Identification of para-Substituted Benzoic Acid Derivatives as Potent Inhibitors of the Protein Phosphatase Slingshot

Article abstract of DOI:10.1002/cmdc.201500454

Slingshot proteins form a small group of dual-specific phosphatases that modulate cytoskeleton dynamics through dephosphorylation of cofilin and Lim kinases (LIMK). Small chemical compounds with Slingshot-inhibiting activities have therapeutic potential against cancers or infectious diseases. However, only a few Slingshot inhibitors have been investigated and reported, and their cellular activities have not been examined. In this study, we identified two rhodanine-scaffold-based para-substituted benzoic acid derivatives as competitive Slingshot inhibitors. The top compound, (Z)-4-((4-((4-oxo-2-thioxo-3-(o-tolyl)thiazolidin-5-ylidene)methyl)phenoxy)methyl)benzoic acid (D3) had an inhibition constant (K_i) of around 4 μm and displayed selectivity over a panel of other phosphatases. Moreover, compound D3 inhibited cell migration and cofilin dephosphorylation after nerve growth factor (NGF) or angiotensin II stimulation. Therefore, our newly identified Slingshot inhibitors provide a starting point for developing Slingshot-targeted therapies.

Full text of DOI:10.1002/cmdc.201500454

DOI: 10.1002/cmdc.201500454
Communications
Identification of para-Substituted Benzoic Acid Derivatives
as Potent Inhibitors of the Protein Phosphatase Slingshot
Kang-shuai Li,[a, c] Peng Xiao,[a, b] Dao-lai Zhang,[a, e] Xu-Ben Hou,[b] Lin Ge,[a] Du-xiao Yang,[a]
Hong-da Liu,[a] Dong-fang He,[a] Xu Chen,[c] Ke-rui Han,[d] Xiao-yuan Song,[f] Xiao Yu,[c]
Hao Fang,[a] and Jin-peng Sun*[a]
Slingshot proteins form a small group of dual-specific phos-
phatases that modulate cytoskeleton dynamics through de-
phosphorylation of cofilin and Lim kinases (LIMK). Small chemi-
cal compounds with Slingshot-inhibiting activities have thera-
peutic potential against cancers or infectious diseases. Howev-
er, only a few Slingshot inhibitors have been investigated and
reported, and their cellular activities have not been examined.
In this study, we identified two rhodanine-scaffold-based para-
substituted benzoic acid derivatives as competitive Slingshot
inhibitors. The top compound, (Z)-4-((4-((4-oxo-2-thioxo-3-(o-
tolyl)thiazolidin-5-ylidene)methyl)phenoxy)methyl)benzoic acid
(D3) had an inhibition constant (Ki) of around 4 mm and dis-
played selectivity over a panel of other phosphatases. More-
over, compound D3 inhibited cell migration and cofilin de-
phosphorylation after nerve growth factor (NGF) or angiotensi-
n II stimulation. Therefore, our newly identified Slingshot inhib-
itors provide a starting point for developing Slingshot-targeted
therapies.
entiation, gene expression, and cellular metabolism.[1] In cells,
protein kinases and protein phosphatases work together to
precisely control the balance of the protein phosphorylation
network to regulate different life activities. The dual-specific
phosphatase Slingshots (SSHs) form a particular group of phos-
phatases which regulates cytoskeleton rearrangement through
dephosphorylation of cofilin at its Ser-3, as well as of Lim kin-
ases (LIMKs) at Thr-508 (LIMK1) or Thr-505 (LIMK2).[2] Because
dephosphorylated cofilin mediates actin depolymerization,
which is a key step in the metastasis of cancer cells as well as
the entry of infectious Salmonella into mammalian cells, down-
regulation of Slingshot phosphatase activity by small molecule
inhibitors can be a promising therapeutic strategy for cancers
or salmonella-related diseases such as mild food poisoning or
life-threatening typhoid fever.[2a,d,3]
Until now, only one report has identified several inhibitors of
Slingshot 1 using virtual screening and homology modeling of
the phosphatase. The best inhibitor among these compounds
displays an IC50 of around 3 mm toward Slingshot 1; however,
the inhibition mode of these compounds have not been deter-
mined, and their selectivity toward other phosphatases have
not been tested. The cellular activities of these compounds
have not been determined either. Thus, there is a need to de-
velop new slingshot inhibitors with good selectivity and cellu-
lar activity to evaluate their therapeutic potential.
Reversible protein phosphorylation plays an important role in
controlling various cellular functions such as cell growth, differ-
[a] Dr. K.-s. Li,+ Dr. P. Xiao,+ Dr. D.-l. Zhang, Dr. L. Ge, Dr. D.-x. Yang,
Dr. H.-d. Liu, Dr. D.-f. He, Prof. H. Fang, [. P. J.-p. Sun
Key Laboratory Experimental Teratology of the Ministry of Education (MOE)
and Department of Biochemistry and Molecular Biology
School of Medicine, Shandong University, Jinan, Shandong 250012 (China)
E-mail: sunjinpeng@sdu.edu.cn
Recently, we have uncovered nine specific lymphoid-specific
tyrosine phosphatase (LYP) inhibitors through target–ligand in-
teraction-based virtual screening methods.[4] Based on the pre-
dicted binding modes and the structure–activity relationship
analysis of these in-silico-identified LYP inhibitors, we found
that the benzoic acid group could bind into the active site of
LYP and contribute to the inhibitory activity against it. Consid-
ering the structural conservation of the active site of the tyro-
sine phosphatase superfamily, compounds containing the ben-
zoic acid fragment might also inhibit the activity of Slingshots.
In the present study, we synthesized a series of rhodanine–
benzoic acid derivatives and screened these compounds for
potential Slingshot inhibitor leads. Additionally, several com-
mercial compounds containing benzoic acid groups were se-
lected from our previous virtual screening study and were
tested for Slingshot inhibitory activities. As results, we found
two rhodanine–benzoic acid derivatives are inhibitors of Sling-
shot 2 with inhibition constants (Ki) less than 20 mm. These in-
hibitors are competitive inhibitors and further studies revealed
that one of the inhibitors shows selectivity toward other phos-
[b] Dr. P. Xiao,+ Dr. X.-B. Hou
Department of Medicinal Chemistry
Key Laboratory of Chemical Biology of Natural Products (MOE)
School of Pharmacy, Shandong University, Jinan, Shandong 250012 (China)
[c] Dr. K.-s. Li,+ Dr. X. Chen, Prof. X. Yu
Department of Physiology, School of Medicine
Shandong University, Jinan, Shandong 250012 (China)
[d] Dr. K.-r. Han
Mailman School of Public Health
Columbia University, New York, NY 10032 (USA)
[e] Dr. D.-l. Zhang
School of Pharmacy, Binzhou Medical University
Yantai, 264003 (China)
[f] Prof. X.-y. Song
Key Laboratory of Brain Function and Disease
Chinese Academy of Sciences (CAS) and
School of Life Sciences, University of Science and Technology of China
Hefei, 230027 (China)
[+] These authors contributed equally to this work.
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201500454.
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phatase and effectively block the cell migration through inhib-
iting Slingshot activity in cells.
thiazolidin-5-ylidene)methyl)phenoxy)methyl)benzoic acid (D3)
and (Z)-4-((4-((3-(1-carboxy-2-(1H-indol-3-yl)ethyl)-4-oxo-2-thio-
We first synthesized the rhodanine-scaffold-based phospha-
tase inhibitor library. Target compounds D3, D17, D25, and
D16 were prepared according to the synthetic routes as
shown in Scheme 1. To synthesize target compounds, 4-substi-
tuted benzaldehydes were first prepared through coupling
and/or substitution reactions. Afterwards, 3-substituted rhoda-
nines were generated by a three-step reaction procedure.
Then, the aldol reaction of the two intermediates afforded
target compounds D3, D17, D25, and D16. The target com-
pound D1 was synthesized through a simple substitution reac-
tion as shown in Scheme 2.
xothiazolidin-5-ylidene)methyl)phenoxy)methyl)benzoic
acid
(D17), as seen in Figure 1A,B. Using a fixed pNPP concentra-
tion of 11 mm (the Michaelis constant, KM, of Slingshot 2), the
IC50 of D3 was determined as 8.2Æ2.6 mm and the IC50 for D17
was determined as 42Æ6.1 mm, respectively (Supplemental
Figure 1 in the Supporting Information).
To determine the inhibition modes of D3 and D17, we per-
formed Michaelis–Menten kinetic studies with recombinant
Slingshot 2 using varying inhibitor and substrate concentra-
tions. The Lineweaver–Burk plots revealed that both com-
pounds D3 and D17 are competitive inhibitors of Slingshot 2
with a calculated Ki of 3.9Æ0.3 mm and 18.9Æ0.3 mm, respec-
tively (Figure 1C,D). D3 is a reversible inhibitor and inhibits
Slingshot 2 approximately fourfold better than D17; we there-
fore selected compound D3 for further study (Figure 1C,D and
Supplemental Figure 2 in the Supporting Information).
Compounds that contained a benzoic acid group were se-
lected from our in-house compound library and screened for
inhibition of phosphatase activity of the catalytic domain of
Slingshot 2 (residues 233–490 ) (Table 1). At 50 mm, four com-
pounds decreased Slingshot 2 activity against a small artificial
substrate, para-nitrophenylphosphate (pNPP), by more than
20%, and two of them showed inhibition rates over 70%.
These two most potent compounds were para-substituted
benzoic acid derivatives: (Z)-4-((4-((4-oxo-2-thioxo-3-(o-tolyl)-
To determine the selectivity of D3 toward other phosphatas-
es, we purified a panel of other protein phosphates including
PTP-Meg2, PTP-N18 (BDP1), PRL-1, MKP3, PPM1A, PPM1G, and
PP1. The purities of these proteins were confirmed by electro-
Scheme 1. Synthesis of D3, D17, D25, and D16. Reagents and conditions: a) K2CO3, acetone, reflux, 8 h, 3: 63%, 7: 52%; b) NaHCO3, THF, 08C, 4 h, 55%;
c) 1) CS2, Et3N, EtOH, 08C, 4 h, 2) ClCH2COONa, H2O, 08C, 12 h, 3) 6m HCl, 858C, 4 h, 9a: 72%, 9b: 36%, 9c: 47%; d) NH4Ac, HAc, reflux, 0.5 h, D3: 59%, D17:
57%, D25: 68%, D16: 51%.
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ed with varying concentrations
of D3 were overlaid in the upper
chamber of a transwell assay
plate, while NGF was present in
the lower chamber as chemoat-
tractant. After 24 h, the number
of cell foci that transmigrated to
the lower chamber, past the mi-
cropores of the cell-permeable
membrane, was counted. Incu-
Scheme 2. Synthesis of D1. Reagents and conditions: a) K2CO3, acetone, reflux, 8 h, 34%.
phoresis. Ki assays were performed according to procedures
described previously, and the results are shown in Supplemen-
tal Figure 3 in the Supporting Information and summarized in
Table 2. Selectivity of D3 for Slingshot 2 ranged from 2.9-fold
over PPM1A and up to >12.8-fold over PPM1G and PP1. We
also compared the inhibitory activity of D3 toward Slingshot 2
and Slingshot 1, using the catalytic domain of Slingshot 2 (resi-
dues 305–450) and the catalytic domain of Slingshot 1 (resi-
dues 309–460), because longer Slingshot 1 constructs were not
soluble in an E. coli expression system. The results indicate that
D3 has similar inhibitory activities toward both Slingshot 1 and
Slingshot 2 (Table 2).
bating cells with D3 at 5 mm significantly decreased the NGF-
induced cell migration, consistent with its inhibitory role in co-
filin dephosphorylation (Figure 2C,D and Figure 3). In contrast,
the compound A14, which has a similar scaffold but lacks the
Slingshot inhibition activity, had no effect on NGF-stimulated
cell migration (Figure 3). Moreover, in HEK293 cells overex-
pressed AT1R, the effect of D3 on cell migration was totally
abolished by knockdown of both Slingshot 1 and Slingshot 2
(Supplemental Figure 5 in the Supporting Information). Taken
together, these results confirm the cellular activity of com-
pound D3 in the inhibition of cell migration by targeting Sling-
shot. Thus, D3 can be selected as a candidate for further anti-
tumor drug development.
Slingshot is a cofilin phosphatase and regulates cytoskeleton
dynamics through dephosphorylation of cofilin at its Ser-3 po-
sition.[1f,5] In particular, previous studies have shown that cofilin
phosphorylation was significantly decreased after nerve
growth factor (NGF) stimulation, and knockdown of Slingshots
significantly reversed this process in a PC12 (pheochromocyto-
ma) cell model.[6] We therefore used PC12 cells to examine the
cellular activity of compound D3 in NGF signaling. PC12 cells
were preincubated with compound D3 for 45 min and then
stimulated with 100 ngmLÀ1 NGF. As shown in Figure 2, NGF-
induced dephosphorylation of cofilin were significantly
blocked by compound D3 at 15 and 30 min. These results con-
firm that compound D3 inhibited Slingshot activity in PC12
cells. We next examined whether D3 regulated cofilin dephos-
phorylation through direct inhibition of Slingshot. In HEK293
(human embryonic kidney) cells transfected with Flag-tagged
AT1R, 20 mm angiotensin II-induced cofilin dephosphorylation
was specifically blocked by compound D3 (Supplemental
Figure 4 in the Supporting Information). Knockdown of both
Slingshot 1 and Slingshot 2 eliminated the effect of D3 on cofi-
lin dephosphorylation (Supplemental Figure 4 in the Support-
ing Information). Therefore, compound D3 prevented cofilin
dephosphroylation through inhibition of Slingshot.
In conclusion, Slingshot phosphatases are key regulators of
cell skeleton rearrangement during cancer cell migration and
bacterial infection. Inhibition of Slingshot by small chemical
compounds has therapeutic potential to treat cancer and cer-
tain infectious diseases. However, only few compounds have
been identified to be Slingshot inhibitors in the micromolar
range.[1a] The selectivity toward other phosphatases and the
cellular activity of these Slingshot inhibitors reported so far
have not been investigated. Therefore, the potential of these
compounds as leads to develop therapeutic methods targeting
Slingshot is still unknown. Here, we have identified two benzo-
ic acid derivatives as competitive Slingshot inhibitors with Ki
values below 20 mm in vitro. Biochemical and cellular studies
demonstrated that the most potent inhibitor, D3, inhibited
Slingshot-mediated cell migration in PC12 cells and showed
certain selectivity toward several other phosphatases There-
fore, compound D3 is a good lead for more potent and selec-
tive Slingshot inhibitors for therapeutic applications. Future re-
search dealing with crystallographic studies of the D3/Sling-
shot complex or structure–activity relationship studies could
help us understand the structural basis of D3-based Slingshot
inhibition and help us design better inhibitors.
Slingshot promotes cancer cell migration and facilitates the
entry of infectious Salmonella into mammalian cells by de-
phosphorylation of cofilin. Studies have shown that knock-
down of Slingshot expression by small interfering RNAs
(siRNAs) significantly decreased the lysophosphatidic acid
(LPA)-induced cell migration of rat ascites hepatoma (MM1)
cells and their motility in two-dimensional culture. Therefore,
a Slingshot inhibitor could be developed to treat cancer cells
or infectious diseases by inhibition of cell migration or occlu-
sion of cytoskeleton dynamics.
Experimental Section
Materials and equipment. pNPP was purchased from Sangon Bio-
tech Co., Ltd. Ni-NTA agarose was from Amersham Pharmacia Bio-
tech. Anti-cofilin/pS3 antibody was purchased from Santa Cruz.
The mouse anti-GAPDH monoclonal antibody was from ZSGB-BIO
Co. The NGF was purchased from Sino Biological Inc. All other
chemicals and reagents were from Sigma. H and 13C NMR spectra
1
were obtained on a Bruker spectrometer (600 and 300 MHz, Bruker
Corp., CA, USA). Chemical shift values (d) are expressed in parts
per million (ppm) relative to TMS internal standard, and significant
data are reported in order of multiplicity (s=singlet, d=doublet,
We then examined the effects of compound D3 in cell mi-
gration using the transwell migration assay. PC12 cells pretreat-
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Table 1. Initial screening of the benzoic acid derivatives toward inhibition of Slingshot 2 phosphatase activity.
Compound
Structure
Molecular weight
Inhibition [%]
D3[b]
461.55
558.62
86Æ5
D17[b]
75Æ6
39Æ3
B7[a]
439.46
D1[b]
358.34
29Æ6
D25[b]
D16[b]
B16[a]
A14[a]
518.60
535.59
527.75
495.94
19Æ3
15Æ4
9Æ3
1Æ1
A11[a]
541.34
426.49
–
–
A13[a]
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Table 1. (Continued)
Compound
Structure
Molecular weight
Inhibition [%]
B15[a]
357.38
384.38
–
B22[a]
–
The compounds are sorted by their inhibition rates for Slingshot 2. [a] Purchased from commercial sources; [b] synthesized compounds. All measurements
were carried out at pH 7.0, 258C, in DMG buffer with enzyme concentration of 100 nm as described in the Experimental Section. All experiments were per-
formed in triplicate and the data are expressed as meanÆSD.
t=triplet, m=multiplet) and number of protons. High-resolution
atmospheric-pressure electrospray ionization mass spectrometry
(HRMS (AP-ESI)) was carried out on an Agilent 6510 quadrupole
time-of-flight LC–MS instrument (Agilent Technologies, CA, USA).
(Z)-4-((4-((3-(1-Carboxy-2-(1H-indol-3-yl)ethyl)-4-oxo-2-thioxo-
thiazolidin-5-ylidene)methyl)phenoxy)methyl)benzoic acid
(D17): Brown solid (0.16 g, 57%); mp: 207–2098C; 1H NMR
(300 MHz, [D6]DMSO): d=13.07 (brs, 2H), 10.77 (s, 1H), 7.97 (d, J=
8.4 Hz, 2H), 7.74 (s, 1H), 7.60-7.56 (m, 4H), 7.48 (d, J=7.8 Hz, 1H),
7.26 (d, J=8.1 Hz, 1H), 7.19 (d, J=8.4 Hz, 2H), 7.04 (m, 1H), 7.00
(m, 1H), 6.90 (m, 1H), 5.85 (brs, 1H), 5.30 (s, 2H), 3.76 (m, 1H),
3.61 ppm (m, 1H); 13C NMR (75 MHz, [D6]DMSO): d=192.9, 168.8,
167.0, 166.6, 160.3, 141.2, 135.9, 132.9, 130.5, 129.4, 127.4, 127.1,
125.7, 123.3, 120.7, 118.2, 117.8, 115.9, 111.2, 109.7, 68.9, 23.2,
14.0 ppm; HRMS (AP-ESI) m/z [M+H]+ calcd for C29H22N2O6S2:
559.0992, found 559.0993.
General procedure for the synthesis of the rhodanine-scaffold-
based phosphatase inhibitor library. Target compounds D3, D17,
D25, and D16 were prepared according to the synthetic routes
shown in Scheme 1. 4-hydroxybenzaldehyde (1, 0.61 g) was dis-
solved in acetone (100 mL). Afterwards, K2CO3 (2.07 g) and 4-(bro-
momethyl)benzoic acid (2, 1.08 g) were added, and the mixture
was left at reflux for 12 h to get intermediate 3. The coupling reac-
tion of bromoacetyl bromide (4, 12.8 g) and 4-(aminomethyl)ben-
zoic acid (5, 8.0 g) afforded 6. After that, 7 was synthesized follow-
ing the same substitution reaction as for 3. Next, the intermediate
N-substituted rhodanines 9a–9c were prepared following the
same procedure as in literature.[9] In brief, under ice-bath condi-
tions, substituted amine (8a–8c, 5 mmol) and triethylamine
(2.53 g) were dissolved in EtOH (1 mL) followed by dropwise addi-
tion of CS2 (0.76 mL). The resulting precipitate was obtained by fil-
tration and washed with water. Then it was added gradually to
a solution of sodium chloroacetate (0.64 mL, 5.5 mm). After stirring
overnight at room temperature, HCl (7 mL, 6m) was added to the
mixture and stirred again at 858C for 4 h to obtain 9a–9c. After all
the intermediates were synthesized, the target compounds D3,
D17, D25, and D16 were afforded through the aldol condensation
reaction of 3 (or 7) and the appropriate intermediate 9 in HOAc
with the addition of NH4OAc.[9]
Constructs. The full-length human Slingshot 1 cDNA was a gift
from Dr. Robert. J. Lefkowitz of Duke University (Durham, NC, USA)
and have been described previously.[7] The constructs of histidine-
tagged phosphatases, namely His-PPM1A, His-PPM1G, His-PRL-1,
His PTP-MEG2(277-583), His-MKP3, and His-PTPN18 (catalytic
domain) have been described previously.[1c,d,8]
Protein expression and purification. The catalytic domain of
Slingshot 2 (residues 305–450 and 233–490) and Slingshot 1 (resi-
dues 309–460) with an N- terminal his tag was prepared and used
for in vitro studies. BL21 (DE3) E. coli cells were transformed with
the expression plasmids and cultured in lysogeny broth (LB)
medium with shaking at 378C. The culture temperature was adjust-
ed to 188C at OD600 =0.6, and expression was induced for 12 h
with 0.3 mm isopropyl b-D-1-thiogalactopyranoside (IPTG) once
OD600 reached 0.8. The cells were harvested by centrifugation and
resuspended in lysis buffer (50 mL, 20 mm Tris pH 8.0, 150 mm
NaCl). After centrifugation, the supernatant was incubated with
Ni2+-NTA resin with end-to-end mixing at 48C for 1 h. The beads
were collected and washed 3 times with buffer (20 mL, 20 mm Tris
pH 8.0, 150 mm NaCl, and 5 mm imidazole) and eluted with an imi-
dazole gradient (20 mm Tris pH 8.0, 150 mm NaCl, and 20–200 mm
imidazole). After purification, the protein was further concentrated
by ultrafiltration and stored at À808C. Expression and purification
of the other His-tagged proteins were performed as previously de-
scribed.
As shown in Scheme 2, target compound D1 was synthesized fol-
lowing a similar route as in Scheme 1.
(Z)-4-((4-((4-Oxo-2-thioxo-3-(o-tolyl)thiazolidin-5-ylidene)me-
thyl)phenoxy)methyl)benzoic acid (D3): Yellow solid (0.14 g,
59%); mp: 276–2788C; 1H NMR (600 MHz, [D6]DMSO): d=12.55
(s,1H), 7.98 (d, J=7.8 Hz, 2H), 7.85 (s,1H), 7.70 (d, J=7.8 Hz, 2H),
7.59 (d, J=7.8 Hz, 2H), 7.43 (m,2H), 7.38 (m, 1H), 7.34 (d, J=
7.8 Hz, 1H), 7.24 (d, J=7.8 Hz, 2H), 5.33 (s, 2H), 2.06 ppm (s, 3H);
13C NMR (100 MHz, [D6]DMSO): d=192.9, 167.0, 166.7, 160.5, 141.4,
135.8, 134,3, 133,4, 133.0, 130.9, 130.4, 129.8, 129.5, 129.0, 127.5,
127.2, 125.9, 119.9, 116.0, 69.0, 16.8 ppm; HRMS (AP-ESI) m/z
[M+H]+ calcd for C25H19NO4S2: 462.0828, found 462.0829.
Chemical library screening. The collected compounds were
screened in a 96-well format. To initiate the reaction, 100 nm Sling-
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Table 2. Selectivity of compound D3 toward a panel of protein phospha-
tases.
Enzymes
Ki [mm][a]
Ratio toward SSH2
SSH2 (233-490)
PPM1A
PRL-1
PTPMEG2
PTPN18
MKP3
PPM1G
PP1
SSH2 (305-450)
SSH1 (309-460)
3.9Æ0.3
11.4Æ1.0
13.8Æ2.0
14.6Æ1.0
20.4Æ2.3
21.6Æ2.7
1.0
2.9
3.5
3.7
5.2
5.5
>50
>50
8.9Æ0.7
7.4Æ0.9
>12.8
>12.8
2.3
1.9
[a] All measurements were assayed using pNPP as a substrate at pH 7.0,
258C, in DMG buffer with ionic strength adjusted to 0.15m. All experi-
ments were performed in triplicate, and the data are expressed as
meanÆSD.
Figure 1. Chemical structures and kinetic studies of D3 and D17 for their
inhibitory activity against Slingshot 2. A–B) Chemical structures of the two
most potent compounds from the screening library. A) Chemical structure of
compound D3. B) Chemical structure of compound D17. C–D) Kinetic stud-
ies of the inhibition mode of D3 and D17. pNPP concentrations were 3.22,
4.83, 7.24, 10.9, 16.3, 24.4, 36.7, and 55 mm, respectively. C) Lineweaver–Burk
plot for compound D3-mediated SSH2 inhibition using pNPP as a substrate.
D) Lineweaver–Burk plot for compound D17-mediated SSH2 inhibition using
pNPP as a substrate.
Figure 2. D3 attenuates NGF-induced cofilin dephosphorylation at pSer3 po-
sition in PC12 cells. A) Effects of compound D3 (5 mm) on the NGF-induced
cofilin dephosphorylation as detected by the anti-pS3-cofilin antibody. The
GAPDH level was used as a control. B) Statistical analysis of the phosphoryla-
tion of cofilin pS3 in PC12 cells treated with NGF. All experiments were re-
peated in at least triplicate. ns: no significant difference was observed be-
tween DMSO and D3 treated groups. ##p<0.01, NGF treated cells were com-
pared with control cells. *p<0.05; **p<0.01; D3-treated cells were com-
pared with DMSO-treated cells.
shot 2 was added to a 3,3-dimethylglutarate (DMG) buffer consist-
ing of 11 mm pNPP, 50 mm compound, 50 mm DMG, pH7.0, 1 mm
EDTA, and 2 mm dithiothreitol (DTT) with an ionic strength of
0.15m, adjusted by NaCl. The final volume for each well was
120 mL. The reaction mixture was incubated at 378C and continu-
ously measured at 405 nm using a Molecular Devices EMax preci-
sion microplate reader (Sunnyvale, CA, USA). Compounds with an
inhibition rate of 50% or more were further tested for IC50 and Ki
studies. All assays were performed in triplicate.
0.15m adjusted by NaCl). The KM value of Slingshot 2 toward pNPP
hydrolysis (11 mm for pNPP) was used to determine the IC50. The
reaction was detected by monitoring the absorbance of pNPP at
405 nm. The IC50 values were obtained by fitting the data to Equa-
tion (1) using GraphPad Prism 5 (GraphPad Software, Inc., La Jolla,
CA, USA) as follows:
IC50 measurements. Kinetic assays for Slingshot 2-catalyzed pNPP
hydrolysis in the presence of small-molecular inhibitors were mea-
sured as previously described.[4] The effect of each inhibitor on the
Slingshot 2-catalyzed pNPP hydrolysis was determined at 258C in
reaction buffer (50 mm DMG buffer with an ionic strength of
AI ¼ A0  IC50=ðIC50 þ ½IŠÞ
ð1Þ
Ki measurements. The phosphatase-catalyzed hydrolysis of pNPP
in the presence of inhibitors was assayed at 258C. The reaction
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peated at least in triplicate. Data analysis was conducted with
GraphPad Prism software.
Data analysis and software. The data were analyzed using ImageJ
and GraphPad Prism software. All experiments were performed in
triplicate, and the data are expressed as meanÆSD. Statistical com-
parisons were performed with ANOVA tests using GraphPad
Prism 5.
Acknowledgements
This work was supported by grants from the National Key Basic
Research Program of China (2013CB967700 to X.Y.,
2012CB910402 to J.-P.S.), the National Natural Science Founda-
tion of China (31100580, 31470789 to J.-P.S.; 31270857 to X.Y.),
the Shandong Natural Science Fund for Distinguished Young
Scholars (JQ201320 to X.Y., JQ201517 to J.-P.S.; JQ201319 to H.F.),
the Program for New Century Excellent Talents in University
(NCET-12-0337 to H.F.), the Projects of International Cooperation
and Exchanges Ministry of Science and Technology of China
(2013DFG32390 to X.-Y.S.), and the Program for Changjiang
Scholars and Innovative Research Team in University (IRT13028).
The authors declare no conflict of interest.
Figure 3. Compound D3 inhibits the NGF-induced migration of PC12 cells.
The transwell assay was used to examine the D3 effects on NGF-induced cell
migration. The PC12 cells were preincubated with D3 (5 mm in 0.2% DMSO),
A14 (5 mm in 0.2% DMSO), or control vehicle (0.2% DMSO) for 45 min. Ap-
proximately 1”105/100 mL cells were added to each upper chamber, and the
100 ngmLÀ1 NGF was incubated in the lower chamber as a chemoattractant.
After 24 h, the number of migrated cells was counted. **p<0.01, D3-treated
cells were compared with DMSO-treated cells (control); ns: no significance
was found between D3-treated cells and DMSO-treated cells (control). All
statistics shown represent the meanÆSEM from at least three independent
experiments.
was initiated by addition of pNPP (ranging from 0.2 to 5 KM) to a re-
action mixture containing different phosphatases and various fixed
concentrations of inhibitors, and stopped by the addition of 1m
NaOH. The inhibition constant Ki and inhibition pattern were evalu-
ated by fitting the data to the Michaelis–Menten equation (or Line-
weaver–Burk equation) for competitive inhibition [Eq. (2)–(3)],
using linear regression and GraphPad Prism as follows:
Keywords: anticancer · cell migration · nerve growth factor
(NGF) · phosphatase inhibitors · Slingshot
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1=v ¼ KMobs=ðVmax  ½SubstrateŠÞ þ 1=Vmax
KMobs ¼ KMð1 þ ½InhibitorŠ=KiÞ
ð2Þ
ð3Þ
Cell culture and Immunoblotting. PC12 cells were cultured as pre-
viously described.[8b] Cells were preincubated with 5 mm (final con-
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with 100 ngmLÀ1 NGF for 0, 15, or 30 min. The stimulation was ter-
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lysis buffer (50 mm Tris pH 7.5, 150 mm NaCl, 10 mm NaF, 2 mm
EDTA, 10% glycerol, 1% NP-40, 0.25% sodium deoxycholate, 1 mm
NaVO4, 1 mm phenylmethylsulfonyl fluoride, 0.3 mm aprotin,
130 mm bestiatin, 1 mm leupeptin, and 1 mm pepstatin) for 15 min.
The lysates were then centrifuged at 12000 rpm for 15 min. The
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Received: October 4, 2015
Published online on November 10, 2015
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