Apoptosis is an evolutionarily conserved process of programmed cell suicide mediated by a cascade of proteolytic enzymes called caspases. Once activated in response to pro-apoptotic signals, caspases cleave protein substrates leading to the eventual disassembly of the cell.
Early research studies using short peptide sequences linked to an aldehyde or fluoromethyl ketone (FMK) reactive group identified a number of peptide sequences preferentially targeted by different caspase enzymes. When linked to an FMK reactive group, the resulting peptide-FMK molecule will form an irreversible, covalent thioether adduct with the reactive SH-site of caspase enzymes.
ImmunoChemistry Technologies utilized these findings to develop the often-cited FLICA® (FLuorescence Labeled Inhibitors of CAspases) caspase inhibitor and apoptosis detection reagents, both of which have been widely used for years as simple and reliable methods for screening apoptosis in live cells and tissues. These reagents consist of short peptide sequences with varying caspase specificities, a fluorescent label, such as carboxyfluorescein (FAM), and an FMK reactive moiety. The resulting fluorescent, cell-permeant, non-cytotoxic caspase inhibitor reagents can be added to cell culture media for whole cell apoptosis detection. These labeled caspase inhibitors will cross the cell membrane and form irreversible covalent bonds with activated caspase enzymes inside apoptotic cells, which can then be differentiated from non-apoptotic cells by their retained fluorescence.
ImmunoChemistry Technologies has released a novel set of inhibitor reagents that employ an O-phenoxy (OPH) reactive group in place of an FMK group. In a manner analogous to the FMK class of cysteine reactive compounds, the OPH caspase inhibitors form a stable, covalent thioether adduct with the reactive SH-site of caspase enzymes present in apoptotic cells. OPH inhibitor compounds have the benefit of being extremely non-cytotoxic, display enhanced stability characteristics compared to the FMK analogs, and have been optimized to provide a high level of performance for in vitro applications.