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Schorpp, K.K. ; Rothenaigner, I. ; Maier, J.* ; Traenkle, B.* ; Rothbauer, U.* ; Hadian, K.

A multiplexed high-content screening approach using the chromobody technology to identify cell cycle modulators in living cells.

J. Biomol. Screen. 21, 965-977 (2016)

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DOI

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	Open Access Green

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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.

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Publication type Article: Journal article

Document type Scientific Article

Corresponding Author

Keywords Cell Cycle ; Chromobodies ; High-content Screening ; Multiplexing ; Phenotypic Drug Discovery; Cancer; Dynamics; Protein; Live; Proliferation; Nanobodies; Molecules; Division; Biology; Pcna

ISSN (print) / ISBN 1087-0571

e-ISSN 1552-454X

Journal Journal of Biomolecular Screening

Quellenangaben Volume: 21, Issue: 9, Pages: 965-977

Publisher Sage

Publishing Place Thousand Oaks

Non-patent literature Publications

Reviewing status Peer reviewed

Institute(s) Institute of Molecular Toxicology and Pharmacology (TOXI)