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Updated: Apr 13, 2026
Identifying Protein-protein Interaction Sites Using Peptide Arrays
Published on: November 18, 2014
Identification of a potent PCNA-p15-interaction inhibitor by autodisplay-based peptide library screening
Sarah Hardebeck1, Natalie Jácobo Goebbels1, Caroline Michalski1
1University of Münster, Institute of Pharmaceutical and Medicinal Chemistry, Münster, Germany.
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 × 106 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-p1551-70 interaction with a half maximal inhibitory activity of 16.2 μM, characterizing P3 as a potent inhibitor of the PCNA-p15 interaction.
Insights
Researchers developed a novel autodisplay screening method to find peptide inhibitors targeting proliferating cell nuclear antigen (PCNA). A peptide variant, P3, showed high affinity and inhibited PCNA interactions, offering potential for cancer therapy.
Area of Science:
- Molecular Biology
- Biochemistry
- Cancer Research
Background:
- Proliferating cell nuclear antigen (PCNA) is crucial for DNA metabolism, replication, and repair.
- High PCNA expression in tumors makes it a significant target for cancer therapeutics.
Purpose of the Study:
- To develop an autodisplay-based screening method for identifying peptide inhibitors of PCNA.
- To discover and characterize novel peptides that interact with and inhibit PCNA function.
Main Methods:
- Construction and surface display of a 12-mer randomized peptide library on Escherichia coli using autodisplay.
- Enrichment of PCNA-binding peptides via fluorescence-activated cell sorting (FACS) using fluorescently labeled PCNA.
- Characterization of peptide-PCNA interactions using flow cytometry and assessment of inhibitory activity against PCNA-protein interactions.
Main Results:
- Discovery of five peptide variants with affinity for mScarlet-I-PCNA.
- Identification of peptide P3 (TCPLRWITHDHP) with a dissociation constant (Kd) of 0.62 μM for PCNA.
- P3 demonstrated potent inhibition of the PCNA-p15 interaction, with a half-maximal inhibitory concentration (IC50) of 16.2 μM.
Conclusions:
- The autodisplay method successfully identified high-affinity PCNA-interacting peptides.
- Peptide P3 is a potent inhibitor of PCNA-p15 interactions, highlighting its potential as a therapeutic agent for cancers with high PCNA expression.
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