ACS Medicinal Chemistry Letters
Letter
(sodium dodecyl sulfate) sample buffer. MKN-45 and BaF3/TPR-Met
cells were treated with indicated anthraquinone derivatives for 2 h at
37 °C and then lysed in 1× SDS sample buffer. These cell lysates were
subsequently resolved by 10% SDS−polyacrylamide gel electro-
phoresis and transferred to nitrocellulose membranes. The membranes
were first probed with phospho-c-Met, c-Met (both from Cell
Signaling Technology), and glyceraldehyde 3-phosphate dehydrogen-
ase (KangChen Biotech) antibody and then with anti-rabbit or -mouse
IgG horseradish peroxidase. Immunoreactive proteins were detected
using ECL Plus (GE Healthcare), and images were captured with
ImageQuant LAS 4010 (GE Healthcare).
Surface Plasmon Resonance (SPR) Analysis. The kinetics of
the binding reactions between 2a and HGF was determined using an
XPR36 (Bio-Rad) SPR apparatus. HGF was immobilized on GLH
sensor chips according to the amine coupling protocol described in the
application handbook. To assess real-time binding capacity, 200 μL of
2a was injected over the sensor chip surface immobilized with HGF.
The chip was then washed with HBS-EP buffer for 5 min. The sensor
chip surface was regenerated using 100 μL of NaCl (2 M). All binding
experiments were performed at 25 °C with a constant flow rate of 50
μL of HBS-EP/min. To correct for nonspecific binding and changes in
the bulk refractive index, a blank channel without HGF was employed
as the control for each experiment. Sensorgrams for all binding
interactions were recorded in real time and analyzed after they had
been subtracted from the blank channel. Changes in mass due to the
binding response were recorded as resonance units. Binding kinetics
and affinities were computed by equilibrium analysis using Bio-Rad
ProteOn Manager version 2.0.1.
the mode of binding of compound 2a with HGF, a molecular
docking simulation was performed. The theoretical result shows
that compound 2a probably bound the N-terminal heparin of
HGF, forming stable electrostatic and cation−π interactions
with each other. The detailed information is provided as
Supporting Information.
Many anthraquinone derivatives, including laxatives, anti-
malarials, and antineoplastics, have been developed as drugs,
indicating that the scaffold has favorable pharmacological
characteristics.18−20 Our study is the first to discover that
anthraquinone derivatives are potent c-Met kinase inhibitors,
which further enriches the structural space of small-molecule
compounds. Among three cell lines tested, anthraquinone
derivatives exclusively displayed potency toward A549 cells, in
which the activation of c-Met kinase is stimulated by the HGF
ligand, indicating that anthraquinone derivatives can block the
extracellular HGF-dependent pathway. The most potent
compound, 2a, displayed high binding affinity for HGF, with
a KD value of 1.95 μM. Until now, the marked expression of
HGF has been detected in many lung cancers. HGF induces
gefitinib resistance in the epidermal growth factor receptor of
mutation-positive non-small cell lung cancer (NSCLC) by
activating c-Met kinase signaling.9 Further studies of combina-
tion therapy with HGF antibodies and small-molecule kinase
inhibitors are warranted in NSCLC patients with HGF-induced
resistance. It is worth designing potential inhibitors targeting
kinase and HGF in the meantime. Therefore, the dual roles of
anthraquinone derivatives against c-Met kinase are clinically
relevant and will shed new light on mechanism-based inhibitor
design.
ASSOCIATED CONTENT
* Supporting Information
■
S
Mode of binding of compound 2a with HGF fragment NK2,
general methods, and synthesis and spectral characterization
data of the target compounds. This material is available free of
EXPERIMENTAL PROCEDURES
■
Kinase Enzyme-Linked Immunosorbent Assay (ELISA).
Tyrosine kinase activities were evaluated according to the reported
protocol. During the ELISA, 20 μg/mL poly(Glu, Tyr) 4:1 (Sigma)
was precoated as a substrate on 96-well plates. Approximately 50 μL of
a 5 μM ATP solution diluted in kinase reaction buffer [50 mM HEPES
(pH 7.4), 50 mM MgCl2, 0.5 mM MnCl2, 0.2 mM Na3VO4, and 1 mM
dithiothreitol] was then added to each well. Various concentrations of
compounds diluted in 10 μL of 1% dimethyl sulfoxide (DMSO) (v/v)
were added to each reaction well; 1% DMSO (v/v) was used as the
negative control.
AUTHOR INFORMATION
Corresponding Author
■
*Telephone: +86-21-50806600. Fax: +86-21-50807188. E-mail:
Author Contributions
Z.L., J.A., and X.D. contributed equally to this study.
Funding
The kinase reaction was initiated by the addition of purified tyrosine
kinase proteins diluted in 40 μL of a kinase reaction buffer solution.
After incubation for 60 min at 37 °C, the plate was washed three times
with phosphate-buffered saline (PBS) containing 0.1% Tween 20 (T-
PBS). Approximately 100 μL of anti-phosphotyrosine (PY99)
antibody [diluted 1:500 in 5 mg/mL bovine serum albumin (BSA)
T-PBS] was added to the plate. After incubation for 30 min at 37 °C,
the plate was washed three times. A solution of 100 μL of horseradish
peroxidase-conjugated goat anti-mouse immunoglobulin G (IgG)
(diluted 1:2000 in 5 mg/mL BSA T-PBS) was added to the plate,
which was then reincubated at 37 °C for 30 min and washed as
described above. Finally, 100 μL of a solution of 0.03% H2O2 and 2
mg/mL o-phenylenediamine in 0.1 mM citrate buffer (pH 5.5) was
added to the plate. Samples were incubated at room temperature until
color emerged. The reaction was terminated by the addition of 50 μL
of 2 M H2SO4, and the plate was read using a multiwell
spectrophotometer (VERSAmax, Molecular Devices, Sunnyvale, CA)
at 490 nm. The inhibition rate (%) was calculated using the expression
(1 − A490/A490 control) × 100%. IC50 values were calculated from the
inhibition curves.
We gratefully acknowledge the financial support from the 863
program (2012AA020302), State Key Program of Basic
Research of China Grant 2009CB918502, the National Science
and Technology Major Project “Key New Drug Creation and
Manufacturing Program” (2013ZX09507-004), and the Na-
tional Natural Science Foundation of China (81025017,
30725046, 81102461, 81021062, 91029704, and 21210003).
Notes
The authors declare no competing financial interest.
REFERENCES
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(1) Yap, T. A.; de Bono, J. S. Targeting the HGF/c-Met axis: State of
play. Mol. Cancer Ther. 2010, 9 (5), 1077−9.
(2) Liu, X.; Newton, R. C.; Scherle, P. A. Developing c-MET pathway
inhibitors for cancer therapy: Progress and challenges. Trends Mol.
Med. 2010, 16 (1), 37−45.
(3) Liu, X.; Newton, R. C.; Scherle, P. A. Development of c-MET
pathway inhibitors. Expert Opin. Invest. Drugs 2011, 20 (9), 1225−41.
(4) Gherardi, E.; Birchmeier, W.; Birchmeier, C.; Woude, G. V.
Targeting MET in cancer: Rationale and progress. Nat. Rev. Cancer
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Western Blot Analysis. Cells were cultured under regular growth
conditions to the exponential growth phase. A549 cells were serum-
starved for 24 h, treated with indicated anthraquinone derivatives for 2
h at 37 °C, incubated with HGF for 15 min, and then lysed in 1× SDS
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dx.doi.org/10.1021/ml4000047 | ACS Med. Chem. Lett. 2013, 4, 408−413