Publications by authors named "David J Powell"

10 Publications

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DDI2 Is a Ubiquitin-Directed Endoprotease Responsible for Cleavage of Transcription Factor NRF1.

Mol Cell 2020 07 9;79(2):332-341.e7. Epub 2020 Jun 9.

Mechanisms of Transcription Laboratory, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK. Electronic address:

The Ddi1/DDI2 proteins are ubiquitin shuttling factors, implicated in a variety of cellular functions. In addition to ubiquitin-binding and ubiquitin-like domains, they contain a conserved region with similarity to retroviral proteases, but whether and how DDI2 functions as a protease has remained unknown. Here, we show that DDI2 knockout cells are sensitive to proteasome inhibition and accumulate high-molecular weight, ubiquitylated proteins that are poorly degraded by the proteasome. These proteins are targets for the protease activity of purified DDI2. No evidence for DDI2 acting as a de-ubiquitylating enzyme was uncovered, which could suggest that it cleaves the ubiquitylated protein itself. In support of this idea, cleavage of transcription factor NRF1 is known to require DDI2 activity in vivo. We show that DDI2 is indeed capable of cleaving NRF1 in vitro but only when NRF1 protein is highly poly-ubiquitylated. Together, these data suggest that DDI2 is a ubiquitin-directed endoprotease.
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http://dx.doi.org/10.1016/j.molcel.2020.05.035DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7369636PMC
July 2020

Assaying kinase activity of the TPL-2/NF-κB1 p105/ABIN-2 complex using an optimal peptide substrate.

Biochem J 2018 01 11;475(1):329-340. Epub 2018 Jan 11.

Immune Cell Signalling Laboratory, The Francis Crick Institute, London NW1 1AT, U.K.

The MKK1/2 kinase tumour progression locus 2 (TPL-2) is critical for the production of tumour necrosis factor alpha (TNFα) in innate immune responses and a potential anti-inflammatory drug target. Several earlier pharmaceutical company screens with the isolated TPL-2 kinase domain have identified small-molecule inhibitors that specifically block TPL-2 signalling in cells, but none of these have progressed to clinical development. We have previously shown that TPL-2 catalytic activity regulates TNF production by macrophages while associated with NF-κB1 p105 and ABIN-2, independently of MKK1/2 phosphorylation via an unknown downstream substrate. In the present study, we used a positional scanning peptide library to determine the optimal substrate specificity of a complex of TPL-2, NF-κB1 p105 and ABIN-2. Using an optimal peptide substrate based on this screen and a high-throughput mass spectrometry assay to monitor kinase activity, we found that the TPL-2 complex has significantly altered sensitivities versus existing ATP-competitive TPL-2 inhibitors than the isolated TPL-2 kinase domain. These results imply that screens with the more physiologically relevant TPL-2/NF-κB1 p105/ABIN-2 complex have the potential to deliver novel TPL-2 chemical series; both ATP-competitive and allosteric inhibitors could emerge with significantly improved prospects for development as anti-inflammatory drugs.
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http://dx.doi.org/10.1042/BCJ20170579DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5763956PMC
January 2018

Design and Implementation of High-Throughput Screening Assays.

Methods Mol Biol 2016 ;1439:1-32

Alternative Drug Discovery, GSK Pharmaceuticals, 1250 South Collegeville Road, Upper Providence, PA, 19426, USA.

HTS remains at the core of the drug discovery process, and so it is critical to design and implement HTS assays in a comprehensive fashion involving scientists from the disciplines of biology, chemistry, engineering, and informatics. This requires careful consideration of many options and variables, starting with the choice of screening strategy and ending with the discovery of lead compounds. At every step in this process, there are decisions to be made that can greatly impact the outcome of the HTS effort, to the point of making it a success or a failure. Although specific guidelines should be established to ensure that the screening assay reaches an acceptable level of quality, many choices require pragmatism and the ability to compromise opposing forces.
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http://dx.doi.org/10.1007/978-1-4939-3673-1_1DOI Listing
December 2017

HIV proteinase inhibitors target the Ddi1-like protein of Leishmania parasites.

FASEB J 2011 May 25;25(5):1729-36. Epub 2011 Jan 25.

Cardiff School of Biosciences, Cardiff University, Cardiff, UK.

HIV proteinase inhibitors reduce the levels of Leishmania parasites in vivo and in vitro, but their biochemical target is unknown. We have identified an ortholog of the yeast Ddi1 protein as the only member of the aspartic proteinase family in Leishmania parasites, and in this study we investigate this protein as a potential target for the drugs. To date, no enzyme assay has been developed for the Ddi1 proteins, but Saccharomyces cerevisiae lacking the DDI1 gene secrete high levels of protein into the medium. We developed an assay in which these knockout yeast were functionally complemented to low secretion by introduction of genes encoding Ddi1 orthologs from Leishmania major or humans. Plasmid alone controls gave no complementation. Treatment of the Ddi1 transformants with HIV proteinase inhibitors showed differential effects dependent on the origin of the Ddi1. Dose responses allowed calculation of IC(50) values; e.g., for nelfinavir, of 3.4 μM (human Ddi1) and 0.44 μM (Leishmania Ddi1). IC(50) values with Leishmania constructs mirror the potency of inhibitors against parasites. Our results show that Ddi1 proteins are targets of HIV proteinase inhibitors and indicates the Leishmania Ddi1 as the likely target for these drugs and a potential target for antiparasitic therapy.
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http://dx.doi.org/10.1096/fj.10-178947DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3739880PMC
May 2011

The retroviral proteinase active site and the N-terminus of Ddi1 are required for repression of protein secretion.

FEBS Lett 2011 Jan 19;585(1):139-42. Epub 2010 Nov 19.

Cardiff School of Biosciences, Cardiff University, Park Place, Cardiff, UK.

The Ddi1 protein of the yeast Saccharomyces cerevisiae is involved in numerous interactions with the ubiquitin system, which may be mediated by its N-terminal ubiquitin like domain and its C-terminal ubiquitin associated domain. Ddi1 also contains a central region with all the features of a retroviral aspartic proteinase, which was shown to be important in cell-cycle control. Here we demonstrate an additional role for this domain, along with the N-terminal region, in protein secretion. These results further substantiate the hypothesis that Ddi1 functions in vivo as a catalytically-active aspartic proteinase.
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http://dx.doi.org/10.1016/j.febslet.2010.11.026DOI Listing
January 2011

Development of an insect-cell-based assay for detection of kinase inhibition using NF-kappaB-inducing kinase as a paradigm.

Biochem J 2009 Apr;419(1):65-73

Biological Reagents & Assay Development, GlaxoSmithKline R&D, New Frontiers Science Park, Third Avenue, Harlow, Essex, CM19 5AW, UK.

Identification of small-molecule inhibitors by high-throughput screening necessitates the development of robust, reproducible and cost-effective assays. The assay approach adopted may utilize isolated proteins or whole cells containing the target of interest. To enable protein-based assays, the baculovirus expression system is commonly used for generation and isolation of recombinant proteins. We have applied the baculovirus system into a cell-based assay format using NIK [NF-kappaB (nuclear factor kappaB)-inducing kinase] as a paradigm. We illustrate the use of the insect-cell-based assay in monitoring the activity of NIK against its physiological downstream substrate IkappaB (inhibitor of NF-kappaB) kinase-1. The assay was robust, yielding a signal/background ratio of 2:1 and an average Z' value of >0.65 when used to screen a focused compound set. Using secondary assays to validate a selection of the hits, we identified a compound that (i) was non-cytotoxic, (ii) interacted directly with NIK, and (iii) inhibited lymphotoxin-induced NF-kappaB p52 translocation to the nucleus. The insect cell assay represents a novel approach to monitoring kinase inhibition, with major advantages over other cell-based systems including ease of use, amenability to scale-up, protein expression levels and the flexibility to express a number of proteins by infecting with numerous baculoviruses.
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http://dx.doi.org/10.1042/BJ20081646DOI Listing
April 2009

No time to reinvent the wheel.

Authors:
David J Powell

Behav Healthc 2006 Oct;26(10):10, 12

Intermational Center for Health Concerns, Inc.

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October 2006

It's time for a national approach on staff development.

Authors:
David J Powell

Behav Healthc 2006 Mar;26(3):42-4

International Center for Health Concerns Inc.

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March 2006

Staphylococcus aureus DNA ligase: characterization of its kinetics of catalysis and development of a high-throughput screening compatible chemiluminescent hybridization protection assay.

Biochem J 2004 Nov;383(Pt. 3):551-9

Assay Development and Compound Profiling, GlaxoSmithKline Pharmaceuticals, New Frontiers Science Park (North), Third Avenue, Harlow, Essex CM19 4AW, UK.

DNA ligases are key enzymes involved in the repair and replication of DNA. Prokaryotic DNA ligases uniquely use NAD+ as the adenylate donor during catalysis, whereas eukaryotic enzymes use ATP. This difference in substrate specificity makes the bacterial enzymes potential targets for therapeutic intervention. We have developed a homogeneous chemiluminescence-based hybridization protection assay for Staphylococcus aureus DNA ligase that uses novel acridinium ester technology and demonstrate that it is an alternative to the commonly used radiometric assays for ligases. The assay has been used to determine a number of kinetic constants for S. aureus DNA ligase catalysis. These included the K(m) values for NAD+ (2.75+/-0.1 microM) and the acridinium-ester-labelled DNA substrate (2.5+/-0.2 nM). A study of the pH-dependencies of kcat, K(m) and kcat/K(m) has revealed values of kinetically influential ionizations within the enzyme-substrate complexes (kcat) and free enzyme (kcat/K(m)). In each case, the curves were shown to be composed of one kinetically influential ionization, for k(cat), pK(a)=6.6+/-0.1 and kcat/K(m), pK(a)=7.1+/-0.1. Inhibition characteristics of the enzyme against two Escherichia coli DNA ligase inhibitors have also been determined with IC50 values for these being 3.30+/-0.86 microM for doxorubicin and 1.40+/-0.07 microM for chloroquine diphosphate. The assay has also been successfully miniaturized to a sufficiently low volume to allow it to be utilized in a high-throughput screen (384-well format; 20 microl reaction volume), enabling the assay to be used in screening campaigns against libraries of compounds to discover leads for further drug development.
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http://dx.doi.org/10.1042/BJ20040054DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1133749PMC
November 2004

Inhibition of human napsin A.

Protein Pept Lett 2003 Feb;10(1):35-42

School of Biosciences, Cardiff University, P.O. Box 911, Cardiff CF10 3US, Wales, UK.

The newly-discovered human aspartic proteinase, napsin A was not susceptible to protein inhibitors from potato, squash or yeast but was weakly inhibited by the 17 kDa polypeptide from Ascaris lumbricoides and potently by isovaleryl and lactoyl-pepstatins. A series of synthetic inhibitors was also investigated which contained in the P(1)-P(1)' positions the dipeptide analogue statine or its phenylalanine or cyclohexylalanine homologues and in which the residues occupying P(4)-P(3)' were varied systematically. On this basis, the active site of napsin A can be readily distinguished from other human aspartic proteinases.
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http://dx.doi.org/10.2174/0929866033408237DOI Listing
February 2003