Novel inhibitors targeting PPM1D phosphatase potently suppress cancer cell proliferation

Article abstract of DOI:10.1016/j.bmc.2015.08.042

Protein phosphatase magnesium-dependent 1δ (PPM1D, Wip1) is a p53 inducible serine/threonine phosphatase. PPM1D is a promising target protein in cancer therapy since overexpression, missense mutations, truncating mutations, and gene amplification of PPM1D

Full text of DOI:10.1016/j.bmc.2015.08.042

Accepted Manuscript
Novel Inhibitors Targeting PPM1D Phosphatase Potently Suppress Cancer Cell
Proliferation
Sari Ogasawara, Yuhei Kiyota, Yoshiro Chuman, Ayano Kowata, Fumihiko
Yoshimura, Keiji Tanino, Rui Kamada, Kazuyasu Sakaguchi
PII:
S0968-0896(15)30024-9
DOI:
http://dx.doi.org/10.1016/j.bmc.2015.08.042
BMC 12546
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
3 August 2015
27 August 2015
29 August 2015
Please cite this article as: Ogasawara, S., Kiyota, Y., Chuman, Y., Kowata, A., Yoshimura, F., Tanino, K., Kamada,
R., Sakaguchi, K., Novel Inhibitors Targeting PPM1D Phosphatase Potently Suppress Cancer Cell Proliferation,
Bioorganic & Medicinal Chemistry (2015), doi: http://dx.doi.org/10.1016/j.bmc.2015.08.042
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Novel Inhibitors Targeting PPM1D Phosphatase Potently Suppress Cancer Cell
Proliferation
a+
a+
a
b
b
Sari Ogasawara , Yuhei Kiyota , Yoshiro Chuman , Ayano Kowata , Fumihiko Yoshimura ,
b
a
a*
Keiji Tanino , Rui Kamada , Kazuyasu Sakaguchi
a
Laboratory of Biological Chemistry, Department of Chemistry, Faculty of Science, Hokkaido
b
University, Sapporo 060-0810, Japan, Laboratory of Organic Chemistry II, Department of
Chemistry, Faculty of Science, Hokkaido University, Sapporo 060-0810, Japan
These authors contributed equally to this work.
+
*
Corresponding author.
Kazuyasu Sakaguchi,
Laboratory of Biological Chemistry, Department of Chemistry, Faculty of Science, Hokkaido
University, North 10, West 8, Kita-ku, Sapporo 060-0810, Japan.
Tel: +81-11-706-2698; Fax: +81-11-706-4683; E-mail: kazuyasu@sci.hokudai.ac.jp

Abstract
Protein phosphatase magnesium-dependent 1δ (PPM1D, Wip1) is a p53 inducible
serine/threonine phosphatase. PPM1D is a promising target protein in cancer therapy since
overexpression, missense mutations, truncating mutations, and gene amplification of PPM1D are
reported in many tumors, including breast cancer and neuroblastoma. Herein, we report that a
specific inhibitor, SL-176 that can be readily synthesized in 10 steps, significantly inhibits
proliferation of a breast cancer cell line overexpressing PPM1D and induces G2/M arrest and
apoptosis. SL-176 decreases PPM1D enzyme activity potently and specifically in vitro. These
results demonstrate that SL-176 could be a useful lead compound in the development of effective
anti-cancer agents.

1. Introduction
The gene PPM1D encoding serine/threonine protein phosphatase PPM1D (also known as
Wip1 and PP2Cδ) is located on the chromosomal region 17q23, which is frequently amplified in
1
many cancers. Overexpression of PPM1D and amplification of the PPM1D gene have been
reported in various cancers, including breast cancer, hepatocellular carcinoma, pancreatic
2-6
adenocarcinoma, ovarian carcinoma and neuroblastoma. PPM1D was identified as a protein
7
induced by p53 following activation in response to irradiation. It has been reported that PPM1D
8, 9
regulates the p53 pathway in a negative feedback loop by dephosphorylating p53 and other
9, 10
important factors involved in cell cycle regulation.
Consequently, overexpression, missense
mutations, and truncating mutations of PPM1D are associated with reduced activation of the
11, 12
regulatory factors and have been implicated in carcinogenesis.
Additionally, amplification of
PPM1D and overexpression of PPM1D in these cancers are significantly associated with poor
clinical prognosis. This suggests that analyzing alterations in copy number and expression level is
2, 5, 6
important for proper clinical prognosis and selection of therapeutic options.
Various PPM1D
inhibitors, including peptidic inhibitors derived from substrate sequences and small chemical
13-17
inhibitors, were reported.
Among them, GSK2830371 was demonstrated very strong
inhibition of cell growth in cell lines overexpressing PPM1D with wild-type TP53, including
1
3-17
breast cancer and neuroblastoma.
However, cell lines with mutant TP53 were shown to be
resistant to GSK2830371. Because PPM1D overexpression has been observed in cell lines with
TP53 mutation, it is required to develop inhibitors that suppress cancer cell growth regardless of
18
TP53 status. Recently, we demonstrated that the PPM1D inhibitor, SPI-001, suppressed the
19
proliferation of a breast cancer cell line. The combined administration of SPI-001 and
doxorubicin suppressed the cell viability of colorectal carcinoma cells overexpressing C-terminal
20
truncated PPM1D. Thus, PPM1D is a therapeutic target and SPI-001 is a promising lead
candidate for additional anti-cancer agents. However, SPI-001 is a challenging molecule to
synthesize that involves 33 steps. Hence, analogs of SPI-001 that require simpler synthetic routes
are more attractive and viable lead candidates. Here, we report that a novel PPM1D inhibitor,
SL-176, effectively prevents cancer cell proliferation.

2. Results and Discussion
We designed simplified and more compact analogs of SPI-001. These analogs, SL-175 and
SL-176 were synthesized in 10 steps from a commercially available starting material (Scheme
S1). Both analogs share a trans decalin framework and bulky hydrophobic groups. These analogs
differ from the hydrophilic groups they bear wherein SL-175 features a hydroxyl functionality, in
contrast to the carboxylic acid moiety of SL-176 (Fig. 1).
Fig. 1. Chemical structures of SL-175 and SL-176.
We examined the effect of these compounds on the proliferation of PPM1D overexpressing
MCF-7 breast cancer cells (Fig. 2 and Table 1). The half maximal inhibitory concentration (IC50
)
value of SL-176, which has a carboxyl group, for the growth inhibition was 7.4 µM. Also, IC50
values of SL-175, which has a hydroxyl group, SPI-001, and GSK2830371 for the growth
inhibition were 36.1 µM, 26.9 µM, and 9.5 µM, respectively. SL-176 has three-times more potent
inhibitory activity than SPI-001. SL-176 completely suppressed cell proliferation over 20 μM
concentration. In contrast, GSK2830371 did not inhibit cell growth completely even in high
concentration. SL-176 showed the strongest inhibition of MCF-7 proliferation. Next, the
inhibitory activity of these compounds against PPM1D phosphatase activity was examined using
recombinant His-PPM1D(1-420) and a phosphorylated peptide derived from p53 as the substrate
(Fig. 3Aand Table 1). The IC50 value of SL-176 against PPM1D phosphatase activity was 110 nM
whereas GSK2830371 showed an IC50 value of 86.3 nM. SL-175 inhibited PPM1D
dephosphorylation twice the IC50 of SL-176. These results indicated that SL-176 had extremely

strong inhibitory effects on both in vitro and in vivo. In order to analyze the inhibitory activity of
SL-176 in more detail, we examined the mode of enzyme inhibition (Fig. 3B). The data of SL-176
were fitted by global fitting analysis in noncompetitive inhibition. Moreover, to confirm
specificity of SL-176 for PPM1D, we performed an in vitro phosphatase assay using three
recombinant enzymes and phosphorylated peptides of their respective substrate (Fig. 3C). SL-176
inhibited His-PPM1D(1-420) phosphatase activity at sub-micromolar concentrations, whereas
neither His-PPM1A, a PPM1 phosphatase family member, nor the non-PPM1 phosphatase family
members, PP1 or PP2A, were inhibited by SL-176. This result showed that SL-176 had high
specificity for PPM1D phosphatase in vitro. Next, we analyzed the PPM1D specificity at the
cellular level using H1299, p53-null cell line, which expresses normal level of PPM1D, and
PPM1D-overexpressed cell line, namely H1299(PMD-9), which we established by transgenic
HA-PPM1D (Fig. 3D and Fig. S1A). SL-176 showed a much more potent inhibition of cell
proliferation against H1299(PMD-9) cells than the H1299 cells. On the other hand, GSK2830371
was not effective in growth inhibition of both H1299 and H1299(PMD-9) (Fig. S1B). These
results suggested that SL-176 selectively inhibited cell proliferation in p53-null cells with
overexpression of PPM1D.
Fig. 2. Growth inhibition of the compounds on PPM1D overexpressing cell line MCF-7. MCF-7
cells were incubated with each compound (circle, SL-175; square, SL-176; triangle, SPI-001;
diamond, GSK2830371). Cell numbers were measured after 3 days. Data represent the mean ±
S.E., n ≥ 3.

Table 1 The effects of the inhibitors on growth inhibition of MCF-7 cells and in vitro phosphatase
activity of PPM1D
Inhibitory activity
Compounds
MCF-7 cell proliferation
IC50 (μM)*
In vitro phosphatase activity
IC50 (nM)*
SL-175
SL-176
36.1 ± 2.89
215 ± 28.8
7.4 ± 0.72
110 ± 12.9
SPI-001
26.9 ± 2.07
86.9 ± 8.43
GSK2830371
9.5 ± 0.86
86.3 ± 8.80
*The IC50 value of each compound against in vitro phosphatase assay using His-PPM1D(1-420) and MCF-7 cell
proliferation. The standard errors of fittings are indicated.
Fig. 3. Inhibitory activity of the compounds against in vitro phosphatase activity. (A) In vitro
phosphatase assay was performed with 2 nM recombinant His-PPM1D(1-420) (circle, SL-175;
square, SL-176; triangle, SPI-001; diamond, GSK2830371). Data represent the mean ± S.D., n =
9. (B) Lineweaver–Burk plot of SL-176 inhibition. Global fitting suggested noncompetitive

inhibition mode (the inhibition constant (Ki) was 0.228 ± 0.025 µM, circle, vehicle; square, 0.050
µM; triangle, 0.100 µM; inverted triangle, 0.150 µM). Data represent the mean ± S.D., n = 9.
(C) In vitro phosphatase assay using PPM1A (circle), PP1 (square) and PP2A (diamond). Data
represent the mean ± S.D., n = 3. (D) The effects of SL-176 on PPM1D overexpressed- or
normal-level cell proliferation (circle, H1299; square, H1299(PMD-9)) Data represent the mean
±
S.E., n = 6.
The single administration of SL-176 induced an increase in the phosphorylation level of p53
at Ser15 in MCF-7 cells (Fig. 4A). In the absence of SL-176, the phosphorylation level of p53
slightly increased in response to UV-induced DNA damage in MCF-7 cells. As expected, the
treatment of SL-176 with UV irradiation significantly increased the phosphorylation level of p53
and protein expression level of p53. This showed that SL-176 inhibited PPM1D in the cell and
induced the activation of p53. It has been reported that up-regulation of phosphorylated p53
2
1
induces G2/M arrest and apoptosis. Therefore, we analyzed the cell cycle of MCF-7 cells using
flow cytometry (Fig. 4B). Treatment with SL-176 increased the amount of cells in the G2/M and
sub-G1 phase. In SL-176-treated cells, the ratio of Annexin V positive cells was higher than
control cells (Fig. 4C). These results suggested that SL-176 induced cell cycle arrest at G2/M and
apoptosis via activation of p53 by PPM1D inhibition.

Fig. 4. The effects of SL-176 on p53 pathway and cell proliferation in MCF-7. (A) The effect of
2
SL-176 was measured in MCF-7 cells by UV-irradiation (15 J/m ). Phosphorylation level of p53
was analyzed by western blotting. (B) Cell cycle analysis was performed with flow cytometry. (C)
The ration of Annexin V positive cells was measured by counting the number of stained cells in
the images. Data represent the mean ± S.D., n = 3.
In cancer cells overexpressing PPM1D, the inactivation of p53 via hypophosphorylation
was observed. The administration of a PPM1D inhibitor induces an increase in the
phosphorylation level of p53 and the subsequent activation of p53 leads to growth inhibition.
However, inhibition of cell proliferation was observed in a p53-null cell line where PPM1D was
overexpressed in this report. Specifically, Natrajan et al reported that a PPM1D inhibitor
1
8
represses the proliferation of p53-mutated cells in which PPM1D is overexpressed. It has been
suggested that a p53-independent pathway exists, which might explain why therapeutic agents

targeting PPM1D can inhibit cell proliferation in p53-null cell, but this needs to be elucidated
2
2
further.
In this report, we clearly demonstrated that SL-176 is a highly potent inhibitor that can
effectively suppress proliferation of PPM1D-overexpressing cancer cells, regardless of the p53
status. SL-176 completely suppressed over 20 μM concentration. GSK2830371 showed some
inhibition in a low concentration; however, it did not inhibit cell growth completely even in high
concentration. Therefore, the inhibition curve of GSK2830371 showed a mild shift (Fig. 2). The
17, 23
administration of GSK2830371 was reported to induce abolishment of PPM1D protein.
On
the other hand, SL-176 did not affect the PPM1D level, probably resulting in the effective
inhibition of cell proliferation.
The remarkable inhibitory activity of SL-176 offers an opportunity for new chemotherapy
options. RNA overexpression and gene amplification of PPM1D has been reported in many
cancers, and the inhibition of PPM1D showed significant suppression of cell proliferation in
breast cancer and neuroblastoma cell lines. In HCT-116 human colorectal carcinoma cells that
possess a C-terminal truncated PPM1D, we revealed that combination treatment with a PPM1D
inhibitor SPI-001 and doxorubicin caused cancer cell death, suggesting that treatments targeting
20
PPM1D could be useful for treating various cancers. Therefore, we propose that SL-176 is an
excellent candidate to develop as anticancer agents.
In conclusion, we developed the potent and specific PPM1D inhibitor SL-176 that contains a
different backbone structure compared with other PPM1D inhibitors that were previously
reported. The newly designed analog SL-176 can be readily synthesized in only 10 steps (30%
overall yield) from 2-cyclohexen-1-one with inexpensive chemicals, whereas SPI-001 required
33 steps. SL-176 specifically inhibited the phosphatase activity of PPM1D, suppressed the cancer
cell proliferation regardless of the p53 status with strong inhibitory activity. It is known that
2
4
compounds with a molecular weight < 500 are suitable for high absorption. The molecular
weights of SL-176 and SPI-001 are 456.8 and 541.0, respectively. In addition, calculated
24
distribution coefficient (ClogD) < 5 is a key factor to estimate good cell membrane permeability.
The ClogD of SL-176 in neutral aqueous solution is 3.89, whereas that of SPI-001 is 6.06. These
parameters also support the strong inhibitory activity of SPI-001 against cancer cell growth.
Taken together, we succeeded in developing a new PPM1D inhibitor SL-176 by simplifying the
structure of SPI-001. As a result of its strong and specific inhibitory activity against PPM1D,
SL-176 should be used as a powerful tool for studying PPM1D in vitro and in vivo.

3. Experimental Section
Synthetic methods, characterization of new compounds and experimental methods are
provided in the Supplementary Material.
Acknowledgements
This work was supported in part by Grants-in-Aid for Scientific Research (B) (No.
4310152) from JSPS (to K.S.) and Grant-in-Aid for Scientific Research (C) (No. 40372263)
2
from JSPS (to Y.C.). We thank J.I. Janairo for providing valuable comments and suggestions.
Keywords
dephosphorylation; anti-tumor; protein phosphatase; inhibitors; apoptosis

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Graphical abstract