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.

PHA-680632 is a novel and potent inhibitor of Aurora kinases, a small family of serine/threonine kinases regulating mitosis in chromosome segregation and cell division, that inhibits the activity of all three Aurora kinases with 50% inhibition concentration IC₅₀values of 27 nM, 135 nM and 120 nM for Aurora A, B and C kinases respectively. PHA-680632 also cross-reacts with other kinases, including FGFR1, FLT3, LCK, PLK1, STLK2, VEGFR2 and VEGFR3, to a lesser extent and exhibits much higher IC₅₀values (390 to 5500 nM) compared to Aurora kinases. Moreover, PHA-680632 potently inhibits proliferation in a wide range of cancer cell types, including HCT116, A2780, HL60 and Hela cells, with IC₅₀values ranging from 0.06 to 7.15 μM.

Reference

[1].Soncini C, Carpinelli P, Gianellini L, Fancelli D, Vianello P, Rusconi L, Storici P, Zugnoni P, Pesenti E, Croci V, Ceruti R, Giorgini ML, Cappella P, Ballinari D, Sola F, Varasi M, Bravo R, Moll J. PHA-680632, a novel Aurora kinase inhibitor with potent antitumoral activity. Clin Cancer Res. 2006 Jul 1;12(13):4080-9.