title: A Generic Platform for Cellular Screening Against Ubiquitin Ligases.
abstract: Ubiquitin signalling regulates most aspects of cellular life, thus deregulation of ubiquitylation has been linked with a number of diseases. E3 ubiquitin ligases provide substrate selectivity in ubiquitylation cascades and are therefore considered to be attractive targets for developing therapeutic molecules. In contrast to established drug target classes, such as protein kinases, GPCRs, hormone receptors and ion channels, ubiquitin drug discovery is in its early stages. This is, in part, due to the complexity of the ubiquitylation pathways and the lack of robust quantitative technologies that allow high-throughput screening of inhibitors. Here we report the development of a Ubiquitin Ligase Profiling system, which is a novel and generic cellular technology designed to facilitate identification of selective inhibitors against RING type E3 ubiquitin ligases. Utilization of this system requires a single co-transfection of cells with assay vectors, thereby enabling readout of E3 ubiquitin ligase catalytic activity within the cellular environment. Therefore, our robust high-throughput screening platform offers novel opportunities for the development of inhibitors against this difficult-to-target E3 ligase enzyme class.
authors.affiliation: Discovery Sciences, AstraZeneca, Alderley Park, Cheshire, SK10 4TG, UK.
authors.forename: Timurs
authors.initials: T
authors.lastname: Maculins
authors.affiliation: Discovery Sciences, AstraZeneca, Darwin Building, Cambridge Science Park, Milton Road, Cambridge, CB4 0WG, UK.
authors.forename: Nikki
authors.initials: N
authors.lastname: Carter
authors.affiliation: Discovery Sciences, AstraZeneca, Darwin Building, Cambridge Science Park, Milton Road, Cambridge, CB4 0WG, UK.
authors.forename: Thierry
authors.initials: T
authors.lastname: Dorval
authors.affiliation: Oncology iMED, AstraZeneca, Alderley Park, Cheshire, SK10 4TG, UK.
authors.forename: Kevin
authors.initials: K
authors.lastname: Hudson
authors.affiliation: Oncology iMED, Darwin Building, Cambridge Science Park, Milton Road, Cambridge, CB4 0WG, UK.
authors.forename: J Willem M
authors.initials: JW
authors.lastname: Nissink
authors.affiliation: Centre for Gene Regulation and Expression, Sir James Black Centre, College of Life Sciences, University of Dundee, Dow Street, Dundee, DD1 5EH, UK.
authors.forename: Ronald T
authors.initials: RT
authors.lastname: Hay
authors.affiliation: Discovery Sciences, AstraZeneca, Alderley Park, Cheshire, SK10 4TG, UK.
authors.forename: Husam
authors.initials: H
authors.lastname: Alwan
mesh_terms: D045744:Cell Line, Tumor; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D055808:Drug Discovery; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000737:chemistry* / Q000378:metabolism; D057809:HEK293 Cells; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D009581:Nitrofurans / Q000494:pharmacology; D010006:Osteoblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D011401:Promoter Regions, Genetic; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D013450:Sulfones / Q000494:pharmacology; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D025801:Ubiquitin / Q000235:genetics* / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D054875:Ubiquitination
chemical_list: C000604167:2-((4-methylphenyl)sulfonyl)-5-nitrofuran; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; C052130:GAL4 protein, S cerevisiae; D009581:Nitrofurans; D029701:Saccharomyces cerevisiae Proteins; D013450:Sulfones; D014157:Transcription Factors; D025801:Ubiquitin; D008156:Luciferases; D044767:Ubiquitin-Protein Ligases