title: Development and application of a GFP-FRET intracellular caspase assay for drug screening.
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.
authors.affiliation: Aurora Biosciences Corporation, San Diego, CA 92121, USA. pollockb@aurorabio.com
authors.forename: J
authors.initials: J
authors.lastname: Jones
authors.forename: R
authors.initials: R
authors.lastname: Heim
authors.forename: E
authors.initials: E
authors.lastname: Hare
authors.forename: J
authors.initials: J
authors.lastname: Stack
authors.forename: B A
authors.initials: BA
authors.lastname: Pollok
mesh_terms: D000595:Amino Acid Sequence; D000906:Antibodies / Q000276:immunology / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D015374:Biosensing Techniques; D053148:Caspase 3; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004735:Energy Transfer; D004789:Enzyme Activation / Q000187:drug effects; D005434:Flow Cytometry; D005453:Fluorescence; D017930:Genes, Reporter / Q000235:genetics; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D019169:Jurkat Cells; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D011993:Recombinant Fusion Proteins; D015203:Reproducibility of Results; D014162:Transfection; D019014:fas Receptor / Q000276:immunology
chemical_list: D000906:Antibodies; D061945:Caspase Inhibitors; D015853:Cysteine Proteinase Inhibitors; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D019014:fas Receptor; D049452:Green Fluorescent Proteins; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases