Received for publication, January 25, 2001, and in revised form, February 28, 2001
Cells can respond to DNA damage by activating
checkpoints that delay cell cycle progression and allow time for DNA
repair. Chemical inhibitors of the G2 phase DNA
damage checkpoint may be used as tools to understand better how the
checkpoint is regulated and may be used to sensitize cancer cells to
DNA-damaging therapies. However, few inhibitors are known. We
used a cell-based assay to screen natural extracts for G2
checkpoint inhibitors and identified debromohymenialdisine (DBH) from a
marine sponge. DBH is distinct structurally from previously known
G2 checkpoint inhibitors. It inhibited the G2
checkpoint with an IC50 of 8 µM and showed
moderate cytotoxicity (IC50 = 25 µM) toward
MCF-7 cells. DBH inhibited the checkpoint kinases Chk1
(IC50 = 3 µM) and Chk2 (IC50 = 3.5 µM) but not ataxia-telangiectasia mutated (ATM),
ATM-Rad3-related protein, or DNA-dependent protein
kinase in vitro, indicating that it blocks two major
branches of the checkpoint pathway downstream of ATM. It did not cause
the activation or inhibition of different signal transduction proteins,
as determined by mobility shift analysis in Western blots, suggesting
that it inhibits a narrow range of protein kinases in
vivo.
 |
INTRODUCTION |
DNA damage activates signal transduction pathways called
checkpoints, which delay cell cycle progression and allow more time to
repair DNA (1-3). Checkpoints arrest cells in the G1 phase to prevent replication of damaged DNA and in the G2 phase
to prevent the segregation of damaged chromosomes during mitosis.
The G2/M transition is controlled by the Cdc2 protein
kinase. During G2 arrest, Cdc2 is inactivated through
phosphorylation of Thr-14 and Tyr-15 in its ATP-binding site by protein
kinases including Wee1 and Myt1 (4, 5). Entry into mitosis requires dephosphorylation of these sites by Cdc25 phosphatases. According to
our current understanding of the G2 checkpoint, DNA damage activates the ATM,1 and ATR
members of the phosphoinositide kinase family (6, 7). A signal then is
transmitted through the downstream protein kinases Chk1 and Chk2
(6-11), which are able to phosphorylate Cdc25 on Ser-216. This
phosphorylation is thought to directly prevent Cdc25 from activating
Cdc2 kinase (12) or to separate Cdc25 from Cdc2 kinase by promoting the
association of Cdc25 with 14-3-3 proteins (10, 13-16). Chk1 and Chk2
also can phosphorylate and activate Wee1, a kinase that catalyzes Cdc2
inhibitory phosphorylation (17, 18). Chk1 is required for initiating
G2 arrest (19, 20). Chk2 can phosphorylate p53 on Ser-20
in vitro (21-23), and p53 targets such as p21 and 14-3-3
have roles in maintaining G2 arrest (24, 25). These results
as well as experiments with knockout mice (21) are consistent with a
role of Chk2 in maintaining G2 arrest.
Our understanding of checkpoints stems mainly from genetic studies in
yeast and mice, in vitro studies with amphibian egg extracts, and studies of human syndromes associated with predisposition to cancer. Compounds that inhibit the G2 checkpoint may be
useful additional tools to study the checkpoint mechanism in mammalian systems. G2 checkpoint inhibitors also may be valuable in
cancer therapy to enhance the effectiveness of DNA-damaging agents in tumors with a defective G1 DNA damage checkpoint, such as
those with mutated p53 (26-29). However, few G2 checkpoint
inhibitors are known. Those found so far include caffeine and
1-substituted caffeine analogs (30-35), 2-aminopurine, and
6-dimethylaminopurine (36), staurosporine, 7-hydroxystaurosporine,
SB-218078 (37-39), and isogranulatimide (29). All have been shown to
enhance the cytotoxicity of DNA-damaging agents (29, 39). Staurosporine is a broad specificity protein kinase inhibitor, and
7-hydroxystaurosporine is an in vitro inhibitor of several
protein kinases (40-42) including Chk1 (43, 44).
7-Hydroxystaurosporine is being evaluated in phase I clinical trials
for the treatment of cancer (45). Caffeine and caffeine analogs have
many pharmacological activities (35) including in vitro
inhibition of ATM and ATR protein kinase activity (3, 12, 46-48), but
they are not considered drug candidates.
To find new G2 checkpoint inhibitors, we used a cell-based
assay (29) to screen marine invertebrate extracts. From the sponge Stylissa flabeliformis, we have isolated
debromohymenialdisine (DBH), a compound structurally distinct from
previously known G2 checkpoint inhibitors. We have
characterized the G2 checkpoint inhibitory activity of DBH
and analogs, and we describe its effects on checkpoint and signal
transduction protein kinases.
 |
EXPERIMENTAL PROCEDURES |
Extraction and Isolation of DBH and Related
Compounds--
Specimens of S. flabeliformis were collected
in the waters off Motupore Island in Papua New Guinea. The samples were
frozen on site and transported to Vancouver over dry ice. A frozen
sample (87 g wet weight) was thawed and extracted exhaustively with
methanol. The methanol extract was filtered and concentrated in
vacuo to give a dark brown solid. This crude extract was
fractionated on a Sephadex LH-20 column using methanol as the eluent.
Active fractions were identified using the G2 checkpoint
inhibition assay (29) and subjected further to silica gel-flash
chromatography using stepwise gradient elution
(CH2Cl2 to 1:1
CH2Cl2/methanol saturated with
NH3). Further purification was achieved by repeated
fractionation on reversed-phase HPLC using 80:20:0.05
water/methanol/trifluoroacetic acid as the eluent. The pure fractions
of DBH and hymenialdisine were converted from trifluoroacetic salts to
the hydrochloride salts by the addition of 3 N HCl followed
by concentration under reduced pressure. Debromopyrrololactam was
obtained by subjecting DBH to a 5% K2CO3
solution followed by concentration in vacuo. It then was
purified using HPLC with 80:20 water/methanol as the eluent. The
related compounds 2-aminoimidazole and
2-amino-4,5-imidazole-dicarbonitrile were obtained from Aldrich.
G2 Checkpoint Inhibition Assay--
The assay was
performed as described in Ref. 29 using human mammary tumor MCF-7 cells
expressing a dominant negative mutant p53 (mp53) gene. The number of
cells that escaped G2 arrest and became trapped in mitosis
was determined by enzyme-linked immunosorbent assay using the TG-3
antibody (29, 49) or by counting mitotic cells using fluorescence
microscopy (50).
MTT Cell Proliferation Assay--
MCF-7 mp53 cells were seeded
at 1000 cells/well in 96-well plates, grown overnight, and treated or
not treated with DBH for 24 h. The drug was removed, and cells
were allowed to grow in fresh medium until those not treated with the
drug approached confluence, which was typically 4-6 days. Cell
proliferation was measured as follows: 25 µl of a 5 mg/ml solution of
3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide in
phosphate-buffered saline was added to cells in the presence of 100 µl of cell culture medium. After a 2-h incubation at 37 °C, 100 µl of 20% sodium dodecyl sulfate dissolved in
dimethylformamide/water (1:1), pH 4.7, was added, and the absorbance at
570 nm was measured after overnight incubation.
Cell and Nuclear Extracts and Western Blotting--
Cells were
harvested by trypsinization, and cell pellets were washed once with
phosphate-buffered saline containing 1 mM
phenylmethylsulfonyl fluoride. Cells were suspended in 20 volumes of
ice-cold lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 1 mM
phenylmethylsulfonyl fluoride, 2 µg/ml each of leupeptin, pepstatin,
and aprotinin, 30 µg/ml DNase I and RNase A, 1 mM
dithiothreitol, and 1 mM sodium orthovanadate) and lysed by
pipeting up and down 20 times, incubating on ice for 15 min, pipeting
up and down again 20 times, and leaving on ice for another 15 min.
Lysates were cleared by centrifugation at 15,000 × g
for 15 min at 4 °C. Nuclei were prepared as described in Ref. 51 and
lysed in SDS-sample buffer. Fifty µg of protein was resolved by 10%
SDS-polyacrylamide gel electrophoresis, transferred onto nitrocellulose
by electroblotting, and blocked in 5% Carnation dry milk in 10 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.5%
Tween 20 for 1 h at room temperature. The nitrocellulose membrane
was probed with Cdc2 rabbit antiserum (KAP-CC001, StressGen
Biotechnologies, Victoria, Canada) in blocking solution overnight at
4 °C. The membrane was washed three times and incubated with
horseradish peroxidase-conjugated goat antibodies to rabbit IgG
(13858-014, Life Technologies, Inc.) in blocking solution for 2 h
at room temperature. After washing, the antigen·antibody complexes
were visualized by chemiluminescence (SuperSignal, Pierce) and exposed
on film (X-OMAT, Eastman Kodak Co.). Signal transduction kinases were
analyzed by immunoblotting by Kinexus Bioinformatics (Vancouver, Canada).
Protein Kinase Assays--
Cdc2 was immunoprecipitated, and its
kinase activity was assayed as described in Ref. 52. ATM and ATR were
immunoprecipitated from human lymphoblastoid BT cells using antibody
Ab-3 and Ab-1 (Oncogene Research Products) respectively, and their
kinase activities assayed using 10 mM HEPES, pH 7.5, 50 mM
-glycerophosphate, 50 mM NaCl, 10 mM MnCl2, 10 µM ATP, and 5 µCi
[32P]ATP per assay as described in Ref. 53. DNA-PK
catalytic subunit and Ku70/80 were purified from human placenta,
reconstituted to give a 1:1 molar ratio of DNA-PK catalytic subunit to
Ku, and assayed as described in Ref. 54. The activity of recombinant glutathione S-transferase-tagged Chk2 was assayed in 50 mM Tris, pH 8.0, 50 mM KCl, 5% glycerol, 10 mM MnCl2, 0.5 µg of recombinant His-tagged
PHAS-1 (Stratagene), 0.1 µg of Chk2, and 10 µM ATP containing 5 µCi [32P]ATP. Reactions were for 30 min at
30 °C. The kinase activity of glutathione
S-transferase-tagged baculovirus-expressed human Chk1 was
assayed as described in Ref. 55 using the peptide GLYRAPSMPENLNRK (derived from residues 210-223 of human Cdc25C containing an S214A substitution) as a substrate.
Immunofluorescence Microscopy--
MCF-7 mp53 cells were
prepared for immunofluorescence as described in Ref. 56. Antibody
dilutions used were 1:100 for Cdc25C (sc-327, Santa Cruz Biotechnology,
Santa Cruz, CA) and 1:400 for Cdc2. Cy3-conjugated anti-mouse
(115-165-006, Jackson ImmunoResearch Laboratories, West Grove, PA) or
anti-rabbit (C-2306, Sigma) secondary antibodies were used at 1:750.
The coverslips were immersed also in 0.1 µg/ml bisbenzimide to stain
the DNA.
 |
RESULTS |
Isolation and Identification of DBH--
A cell-based assay for
G2 checkpoint inhibition (29) was used to screen several
thousand crude organic extracts from marine invertebrates. An extract
from a sponge collected in Papua New Guinea showed activity. A compound
with strong G2 checkpoint inhibitory activity was isolated
by chromatographic procedures (see "Experimental Procedures") using
the cell-based assay to direct purification. The active compound was
identified as DBH (Fig. 1) by analysis of
its mass spectrometry and NMR data and comparison with published values
(57). DBH was identified first in 1980 as a yellow compound from the
sponge Phakellia flabelata (57). The extract also contained minor amounts of the structurally related compounds hymenialdisine, debromoaxinohydantoin, and debromopyrrololactam (Fig. 1).
Activity Profile of DBH and Analogs--
The activity profile of
DBH as a G2 checkpoint inhibitor is shown in Fig.
2A. MCF-7 mp53 cells arrested
in the G2 phase after exposure to ionizing radiation were
incubated with DBH for 8 h in the presence of nocodazole to trap
cells in mitosis. The number of mitotic cells was counted by
microscopy. DBH showed dose-dependent G2
checkpoint inhibition with an IC50 of 8 µM
and maximal activity at 40 µM. Higher concentrations
caused a decrease in activity, a phenomenon also seen with other
checkpoint inhibitors (29, 35) and believed to be caused by the
inhibition of enzymatic activities required for cell cycle progression
(45).

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Fig. 2.
Inhibition of G2 checkpoint and
of cell proliferation by DBH. A, MCF-7 mp53 cells
arrested in G2 phase by ionizing radiation were treated
with different concentrations of DBH together with nocodazole for
8 h. G2 checkpoint inhibition was determined by
counting the number of mitotic cells by microscopy. B, MCF-7
mp53 cells were exposed to different concentrations of DBH for 20 h, and cell proliferation was assayed 3 days later as described under
"Experimental Procedures."
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We next determined the effect of DBH on cell proliferation. Cycling
cells were incubated with DBH for 20 h, the compound was washed
away, and the cells were allowed to grow. Cell proliferation was
determined after 3 days using the MTT assay. DBH inhibited cell
proliferation but not potently, with an IC50 of 25 µM (Fig. 2B).
Hymenialdisine was active also as a checkpoint inhibitor with an
IC50 of 6 µM (Table
I). Debromoaxinohydantoin and
debromopyrrololactam, which were present also in the sponge extract,
and the related compounds 2-aminoimidazole and
2- amino-4,5-imidazole-dicarbonitrile obtained from commercial
sources showed no activity at all concentrations tested (Table I). Only
DBH was isolated in sufficient quantities for the additional studies
described below.
Activation of Cdc2 Kinase by DBH--
When cells are
arrested in the G2 phase by DNA damage, Cdc2 kinase is
maintained in an inactive state by phosphorylation of Thr and Tyr
residues in its ATP-binding site. Entry into mitosis requires the
dephosphorylation of these residues by Cdc25 protein phosphatases. We
first determined whether DBH overcomes G2 arrest by
affecting the phosphorylation and the protein kinase activity of Cdc2.
Extracts were prepared from cells arrested in mitosis by treatment with
nocodazole, from cells arrested in G2 phase after exposure
to ionizing radiation, or from cells arrested in G2 phase and then treated with 40 µM DBH for up to 6 h.
Cellular proteins were separated by SDS-polyacrylamide gel
electrophoresis and analyzed by immunoblotting using a Cdc2 antibody.
Depending on its inhibitory phosphorylation state, Cdc2 may be detected
as slow migrating bands corresponding to phosphorylated inactive kinase
and a faster migrating band corresponding to hypophosphorylated active
Cdc2. As shown in Fig. 3A,
cells arrested in mitosis with nocodazole had no detectable
phosphorylated inhibited Cdc2, whereas cells arrested in G2
after irradiation contained phosphorylated Cdc2. Cells arrested in
G2 and incubated with DBH showed a
time-dependent decrease in the amount of phosphorylated
Cdc2, with the slowest migrating band essentially becoming undetectable
by 6 h. The well characterized G2 checkpoint inhibitor
caffeine also caused a decrease of phosphorylated Cdc2 (Fig.
3A).

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Fig. 3.
Effect of DBH on the phosphorylation and
activity of Cdc2 kinase. A, Cdc2 immunoblot from cells
arrested in the M or G2 phase and from cells arrested in
the G2 phase and exposed to 40 µM DBH or 2 mM caffeine (caf) for 2, 4, or 6 h.
B, histone H1 kinase activity of Cdc2 immunoprecipitated
from cells arrested in the G2 or M phase or from
G2-arrested cells exposed to DBH or caffeine for 4 h.
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We next examined the activity of immunoprecipitated Cdc2 using histone
H1 as a substrate. Cdc2 activity was high in cells arrested in mitosis
and very low in cells arrested in G2 phase by irradiation
(Fig. 3B). DBH caused a concentration-dependent activation of Cdc2. DBH at 4 and 40 µM activated Cdc2 to
levels activated by 0.2 and 2 mM caffeine, respectively
(Fig. 3B). Taken together, these results indicate that DBH
overcomes G2 arrest by interfering with the inhibitory
phosphorylation of Cdc2 kinase and causing its activation.
Inhibition of Chk1 and Chk2 by DBH--
Hymenialdisine was shown
recently to inhibit several protein kinases in vitro (58).
However, this study did not test kinases involved in the G2
checkpoint. The checkpoint inhibitor 7-hydroxystaurosporine inhibits
Chk1 but not Chk2 (43, 44), and caffeine inhibits ATM and ATR (12,
46-48). Therefore, we next examined whether DBH inhibits checkpoint kinases.
The effect of DBH on the protein kinase activity of purified DNA-PK was
determined using a synthetic peptide derived from p53 as described
previously (59). More than 70% of the protein kinase activity of
DNA-PK was retained in the presence of 20 µM DBH, whereas
the known DNA-PK inhibitor wortmannin (1 µM) effectively abolished DNA-PK activity (Fig.
4A). To determine the effect
of DBH on the protein kinase activity of ATM and ATR, the proteins were
immunoprecipitated from human lymphoblastoid cells and assayed in the
presence of different concentrations of DBH. DBH did not cause
significant inhibition of ATM and ATR in these assays, suggesting that
it does not act on phosphatidylinositol 3-kinase-like enzymes (Fig.
4B).

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Fig. 4.
DBH does not inhibit the kinases ATM, ATR, or
DNA-PK. A, purified DNA-PK catalytic subunit (0.03 µg) and Ku70/80 (0.01 µg) were combined and assayed under standard
assay conditions (50 mM Tris-HCl, pH 8.0, 50 mM
KCl, 10 mM MgCl2, 10 µg/ml sonicated calf
thymus DNA, 1 mM dithiothreitol, 0.2 mM EGTA,
200 µM ATP containing 1 µCi of
[ -32P]ATP, and 0.25 mM substrate peptide
(PESQEAFADLWKK) (59). Incubations were for 5 min at 30 °C. Where
indicated, DBH (open circles) or wortmannin (closed
circles) was added at the concentrations indicated prior to the
addition of ATP. B, ATM or ATR was immunoprecipitated from
human lymphoblastoid BT cells and assayed for protein kinase activity
as described in Ref. 53 using PHAS-I as a substrate. The kinases were
incubated with DBH at the concentrations indicated. Their activity was
measured using PHAS-1 as a substrate and visualized by
autoradiography.
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The effect of DBH on Chk2 activity was also measured. As shown in Fig.
5, DBH was a potent inhibitor of Chk2
kinase activity with an IC50 of 3.5 µM. DBH
also inhibited autophosphorylation of Chk2 (data not shown). The
activity of human recombinant Chk1 was assayed using a Cdc25C-derived
peptide as a substrate (55) in the presence of different concentrations
of DBH. DBH inhibited Chk1 with an IC50 of 3 µM (Fig. 6). To determine
whether DBH inhibits the activity of Chk1 by competing with ATP, Chk1
activity was assayed with varying concentrations of ATP and with
different concentrations of DBH. As shown in Fig. 6 (inset),
DBH inhibits Chk1 competitively with respect to ATP.

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Fig. 5.
DBH inhibits Chk2 in
vitro. A, recombinant Chk2 was assayed as
described under "Experimental Procedures" using PHAS-I as a
substrate. Samples were analyzed by autoradiography after
electrophoresis on SDS-polyacrylamide gels. B, Chk2 kinase
activity was calculated using a PhosphorImager with Fuji MacBas
software and plotted as percentage of activity in the absence of
DBH.
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Fig. 6.
DBH inhibits Chk1 in
vitro. Recombinant Chk1 was incubated with the
indicated concentrations of DBH or with DBH in the presence of
different concentrations of ATP (inset), and its kinase
activity was measured using a peptide substrate containing the Cdc25C
Ser-216 phosphorylation site.
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Effect of DBH on the Intracellular Localization of Cell Cycle
Regulatory Proteins--
In the yeast Schizosaccharomyces
pombe, the Chk1 protein kinase is essential for DNA
damage-induced G2 arrest (60). Chk1 can phosphorylate Cdc25
(13), creating a binding site for 14-3-3 proteins. The Cdc25·14-3-3
complexes localize to the cytoplasm, in which they are separated from
the nuclear pools of Cdc2 kinase. This provides an attractive model for
checkpoint arrest in organisms such as fission yeast, in which Cdc2
kinase is exclusively nuclear and mitosis is closed. However, in human
cells at least some Cdc2-cyclin B and Cdc25C localizes to the cytoplasm
in interphase (15), and it is not clear how phosphorylation-induced
retention of Cdc25·14-3-3 complexes in the cytoplasm could contribute
to G2 arrest. Cdc25C and cyclin B enter the nucleus just
prior to mitosis. One possibility is that 14-3-3 binding to Cdc25C
prevents this event. Because of the possible importance of cellular
compartmentalization in checkpoint arrest, we examined the effects of
irradiation and DBH treatment on the intracellular localization of a
variety of cell cycle regulatory proteins implicated in the control
of the G2/M transition.
Immunofluorescence microscopy was performed on cycling MCF-7 mp53
cells, G2-arrested cells, or G2-arrested cells
treated for 3 h with 40 µM DBH. We observed no
changes in the cellular localization of Cdc2, Cdc25C, cyclin A, cyclin
B, Cdk2, Cdc25B, Wee1, and 14-3-3 (data not shown) between cycling
cells and irradiated G2-arrested cells. Although subtle
changes in cellular distribution may have been missed using this
technique, these data indicate that in this cell line, G2
arrest is not accompanied by a major change in the nuclear
versus cytoplasmic distribution of these proteins. DBH
treatment of G2-arrested cells caused no significant
changes in the distribution of Cdc2, Cdc25C, cyclin A, cyclin B, Cdk2, Cdc25B, Wee1, and 14-3-3 in cells that did not yet show morphological evidence of having entered mitosis (data not shown). Similarly, the
distribution of these proteins in cells forced to enter mitosis by DBH
was not different from that of cells in a normal mitosis (data not shown).
Effect of DBH on Other Signal Transduction Kinases--
Signal
transduction pathways are controlled typically by kinase
phosphorylation cascades. Many of these kinases undergo mobility changes in SDS-polyacrylamide gel electrophoresis after
phosphorylation, reflecting the activation state of the pathways. To
determine whether DBH affects many or a few kinases in vivo,
its effects on the abundance and mobility of 24 kinases were evaluated.
Cycling MCF-7 mp53 cells and G2-arrested cells treated or
not treated with 40 µM DBH for 2 h were harvested.
Cell extracts were prepared in buffer containing phosphatase and
protease inhibitors, and proteins were separated using
SDS-polyacrylamide gel electrophoresis. The proteins then were
subjected to immunoblotting with a panel of antibodies that recognize
kinases that undergo mobility shift when activated or inhibited by
phosphorylation: CaMK4, Cdk2, CK1
, CK2
, Erk1, Erk2, p38 Hog
mitogen-activated protein kinase, Mek1, Mek2, Mek3, Mek4, Mek5, Mekk3,
Pim1, PKB
, PKC
, PKC
, PKC
, Rsk1, p70S6k, p46
SAPK, p54 SAPK, and Tak1. All the antibodies revealed clear bands at
the expected molecular masses of their respective kinases, but none
showed changes in abundance or mobility after treatment with DBH (data
not shown). This shows that DBH is not a broad spectrum protein kinase
inhibitor in vivo.
 |
DISCUSSION |
This study identifies DBH and hymenialdisine as new G2
checkpoint inhibitors. These compounds show no obvious structural
resemblance to previously described checkpoint inhibitors. The bulky
bromine substituent at position 2 in hymenialdisine that is absent in DBH does not influence G2 checkpoint inhibition
significantly, the two compounds having IC50 values of 6 and 8 µM, respectively (Table I). Both the pyrrololactam
and the aminoimidazole moieties are required because 2-aminoimidazole
and 2-amino-4,5-imidazole-dicarbonitrile (which lack the pyrrolactam
moiety) and debromopyrrololactam (which lacks the aminoimidazole
moiety) are without G2 checkpoint inhibition activity
(Table I). Debromoaxinohydantoin is very similar structurally to DBH
(Fig. 1) but has negligible activity (Table I), indicating that a
precise orientation of the carbonyl and amino groups in aminoimidazolidinone is important.
Hymenialdisine is a protein kinase inhibitor in vitro (58),
and our in vivo structure-activity data for G2
checkpoint inhibition are consistent with inhibition of a protein
kinase as the mechanism of action. A crystal structure of a complex of
hymenialdisine and the protein kinase Cdk2 shows multiple hydrogen
bonds and Van der Waals contacts between the aminoimidazolidinone and
residues in the ATP binding pocket of the kinase (58). These would not occur with the analog axinohydantoin, which is two orders of magnitude less potent as an in vitro protein kinase inhibitor than
hymenialdisine (58). This agrees with our finding that
debromoaxinohydantoin shows no G2 checkpoint inhibition
activity. In the Cdk2·hymenialdisine complex, the bromine at position
2 faces the entrance of the ATP binding pocket, where it contributes
somewhat to binding affinity but would not be required for binding
(58). This agrees with our finding that hymenialdisine and DBH has
similar G2 checkpoint inhibition activities.
Hymenialdisine inhibits a number of kinases in vitro:
cyclin-dependent kinases, GSK-3, and CK1 at nanomolar
concentrations and several more kinases at micromolar concentrations
(58). However, we show that in vivo neither hymenialdisine
nor DBH have such broad activity. First, although hymenialdisine can
inhibit Cdc2 kinase at nanomolar concentrations in vitro,
in vivo hymenialdisine and DBH are checkpoint inhibitors and
actually activate Cdc2 kinase (Fig. 3 and data not shown). Second,
although hymenialdisine can inhibit Cdk2, -3, -4, -5, and -6 at
submicromolar concentrations in vitro, in vivo
DBH is only a poor inhibitor of cell proliferation (IC50 = 25 µM, Fig. 2), indicating that it does not inhibit these kinases in vivo. Third, DBH has no effect on the
phosphorylation state of 24 signal transduction kinases in
vivo. Different factors could explain higher selectivity in
vivo than in vitro. For example, DBH could reach
different concentrations in different subcellular compartments,
resulting in preferential inhibition of kinases within certain
compartments. Certain protein kinases also may be part of
macromolecular complexes that facilitate or hinder DBH binding.
We propose that the checkpoint kinases Chk1 and Chk2 are the targets of
DBH involved in the G2 checkpoint. Preliminary data indicate that DBH has little or no effect on the activity of Wee1 in vitro at concentrations up to 10 µM (data
not shown). However, Myt1 cannot be excluded as a target because it was
not tested. The concentration of DBH required to inhibit Chk1 and Chk2
in vitro (IC50 = 3 and 3.5 µM,
respectively) is close to that required for inhibition of the
checkpoint in vivo (IC50 = 8 µM).
Both kinases probably cooperate in maintaining Cdc25C phosphorylated at
Ser-216 after DNA damage, and it seems likely that DBH owes its high
efficacy as a checkpoint inhibitor to inhibition of both kinases.
Structural and functional redundancy of kinases is common in mammalian
signal transduction pathways. It may be generally the case that agents with broader specificity will have more impact on complex signaling pathways than highly selective agents.
Irradiation and DBH treatment caused no noticeable alteration of the
cellular distribution of the major cell cycle regulatory proteins Cdc2,
Cdc25C, cyclin A, cyclin B, Cdk2, Cdc25B, Wee1, and 14-3-3 in MCF-7
mp53 cells. In addition, we saw no evidence of irradiation causing a
physical separation of Cdc2-cyclin B from Cdc25C or of DBH reversing
such a separation. This may indicate that the major function of the
phosphorylation of Ser-216 in Cdc25C by Chk1 and Chk2 is not to
separate Cdc25C from Cdc2 kinase physically.
The evidence that DBH inhibits two checkpoint kinases in
vitro and is a more specific kinase inhibitor in vivo
than in vitro may be relevant to its consideration as a drug
candidate. DBH showed moderate cytotoxicity (IC50 = 25 µM) toward cells not exposed to DNA damage, roughly
3-fold higher than the concentration required for G2
checkpoint inhibition (IC50 = 8 µM). This
difference may be sufficient to provide the therapeutic window required
for achieving G2 checkpoint inhibition in animal models
without excessive toxicity. Indeed, hymenialdisine has been reported to
slow down joint deterioration and cartilage degradation-associated
osteoarthritis in animal models (61), implying that concentrations
sufficiently high to modulate cellular responses are achievable in
animals. If hymenialdisine and DBH can be shown to modulate checkpoint
function in animal models, they may merit consideration as drug
candidates for cancer therapy.
We thank Mike LeBlanc for collecting sponges
in Papua New Guinea, Steven Pelech and Kinexus Bioinformatics for
immunoblot assays, Ruiqiong Ye for excellent technical assistance, and
Hilary Anderson for critical evaluation of the manuscript.
The abbreviations used are:
ATM, ataxia-telangiectasia mutated protein;
DBH, debromohymenialdisine;
HPLC, high pressure liquid chromatography;
mp53, mutant p53;
MTT, 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium
bromide;
PK, protein kinase;
DNA-PK, DNA-dependent protein
kinase;
SAPK, stress-activated protein kinase;
ATR, ATM-Rad3-related
protein.
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