Biochemical and in vivo characterization of a small, membrane-permeant, caspase-activatable far-red fluorescent peptide for imaging apoptosis

Apoptosis is an important process involved in diverse developmental pathways, homeostasis, and response to therapy for a variety of diseases. Thus, noninvasive methods to study regulation and to monitor cell death in cells and whole animals are desired. To specifically detect apoptosis in vivo, a novel cell-permeable activatable caspase substrate, TcapQ₆₄₇, was synthesized and K_m, k_cat, and K_i values were biochemically characterized. Specific cleavage of TcapQ₆₄₇ by effector caspases was demonstrated using a panel of purified recombinant enzyme assays. Of note, caspase 3 was shown to cleave TcapQ₆₄₇ with a k _cat 7-fold greater than caspase 7 and 16-fold greater than caspase 6. No evidence of TcapQ₆₄₇ cleavage by initiator caspases was observed. In KB 3-1 or Jurkat cells treated with cytotoxic agents or C ₆-ceramide, TcapQ₆₄₇ detected apoptosis in individual- and population-based fluorescent cell assays in an effector caspase inhibitor-specific manner. Further, only background fluorescence was observed in cells incubated with dTcapQ₆₄₇, a noncleavable all D-amino acid control peptide. Finally, in vivo experiments demonstrated the utility of TcapQ₆₄₇ to detect parasite-induced apoptosis in human colon xenograft and liver abscess mouse models. Thus, TcapQ₆₄₇ represents a sensitive, effector caspase-specific far-red "smart" probe to noninvasively monitor apoptosis in vivo.