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Curr Protoc Neurosci. 2017 Apr 10;79:5.32.1-5.32.27. doi: 10.1002/cpns.27.

Quantitative High-Throughput Screening Using a Coincidence Reporter Biocircuit.

Author information

1
National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, Maryland.
2
National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland.

Abstract

Reporter-biased artifacts-i.e., compounds that interact directly with the reporter enzyme used in a high-throughput screening (HTS) assay and not the biological process or pharmacology being interrogated-are now widely recognized to reduce the efficiency and quality of HTS used for chemical probe and therapeutic development. Furthermore, narrow or single-concentration HTS perpetuates false negatives during primary screening campaigns. Titration-based HTS, or quantitative HTS (qHTS), and coincidence reporter technology can be employed to reduce false negatives and false positives, respectively, thereby increasing the quality and efficiency of primary screening efforts, where the number of compounds investigated can range from tens of thousands to millions. The three protocols described here allow for generation of a coincidence reporter (CR) biocircuit to interrogate a biological or pharmacological question of interest, generation of a stable cell line expressing the CR biocircuit, and qHTS using the CR biocircuit to efficiently identify high-quality biologically active small molecules.

KEYWORDS:

assay development; coincidence reporter; quantitative high-throughput screening

PMID:
28398644
PMCID:
PMC5510169
DOI:
10.1002/cpns.27
[Indexed for MEDLINE]
Free PMC Article

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