title: Developing peptide-based multivalent antagonists of proliferating cell nuclear antigen and a fluorescence-based PCNA binding assay.
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.
authors.affiliation: Department of Chemistry and Biochemistry, University of Delaware, Newark, DE 19716, USA.
authors.forename: William P
authors.initials: WP
authors.lastname: Bozza
authors.forename: Kun
authors.initials: K
authors.lastname: Yang
authors.forename: Jialiang
authors.initials: J
authors.lastname: Wang
authors.forename: Zhihao
authors.initials: Z
authors.lastname: Zhuang
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D049109:Cell Proliferation / Q000187:drug effects; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology; D004261:DNA Replication / Q000187:drug effects; D005453:Fluorescence; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology; D018809:Proliferating Cell Nuclear Antigen / Q000032:analysis* / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation
chemical_list: D050759:Cyclin-Dependent Kinase Inhibitor p21; D010455:Peptides; D018809:Proliferating Cell Nuclear Antigen