Developing peptide-based multivalent antagonists of proliferating cell nuclear antigen and a fluorescence-based PCNA binding assay
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Published on: March 24, 2017
William P Bozza1, Kun Yang, Jialiang Wang
1Department of Chemistry and Biochemistry, University of Delaware, Newark, DE 19716, USA.
Abstract:
Proliferating cell nuclear antigen (PCNA) is a critical player in cell proliferation. It interacts with a myriad of cellular proteins in genomic DNA replication and cell cycle control. This makes PCNA an attractive target for developing antiproliferative therapeutics. Indeed, the binding of a human tumor suppressor protein, p21, to PCNA contributes to its antiproliferative effect in cells. In this work, we report a fluorescence polarization-based binding assay for determining the affinity between the p21 peptide and human PCNA. To improve the potency of the p21-based PCNA antagonist, we exploited the homotrimeric structure of PCNA and developed multivalent peptide-based PCNA antagonists. The di- and trivalent p21-based antagonists bind to PCNA with low nanomolar dissociation constant. Moreover, we show that the multivalent PCNA antagonists inhibited PCNA-dependent DNA synthesis in a human cell extract with improved avidity when compared with the monovalent p21 peptide. The fluorescence polarization assay holds promise for the discovery of potent small-molecule PCNA inhibitors given its ready adaptability to a high-throughput screening format.
Insights
Researchers developed multivalent peptide antagonists targeting proliferating cell nuclear antigen (PCNA) for cancer therapy. These novel compounds show enhanced binding affinity and inhibit DNA synthesis, offering a promising avenue for antiproliferative drug discovery.
Area of Science:
- Molecular Biology
- Biochemistry
- Drug Discovery
Background:
- Proliferating cell nuclear antigen (PCNA) is crucial for DNA replication and cell cycle progression.
- PCNA's role in proliferation makes it a key target for antiproliferative therapeutics.
- The tumor suppressor protein p21 inhibits PCNA, demonstrating antiproliferative potential.
Purpose of the Study:
- To develop a fluorescence polarization assay for quantifying p21 peptide-PCNA binding affinity.
- To design and synthesize multivalent peptide-based antagonists targeting the homotrimeric structure of PCNA.
- To evaluate the binding avidity and inhibitory effects of these novel antagonists on PCNA-dependent DNA synthesis.
Main Methods:
- Development of a fluorescence polarization-based binding assay to measure PCNA-peptide interactions.
- Design and synthesis of di- and trivalent peptide antagonists based on the p21 interaction motif.
- Assessment of PCNA antagonist avidity and inhibition of DNA synthesis in human cell extracts.
Main Results:
- Di- and trivalent p21-based antagonists exhibit low nanomolar dissociation constants for PCNA.
- Multivalent antagonists demonstrate significantly improved avidity compared to monovalent p21 peptide.
- Inhibition of PCNA-dependent DNA synthesis was observed in human cell extracts using these antagonists.
Conclusions:
- Multivalent peptide antagonists are potent inhibitors of PCNA function.
- The developed fluorescence polarization assay is suitable for high-throughput screening of PCNA inhibitors.
- These findings pave the way for developing novel antiproliferative therapeutics targeting PCNA.
