SNS-314, a novel small-molecule Aurora kinase inhibitor, induces cell-cycle
defects and potently suppresses tumor growth
K. Gamo, B. Belmont, D. Stockett, B. Tangonan, M. Romanowski, W. Lew, S. Baskaran, C. Yu, E. Hanan, J. Oslob, M. Bui, M. Zhong, S. Heumann, D. Allen, W. Shen, J. Hogan,
J. Kumer, A. Howlett, T. O’Brien, W. M. Flanagan
Sunesis Pharmaceuticals, South San Francisco, CA 94080
Abstract SNS-314 is a potent Aurora kinase inhibitor SNS-314 disrupts mitosis
Aurora kinases constitute a family of serine-threonine kinases that are strongly associated with 70 Reduction in phospho-Histone H3 Formation of large polyploid cells
cancer. Aurora A and B are essential in mitosis. Perturbation of their activity leads to multiple Aurora A HTRF Aurora B HTRF
Mean Average pHH3
% Relative Enzyme
% Relative Enzyme
defects in mitosis including aberrant centrosome duplication, misalignment of chromosomes, IC50= 8.9 nM IC50= 20 nM
inhibition of cytokinesis, and disruption of the spindle checkpoint. The role of Aurora C is
IC50 = 9nM
unclear; however, Aurora C can complement Aurora B kinase activity in mitosis. SNS-314 is an 40
ATP-competitive, selective, and potent nanomolar inhibitor of Aurora kinases in vitro. A co-
50 50 30
crystal structure of SNS-314 with Aurora A confirms that SNS-314 engages the purine-binding
pocket of Aurora. SNS-314 inhibits cellular proliferation in the HCT116 colorectal carcinoma 20
cell line with an EC50 of ~5 nM. Analysis of DNA content and indirect immunofluoresence 10
demonstrates that SNS-314 induces defects in cytokinesis and spindle checkpoint that are -7 -6 -5 -4 -3 -2 -1 0 1 2
0 0 α-tubulin
consistent with Aurora kinase inhibition. Phosphorylation of Histone H3 on serine 10, a known Log Compound Concentration (uM)
Aurora B cellular target, is inhibited with an EC50 of ~9 nM following treatment of cells with
Aberrant mitosis (16 h)
SNS-314. Administration of SNS-314 to HCT116 tumor bearing mice potently suppresses
tumor growth. Analysis of SNS-314 treated tumors confirms that the anti-tumor activity is Log [SNS-314] (uM) Log [SNS-314] (uM)
consistent with Aurora kinase inhibition. SNS-314 is a potent small-molecule inhibitor of Aurora
Representative SNS-314 IC50 curves for Aurora A and B using a Homogenous Time-Resolved
kinase that is being developed as a novel anti-cancer therapeutic agent.
Fluorescence (HTRF)-based biochemical assay. 36nM SNS-314
16 nM SNS-314
SNS-314 is a novel, small-molecule (< 450 Da), multi-Aurora kinase inhibitor. Aurora kinases Summary of IC50 data for SNS-314 versus Aurora A, B, and C.
play a central role during mitosis and cytokinesis, and disruption of their activity leads to Cells were treated with SNS-314 for 72 h and
imaged under identical conditions.
aberrant mitotic cell division, aneuploidy, and apoptosis. Thus, a small-molecule antagonist of
Aurora kinase activity is predicted to be an effective anti-cancer agent. SNS-314 exhibits Aurora A Aurora B Aurora C
potent anti-tumor activity in multiple xenograft models and will enter phase 1 clinical trials for
the treatment of patients with advanced solid tumors in 2007.
SNS-314 shows potent anti-tumor activity
Avg. IC50 (nM) 9.0 31 3.4 consistent with Aurora kinase inhibition
Methods SD 2.0 7.0 n/a
HCT116 colorectal carcinoma model
Kinase assays: SNS-314 was tested for inhibitory activity against a panel of 219 kinases 1500 biw x 3
(Upstate Biotechnology, Dundee, UK). All screens were performed by incubating the kinase, n 9 10 1
Tumor Volume (mm )
SNS-314, and radiolabeled ATP for typically 30-60 minutes. The final ATP concentration in the 1250
reaction was within 15 μM of the Km for ATP, as calculated by Upstate. SNS-314 is a potent multi-Aurora kinase inhibitor.
Aurora biochemical assays: A Homogenous Time-Resolved Fluorescence (HTRF)-based 1000 Vehicle
biochemical IC50 assay from Cisbio (Bedford, MA) was used to test for the kinase activity of the
three isoforms of Aurora—Aurora A, B, and C—in the presence of SNS-314. A biotin conjugated Co-crystal structure of SNS-314 with Aurora A 750
150 mg/kg biw x 3
histone H3 peptide (Upstate Biotechnology) was used as a substrate.
Crystallography: Diffraction-quality crystals of Aurora-A in complex with inhibitors were 500
obtained by hanging-drop vapor diffusion at 20-25°C. Diffraction data were collected under
standard cryogenic conditions on RAXIS-IV, processed and scaled by using CrystalClear from 250
Rigaku/Molecular Structure Corporation. The structures were determined from single-wavelength
native diffraction experiments by molecular replacement with AMoRe using a search model from 0
a previously determined structure. 10 20 30 40 50 60 70
Cell biology: Phospho-Histone H3 (serine 10)– analysis of phospho-Histone H3 levels was Days Post-Implantation
performed on adherent cells using high-content screening methodology. Briefly, HCT116 cells SNS-314 shows strong anti-tumor activity in HCT116 xenografts on an intermittent dosing
were plated in growth medium on 96-well poly-L-lysine plates. After overnight growth at 37°C,
schedule. The red arrows indicate the treatment days.
various concentrations of SNS-314 were added to each well for a 1 h treatment. Cells were Mice treated with SNS-314 demonstrate a 95.6% tumor growth inhibition at day 36 and
processed according to standard procedures. Images were captured and pHH3 staining was showed a 32.5 day tumor growth delay.
analyzed using the Target Activation application and ArrayScan VTITM instrument (Cellomics,
Inc.). Data points taken from the parameter Mean_AveIntenCh2 were graphed in GraphPrism
SNS-314 induces large polyploid cells in HCT116 tumors
and fitted into an IC50 equation.
Fluorescent Imaging: Slides were examined on a LEICA DMIRE2 fluorescent microscope with a Day 4 Day 11 Day 18 Day 25
63x oil immersion objective. Images were captured on a LEICA DFC300FX CCD camera and
analyzed using Image-Pro software. DFG
Pharmacology: Mice (nu/nu) were subcutaneously implanted with HCT-116 colorectal Vehicle
carcinoma cells in the right hind flank with 200 μl of a 2.5 x 107 cell/ml suspension (1:1 DPBS
Crystal structure of Aurora-A with SNS-314. The crystal structure reveals the location of
with cells:Matrigel). When tumors reached an average volume of 198 mm3, mice were
SNS-314 in the ATP binding pocket and extending into the substrate binding groove.
randomized into groups and treated with vehicle or SNS-314. All animal experiments were in
accordance with protocols approved by the Sunesis Pharmaceuticals, Inc. Institutional Animal
Care and Use Committee and in accordance with local state and Federal regulations. Following
treatment with SNS-314, tumors were harvested, placed in Streck fixative, paraffin embedded,
sectioned, and transferred to slides. Tumor sections were stained with hematoxylin and eosin
SNS-314 blocks proliferation in a broad panel of SNS-
(H&E). tumor cell lines 314
SNS-314 is a selective Aurora kinase inhibitor Cell Line Tumor Type BrdU IC50 (nM)
biw x 3
SNS-314 was tested against a panel of 219 kinases to determine its selectivity profile. HCT116 Colon 6.4 Large polyploid cells consistent with Aurora B kinase inhibition appear by Day 4 and persist
for at least 25 days after treatment initiation. All images were taken at 40x magnification.
HT29 Colon 24
Target IC50* (nM) Calu 6 Lung 13
Summary and Conclusions
Aurora A 1 H1299 Lung 4.0
SNS-314 is a highly selective and potent multi-Aurora kinase inhibitor
TrkB 5 PC3 Prostate 4.4
that binds to the active form of Aurora in an extended conformation.
TrkA 12 A2780 Ovarian 1.8
SNS-314 shows low nanomolar anti-proliferative activity in a broad
Flt4 14 MDA-MB-231 Breast 8.1
panel of cancer cell lines.
Fms 15 HeLa Cervical 9.3
MiaPaCa Pancreatic 9.1
SNS-314 demonstrates potent anti-tumor activity in a human
colorectal carcinoma model that is consistent with Aurora B inhibition
Axl 84 A375 Melanoma 5.9 as the mechanism of action.
* Radiometric kinase assay Cellular proliferation was assessed using the Cell Proliferation ELISA, BrdU kit (Roche) including The combination of potency, selectivity and robust in vivo activity
reagents, according to the kit protocol. Cells were treated with compound for 96 h and labeled with coupled to flexible intermittent dose schedule suggest that SNS-314
SNS-314 is a highly selective Aurora kinase inhibitor may be a best in class Aurora kinase inhibitor for the treatment of
BrdU for 2 h before preparation for analysis.
Only 7 kinases out of the 219 show IC50 less than or equal to 100 nM. The respective IC50
values for the 7 kinases are shown in the table.
diverse human malignancies.
Fourteen other kinases were had an IC50 value between 0.100 and 1 μM.
SNS-314 showed at least a 1000-fold selectivity over the remaining 197 kinases; 197 An IND has been submitted for SNS-314 and a phase I clinical trial for
kinases had IC50> 1 μM. the treatment of patients with advanced solid tumors is planned in
These data suggest that SNS-314 has a low potential for off-target kinase related 2007.