Discovery and structural insight of a highly selective protein kinase inhibitor hit through click chemistry

Article abstract of DOI:10.1039/c1cc15851a

Novel bisaryl maleimide derivatives to mimic natural kinase inhibitors were prepared through click chemistry. A highly selective hit was discovered in a 124-kinase-assay, and docking studies revealed a π-π stacking interaction with the Phe67 at the P-loop of GSK-3β kinase. The Royal Society of Chemistry 2012.

Full text of DOI:10.1039/c1cc15851a

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COMMUNICATION
Discovery and structural insight of a highly selective protein kinase
inhibitor hit through click chemistryw
Guoxian Gu,z Huihui Wang,z Pi Liu,z Chenzeng Fu, Zhonghua Li, Xuefeng Cao, Yunping Li,
Qinghong Fang, Feng Xu, Jie Shen* and Peng George Wang*
Received 21st September 2011, Accepted 10th November 2011
DOI: 10.1039/c1cc15851a
Novel bisaryl maleimide derivatives to mimic natural kinase
inhibitors were prepared through click chemistry. A highly
selective hit was discovered in a 124-kinase-assay, and docking
studies revealed a p–p stacking interaction with the Phe67 at the
P-loop of GSK-3b kinase.
Protein kinases constitute a large family of more than 500
enzymes to control a plethora of signal transduction processes
within cells.1,2 Many human diseases are associated with
kinase dysfunction, and therefore selective protein kinase
inhibitors are of great potential for therapeutic applications.3
Indolocarbazole and structurally related natural products
(Fig. 1) are a family of non-selective ATP competitive inhibitors.4
Extensive efforts have been made to derivatize these natural
indolocarbazole products to afford potent inhibitors with improved
selectivity. Some bisaryl maleimide derivatives (Fig. 2), which can
be considered as the ‘‘open’’ form of natural indolocarbazole
scaffold, demonstrated better selectivity.5–7 For example,
Enzastaurin and Ruboxistaurin are two PKC selective kinase
inhibitors under clinical trials.8
Fig. 2 Bisaryl maleimides: open form of the indolocarbazole scaffold.
Moreover, the quickly constructed and structurally diversified
novel bisaryl maleimides (Fig. 2) would provide useful molecular
probes for elucidating the structural features of kinases and for
highlighting the future designs of inhibitors thereof.
In the initial stage, seven click bisaryl maleimides were prepared
(Scheme 1). Compound 2 was obtained by coupling indole
magnesium bromide with N-methyl-dichloromomaleimide in
tetrahydrofuran (THF) solution. N-Methyl-dichloromomaleimide
was obtained from maleic anhydride through two steps according
to the procedure reported by Relles.13 Then, the Sonogashira
reaction was employed to give alkyne precursor 3 in 75%
separation yield. After de-silylation with one equivalent tetra-
butylammonium fluoride (TBAF), alkyne precursor 3 reacted
with azides to give the middle compound 4a–4g in the presence
Being highly efficient and convenient, the Cu(I)-mediated
Huisgen cycloaddition reaction, the premier example of ‘‘click
chemistry’’,9 became a powerful tool for hit discovery in the
past decade.10–12 Herein, it was employed to accomplish the
goal of finding a novel selective hit against human kinases.
Fig. 1 Natural indolocarbazole derivatives as kinase inhibitors.
College of Pharmacy and Tianjin Key Laboratory of Molecular
Research, State Key Laboratory of Medicinal Chemical Biology,
Nankai University, Tianjin300071, P. R. China.
Scheme 1 Synthesis of 1a–1g. Reagents and conditions: (i) (a) EtMgBr,
THF, 60 1C, 1 hour; (b) 2,3-dichloro-N-methylmaleimide, 65 1C, 1 hour,
52%; (ii) Pd(PPh3)4, CuI, trimethylsilylacetylene, THF, Et3N, room
temperature, 75%; (iii) (a) TBAF, methanol, THF; (b) R–N3, sodium
ascorbate, CuSO4(H2O)5, room temperature, 22%–71%; (iv) NaOH,
room temperature, 2 hours; HCl, pH = 1, room temperature, overnight.
(v) NH4OAc, DMF, 140 1C, 1 hour, 28%–81% (two steps).
E-mail: jieshen@nankai.edu.cn
w Electronic supplementary information (ESI) available: Details for
assay conditions; synthesis and characterization; biological testing. See
DOI: 10.1039/c1cc15851a
z These authors contributed equally to this work.
c
2788 Chem. Commun., 2012, 48, 2788–2790
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Table 1 MTT assays of ‘‘click’’ bisaryl maleimides
In vitro cytotoxicity IC50/mM
which only showed high potency against three kinases in a
124-kinase panel.
To the best of our knowledge, there is no bisaryl maleimide
derivative in publications to show such surprisingly high
selectivity in an assay panel of more than 100 kinases. Some
‘‘selective’’ inhibitors, which had a bisaryl maleimide scaffold,
turned out not so selective when the number of assayed
kinases was increased.16 It was therefore of great interest to
understand the structural origin for the high selectivity of
compound 1c. Prior to the computational studies, more assay
experiments were carried out. The ATP-competition experiment
confirmed compound 1c as an ATP competitive inhibitor (Fig. 4).
Compounds 1d, 1e and 1g were also assayed against GSK-3b,
and these three compounds were all one to two magnitude less
potent than the hit compound 1c (Table 3).
SH-SY5Ya
A549b
NCI-H460c
K562d
Compounds
1a
1b
1c
1d
1e
1f
1g
450
450
1.83
450
5.42
1.77
45
450
450
2.43
450
3.95
0.82
450
0.17
450
450
13.31
450
13.48
3.60
450
450
9.82
450
10.54
2.22
450
0.08
450
0.02
Doxorubicin
0.06
a
b
Human derived neuroblastoma. Carcinomic human alveolar basal
c
d
epithelial cell. Human non-small cell lung cancer cells. Human
immortalised myelogenous leukaemia.
of copper sulfate pentahydrate and sodium ascorbate. The
N-methylated compound 4 was treated with aqueous NaOH
followed by acidification with hydrochloric acid affording
anhydride 5. Then the anhydride 5 was heated with ammonium
acetate to afford 1a–1g in moderate yields.
Docking studies of compounds 1c, 1d, 1e and 1g to GSK-3b
crystal structure (PDB code 1Q41) were performed, and
consistent computational results to the assay data were
obtained. As shown in Fig. 5, the active binding site of
GSK-3b is bound by the hinge loop between the N-lobe and
C-lobe, the P-loop which would contact with the phosphate
moiety of ATP, the C-helix at the N-lobe, and the A-loop
which would take a key role to activate ATP.17 Compound 1c,
when docked with GSK-3b, forms three hydrogen-bonds with
the protein: the –NH of the indole ring with the backbone of
Ile62, the –NH of the maleimide with the backbone of Tyr134,
and the carbonyl group of maleimide with Val135. A distinct
feature of this protein structure (PDB code 1Q41) is that
Phe67 flips inwards the active site, and therefore Phe67 is
around 3 to 4 A away from the phenyl moiety of 1c. Such a
distance is suitable for the p–p interaction. Obviously, either
shortening the chain length between the phenyl ring and the
triazole ring (e.g. compounds 1d and 1e), or improperly
freezing the chain (e.g. compound 1g) would attenuate such a
p–p interaction. It should be addressed that Phe67 completely
extends outwards the active site when GSK-3b binds with ATP
derivatives (PDB code 1PYX). This means the inwards flipped
Phe67 might cause a dramatic conformation change of P-loop,
To quickly verify the physiological activities of the newly
prepared click bisaryl maleimides 1a–1g, MTT assays were
performed utilizing four cancer cell lines (Table 1). Three
compounds 1c, 1f and 1g could effectively inhibit in vitro
tumor cell growth at low micromolar level. These three
compounds shared similar molecular structures which all had a
three-atom chain between the maleimide ring and phenyl ring.
Therefore, only compound 1c was selected as a representative to
enter into kinase inhibition assay (Fig. 3). In the initial one-dose
screening, only four kinases of the 124-kinase panel were
significantly inhibited by 1c at 500 nM. IC50 values of these
four kinases were then determined (Table 2). ZAP70 was identified
as a false positive since its IC50 value was only 1.66 mM.15 Taken
together, a highly selective hit compound 1c was identified
Fig. 4 ATP-competition assay of compound 1c against GSK-3b.14
Fig. 3 A radioactive 33P-ATP filter-binding assay containing 124 kinases
against 1c at 500 nM.14
Table
3
GSK-3b inhibition activity of selected ‘‘click’’ bisaryl
Table 2 Kinase inhibition activity of 1ca
maleimidesa
GSK-3b
100 ꢀ 20
CLK2
ZAP70
VEGFR2
1c
1d
1e
1g
1c
350 ꢀ 0
1660 ꢀ 610
200 ꢀ 50
GSK-3b 0.10 ꢀ 0.02 34.89 ꢀ 6.50 1.73 ꢀ 0.38 1.49 ꢀ 0.34
IC50 data in mM. Curves obtained by measuring 10 concentrations in
duplicate (3 nM, 10 nM, 30 nM, 100 nM, 300 nM, 1 mM, 3 mM, 10 mM,
30 mM, 100 mM). The full 6 curves are included in ESI.
a
a
IC50 data in nM. Curves obtained by measuring 10 concentrations in
duplicate (3 nM, 10 nM, 30 nM, 100 nM, 300 nM, 1 mM, 3 mM, 10 mM,
30 mM, 100 mM). The full 8 curves are included in ESI.
c
This journal is The Royal Society of Chemistry 2012
Chem. Commun., 2012, 48, 2788–2790 2789

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This work was supported by the National Basic Research
Program of China (973 Program, No. 2010CB529100) and
National Natural Science Foundation of China (No. 91013013).
Notes and references
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Fig. 5 Docking mode of compound 1c into the ATP-site of GSK-3b.
Autodock4.2 was applied to conduct the docking studies of GSK3b
(PDB code: 1Q41) with hit compound 1c. Experiment parameters: grid
spacing = 0.375, box size = 22.5 ꢁ 22.5 ꢁ 22.5, grid center =
38 : 6 : 32, GA runs = 50, population size = 200, quaternion = 30.0,
torsion = 30.0. Other parameters are set to default.
and in turn the dramatic topological change of the ATP binding
site. Such a mechanism of deformed P-loop was recently utilized
for computational design of a selective kinase inhibitor.18 This
mechanism might also account for the observed high selectivity
of compound 1c in 124-kinase assay. Nevertheless, at this stage,
other possible mechanisms to explain the selectivity could not
be ruled out. For example, the Phe201 residue on the flexible
A-loop might take an inactive ‘‘Phe-out’’ conformation19 to
contact with the phenyl ring of 1c. Dynamic simulation studies
and hit-to-lead optimization work are currently ongoing to
further elucidate these questions.
13 H. M. Relles, J. Org. Chem., 1972, 37, 3630–3637.
14 The assay experiments were performed by National Centre for
Protein Kinase Profiling, University of Dundee, and the detailed
procedures are attached in ESIw.
In summary, a novel kinase inhibitor 1c, which targeted only
three out of 124 human kinases, was identified. This is the second
hit-discovery project from our research laboratory utilizing click
chemistry. In both cases, the triazole ring does not take a simple
role of linker fragment. In the histone deacetylase inhibitor
project, the triazole ring forms a p–p stacking interaction within
the narrow ‘‘tube-like’’ binding pocket;20 and in this work, the
triazole ring is well designed to construct a novel scaffold to
mimic natural indolocarbazole derivatives. The success of these
two examples implicates that the combination of click chemistry
with rational design would be a powerful approach for hit
discovery in medicinal chemistry.
15 It is of no surprise since large variation was seen for ZAP70 in the
one-dose assay, Fig. 3.
16 J. Bain, H. McLauchlan, M. Elliott and P. Cohen, Biochem. J.,
2003, 371, 199.
17 R. Li and J. A. Stafford, Kinase inhibitor drugs, Wiley, Hoboken,
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18 C. R. Guimaraes, B. K. Rai, M. J. Munchhof, S. Liu, J. Wang, S. K.
Bhattacharya and L. Buckbinder, J. Chem. Inf. Model., 2011, 51, 1199.
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20 J. Hou, C. Feng, Z. Li, Q. Fang, H. Wang, G. Gu, Y. Shi, P. Liu, F. Xu,
Z. Yin, J. Shen and P. G. Wang, Eur. J. Med. Chem., 2011, 46, 3190.
c
2790 Chem. Commun., 2012, 48, 2788–2790
This journal is The Royal Society of Chemistry 2012