Rhodanine-based PRL-3 inhibitors blocked the migration and invasion of metastatic cancer cells

Article abstract of DOI:10.1016/j.bmcl.2013.04.092

PRL-3, phosphatase of regenerating liver-3, plays a role in cancer progression through its involvement in invasion, migration, metastasis, and angiogenesis. We synthesized rhodanine derivatives, CG-707 and BR-1, which inhibited PRL-3 enzymatic activity wi

Full text of DOI:10.1016/j.bmcl.2013.04.092

Bioorganic & Medicinal Chemistry Letters 23 (2013) 3769–3774
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Rhodanine-based PRL-3 inhibitors blocked the migration
and invasion of metastatic cancer cells
Garam Min a, Su-Kyung Lee a, Hye-Nan Kim a, Young-Min Han a, Rhan-Hee Lee a, Dae Gwin Jeong b,
Dong Cho Han a, , Byoung-Mog Kwon a,
⇑
⇑
a Laboratory of Chemical Genomics and Biology, Korea Research Institute of Bioscience and Biotechnology, University of Science and Technology, 125 Gwahakro, Yoosunggu, Daejeon
305-600, Republic of Korea
b Proteomics Research Center, Korea Research Institute of Bioscience and Biotechnology, University of Science and Technology, 125 Gwahakro, Yoosunggu, Daejeon 305-600,
Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
PRL-3, phosphatase of regenerating liver-3, plays a role in cancer progression through its involvement in
invasion, migration, metastasis, and angiogenesis. We synthesized rhodanine derivatives, CG-707 and BR-
1, which inhibited PRL-3 enzymatic activity with IC50 values of 0.8 lM and 1.1 lM, respectively. CG-707
and BR-1 strongly inhibited the migration and invasion of PRL-3 overexpressing colon cancer cells with-
out exhibiting cytotoxicity. The specificity of the inhibitors on PRL-3 phosphatase activity was confirmed
by the phosphorylation recovery of known PRL-3 substrates such as ezrin and cytokeratin 8. The com-
pounds selectively inhibited PRL-3 in comparison with other phosphatases, and CG-707 regulated epithe-
lial-to-mesenchymal transition (EMT) marker proteins. The results of the present study reveal that
rhodanine is a specific PRL-3 inhibitor and a good lead molecule for obtaining a selective PRL-3 inhibitor.
Ó 2013 Elsevier Ltd. All rights reserved.
Received 23 March 2013
Revised 21 April 2013
Accepted 30 April 2013
Available online 9 May 2013
Keywords:
PRL-3
Migration
Invasion
Metastasis
Rhodanine
Tumor progression is a complex process that includes malig-
nant transformation, proliferation, invasion, and metastasis of can-
cer cells. Cancer cell invasion and metastasis are critical processes,
and more than 90% of cancer deaths have been attributed to the
metastatic spread of the disease rather than to the primary tu-
mor.1–3 Decades of intensive research have been focused on the
search for therapeutic solutions targeting cancer cell migration,
invasion, and angiogenesis.4–6 From a clinical standpoint, the only
major advance in the past 20 years has been the development of
bone metastasis-targeting bisphosphonates and RANK-ligand
inhibitors.
PRL-3, phosphatase of regenerating liver-3, belongs to the pro-
tein tyrosine phosphatase (PTP) family which regulates phosphor-
ylation and dephosphorylation of many biologically significant
molecules. The malfunction of PTP family members leads to vari-
ous diseases including cancer, neurological disorder, and diabetes.
Members of the PRL family, consisting of PRL-1, PRL-2, and PRL-3,
are closely related and share the specific sequence CX5R which is
the PTP active site. PRL-3 is known to affect the cell cycle, cell pro-
liferation, and differentiation, and it plays a role in cancer progres-
sion through its involvement in cancer cell invasion, migration,
metastasis, and angiogenesis.7,8 A relationship between PRL-3
and metastatic behaviors has been investigated since PRL-3 was
shown to be expressed in 100% of metastatic colorectal tumor sam-
ples by Saha et al., in 2001.9 PRL-3 expression was high in other
metastatic tumors, but was low in non-metastatic carcinomas
and normal epithelial cells. Increasing evidences from recent stud-
ies suggests that PRL-3 is an important regulator of cancer metas-
tasis. Researches on PRL-3 have also tended to focus on metastasis-
associated characteristics such as its stimulating effect on cell
migration and invasion.10 Recent studies showed that PRL-3 knock-
down inhibited metastasis developments by decreasing the pri-
mary tumor size and also inhibited the invasion and growth of
cancer cells.11,12 Therefore, PRL-3 appears to be a diagnostic mar-
ker and a promising therapy target in cancer, especially colorectal
cancer.
Rhodanine- and 1,3-thiazolidine-2,4-dione-based compounds
have become a very important group of heterocyclic compounds
in drug development, ever since the introduction of various glitaz-
ones into clinical use for the treatment of type II diabetes mellitus.
Rhodanines have been reported to possess antibacterial, anti-
fungal, antiviral, antimalarial, insecticidal, herbicidal, antitumor,
anti-inflammatory and cardiotonic activities.13,14 These rhodanine
derivatives have been reported to inhibit cancer cell migration
and the enzymatic activity of PRL-3.15 In an effort to design and
⇑
Corresponding authors. Address: Korea Research Institute of Bioscience and
Biotechnology, 52 Uendong, Yoosung, Daejeon 305-806, Republic of Korea. Tel.: +82
42 860 4557; fax: +82 42 861 2675 (B.-M.K.); tel.: +82 42 860 4568; fax: +82 42 861
2675 (D.C.H.).
E-mail addresses: kwonbm@kribb.re.kr (D.C. Han), dchan@kribb.re.kr (B.-M.
Kwon).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.04.092

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G. Min et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3769–3774
Table 1
synthesize new rhodanine compounds as potential PRL-3 inhibi-
tors, we have found that cinnamaldehyde-based rhodanines dra-
matically inhibit PRL-3 activity. A subsequent cell-based assay
revealed that this structural modification allowed the compounds
to inhibit human cancer cell migration and invasion. One such rho-
danine derivative (CG-707) showed a strong ability to inhibit PRL-3
activity and cancer cell migration but did not exhibit apparent cel-
lular toxicity in cell proliferation assays. This finding prompted us
to design a series of rhodanine derivatives by varying the substitu-
tion groups on the cinnamaldehyde. We report here the prepara-
tion and biological activity evaluation of these new derivatives
and a known PRL-3 inhibitor BR-1.
As shown in Figure 1, the designed derivatives were synthesized
following the general procedures as detailed below. The condensa-
tion of rhodanine or thiazolidinedione with 2-substituted cinn-
amaldehydes by ammonium acetate and acetic acid afforded
quantitative yield of rhodanines in refluxing toluene. After filtra-
tion, the product was recrystallized from acetone and hexane.
To determine the effects of the synthesized rhodanine deriva-
tives on PRL-3 activity, we performed an in vitro DiFMUP assay
with purified PRL-3 protein.16,17 DiFMUP is a substrate for alkaline
and acid phosphatase and its dephosphorylation yields fluores-
cence. Purified PRL-3 protein solution was added to the substrate
and absorbance was read to measure the enzyme activity. As
shown in Table 1, most of the synthetic derivatives displayed mod-
erate or potent inhibitory activity against PRL-3, and 8 derivatives
Biological activities of rhodanine derivatives
Compound
PRl-3 inhibitiona
Migration inhibitionb
Cell viabilityc
BR-1
65.2
51.9
30.6
49.5
52.8
63.1
40.8
78.8
52.9
40.3
64.2
35.2
53.9
37.4
41.7
39.4
47.1
22.5
3.8
95
65
40
65
73
78
80
98
82
71
80
68
80
81
69
62
45
50
45
98.1
96.3
99.2
87.9
75.5
78.8
41.5
99.5
87.1
72.3
83.1
97.1
81.9
96.6
98.8
96.5
105.4
37.7
37.2
CG-701
CG-702
CG-703
CG-704
CG-705
CG-706
CG-707
CG-708
CG-709
CG-710
CG-711
CG-712
CG-713
CG-714
CG-715
CG-716
CG-717
CG-718
a
b
c
Inhibitory activity against PRL-3 at 5
Inhibition of DLD-1(PRL-3) cell migration at 10
Cell viability of DLD-1(PRL-3) cells at 10 g/ml (%).
lg/ml (%).
lg/ml (%).
l
inhibit PRL-3, because PRL-1, PRL-2, and PRL-3 share 76–87% ami-
no acid sequence identity.18
showed greater than 50% inhibition at a concentration of 5
l
g/ml,
To determine the effects of the synthesized rhodanine deriva-
tives on cancer cell migration, we prepared PRL-3 overexpressing
DLD-1 colon tumor cells [DLD-1 (PRL-3)] because PRL-3 induces
the invasion of human tumor cells.19 We performed a transwell
migration assay on each compound using the DLD-1 (PRL-3) cells.
The cells were seeded at a density of 5 Â 104 in serum-free med-
ium in the upper chamber, with the lower chamber containing
5% FBS medium, and their ability to migrate in the presence and
absence of the compounds was measured by counting the total
except the thiazolidinedione derivative (CG-718). Both CG-707 and
BR-1 dose-dependently inhibited the dephosphorylation of DiF-
MUP by PRL-3 as shown in Figure 2. CG-707 and BR-1 strongly
inhibited PRL-3 phosphatase activity in a dose-dependent manner
with IC50 values of 0.8 and 1.1 lM, respectively (Fig. 2). These data
demonstrate that rhodanines are good lead molecules for PRL-3
inhibitors. Although we did not test the inhibitory activity of the
compounds against PRL-1 and 2, rhodanines may not selectively
O
O
A
Rhodanine or thiazolidinedione
NH4OAc,AcOH,Toluene,Reflux
H
NH
S
R
R
X
X =S or O
B
O
O
O
O
O
O
NH
S
NH
S
NH
S
NH
S
NH
S
S
S
S
O
NH
S
S
O
S
O
CH3
O
Br
O
CF3
S
O
OH
CG-701
CG-702
CG-703
CG-704
CG-705
CG-706
O
O
O
O
O
Br
NH
S
NH
O
NH
S
NH
S
NH
S
S
S
S
S
O
O
S
O
Br
S
OCH3
O
OCH3
NH
NH
S
O2S
S
O
Cl
CG-707
CG-708
CG-709
CG-710
CG-711
CG-712
O
O
O
O
NH
S
O
Br
O
NH
S
S
NH
O
NH
S
S
S
NH
S
O
NH
S
O
Br
S
O
S
S
OCH3
N
N
Br
CG-713
CG-714
CG-715
CG-716
CG-717
BR-1
Figure 1. (A) Synthetic scheme. (B) Structures of the rhodanine derivatives.

G. Min et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3769–3774
3771
90
80
70
60
50
40
30
pounds with an amide (CG-11) or a pyridine ring (CG-15 and 16)
only moderately inhibited PRL-3 activity and DLD-1 (PRL-3) cell
migration.
CG-707
The recovery of phosphorylation was examined in an attempt to
confirm the inhibition of PRL-3 phosphatase activity by CG-707
and BR-1. Two PRL-3 substrates were used: ezrin and cytokeratin
8. Ezrin, a direct target of PRL-3, interacts with radixin and moesin,
constituting the ERM complex, which connects the actin cytoskel-
eton with cell surface receptor. Phosphorylation at amino acid
Thr567 activates ezrin and controls the structural transitions of
this protein, which plays a pivotal role in tumor progression and
metastasis, therefore, we detected the residue phosphorylation of
the Thr567 residue.20,21 Cytokeratin 8 (KRT8) is reported to colo-
calize and interact with PRL-3 at lamellipodias, participating in cel-
BR-1
0.1
0.5
1
2
5
10
lular movement.22 Treatment with 20
lM CG-707 or BR-1 in DLD-1
Concentration (μM)
(PRL-3) cells increased the level of phospho-ezrin more than three-
fold when compared with DMSO-treated cells. CG-707 and BR-1
also demonstrated the ability to rescue phospho-cytokeratin 8
which is a PRL-3 mediated effector protein (Fig. 4). These data sug-
gests that CG-707 and BR-1 specifically inhibited the phosphatase
activity of PRL-3.
Figure 2. Inhibition of PRL-3 phosphatase activity by CG-707 and BR-1 was
observed using a DiFMUP assay.
number of cells in the lower chamber after 20 h. As shown in Ta-
ble 1, most of the synthetic derivatives strongly inhibited the
The expression of PRL-3 has been shown to increase cell migra-
tory and invasive ability. We reported that DLD-1 colon cancer
cells overexpressing PRL-3 exhibited increased migration and inva-
sion rates when compared with DLD-1 cells.22 To observe the rela-
tionship between PRL-3 expression level and the anti-migratory
activity of CG-707 and BR-1, the two compounds were treated to
cell lines other than DLD-1 (PRL-3). The numbers of cells seeded
were varied depending on the size and mobility of each cell line.
The migration assay was stopped when the cells in a control well
had moved and penetrated the membrane to approximately 70%
confluence. As shown in Figure 5A, CG-707 and BR-1 inhibited
the migration of DLD-1, A375P, MIA PaCa-2, and AsPC-1 cells
which represent a colon cancer cell line, a melanoma cell line,
and 2 pancreatic cancer cell lines, respectively.17 However, the
compounds did not affect the migration of DU 145 or MDA-MB-
231, which have relatively low level of PRL-3 expression and are
derived from prostate cancer and breast cancer, respectively. CG-
707 and BR-1 strongly blocked the migration of A375P and MIA
PaCa-2 cells which have higher endogenous PRL-3 expression lev-
els than the cell lines such as DU 145 and MDA-MB-231, that were
not affected by the compounds. These results showed that the anti-
migratory effects of the compounds correlated with the PRL-3
expression level of each cell lines (Fig. 5B).
migration of DLD-1 (PRL-3) cells at a 10 lg/ml concentration. The
thiazolidinedione derivative (CG-18) displayed moderate anti-
migratory activity but this compound causes cell cytotoxicity.
Given the potent anti-migration efficacy demonstrated by most
of the rhodanine derivatives, we decided to study the dose-re-
sponse of the most potent derivatives to obtain their IC50 values
for suppressing the transwell migration of the DLD-1 (PRL-3) cells.
As shown in Figure 3A, CG-707 and BR-1 inhibited the migration of
DLD-1 (PRL-3) cells in a dose-dependent manner with IC50 values
of 5 and 7
lM, respectively. After the cells were treated with
20 M of the compounds, more than 90% of cell migration was
l
blocked and cell invasion was blocked by over 60% at the same
concentration (Fig. 3B).
We next performed proliferation assays on DLD-1 (PRL-3) can-
cer cells treated with the compounds to rule out any indirect ef-
fects on cell migration and invasion due to cytotoxicity. DLD-1
(PRL-3) cells were allowed to grow for 24 h in 96-well plates in
the presence or absence of the synthetic compounds at a concen-
tration of 10 lg/ml. The cell toxicity data for all derivatives are
listed in Table 1, along with the anti-migration data for compari-
son. The 14 compounds all inhibited cell migration by over 60%
(62–98%) without significant cell toxicity when the cells were trea-
ted with the derivatives at a dose of 10
l
g/ml, the exception was
Our primary screen revealed a class of rhodanine molecules as
potent PRL inhibitors. The structure and activities of selected com-
pounds, CG-707 and BR-1, are shown in Figures 1 and 2. These
CG-6, which significantly inhibited cell growth by approximately
60%. A few other anti-migratory derivatives greatly inhibited
DLD-1 (PRL-3) cell proliferation. For example, CG-17 and CG-18
blocked cell migration (approximately 50%) and also inhibited
the cell proliferation (approximately 63%). For these derivatives,
cytotoxicity may have partially contributed to their overall anti-
migratory effect. However, the potent PRL-3 inhibitors, CG-707
and BR-1, did not inhibit the proliferation of DLD-1 (PRL-3) cells,
compounds exhibited similar IC50 values, 0.8
lM for CG-707 and
1.1 M for BR-1 in PRL-3 phosphatase assays, respectively. To
l
examine the selectivity of these compounds, we further examined
the inhibitory activities of the rhodanines by performing a phos-
phatase assay with 9 other phosphatases that represent different
classes of phosphatases, including tyrosine and dual-specific phos-
phatases. As shown in Table 2, using DiFMUP as the substrate for
all of the protein phosphatase (PTPase) assays, the compounds dis-
played excellent selectivity for the PRL-3 phosphatase, but showed
minimal effects on other phosphatases.
even at concentration as high as 50 lM (Fig. 3C). These results indi-
cate that these compounds significantly inhibit cell migration and
invasion without cytotoxicity and may be good lead molecules for
designing a new PRL-3 inhibitor.
Despite of having very limited existing structure–activity rela-
tionships (SAR), the compounds with benzyl group showed more
promising activity against PRL-3 compared with the other com-
pounds, and a dramatic loss of inhibitory potency was seen when
the rhodanine group was substituted by a thiazolidinedione (CG-
18). The replacement of the oxybenzyl group with a sulfonylbenzyl
(CG-10) provided more promising starting points for the design of
a new PRL-3 inhibition, because CG-10 strongly inhibited both PRL-
3 activity and the migration of DLD-1 (PRL-3) cells. However, com-
EMT, epithelial-to-mesenchymal transition, is a critical step in
the metastatic progression and invasion of cancer cells during
which epithelial cells gain fibroblastic properties and lose cell adhe-
sive ability. A decreased level of the epithelial cell marker protein,
E-cadherin, resulted in a loss of cell-cell adhesion junctions and
was followed by an increased invasiveness of tumor cells. It has
been reported that PRL-3 is associated with EMT, where the overex-
pression of PRL-3 promotes the mesenchymal transition.23 To
observe the modulation of EMT marker proteins, such as E-cadherin

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G. Min et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3769–3774
A
Compound: CG-707
Compound: BR-1
DLD-1(PRL-3)
DLD-1(PRL-3)
1μM
5μM
7μM
1μM
5μM
7μM
10μM
15μM
20μM
10μM
15μM
20μM
B
C
200
180
160
140
120
100
80
CG-707
BR-1
DMSO (0.1%)
60
40
20
0
CG-707 10 μM
CG-707 20 μM
0
1
2
5
10 20 50 100
Concentration of compounds
(μM)
BR-1 10 μM
BR-1 20 μM
Figure 3. CG-707 and BR-1 inhibit the migration and invasion of DLD-1 cells overexpressing PRL-3. (A) The cell migration assay was performed with DLD-1 (PRL-3) cells that
were treated with various concentrations of the compounds for 20 h. Migrating cells were stained with crystal violet and were counted under a light microscope. (B) The cell
invasion assay was performed with DLD-1 (PRL-3) cells which were treated with various concentration of the compound for 20 h. Cell invasion was investigated using
Matrigel-coated 8.0 lm filter invasion chambers. Invaded cells were stained with crystal violet and were counted under a light microscope. (C) The proliferation assay with
DLD-1 (PRL-3) cells in the presence of the compound. The cells were seeded at a density of 5000 cells per well in 96-well plates in RPMI 1640 medium containing 10% FBS. The
cells were incubated with fresh complete medium containing the compounds or 0.1% DMSO. After 24 h of incubation, the cell proliferation reagent WST-1 (Roche Applied
Science) was added to each well. WST-1 formazan was quantitatively measured at 450 nm using an enzyme-linked immunosorbent assay reader (Bio-Rad). Each experiment
was repeated three times, and each value is expressed as the mean SD. Statistical significance (p value) is p < 0.01.
and Snail, by CG-707, we observed changes in the EMT marker
expression levels in DLD-1(PRL-3) cells. As shown in Figure 6, the
E-cadherin expression level was decreased and Snail expression le-
vel was increased in DLD-1 (PRL-3) cells. In a phosphatase-activity-
mutated cell line, DLD-1 (C104S), E-cadherin expression was in-
creased and Snail expression was decreased in contrast to PRL-3
overexpressing cells. When the DLD-1 (PRL-3) cells were treated
with the rhodanine compound, E-cadherin expression increased

G. Min et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3769–3774
3773
Table 2
BR-1
CG707
Selectivity assays of CG-707 and BR-1 against PRL-3 and 9 other phosphatases using
DiFMUP as a substrate
DMSO
(0.1%)
10 μM 20 μM
10 μM 20 μM
Phosphatasea
BR-1
CG-707
P-Ezrin
PRL-3
CD45
DUSP22
LMPTP-A
MKP5
PP2A
PTP-1B
SHP-2
TCPTP
VHR
64
12
30
0
5
0
11
27
9
73
11
10
6
1
29
8
18
1
3
1.00
1.98
4.36
2.76
3.60
Ezrin
7
YopH
14
9
P-KRT8
a
Inhibitory activity against phosphatases at 5 lM (%).
1.00
2.13
1.74
0.83
1.29
to block cell migration and invasion was confirmed by the changes
in the expression level of EMT markers.
β-Actin
In summary, we have designed and synthesized 18 rhodanine
derivatives, based on BR-1, to be novel anti-migration and anti-
invasion agents. Structural modification of the substitution groups
on the cinnamaldehyde group led to the identification of several
potent PRL-3 inhibitors that strongly suppressed cell migration in
metastatic cancer cells. The compounds rescued the phosphoryla-
tion of ezrin and KRT8, which are known PRL-3 substrates. More
importantly, CG-707 and BR-1 exhibited no apparent cytotoxicity
and displayed a selectivity that inhibited cancer cell migration in
proportion to the level of PRL-3 expression. These data support
the idea that the anti-migration activity of these compounds may
be a result of the inhibition of PRL-3 activity in cells. Potent PRL3
inhibitors also selectively inhibited PRL-3, when compared other
phosphatases, and modulated the expression of EMT marker
proteins. Thus, our study provides a novel type of small molecule
Figure 4. The phosphorylation rescue of ezrin and cytokeratin 8 (KRT8) after CG-
707 or BR-1 treatment in DLD-1 (PRL-3) cells. The lysates of cells that were treated
with the compound were prepared using RIPA lysis buffer (50 mM Tris, pH 7.0,
150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 1% deoxycholic acid, 0.1% SDS, 30 mM
Na2HPO4, 50 mM NaF, and 1 mM NaVO4) containing a protease inhibitor cocktail
(Roche Applied Science). The protein (45 lg) was resolved using 7.5 or 10% SDS–
PAGE and transferred to a PVDF membrane (Roche Applied Science). The membrane
was blocked with 5% nonfat dried milk in TBS-T (50 mM Tris–HCl, pH 7.6, 150 mM
NaCl, and 0.1% Tween 20) and was probed with primary antibodies (Cell Signaling
Technology) for 2–2.5 h. The blot was washed, exposed to HRP-conjugated anti-
rabbit IgG for 1.5 h, and examined using chemiluminescence POD reagents (Roche
Applied Science).
and snail expression decreased in a similar pattern to that seen in
the phosphatase-activity-mutated cell line. The ability of CG-707
BR-1 20 μM
A
BR-1 10 μM
CG707 10 μM
DMSO
CG707 20 μM
DLD-1
A375P
MIA PaCa-2
DU 145
MDA-MB-231
AsPc-1
B
A375P AsPC-1
DLD-1
DU 145
MIA PaCa-2 MDA-MB-231
PRL-3
β-Actin
Figure 5. (A) Migration assay of CG 707 and BR-1 in various cancer cell lines. (B) Expression level of PRL-3 in various tumor cells.

3774
G. Min et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3769–3774
DLD-1
(c104s)
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This work was supported by the Korea Research Institute of Bio-
science and Biotechnology Research Initiative Program and the Bio
& Medical Technology Development Program of the National Re-
search Foundation funded by the Korean government.