A high through-put screen for small molecules modulating MCM2 phosphorylation identifies Ryuvidine as an inducer of the DNA damage response

Jennifer FitzGerald; Laura S Murillo; Gemma O'Brien; Enda O'Connell; Aisling O'Connor; Kevin Wu; Guan-Nan Wang; Michael D Rainey; Alessandro Natoni; Sandra Healy; Michael O'Dwyer; Corrado Santocanale

doi:10.1371/journal.pone.0098891

A high through-put screen for small molecules modulating MCM2 phosphorylation identifies Ryuvidine as an inducer of the DNA damage response

PLoS One. 2014 Jun 5;9(6):e98891. doi: 10.1371/journal.pone.0098891. eCollection 2014.

Affiliations

¹ Centre for Chromosome Biology and National Centre for Biomedical Engineering Science, School of Natural Sciences, National University of Ireland Galway, Galway, Ireland.
² 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.
³ Screening Core, National Centre for Biomedical Engineering Science, Biosciences, National University of Ireland Galway, Galway, Ireland.
⁴ School of Medicine, National University of Ireland Galway, Galway, Ireland.

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.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Cell Cycle Proteins / metabolism
DNA Damage / drug effects*
DNA Replication / drug effects
Drug Discovery*
Enzyme Activation / drug effects
Enzyme Inhibitors / chemistry
Enzyme Inhibitors / pharmacology*
HeLa Cells
High-Throughput Screening Assays / methods*
Humans
Minichromosome Maintenance Complex Component 2 / metabolism*
Phosphorylation / drug effects
Protein Serine-Threonine Kinases / metabolism
Reproducibility of Results
Small Molecule Libraries

Substances

Cell Cycle Proteins
Enzyme Inhibitors
Small Molecule Libraries
CDC7 protein, human
Protein Serine-Threonine Kinases
Minichromosome Maintenance Complex Component 2

Grants and funding

This work was partially supported by Science Foundation Ireland (http://www.sfi.ie<http://www.sfi.ie/>) grant 08/IN.1/B2064 and the Irish Health Research Board (http://www.hrb.ie<http://www.hrb.ie/>) grant HRA/2009/89. Screening Core and Flow Cytomerty facilities are supported by HEA (www.hea.ie<http://www.hea.ie>) PRTLI5 Advancing Medicine through Discovery programme. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.