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Journal of Biomolecular Screening
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Development and Application of a GFP-FRET Intracellular Caspase Assay for Drug Screening

Jay Jones

Aurora Biosciences Corporation, San Diego, CA

Roger Heim

Aurora Biosciences Corporation, San Diego, CA

Eric Hare

Aurora Biosciences Corporation, San Diego, CA

Jeffrey Stack

Aurora Biosciences Corporation, San Diego, CA

Brian A. Pollok

Aurora Biosciences Corporation, San Diego, CA

Apoptosis is a crucial biological process, and activation of caspase endoproteases is essential for proper regulation and execution of apoptosis. Because caspases also appear to be central players in several pathological states, there is a practical need within the biopharmaceutical research community for facile, noninvasive cellular assays for the discovery of compounds that modulate caspase activity. Tandem molecules of green fluorescent protein (GFP) stably expressed within cells can serve as a genetically encoded sensor of protease activity. Using this technology, we have developed a stable cellular system for the screening of agents that modulate activation of the caspase cascade. This assay technology allows for the real-time monitoring of apoptosis in situ, using conventional fluorescent plate reader detection. By applying this assay system to an actual compound screen, small-molecule inducers of cell apoptosis were reliably identified. Follow-up pharmacology confirmed that the rank-order potency of primary hits using the intracellular GFP assay corresponded to that found using a conventional, cell lysis-based assay method.

Journal of Biomolecular Screening, Vol. 5, No. 5, 307-317 (2000)
DOI: 10.1177/108705710000500502
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