Work

Synthesis of Novel Caspase Inhibitors for Characterization of the Active Caspase Proteome in Vitro and in Vivo

Year: 2006

Type: article

Abstract: Caspases are cysteine proteases that are essential for cytokine maturation and apoptosis. To facilitate the dissection of caspase function in vitro and in vivo, we have synthesized irreversible caspase inhibitors with biotin attached via linker arms of various lengths (12a-d) and a 2,4-dinitrophenyl labeled inhibitor (13). Affinity labeling of apoptotic extracts followed by blotting reveals that these affinity probes detect active caspases. Using the strong affinity of avidin for biotin, we have isolated affinity-labeled caspase 6 from apoptotic cytosolic extracts of cells overexpressing procaspase 6 by treatment with 12c, which contains biotin attached to the N(epsilon)-lysine of the inhibitor by a 22.5 A linker arm, followed by affinity purification on monomeric avidin-sepharose beads. Compound 13 has proven sufficiently cell permeable to rescue cells from apoptotic execution. These novel caspase inhibitors should provide powerful probes for the study of the active caspase proteome during apoptosis both in vitro and in vivo.

Source: Journal of Medicinal Chemistry

DOI: 10.1021/jm060385h

Authors: Alexander J. Henzing, Helen Dodson, Joel M. Reid, Scott H. Kaufmann, Robert L. Baxter, William C. Earnshaw

Institutions: Mayo Clinic, Wellcome Centre for Cell Biology, University of Edinburgh

Language: English

Topic: Cell death mechanisms and regulation

Subfield: Molecular Biology

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

SDG: Good health and well-being

Open Access status: green

Funders: Wellcome Trust, National Cancer Institute