A cell permeable bimane-constrained PCNA-interacting peptide.

Abstract

The human sliding clamp protein known as proliferating cell nuclear antigen (PCNA) orchestrates DNA-replication and -repair and as such is an ideal therapeutic target for proliferative diseases, including cancer. Peptides derived from the human p21 protein bind PCNA with high affinity <i>via</i> a 3₁₀-helical binding conformation and are known to shut down DNA-replication. Here, we present studies on short analogues of p21 peptides (143-151) conformationally constrained with a covalent linker between <i>i</i>, <i>i</i> + 4 separated cysteine residues at positions 145 and 149 to access peptidomimetics that target PCNA. The resulting macrocycles bind PCNA with <i>K</i> _D values ranging from 570 nM to 3.86 μM, with the bimane-constrained peptide <b>7</b> proving the most potent. Subsequent X-ray crystallography and computational modelling studies of the macrocyclic peptides bound to PCNA indicated only the high-affinity peptide <b>7</b> adopted the classical 3₁₀-helical binding conformation. This suggests the 3₁₀-helical conformation is critical to high affinity PCNA binding, however NMR secondary shift analysis of peptide <b>7</b> revealed this secondary structure was not well-defined in solution. Peptide <b>7</b> is cell permeable and localised to the cell cytosol of breast cancer cells (MDA-MB-468), revealed by confocal microscopy showing blue fluorescence of the bimane linker. The inherent fluorescence of the bimane moiety present in peptide <b>7</b> allowed it to be directly imaged in the cell uptake assay, without attachment of an auxiliary fluorescent tag. This highlights a significant benefit of using a bimane constraint to access conformationally constrained macrocyclic peptides. This study identifies a small peptidomimetic that binds PCNA with higher affinity than previous reported p21 macrocycles, and is cell permeable, providing a significant advance toward development of a PCNA inhibitor for therapeutic applications.