Targeting the Genome-Stability Hub Ctf4 by Stapled-Peptide Design

Area of Science:

• Molecular Biology
• Biochemistry
• Drug Discovery

Background:

• Synthetic lethality is a promising chemotherapeutic strategy targeting genomic instability.
• The Ctf4/AND-1 protein complex is crucial for DNA replication, repair, and chromosome segregation.
• Targeting Ctf4/AND-1 offers a novel synthetic lethality approach for cancer therapy.

Purpose of the Study:

• To design and validate stapled peptides targeting the Ctf4 protein hub.
• To develop small-molecule inhibitors for the human AND-1 protein, an orthologue of Ctf4.
• To explore the potential of targeting Ctf4/AND-1 for cancer treatment.

Main Methods:

• Design and optimization of double-click stapled peptides based on the Sld5 Ctf4-interacting peptide (CIP).
• Screening of stapling positions to identify peptides with high binding affinity.
• X-ray crystallography to determine the binding mode of the stapled peptide to Ctf4.
• Yeast extract assays to assess the displacement of DNA polymerase α from the replisome.

Main Results:

• An unorthodox i,i+6 stapled peptide demonstrated submicromolar binding affinity to Ctf4.
• The crystal structure confirmed the interaction mode between the stapled peptide and Ctf4.
• The stapled peptide successfully displaced DNA polymerase α from the replisome in yeast extracts.
• Proof-of-principle for developing small-molecule inhibitors of the human AND-1 protein.

Conclusions:

• Stapled peptides targeting Ctf4 represent a viable strategy for developing novel cancer therapeutics.
• This work validates Ctf4/AND-1 as a druggable target for synthetic lethality-based cancer treatments.
• The developed stapled peptide serves as a foundation for future drug development against AND-1.