title: Correlating the fractional inhibition of caspase-3 in NT2 cells with apoptotic markers using an active-caspase-3 enzyme-linked immunosorbent assay.
abstract: A rapid and quantitative method for measuring the activity and fractional inhibition of enzymes within their natural cellular environment remains an unmet need in drug discovery. We describe the use of a nonradioactive quantitative enzyme-linked immunosorbent assay (ELISA) for measuring intracellular caspase activity that is amenable to robotic automation. The ELISA specifically detects active-caspase-3 and was used to correlate the in-cell activity of caspase-3 with the progress of caspase-3-mediated events under varying concentrations of caspase-3 inhibitors in NT2 cells. We examined the cleavage of endogenous substrates (poly(ADP-ribose)polymerase and alphaII-spectrin), the extent of DNA fragmentation, and the autocatalytic removal of the caspase-3 prodomain as markers of caspase-3 activity. To impart inhibition of the downstream markers, a greater level of caspase-3 inhibition was required. Although the functional markers were found not to accurately predict intracellular caspase-3 activity, we found that the inhibition of intracellular caspase-3 was highly correlated (R(2) = 0.96) to the inhibition of DNA fragmentation. Also, by comparing the potency of the different inhibitors against the intracellular enzyme versus the purified enzyme, the effects of inhibitor functional groups on whole-cell activity were addressed.
authors.affiliation: Department of Biochemistry and Molecular Biology/Medicinal Chemistry, Merck Frosst Centre for Therapeutic Research, Kirkland, Que., Canada. paul_tawa@merck.com
authors.forename: Paul
authors.initials: P
authors.lastname: Tawa
authors.forename: Andre
authors.initials: A
authors.lastname: Giroux
authors.forename: Erich
authors.initials: E
authors.lastname: Grimm
authors.forename: Yongxin
authors.initials: Y
authors.lastname: Han
authors.forename: Donald W
authors.initials: DW
authors.lastname: Nicholson
authors.forename: Steven
authors.initials: S
authors.lastname: Xanthoudakis
mesh_terms: D017209:Apoptosis; D001710:Biotin / Q000031:analogs & derivatives / Q000494:pharmacology; D053148:Caspase 3; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism*; D002460:Cell Line; D003374:Coumarins / Q000494:pharmacology; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D053938:DNA Fragmentation / Q000187:drug effects; D004151:Dipeptides / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D020128:Inhibitory Concentration 50; D009842:Oligopeptides / Q000494:pharmacology; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D012680:Sensitivity and Specificity; D013049:Spectrin / Q000037:antagonists & inhibitors
chemical_list: C112287:Ac-aspartyl-glutamyl-valyl-aspartyl-aminomethylcoumarin; D061945:Caspase Inhibitors; D003374:Coumarins; D015853:Cysteine Proteinase Inhibitors; D004151:Dipeptides; D009842:Oligopeptides; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; C509807:z-VK(biotin)D(OMe)-fmk; D013049:Spectrin; D001710:Biotin; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases