Development and application of a GFP-FRET intracellular caspase assay for drug screening

Apoptosis is a crucial biological process, and activation of caspase endopr oteases is essential for proper regulation and execution of apoptosis, Beca use caspases also appear to be central players in several pathological stat es, there is a practical need within the biopharmaceutical research communi ty for facile, noninvasive cellular assays for the discovery of compounds t hat modulate caspase activity. Tandem molecules of green fluorescent protei n (GFP) stably expressed within cells can serve as a genetically encoded se nsor of protease activity. Using this technology, we have developed a stabl e cellular system for the screening of agents that modulate activation of t he caspase cascade. This assay technology allows for the real-time monitori ng of apoptosis in situ, using conventional fluorescent plate reader detect ion. By applying this assay system to an actual compound screen, small-mole cule inducers of cell apoptosis were reliably identified. Follow-up pharmac ology confirmed that the rank-order potency of primary hits using the intra cellular GFP assay corresponded to that found using a conventional, cell ly sis-based assay method.