title: A High-Throughput Screening Strategy for Development of RNF8-Ubc13 Protein-Protein Interaction Inhibitors.
abstract: The ubiquitin-proteasome system plays an essential role in a broad range of cellular signaling pathways. Ubiquitination is a posttranslational protein modification that involves the action of an enzymatic cascade (E1, E2, and E3 enzymes) for the covalent attachment of ubiquitin to target proteins. The emerging knowledge of the molecular mechanisms and correlation of deregulation of the ubiquitin system in human diseases is uncovering new opportunities for therapeutics development. The E3 ligase RNF8 acts in cooperation with the heterodimeric E2 enzyme Ubc13/Uev1a to generate ubiquitin conjugates at the sides of DNA double-strand breaks, and recent findings suggest RNF8 as a potential therapeutic target for the treatment of breast cancer. Here, we present a novel high-throughput screening (HTS)-compatible assay based on the AlphaScreen technology to identify inhibitors of the RNF8-Ubc13 protein-protein interaction, along with a follow-up strategy for subsequent validation. We have adapted the AlphaScreen assay to a 384-well format and demonstrate its reliability, reproducibility, and suitability for automated HTS campaigns. In addition, we have established a biochemical orthogonal homogeneous time-resolved fluorescence (HTRF) assay in HTS format and a cellular microscopy-based assay allowing verification of the primary hits. This strategy will be useful for drug screening programs aimed at RNF8-Ubc13 modulation.
authors.affiliation: 1 Assay Development and Screening Platform, Institute of Molecular Toxicology and Pharmacology, Helmholtz Zentrum München für Gesundheit und Umwelt, Neuherberg, Germany.
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Weber
authors.affiliation: 1 Assay Development and Screening Platform, Institute of Molecular Toxicology and Pharmacology, Helmholtz Zentrum München für Gesundheit und Umwelt, Neuherberg, Germany.
authors.forename: Ina
authors.initials: I
authors.lastname: Rothenaigner
authors.affiliation: 1 Assay Development and Screening Platform, Institute of Molecular Toxicology and Pharmacology, Helmholtz Zentrum München für Gesundheit und Umwelt, Neuherberg, Germany.
authors.forename: Stefanie
authors.initials: S
authors.lastname: Brandner
authors.affiliation: 1 Assay Development and Screening Platform, Institute of Molecular Toxicology and Pharmacology, Helmholtz Zentrum München für Gesundheit und Umwelt, Neuherberg, Germany.
authors.forename: Kamyar
authors.initials: K
authors.lastname: Hadian
authors.affiliation: 1 Assay Development and Screening Platform, Institute of Molecular Toxicology and Pharmacology, Helmholtz Zentrum München für Gesundheit und Umwelt, Neuherberg, Germany.
authors.forename: Kenji
authors.initials: K
authors.lastname: Schorpp
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology*; D045744:Cell Line, Tumor; D003546:Cysteine Endopeptidases / Q000494:pharmacology*; D004247:DNA / Q000235:genetics / Q000378:metabolism; D053903:DNA Breaks, Double-Stranded; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D000072017:Deubiquitinating Enzymes; D057166:High-Throughput Screening Assays; D006801:Humans; D010006:Osteoblasts / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D011499:Protein Processing, Post-Translational; D015203:Reproducibility of Results; D015398:Signal Transduction; D013050:Spectrometry, Fluorescence; D014157:Transcription Factors / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D025801:Ubiquitin / Q000235:genetics / Q000378:metabolism; D044763:Ubiquitin-Conjugating Enzymes / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination
chemical_list: D000970:Antineoplastic Agents; D004268:DNA-Binding Proteins; C486521:RNF8 protein, human; D014157:Transcription Factors; D025801:Ubiquitin; D004247:DNA; C471607:UBE2N protein, human; C471624:UBE2V1 protein, human; D044763:Ubiquitin-Conjugating Enzymes; D044767:Ubiquitin-Protein Ligases; D000072017:Deubiquitinating Enzymes; C538826:OTUB1 protein, human; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex
keywords: AlphaScreen; RNF8; Ubc13; high-throughput screening; ubiquitin E3 ligases