Characterization of a highly selective inhibitor of the Aurora kinases

Article abstract of DOI:10.1016/j.bmcl.2017.08.016

Aurora kinases play an essential role in mitosis and cell cycle regulation. In recent years Aurora kinases have proved popular cancer targets and many inhibitors have been developed. The majority of these clinical candidates are multi-targeted, rendering them inappropriate as tools for studying Aurora kinase mediated signaling. Here we report discovery of a highly selective inhibitor of Aurora kinases A, B and C, with potent cellular activity and minimal off-target activity (PLK4). The X-ray co-crystal structure of Aurora A in complex with compound 2 is reported, and provides insights into the structural determinants of ligand binding and selectivity.

Full text of DOI:10.1016/j.bmcl.2017.08.016

Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Characterization of a highly selective inhibitor of the Aurora kinases
Fleur M. Ferguson , Zainab M. Doctor a,h, Apirat Chaikuad b,d, Taebo Sim e,f, Nam Doo Kim g,
a,h
b,c,d
a,
⇑
Stefan Knapp
a
, Nathanael S. Gray
Department of Cancer Biology, Dana-Farber Cancer Institute, Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA
Structural Genomics Consortium, University of Oxford, Old Road Campus Research Building, Roosevelt Drive, Oxford OX3 7DQ, United Kingdom
German Cancer Consortium (DKTK), Frankfurt site, Germany
Institute for Pharmaceutical Chemistry and Buchmann Institute for Life Sciences, Goethe University, Max-von Laue Str. 9, 60438 Frankfurt am Main, Germany
Chemical Kinomics Research Center, Korea Institute of Science and Technology, Republic of Korea
b
c
d
e
f
KU-KIST Graduate School of Converging Science and Technology, Korea University, Republic of Korea
Daegu-Gyeongbuk Medical Innovation Foundation, Republic of Korea
g
a r t i c l e i n f o
a b s t r a c t
Article history:
Aurora kinases play an essential role in mitosis and cell cycle regulation. In recent years Aurora kinases
have proved popular cancer targets and many inhibitors have been developed. The majority of these clin-
ical candidates are multi-targeted, rendering them inappropriate as tools for studying Aurora kinase
mediated signaling. Here we report discovery of a highly selective inhibitor of Aurora kinases A, B and
C, with potent cellular activity and minimal off-target activity (PLK4). The X-ray co-crystal structure of
Aurora A in complex with compound 2 is reported, and provides insights into the structural determinants
of ligand binding and selectivity.
Received 20 July 2017
Revised 8 August 2017
Accepted 9 August 2017
Available online xxxx
Keywords:
Aurora kinase
Selective kinase inhibitor
Pan-Aurora inhibitor
Mitosis
Ó 2017 Published by Elsevier Ltd.
Cancer
The Aurora kinases are a family of cell-cycle regulated serine/
threonine kinases which are primarily active during mitosis.
cells, as its primary role is in male meiosis during
spermatogenesis.
1
2,6
These homologous kinases effect distinct processes via differential
expression, localization and interaction partners. Aurora A local-
izes at the centrosome during interphase, and localizes at mitotic
poles and to the spindle throughout mitosis.2 Aurora A regulates
The Aurora kinases are implicated in a variety of hematological
and solid cancers. Aurora A and B are frequently overexpressed in
cancer, and have been associated with aneuploidy and poor prog-
7,8
nosis. Aurora C has also been show to function as an oncogene
1
and overexpression has been reported in thyroid cancer tissues.6
The Aurora kinases have therefore become attractive drug targets
with more than ten Aurora inhibitors undergoing clinical trials.9
In solid tumors, on-target bone marrow toxicity has precluded
the use of Aurora inhibitors in cancer therapy. This is hypothesized
to be due to the slower proliferation rate of cells in solid tumors
relative to those in the bone marrow, and the requirement for drug
exposure through several cell cycles before the maximal cytotoxic
progression of mitosis and promotes centrosome maturation. Aur-
ora B localizes to centromeres during metaphase as part of the
chromosomal passenger complex (CPC) and remains associated
with the central mitotic spindle during anaphase.3 The CPC regu-
lates chromosome condensation, via phosphorylation of histone
H3, the spindle assembly checkpoint (SAC)4 and cytokinesis.
Unlike Aurora A and B, Aurora C is not expressed in all dividing
5
9
effects are realized. Acute hematological tumors, such as Acute
Myeloid Leukaemia (AML), have higher proliferation rates and
have shown more promising response rates to Aurora kinase inhi-
bitors in the clinic.
Abbreviations: PLK4, Polo-like kinase 4; FACS, fluorescence activated cell
sorting; DMSO, dimethylsulfoxide; AML, acute myeloid leukemia; T-ALL, T-cell
acute lymphoblastic leukemia; ABL1, Abelson Tyrosine-Protein Kinase 1; LRRK2,
Leucine Rich Repeat Kinase 2; BRD4, Bromodomain-Containing Protein 4; ERK5,
Mitogen-activated protein kinase 7; LC-MS/MS, Liquid Chromatography–Mass
Spectrometry/Mass Spectrometry.
1
0–12
Many Aurora kinase inhibitors developed to date display poly-
9
pharmacology, targeting numerous kinases. Whilst this may con-
tribute to clinical efficacy, it does not facilitate detailed study of
Aurora kinase mediated signaling and cellular processes. Recently,
Carry et al. reported an exquisitely selective pan-Aurora inhibitor,
⇑
Corresponding author.
E-mail address: Nathanael_Gray@dfci.harvard.edu (N.S. Gray).
These authors contributed equally.
h
http://dx.doi.org/10.1016/j.bmcl.2017.08.016
960-894X/Ó 2017 Published by Elsevier Ltd.
0
Please cite this article in press as: Ferguson F.M., et al. Bioorg. Med. Chem. Lett. (2017), http://dx.doi.org/10.1016/j.bmcl.2017.08.016

2
F.M. Ferguson et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
SAR156497. This compound displayed efficacy but had a narrow
therapeutic window in colon adenocarcinoma xenograft studies.
SAR156497 was used as a control compound in our experiments, is
one of the most selective cell permeable pan-Aurora inhibitors
described to date.
and exhibited similar potency and selectivity to compound 1. Both
compounds have moderate off-target activity on PLK4 (IC50 com-
pound 1 = 43 nM, IC50 compound 2 = 82 nM) but otherwise display
excellent kinome selectivity. The ABL1 Y253 F activity of com-
pound 2 from the kinome screen was not reproduced in a biochem-
1
3
Here we report discovery of highly selective pan-Aurora kinase
inhibitors through phenotypic screening. The co-crystal structure
of compound 2 bound in the ATP binding site of Aurora A is
described, and provides insight into the possible structural basis
for the selectivity of the interaction.
ical kinase assay and is likely to be
a
false positive
(IC50 > 10,000 nM). Inhibitors based on this scaffold have been
reported to bind to LRRK2 and BRD4, proteins not covered in the
selectivity panel. Compounds 1 and 2 were tested in biochemical
assays against these targets and gratifyingly both molecules were
inactive (Supporting Table 1).
To identify anti-leukemic compounds, a library of pyrimido-
benzodiazepinones was tested for anti-proliferative activity in Jur-
kat cells, a T-cell acute lymphocytic leukemia (T-ALL) cell line. We
previously used this phenotypic screening and profiling approach
We have previously described pan-Aurora inhibitors based
1
5
upon the same chemical scaffold (XMD12-1). However, com-
pounds 1 and 2 have significantly improved kinome selectivity
(Fig. 1), making them some of the most selective pan-Aurora kinase
inhibitors reported to date.
to identify PI3K-d/c
inhibitors.14 Compound 1 was identified as a
cytotoxic molecule with an IC50 of 370 nM. Kinase profiling against
a panel of 468 human wild-type and mutant kinases using KINO-
The cellular activity of compounds 1 and 2 were assessed by
their ability to inhibit the phosphorylation of biomarkers of Aurora
A and Aurora B by western blot in HCT116 cells. Compounds 1 and
2 were cell permeable and inhibited Aurora A auto-phosphoryla-
tion and Aurora B mediated histone H3 S10 phosphorylation at
concentrations below 40 nM (Fig. 2A, C, Supporting Fig. 1). These
compounds showed improved cellular inhibition of Aurora A
auto-phosphorylation and histone H3 S10 phosphorylation com-
Ò
MEscan at 1
l
M revealed that compound 1 is a highly selective
pan-Aurora inhibitor with exclusively on-target activity detected
across the panel at 1 M compound concentration (Fig. 1). The
l
Aurora A, B and C activity was confirmed using biochemical kinase
assays (Fig. 2C).
The Aurora kinases do not contain cysteine residues proximal to
their ATP binding sites, therefore we hypothesized that the interac-
tion of compound 1 with Aurora kinases is reversible, and that the
acrylamide functionality could be removed without loss of
potency. To confirm this we incubated compound 1 at 10-fold
excess, with recombinant purified Aurora A protein, and observed
no labeling by LC-MS/MS analysis. Compound 2 was synthesized
1
3
pared to the control compound SAR156497.
To investigate the effects of compounds 1 and 2 on the cell cycle,
we performed FACS cell cycle analysis in HCT116 cells after 48 h of
500 nM compound treatment (Fig. 2B, Supporting Fig. 2). Treat-
ment with compounds 1, 2, or the control pan-Aurora inhibitor
XMD12-1
Compound 1
Compound 2
Fig. 1. A) Chemical structure and kinome-wide selectivity plot of compounds 1 and 2 compared to previously reported benzodiazepinone inhibitor XMD12-1. Assay
performed by DiscovRX at 1 M compound concentration. On target activity (Aurora A/B/C) represented by blue circles, off target activity represented by red circles. Chemical
l
modifications to the core (red), and aniline (blue), are highlighted.
Please cite this article in press as: Ferguson F.M., et al. Bioorg. Med. Chem. Lett. (2017), http://dx.doi.org/10.1016/j.bmcl.2017.08.016

F.M. Ferguson et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
3
A
SAR156497
Compound 1
Compound 2
pH3 S10
H3
pH3 S10
H3
tubulin
tubulin
pAurora A
pAurora A
Aurora A
vinculin
Aurora A
vinculin
B
C
Biochemicala
Aurora B
IC50 (nM)
Cellularb
Aurora A
IC50 (nM)
Aurora C
IC50 (nM)
Aurora A
Aurora B
Compound
EC50 (nM) EC50 (nM)
SAR156497 75 ± 26
34 ± 4.6
29 ± 2.0
41 ± 4.0
91 ± 17
63 ± 8.1
53 ± 2.4
106
5.8
38
27
7.1
11
1
2
44 ± 4.5
52 ± 3.0
Fig. 2. A) Western blot showing dose dependent inhibition of pAurora A and histone H3 pS10 marks in HCT116 cells. B) FACS analysis after 48 h treatment with 500 nM
compound concentration in HCT116 cells. Data shown is average of three replicates ± standard error. C) Biochemical IC50 and cellular EC50 values of pan-Aurora inhibitors.
a
b
Z’LYTE kinase assay, measurements performed at km ATM concentration for each kinase. Measurements are shown ± standard error. Cellular EC50 based on 4 h treatment
followed by western blot analysis of pAurora A and histone H3 pS10, representative western blot of three replicate experiments shown in this Figure, dose-response curves
shown in Supporting Fig. 1.
SAR156497 led to failed cytokinesis and accumulation of cells that
contain 4N DNA (polyploidy), consistent with cellular Aurora B
inhibition.
To assess the cytotoxicity of lead compound 1, we treated a
panel of human cancer cell lines in dose response, and assessed cell
viability after 72 h (Table 1, Supporting Fig. 3). The HeLa cell line is
highly sensitive to pan-Aurora inhibition, the IC50 of compounds 1,
of 7.5 and 2.9 min respectively, indicating these compounds may
be unsuitable for in vivo studies.
1
6
In order to gain structural insights into the mode of binding and
to rationalize the selectivity of the molecules, we solved the co-
crystal structure of compound 2 in complex with Aurora A at a res-
olution of 2.6 Å (Fig. 3, PDB: 5ONE). The structure of the complex of
compound 2 with Aurora A revealed the expected ATP competitive
binding mode of pyrimido-benzodiazepinone inhibitors, compris-
ing two hydrogen bonds with the main chain amide and carbonyl
group of the hinge residue A213. A similar binding mode was
observed in docking studies for compound 1 (Supporting Fig. 4).
2
and control compound SAR156497 are below 10 nM in this cell
line. In HCT116, PC3 and HEK293 cells, compound 1 is more potent
than SAR156497, consistent with it’s improved cellular inhibition
of the Aurora kinases signaling seen in Fig. 2A, C.16 When com-
pound 2 was tested in the cell line panel, it was highly toxic in
HeLa cells. In other cells lines, cell viability loss plateaued at 50%,
The 4-propanoylaminobenzamid moiety is rests over the aD helix,
with the propyl group oriented towards the solvent exposed area.
The kink created by the benzodiazepinone ring inserts the aro-
matic toluene portion of this core ring system into a deep
hydrophobic pocket created by L263 and an unusual inactive con-
formation of the activation segment which assumed a DFG ‘‘in”
conformation with residues C-terminal to the DFG motif packing
against the aromatic ring of the benzodiazepinone moiety. The
methyl group addition to the scaffold cannot be accommodated
in this region by many kinases, including ERK5, which contributes
when tested at concentrations up to 33.3
cell cycle arrest (Supporting Fig. 3).
lM, indicating possible
It is known that the time from mitotic arrest to apoptotic cell
death is highly variable between cancer cell lines, which may
account for some of these differences.17 Compound 1 is more active
against Aurora A (cellular), and PLK4 (biochemical) than compound
2, which may account for it’s additional toxicity in this assay. Fur-
thermore, compound 1 and SAR156497 may have additional
uncharacterized off-target activity that contributes to their
toxicity.
To test the compounds metabolic stability, we measured the
half-life of compounds 1 and 2 in mouse hepatic microsomes
1
8
to the selectivity of compounds 1 and 2.
Comparison of the binding mode of compound 2 with that of
the Aurora A specific inhibitor Alisertib (PDB: 2X81) revealed that
both inhibitors share the same hinge binding interactions.
However, the different coupling of the aromatic ring systems to
(
1 mg/mL) and found them to have modest stability, with half-lives
Please cite this article in press as: Ferguson F.M., et al. Bioorg. Med. Chem. Lett. (2017), http://dx.doi.org/10.1016/j.bmcl.2017.08.016

4
F.M. Ferguson et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
Table 1
Anti-proliferative activity of pan-Aurora inhibitors.
Compound
HCT116
HeLa
HEK293
PC3
IC50 (nM)
IC50 (nM)
IC50 (nM)
IC50 (nM)
SAR156497
3700 ± 2000
840 ± 240
ꢀ100
6.0 ± 1.5
<1.0
<1.0
5900 ± 1800
250 ± 48
ꢀ100
>10,000
1
2
990 ± 1500
a
a
a
ꢀ100
Cell Titer Glo cell viability assay, measurements were performed in triplicate and are shown ± standard error.
a
Loss of cell viability plateaued at 50%.
A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2017.08.
0
16.
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Acknowledgments
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We would like to acknowledge funding from NIH R01 U54
HL127365, the Linde center for chemical biology and the KU-KIST
Graduate School of Converging Science and Technology Program.
Please cite this article in press as: Ferguson F.M., et al. Bioorg. Med. Chem. Lett. (2017), http://dx.doi.org/10.1016/j.bmcl.2017.08.016