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Updated: Aug 25, 2026
Parallel High Throughput Single Molecule Kinetic Assay for Site-Specific DNA Cleavage
Published on: May 6, 2020
A caspase active site probe reveals high fractional inhibition needed to block DNA fragmentation
Nathalie Méthot1, John P Vaillancourt, JingQi Huang
1Merck Frosst Centre for Therapeutic Research, Merck Research Laboratories, Montréal, Québec H9H 3L1, Canada.
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.
Insights
Developing novel caspase inhibitors requires understanding their precise role in apoptosis. This study reveals that significant caspase activity must be inhibited to prevent DNA fragmentation, informing future therapeutic strategies.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- Apoptotic markers include caspase substrate cleavage products and phenotypic changes.
- Pharmacological caspase inhibitors prevent alphaII-spectrin cleavage and DNA fragmentation.
- Higher inhibitor concentrations are needed to block DNA fragmentation compared to spectrin cleavage.
Purpose of the Study:
- Investigate the differential inhibition of apoptosis markers.
- Develop and utilize a novel radiolabeled caspase inhibitor probe.
- Quantify the caspase active site occupancy required to inhibit DNA fragmentation.
Main Methods:
- Utilized a novel radiolabeled caspase inhibitor, [(125)I]M808, as an active site probe.
- Developed an active site occupancy assay to measure fractional inhibition.
- Assessed caspase activity in cell extracts, animal tissue extracts, and living cells, including septic mice.
Main Results:
- [(125)I]M808 irreversibly bound to active caspases with high sensitivity.
- The probe detected active caspases in various biological samples and in vivo.
- Up to 40% caspase active site occupancy did not affect DNA fragmentation.
- 65-75% active site occupancy was required to inhibit half of the DNA cleaving activity.
Conclusions:
- A high and persistent fractional inhibition of caspase activity is necessary for effective caspase inhibition therapies.
- The findings provide critical insights into the quantitative requirements for blocking apoptosis-induced DNA fragmentation.
- This research advances the understanding of caspase function in apoptosis and guides the development of targeted therapeutics.
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