6):1067-77

Discovery Research, GlaxoSmithKline, Third Ave, Harlow, Essex, CM19 5AW, United Kingdom.

Mitogen and stress-activated kinase-1 (MSK1) is a serine/threonine protein kinase that is activated by either p38 or p42ERK MAPKs in response to stress or mitogenic extracellular stimuli. MSK1 belongs to a family of protein kinases that contain two distinct kinase domains in one polypeptide chain. We report the 1.8 A crystal structure of the N-terminal kinase domain of MSK1. The crystal structure reveals a unique inactive conformation with the ATP binding site blocked by the nucleotide binding loop. This inactive conformation is stabilized by the formation of a new three-stranded beta sheet on the N lobe of the kinase domain. The three beta strands come from residues at the N terminus of the kinase domain, what would be the alphaB helix in the active conformation, and the activation loop. The new three-stranded beta sheet occupies a position equivalent to the N terminus of the alphaC helix in active protein kinases.
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http://dx.doi.org/10.1016/j.str.2004.02.040DOI Listing
June 2004

The antimicrobial natural product chuangxinmycin and some synthetic analogues are potent and selective inhibitors of bacterial tryptophanyl tRNA synthetase.

Bioorg Med Chem Lett 2002 Nov;12(21):3171-4

GlaxoSmithKline, New Frontiers Science Park, Third Avenue, Harlow, Essex CM19 5AW, UK.

The antimicrobial natural product chuangxinmycin has been found to be a potent and selective inhibitor of bacterial tryptophanyl tRNA synthetase (WRS). A number of analogues have been synthesised. The interaction with WRS appears to be highly constrained, as only sterically smaller analogues afforded significant inhibition. The only analogue to show inhibition comparable to chuangxinmycin also had antibacterial activity. WRS inhibition may contribute to the antibacterial action of chuangxinmycin.
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http://dx.doi.org/10.1016/s0960-894x(02)00604-2DOI Listing
November 2002

Expression and purification of functional JNK2beta2: perspectives on high-level production of recombinant MAP kinases.

Protein Expr Purif 2002 Feb;24(1):25-32

GlaxoSmithKline, New Frontiers Science Park (North), Coldharbour Road, Harlow, Essex CM19 5AD, United Kingdom.

The mitogen-activated protein (MAP) kinases are a group of serine/threonine kinases that mediate intracellular signal transduction in response to environmental stimuli including stress, growth factors, and various cytokines. Of this family, the c-Jun N-terminal kinases (JNKs) are members which, depending on cell type, have been shown to activate the transcription of genes involved in the inflammatory response, apoptosis, and hypertrophy. Here we report the use Baculovirus/Sf9 cells to produce milligram quantities of recombinant JNK2beta2 substrate which could be purified to >90% as judged by SDS-PAGE. In addition, we report a novel method for the site-specific biotinylation for this enzyme and demonstrate that the biotinylated product is an authentic substrate of the upstream kinases MKK4 and 7 and can phosphorylate a downstream target, ATF-2. We also show that the phosphorylated product can be captured efficiently on streptavidin-coated beads for use in scintillation proximity assays.
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http://dx.doi.org/10.1006/prep.2001.1544DOI Listing
February 2002