title: A site-selective, irreversible inhibitor of the DNA replication auxiliary factor proliferating cell nuclear antigen (PCNA).
abstract: Proliferating cell nuclear antigen (PCNA) assumes an indispensable role in supporting cellular DNA replication and repair by organizing numerous protein components of these pathways via a common PCNA-interacting sequence motif called a PIP-box. Given the multifunctional nature of PCNA, the selective inhibition of PIP-box-mediated interactions may represent a new strategy for the chemosensitization of cancer cells to existing DNA-directed therapies; however, promiscuous blockage of these interactions may also be universally deleterious. To address these possibilities, we utilized a chemical strategy to irreversibly block PIP-box-mediated interactions. Initially, we identified and validated PCNA methionine 40 (M40) and histidine 44 (H44) as essential residues for PCNA/PIP-box interactions in general and, more specifically, for efficient PCNA loading onto chromatin within cells. Next, we created a novel small molecule incorporating an electrophilic di-chloro platinum moiety that preferentially alkylated M40 and H44 residues. The compound, designated T2Pt, covalently cross-linked wild-type but not M40A/H44A PCNA, irreversibly inhibited PCNA/PIP-box interactions, and mildly alkylated plasmid DNA in vitro. In cells, T2Pt persistently induced cell cycle arrest, activated ATR-Chk1 signaling and modestly induced DNA strand breaks, features typical of cellular replication stress. Despite sustained activation of the replication stress response by the compound and its modestly genotoxic nature, T2Pt demonstrated little activity in clonogenic survival assays as a single agent, yet sensitized cells to cisplatin. The discovery of T2Pt represents an original effort directed at the development of irreversible PCNA inhibitors and sets the stage for the discovery of analogues more selective for PCNA over other cellular nucleophiles.
authors.affiliation: Department of Chemical Biology and Therapeutics, St. Jude Children's Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA.
authors.forename: Benjamin J
authors.initials: BJ
authors.lastname: Evison
authors.affiliation: Department of Chemical Biology and Therapeutics, St. Jude Children's Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA.
authors.forename: Marcelo L
authors.initials: ML
authors.lastname: Actis
authors.affiliation: Department of Chemical Biology and Therapeutics, St. Jude Children's Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA.
authors.forename: Sean Z
authors.initials: SZ
authors.lastname: Wu
authors.affiliation: Protein Production Facility, St. Jude Children's Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA.
authors.forename: Youming
authors.initials: Y
authors.lastname: Shao
authors.affiliation: Protein Production Facility, St. Jude Children's Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA.
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Heath
authors.affiliation: Department of Chemical Biology and Therapeutics, St. Jude Children's Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA.
authors.forename: Lei
authors.initials: L
authors.lastname: Yang
authors.affiliation: Department of Chemical Biology and Therapeutics, St. Jude Children's Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA. Electronic address: naoaki.fujii@stjude.org.
authors.forename: Naoaki
authors.initials: N
authors.lastname: Fujii
mesh_terms: D001665:Binding Sites; D002219:Carbamates / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology; D059447:Cell Cycle Checkpoints / Q000187:drug effects; D002460:Cell Line; D004249:DNA Damage / Q000187:drug effects; D004261:DNA Replication / Q000187:drug effects; D005454:Fluorescence Polarization; D006367:HeLa Cells; D006801:Humans; D062105:Molecular Docking Simulation; D016297:Mutagenesis, Site-Directed; D009944:Organoplatinum Compounds / Q000138:chemical synthesis / Q000737:chemistry* / Q000494:pharmacology; D018809:Proliferating Cell Nuclear Antigen / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics
chemical_list: D002219:Carbamates; D009944:Organoplatinum Compounds; D018809:Proliferating Cell Nuclear Antigen; D011994:Recombinant Proteins; C000599471:T2Pt compound
keywords: Chemotherapy; Irreversible inhibitor; PCNA protein–protein interaction; Platinum complex; Small molecule