In vitro activity: CCT007093 selectively and potently inhibits human tumor cell lines (MCF-7, KPL-1, and MCF-3B) that overexpress PPM1D. CCT007093 induces cell death via the activation of p38 kinase activity. CCT007093 in combination with paclitaxel, results in synergistic inhibition of the four paclitaxel-resistant triple-negative breast cancer (TNBC) cell lines. CCT007093 selectively promote apoptosis in breast cancer cells and skin transformed keratinocytes that ectopically expressed Wip1, while attenuates the UV-mediated apoptotic response in both skin keratinocytes and a Wip1-null cell model.

Kinase Assay: Recombinant PPM1D (20-50 pmol) is diluted in Tris buffer (50 mM, pH 8), NaCl (100 mM), β-mercaptoethanol (1 mM) or DTT (1 mM) and treated with MnCl2 (0, 1, 10 and 20 mM) or MgCl2 (0 and 40 mM). Where appropriate, inhibitors of PPM1D (10-50 μM) are added and the assay mix incubated for 30 min at room temperature. Recombinant phospho-P38 (200 pmol) is then added and the mixture incubated at 37°C for 1 h. The reaction is quenched by the addition of excess ethylenediaminetetraacetic acid (EDTA), sodium dodecyl sulphate-sample loading buffer and boiling for 5 min at 95°C followed by gel electrophoresis and western blotting.

Cell Assay: Cells (MCF-7, KPL-1, and MCF-3B cells) are transfected with a pSUPER plasmid and an additional plasmid expressing the blasticidin resistance gene (pEFBsd) in a molar ratio of 10:1. Cells are plated in 10 cm plates 24 h after transfection. Blasticidin selection (5 μg/ml) is initiated 48 h post-transfection and replenished every 3 days. Colonies are fixed in methanol and stained with crystal violet after 14 days. Colonies are quantified on a Colcount and the surviving fraction (SF) determined.