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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)
Postprint DOI PMC
Open Access Green
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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Publikationstyp Artikel: Journalartikel
Dokumenttyp Wissenschaftlicher Artikel
Korrespondenzautor
Schlagwörter 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
Zeitschrift Journal of Biomolecular Screening
Quellenangaben Band: 21, Heft: 9, Seiten: 965-977 Artikelnummer: , Supplement: ,
Verlag Sage
Verlagsort Thousand Oaks
Nichtpatentliteratur Publikationen
Begutachtungsstatus Peer reviewed
Institut(e) Institute of Molecular Toxicology and Pharmacology (TOXI)