ZM 447439

In vitro, ZM-447439 selectively inhibits recombinant human Aurora A and B with IC50 values of 110 and 130 nM, respectively, while other protein kinases of diverse structural types including the mitotic kinases CDK1 and PLK1 are inhibited with IC50 values >10 μM. ^[1] Aurora kinase inhibitor, ZM-447439 time- and dose-dependently inhibits the growth of all three cell lines with IC50 values of 3 μM (BON), 0.9 μM (QGP-1) and 3 μM (MIP-101) after 72 hours of continuous exposure. In addition, ZM-447439 potently induces cell apoptosis by promoting DNA fragmentation and caspase 3 and 7 activation, and arrests GEP-NET cells in the G₀ /G₁and G₂/M phase of the cell cycle. ^[2] In mouse embryo, inhibition of Aurora kinase activity by ZM-447439 results in abnormalities during mitosis by regulating the phosphorylation of histone H3 serine 10 (H3S10Ph) from G2 to metaphase with different perturbations in each embryonic cycle. ^[3] A recent study shows that ZM-447439 exhibits growth inhibitory and proapoptotic effect on cervical cancer SiHa cells, and enhances the chemosensitivity. ^[4]

• In vitro kinase assays

  Recombinant Aurora A and B are expressed as NH₂-terminal His₆-tagged fusion proteins using a baculovirus expression system. Aurora A is purified by affinity chromatography using Ni-NTA agarose, and Aurora B is purified by ion exchange chromatography using CM Sepharose Fast Flow. 1 ng purified recombinant enzyme is added to a reaction cocktail containing 25 mM Tris-HCl, pH 7.5, 12.5 mM KCl, 2.5 mM NaF, 0.6 mM DTT, 6.25 mM MnCl₂, 10 μM peptide substrate, 10 μM for Aurora A or 5 μM ATP for Aurora B, and 0.2 μCi γ-[³³P]ATP (specific activity ≥2,500 Ci/mmol), and is then incubated at RT for 60 minutes. Reactions are stopped by addition of 20% phosphoric acid, and the products are captured on P30 nitrocellulose filters and assayed for incorporation of ³³P with a Betaplate^TM counter. No enzyme and no compound control values are used to determine the concentration of ZM447439, which gave 50% inhibition of enzyme activity. Further details are available on request from Nicholas Keen.

• Cell lines

  BON, QGP-1 and MIP-101 cells

• Concentrations

  0-5 μM

• Incubation Time

  72 hours

• Method

  Cell number is evaluated by crystal violet staining. In brief, cells in 96-well plates are fixed with 1% glutaraldehyde. Then cells are stained with 0.1% crystal violet. The unbound dye is removed by washing with water. Bound crystal violet is solubilized with 0.2% Triton X-100. Light extinction which increases linearly with the cell number is analyzed at 570 nm using an ELISA reader.