Dual-specificity phosphatase CDC25A/B inhibitor identified from a focused library with nonelectrophilic core structure

Article abstract of DOI:10.1021/ml2002778

Focused libraries of enamine derivatives with a nonacidic, nonelectrophilic core structure were screened for inhibitors of dual-specificity protein phosphatases, and an o-hydroxybenzyl derivative RE44 (10d) was identified as a selective inhibitor of CDC25A/B. This inhibitor induced cell-cycle arrest of tsFT210 cells at the G2/M phase and inhibited dephosphorylation of the CDC25B substrate CDK1. Unlike most quinone-based inhibitors, 10d does not generate reactive oxygen species.

Full text of DOI:10.1021/ml2002778

Letter
pubs.acs.org/acsmedchemlett
Dual-Specificity Phosphatase CDC25A/B Inhibitor Identified from a
Focused Library with Nonelectrophilic Core Structure
Ayako Tsuchiya,† Go Hirai,† Yusuke Koyama,† Kana Oonuma,† Yuko Otani,† Hiroyuki Osada,†
and Mikiko Sodeoka*,†,‡
†RIKEN Advanced Science Institute, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan
‡Sodeoka Live Cell Chemistry Project, ERATO, Japan Science and Technology Agency, 2-1 Hirosawa, Wako-shi, Saitama 351-0198,
Japan
S
* Supporting Information
ABSTRACT: Focused libraries of enamine derivatives with a nonacidic, nonelectrophilic core structure
were screened for inhibitors of dual-specificity protein phosphatases, and an o-hydroxybenzyl derivative
RE44 (10d) was identified as a selective inhibitor of CDC25A/B. This inhibitor induced cell-cycle arrest
of tsFT210 cells at the G2/M phase and inhibited dephosphorylation of the CDC25B substrate CDK1.
Unlike most quinone-based inhibitors, 10d does not generate reactive oxygen species.
KEYWORDS: dual-specificity protein phosphatases, CDC25, inhibitor, reactive oxygen species, cell cycle, focused library
ell cycle progression and transition between phases of the
cell cycle are controlled by the activation of cyclin-
cysteine residue in the catalytic P-loop structure.13 It has been
shown that reactive oxygen species (ROS) are generated in
cultured cells treated with o- and p-quinone derivatives.13,14
The activity of CDC25B is modulated by reversible or
irreversible redox reaction,15 and the cysteine residue on the
P-loop of CDC25B was suggested to be more reactive to
oxidant than usual cysteine residues. However, nonspecific
oxidation of relatively reactive cysteine residues of proteins
involved in cellular signaling, including other DSPs and protein
tyrosine phosphatases (PTPs), as well as many other redox
sensor proteins, by ROS would be likely to seriously disrupt cell
function.16 Therefore, the potential toxicity to normal cells and
tissues caused by ROS derived from usual quinoid inhibitors
would be problematic in biological studies. Although inhibitors
such as a H3217 that do not oxidize cysteine residues or
generate ROS have been reported, it is expected that
nonquinoid inhibitors with high enzyme selectivity would
have great potential for the development of pharmaceutical
agents and useful biological tools. Herein, we report a novel
class of inhibitor, RE44 (10d), which has potent and selective
inhibitory activity toward CDC25A/B without generation of
ROS.
C
dependent kinases (CDKs), and alterations in CDKs activities
are involved in tumor-associated cell-cycle aberration.1 Dual-
specificity protein phosphatase (DSP) CDC25s (CDC25A,
CDC25B, and CDC25C) have the unique function of
activating CDKs by dephosphorylating specific phospho-
tyrosine and phospho-threonine residues on CDKs.2,3
CDC25s are important checkpoint regulators for handling
DNA damage caused by UV light, ionizing irradiation, or
chemicals in eukaryotic cells. Thus, misregulation of CDC25s
can trigger genomic instability. Furthermore, CDC25A and
CDC25B have been suggested to be oncogenic and are
overexpressed in various cancer cells.3 Therefore, inhibitors of
CDC25s would be promising candidates for cancer therapy. As
has been reviewed recently,4−6 powerful CDC25s inhibitors
with antitumor activity have been found. On the other hand,
although all three CDC25 isoforms appear to participate in the
regulation of G1-S and G2-M transitions and mitosis, the
precise functions of individual CDC25 isoforms still remain to
be fully established. This is at least partly due to the lack of
inhibitors with high enzyme specificity as well as isoform
specificity.2
Many of the reported inhibitors of CDC25s have highly
acidic, electrophilic, or quinone-based structures and were
discovered through screening of natural products or synthetic
libraries.4 Among the p-quinoid derivatives, NSC663284 (1),7
NSC95397 (2),8 adociaquinone B,9 and IRC-08386410 are
representative potent inhibitors, and this class of inhibitors
generally inhibits all three isoforms of CDC25 in an unselective
manner. These compounds cause remarkable inhibition of
tumor cell proliferation via irreversible inhibition of CDC25s by
electrophilic modification11,12 or oxidation of the thiolate of the
We have previously reported the development of inhibitors
for DSPs/PTPs by screening of a 3-acyltetronic acid library (3)
based on RK-682,18 in which the 3-acyltetronate 4 acts as a
phosphate-mimicking core structure (Figure 1). However, the
anion-delocalized core 4 resulted in poor cell permeability and
enzyme selectivity of this class of inhibitors. Recently, we
addressed these problems by designing and synthesizing novel
Received: November 28, 2011
Accepted: February 15, 2012
Published: February 15, 2012
© 2012 American Chemical Society
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Letter
(Me, Cl, F, OH, OMe, and NMe2) at each position of the
aromatic ring (ortho, meta, and para) were employed to
examine the effect of the substituent on the enzyme selectivity.
Enamine derivatives 8−10 (Figure 1) were formed cleanly as
inseparable and interconvertible mixtures of the E- and Z-
isomers (about 1.3:1) at the enamine double bond. Inhibitory
activities of all of the synthesized compounds for PTP1B (as a
representative of PTPs) and DSPs (CDC25s, VHR, MKP-1,
and MKP-3) were evaluated in vitro by utilizing purified
phosphatase proteins as previously described.19 Only a few
compounds (8c, 9a, 9d, and 10c) weakly inhibited PTP1B and
CDC25C, but generally, the benzyl-substituted RE derivative
showed a strong preference for the inhibition of VHR and
CDC25A/B. As we had expected, the nature of the substituent
on the aromatic ring affected the selectivity between VHR and
CDC25A/B. Introduction of a m-substituent, such as methyl or
chloro, decreased the inhibitory activity for CDC25A/B and
increased VHR-inhibitory potency, and thus, we were able to
obtain the selective VHR inhibitors 8a (RE12) and 8b. In
contrast, p- or o-methyl or chloro substituent (9a, 10a, 9b, and
10b) did not result in similar changes of inhibitory potency
toward VHR and CDC25A/B. Fluoro derivatives 8c, 9c, and
10c showed a slight increase of CDC25A inhibition as
compared to the nonsubstituted 7 but still inhibited VHR
more efficiently. To our delight, introduction of a hydroxyl
group improved the selectivity for CDC25B. The m-hydroxyl
derivative showed increased inhibitory activity for CDC25B,
but similar inhibition profiles were not seen in the cases of the
m-OMe derivative 8e and m-NMe2 derivative 8f. Although
substitution of the p-position with a hydroxyl group (9d) was
not effective for further improvement of the selectivity,
interestingly, the o-hydroxyl derivative 10d (RE44) showed
much less potent inhibition of VHR and stronger inhibition of
CDC25B. Such a substituent effect was not observed with
methoxy and dimethylamino groups at the p- and o-positions
(9e, 9f, 10e, and 10f). Finally, the inhibitory selectivity of
compound 10d for CDC25B over CDC25A and VHR was
found to be the highest among the compounds tested.
Compound 10d should be a promising candidate as a biological
tool for studying the functions of CDC25B, if it operates at the
cell level.
Both CDC25s and VHR phosphatases are involved in the
regulation of the cell cycle,21 and inhibition of the enzymatic
activity of these proteins is expected to have an antiproliferative
effect on cultured cells. Therefore, we tested the inhibitory
effect of all of the synthesized compounds on proliferation of
HL-60 cells. All compounds inhibited the proliferation of HL-
60 cells, and the IC50 values of most of the compounds were
around 10 μM. However, it is noteworthy that compounds 8d,
9d, and 10d possessing a hydroxyl group on the aromatic ring,
which showed greater inhibitory potency for CDC25B as
described above, also inhibited HL-60 cell proliferation more
efficiently (Table 1).
With these DSP inhibitors having distinct selectivity for
CDC25A/B and VHR in hand, we next evaluated the effect of
compounds 7 (RE1: dual CDC25A/B and VHR inhibitor), 8a
(RE12: selective VHR inhibitor), and 10d (RE44: selective
CDC25A/B inhibitor) on the cell-cycle progression of tsFT210
cells.22 The temperature-sensitive FT210 cell line has been
widely used for cellular studies of CDC25s inhibitors. Although
tsFT210 cells can grow normally at the permissive temperature
of 32 °C and show a normal cell-cycle distribution, the cell
cycle can be easily arrested at mid to late G2 phase, because
Figure 1. Structures of 1, 2, 3-acyltetronic acid derivatives (3) and
their anionic form (4), and RE derivatives (5) and their mesomeric
structure (6) as well as compounds in the second-generation RE
library (7−10).
RK-682 enamine (RE) derivatives 5.19 An RE library 5 and a 3-
acyltetronic acid library 3 were constructed based on the
focused library concept; that is, a series of derivatives having a
core phosphate-mimicking structure were systematically
synthesized and evaluated as protein phosphatases inhibitors.
In the case of the RE library, the most distinctive feature is the
neutral phosphate-mimicking and electron-delocalized core
structure (6), which results in improvement of cell permeability
and high selectivity for DSPs. In evaluation of the first-
generation library of RE derivatives, we found that 7 (RE1)
having a benzyl group as R3 inhibited both VHR and CDC25A/
B but not CDC25C, MKPs, or PTP1B. Furthermore, a highly
selective inhibitor of VHR, RE12 (8a), was obtained by the
introduction of a methyl group. These results strongly indicated
a critical role of the substituent on the benzene ring of 7 for the
discrimination of DSPs. Therefore, we next focused on a
selective inhibitor of CDC25s and prepared a second-
generation library of compounds with one substituent on the
phenyl ring of 7.
This library was prepared by heating of 3 [R1 = (CH2)14CH3,
R2 = CH2OH] with commercially available substituted
benzylamines in the presence of a catalytic amount of p-
toluenesulfonic acid (pTsOH) (Figure 1).20 This time,
benzylamines having one of six relatively small substituents
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ACS Medicinal Chemistry Letters
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Table 1. Inhibition of CDC25s and VHR by RE Derivatives
IC50 values (SD) (μM)
b
b
b
b
b
b
c
d
compd
CDC25A
CDC25B
CDC25C
VHR
MKP-1
MKP-3
PTP1B growth inhibition of HL-60 cells IC50 (range) (μM)
a
7
16.6 (1.3)
ND
8.42 (0.53)
ND
ND
ND
ND
11.4 (0.47)
1.63 (0.23)
1.43 (0.06)
4.69 (0.67)
11.1 (0.36)
5.53 (0.79)
5.13 (0.70)
2.68 (0.27)
3.63 (0.27)
8.41 (1.1)
9.93 (0.27)
6.77 (1.3)
12.0 (1.2)
8.99 (0.13)
10.2 (2.3)
8.93 (0.73)
24.9 (2.2)
6.60 (0.97)
5.19 (0.65)
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
83.7
ND
ND
ND
ND
ND
ND
68.1
ND
ND
ND
ND
ND
ND
ND
ND
9.51 (1.97)
9.29 (0.05)
12.0 (3.50)
10.8 (1.23)
2.09 (0.06)
8.87 (2.66)
5.92 (0.66)
19.1 (2.94)
18.2 (2.36)
8.25 (0.07)
2.45 (0.01)
17.8 (0.52)
10.9 (0.53)
7.09 (0.26)
11.4 (2.81)
7.88 (0.20)
2.44 (0.16)
11.1 (2.22)
7.03 (0.36)
a
8a
8b
ND
ND
8c
6.22 (0.27)
15.4 (3.3)
10.2 (0.14)
11.4 (1.3)
7.35 (0.44)
6.25 (0.40)
10.7 (1.2)
6.86 (1.4)
11.7 (0.33)
8.67 (0.82)
12.7 (0.43)
12.3 (0.62)
12.6 (0.19)
13.5 (3.4)
19.0 (1.2)
8.50 (0.88)
5.13 (0.42)
3.15 (0.46)
8.31 (1.6)
6.55 (0.39)
3.33 (0.11)
5.52 (1.1)
13.1 (4.0)
4.23 (0.33)
23.8 (1.1)
6.23 (0.37)
8.61 (0.55)
13.5 (0.19)
7.53 (1.6)
4.26 (0.17)
12.1 (0.90)
5.75 (0.43)
78.2 (3.5)
ND
8d
8e
ND
8f
ND
9a
56.7 (3.0)
ND
9b
9c
ND
9d
9e
44.1 (1.7)
ND
9f
ND
10a
10b
10c
10d
10e
10f
ND
ND
47.1 (11.4)
ND
ND
ND
a
b
c
d
Ref 19. IC50 values were determined from three independent experiments. IC50 values were determined from a single experiment. IC50 values
were determined from two independent experiments. ND: IC50 > 100 μM.
Figure 2. (A) Cell-cycle analysis of tsFT210 cells in the absence or presence of test compounds. (a) G2/M-arrested cells after a temperature shift for
17 h at 39 °C. (b) DMSO-treated cells after a temperature shift for 4 h at 32 °C. (c) Cells treated with 100 nM nocodazole. (d−l) Cells treated with
5−50 μM RE derivatives 7, 8a, and 10d. (B) Effect of RE derivatives (7, 8a, and 10d) on CDK1 phosphorylation status.
two point mutations on the cdc2 gene of this cell line cause
inactivation of the gene product, CDK1, at the restrictive
temperature of 39 °C.23 In fact, as shown in Figure 2Aa,
incubation of tsFT210 cells at 39 °C for 17 h resulted in
synchronization at G2 phase. The populations of the various
phases of the cell cycle are summarized in the Supporting
Information.20 Release of the G2-synchronized cells was
performed by incubation for 4 h at 32 °C in the presence or
absence of compounds (Figure 2Ab−l). Treatment of 7 and
10d predominantly maintained the G2/M arrest of tsFT210
cells in a concentration-dependent manner (Figure 2Ad−f,j−l)
as compared with the vehicle control (Figure 2Ab). Moreover,
compound 8a, which is a selective inhibitor of VHR without
inhibitory potency for CDC25s in vitro, had little effect on
tsFT210 cells (Figure 2Ag−i), as expected. These results
suggested that inhibition potency for CDC25 phosphatases is
well correlated with G2/M-arresting activity in tsFT210 cells.
To confirm CDC25s inhibition at the cell level, the
phosphorylation status of CDK1, which is a substrate of
CDC25s, was analyzed by Western blotting (Figure 2B).
Inhibition of CDC25s should induce hyperphosphorylation of
CDK1 protein, resulting in deactivation of CDK1 kinase and
cell-cycle arrest. At the restrictive temperature of 39 °C,
hyperphosphorylation of CDK1 was observed (lane a). In the
vehicle control at the permissive temperature of 32 °C, CDK1
proteins were dephosphorylated without change of the total
amount of CDK1 proteins (lane b). In accordance with the
results of cell-cycle analysis and inhibitory activity for CDC25s
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ACS Medicinal Chemistry Letters
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in vitro, 7 and 10d concentration dependently inhibited the
dephosphorylation of CDK1 (lanes d−f and lanes j−l). In
contrast, 8a did not inhibit the dephosphorylation of CDK1
even at 50 μM (lanes g−i). These results indicated that the 7
and 10d acted as CDC25s inhibitors at the cell level and
confirm that small differences in the chemical structure of RE
derivatives markedly influence the selectivity of inhibition of
CDC25s at the cell level. Moreover, potent cytostatic effects of
7 and 10d lacking CDC25C inhibitory activity suggest that
CDC25C may not be important for the G2/M transition in
tsFT210 cells. It is noteworthy that sub-G1 phase cells, which
are indicative of apoptotic cell death, were not increased by
high concentrations of the RE derivatives (Figure 2A and
Supporting Information). Thus, the cytotoxicity of these
compounds appears to be low, which would be very
advantageous for the use of these inhibitors in cellular studies
on CDC25s functions.
As mentioned above, ROS generation and electrophilic
reactivity are important in the inhibition of CDC25s by quinoid
1.13 A decrease of inhibitory potency of 1 was observed when
the concentration of DTT was increased in the assay buffer
used for the evaluation of inhibitory activity against CDC25s,
probably due to direct reaction of DTT with ROS generated by
1 and/or with electrophilic 1 itself. RE derivatives have a
characteristic enamine structure conjugated with two carbonyl
groups and are not expected to generate ROS or to react with
thiol functionality as an electrophile. However, to completely
exclude the possibility of ROS generation or an electrophilic
mechanism for the CDC25s inhibition by RE derivatives, we
examined the effect of DTT concentration on the inhibitory
potency of RE derivatives. In accordance with the previous
report,13 a higher concentration of DTT resulted in an increase
of IC50 value of 1 for CDC25A/B (Figure 3A), although only a
slight change of enzymatic activity of CDC25A/B itself was
observed in the presence of a large amount of DTT. In contrast,
the inhibitory activities of 7 and 10d for CDC25s were
independent of DTT concentration. These results support the
idea that neither electrophilic addition of RE derivatives to the
thiol group in the catalytic site nor ROS generation is involved
in the inhibition of CDC25s by RE derivatives. Finally, we
measured the ROS level of cells untreated or treated with
inhibitors, utilizing the ROS indicator H2DCFDA.20 Upon
pretreatment of tsFT210 cells with H2DCFDA at the restrictive
temperature of 39 °C for 1 h, followed by treatment with 1 (5
μM or 15 μM) at the permissive temperature of 32 °C for 30
min, a 3- (5 μM 1) or 4.4-fold (15 μM 1) increase of
fluorescence intensity as compared to the vehicle control was
observed; this is characteristic of an oxidative burst (Figure 3B).
In contrast, 7, 8a, and 10d did not induce a significant change
of the fluorescence intensity even at the concentration of 50
μM (Figure 3B). These results strongly suggest that RE
derivatives do not generate ROS, and thus, participation of
ROS in the cell cycle-arresting effect of RE derivatives can be
excluded.
Figure 3. (A) Inhibition of CDC25A and CDC25B activities in vitro
in the presence of DTT. (B) ROS generation in tsFT210 cells by RE
derivatives (7, 8a, and 10d, 50 μM) and 1 (15 μM).
CDC25C even at 100 μM. This significant subtype selectivity
among CDC25s should make RE44 a useful tool for biological
research concerning CDC25A/B, especially in combination
with the VHR-selective inhibitor RE12. These inhibitors are
also expected to be new lead molecules for candidate
therapeutic agents. Work to enhance the cellular activity and
to develop more specific inhibitors able to discriminate the
subtypes of CDC25s is under way.
ASSOCIATED CONTENT
■
S
* Supporting Information
Synthesis of compounds, experimental procedure, character-
ization of new compounds, 1H NMR, 13C NMR, and biological
experiment. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Fax: +81-48-462-4666. E-mail: sodeoka@riken.jp.
Funding
This work was supported in part by Project Funding from
RIKEN and a grant from Uehara Memorial Foundation.
In conclusion, we have discovered a CDC25A/B-selective
inhibitor, RE44 (10d), by screening of an RE-focused library
with a nonacidic and nonelectrophilic enamine core structure.
Moreover, it was demonstrated that small structural differences
of RE derivatives could control the selectivity of inhibition for
DSPs, and the enzyme selectivity was correlated with the
activity in tsFT210 cells. Although the cellular activity of RE44
was moderate24 and the specificity over VHR and CDC25A is
not complete, it is noteworthy that RE44 did not inhibit
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Dr. Yushi Futamura (RIKEN-ASI, Japan) for helpful
discussions about the cell-based assay. We also thank the
Material Characterization Team (RIKEN-ASI, Japan) for
elemental analysis of the synthesized compounds.
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ACS Medicinal Chemistry Letters
Letter
(19) Hirai, G.; Tsuchiya, A.; Koyama, Y.; Otani, Y.; Oonuma, K.;
Dodo, K.; Simizu, S.; Osada, H.; Sodeoka, M. Development of a
Vaccinia H1-related (VHR) phosphatase inhibitor with a nonacidic
phosphate-mimicking core structure. ChemMedChem 2011, 6, 617−
622.
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