A Generic Platform for Cellular Screening Against Ubiquitin Ligases

• Published in: Scientific reports Vol. 6; no. 1; p. 18940
• Main Authors: Maculins, Timurs, Carter, Nikki, Dorval, Thierry, Hudson, Kevin, Nissink, J. Willem M., Hay, Ronald T., Alwan, Husam
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 08.01.2016; Nature Publishing Group
• Subjects: 49; 631/154/1435/2163; 631/154/555; Cell Line, Tumor; Deregulation; DNA-Binding Proteins - genetics; DNA-Binding Proteins - metabolism; Drug Discovery; Enzyme Inhibitors - pharmacology; Gene Expression Regulation; Genes, Reporter; Genetic Vectors - chemistry; Genetic Vectors - metabolism; HEK293 Cells; High-throughput screening; High-Throughput Screening Assays - methods; Humanities and Social Sciences; Humans; Inhibitors; Ion channels; Kinases; Luciferases - genetics; Luciferases - metabolism; multidisciplinary; Nitrofurans - pharmacology; Osteoblasts - cytology; Osteoblasts - drug effects; Osteoblasts - metabolism; Promoter Regions, Genetic; Protein kinase; Saccharomyces cerevisiae Proteins - genetics; Saccharomyces cerevisiae Proteins - metabolism; Science; Signal transduction; Sulfones - pharmacology; Transcription Factors - genetics; Transcription Factors - metabolism; Transfection; Ubiquitin; Ubiquitin - genetics; Ubiquitin - metabolism; Ubiquitin-protein ligase; Ubiquitin-Protein Ligases - antagonists & inhibitors; Ubiquitin-Protein Ligases - genetics; Ubiquitin-Protein Ligases - metabolism; Ubiquitination
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/srep18940

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.