Inhibition of Cdc25 phosphatases by indolyldihydroxyquinones

Article abstract of DOI:10.1021/jm0300835

Overexpression of the Cdc25A and Cdc25B dual-specificity phosphatases correlates with a wide variety of cancers, making the Cdc25s attractive drug targets for anticancer therapies. However, the search for good lead molecules has been hampered by the reactivity of the active site thiolate anion and the flat solvent-exposed active site region. We describe here the indolyldihydroxyquinones, a new class of inhibitors of Cdc25 that bind reversibly to the active site with submicromolar potency. Structure-activity relationships in the 50 derivatives of the lead molecule 2,5-dihydroxy-3-(1H-indol-3-yl)[1,4]benzoquinone show interesting and consistent trends identifying features required for inhibition of all three isoforms of Cdc25. The compounds do not show time-dependent inhibition, indicating that they form neither covalent adducts with nor oxidize the active site thiol. Our best compounds, 2,5-dihydroxy-3-(7-farnesyl-1H- indol-3-yl)[1,4]benzoquinone and 2,5-dihydroxy-3-(4,6-dichloro-7-farnesyl-1H-indol-3-yl)[1,4]- benzoquinone, are competitive with substrate for the active site and yield K_is of 640 and 470 nM, respectively. Binding of the indolylhydroxyquinones is diminished by three, but not by six other, specific mutations in the active site region. Additionally, the flexible C-terminal tail required for binding of protein substrate is also required for binding derivatives with hydrophobic modifications at the 7-position. The indolyldihydroxyquinones compete effectively with the protein substrate for Cdc25 in vitro and lead to rapid cell death in vivo. Thus, the indolyldihydroxyquinones will serve as useful lead molecules for drug discovery and further cell-based studies on the role of Cdc25s in cell cycle control.

Full text of DOI:10.1021/jm0300835

2580
J . Med. Chem. 2003, 46, 2580-2588
In h ibition of Cd c25 P h osp h a ta ses by In d olyld ih yd r oxyqu in on es
J ungsan Sohn, Brendan Kiburz, Zhitao Li, Liu Deng, Alexias Safi, Michael C. Pirrung, and J ohannes Rudolph*
Departments of Biochemistry and Chemistry, Duke University, and Duke University Medical Center,
Durham, North Carolina 27710
Received February 19, 2003
Overexpression of the Cdc25A and Cdc25B dual-specificity phosphatases correlates with a wide
variety of cancers, making the Cdc25s attractive drug targets for anticancer therapies. However,
the search for good lead molecules has been hampered by the reactivity of the active site thiolate
anion and the flat solvent-exposed active site region. We describe here the indolyldihydrox-
yquinones, a new class of inhibitors of Cdc25 that bind reversibly to the active site with
submicromolar potency. Structure-activity relationships in the 50 derivatives of the lead
molecule 2,5-dihydroxy-3-(1H-indol-3-yl)[1,4]benzoquinone show interesting and consistent
trends identifying features required for inhibition of all three isoforms of Cdc25. The compounds
do not show time-dependent inhibition, indicating that they form neither covalent adducts
with nor oxidize the active site thiol. Our best compounds, 2,5-dihydroxy-3-(7-farnesyl-1H-
indol-3-yl)[1,4]benzoquinone and 2,5-dihydroxy-3-(4,6-dichloro-7-farnesyl-1H-indol-3-yl)[1,4]-
benzoquinone, are competitive with substrate for the active site and yield Kis of 640 and 470
nM, respectively. Binding of the indolylhydroxyquinones is diminished by three, but not by six
other, specific mutations in the active site region. Additionally, the flexible C-terminal tail
required for binding of protein substrate is also required for binding derivatives with
hydrophobic modifications at the 7-position. The indolyldihydroxyquinones compete effectively
with the protein substrate for Cdc25 in vitro and lead to rapid cell death in vivo. Thus, the
indolyldihydroxyquinones will serve as useful lead molecules for drug discovery and further
cell-based studies on the role of Cdc25s in cell cycle control.
Cancer at its most basic level is a rampant and
undesired growth of cells that leads to a serious and
life-threatening disruption of the normal function of
multicellular organisms. For this reason, much effort
has been devoted to understanding the processes of
normal cell cycle regulation and how disruption of these
processes contributes to uncontrolled cell growth. In the
elucidation of these control mechanisms, the cyclin-
dependent kinases (Cdks)1 were found to be the central
regulators of the eukaryotic cell cycle.1 The kinase
activity of the Cdks is directly responsible for the
initiation and progression of successive phases of the
cell cycle by modification of proteins responsible for the
activation of numerous structural and regulatory genes.
Given their central role in controlling cell cycle progres-
sion, it was not surprising to find that numerous other
regulatory proteins modulate the activity of these
protein kinases. Among these regulatory proteins, the
Cdc25 phosphatases were found to be particularly
important in that they are responsible for the final
activation step of the Cdks by their dephosphorylation
of two inhibitory phosphorylation sites on the Cdks.2
There exist three phase-specific isoforms of Cdc25 in
human cells. Cdc25A is responsible for the initiation of
DNA synthesis in the S-phase of the cell cycle whereas
Cdc25B and Cdc25C are regulators of the G2/M transi-
tion.
Cdc25B showed that they were proto-oncogenes and
targets of c-myc.3,4 Overproduction of both Cdc25A and
Cdc25B has been found in a high percentage of breast
cancer patients, where elevated expression levels cor-
relate with reduced life expectancy.3,5 Cdc25 overex-
pression has also been associated with gastric carcino-
mas,6 colon cancer,7,8 non small cell lung carcinoma,9
and aggressive non-Hodgkin’s lymphomas.10 Additional
support for the importance of Cdc25 phosphatase activ-
ity has come from forced overexpression of Cdc25B in
mouse models11,12 and studies correlating Cdc25 inhibi-
tion by small molecules with growth arrest or cell cycle
blocks.13-17
Because of their important role in cell cycle regulation
and their correlation with a wide variety of cancers, the
Cdc25s have been attractive targets for drug develop-
ment.18,19 However, despite extensive efforts by both
academic and corporate researchers, there has been a
dearth of potent, specific, active site-directed inhibitors
of Cdc25 for use in studies of Cdc25 function or in the
development of pharmaceuticals. For many years, so-
dium orthovanadate, a broad-spectrum protein phos-
phatase inhibitor, was the only available Cdc25 phos-
phatase inhibitor, and it is still being used in many cell-
based assays to achieve cell cycle blocks. Inhibitors
derived from natural products such as dysidiolide 16,20,21
,
sulfiricin,22 and vitamin K-derivatives 13,15,23 are either
irreproducible, weak, or form irreversible adducts with
Cdc25. Other inhibitors derived from combinatorial
libraries based on Ser/Thr kinase inhibitors,24 libraries
of dipeptidyl phosphonates,25 or steroidal derivatives17,26
have been described that do not break the nanomolar
As important cell cycle regulators, Cdc25A and Cdc25B
have been consistently linked to cancer in a variety of
ways. The initial discovery of human Cdc25A and
* To whom correspondence should be addressed. E-mail: rudolph@
biochem.duke.edu.
10.1021/jm0300835 CCC: $25.00 © 2003 American Chemical Society
Published on Web 05/21/2003

Inhibition of Cdc25 Phosphatases
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13 2581
Sch em e 1. Synthetic Route to Compounds 8J and 8L
barrier, lack specificity toward Cdc25, and/or show
noncompetitive inhibition. Even apparently more potent
compounds, such as the quinolinediones27 and the
naphthoquinones,28 derived from a screen of the Na-
tional Cancer Institute Diversity Set, exhibit mixed
inhibition kinetics and appear at least in part to be
irreversible.29
indol-3-yl)[1,4]benzoquinone), reversible active-site
directed inhibition, submicromolar binding constants,
and effective inhibition of the natural protein substrate
make this family of compounds an attractive lead for
the development of specific Cdc25 inhibitors.
Exp er im en ta l Section
The lack of real success in the discovery of reversible
active-site directed inhibitors for Cdc25 can be at-
tributed to two defining characteristics of this phos-
phatase. First, Cdc25 as a dual-specificity phosphatase
containing the active site motif HCX5R is highly sus-
ceptible to inactivation by covalent modification and
oxidation. In this motif, H is a highly conserved histidine
residue, C is the catalytic cysteine, the five X residues
form a loop whose backbone amides hydrogen bond to
the phosphate of the substrate, and R is a highly
conserved arginine that is required for binding of the
phosphate and transition state stabilization. The cata-
lytic cysteine forms a covalent phosphocysteine inter-
mediate in a two-step reaction mechanism and exists
as a thiolate anion in the free enzyme.30 This active-
site thiolate is the source of Cdc25s susceptibility to
covalent modification and oxidation.31 Thus, assays of
Cdc25 in the absence of reducing agents or containing
reactive compounds are highly likely to demonstrate
covalent and irreversible inhibition, a trait generally
avoided in drug discovery and inhibitor development.
The second reason Cdc25 has proven to be a difficult
target in the development of inhibitors is the open and
somewhat featureless architecture of its active site. The
crystal structures of the catalytic domains of Cdc25A32
and Cdc25B33 show an exposed active site region not
suitable for structure-based design of compounds with
complementary binding surfaces. There are no apparent
phospho-threonine or phospho-tyrosine binding pockets,
let alone two adjacent pockets as suggested by the
preference of Cdc25 for bis-phosphorylated protein
substrate.34 The open architecture of the active site is
reflected in the low and nonspecific activity of Cdc25
toward phospho-peptide substrates and suggests that
peptide mimetics are not a viable route to Cdc25
inhibition.34
Com p ou n d s. Synthesis, purification, and characterization
of all the indolyldihydroxyquinones derivatives except 8J and
8L have been described previously.35 Syntheses of 8J and 8L
were performed using materials and methods as described
previously,35 as shown in Scheme 1, and as further elucidated
below. NSC668394 (Figure 1) was obtained from the Drug
Synthesis & Chemistry Branch, Developmental Therapeutics
Program, Division of Cancer Treatment and Diagnosis, Na-
tional Cancer Institute.
2-Ch lor o-6-n itr op h en yla m in e.36 1-Chloro-3-nitrobenzene
(3.1546 g, 20 mmol) and methoxyamine hydrochloride (2.02
g, 24 mmol) were dissolved in DMF (15 mL). The solution was
added dropwise in 15 min to a suspension of cuprous chloride
(209.5 mg, 2.1 mmol) and potassium tert-butoxide (11.07 g,
98.6 mmol) in DMF (35 mL) cooled in a 15 °C bath. The cold
bath was removed, and the mixture was stirred for 1 h. The
reaction was quenched with aqueous saturated ammonium
chloride and extracted with ethyl acetate. After being washed
with brine, the organic layer was separated and dried over
Na2SO4, and the solvent was removed under vacuum. After
flash column chromatography, the desired product was
obtained as a light yellow solid (1.601 g, 46%). mp 72-73 °C.
1H NMR (CDCl3): 8.06 (1H, dd, J ) 8.7, 1.5 Hz), 7.50 (1H, dd,
J ) 7.8, 1.5 Hz), 6.64 (1H, dd, J ) 8.7, 7.8 Hz), 6.4-6.5 (2H,
br s).
1-Ch lor o-2-iod o-3-n itr oben zen e.37 2-Chloro-6-nitrophe-
nylamine (1.87 g, 10.8 mmol) was dissolved in HCl (37%, 20
mL) and cooled in an ice bath. Then a solution of NaNO2 (1.27
g, 18.4 mmol) in water (8 mL) was added very slowly with
stirring. After 15 min the mixture was filtered through glass
wool into a solution of KI (7.8 g, 47 mmol) in water (20 mL).
The resulting orange mixture was stirred at room tempera-
ture overnight, then extracted with ethyl acetate and subse-
quently washed twice with 10% NaOH and once with brine.
The organic layer was dried over Na2SO4, and the solvent was
removed under vacuum. After flash column chromatography,
the desired product was obtained as a yellow solid (2.29 g,
75%). mp 101-102 °C. 1H NMR (CDCl3): 7.66 (1H, dd,
J ) 7.8, 1.8 Hz), 7.52 (1H, dd, J ) 7.8, 1.8 Hz), 7.42 (1H, t, J
) 7.8 Hz).
1-Ch lor o-2-(3-m eth ylbu t-2-en yl)-3-n itr oben zen e. 1-Chlo-
ro-2-iodo-3-nitrobenzene (711.8 mg, 2.5 mmol) and Pd(PPh3)4
(580.3 mg, 0.5 mmol) were dissolved in DMF (10 mL). Argon
was bubbled through the solution for 10 min to remove oxygen.
Tributyl(3-methyl-but-2-enyl)stannane (1.06 g, 2.95 mmol) was
added dropwise. The solution was then heated to 90 °C. After
incubation overnight, the mixture was diluted with ethyl
acetate and washed with saturated aqueous KF solution twice.
In this manuscript we describe the discovery and
characterization of a novel class of compounds, the
indolyldihydroxyquinones, that inhibit the Cdc25 phos-
phatases with surprising potency. Extensive and con-
sistent structure-activity relationships with 50+ de-
rivatives of the parent molecule (2,5-dihydroxy-3-(1H-

2582 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13
Sohn et al.
The organic layer was dried over Na2SO4, and the solvent was
removed under vacuum. After flash column chromatography,
the desired product was obtained as a light yellow oil (496.5
mg, 88%). 1H NMR (CDCl3): 7.63 (1H, dd, J ) 8.1, 1.2 Hz),
7.58 (1H, dd, J ) 8.1, 1.2 Hz), 7.27 (1H, t, 8.1 Hz), 5.05-5.14
(1H, m), 3.66 (2H, d, J ) 6.6 Hz), 1.74 (3H, m), 1.70 (3H, m).
13C NMR (CDCl3): 151.4, 136.4, 134.4, 133.8, 133.5, 127.3,
122.6, 119.1, 28.7, 25.6, 17.5.
solution twice. The organic layer was dried over Na2SO4, and
the solvent was removed under vacuum. After flash column
chromatography, the desired product was obtained as a light
1
yellow oil (565.9 mg, 83%). H NMR (CDCl3): δ 7.62 (1H, d, J
) 2.4 Hz), 7.57 (1H, d, J ) 2.1 Hz), 4.8-5.6 (1H, m), 3.60 (2H,
d, J ) 6.6 Hz), 1.71 (3H, m), 1.67 (3H, m). 13C NMR (CDCl3):
δ 151.6, 137.1, 134.9, 134.5, 133.2, 132.6, 122.9, 118.6, 28.4,
25.6, 18.1.
6-Ch lor o-7-(3-m eth ylbu t-2-en yl)-1H-in d ole. 1-Chloro-2-
(3-methylbut-2-enyl)-3-nitrobenzene (184.7 mg, 0.82 mmol)
was dissolved in THF (8 mL) and cooled to -48 °C. Under a
N2 stream ,vinylmagnesium bromide (0.8 M, 4.5 mL, 3.6 mmol)
was added in one portion. After being stirred for 30 min at
the same temperature, the reaction was quenched with
saturated aqueous NH4Cl before allowing the mixture to warm
to room temperature and extracting it with ethyl acetate. The
organic layer was washed with saturated aqueous NH4Cl and
brine and dried over Na2SO4, and the solvent was removed
under vacuum. After flash column chromatography, the de-
sired product was isolated as a light yellow oil (68 mg, 38%).
1H NMR (CDCl3): 8.21 (1H, br s), 7.43 (1H, d, J ) 8.7 Hz),
7.19 (1H, dd, J ) 3.0, 2.4 Hz), 7.14 (1H, d, J ) 8.4 Hz), 6.54
(1H, dd, J ) 3.0, 2.1 Hz), 5.27-5.38 (1H, m), 3.75 (2H, d, J )
6.9 Hz), 1.90 (3H, m), 1.78 (3H, m). 13C NMR (CDCl3): 136.1,
133.8, 127.2, 126.8, 124.8, 121.8, 121.7, 121.5, 119.6, 103.3,
28.4, 25.9, 18.4.
3-[6-Ch lor o-7-(3-m et h ylb u t -2-en yl)-1H -in d ol-3-yl]-2,5-
d ih yd r oxy-[1,4]ben zoqu in on e (8J ). 6-Chloro-7-(3-methyl-
but-2-enyl)-1H-indole (33 mg, 0.15 mmol) was dissolved in
acetonitrile (0.2 mL) and THF (1 mL) at room temperature.
H2SO4 (8 µL, 0.15 mmol) was added, followed by 2,5-dichloro-
[1,4]benzoquinone (53.1 mg, 1.0 mmol) under a N2 stream. The
solution was stirred for 24 h followed by addition of silver
carbonate (∼50 wt % on Celite, 165 mg, 0.3 mmol). After 2 h,
the mixture was diluted with ethyl acetate and filtered. The
filtrate was washed with saturated aqueous NaHCO3 and
brine. The organic layer was dried over Na2SO4, and the
solvent was removed under vacuum. After flash chromatog-
raphy, the desired product was isolated and dissolved in
methanol (3 mL). The solution was brought to reflux and 10%
NaOH (1.5 mL) was added dropwise. After 30 min, the solution
was poured into cold water (20 mL), acidified with 10% H2-
SO4, and extracted with ethyl acetate. The organic layer was
washed with brine and dried over Na2SO4, and the solvent was
removed under vacuum. After flash column chromatography
(oxalic acid-coated silica gel), the desired product was isolated
as a red solid (10 mg, two-step yield 19%). 1H NMR (d6-
acetone): 10.55 (1H, br s), 9.0-10.2 (2H, br s), 7.39 (1H, d, J
) 3.0 Hz), 7.35 (1H, d, J ) 8.4 Hz), 7.04 (1H, d, J ) 8.4 Hz),
6.01 (1H, s), 5.19-5.27 (1H, m), 3.76 (1H, d, J ) 6.9 Hz), 1.83
(3H, d, J ) 0.9 Hz), 1.67 (3H, d, J ) 0.9 Hz).
4,6-Dich lor o-7-(3-m eth ylbu t-2-en yl)-1H-in d ole.
1,5-
Dichloro-2-(3-methylbut-2-enyl)-3-nitrobenzene (582.4 mg, 2.2
mmol) was dissolved in THF (16 mL) and cooled to -48 °C
under N2. Vinylmagnesium bromide (0.8 M, 8.4 mL, 6.7 mmol)
was added in one portion. After being stirred for 30 min, the
reaction was quenched with saturated aqueous NH4Cl before
allowing the mixture to warm to room temperature. After
extraction with ethyl acetate, the organic layer was washed
with saturated aqueous NH4Cl and brine and dried over Na2-
SO4, and the solvent was removed under vacuum. After flash
column chromatography, the desired product was isolated as
a light yellow oil (220 mg, 39%). 1H NMR (CDCl3): δ 8.29 (1H,
br s), 7.19 (1H, dd, J ) 2.4, 3.3 Hz), 7.15 (1H, s), 6.59 (1H, dd,
J ) 2.1, 3.3 Hz), 5.2-5.3 (1H, m), 3.67 (2H, d, J ) 6.9 Hz),
1.84 (3H, m), 1.74 (3H, m). 13C NMR (CDCl3): δ 136.0, 134.0,
126.7, 125.5, 125.0, 124.0, 120.8, 120.7, 120.5, 101.8, 27.8, 25.6,
18.1.
3-[4,6-Dich lor o-7-(3-m eth ylbu t-2-en yl)-1H-in d ol-3-yl]-
2,5-d ih yd r oxy-[1,4]ben zoqu in on e (8L). 4,6-Dichloro-7-(3-
methylbut-2-enyl)-1H-indole (130.4 mg, 0.5 mmol) was dis-
solved in acetonitrile (2 mL) and THF (0.5 mL) at room
temperature under N2. H2SO4 (26 µL, 0.5 mmol) was added,
followed by 2,5-dichloro-[1,4]benzoquinone (176.7 mg, 1.0
mmol). The solution was stirred for 5 d, and then silver
carbonate (∼50 wt. % on Celite, 550 mg, 1 mmol) was added.
After 2 h, the mixture was diluted with ethyl acetate and
filtered. The filtrate was washed with saturated aqueous
NaHCO3 and brine. The organic layer was dried over Na2SO4,
and the solvent was removed under vacuum. After flash
chromatography, the desired product was isolated and dis-
solved in methanol (3 mL). The solution was brought to reflux
and 10% NaOH (1.5 mL) was added dropwise. After 30 min,
the solution was poured into cold water (20 mL), acidified with
10% H2SO4, and extracted with ethyl acetate. The organic layer
was washed with brine, and dried over Na2SO4, and the solvent
was removed under vacuum. After flash column chromatog-
raphy (oxalic acid-coated silica gel), the desired product was
1
isolated as a red solid (27 mg, two-step yield 15%). H NMR
(CDCl3): δ 10.74 (1H, br s), 8.4-10.0 (2H, br s), 7.39 (1H, d, J
) 3.0 Hz), 7.07 (1H, s), 6.04 (1H, s), 5.16-5.25 (1H, m), 3.74
(1H, d, J ) 6.9 Hz), 1.82 (3H, d, J ) 0.9 Hz), 1.68 (3H, d, J )
1.2 Hz).
En zym es. Cdc25A, Cdc25B, and Cdc25C were prepared as
previously described.34,39,40 All three isoforms are prepared as
native proteins consisting of the catalytic domains starting
from residue 323 for Cdc25A, 377 for Cdc25B, and residue 280
for Cdc25C and terminating at the natural C-termini. The
truncated catalytic domain of Cdc25B has been described
previously.39 Site-directed mutations of Cdc25B were gener-
ated using the appropriate primers and site-directed mutagen-
esis as described previously.39 Expression, purification, and
detailed characterization of F475A, R482L, R492L, Y528F,
M531A, N532A, and R544L will be described elsewhere
(manuscript in preparation).
In h ibition Assa ys. Cdc25 was assayed using 3-O-meth-
ylfluorescein phosphate (mFP) as a substrate in time-depend-
ent assays monitoring product formation at 477 nm following
the reaction under initial velocity conditions (2-5% turnover).
IC50 determinations were performed using eight different
concentrations of inhibitor at an mFP concentration of 50 µM
(∼5 × Km) and the data were fitted to eq 1. Ki determinations
were performed using mFP concentrations from 2 to 150 µM
and inhibitor concentrations as indicated in the figure legends.
The data were fitted to models for competitive, uncompetitive,
and noncompetitive inhibition using Cleland’s equations in the
Kinet-A-Syst package.41 Assays using the protein substrate
1,5-Dich lor o-2-iod o-3-n itr oben zen e.38 2,4-Dichloro-6-ni-
trophenylamine (2.14 g, 10.3 mmol) was dissolved in HCl (37%,
20 mL) and cooled in an ice bath. A solution of NaNO2 (1.27 g,
18.4 mmol) in water (8 mL) was added very slowly with
stirring. After 15 min the mixture was filtered through glass
wool into a solution of KI (7.5 g, 45.2 mmol) in water (20 mL).
The resulting orange mixture was stirred at room-temperature
overnight and extracted with ethyl acetate. The organic layer
was washed twice with 10% NaOH and once with brine. It
was then dried over Na2SO4, and the solvent was removed
under vacuum. After flash column chromatography, the de-
sired product was obtained as a yellow solid (2.42 g, 76%). Mp
1
56-57 °C. H NMR (CDCl3): δ 7.63 (1H, d, J ) 2.1 Hz), 7.50
(1H, d, J ) 2.4 Hz). MS (EI): calcd for C6H2Cl2INO2: 316.85,
found 317.
1,5-Dich lor o-2-(3-m eth ylbu t-2-en yl)-3-n itr oben zen e. 1,5-
Dichloro-2-iodo-3-nitrobenzene (830.5 mg, 2.6 mmol) and Pd-
(PPh3)4 (604.5 mg, 0.5 mmol) were dissolved in DMF (10 mL).
Argon was bubbled through the solution for 10 min to remove
oxygen. Tributyl(3-methyl-but-2-enyl)stannane (1.28 g, 3.6
mmol) was added dropwise. The solution was then heated to
65 °C. After incubation overnight, the mixture was diluted
with ethyl acetate and washed with saturated aqueous KF

Inhibition of Cdc25 Phosphatases
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13 2583
Ta ble 1. Percent Inhibition of Cdc25A, Cdc25B, and Cdc25C at 50 µM Indolyldihydroxyquinonesa
compd
modification
Cdc25A Cdc25B Cdc25C
compd
modification
6-benzyloxy
6-methyl
7-fluoro
7-chloro
7-bromo
7-methyl
7-propyl
7-prenyl
7-geranyl
7-farnesyl
7-benzyl
7-(2-methyl-benzyl)
7-tert-butyl
7-phenyl
Cdc25A Cdc25B Cdc25C
2A
2B
2C
2D
2E
2F
2G
2H
3A
3B
3C
3D
3E
3F
3G
3H
4A
4B
4C
4D
4F
4G
4H
5A
5B
none
33
18
24
15
22
7
75
42
9
69
19
5
14
0
0
0
0
22
18
21
0
5C
5D
5E
5F
5G
5H
6A
6B
6C
6D
6E
6F
6G
6H
7A
7B
7C
7D
7E
7F
7G
7H
8A
8B
8C
82
66
100
0
11
0
97
16
67
23
45
0
92
28
68
54
11
1
1-methyl
2-methyl
2-ethyl
36
32
13
17
11
11
38
0
5
21
6
21
0
9
2-cyclopropyl
2-isopropyl
2-(1-methylcyclopropyl)
2-tert-butyl
2-(1-methylcyclohexyl)
2-phenyl
2-(1,1, dimethyl-allyl)
4-fluoro
4-chloro
0
72
100
86
96
100
100
93
99
0
98
18
0
0
0
23
99
95
89
97
97
33
92
15
97
20
0
79
84
96
85
98
94
37
91
13
85
25
31
30
0
30
44
40
12
20
22
51
40
48
39
39
59
83
0
0
4-bromo
54
19
2
28
23
45
26
0
4-methoxy
4-benzyloxy
4-methyl
5-fluoro
5-chloro
7-methoxy
7-benzyloxy
2,5-dimethyl
2-methyl-5-methoxy
2-methyl-5-chloro
2,6-dimethyl
2,7-dimethyl
5,6-methylenedioxy
5,6-dimethoxy
6,7-dimethyl
benzo[g]
32
32
55
0
42
16
0
5-bromo
5-methoxy
5-benzyloxy
5-methyl
6-fluoro
6-chloro
0
0
100
100
97
100
50
61
90
92
26
55
48
81
99
54
9
88
19
32
79
63
20
20
75
99
a
Each compound was tested in a time-dependent assay and compared with a simultaneous control reaction containing no inhibitor. By
testing selected compounds in duplicate the average error is estimated at (7%.
Cdk2-pTpY/CycA were performed as IC50’s using eight differ-
ent inhibitor concentrations in time-dependent assays with 300
nM Cdk2-pTpY/CycA in a modification of the previously
described procedure.34 To avoid assaying compounds in the
presence of 1 mg/mL bovine serum albumin, this carrier
protein was added after the TCA quench. Assays using protein
tyrosine phosphatase 1B, a generous gift from Zhong-Yin
Zhang (Albert Einstein College of Medicine), were performed
in the same buffer as for Cdc25, and product formation was
monitored at 410 nm using 200 µM p-nitrophenyl phosphate
as a substrate.
Cell-Ba sed Assa ys. HEK293 cells were cultured on poly-
L-lysine coated plates in MEM media supplemented with 10%
fetal bovine serum and 0.1 mM nonessential amino acids. Cell
synchronization in G1/S was achieved using a double thymi-
dine block.42 Cell lysis was performed in RIPA buffer supple-
mented with protease and phosphatase inhibitors. The phospho-
Cdk-specific antibody was obtained from Cell Signaling
Laboratories. Cell staining for apoptosis using TUNEL was
performed according to the manufacturer’s instructions (Boe-
hringer Mannheim). Caspase activation was measured using
the Apo-ONE homogeneous caspase-3/7 assay and 50000
HEK293 cells/well according to the manufacturer’s instructions
(Promega).
(Figure S1, Supporting Information). The percent inhi-
bition at 50 µM ranged from 0 to 100%, indicating a
broad range of inhibitory activities for this class of
compounds (Table 1). Using protein tyrosine phos-
phatase 1b (PTP1b) and compounds 2A and 6B, we saw
no significant inhibition at 50 µM, indicating that this
family of compounds is not a general nonspecific inhibi-
tor of cysteine phosphatases (data not shown).
We next tested selected compounds using eight dif-
ferent inhibitor concentrations ranging from 0.1 to 50
µM to address in more detail the interactions of these
compounds with Cdc25. Again, all activities were linear
in the presence of the compounds. These studies provide
reproducible IC50s along with Hill slopes and display
some differentiation among the three Cdc25s (Figure
S2, Supporting Information, and Table 2). To compare
our compounds against other reported inhibitors of
Cdc25, we tested NSC668394 (Figure 1) under our
assay conditions and obtained IC50 ranging from 15 to
35 µM (Table 2). The origin of the 50-fold discrepancy
with the published report is unknown.27
Resu lts
On the basis of the significantly improved potency for
modifications in the 7-position and the favorable Hill
slope characteristics of chloro-modifications to the indole
ring, we generated two second generation compounds,
namely 8J and 8L. Compounds 8J and 8L were
subjected to the same eight-point IC50 tests as the other
compounds and displayed improved potency toward
Cdc25B and Cdc25C but not Cdc25A (Table 2).
The core molecule in the library is 2,5-dihydroxy-3-
(1H-indol-3-yl)[1,4]benzoquinone (2A) (Figure 1). The
library of 50 derivatives with a variety of single and
double modifications of differing positions on the indole
ring are listed in Table 1. We first screened the library
of 50 compounds against Cdc25A, Cdc25B, and Cdc25C
by measuring the amount of inhibition seen against the
artificial substrate mFP (50 µM). Although formation
of methylfluorescein can be readily measured by fluo-
rescence spectroscopy, we have chosen to perform our
assays using UV-vis spectroscopy to avoid potential
artifacts induced by fluorescence quenching of meth-
ylfluorescein by the inhibitors. All measured activities
in the presence of inhibitors were linear over the time
course of the assay indicating that these compounds
were not causing time-dependent inactivation of Cdc25
To address specifically in more depth the question of
reversibility, we further tested compounds 2A, 6B, and
8L. Following preincubation of 320 nM Cdc25B with
4-50 µM inhibitor for varying lengths of time, the
enzyme was diluted 100-fold into a reaction mixture
containing 50 µM mFP. In comparison to control experi-
ments lacking inhibitor, there was no significant loss
in activity, in particular for 6B and 8L, indicating that
these inhibitors are reversible (Figure 2). Additionally,

2584 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13
Sohn et al.
Ta ble 2. Eight-Point IC50’s (µM) of Selected
Indolyldihydroxyquinones against Cdc25A, Cdc25B, and
Cdc25Ca
compd
modification
none
2-cyclopropyl
4-chloro
Cdc25A Cdc25B Cdc25C
2A
2E
3E
4D
4G
4H
5B
6A
6B
6C
6D
6E
6F
6G
6H
7B
8C
8J
25
35
28
18
>50
>50
30
>50
>50
5-bromo
5.8
n.d.
n.d.
5-benzyloxy
5-methyl
6-chloro
7-propyl
7-prenyl
7-geranyl
7-farnesyl
7-benzyl
7-(2-methyl-benzyl)
7-tert-butyl
7-phenyl
7-benzyloxy
benzo[g]
5.5
8.1
4.2
4.2
4.1
1.0
2.4
2.3
1.0
7.2
2.5
4.2
2.5
4.0
5.0
n.d.
n.d.
14
59
5.6
n.d.
2.4
2.3
1.0
7.2
2.5
4.2
n.d.
n.d.
40
>50
15
10
3.5
15
3.5
>50
27
6.9
15
11
8.8
35
F igu r e 2. Reversibility of Cdc25 Inhibition. Cdc25B was
incubated with an excess of inhibitor at the concentration
indicated for varying lengths of time, as indicated. The
remaining activity was measured by dilution of the enzyme
100-fold into a reaction mixture and measuring the activity
with mFP as a substrate. The data represent the average of
2-5 separate determinations, depending on the compound
tested.
24
7.2
2.3
6-chloro-7-prenyl
4,6-dichloro-7-prenyl
8L
NSC668394
15
35
a
The values shown are the mean of three separate determina-
tions with errors averaging 20%.
Ta ble 3. Ki Values Obtained from Fits to the Equation
Describing Competitive Inhibition for Selected
Indolyldihydroxyquinones Using MFP as a Substrate and IC50
Determinations Using Cdk2-pTpY/CycA as a Substrate against
Cdc25B
IC50 vs
copmd
modification
Ki vs mFP
Cdk2-pTpY/CycA
2A
6B
8L
none
13.0 ( 1.9 µM
0.64 ( 0.13 µM
0.43 ( 0.07 µM
>50 µM
28.2 ( 0.6 µM
8.6 ( 3.1 µM
6-prenyl
4,6-dichloro-
7-prenyl
conditions and the equation that applies to reversible
competitive inhibitors.43
To provide further confirmation that the inhibitors
were active site directed and in order to map the binding
site for these inhibitors, we tested the activity of
compound 6B in eight-point IC50 assays using nine
different site-directed mutants of Cdc25B. All but
Arg492 are located in close proximity to the active site
thiol as seen in Figure 3. Additionally, we tested the
activity toward a truncated form of Cdc25B lacking the
last 17 residues. The mutants E474Q, F475A, and
R482L and the truncated mutant showed significantly
altered activity toward 6B without demonstrating sig-
nificantly altered activity toward the small molecule
substrate mFP (Table 4). When we further tested the
three site-directed mutants with altered potencies using
the parent molecule 2A, we observed similar losses in
potency (Table 4). In contrast, the truncated mutant
showed no significant difference in its inhibition by 2A
or 6B (Table 4).
Given the much higher reactivity of Cdc25 toward its
protein substrate Cdk2-pTpY/CycA in comparison to the
artificial substrate mFP,34 we tested the activity of
compounds 2A, 6B, and 8L using the native protein
substrate. The IC50 were found to be >50 µM, 28.2 (
0.6 and 8.6 ( 3.1, respectively (Table 3).
Finally, to evaluate the in vivo effect of these com-
pounds, we tested these compounds on the human cell
line HEK293, embryonic kidney cells. Incubation of 50
but not 25 µM of 6B in the absence of serum led to cell
death within 4-6 h as indicated by rounding up of cells
F igu r e 1. Structures of Cdc25 inhibitors. The unmodified
indolyldihydroxyquinone that forms the core structure for the
compounds described in this report is 2A. The carbons on the
indole ring are numbered in accord with the substitutions
indicated in Table 1. Compound 5, NSC668394, and NSC95397
have been described previously.15,27,28
incubation of 1 µM of 8J in the presence of 10 µM
Cdc25B did not change the UV-vis absorbance spectra
of 8J , suggesting that a reversible covalent adduct is
not formed (data not shown). Control experiments using
NSC668394 (25 µM) or hydrogen peroxide (200 µM) that
modify the active site cysteine by alkylation or oxida-
tion, respectively, showed time-dependent loss of activ-
ity (Figure 2).
To elucidate the kinetic mechanism of inhibition, we
tested compounds 2A, 6B, and 8L by varying the
concentrations of both the substrate mFP and the
inhibitors. All the compounds exhibited competitive
inhibition (Figure S3, Supporting Information). No
adequate fits could be obtained when the primary data
were fitted to equations for noncompetitive or uncom-
petitive inhibition as judged by variance, relative errors
in fitted values (>100%), and/or prediction of negative
inhibition constants. As shown in Table 3, the inhibition
constants (Ki), equal to the dissociation constants (Kd)
for competitive inhibitors, are approximately 5-10-fold
lower than the IC50 values, consistent with the assay

Inhibition of Cdc25 Phosphatases
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13 2585
F igu r e 3. Active site region of Cdc25B. Mutated residues near
the active site are labeled and shown as sticks, with the
remainder of the protein shown as a cartoon. (Arg492 is not
seen in this view.) A sulfate ion as seen in the crystal structure
of the catalytic domain of Cdc25B is shown bound in the active
site pocket. The coordinates of Cdc25B were taken from 1qb0
and the figure prepared using RasMol.33
F igu r e 4. Rapid apoptosis as measured by caspase 3/7
activation is caused by treatment with 6B for 4 h at 50 µM,
but not 25 µM following. The negative control contains only
DMSO and the positive control consists of overnight incubation
with 5 µM staurosporine.
Ta ble 4. Eight-Point IC50’s (µM) vs Various Mutants of
Cdc25B for Compounds 2A and 6Ba
feature based on the following observations regarding
the extended aromatic ring structure. First, although
Cdc25 has good activity toward the aromatic substrate
mFP, burst kinetics have shown that the affinity (Kd)
for mFP is very weak (>1 mM).39,44 Thus, the high
reactivity toward mFP probably relies mostly on the low
pKa of the leaving group (pKa ) 4.5) and the unusual
pH-dependence of this enzyme, not on any intrinsic
affinity for large aromatic ring structures.30,39 Second,
in overlaying the quinone rings of our indolyldihydrox-
yquinones with those of NSC668394 or NSC95397, for
example, the extended aromatic structures, the indole
ring in the former and the naphthoquinone ring in the
latter, do not overlap. Similarly, overlaying the aromatic
parts of these two classes of molecules yields highly
divergent structures in three-dimensional space.
Although the quinone ring appears to be the common
feature between the previously described Cdc25 inhibi-
tors and our indolyldihydroxyquinones, we believe that
the detailed mode of inhibition is fundamentally differ-
ent by being reversible and noncovalent. The broadly
known electrophilic properties of quinones suggest the
possibility of a reaction with nucleophilic groups such
as the essential thiolate in the active site of Cdc25.
Alternatively, the ability of quinones to be readily
reduced might be related to their activity toward Cdc25,
as large classes of anticancer agents (such as anthro-
quinones) are known to undergo reductive activation,
and the reactions of quinones with thiols have been
investigated.45,46 However, the reactivity of the in-
dolyldihydroxyquinones and the naphthoquinones are
divergent in both these respects. First, the former are
quite electron rich, with two electron-donating hydroxy
groups and an electron-donating indole substitutent
making them much less likely to accept nucleophiles.47
Second, the redox properties of the indolyldihydrox-
yquinones have been investigated. It has been reported
that the indolyldihydroxyquinones do not sustain redox
cycling (cyclic voltammetry) in aqueous DMF.48 For
example, we have observed that the chemically similar
molecule demethylasterriquinone B1 is oxidized at
mutant
WT
IC50 vs 6B
IC50 vs 2A
6.4
>50
>50
6.5
40
11
15
6.5
3.9
12
18
200
76
n.d.
73
n.d.
n.d.
n.d.
n.d.
n.d.
34
E474Q
F475A
E478Q
R482L
R492L
Y528F
M531A
N532A
R544L
truncated
33
a
The values shown are the mean of two separate determina-
tions with errors averaging 28%.
and their loss of adhesion to the plate. HEK293 cells
assayed for apoptosis by monitoring caspase activation
showed a significant increase in caspase activity fol-
lowing 4 h of treatment with 50 µM, but not 25 µM, of
6B (Figure 4). Similarly, staining of HEK293 cells with
TUNEL following 4 h of treatment with 6B showed 10%
of the cells in late apoptosis compared to 0% in the
control (data not shown). Attempts to correlate these
cellular effects with phosphorylation levels on the Cdks
showed no significant differences with control samples
using either commercial or in-house phospho-Cdk-
specific antibodies using unsynchronized or synchro-
nized cultures.
Discu ssion
We have discovered a novel class of compounds that
appear to be excellent lead molecules for the design of
specific and potent inhibitors of Cdc25 phosphatases.
These indolyldihydroxyquinones share some similarity
yet critical differences with other recently described
quinone inhibitors of Cdc25 (Figure 1).15,27-29 Interest-
ingly, all of these compounds share a substituted
quinone and an extended aromatic ring structure. This
suggests a paradigm for inhibition of Cdc25 based on
either one or both of these shared features. We believe
that the quinone ring is the only important common

2586 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13
Sohn et al.
+0.18 V and +0.394 V vs Ag+ at a glassy carbon
electrode in MeOH with lithium perchlorate as the
supporting electrolyte. We see it reduced at a Pt
electrode at -0.58 V vs Ag+. None of these potentials
are close to those of biologically relevant redox cofactors.
It has also been observed that demethylasterriquinone
B1 is reduced by hydrogen sulfide but readily converted
back to the dihydroxybenzoquinone oxidation state in
air.48 Last, the indolyldihydroxyquinones have strong
intramolecular hydrogen bonds further significantly
reducing their reactivity.49 It is for these reasons that
we believe our compounds are significantly different
than the previously described quinones and thus do not
show time-dependent inhibition of Cdc25 (see below).
That is, although the initial affinity toward the active
site may be governed by recognition of the quinone ring,
the final mode of binding is significantly different as
the indolyldihydroxyquinones do not covalently modify
any active site residues and are therefore classical
reversible inhibitors.
substrate. Attempts to cocrystallize selected indolyldihy-
droxyquinones with the catalytic domain are underway
and should yield insight into the details of the protein-
protein interaction involved in recognition of the Cdk2-
pTpY/CycA substrate.
Given the high sequence identity among the three
Cdc25 isoforms, especially in the active site region, it
was not surprising to find that the structure-activity
relationships we observed applied to all three Cdc25s.
However, we did detect a difference among the three
isoforms for modifications at the 5-position. Three
compounds with substitutions of bromo- (4D), benzy-
loxy- (4G), and methyl- (4H) are significantly improved
in their inhibition of Cdc25A compared to Cdc25B and
Cdc25C. Interestingly, this increased potency requires
an intact C-terminal tail, as assays with a truncated
form of Cdc25A do not show a similar increase in
potency with these modifications at the 5-position (12%,
40%, and 17% remaining activity at 50 µM compound
for 4D, 4G, and 4H, respectively). This provides further
support that the 5-position is aimed outward toward
solvent, and in this case makes isoform-specific contacts
with the relatively divergent C-terminal tail of Cdc25A.
From the initial screen of the library one can derive
interesting structure-activity relationships, both in
terms of the overall effects of modifications on the indole
ring as well as specific differences between the three
different isoforms of Cdc25. We discuss first the effects
that appear to be applicable to Cdc25 inhibition by the
indolyldihydroxyquinones in general (Table 1, Table 2).
Modification of the nitrogen of the indole by a methyl
group appears to be only slightly detrimental and
further explorations at this position need to be under-
taken. Modification of the 2- or 4-position by anything
ranging from a methyl to a tert-butyl to a halide to the
aromatic phenyl group leads to a significant and con-
sistent loss of potency. Given that these modifications,
in particular substituents at the 2-position, greatly
increase the barrier to rotation around the biaryl bond
and force the dihedral angle to 90°, these results suggest
that some angle other than 90° may be required for
optimal positioning onto Cdc25.35 These self-consistent
results among monosubstitutions at the 2-position are
further confirmed by the loss of potency seen in the
compounds with a second modification in addition to a
methyl at the 2-position (e.g., 7C-G). Modifications at
the 5-position appear to have little effect, except for
Cdc25A (see below), as if this position is pointed away
from the active site and out into the solvent. Modifica-
tions at the 6-position show a significant increase in
potency for the halides and benzyloxy, whereas a methyl
group is detrimental. At the 7-position, all substituents
of size greater than propyl appear to increase potency
significantly. As exemplified by 6B (Table 4), this
increase in potency for modification at the 7-position
requires an intact C-terminus on Cdc25B. This suggests
that the C-terminal tail, not seen in the crystal struc-
tures of either Cdc25A or Cdc25B, forms an important
part of the binding site for these inhibitors. This is
particularly intriguing given that we have previously
shown that the C-terminal tail is not important for
activity with small molecule substrates, but is required
for interaction with the protein substrate.40 It is of
interest to understand how the indolyldihydroxyquino-
nes compounds take advantage of this C-terminal tail
in their binding, as they may mimic the not yet
understood interaction between Cdc25 and its protein
IC50 determinations using eight different substrate
concentrations were used to further evaluate selected
compounds from the library as well as the second
generation compounds 8J and 8L. The IC50s we found
under identical assay conditions are among the most
potent reported for Cdc25 in the literature at this time
(Table 2). The IC50 determinations also yield Hill slope
data that can point to potential artifacts such as
multiple inhibitors binding to each protein and/or
protein or inhibitor aggregation.50-52 We noted reason-
able Hill slopes (0.7-1.5) with all of the compounds,
except those with very large hydrophobic substituents
at the 7-position. In particular, substitution at this
position with farnesyl (6D), phenyl (6H), and benzyloxy
(7B), although giving a nice increase in potency over
the parent molecule 2A, yielded Hill slopes greater than
3. Given that such compounds are difficult to work with
and that the origin of the Hill slope can have diverse
causes, we avoided further experimentation with these
compounds. Fortunately, addition of the smaller prenyl
group (6B) still generated much of the increase in
potency over the parent molecule 2A while retaining a
reasonable Hill slope (1.2-2.0). The compounds 8J and
8L were designed to test the effects of making multiple
beneficial substitutions. Although simultaneous incor-
poration of a 6-chloro and a 7-prenyl in compound 8J
did not show a significant increase in activity against
any of the Cdc25s, the addition of a 4-chloro to the
6-chloro-7-prenyl in 8L did show increased potency
toward Cdc25B and Cdc25C, but not Cdc25A. We did
note an increased potency for 8J over 6B (IC50 ) 11
µM vs 33 µM) with the truncated form of Cdc25B,
confirming the benefit of the 6-chloro substitution seen
for 5B. These experiments demonstrate that not all
multiple substitutions of the indolyldihydroxyquinones
follow simple additivity rules. Thus we are pursuing
larger and more diverse libraries so as to rapidly screen
further combinations.
Time-dependent inhibition is a significant issue when
working with protein tyrosine phosphatases and dual-
specificity phosphatases containing an active site thi-

Inhibition of Cdc25 Phosphatases
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13 2587
olate anion. Although we observed linear rates of
reaction in the presence of inhibitors, we specifically
tested for time-dependent inhibition in a separate series
of experiments so as to ensure that our determination
of Ki’s in subsequent experiments would not become
titrations of enzyme concentrations. Incubation of com-
pounds 2A, 6B, or 8L in excess over enzyme at concen-
trations greater than the Ki with Cdc25B followed by
dilution into assay buffer led to an almost complete
recovery of activity (Figure 2). Thus, the indolyldihy-
droxyquinones appear to behave significantly different
than the naphthoquinones that showed significant loss
of activity in related experiments.28
physiological substrate that is 2 orders of magnitude
better than the artificial mFP substrate as determined
by kcat/Km measurements.34 In associated control experi-
ments we found that our class of compounds exhibit
significant nonspecific binding to bovine serum albumin,
a carrier protein normally used in the assay with the
Cdk2-pTpY/CycA substrate. Thus, if this class of com-
pounds is to be pursued further for drug development,
it may be necessary to incorporate a modification that
reduces nonspecific protein binding.53 Incorporation of
such simple modifications may be possible on the
5-position of the indole ring, which was seen to tolerate
a wide variety of modifications (Table 1). Given that
demethylasterriquinones with symmetrical indole rings
attached to the remaining unmodified position of the
quinone ring are also potent Cdc25 inhibitors (data not
shown), modifications that reduce nonspecific protein
binding could also be added on the quinone ring.
Our in vivo test of the indolyldihydroxyquinones
showed effective killing of cells at 50 µM, but not 25
µM, of 6B that may be mediated by apoptotic mecha-
nisms that are under further investigation. This cell
killing was not accompanied by an increase in phospho-
rylation of the Cdks, pointing to a mechanism of cell
death mediated by some as yet unknown mechanism.
In summary, we have discovered the indolyldihydrox-
yquinones as a new class of submicromolar inhibitors
of the Cdc25phosphatases that bind at the active site
and are reversible. Additionally, they show activity in
competition with the natural protein substrate in vitro
and rapidly lead to cell death. Thus, these compounds
serve as attrractive lead molecules for drug development
efforts against the Cdc25 phosphatase, an important
anticancer target.
Given the open architecture of the active site region
of Cdc25, it has been difficult to envision tight binding
of a small molecule near the catalytic cysteine. There-
fore, two other types of experiments were completed to
determine if the binding of the indolyldihydroxyquino-
nes was occurring at the active site of Cdc25. First,
classical Ki determinations performed by simultaneous
variation of substrate and inhibitor were found to fit
only to equations for competitive inhibition (Table 3).
This is in stark contrast to other published inhibitors
of Cdc25 that appear to bind via a mixed inhibition
mechanism that include a time-dependent compo-
nent.27,28 Therefore 6B and 8L present the first true
submicromolar small molecule binders of Cdc25. Second,
using a series of site-directed mutants located near the
active site and in the water pocket adjacent to the
catalytic site of Cdc25B (Figure 3), we found that
mutation of Glu474, Phe475, and Arg482, but not
Glu478, Arg492, Tyr528, Met531, or Asn532, reduced
the amount of inhibition seen for the indolyldihydrox-
yquinone class of inhibitors (Table 4). Glu474 and
Phe475 are a part of the HCX5R loop that cradles the
phosphate of the substrate. We have recently provided
evidence that the former plays a critical role as a
catalytic base in the reaction mechanism.30 Interest-
ingly, Arg482 is one of the two residues suggested
previously by molecular modeling to be required for the
binding of the large anionic quinone part of the naph-
thoquinone inhibitors.28 These data thus bolster this
particular docking model, provide strong support for the
conclusion that these compounds are active-site di-
rected, and provide further evidence for the concept of
the quinone moiety serving as a paradigm for Cdc25
inhibition.
Ack n ow led gm en t. This work was supported by
NIH GM61822-01 grant to J ohannes Rudolph and NIH
AI48521 grant to Michael Pirrung.
Abbr evia tion s
PTPase, protein tyrosine phosphatase; DSP, dual-
specificity phosphatase; Cdk, cyclin-dependent kinase;
Cyc, cyclin; Cdk2-pTpY/CycA, Cdk2/CycA complex phos-
phorylated on Thr14 and Tyr15; mFP, 3-O-methylfluo-
rescein phosphate.
Su p p or tin g In for m a tion Ava ila ble: Figures S1, S2, and
S3, showing representative kinetic inhibition data, are avail-
able free of charge via the Internet at http://pubs.acs.org.
Given our promising results with the indolyldihy-
droxyquinones as potent and reversible inhibitors of
Cdc25, we sought to extend the experiments to more
physiologically relevant assays. We monitored the effect
of selected compounds on Cdc25s ability to dephospho-
rylate its native substrate, Cdk2-pTpY/CycA, in vitro.
This in vitro assay allowed us to determine the potency
of our compounds without concern for membrane per-
meability or cellular stability. The parent molecule (2A),
that apparently does not interact with the C-terminus
of Cdc25, was not effective in competing with the
natural substrate, where we have shown that the
interaction with the C-terminal tail is important.40
Although somewhat less potent than toward mFP, 6B
and in particular 8L showed good inhibition of Cdc25
with IC50’s of 20 µM and 8 µM, respectively (Table 3).
Thus, these compounds can compete effectively with the
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