Discovery of a broad spectrum antiproliferative agent with selectivity for DDR1 kinase: Cell line-based assay, kinase panel, molecular docking, and toxicity studies

Article abstract of DOI:10.3109/14756366.2015.1004057

Herein, we report compound KST9046, a new agent possessing quinazoline-urea scaffold. Preliminary biological evaluation done by the National Cancer Institute (NCI), USA, showed a great inhibitory effect of KST9046 over the 60 cell-line tumor panel. Accord

Full text of DOI:10.3109/14756366.2015.1004057

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ISSN: 1475-6366 (print), 1475-6374 (electronic)
J Enzyme Inhib Med Chem, Early Online: 1–9
2015 Korea Institute of Science and Technology. Published by Taylor & Francis. DOI: 10.3109/14756366.2015.1004057
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SHORT COMMUNICATION
Discovery of a broad spectrum antiproliferative agent with selectivity
for DDR1 kinase: cell line-based assay, kinase panel, molecular docking,
and toxicity studies
Ahmed Elkamhawy1,2, Jung-eun Park1, Nam-Chul Cho3,4, Taebo Sim1,2,5, Ae Nim Pae3, and Eun Joo Roh1,2
2
1Chemical Kinomics Research Center, Korea Institute of Science and Technology (KIST), Seoul, South Korea, Department of Biological Chemistry,
3
Korea University of Science and Technology (UST), Daejeon, South Korea, Center of Neuromedicine, Korea Institute of Science and Technology
4
5
(KIST), Seoul, South Korea, Department of Biotechnology, Yonsei University, Seoul, South Korea, and KU-KIST Graduate School of Converging
Science and Technology, Seoul, South Korea
Abstract
Keywords
Herein, we report compound KST9046, a new agent possessing quinazoline-urea scaffold.
Preliminary biological evaluation done by the National Cancer Institute (NCI), USA, showed a
great inhibitory effect of KST9046 over the 60 cell-line tumor panel. Accordingly, it was
selected for a dose-response assay; a broad spectrum antiproliferative activity with GI50 ranging
from 1.3 to 3.9 mM was exerted. To explore a potential kinase inhibitory effect, KST9046 was
applied at a single dose of 10 mM against a kinase panel of 347 different enzymes representing
450% of the predicted human protein kinome. Interestingly, selective inhibition of 76% was
observed on DDR1 kinase. Further, KST9046 showed an IC50 value of 4.38 mM for DDR1.
A molecular docking model presented KST9046 as a potential type III inhibitor for DDR1 kinase
with an allosteric mode of interaction, which may offer an explanation for its selectivity. As
further investigation, CYP450 assay was carried out for KST9046, it showed a promising toxicity
profile against four different isoforms. Based on these findings, KST9046 can be further
evaluated as a promising safe new hit for the development of broad spectrum anticancer
agents with a selectivity for DDR1 kinase.
CYP450, DDR1 kinase inhibitor, kinase panel,
molecular docking, NCI panel
History
Received 15 September 2014
Revised 23 December 2014
Accepted 24 December 2014
Published online 25 March 2015
Introduction
small molecules as drugs or as tools for chemical biology
research. Based on the mechanism of action, there are four types
of kinase inhibitors; Type I which interacts directly with the ATP
binding pocket via the hinge region. This type lacks the selectivity
because the ATP-binding pocket is highly conserved among
members of the kinase family. Type II binds to the hinge region as
well as in a pocket created by the DFG residues of the activation
loop in ‘‘DFG-out’’ conformation. As a result, they are able to
achieve a higher level of selectivity compared to type I. The
highest selectivity profile could be expected by both; type III
(allosteric modulators exclusively bind in a pocket adjacent to the
ATP site, without making any interactions with the hinge region)
and type IV (allosteric modulators bind to a remote allosteric site
on the kinase)5. In this sense, kinase inhibitors possessing an
allosteric mode of action could exhibit the highest degree of
selectivity because they bind to less conserved allosteric sites
outside the ATP pocket and exploit regulatory mechanisms that
are unique to a specific kinase6.
Cancer is thought to reflect a multi-step process, resulting from an
accumulation of inherited and/or acquired defects in genes
involved in cell survival and proliferation. The human genome
encodes approximately 500 predicted protein kinases, many of
them are participating in signal transduction pathways that
regulate cell growth and survival1. Consequently, abnormal
phosphorylation due to kinases overexpression can lead to
numerous types of cancer2–4. Currently, several protein kinase
inhibitors have been approved by the FDA for the treatment of
different types of both solid and non-solid tumors of various
origins and types. However, many kinase inhibitors are in fact not
selective for one particular kinase but target several kinases,
which potentially increases the risk of unwanted effects and
toxicity.
Achieving inhibitor selectivity for particular protein kinases
often remains a significant challenge in the development of new
Discoidin domain receptor 1 (DDR1), binds to several
collagens and widely expressed in epithelial cells of different
tissues. Recent reports indicated altered expression of DDR1
kinase in various human cancers, including brain, ovary, breast,
lung and esophagous cancers, suggesting a potential role of DDR1
Address for correspondence: Dr Eun Joo Roh, Future Convergence
Research Division, Chemical Kinomics Research Center, Korea Institute
of Science and Technology, Hwarang-ro 14-gil 5, Seongbuk-gu, Seoul
136-791, South Korea. E-mail: r8636@kist.re.kr

2
A. Elkamhawy et al.
J Enzyme Inhib Med Chem, Early Online: 1–9
in tumor progression7–11. Although the exact mechanisms by compound was tested over 347 different kinases representing
which this receptor may contribute to oncogenesis is unknown, it 450% of the predicted human protein kinome, and it showed
has been demonstrated that it may act as a critical regulator of cell selectivity for DDR1 kinase.
proliferation, adhesion, migration and subsequent tumor metas-
tasis12. Hence, inhibition of DDR1 function may provide a
potential approach to selectively enhance treatment of such
tumors.
In a previous work reported by our group, novel quinazoline-
Materials and methods
Chemistry
All reactions and manipulations were performed in nitrogen
atmosphere using standard Schelenk techniques. Reagents and
solvents were purchased from Aldrich Co. (St. Louis, MO) and
Tokyo Chemical Industry Co. (Tokyo, Japan) and used without
purification. Thin-layer chromatography was performed with
Merck (White House Station, NJ) silica gel 60 F254 pre-coated
glass sheets. Column chromatography was performed on Merck
Silica Gel 60 (230–400 mesh) and the eluting solvents are noted
urea derivatives were synthesized and biologically evaluated
as modulators for Ab-induced mitochondrial dysfunction13.
Since several quinazoline derivatives have been approved by the
FDA as kinase inhibitory anticancer drugs, such as Gefitinib,
Erlotinib, Lapatinib and Vandetanib, we found that the novel
quinazoline-urea chemical structure with its unique substitution
pattern on positions C6 and C7 (Figure 1), which has not been
tested before for either antiproliferative or kinase inhibition effect,
is worthy to be evaluated. Herein, we report the discovery of
1
as mixed solvent with given volume-to-volume ratios. H NMR
400 MHz was measured on Bruker Avance 400 (Billerica, MA),
and chemical shifts and coupling constants are presented in parts
per million relative to Me4Si and Hertz, respectively.
Abbreviations are as follows: s, singlet; d, doublet; t, triplet; m,
multiplet. High-resolution spectra were performed on Waters
(Milford, MA) ACQUITY UPLC BEH C18 1.7m–Q-TOF
SYNAPT G2-Si High Definition Mass Spectrometry.
compound KST9046,
a new agent with quinazoline-urea
scaffold. The synthetic and screening protocols for the new
agent are illustrated in details. Preliminary in vitro anticancer
assay done by the National Cancer Institute (NCI), USA, showed
a broad spectrum activity of the new compound in low
micromolar range over all of the 60 NCI tumor cell-lines.
Accordingly, the kinase inhibitory activity of the synthesized
FDA approved quinazoline-based anticancer drugs
F
NH
Cl
NH
N
O
O
O
O
N
N
O
O
N
N
O
Gefitinb
Erlotinib
Br
O
O
S
F
NH
N
HN
Cl
NH
N
O
F
O
O
N
O
N
N
Vandetanib
Lapatinib
F
6,7-disubstituted
new quinazoline agent
- Broad spectrum antiproliferative activity against NCI
60 cell-line panel with GI50 range of 1.3 µM to 3.9 µM.
- Selective inhibition for DDR1 kinase: IC50 = 4.38 µM.
O
N
O
N
N
H
N
H
Cl
KST9046
Figure 1. Quinazoline-based anticancer drugs and KST9046 chemical structure.

DOI: 10.3109/14756366.2015.1004057
Discovery of a broad spectrum antiproliferative agent with selectivity for DDR1 kinase
3
Preparation of the intermediates 2, 3, 4 and 5
each of the drug concentrations levels. Percentage growth
inhibition is calculated as:
The synthetic procedures and compounds’ characterization were
carried out as reported by our research group13.
½ðTi ꢁ TzÞ=ðC ꢁ TzÞꢂ ꢃ 100 for concentrations for which Ti ꢄ Tz
½ðTi ꢁ TzÞ=Tzꢂ ꢃ 100 for concentrations for which Ti 5 Tz:
Preparation of compound 1-(3-chlorophenyl)-3-(6-(3-fluoroben-
zyloxy)quinazolin-7-yl)urea (KST9046)
Three dose–response parameters are calculated for each
experimental agent. Growth inhibition of 50% (IC50) is calculated
by using [(Ti ꢁ Tz)/(C ꢁ Tz)] ꢃ 100 ¼ 50, which is the drug
concentration resulting in a 50% reduction in the net protein
increase (as measured by SRB staining) in control cells during the
drug incubation. The drug concentration resulting in total growth
inhibition (TGI) is calculated from Ti ¼ Tz. The LC50 (concen-
tration of drug resulting in a 50% reduction in the measured
protein at the end of the drug treatment as compared to that at the
beginning) indicating a net loss of cells following treatment is
calculated from [(Ti ꢁ Tz)/Tz] ꢃ 100 ¼ ꢁ50. Values are calculated
for each of these three parameters if the level of activity is
reached; however, if the effect is not reached or is exceeded, the
value for that parameter is expressed as greater or lesser than the
maximum or minimum concentration tested.
A mixture of quinazolinamine 5 (1.0 mmol) and 3-chlorophenyl
isocyanate (1.2 mmol) in anhydrous THF (10 mL) was stirred at
85 ꢀC overnight. The mixture was evaporated under reduced
pressure and the residue was purified by flash column chroma-
tography (silica gel, methylene chloride/ethyl acetate 1:4 v/v).
1
Yield 62%; mp: 147–148 ꢀC, H NMR (400 MHz, DMSO-d6) d
[ppm]: 9.96 (s, 1H), 9.26 (s, 1H), 9.08 (s, 1H), 8.85 (s, 1H), 8.75
(s, 1H), 7.79 (s, 1H), 7.66 (s, 1H), 7.53–7.43 (m, 3H), 7.37–7.29
(m, 2H), 7.22 (t, J ¼ 8.2 Hz, 1H), 7.09 (d, J ¼ 7.6 Hz, 1H), 5.50 (s,
2H). HRMS (ES+): m/z calculated for C22H16ClFN4O2: 445.0844
[M + Na]+. Found 445.0848.
NCI-cell line screening
Cell-line screening was applied at the National Cancer Institute
(NCI), Bethesda, MC, USA14, by applying the following
procedure. The human tumor cell lines of the cancer screening
panel are grown in RPMI 1640 medium containing 5% fetal
bovine serum and 2 mM L-glutamine. For a typical screening
experiment, cells are inoculated into 96-well microtiter plates in
100 mL at plating densities ranging from 5000 to 40 000 cells/
well depending on the doubling time of individual cell lines.
After cell inoculation, the microtiter plates are incubated at
37 ꢀC, 5% CO2, 95% air and 100% relative humidity for 24 h
prior to addition of experimental drugs. After 24 h, two plates of
each cell line are fixed in situ with TCA, to represent a
measurement of the cell population for each cell line at the time
of drug addition (Tz). Experimental drugs are solubilized in
dimethyl sulfoxide at 400-fold of the desired final maximum test
concentration and stored frozen prior to use. At the time of drug
addition, an aliquot of frozen concentrate is thawed and diluted
to twice the desired final maximum test concentration with a
complete medium containing 50 mg/mL gentamicin. Additional
four, 10-fold or 1/2 log serial dilutions are made to provide a
total of five drug concentrations plus control. Aliquots of 100 mL
of these different drug dilutions are added to the appropriate
microtiter wells already containing 100 mL of medium, resulting
in the required final drug concentrations. Following drug
addition, the plates are incubated for an additional 48 h at
37 ꢀC, 5% CO2, 95% air and 100% relative humidity. For
adherent cells, the assay is terminated by the addition of cold
TCA. Cells are fixed in situ by the gentle addition of 50 mL of
cold 50% (w/v) TCA (final concentration, 10% TCA) and
incubated for 60 min at 4 ꢀC. The supernatant is discarded, and
the plates are washed five times with tap water and air dried.
Sulforhodamine B (SRB) solution (100 mL) at 0.4% (w/v) in 1%
acetic acid is added to each well, and plates are incubated for
10 min at room temperature. After staining, unbound dye is
removed by washing five times with 1% acetic acid and the
plates are air dried. Bound stain is subsequently solubilized with
10 mM trizma base, and the absorbance is read on an automated
plate reader at a wavelength of 515 nm. For suspension cells, the
methodology is the same except that the assay is terminated by
fixing settled cells at the bottom of the wells by gently adding
50 mL of 80% TCA (final concentration, 16% TCA). Using the
seven absorbance measurements [time zero (Tz), control growth
(C) and test growth in the presence of drug at the five
concentration levels (Ti)], the percentage growth is calculated at
Kinase screening
Kinase assays were performed at Reaction Biology Corporation
(Malvern, PA) using the ‘‘HotSpot’’ assay platform15. Kinase
assay protocol; reaction buffer: base reaction buffer; 20 mM
Hepes (pH 7.5), 10 mM MgCl2, 1 mM EGTA, 0.02% Brij35,
0.02 mg/mL BSA, 0.1 mM Na3VO4, 2 mM DTT, 1% DMSO.
Required cofactors were added individually (if needed) to each
kinase reaction. Reaction procedure: To a freshly prepared buffer
solution was added any required cofactor for the enzymatic
reaction, followed by the addition of the selected kinase at a
concentration of 20 mM. The contents were mixed gently, then
the compound under test (KST9046) dissolved in DMSO was
added to the reaction mixture in the appropriate concentration.
339-ATP (specific activity 500 mCi/mL) was added to the
mixture in order to initiate the reaction, and the mixture was
incubated at room temperature for 2 h. Initial screening over 347
kinases: KST9046 was tested by single dose duplicate made at a
concentration of 10 mM. Staurosporine was used as a control
compound in a 5-dose IC50 mode with 10-fold serial dilutions
starting at 20 mM. Reaction was carried out at 10 mM ATP
concentration. Testing against DDR1 kinase: KST9046 was
tested in a 10-dose IC50 mode with three-fold serial dilutions
starting at 20 mM. Staurosporine was used as
a control
compound in a 10-dose IC50 mode with five-fold serial dilutions
starting at 20 mM. Reaction was carried out at 10 mM ATP
concentration.
Molecular docking
We retrieve co-crystallized DDR1 kinase with Imatinib (PDB
code: 4BKJ) from PDB bank. Compound was drawn with
Chemdraw14 and then converted into 3D structure. The protein
and ligand were prepared using ProteinPrepareWizard and
¨
Ligprep module in Maestro 9.7 (Schrodinger, LLC, New York,
NY), respectively. After removing water and adding hydrogen,
protein was neutralized and then optimized with energy mini-
mization on only hydrogens. Ligand was prepared with proton-
ation at pH 7.4 and energy minimization. InduceFit docking
module was employed to predict binding mode of ligand
into DDR1 kinase with XP-Gscore. The pose with the lowest
XP-Gscore was selected as the best docking pose and used for the
analysis of binding mode.

4
A. Elkamhawy et al.
J Enzyme Inhib Med Chem, Early Online: 1–9
CYP 450 metabolic stability test
Preliminary in vitro assay was performed against a full panel of
approximately 60 human tumor cell lines representing leukemia,
melanoma and cancers of lung, colon, brain, breast, ovary, kidney
and prostate in accordance with the protocol of the NCI. The
compound was added at a single dose concentration of 10 mM, and
the culture was incubated for 48 h. End point determinations were
made with a protein binding dye, Sulforhodamine B (SRB).
Results for the compound were reported as a mean graph of the
percent growth inhibition (GI %) of the treated cells when
compared to the untreated control cells14,16,17. As shown in
Table 1, the compound exerted great inhibitions up to lethal
effects over almost all of the 60 NCI cell-lines. Compound
KST9046 (NCS: 777737) satisfied pre-determined threshold
growth inhibition criteria and further selected for NCI full panel
five-dose assay at 10-fold dilutions of five different concentra-
tions (0.01, 0.1, 1, 10 and 100 mM). The result of tested compound
is given by three response parameters (GI50, TGI and LC50) for
each cell line from log concentration versus % growth inhibition
curves on nine cancer disease (Figure 2). The GI50 value (growth
inhibitory activity) corresponds to the concentration of the
compound causing 50% decrease in net cell growth, the TGI
value (cytostatic activity) is the concentration of the compound
resulting in total growth inhibition and LC50 value (cytotoxic
activity) is the concentration of the compound causing net 50%
loss of initial cells at the end of the incubation period of 48 h.
Furthermore, a mean graph midpoint (MG-MID) is calculated
giving an averaged activity parameter overall cell lines. The title
compound under investigation exhibited remarkable anticancer
activity against all the tested cell lines representing nine different
subpanels with GI50 values between 1.3 and 3.9 mM (Table 1).
Cytochrome P-450 enzyme inhibition was tested using the P450-
gloÔ assay kit (Promega, Madison, WI) according to the
protocols provided by the manufacturer. First, the compounds
were diluted into 4ꢃ the final test concentrations. Each CYP
membrane and the corresponding luciferin-tagged substrate were
diluted with water. Equal volume (6.25 mL) of the 4ꢃ sample and
4ꢃ CYP were mixed in a 384-well white plate and pre-incubated
at room temperature for 10 min. Then, the plate was stored for 20–
30 min after adding 12.5 mL of a 2ꢃ NADPH generation system.
Luminescence was detected in Fusion-alphaÕ (PerkinElmer,
Waltham, MA) after 20 min stabilization with a luciferin detection
reagent.
Results and discussion
Chemistry
Synthesis of the target compound was achieved according to the
sequence illustrated in Scheme 1. Heating 2,4-dinitrophenol 1
with 3-fluorobenzyl bromide in the presence of anhydrous K2CO3
and catalytic amount of potassium iodide in acetonitrile afforded
the corresponding dinitrobenzene derivative 2 which by catalytic
hydrogenation at room temperature using 10% Pt/C in methanol
gave the diamine analogue 3. The dicarbamate derivative 4 was
obtained by stirring compound 3 with ethyl chloroformate in the
presence of triethylamine (TEA) in tetrahydrofuran (THF) at
room temperature. Cyclization to quinazolineamine 5 was carried
out by treatment of intermediate 4 with hexamethylenetetramine
(HMTA) in the presence of trifluoroacetic acid (TFA) at room
temperature, and subsequent heating with 10% aqueous-ethanolic
KOH (1:1) and K3Fe(CN)613. Reaction of the amino group in
compound 5 with 3-chlorophenyl isocyanate in THF at 85 ꢀC
provided the new quinazoline-urea derivative KST9046.
In vitro kinase profile screening
In order to investigate a possible kinase inhibitory activity of the
new compound, it was tested over a panel of 347 different kinases
at Reaction Biology Corporation (Malvern, PA)15. The screening
results have revealed that a remarkable inhibitory activity was
selectively shown at DDR1 kinase only. The compound was tested
initially at a single dose concentration of 10 mM. At this
concentration, 76% inhibition of the enzymatic activity of
Full NCI 60 cell panel assay
The new agent was selected by the NCI, USA, for in vitro disease-
oriented human cells screening panel assay for anticancer activity,
under the Developmental Therapeutic Program (DTP).
Scheme 1. Reagents and conditions: (i) 3-fluorobenzyl bromide, K2CO3, KI, CH3CN, 75 ꢀC, 8 h; (ii) H2, 10% Pt/C, CH3OH, rt, 6 h; (iii) Ethyl
chloroformate, TEA, THF, rt, 2 h; (iv) (a) HMTA, TFA, rt, 1 h, (b) 10% KOH aqueous ethanolic (1:1), K3Fe(CN)6, 100 ꢀC, 4 h; (v) 3-chlorophenyl
isocyanate, THF, 85 ꢀC, overnight.

DOI: 10.3109/14756366.2015.1004057
Discovery of a broad spectrum antiproliferative agent with selectivity for DDR1 kinase
5
Table 1. % Growth inhibition at 10 mM, GI50, TGI and LC50 of compound KST9046 over the NCI cell-line panel.
% Growth
inhibition
Tumor cell lines
GI50 (mM)
TGI (mM)
LC50 (mM)
Leukemia
CCRF-CEM
HL-60(TB)
K-562
MOLT-4
RPMI-8226
SR
97.19
92.51
91.78
95.16
L
2.33
2.20
2.52
1.72
1.30
1.98
–
–
4100
4100
4100
4100
4100
4100
4100
–
–
–
L
Non-small cell lung cancer
A549/ATCC
HOP-62
97.74
L
L
62.86
79.43
84.46
95.64
L
2.88
2.10
1.88
2.91
2.96
2.79
2.48
1.68
–
5.60
6.88
9.19
–
–
–
3.81
4100
4100
4100
4100
4100
4100
4100
–
HOP-92
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
Colon cancer
COLO 205
HCC-2998
HCT-116
HCT-15
L
1.81
3.90
2.61
2.72
2.93
1.77
2.79
–
4100
4100
4100
–
–
4100
4100
4100
4100
–
64.30
91.37
99.23
99.26
90.92
76.07
HT29
KM12
SW-620
–
–
4100
CNS cancer
SF-268
SF-295
SF-539
SNB-19
73.45
92.78
64.04
77.06
78.90
93.01
2.37
2.27
2.28
2.91
1.81
3.28
–
–
–
4100
4100
4100
4100
8.73
7.42
3.98
4100
SNB-75
U251
4100
Melanoma
LOX IMVI
MALME-3 M
M14
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
Ovarian cancer
IGROV1
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
Renal cancer
786-0
78.59
89.16
L
99.54
L
79.13
L
L
2.93
2.41
2.45
1.48
2.04
2.20
1.61
2.44
1.68
–
–
–
4.34
4.23
–
2.98
–
4100
4100
4100
4100
–
4100
–
4100
–
L
3.50
81.01
87.42
77.55
70.37
96.48
86.36
71.87
2.59
1.75
2.56
2.72
2.76
2.97
2.45
–
4100
–
4.17
7.34
4100
7.98
7.71
6.13
4100
4100
4100
4100
4100
60.99
L
3.00
1.75
2.58
3.03
–
2.53
2.63
2.17
4100
5.96
–
4100
–
–
7.98
–
4100
4100
4100
4100
4100
4100
4100
4100
A498
ACHN
CAKI-1
RXF 393
SN12C
TK-10
96.53
85.36
68.53
78.26
L
UO-31
90.63
Prostate cancer
PC-3
DU-145
90.43
81.09
2.22
3.38
–
4100
4100
4100
Breast cancer
MCF7
MDA-MB-231/ATCC
HS 578T
BT-549
93.96
72.76
54.14
55.22
L
2.02
2.54
2.62
2.34
1.75
2.31
–
4100
4100
4100
4100
4100
4100
7.80
7.02
7.41
4.37
–
T-47D
MDA-MB-468
L
L: lethal, –: not tested.

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A. Elkamhawy et al.
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DOI: 10.3109/14756366.2015.1004057
Discovery of a broad spectrum antiproliferative agent with selectivity for DDR1 kinase
7
DDR1 kinase was observed, while the inhibition in activity was between the urea NH groups and the carboxylic side chain of
below 25% in all of the other kinases, and in the range of 41–53% Glu672 residue in the aC helix, while another H-bond was noticed
in four kinases only (ARAF, FLT3, LIMK1 and MLK2) as between the urea carboxylic group and NH moiety of Asp784
summarized in Table 2 (for full data screening results, see residue in DFG motif.
supplementary file).
Further, we docked known type II DDR1 inhibitors; Imatinib
Further, KST9046 was tested over DDR1 kinase in a 10-dose (Figure 5A) and DDR1-1 N-1 (Figure 5B)21 to the catalytic
IC50 mode and showed an IC50 value of 4.38 mM. However, the domain of DDR1 and compared the binding modes with that of
potency of the compound over DDR1 kinase cannot justify for its KST9046. As expected, Imatinib and DDR1-1 N-1 exhibited
strong and broad spectrum anticancer activity, and this suggests similar bonds to Glu672 and Asp784 residues via their amide
presence of other underlying mechanisms that may control the linker as indicated in Figure 5. Hence, it was clear that although
activity of this new hit against cancer.
KST9046 missed H-bonds formation with the hinge region, which
Since DDR1 and DDR2 have extensive homology, we have was achieved by type II inhibitors, it could successfully fit and
searched the literature for type III DRR1 and/or DDR2 inhibitors. bound to the hydrophobic pocket in the DFG motif.
Although only a few DDR2 inhibitors are known, a high structural
It is worth pointing out that some other interactions with
similarity was noted between KSR9046 and a recently reported DDR1 binding pocket were exhibited by KST9046; the quinazo-
type III DDR2 inhibitor discovered through high-throughput line core formed a hydrophobic binding with Leu757 and His764
screening as illustrated in Figure 318. Both compounds possess a residues in the aE helix, in addition to ꢀ-anion interaction with
heterocyclic system, a hydrophobic chlorophenyl moiety and a Asp784 residue in DFG motif. Moreover, the 3-chlorophenyl
hydrogen bond donor-acceptor pair (urea linker). This finding moiety attached to the urea linker also showed another hydro-
could support KST9046 as type III-like compound with possible phobic interaction with Ala653 and Lys655 in b3 strand, Thr701
allosteric modulation for DDR1.
in b5 strand, and Ile685 residues in b4 strand.
Of special interest, the binding affinity of the illustrated
docking pose of KST9046 has achieved XP-Gscore of
ꢁ8.962 kcal/mol. From this model, type III kinase inhibitory
allosteric mechanism of action is suggested to be a possible
binding mode of KST9046 with DDR1 kinase which may offer an
explanation of its selectivity.
Molecular docking
¨
A molecular docking study using Maestro 9.7 (Schrodinger, LLC,
New York) program19 was performed in order to predict the
binding mode of KST9046 with the catalytic domain of DDR1
enzyme. The docking model was carried out using X-ray cocrystal
structure of DDR1 kinase with Imatinib (PDB: 4BKJ)20, which is
DFG-out conformation of DDR1 bound with type II inhibitor. In
CYP 450 metabolic stability test
this study, induced Fit Docking XP-Gscore was used for analysis Cytochrome P450 (CYP450) is an enzyme responsible for drug
of the binding mode. metabolism in human. Inhibition of CYP450 at a clinically
As shown in Figure 4, different types of binding interactions relevant concentration causes drug–drug interactions as well as
have been achieved between KST9046 and DDR1 kinase catalytic various side effects. Consequently, KST9046 was subjected to a
domain immediately adjacent to the hinge region; the urea linker metabolic stability test over four isoforms of CYP450 (2C9, 2C19,
formed three H-bonds suggested to be the main keys responsible 2D6 and 3A4). The toxicity of KST9046 over CYP450 isoforms
for the ligand-enzyme interaction; two H-bonds were formed was tested and IC50 values are reported in Table 3. KST9046
exhibited very promising toxicity profile with high IC50 values
compared to the positive control agents used in this experiment.
Table 2. Summary of kinase inhibitory profile of the
The results indicated that KST9046 has no potential inhibitory
new agent KST9046 at 10 mM over a panel of 347
effect over CYP450, which decreases the possibilities of drug–
kinases.
drug interactions as well as unwanted adverse side effects.
Kinase
% Enzyme inhibition at 10 mM
Conclusion
ARAF
DDR1
FLT3
LIMK1
MLK2
Other 342 kinases*
44
76
49
40
53
In conclusion, biological evaluation of KST9046 at NCI, aimed at
screening its potential anticancer activity, resulted in discovery of
a broad spectrum effect over almost all of the 60 NCI cell-lines. In
addition, a selective kinase inhibition for DDR1 enzyme was
disclosed. Currently, in cooperation with other research groups,
detailed in vitro and in vivo pharmacological studies are carried
Less than 25
Figure 3. Structure similarity between
KST9046 and type III DDR2 allosteric
modulator.

8
A. Elkamhawy et al.
J Enzyme Inhib Med Chem, Early Online: 1–9
Figure 4. Docking of compound KST9046 in the binding site of DDR1 kinase catalytic domain (PDB ID: 4BKJ) in 3D style.
Figure 5. Docking of Imatinib (A) and DDR1-1N-1 (B) (type II DDR1 inhibitors) in the binding site of DDR1 kinase catalytic domain in 3D style.
Table 3. IC50 values of compound KST9046 over four different isoforms of CYP450 comparing to positive control agents.
CYP450 Inhibition, IC50 (mM)
Compound
2C9
2C19
2D6
3A4
KST9046
Positive control IC50 (mM)
410
Sulfaphenazole (0.08)
410
Fluoxetine (0.04)
9.6
Quinidine (0.03)
410
Ketoconazole (0.06)
out using compound KST9046 as a promising hit to develop new
This research was supported by a grant (NRF-2011-0028676)
agents targeting glioblastoma multiforme (GBM), the most from the creative/challenging research program of National
common and malignant type of human brain tumor. The results Research Foundation of Korea.
of the current work will be published as soon as done.
Acknowledgements
References
We would like to thank the National Cancer Institute (NCI), Bethesda,
MD, USA, for performing the anticancer testing over the cell lines. We
also appreciate Dr Haiching Ma from Reaction Biology Corporation for
kinase profile screening.
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Declaration of interest
The authors have declared no conflict of interest.

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Supplementary material available online
Supplementary Information.