title: A high through-put screen for small molecules modulating MCM2 phosphorylation identifies Ryuvidine as an inducer of the DNA damage response.
abstract: DNA replication is an essential process for cell division and as such it is a process that is directly targeted by several anticancer drugs. CDC7 plays an essential role in the activation of replication origins and has recently been proposed as a novel target for drug discovery. The MCM DNA helicase complex (MCM2-7) is a key target of the CDC7 kinase, and MCM phosphorylation status at specific sites is a reliable biomarker of CDC7 cellular activity. In this work we describe a cell-based assay that utilizes the "In Cell Western Technique" (ICW) to identify compounds that affect cellular CDC7 activity. By screening a library of approved drugs and kinase inhibitors we found several compounds that can affect CDC7-dependent phosphorylation of MCM2 in HeLa cells. Among these, Mitoxantrone, a topoisomerase inhibitor, and Ryuvidine, previously described as a CDK4 inhibitor, cause a reduction in phosphorylated MCM2 levels and a sudden blockade of DNA synthesis that is accompanied by an ATM-dependent checkpoint response. This study sheds light on the previously observed cytotoxity of Ryuvidine, strongly suggesting that it is related to its effect of causing DNA damage.
authors.affiliation: Centre for Chromosome Biology and National Centre for Biomedical Engineering Science, School of Natural Sciences, National University of Ireland Galway, Galway, Ireland.
authors.forename: Jennifer
authors.initials: J
authors.lastname: FitzGerald
authors.affiliation: Centre for Chromosome Biology and National Centre for Biomedical Engineering Science, School of Natural Sciences, National University of Ireland Galway, Galway, Ireland; School of Medicine, National University of Ireland Galway, Galway, Ireland.
authors.forename: Laura S
authors.initials: LS
authors.lastname: Murillo
authors.affiliation: Centre for Chromosome Biology and National Centre for Biomedical Engineering Science, School of Natural Sciences, National University of Ireland Galway, Galway, Ireland.
authors.forename: Gemma
authors.initials: G
authors.lastname: O'Brien
authors.affiliation: Screening Core, National Centre for Biomedical Engineering Science, Biosciences, National University of Ireland Galway, Galway, Ireland.
authors.forename: Enda
authors.initials: E
authors.lastname: O'Connell
authors.affiliation: Centre for Chromosome Biology and National Centre for Biomedical Engineering Science, School of Natural Sciences, National University of Ireland Galway, Galway, Ireland.
authors.forename: Aisling
authors.initials: A
authors.lastname: O'Connor
authors.affiliation: Centre for Chromosome Biology and National Centre for Biomedical Engineering Science, School of Natural Sciences, National University of Ireland Galway, Galway, Ireland.
authors.forename: Kevin
authors.initials: K
authors.lastname: Wu
authors.affiliation: Centre for Chromosome Biology and National Centre for Biomedical Engineering Science, School of Natural Sciences, National University of Ireland Galway, Galway, Ireland.
authors.forename: Guan-Nan
authors.initials: GN
authors.lastname: Wang
authors.affiliation: Centre for Chromosome Biology and National Centre for Biomedical Engineering Science, School of Natural Sciences, National University of Ireland Galway, Galway, Ireland.
authors.forename: Michael D
authors.initials: MD
authors.lastname: Rainey
authors.affiliation: Centre for Chromosome Biology and National Centre for Biomedical Engineering Science, School of Natural Sciences, National University of Ireland Galway, Galway, Ireland; School of Medicine, National University of Ireland Galway, Galway, Ireland.
authors.forename: Alessandro
authors.initials: A
authors.lastname: Natoni
authors.affiliation: Centre for Chromosome Biology and National Centre for Biomedical Engineering Science, School of Natural Sciences, National University of Ireland Galway, Galway, Ireland.
authors.forename: Sandra
authors.initials: S
authors.lastname: Healy
authors.affiliation: School of Medicine, National University of Ireland Galway, Galway, Ireland.
authors.forename: Michael
authors.initials: M
authors.lastname: O'Dwyer
authors.affiliation: Centre for Chromosome Biology and National Centre for Biomedical Engineering Science, School of Natural Sciences, National University of Ireland Galway, Galway, Ireland.
authors.forename: Corrado
authors.initials: C
authors.lastname: Santocanale
mesh_terms: D018797:Cell Cycle Proteins / Q000378:metabolism; D004249:DNA Damage / Q000187:drug effects*; D004261:DNA Replication / Q000187:drug effects; D055808:Drug Discovery; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D006367:HeLa Cells; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D064111:Minichromosome Maintenance Complex Component 2 / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D015203:Reproducibility of Results; D054852:Small Molecule Libraries
chemical_list: D018797:Cell Cycle Proteins; D004791:Enzyme Inhibitors; D054852:Small Molecule Libraries; C068619:CDC7 protein, human; D017346:Protein Serine-Threonine Kinases; D064111:Minichromosome Maintenance Complex Component 2