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Development and application of a GFP-FRET intracellular caspase assay for drug screening
1Aurora Biosciences Corporation, San Diego, CA 92121, USA. pollockb@aurorabio.com
Abstract:
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.
Insights
Researchers developed a novel cell-based assay using green fluorescent protein (GFP) to easily screen for compounds that modulate apoptosis. This noninvasive method reliably identifies small molecules that induce programmed cell death.
Area of Science:
- Biochemistry
- Cell Biology
- Pharmacology
Background:
- Apoptosis is a critical biological process regulated by caspase endoproteases.
- Caspase dysregulation is implicated in various pathological conditions.
- There is a need for efficient, noninvasive assays to discover caspase modulators in biopharmaceutical research.
Purpose of the Study:
- To develop a stable, genetically encoded cellular system for screening compounds that modulate caspase activity.
- To enable real-time, in situ monitoring of apoptosis using fluorescence detection.
- To validate the assay's efficacy in identifying small-molecule inducers of apoptosis.
Main Methods:
- Development of a stable cell line expressing tandem green fluorescent protein (GFP) molecules as a protease activity sensor.
- Utilizing a fluorescence plate reader for real-time, in situ monitoring of apoptosis.
- Performing a compound screen to identify small-molecule inducers of apoptosis.
- Conducting follow-up pharmacology to compare assay results with conventional methods.
Main Results:
- Successfully developed and implemented a stable cellular system for screening apoptosis modulators.
- The assay allowed for reliable, real-time monitoring of apoptosis in situ.
- Small-molecule inducers of apoptosis were effectively identified through compound screening.
- The intracellular GFP assay demonstrated a strong correlation in potency ranking compared to traditional cell lysis assays.
Conclusions:
- The developed GFP-based assay provides a facile and noninvasive method for screening compounds that modulate caspase activity and apoptosis.
- This assay technology is suitable for high-throughput screening in biopharmaceutical research.
- The findings validate the utility of genetically encoded sensors for studying dynamic cellular processes like apoptosis.