Specific targeting of human caspases using designed ankyrin repeat proteins.

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• Caspases are crucial proteases regulating cell death, differentiation, and proliferation.
• High homology among caspases, particularly at the active site, makes specific targeting challenging.
• Existing peptide-based small molecules often lack specificity due to overlapping cleavage motifs.

Purpose of the Study:

• To develop highly specific targeting agents for human caspases.
• To generate and characterize novel designed ankyrin repeat proteins (DARPins) against caspases 1-9.
• To elucidate the structural basis of DARPin-caspase interactions for enhanced specificity.

Main Methods:

• Selection of DARPins against human caspases 1-9.
• Affinity-based screening to identify high-affinity binders.
• Confirmation of specificity using a subset of caspase family members.
• X-ray crystallography to determine the structure of caspase 8 in complex with DARPin D8.4.

Main Results:

• High-affinity DARPins were identified for caspases 1, 5, 6, and 8, with no binders found for caspase 4.
• Novel DARPins (D1.73, D5.15, D6.11, D8.1, D8.4, and D9.2) demonstrated confirmed specificity.
• Crystal structure revealed DARPin D8.4 binding to non-conserved residues on the large subunit of caspase 8, explaining its specificity.
• Structural analysis identified two primary DARPin binding modes on caspases, involving active site loops or lateral surfaces of the large subunit, both utilizing non-conserved residues.

Conclusions:

• DARPins represent a promising class of highly specific targeting agents for caspases.
• The identified DARPins overcome limitations of traditional substrate analogues in caspase research.
• Structural insights into DARPin-caspase interactions provide a foundation for designing even more specific inhibitors.