title: A Multiplexed High-Content Screening Approach Using the Chromobody Technology to Identify Cell Cycle Modulators in Living Cells.
abstract: Many screening hits show relatively poor quality regarding later efficacy and safety. Therefore, small-molecule screening efforts shift toward high-content analysis providing more detailed information. Here, we describe a novel screening approach to identify cell cycle modulators with low toxicity by combining the Cell Cycle Chromobody (CCC) technology with the CytoTox-Glo (CTG) cytotoxicity assay. The CCC technology employs intracellularly functional single-domain antibodies coupled to a fluorescent protein (chromobodies) to visualize the cell cycle-dependent redistribution of the proliferating cell nuclear antigen (PCNA) in living cells. This image-based cell cycle analysis was combined with determination of dead-cell protease activity in cell culture supernatants by the CTG assay. We adopted this multiplex approach to high-throughput format and screened 960 Food and Drug Administration (FDA)-approved drugs. By this, we identified nontoxic compounds, which modulate different cell cycle stages, and validated selected hits in diverse cell lines stably expressing CCC. Additionally, we independently validated these hits by flow cytometry as the current state-of-the-art format for cell cycle analysis. This study demonstrates that CCC imaging is a versatile high-content screening approach to identify cell cycle modulators, which can be multiplexed with cytotoxicity assays for early elimination of toxic compounds during screening.
authors.affiliation: Assay Development and Screening Platform, Institute of Molecular Toxicology and Pharmacology, Helmholtz Zentrum München, German Research Center for Environmental Health, Neuherberg, Bavaria, Germany.
authors.forename: Kenji
authors.initials: K
authors.lastname: Schorpp
authors.affiliation: Assay Development and Screening Platform, Institute of Molecular Toxicology and Pharmacology, Helmholtz Zentrum München, German Research Center for Environmental Health, Neuherberg, Bavaria, Germany.
authors.forename: Ina
authors.initials: I
authors.lastname: Rothenaigner
authors.affiliation: Pharmaceutical Biotechnology, University of Tuebingen, Tuebingen, Germany.
authors.forename: Julia
authors.initials: J
authors.lastname: Maier
authors.affiliation: Pharmaceutical Biotechnology, University of Tuebingen, Tuebingen, Germany.
authors.forename: Bjoern
authors.initials: B
authors.lastname: Traenkle
authors.affiliation: Pharmaceutical Biotechnology, University of Tuebingen, Tuebingen, Germany Natural and Medical Sciences Institute at the University of Tuebingen, Reutlingen, Germany.
authors.forename: Ulrich
authors.initials: U
authors.lastname: Rothbauer
authors.affiliation: Assay Development and Screening Platform, Institute of Molecular Toxicology and Pharmacology, Helmholtz Zentrum München, German Research Center for Environmental Health, Neuherberg, Bavaria, Germany kamyar.hadian@helmholtz-muenchen.de.
authors.forename: Kamyar
authors.initials: K
authors.lastname: Hadian
mesh_terms: D002453:Cell Cycle / Q000187:drug effects*; D049109:Cell Proliferation / Q000187:drug effects; D005434:Flow Cytometry / Q000379:methods; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D057054:Molecular Imaging / Q000379:methods; D018809:Proliferating Cell Nuclear Antigen / Q000235:genetics; D061905:Single-Domain Antibodies / Q000302:isolation & purification / Q000494:pharmacology*; D054852:Small Molecule Libraries / Q000302:isolation & purification / Q000494:pharmacology*
chemical_list: D018809:Proliferating Cell Nuclear Antigen; D061905:Single-Domain Antibodies; D054852:Small Molecule Libraries
keywords: cell cycle; chromobodies; high-content screening; multiplexing; phenotypic drug discovery