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 inhibits Aurora-A activity in an ATP-competitive manner. MK-5108 shows robust selectivity against the other family kinases Aurora-B (220-fold) and Aurora-C (190-fold) in the biochemical assay. MK-5108 also reveals high selectivity for Aurora-A over other protein kinases. MK-5108 inhibits only one kinase (TrkA) with <100-fold selectivity. MK-5108 may be more Aurora-A selective than MLN8054. Consistent with the induction of pHH3-positive cells, MK-5108 induces accumulation of cells in the G2-M phase. MK-5108 inhibits the proliferation of tumor cells including HCC1143, AU565, MCF-7, HCC1806 and CAL85-1 with an IC50 of 0.42 μM, 0.45 μM, 0.52 μM, 0.56 μM and 0.74 μM, respectively. [1] MK-5108 decreases cell viability in a dose-dependent fashion in all three cell lines including LEIO285, LEIO505 and SK-LSM1 cells with an IC50 of approximately 100 nM. Incubation with MK-5108 in LEIO285 increases the proportion of cells in G2/M at 48 and 72 hours post-treatment. MK-5108 significant increases in Caspase 3/7 activity when compared to DMSO-treated control cultures at both time points. In LEIO505 cells, MK-5108 leads to more cells accumulating at G2/M phases at 24 hours but not 48 hours or 72 hours. MK-5108 arrests ULMS cell lines at M phase MK-5108 decreases the IC50 of gemcitabine in LEIO285 cells, but increases IC50 of gemcitabine in LEIO505 and SK-LMS1 cells. [2]

MK-5108 induces pHH3-positive cells at doses of 16 mg/kg and 32 mg/kg. Plasma concentration of MK-5108 at 8 mg/kg and 16 mg/kg are 1.7 μM and 4.4 μM, respectively. MK-5108 treatment results in the induction of pHH3 in tumor and skin tissues, which starts at 2 hours and reachs a maximum at 4 hours. MK-5108 treatments at 15 mg/kg and 30 mg/kg results in significant tumor growth inhibition with the change in mean tumor volume for the treatment group as a percentage of the mean change in the control group (%T/C) of 10% and ?6% at day 11, and 17% and 5% at day 18, respectively. MK-5108 is well tolerated at both doses, with minimal reduction in body weight. MK-5108 also exhibits significant antitumor activity through intermittent dosing in nude rats bearing SW48 tumors, MK-5108 at 15 mg/kg and 45 mg/kg causes dose-dependent tumor growth inhibition with a %T/C of 35% and 7% at day 10, and 58% and 32% at day 27, respectively. [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)