A high through-put screen for small molecules modulating mcm2 phosphorylation identifies ryuvidine as an inducer of the dna damage response

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

Characterization of pSer40/41MCM2 antibody.

<p>A) HeLa whole cell extract was incubated with Lambda phosphatase in the presence or absence of phosphatase inhibitors. Proteins were analyzed by western blot with the indicated antibodies. B) Equal amounts of protein extract were separated on a single SDS-PAGE gel and transferred onto membranes. Vertical slices of membrane (each with identical protein content) were then incubated with anti-pSer40/41MCM2 antibody in the presence of the indicated competitor peptides (upper panels). Membranes were then re-probed with an anti-MCM2 antibody (lower panels). Identical exposures are shown. C) HeLa cells growing on coverslips were fixed and stained with anti-pSer40/41MCM2 antibody in the presence of the indicated competitor peptides. DNA was counterstained with DAPI. D) Serial sections of normal and cancerous colon tissue were used in IHC with the pSer40/41MCM2 antibody in the presence or absence of competitor peptide. Nuclear positivity is shown by brown color. E) HeLa cells were treated with PHA-767491 for the indicated time. Protein extracts were prepared and analysed by western blot with the indicated antibodies.</p

Ryuvidine elicits a DNA damage response.

<p>A) Protein extracts prepared from HeLa cells treated with either PHA-767491, Ryuvidine, Mitoxantrone or hydroxyurea (HU) for the indicated time were analyzed by western blot with indicated antibodies. B) Formation of γ-H2AXnuclear foci in cells treated with ATM inhibitor KU55933, Mitoxantrone, Ryuvidine alone or in combination was assessed by fluorescence microscopy. Nuclei were stained with DAPI. Representative fields are shown.</p

Confirmation and <i>in vitro</i> characterization of Hit compounds.

<p>A) HeLa cells were treated with the ten compounds that produced the strongest reduction in pSer40/41MCM2 levels in the primary screen. Protein extracts were prepared and levels of residual pSer40/41MCM2 phosphorylation were measured by western blot analysis with the indicated antibodies. B) The same compounds were tested for their ability to inhibit CDC7 kinase activity in <i>in vitro</i> kinase assay. Kinase reactions were performed on a synthetic MCM2 substrate in the absence or presence of the indicated drug and reactions were run on SDS-PAGE gels. CDC7 activity on the synthetic MCM2 substrate was monitored by Western blotting with an anti-pSer108MCM2 antibody. As a control the levels of CDC7 kinase and synthetic MCM2 substrate present in each reaction were also assessed and shown to be similar in all reactions.</p

Ryuvidine and Mitoxantrone reduce CDC7 levels and pSer40/41 MCM2 phosphorylation.

<p>A) Molecular structure of PHA-767491, Ryuvidine and Mitoxantrone. B) HeLa cells were incubated with either CDC7 inhibitor PHA-767491, Ryuvidine or Mitoxantrone at 10 micromolar each for the indicated time. Protein extracts were prepared and analyzed by western blot with the indicated antibodies. C) Levels of Cdc7-independent pSer41MCM2 and Cdc7-dependent pSer40/41MCM2 phosphorylation assessed in HeLa cells treated with the indicated concentration of PHA-767491, Ryuvidine or Mitoxantrone. D) Cells were incubated with either PHA-767491, Ryuvidine or Mitoxantrone at the indicated concentration for nine hours. Protein extracts were then prepared and analyzed by western blot with the indicated antibodies. E) HeLa cells incubated with either PHA-767491, Ryuvidine or Mitoxantrone at 10 micromolar each for nine hours in the presence or absence of 20 micromolar of caspase inhibitor Boc-D-fmk. Levels of Ser40/41MCM2 phosphorylation, CDC7 and MCM2 abundance as well as PARP cleavage, were assessed by western blotting.</p

Screening for modulators of Cdc7-dependent phosphorylation of Ser40/41 MCM2.

<p>Results of the screening of the Johns Hopkins Clinical Compound Library and Tocris kinase inhibitor library using In Cell Western Technology. Each dot represents a compound and the normalized pSer40/41 MCM2 intensity is reported - See text for experimental details. The names of the hits producing the strongest reduction in phosphorylation at Ser40/41MCM2 are indicated.</p