Identification of a potent PCNA‐p15‐interaction inhibitor by autodisplay‐based peptide library screening

Abstract Proliferating cell nuclear antigen (PCNA) is an essential factor for DNA metabolism. The influence of PCNA on DNA replication and repair, combined with the high expression rate of PCNA in various tumours renders PCNA a promising target for cancer therapy. In this context, an autodisplay‐based screening method was developed to identify peptidic PCNA interaction inhibitors. A 12‐mer randomized peptide library consisting of 2.54 × 10 6 colony‐forming units was constructed and displayed at the surface of Escherichia coli BL21 (DE3) cells by autodisplay. Cells exhibiting an enhanced binding to fluorescent mScarlet‐I‐PCNA were enriched in four sorting rounds by flow cytometry. This led to the discovery of five peptide variants with affinity to mScarlet‐I‐PCNA. Among these, P3 (TCPLRWITHDHP) exhibited the highest binding signal. Subsequent flow cytometric analysis revealed a dissociation constant of 0.62 μM for PCNA‐P3 interaction. Furthermore, the inhibition of PCNA interactions was investigated using p15, a PIP‐box containing protein involved in DNA replication and repair. P3 inhibited the PCNA‐p15 51‐70 interaction with a half maximal inhibitory activity of 16.2 μM, characterizing P3 as a potent inhibitor of the PCNA‐p15 interaction.