Molecule

Description

GSK1070916 is a reversible and ATP-competitive inhibitor of Aurora B and Aurora C with IC50 of 3.5 nM and 6.5 nM, respectively.

Targets

IC₅₀

In Vitro

GSK1070916 selectively inhibits Aurora B and Aurora C with K_i of 0.38 nM and 1.5 nM over Aurora A with K_i of 490 nM. Inhibition of Aurora B and Aurora C is time-dependent, with an enzyme-inhibitor dissociation half-life of >480 min and 270 min respectively. In addition, GSK1070916 is also a competitive inhibitor with respect to ATP. ^[1] Human tumor cells treated with GSK1070916 shows dose-dependent inhibition of phosphorylation on serine 10 of Histone H3, a substrate specific for Aurora B. Moreover, GSK1070916 inhibits the proliferation of tumor cells with EC50 values of <10 nm="" in="" over="" 100="" cell="" lines="" spanning="" a="" broad="" range="" of="" tumor="" types,="" with="" a="" median="" ec50="" of="" 8="" nm.="" although="" gsk1070916="" has="" potent="" activity="" against="" proliferating="" cells,="" a="" dramatic="" shift="" in="" potency="" is="" observed="" in="" primary,="" nondividing,="" normal="" human="" vein="" endothelial="" cells.="" furthermore,="" gsk1070916-treated="" cells="" do="" not="" arrest="" in="" mitosis="" but="" instead="" fails="" to="" divide="" and="" become="" polyploid,="" ultimately="" leading="" to="" apoptosis.="">^[2] In another study, it is also reported high chromosome number associated with resistance to the inhibition of Aurora B and C suggests cells with a mechanism to bypass the high ploidy checkpoint are resistant to GSK1070916. ^[3]

In Vivo

GSK1070916 (25, 50, or 100 mg/kg) shows dose-dependent inhibition of phosphorylation of an Aurora B–specific substrate in mice and consistent with its broad cellular activity, has antitumor effects in 10 human tumor xenograft models including breast, colon, lung, and two leukemia models. ^[2]

Clinical Trials

GSK1070916A is under a Phase I Trial to study the side effects and best dose of it in treating patients with advanced solid tumors.

Features

Kinase Assay

The ability of GSK1070916 to inhibit the Aurora enzymes is measured using in vivo kinase assays. The assays measure the ability of Aurora A, Aurora B and Aurora C to phosphorylate a synthetic peptide substrate. Biotin-Ahx-RARRRLSFFFFAKKK-NH2 is used for the Aurora A–TPX2 LEADseeker^TM assay and 5FAM-PKAtide is used for the IMAP^TM assay for all three Aurora kinases. To take into account time-dependent inhibition of Aurora enzymes, Aurora A–TPX2, Aurora B–INCENP and Aurora C–INCENP are incubated with GSK1070916 at various concentrations for 30 min before the reactions are initiated with the addition of substrates. For the Aurora A LEADseeker^TM assay, final assay conditions are 0.5 nM Aurora A–TPX2, 1 μM peptide substrate, 6 mM MgCl₂, 1.5 μM ATP, 0.003 μCi/μL [γ-³³P] ATP in 50 mM Hepes, pH 7.2, 0.15 mg/mL BSA, 0.01% Tween-20, 5 mM DTT and 25 mM KCl. The reactions are incubated at room temperature (25 °C) for 120 min and terminated by the addition of LEADseeker^TM beads in PBS containing EDTA (final concentration 2 mg/mL beads and 25 mM EDTA). The plates are then sealed, and the beads are allowed to settle overnight. Product formation is quantified using a Viewlux Imager. For the IMAP^TM assays, Aurora A–TPX2 (final concentration 1 nM), Aurora B–INCENP (final concentration 2 nM) or Aurora C–INCENP (final concentration 2.5 nM) is added to the compound-containing plates in 5 μL of buffer (25 mM Hepes, pH 7.2, for Aurora A, 25 mM Hepes, pH 7.5, for Aurora B and 20 mM Hepes, pH 7.2, for Aurora C) containing 0.15 mg/mL BSA, 0.01% Tween 20 and 25 mM NaCl. This mixture is incubated at room temperature for 30 min. To start the reaction, 5 μL of a substrate solution is added containing the same Hepes buffer as used for the pre-incubation, 25 mM NaCl, MgCl₂ (2, 4 and 4 mM for Aurora A, B and C respectively), DTT (4, 4 and 2 mM for Aurora A, B and C respectively), ATP (4, 4 and 10 μM for Aurora A, B and C respectively), 200 nM 5FAM-PKAtide, 0.01% Tween 20 and 0.15 mg/mL BSA. The reactions are incubated at room temperature for 120 min for Aurora A and B and 60 min for Aurora C. These reactions are then terminated by the addition of 10 μL of 1:500 (1:600 for Aurora C) Progressive Binding Reagent in 95% Progressive Binding Buffer A and 5% Progressive Binding Buffer B. Plates are incubated at room temperature for approx. 90–120 min (time allowed for equilibrium to be reached). Plates are read in a Molecular Devices Analyst plate reader in fluorescence polarization mode.

Cell Lines

SW48, Colo 201, SW480, WiDr, Colo205, RKO E6, RKO, LoVo, HCT-116, SW620, HT29, W1417, DLD-1, HCT-8, Colo 320HSR, Hep-3B, OVCAR-3, MEC-1 cells

Concentrations

0-15 mM

Incubation Time

6-7 days

Methods

Cells are plated in 96-well plates in the recommended growth media and incubated at 37 °C in 5% CO₂ overnight. The following day, the cells are treated with serial dilutions of GSK1070916. At this time, one set of cells is treated with CellTiter-Glo for a time equal to 0 (T = 0) measurement. Following a 6- to 7-d incubation with compound, cell proliferation is measured using the CellTiter-Glo reagent according to the manufacture's recommended protocol. As inhibition of Aurora B induces endomitosis, the degree of which differs depending on the cell type, an extended compound treatment time is required to accurately reflect the effects on cell viability across a large panel of cell lines. For analysis of cell viability, values from wells with no cells are subtracted for background correction and the data plotted as a percent of the DMSO-treated control samples using Microsoft Excel XLfit4 software. The EC50 values represent the concentration of GSK1070916 where 50% maximal effect is observed.

Animal Models

Mice tumor xenograft models (A549, SW620, HCT116, H460, MCF-7, HL60, K562, Colo205)

Formulation

2% Cremophor EL, 2% N,N-dimethylacetamide, and 96% acidified water (pH 5.0)

Doses

25, 50, or 100 mg/kg

Administration

Administered via i.p. once daily