Quantitative analysis of fluorescent caspase substrate cleavage in intact cells and identification of novel inhibitors of apoptosis

Caspase activation and proteolytic cleavage of specific target proteins rep resents an integral step in the pathway leading to the apoptotic death of c ells. Analysis of caspase activity in intact cells, however, has been gener ally limited to the measurement of end-point biochemical and morphological markers of apoptosis. In an effort to develop a strategy with which to moni tor caspase activity, early in the cell death cascade and in real-time, we have generated cell lines that overexpress recombinant GFP-based caspase su bstrates that display a quantifiable change in their spectral properties wh en cleaved by group II caspases. Specifically, tandem GFP substrates linked by a caspase-sensitive cleavage site show diminished fluorescence resonanc e energy transfer (FRET), as a consequence of cleavage, due to physical sep aration of the GFP moieties in apoptotic cells, We have evaluated the influ ence of different caspase-sensitive linkers on both FRET efficiency and cle avage by caspase-3. We also demonstrate that caspase activity as well as in hibition by pharmacological agents can be monitored, with minimal manipulat ion, in intact adherent cells seeded in a 96-well cell culture dish, Finall y, we have adapted this technology to a high throughput screening platform to identify novel small molecule and cell permeable inhibitors of apoptosis . Based on a biochemical analysis of the compounds identified it is clear t hat this assay can be used to detect drugs which inhibit caspases directly as well as those which target upstream components of the caspase cascade.