title: A caspase active site probe reveals high fractional inhibition needed to block DNA fragmentation.
abstract: Apoptotic markers consist of either caspase substrate cleavage products or phenotypic changes that manifest themselves as a consequence of caspase-mediated substrate cleavage. We have shown recently that pharmacological inhibitors of caspase activity prevent the appearance of two such apoptotic manifestations, alphaII-spectrin cleavage and DNA fragmentation, but that blockade of the latter required a significantly higher concentration of inhibitor. We investigated this phenomenon through the use of a novel radiolabeled caspase inhibitor, [(125)I]M808, which acts as a caspase active site probe. [(125)I]M808 bound to active caspases irreversibly and with high sensitivity in apoptotic cell extracts, in tissue extracts from several commonly used animal models of cellular injury, and in living cells. Moreover, [(125)I]M808 detected active caspases in septic mice when injected intravenously. Using this caspase probe, an active site occupancy assay was developed and used to measure the fractional inhibition required to block apoptosis-induced DNA fragmentation. In thymocytes, occupancy of up to 40% of caspase active sites had no effect on DNA fragmentation, whereas inhibition of half of the DNA cleaving activity required between 65 and 75% of active site occupancy. These results suggest that a high and persistent fractional inhibition will be required for successful caspase inhibition-based therapies.
authors.affiliation: Merck Frosst Centre for Therapeutic Research, Merck Research Laboratories, Montréal, Québec H9H 3L1, Canada.
authors.forename: Nathalie
authors.initials: N
authors.lastname: Méthot
authors.forename: John P
authors.initials: JP
authors.lastname: Vaillancourt
authors.forename: JingQi
authors.initials: J
authors.lastname: Huang
authors.forename: John
authors.initials: J
authors.lastname: Colucci
authors.forename: Yongxin
authors.initials: Y
authors.lastname: Han
authors.forename: Stéphane
authors.initials: S
authors.lastname: Ménard
authors.forename: Robert
authors.initials: R
authors.lastname: Zamboni
authors.forename: Sylvie
authors.initials: S
authors.lastname: Toulmond
authors.forename: Donald W
authors.initials: DW
authors.lastname: Nicholson
authors.forename: Sophie
authors.initials: S
authors.lastname: Roy
mesh_terms: D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis; D001665:Binding Sites; D015153:Blotting, Western; D061945:Caspase Inhibitors; D020169:Caspases / Q000737:chemistry*; D002432:Cecum / Q000473:pathology; D018920:Coculture Techniques; D053938:DNA Fragmentation; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D006801:Humans; D020128:Inhibitory Concentration 50; D007457:Iodine Radioisotopes / Q000378:metabolism; D019169:Jurkat Cells; D007700:Kinetics; D051379:Mice; D008956:Models, Chemical; D011485:Protein Binding; D051381:Rats; D017207:Rats, Sprague-Dawley; D012680:Sensitivity and Specificity; D018805:Sepsis; D013950:Thymus Gland / Q000166:cytology; D014018:Tissue Distribution
chemical_list: C488252:5-iodo-2-methoxybenzylcarbonyl-valyl-aspartic acid fluoromethyl ketone; D000590:Amino Acid Chloromethyl Ketones; D061945:Caspase Inhibitors; D004791:Enzyme Inhibitors; D007457:Iodine Radioisotopes; D020169:Caspases