Discovery of novel chemical scaffolds as RhoA inhibitors using virtual screening, synthesis, and bioactivity evaluation

Article abstract of DOI:10.1039/c6ra11398b

RhoA has been implicated in diverse cellular functions and is a potential cancer therapeutic target. Through virtual screening, we have identified a RhoA inhibitor, DDO-5701. DDO-5701 has an affinity to RhoA at the micromolar level in vitro. By structural modifications, considering the binding activity and synthesis ease of DDO-5701, 17 compounds were designed and synthesized accordingly. Among these compounds, 4 compounds (DDO-5713, DDO-5714, DDO-5715, DDO-5716) exhibited higher RhoA inhibition activities than DDO-5701, while DDO-5716 can effectively reverse the functions of breast cancer cells regulated by RhoA. Thus, the rationally designed small molecule inhibitor of RhoA (DDO-5716) is useful for studying the physiological and pathological roles of Rho GTPase. However, DDO-5701 is an approved drug-proglumide, which makes it and its derived compound DDO-5716 more likely to be well tolerated in humans and could quickly lead to further clinical development.

Full text of DOI:10.1039/c6ra11398b

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PAPER
Discovery of novel chemical scaffolds as RhoA
inhibitors using virtual screening, synthesis, and
bioactivity evaluation†
Cite this: RSC Adv., 2016, 6, 56738
Chao Zhang,ab Hui-Jie Wang,ab Qi-Chao Bao,ab Jin-Lei Bian,ab Ying-Rui Yang,ab
ab
ab
*
*
Qi-Dong You
and Xiao-Li Xu
RhoA has been implicated in diverse cellular functions and is a potential cancer therapeutic target. Through
virtual screening, we have identified a RhoA inhibitor, DDO-5701. DDO-5701 has an affinity to RhoA at the
micromolar level in vitro. By structural modifications, considering the binding activity and synthesis ease of
DDO-5701, 17 compounds were designed and synthesized accordingly. Among these compounds, 4
compounds (DDO-5713, DDO-5714, DDO-5715, DDO-5716) exhibited higher RhoA inhibition activities
than DDO-5701, while DDO-5716 can effectively reverse the functions of breast cancer cells regulated
by RhoA. Thus, the rationally designed small molecule inhibitor of RhoA (DDO-5716) is useful for
studying the physiological and pathological roles of Rho GTPase. However, DDO-5701 is an approved
drug – proglumide, which makes it and its derived compound DDO-5716 more likely to be well
tolerated in humans and could quickly lead to further clinical development.
Received 3rd May 2016
Accepted 27th May 2016
DOI: 10.1039/c6ra11398b
www.rsc.org/advances
the other strategy is targeting the GEF–RhoA interactive surface
essential for the guanine nucleotide exchange reaction also
leading to RhoA inactivation.15,16 To date, both strategies have
led to effective small molecular compounds targeting RhoA
(Fig. 1).
In the current work, we initiated a program aimed at seeking
the small molecule RhoA inhibitors. By using a docking-based
virtual screening approach in conjunction with uorescence
polarization (FP) determination, one novel small molecule
RhoA binder (hit) (DDO-5701) has been discovered and selected
as the starting point for further structural optimization. In total,
17 new compounds have been designed, synthesized, and tested
with biological assays. Finally, four compounds (DDO-5713,
DDO-5714, DDO5715 and DDO-5716) were found to show
higher RhoA inhibition activities compared to DDO-5701, and
compound DDO-5716 was further selected to study the
1. Introduction
RhoA is a small GTPase, belonging to the Ras super family.
RhoA and its downstream effectors are instrumental in regu-
lating cytoskeleton organization, gene expression, cell cycle
progression, cell motility, and other cellular processes.1–3 RhoA
GTPases shuttle between an inactive GDP-bound and an active
GTP-bound form, which allows it to carry out its cellular func-
tions through its downstream effectors mDIA and ROCKs.4–7
Hyper-expressed or activated RhoA is involved in cancer
progression of several human tumours.8–10 Particularly, RhoA
deciency suppresses the invasive type of breast cancer cells,11,12
whereas elevated RhoA activity can induce breast cancer cell
migration, invasion and metastasis.13 RhoA has been suggested
to serve as a potential therapeutic target of breast cancer.
The inactive form of RhoA can turn to the active form of
RhoA in response to a variety of upstream signals, such as
growth factors, cytokines, adhesion molecules and cell intrinsic
oncogenic cues. Within the RhoA signaling module, two strat-
egies of intervention have been proposed for effective suppres-
sion of RhoA activities. One strategy is targeting GTP binding
sites to compete with GTP leading to RhoA inactivation,14 while
aState Key Laboratory of Natural Medicines and Jiangsu Key Laboratory of Drug Design
and Optimization, China Pharmaceutical University, Nanjing, 210009, China. E-mail:
youqd@163.com; xuxiao_li@126.com
bDepartment of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical
University, Nanjing, 210009, China
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c6ra11398b
Fig. 1 Representative structures of known inhibitors of RhoA.
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mechanism of this structure of small molecular compounds 0.01% IGEPAL, 1 mM freshly DTT, pH ¼ 7.5). Compounds were
targeting RhoA. It is useful for studying the physiological added in the FP buffer at the indicated concentrations. For
function of the RhoA subfamily GTPases and for determining control wells, equal DMSO from test compounds was added into
the therapeutic potential of Rho targeting in pathological the wells. Aer incubation at RT for 1 hour, mP values were
conditions.
measured by the SpectraMax Paradigm Microplate Reader
DDO-5701 is an approved drug called proglumide, which (Molecular Devices).
initially has only been used as a drug to reduce gastric motility
2.2.3 Thermal shi assay. The thermal shi assay was
and pancreatic secretion in patients. Proglumide acts as an performed according to the instruction of the Thermal shi dye
antagonist on the cholecystokinin receptor blocking pancreatic kit (ThermoFisher, Cat no. 4461146). Briey, 2.6 mL of GST-
amylase secretion and gall bladder contraction initiated by tagged RhoA protein and 2.5 mL of dye were rst dispensed
gastrin.17 In addition, proglumide has been found to have anti- into each well of a 96-well microplate. Then 0.5 mL of the test
tumor properties in vitro and in vivo in the past few decades.18–22 compound suspended in 14.5 mL of buffer containing 10 mM
Proglumide was regarded as a gastrin receptor blocking agent, MgCl2 were dispensed into each well to generate test ligands at
which could decrease gastrin-derived cell proliferation of 50 mM in an assay volume of 20 mL. For control wells, equal
gastrointestinal tumors. However, our ndings demonstrated DMSO from test compounds was added into the wells. The
that proglumide binding to RhoA, which leads to RhoA inac- resulting amount of protein utilized is usually 1.5 mM. The plate
tivity, may be the main cause of the exhibited anti-tumor activity was subsequently heated from 25 ꢁC to 99 ꢁC in the StepO-
of proglumide.
ne™plus Real-Time PCR System (Applied Biosystems). Fluo-
rescent intensity was recorded in this process. Protein Thermal
Shi™ Soware v1.0 (Applied Biosystems) was utilized to
analyze recording data from StepOne plus system.
2. Methodology
2.1. Computation
2.2.4 p50RhoGAP assay. Inorganic phosphate produced as
a result of GTPase activity was measured by using a p50RhoGAP
assay (Cytoskeleton, Cat no. BK105) and absorbance-based
detection method. Briey, (His)6-tagged RhoA was preloaded
with GTP or DDO-5701 and incubated in the reaction buffer
(provided in the kit) for 20 min at 37 ꢁC. p50GAP was then added
for an additional 20 min at 37 C. Following a 10 min incuba-
tion in CytoPhos reagent (Cytoskeleton), inorganic phosphate
was detected at 650 nm.
2.2.5 Isothermal titration calorimetry (ITC). An ITC200
calorimeter (Malvern) was used to carry out the ITC experiment.
300 mL of GST-tagged RhoA protein was inserted into the sample
cell at a concentration of 30 mM in a buffer containing 20 mM
Tris, pH 7.4, 150 mM NaCl and 10 mM MgCl2. The syringe was
lled with test compound in the same buffer condition. Two
microliter aliquots of a 300 mM solution were titrated into the
sample cell at 25 ꢁC. 180 s intervals and a stirring speed of 1000
rpm were used for the whole procedure of the injection. In
addition, the rst 0.5 mL of the ligand solution was titrated to
2.1.1 Compound database preparation. A subset of the
MayBridge and US drug collection database of commercially
available compounds was used, and the bank of 63 000
compounds was prepared using Prepare Ligands module and
Minimize Ligands module in Discovery Studio 4.0 (DS 4.0).
Multiple conformations of each compound were generated
through the Diverse Conformation Generation protocol with an
energy threshold of 20 kcal molꢀ1 and a maximum of 255
conformations.
ꢁ
2.1.2 Virtual screening. Docking is
a
conventional
structure-based virtual screening method. The molecular
docking studies were performed using two different programs:
LigandFit/DS 4.0 and Genetic Optimization for Ligand Docking
(GOLD 5.1, Cambridge Crystallographic Data Center). In the
present study, a crystal (PDB ID: 1KMQ) was employed for
˚
docking studies. Residues around GNP (radius 8 A) were
dened as the active site. All other parameters were set as
default. The docking was terminated when the top ten solutions
attained root-mean-square deviation (RMSD) values within 1.5
prevent initial interference. All the data obtained from the
1
4
experiment including the association constant (Ka
1/Kd),
˚
A. A similarity search was undertaken using the program
enthalpy value (DH) and entropy value (DS) were analyzed by the
Origin soware package.
Diverse Subset (MOE 2014.09).
2.2.6 Endogenous Rho GTPase activity assay. MCF7 cells
were grown in log phase in a 10 cm dish, which were starved in
2.2. Biology
2.2.1 Recombinant protein production. Recombinant serum-free medium in the presence or absence of DDO-5701
human RhoA (residues 1–193) was expressed in the E. coli DH5a and DDO-5716 at indicated concentrations for 24 hours and
strain as GST fusions by using the pGEX-2T vector (addgene, Cat were subsequently stimulated with LPA for 10 min at a 50 ng
no. 12202). The N-terminal tagged GST fusion protein was mLꢀ1 concentration. MCF7 cells were lysed in lysis buffer con-
puried by glutathione–agarose affinity chromatography.
taining 50 mM Tris, pH ¼ 7.4, 10 mM MgCl2, 0.5 M NaCl, 2%
2.2.2 Fluorescence polarization (FP). We consulted a pub- IGEPAL. Lysates were claried and the protein concentrations
lished paper and made some changes to contribute to our FP were normalized. The GTP bound RhoA in the lysates was pull-
method.23 A total of 1 mM GST-tagged RhoA protein (vector was down by rhotekin RBD beads (Cytoskeleton, Cat no. RT02) and
obtained from addgene, Cat no. 12202) was incubated with a 40 was measured by a respective anti-RhoA (Cytoskeleton, Cat no.
nM BODIPY FL-tagged GTPgS uorescence probe in FP buffer ARH04) western blot. RhoA downstream protein phosphor-
(20 mM Tris, 150 mM NaCl, 10 mM MgCl2, 10% glycerine, MLC/MLC in the lysates was measured by an anti-p-MLC and
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anti-MLC (Cell Signaling, Cat no. 3672, 3674, respectively) with respect to scaffold and chemical class, a diversity analysis
western blot. was undertaken using the program Diverse Subset (MOE
2.2.7 Immunouorescence. Aer overnight serum starva- 2014.09) and subsequently followed with visual inspection to
tion in the presence or absence of DDO-5701 and DDO-5716 at avoid the potential of PAINS. Finally, 34 molecules were
the indicated concentration, MCF7 cells were treated with LPA selected.
at 50 ng mLꢀ1 for 10 min. The cells were washed twice with cold
PBS and xed for 30 min with 4% (w/v) formaldehyde at RT
temperature. Then the xed cells were washed three times with
PBS containing 0.1% Triton X-100, followed by washing three
times with PBS. The xed cells were blocked with 5% (w/v) BSA,
and stained with an appropriate primary antibody (anti-
vinculin, 1 : 200 Abcam, CA, Cat no. ab73412; anti-Ki67, 1 : 50,
Abcam, CA, Cat no. ab16667), followed by Fluor488-conjugated
secondary antibody (Invitrogen, CA, Cat no. A21202). For the
stress ber formation assay, the cells were stained with rhoda-
mine phalloidin for 30 min (Cytoskeleton, Cat no. PHDR1). The
coverslips were mounted in Vectashield mounting medium
with DAPI. The uorescence images were obtained with an
Olympus uorescence microscope.
3.2. Biological assay of the hit molecules
To characterize the inhibition of RhoA by the 34 molecules,
a uorescence polarization (FP) competitive binding assay was
used to measure the binding affinity to RhoA (represented as
IC50 values). We developed our FP methods based on a reported
method with some changes.23 In our FP experiments, we only
used recombinant RhoA protein instead of the RhoA protein
and LARG protein combination reported in that paper, in order
to evaluate the molecules’ affinities for competing with GTP.
Among the 34 molecules, we found compound 34 (DDO-5701)
had the highest affinity to RhoA (IC50 ¼ 0.699 ꢂ 0.12 mM) and we
chose this compound as a hit to further test its affinity to RhoA
(Fig. 3).
2.2.8 Cell proliferation assay. MCF7 cells and MDA-MB-231
cells were seeded in 96-well plates at an initial density of 5000
cells per well and DDO-5701 or DDO-5716 treatment was per-
formed on the second day. On the following days, 10 mL of
thiazolyl blue (MTT) was added to each well and incubated at 37
ꢁC for 4 h. Then the medium was removed carefully and 150 mL
of DMSO was added followed by gentle shaking. The optical
density of the released color was read at 570 nm.
The thermal shi assay (TSA) operates on the principle that
ligand binding alters the thermal stability of proteins.24,25 TSA
provides a measurement of a temperature shi and its setup
allows one to monitor protein denaturation upon heating via
uorescent-based detection. A uorescent dye based probe is
used that preferentially binds the hydrophobic regions of
a protein, which are increasingly exposed during protein
denaturation. When coupled to a real-time PCR setup, moni-
toring the change in uorescence provides a thermal melting
curve or thermogram. The mid-point of the melting curve, i.e.,
the temperature at which 50% of the protein has denatured, is
designated as the melting temperature, Tm, and is a measure of
the protein’s inherent thermal stability.26 A ligand bound to
a protein, e.g. to its active site, has the propensity to increase its
thermal stability (and hence its Tm) through newly formed
ligand–protein interactions. In this manner, a melting curve is
generated, the Tm determined, and the changes in Tm (DTm)
induced by prospective binding ligands can be calculated. DDO-
5701 exhibited DTm values ranging from 3.68–4.08 ꢁC at a 25 mM
concentration, which demonstrated that DDO-5701 had an
affinity to RhoA (Fig. 4A).
3. Results and discussions
3.1. Database screening for potential RhoA inhibitors
To identify novel RhoA inhibitors, we built a simple screening
protocol to carry out the virtual screening (Fig. 2). The May-
Bridge and US drug collection database containing 63 000
compounds were rst searched using the “LigandFit” protocol
in DS. 6300 hits (10% of the ranked compounds) were obtained
and further screened using GOLD docking. Aer the ltering,
we received 315 hits (5% of the ranked compounds) for the next
studies. Because this number included a large degree of similar
compounds and PAINS (pan-assay interference compounds),
RhoA has a weak GTPase activity and is hard to detect, while
p50RhoGAP can increase the GTPase activity of RhoA.27 We
had utilized a commercial kit to detect RhoA GTPase activity
through a simple absorbance based detection method. We
found that increased concentrations of DDO-5701 ranging
from 25–100 mM could compete with preloaded GTP on RhoA
and reduces GTP hydrolysis by puried recombinant RhoA
protein in vitro (Fig. 4B). Taken together, DDO-5701 had an
affinity binding to RhoA and was a potential hit for RhoA
inhibitors.
3.3. Design of new derivatives based on hit compound DDO-
5701 and precursor evaluation
In order to optimize the structure of DDO-5701, the docked pose
of the identied hit was analysed within the RhoA binding
pocket. The general molecular orientation and the spatial
Fig. 2 The screening protocol that was applied to RhoA inhibitors.
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Fig. 3 Nine ligands were found by VS from the database.
location of the chemical features of DDO-5701 are shown in that the carboxyl group of DDO-5701 could bind rmly to the
Fig. 5C and D. The binding mode of the co-crystal GNP of RhoA Thr19 and Gly17 residues by hydrogen bonding interactions,
is shown in Fig. 5A and B, which is featured by the triphosphate which was similar to GNP in complex with RhoA. In addition,
group forming complex hydrogen bonds with the backbone of the carboxyl group could also formed a strong salt bridge with
the surrounding residues as well as a salt bridge with Lys18 for Lys18. Moreover, the propanediamine group could occupy the
further stabilizing the binding pose of GNP.25 The general guanine site formed by Pyr34, Thr19 and Thr37. And the
molecular orientation and the spatial location of the chemical carboxyl attached to the propanediamine could form another
features of DDO-5701 were similar to that of GNP. It was found hydrogen bond with Thr19.
Fig. 4 (A) DDO-5701 has affinity to RhoA detected by TSA. DDO-5701 has exhibited DTm values ranging from 3.68–4.08 ꢁC at a 25 mM
concentration. (B) DDO-5701 reduced RhoA GTPase activity demonstrated by the p50RhoGAP assay.
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Fig. 5 The binding site of GNP (A and B) and DDO-5701 (C and D) with RhoA. The binding pattern was generated from the cocrystal structure
(PDB code: 1KMQ) depicted using MOE 2014.09. The carbon atoms of GNP and DDO-5701 and the key residues in the active site of RhoA were
colored in yellow and purple, respectively.
On the basis of the binding mode of the hit compound DDO-
5701 with RhoA, we designed two series of derivatives of DDO-
Table 1 Inhibition of RhoA by compounds DDO-5701–DDO-5718
5701 to further study the detailed structure activity relationship
(SAR) (DDO-5702–DDO-5718) and several more potent inhibi-
tors were identied (DDO-5713–DDO-5716). We used the FP
competitive binding assay to measure the ability of these
derivatives for the binding affinity to RhoA. As shown in Table 1,
compound DDO-5702–DDO-5709 and DDO-5718 displayed
lower activity, indicating the carboxy group was necessary for
inhibitors of RhoA. The phenomenon could be attributed to the
H-bond and salt bridge disappearance between carboxy and
RhoA. Notably, compound DDO-5716 was the most potent
inhibitor with an IC50 of 0.318 ꢂ 0.11 mM. DDO-5716 could be
a promising RhoA inhibitor for further studies.
Cpd
R1
R2
IC50 (mM)
DDO-5702
DDO-5703
DDO-5704
DDO-5705
DDO-5706
DDO-5707
DDO-5708
DDO-5709
DDO-5710
DDO-5711
DDO-5712
DDO-5713
DDO-5714
DDO-5715
DDO-5716
DDO-5717
DDO-5718
DDO-5701
OH
Et2N
EtNH
Me2N
Piperidin-1-yl
p-Br–PhNH
p-Cl–PhNH
p-MeO–PhNH
Et2N
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OH
OH
OH
OH
OH
80.71 ꢂ 1.31
85.22 ꢂ 1.41
123.1 ꢂ 1.36
131.3 ꢂ 0.82
35.22 ꢂ 1.18
23.95 ꢂ 1.26
24.45 ꢂ 1.31
21.49 ꢂ 0.99
0.904 ꢂ 0.11
1.373 ꢂ 0.21
1.794 ꢂ 0.35
0.561 ꢂ 0.33
0.339 ꢂ 0.21
0.414 ꢂ 0.12
0.318 ꢂ 0.11
1.273 ꢂ 0.24
208.8 ꢂ 2.14
0.712 ꢂ 0.07
3.4. Chemistry
The synthesis routes used to prepare compounds DDO-5702–
DDO-5718 are shown in Scheme 1. Compound DDO-5702.
starting from commercially available glutamic acid-5-methyl
ester (1) and benzoyl chloride (2), provided the product
in 85.1% yield. Then, the target compounds DDO-5703–DDO-
5709 were obtained through the coupling reaction of acid
DDO-5702 with different amines, carried out using 1-(3-
EtNH
Me2N
Piperidin-1-yl
p-Br–PhNH
p-Cl–PhNH
p-MeO–PhNH
OH
OH
OH
OH
Et2N
OH
dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride
(EDCI)/1-hydroxybenzotriazole (HOBt) in CH2Cl2 (Scheme 1).
Hydrolysis of compound DDO-5702 and DDO-5703–DDO-5709
using sodium hydroxide provided the product DDO-5717 and
Et2N
(n-Pr)2N
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Scheme 1 Synthesis of compounds DDO-5702–DDO-5718. Reagents and conditions: (a) (C2H5)2O/H2O, NaHCO3, 0 ꢁC, 12 h. (b) EDCI/HOBt,
CH2Cl2, rt, 12 h. (c) NaOH, EtOH, 50 ꢁC, 2 h.
DDO-5710–DDO-5716 in 56.0–83.8% yield. Compound DDO- although both of these two compounds exhibited a reversible
5718 was obtained through the coupling reaction of acid DDO- 1 : 1 binding stoichiometry, which demonstrated that one
5717 with diethylamine.
compound molecule bound to one molecule of RhoA protein
once. However, as shown in Fig. 6, DDO-5716 exhibited a larger
energy release than DDO-5701 in the whole process of injection,
when these two compounds bound to recombinant RhoA
protein, indicating that DDO-5716 has a better affinity to RhoA
(Kd ¼ 4.7 mM) than DDO-5701 (Kd ¼ 10.9 mM). These data,
together with the IC50 of these two compounds binding to RhoA
protein as measured by FP, strongly suggested that DDO-5716
3.5. The exploration of the biological mechanism of
compound DDO-5716
To further investigate the binding affinity of DDO-5716 to RhoA,
ITC was applied to evaluate the thermodynamic properties of
the binding interaction. According to the curve-tting analysis,
Fig. 6 DDO-5716 has a better affinity to RhoA than DDO-5701 measured by ITC.
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Fig. 7 (A) DDO-5716 treatment affects signaling downstream of RhoA. Western blots are of p-MLC/MLC and relevant controls of MCF7 cells
treated with DDO-5716 or DDO-5701 at indicated concentrations (B) DDO-5716 can reduce RhoA-GTP formation. Blotting of the respective
total-cell lysates was carried out in parallel. Results shown are representative of three independent experiments. (C) Effect of DDO-5716 on cell
stress fiber and focal complex assembly. The cells were stained with rhodamine-phalloidin for F-actin (red) and anti-vinculin for focal adhesion
complexes (green). Images shown are representative of more than 100 cells examined. DDO-5716 can reduce stress fiber and focal complex
formation.
which we derived from DDO-5701 exhibited a better affinity positive control) in different concentrations, followed by stim-
than DDO-5701 to RhoA protein in vitro.
ulation with LPA. As shown in Fig. 7B, DDO-5716 strongly
Elevated RhoA activities have been associated with cancer inhibited RhoA-GTP formation in a dose-dependent manner,
cell hyperproliferative and invasive behaviors.28 Next, we indicating endogenous RhoA activity inhibition at MCF7 cells.
investigated whether DDO-5716 was effective in suppressing To evaluate the ability of DDO-5716 to inhibit RhoA-mediated
RhoA activity in MCF7 cells. MCF7 cells grown in serum-free cell functions, we next examined actin cytoskeleton structures
media were treated with DDO-5716 or DDO-5701 (as the of cells stimulated by serum or LPA in the absence or presence
Fig. 8 DDO-5716 inhibited breast cancer cell proliferation. (A) Cell proliferation was measured by Ki67 immunostaining. Cells were stained with
anti-Ki67 antibodies to detect cell proliferation ability (green), and with DAPI, to detect nuclei (blue). n ¼ 5. (B) The IC50 values of DDO-5701 and
DDO-5716’s inhibition of cell growth were measured by using the thiazolyl blue assay at MCF7 and MDA-MB-231 cell lines. n ¼ 10, bar: SD.
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of DDO-5716. Fig. 7C shows that in the presence of 25 mM DDO- RhoA that is useful in the study of the physiological role of RhoA
5716, stress ber and formation of the MCF7 cells were signif- and for tackling RhoA-mediated pathologies.
icantly reduced, and at the 50 mM concentration, both the stress
ber and focal complex formation of the MCF7 cells were
signicantly reduced, while at the 50 mM concentration of DDO-
5701, we only observed stress ber formation reducing. More-
Acknowledgements
over, Fig. 7A shows DDO-5716 dose-dependently reduced p-MLC This work is supported by the project 81230078, 81502990 and
activities, while p-MLC indicated actin–myosin contraction.29,30 81573346 of National Natural Science Foundation of China;
Given the implicated role of Rho activity in actin cytoskeleton 2013ZX09402102-001-005 and 2014ZX09507002-005-015 of the
organization and adhesion,31 these results indicate that DDO- National Major Science and Technology Project of China
5716 is active in inhibiting RhoA-mediated cellular events.
(Innovation and Development of New Drugs); Specialized
To assess the cellular effect of DDO-5716, we performed a cell Research Fund for the Doctoral Program of Higher Education
proliferation assay using the MTT assay and Ki67 staining (SRFDP, 20130096110002); A Project Funded by the Priority
immunouorescence. As shown in Fig. 8B, DDO-5716 showed Academic Program Development of Jiangsu Higher Education
more effective inhibition of growth of MCF7 and MDA-MB-231 Institutions; A project BK20150691 of Natural Science Founda-
(IC50 ¼ 5.24 ꢂ 0.66 mM, IC50 ¼ 4.37 ꢂ 0.39 mM, respectively) tion of Jiangsu Province; 2016ZPY016 of the Fundamental
than DDO-5701 (IC50 ¼ 10.01 ꢂ 1.02 mM, IC50 ¼ 8.75 ꢂ 0.96 mM, Research Funds for the Central Universities. This work is
respectively). In the presence of 5 mM DDO-5716, Ki67 staining supported by the Project Program of State Key Laboratory of
was signicantly reduced, which indicated reduced cell prolif- Natural Medicines, China Pharmaceutical University (No.
eration, while Ki67 staining was reduced at a 10 mM concen- SKLNMZZCX201611).
tration of DDO-5701 (Fig. 8A). These results indicated that DDO-
5716 was effective in targeting RhoA mediated breast cancer cell
proliferation. The underlying mechanism as to why proglumide
shows anti-cancer properties remains poorly understood. Pro-
References
glumide was initially used as an anti-tumor agent through its
blocking of the cholecystokinin A/B receptor (CCKa/b) or gastrin
receptor. Our ndings indicated that proglumide exhibited
anti-tumor activity in breast cancer cells, which did not show
CCKa/b and gastrin receptor expression. The effect of cell
proliferation inhibition of DDO-5701 (proglumide) may be the
result of its interaction with other receptor targets, but taken
together with our results, proglumide might reduce breast
cancer cell proliferation largely due to its binding to RhoA and
leading to RhoA-GTP formation decreasing.
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4
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