Developing peptide-based multivalent antagonists of proliferating cell nuclear antigen and a fluorescence-based PCNA binding assay

作者：Bozza William P; Yang Kun; Wang Jialiang; Zhuang Zhihao^*

来源：Analytical Biochemistry, 2012, 427(1): 69-78.

DOI：10.1016/j.ab.2012.04.018

• PCNA
• Multivalent binding
• Fluorescence assay
• DNA synthesis
• Antagonist

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.

• 出版日期2012-8-1