PHA-680632

In vitro kinase assays

Inhibition of kinase activity by PHA-680632 was assessed using a scintillation proximity assay format. In this assay, the biotinylated substrate is transphosphorylated by the kinase in presence of ATP traced with γ33-ATP. The phosphorylated substrate is then captured using streptavidin-coated scintillation proximity assay beads and the extent of phosphorylation is evaluated by β-counter after a 4-hour rest for the floatation of the beads on a dense 5 mol/L CsCl solution. In particular a peptide derived from the Chocktide sequence (LRRWSLGL) was used as substrate for Aurora A, whereas the optimized peptide Auroratide1 was employed for Aurora B and C. The assay was run in a robotized format on 96-well plates. The potency of the compound toward Aurora kinases and 29 additional kinases belonging to our Kinase Selectivity Screening panel was evaluated and the relevant IC50s were determined.

Cell lines

HeLa, HCT116, HT29, LOVO, DU145, and NHDF cells

Preparation method

The solubility of this compound in DMSO is > 10 mM. General tips for obtaining a higher concentration: Please warm the tube at 37 ℃ for 10 minutes and/or shake it in the ultrasonic bath for a while. Stock solution can be stored below -20℃ for several months.

Reacting condition

0.5 μM

Applications

PHA680632 in association with radiation led to additive effects in cancer cells, especially in the p53-deficient cells. Combined ionising radiation (IR) and treatment of PHA680632 (100–400 nM) prior to IR led to an enhancement of radiation-induced Annexin V positive cells, micronuclei formation, and Brca1 foci formation only in HCT116 cells with deficient p53, other than the p53 wild-type counterparts. PHA-680632 showed potent anti-proliferative effects in a wide range of cell types with IC50 values of 0.06–7.15 μM, including HeLa, HCT116, HT29, LOVO, DU145, and NHDF cells. PHA-680632 (0.5 μM) caused polyploidy in tumor cells.

Animal models

Mice xenografts models of HL60, A2780, and HCT116 cells, mouse mammary tumor virus v-Ha-ras transgenic mice

Dosage form

Subcutaneous injection, 15–60 mg/kg

Application

HA-680632 (15–60 mg/kg) inhibited tumor growth in mice xenografts models of HL60, A2780, and HCT116 cells, by reducing tumor cell proliferation and increasing apoptosis. PHA-680632 (45 mg/kg) suppressed growth of activated ras-driven mammary tumors in mouse mammary tumor virus v-Ha-ras transgenic mice and results in complete tumor stabilization and partial regression.

Other notes

Please test the solubility of all compounds indoor, and the actual solubility may slightly differ with the theoretical value. This is caused by an experimental system error and it is normal.

References:

[1]. Soncini, C., Carpinelli, P., Gianellini, L., Fancelli, D., Vianello, P., Rusconi, L., ... & Ceruti, R. (2006). PHA-680632, a novel Aurora kinase inhibitor with potent antitumoral activity. Clinical Cancer Research, 12(13), 4080-4089.

[2]. Tao, Y., Zhang, P., Frascogna, V., Lecluse, Y., Auperin, A., Bourhis, J., & Deutsch, E. (2007). Enhancement of radiation response by inhibition of Aurora-A kinase using siRNA or a selective Aurora kinase inhibitor PHA680632 in p53-deficient cancer cells. British Journal of Cancer, 97(12), 1664.