MK-5108 (VX-689) is a highly potent and specific Aurora-A kinase inhibitor with an IC50 value of 0.064 nM.

Aurora A:0.064 nM

MK-5108以ATP竞争性方式抑制Aurora-A活性，对其他家族激酶Aurora-B（220倍）和Aurora-C（190倍）显示出强大的选择性。MK-5108对Aurora-A与其他蛋白激酶的选择性也很高，仅对一种激酶（TrkA）显示出<100倍的选择性，可能比MLN8054对Aurora-A的选择性更高。MK-5108诱导pHH3阳性细胞的产生，与G2-M期细胞堆积一致。在生化检测中，MK-5108抑制包括HCC1143、AU565、MCF-7、HCC1806和CAL85-1在内的肿瘤细胞增殖，其IC50分别为0.42μM、0.45μM、0.52μM、0.56μM和0.74μM。MK-5108以剂量依赖性方式降低包括LEIO285、LEIO505和SK-LSM1细胞线在内的所有三种细胞线的细胞活性，IC50约为100nM。LEIO285细胞在与MK-5108孵育后，48小时和72小时后G2/M期细胞比例增加。与DMSO处理的对照组相比，MK-5108显著提高Caspase 3/7活性。在LEIO505细胞中，MK-5108导致更多细胞在24小时累积在G2/M期，但在48小时或72小时没有此现象。MK-5108在M期阻滞ULMS细胞线，降低LEIO285细胞中gemcitabine的IC50值，但在LEIO505和SK-LMS1细胞中升高。

MK-5108在16 mg/kg和32 mg/kg剂量下诱导pHH3阳性细胞。MK-5108在8 mg/kg和16 mg/kg剂量下的血浆浓度分别是1.7 μM和4.4 μM。MK-5108处理在肿瘤和皮肤组织中诱导pHH3的表达，从2小时开始，4小时达到最大。以15 mg/kg和30 mg/kg剂量进行的MK-5108处理显著抑制肿瘤生长，治疗组平均肿瘤体积变化百分比相对于对照组（%T/C）在第11天分别为10%和-6%，而在第18天分别为17%和5%。MK-5108在这两个剂量下耐受性良好，体重轻微减少。MK-5108还通过间歇给药在裸鼠负载SW48肿瘤模型中展现显著的抗肿瘤活性，15 mg/kg和45 mg/kg剂量的MK-5108引起剂量依赖性的肿瘤生长抑制，第10天的%T/C分别为35%和7%，第27天分别为58%和32%。[1]

Biochemical kinase assays: Recombinant His-tagged human Aurora-A protein is expressed in Escherichia coli and is purified with HisTrap HP column. Purified recombinant human Aurora-B and Aurora-C protein are purchased. Experiments are done in quintuplicate in 96-well plates. The Aurora-A assay reaction is conducted in the presence of 20 μM ATP, 25 μM Tetra-Kemptide [RRR(GLRRASLG)4R-NH2], 1.0 μCi per well [γ-33P]-ATP, 0.1 ng per well Aurora-A in 50 mM Tris-HCl (pH 7.4), 15 mM Mg(OAc)2, and 0.2 mM EDTA at 30°C for 40 minutes. To investigate the inhibition mode of MK-5108 for Aurora-A, the IC50 values of MK-5108 are determined in the presence of different concentrations of ATP. Then, the IC50 value is plotted as a function of ATP concentration to analyze the effect of ATP concentration on the IC50 value of MK-5108. The Aurora-B assay reaction is conducted in the presence of 15 μM ATP, 100 μM Kemptide (GLRRASLG-NH2), 1.0 μCi per well [γ-33P]-ATP, 5.0 ng per well Aurora-B in 50 mM Tris-HCl (pH 7.4), 15 mM Mg(OAc)2, and 0.2 mM EDTA at 30 °C for 20 minuts. The Aurora-C assay reaction is conducted in the presence of 40 μM ATP, 100 μM Kemptide, 1.0 μCi per well [γ-33P]-ATP, 15 ng per well Aurora-C in 10 mM MOPS-NaOH (pH 7.4), 5 mM Mg(OAc)2, 1 mM (±) DTT, and 1 mM EGTA at 30 °C for 20 minutes. After kinase reactions are terminated by adding 2.0% phosphoric acid, Tetra-Kemptide or Kemptide is trapped on the MultiScreen-PH plate. Wells are washed five times with 0.64% phosphoric acid and then monitored for radioactivity in a liquid scintillation counter.

HeLa-S3 cells are synchronized at the G1-S phase boundary by double thymidine block with 2 mM thymidine. Cells are washed and seeded to 96-well cell culture plates. After 4 hours, an equal volume of medium containing MK-5108 is added to each well. Nocodazole (300 nM) is used as a 100% control. The cells are fixed overnight with cold methanol 12 hours after seeding. Then, the cells are stained with rabbit anti-phospho-histone H3 Ser28 antibody and then with anti-rabbit IgG-Cy5. Total nuclei are stained with 10 mg/mL 4′,6-diamidino-2-phenylindole. Immunostained images are acquired using the IN Cell Analyzer1000 with ×10 objective lens. After acquisition of images, data are analyzed. The %pHH3-positive index is determined by measuring the %pHH3-positive cell counts per total nuclei counts for each sample, then by normalizing with respect to nocodazole-treated cells. (Only for Reference)