A cell permeable bimane-constrained PCNA-interacting peptide.

Area of Science:

• Biochemistry
• Medicinal Chemistry
• Molecular Biology

Background:

• Proliferating cell nuclear antigen (PCNA) is crucial for DNA replication and repair, making it a key therapeutic target for cancers.
• Peptides from human p21 protein bind PCNA with high affinity, inhibiting DNA replication via a 3₁₀-helical conformation.

Purpose of the Study:

• To design and synthesize conformationally constrained peptidomimetics targeting PCNA.
• To evaluate the binding affinity, structural basis, and cellular activity of these novel PCNA inhibitors.

Main Methods:

• Synthesis of macrocyclic peptides with covalent linkers between cysteine residues.
• Binding affinity assessment using K_D values.
• Structural analysis via X-ray crystallography and computational modeling.
• Cell permeability and localization studies using confocal microscopy.

Main Results:

• Macrocyclic peptides bound PCNA with K_D values from 570 nM to 3.86 μM.
• The bimane-constrained peptide 7 exhibited the highest affinity and adopted the critical 3₁₀-helical conformation.
• Peptide 7 demonstrated cell permeability and localized to the cytosol of breast cancer cells.
• The bimane linker provided inherent fluorescence for direct imaging.

Conclusions:

• Conformational constraint, particularly the 3₁₀-helical structure, is vital for high-affinity PCNA binding.
• Peptide 7 represents a potent, cell-permeable peptidomimetic inhibitor of PCNA.
• This study advances the development of therapeutic PCNA inhibitors for proliferative diseases.