ZM 447439 is a selective and ATP-competitive inhibitor for Aurora A and Aurora B with IC50 of 110 nM and 130 nM, respectively. It is more than 8-fold selective for Aurora A/B than MEK1, Src, Lck and has little effect against CDK1/2/4, Plk1, Chk1, etc.

Aurora B:130 nM, Aurora A:110 nM

体外实验中，ZM-447439 对重组人源Aurora A和B具有选择性抑制作用，IC50值分别为110 nM和130 nM，而其他结构类型多样的蛋白激酶，包括有丝分裂激酶CDK1和PLK1的IC50值均>10 μM。[1] ZM-447439作为Aurora激酶抑制剂，以时间和剂量依赖的方式抑制三种细胞系的生长，72小时持续暴露后的IC50值分别为3 μM (BON)、0.9 μM (QGP-1) 和3 μM (MIP-101)。此外，ZM-447439 强效诱导细胞凋亡，通过促进DNA碎片化以及caspase 3和7的激活，并使GEP-NET细胞在细胞周期的G0/G1和G2/M阶段停滞。[2] 在小鼠胚胎中，通过ZM-447439抑制Aurora激酶活性，在G2期至中期调控组蛋白H3丝氨酸10位点(H3S10Ph)的磷酸化，影响每个胚胎周期的正常进程。[3] 最近的研究显示，ZM-447439对子宫颈癌SiHa细胞展示出生长抑制和促凋亡效果，并增强对顺铂的化疗敏感性。[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 BetaplateTM 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 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.(Only for Reference)