A WEE1 Inhibitor Analog of AZD1775 Maintains Synergy with Cisplatin and Demonstrates Reduced Single-Agent Cytotoxicity in Medulloblastoma Cells

Article abstract of DOI:10.1021/acschembio.5b00725

The current treatment for medulloblastoma includes surgical resection, radiation, and cytotoxic chemotherapy. Although this approach has improved survival rates, the high doses of chemotherapy required for clinical efficacy often result in lasting neurocognitive defects and other adverse events. Therefore, the development of chemosensitizing agents that allow dose reductions of cytotoxic agents, limiting their adverse effects but maintaining their clinical efficacy, would be an attractive approach to treat medulloblastoma. We previously identified WEE1 kinase as a new molecular target for medulloblastoma from an integrated genomic analysis of gene expression and a kinome-wide siRNA screen of medulloblastoma cells and tissue. In addition, we demonstrated that WEE1 prevents DNA damage-induced cell death by cisplatin and that the WEE1 inhibitor AZD1775 displays synergistic activity with cisplatin. AZD1775 was developed as a WEE1 inhibitor from an initial hit from a high-throughput screen. However, given the lack of structure-activity data for AZD1775, we developed a small series of analogs to determine the requirements for WEE1 inhibition and further examine the effects of WEE1 inhibition in medulloblastoma. Interestingly, the compounds that inhibited WEE1 in the same nanomolar range as AZD1775 had significantly reduced single-agent cytotoxicity compared with AZD1775 and displayed synergistic activity with cisplatin in medulloblastoma cells. The potent cytotoxicity of AZD1775, unrelated to WEE1 inhibition, may result in dose-limiting toxicities and exacerbate adverse effects; therefore, WEE1 inhibitors that demonstrate low cytotoxicity could be dosed at higher concentrations to chemosensitize the tumor and potentiate the effect of DNA-damaging agents such as cisplatin.

Full text of DOI:10.1021/acschembio.5b00725

Articles
pubs.acs.org/acschemicalbiology
A WEE1 Inhibitor Analog of AZD1775 Maintains Synergy with
Cisplatin and Demonstrates Reduced Single-Agent Cytotoxicity in
Medulloblastoma Cells
†
‡
‡
‡
†
,†
Christopher J. Matheson, Sujatha Venkataraman, Vladimir Amani, Peter S. Harris, Donald S. Backos,
‡
†
‡
‡
Andrew M. Donson, Michael F. Wempe, Nicholas K. Foreman, Rajeev Vibhakar, and Philip Reigan*
†Department of Pharmaceutical Sciences, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado Anschutz
Medical Campus, 12850 East Montview Boulevard, V20-2102, Aurora, Colorado 80045, United States
‡
Department of Pediatrics and Section of Pediatric Hematology/Oncology/BMT, University of Colorado Anschutz Medical Campus,
12800 E 19th Ave, Mail Stop 8302, Aurora, Colorado 80045, United States
*
S Supporting Information
ABSTRACT: The current treatment for medulloblastoma includes surgical resection, radiation, and cytotoxic chemotherapy.
Although this approach has improved survival rates, the high doses of chemotherapy required for clinical efficacy often result in
lasting neurocognitive defects and other adverse events. Therefore, the development of chemosensitizing agents that allow dose
reductions of cytotoxic agents, limiting their adverse effects but maintaining their clinical efficacy, would be an attractive approach
to treat medulloblastoma. We previously identified WEE1 kinase as a new molecular target for medulloblastoma from an
integrated genomic analysis of gene expression and a kinome-wide siRNA screen of medulloblastoma cells and tissue. In addition,
we demonstrated that WEE1 prevents DNA damage-induced cell death by cisplatin and that the WEE1 inhibitor AZD1775
displays synergistic activity with cisplatin. AZD1775 was developed as a WEE1 inhibitor from an initial hit from a high-
throughput screen. However, given the lack of structure−activity data for AZD1775, we developed a small series of analogs to
determine the requirements for WEE1 inhibition and further examine the effects of WEE1 inhibition in medulloblastoma.
Interestingly, the compounds that inhibited WEE1 in the same nanomolar range as AZD1775 had significantly reduced single-
agent cytotoxicity compared with AZD1775 and displayed synergistic activity with cisplatin in medulloblastoma cells. The potent
cytotoxicity of AZD1775, unrelated to WEE1 inhibition, may result in dose-limiting toxicities and exacerbate adverse effects;
therefore, WEE1 inhibitors that demonstrate low cytotoxicity could be dosed at higher concentrations to chemosensitize the
tumor and potentiate the effect of DNA-damaging agents such as cisplatin.
edulloblastoma is the most common primary brain
tumor in children. The current multimodal treatment
outcomes, but these highly cytotoxic treatments are far from
optimal. There is increasing evidence that high-dose cisplatin
1
,2
8,9
M
for medulloblastoma of surgical resection, posterior fossa and
craniospinal irradiation, and chemotherapy has improved 5-year
and radiation required to circumvent tumor resistance and
maintain clinical efficacy can result in lasting neurocognitive
defects, stunted growth, deafness, and even secondary
3
,4
survival rates from 3 to >60% over the past 50 years.
10−15
tumors
and that the dose and frequency of cisplatin
Although, there has been considerable improvement in long-
term survival rates, the tumor remains incurable in about a third
of patients while cognitive deficits and other quality of life
8,9
treatment is often limited by nephrotoxicity and ototoxicity.
Therefore, there is a critical need to understand the molecular
pathways in medulloblastoma to identify new molecular targets
(
QoL) measures are often impaired in long-term survivors
following radiation and high-dose chemotherapy to the
5
−7
developing brain.
The cytotoxic agent cisplatin combined
Received: September 9, 2015
Accepted: December 22, 2015
with radiation has been the cornerstone of medulloblastoma
treatment for over 20 years and has produced good clinical
©
XXXX American Chemical Society
A
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that may lead to strategies to chemosensitize the tumor to
safely allow dose reductions of cisplatin while maintaining
clinical efficacy and resulting in improved survival and QoL
outcomes.
cancer types; therefore, our data could be of critical importance
as the off-target toxicities of AZD1775 may limit its utility in
the clinic. Therefore, WEE1 inhibitors which demonstrate low
cytotoxicity could be used at higher concentrations to
chemosensitize the tumor and potentiate the effect of DNA-
damaging agents such as cisplatin, allowing for dose reductions
of DNA-damaging agents and limiting therapy-related adverse
effects.
In order to identify novel molecular targets for medullo-
blastoma therapy, we performed an integrated genomic screen
using pathway analysis of gene expression from 16 medullo-
blastoma patient samples and a kinome-wide siRNA screen of
16
medulloblastoma cells. This combined analysis identified cell
cycle-related kinases in the G2 checkpoint, implicating the G2
checkpoint control as a target for medulloblastoma therapy.
Many cancers possess a deficient G1 checkpoint that impairs
the ability of the cell to halt the cell cycle in order to repair
RESULTS AND DISCUSSION
■
Targeting WEE1 in Medulloblastoma. There is a critical
need to develop new therapeutic strategies that reduce long-
term adverse events that arise from chemotherapy-associated
toxicities while maintaining or improving treatment efficacy in
patients with medulloblastoma. We have previously identified
WEE1 as a promising therapeutic target in medulloblastoma as
it is a focal kinase involved in the G2 checkpoint control that
prevents the accumulation of excessive DNA damage and
17
DNA damage prior to replication. This gives cancer cells a
means to accumulate mutations and propagate irregularities
that are favorable to cancer formation. Therefore, cancer cells
are reliant on the G2 checkpoint to prevent excessive DNA
17,18
damage that leads to apoptosis via mitotic catastrophe.
In
16
normal cells, the G1 checkpoint is not compromised; therefore,
the G2 checkpoint is not burdened with halting the cell cycle
prior to DNA damage repair. This supports that abrogation of
the G2 checkpoint will selectively impact tumorigenesis rather
than normal cell growth. From our genomic analysis, we
identified WEE1 as a focal kinase in two signaling pathways,
and we hypothesized that targeting this kinase for inhibition
subsequent induction of cell death. Furthermore, studies have
shown that strategies targeting the G2 checkpoint may be
effective at inducing cancer-specific synthetic lethality while
32
generally being well-tolerated by normal cells. AZD1775 is a
potent inhibitor of WEE1 kinase activity that is currently
undergoing clinical trials for use in combination therapies to
sensitize tumors to DNA-damaging agents. The assumption is
that AZD1775, as a sensitizing agent, would be relatively
nontoxic when used as a single agent; however, our results
clearly demonstrate that AZD1775 negatively impacts cellular
viability in medulloblastoma cells at nanomolar concentrations.
Analysis of our series of AZD1775 analogs indicated that small
changes in the AZD1775 structure dramatically alter WEE1
inhibitory activity or medulloblastoma cell growth inhibition
independently. Our results also support that WEE1 inhibition
per se is not responsible for the decrease in cell viability
observed with AZD1775.
16
could potentially disrupt multiple tumor survival mechanisms.
WEE1 is a tyrosine kinase that is a critical component of the
ATR-mediated G2 cell cycle checkpoint control that prevents
19
entry into mitosis in response to cellular DNA damage. ATR
phosphorylates and activates CHK1, which in turn activates
WEE1, leading to the selective phosphorylation of cyclin-
dependent kinase 1 (CDK1) at Tyr15, thereby stabilizing the
20,21
CDK1-cyclin B complex and halting cell-cycle progression.
This process confers a survival advantage by allowing tumor
cells time to repair damaged DNA prior to entering
2
2−24
mitosis.
Inhibition of WEE1 abrogates the G2 checkpoint,
Structural Modification of AZD1775 and in Vitro
Kinase Activity. Computational-based modeling of the
predicted interactions of AZD1775 in the ATP-binding domain
of WEE1 (Figure 1) indicated that the 4-methylpiperazinyl and
pyridyl-2-propan-2-ol side chains were orientated toward the
entrance of the binding cavity where a range of substitutions
could be accommodated. From the model, the 4-methylpiper-
azinyl group can interact with Ile305, Tyr378, and Cys379 via
hydrophobic and π-alkyl interactions. To understand the extent
of these interactions, a series of compounds were proposed as
chemical probes that retain the dialkylanilino group, while
sequentially building complexity in this region with dimethy-
lamino, piperadine, morpholine and piperazine N-methyl ester
groups. The pyridyl-2-propan-2-ol substituent in AZD1775 was
not predicted to make significant interactions with the binding
pocket apart from an edge-face π−π interaction with Phe433.
To confirm the amenability of this group to modification, the
commercially available pyridines 2-trifluoromethlpyridine and
2-methoxypyridine were identified that would retain this π−π
interaction, while being structurally diverse from the propan-2-
ol group in AZD1775. All possible combinations of analogs
(11a−n) and AZD1775 were synthesized (Figure 2A and
promoting cancer cells with DNA damage to enter into
unscheduled mitosis and undergo cell death via mitotic
2
5−31
catastrophe.
Therefore, WEE1 inhibition has the potential
to sensitize tumors to DNA-damaging agents, such as cisplatin.
In our previous work, we have shown that WEE1 inhibition
by the small molecule inhibitor AZD1775 (previously known as
MK1775) suppressed cell growth, induced apoptosis, and
16
decreased tumor growth in medulloblastoma. There are
limited structure−activity relationship (SAR) data for
AZD1775. It was developed as a WEE1 inhibitor by Banyu
Pharmaceutical Co. from an initial hit discovered from a high-
throughput screen (HTS), and it is known to have nanomolar
26
activity with at least eight other kinases. Therefore, in the
present study, we have developed a small series of AZD1775
analogs by substituting the side chains around the pyrazolopyr-
imidinone of AZD1775 to establish a SAR for WEE1 inhibition
and further examine the effects of WEE1 inhibition in
medulloblastoma. Interestingly, our AZD1775 analogs that
inhibited WEE1 in the same nanomolar range as AZD1775 did
not exhibit the same potent inhibitory effect on medullo-
blastoma cell growth as single agents, yet these compounds
demonstrated synergy with cisplatin at nontoxic inhibitor
concentrations. Our data support that WEE1 inhibition
sensitizes medulloblastoma cells to cisplatin and indicate that
the cytotoxicity of AZD1775 may be uncoupled from WEE1
inhibition. AZD1775 is currently being evaluated in clinical
trials as a potentiator of DNA-damaging agents for a number of
33
Supporting Information), and the IC50 values for each
compound were determined against WEE1 in an in vitro
recombinant kinase assay (Figure 2B, Supporting Information
Figure S1). All the compounds retaining the pyridyl-2-propan-
2-ol substituent (11a−d) of AZD1775 demonstrated potent
WEE1 inhibition with AZD1775 being the most potent overall.
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16
blastoma cells as a single agent. In order to evaluate the effect
of our inhibitors under these conditions, Daoy cells were
treated with AZD1775 and 11a−11n, at 75 nM and 150 nM,
and cell growth was monitored in real-time using xCELLigence
analysis (ACEA Biosciences Inc.; Figure 3A, Supporting
Information Figure S3). Interestingly, all compounds (11a−
11n), including those with comparable inhibitory activity in the
in vitro kinase assay (11a−d, 11m), exhibited a reduced effect
on cell growth inhibition compared with AZD1775. This
difference in single agent toxicity was also observed between
AZD1775 and the most potently active WEE1 inhibitors (11d
and 11m) by MTS assay (Figure 3B and C). Although all of the
compounds tested had an effect on cell viability, it was apparent
that AZD1775 (Daoy; EC50 = 289 ± 22 nM, ONS-76; EC50
49 ± 64 nM) had an increased single agent effect by MTS
when compared to 11d (Daoy; EC = 791 ± 95 nM, ONS-76;
=
2
50
EC = 868 ± 94 nM) and 11m (Daoy; EC = 558 ± 34 nM,
50
50
ONS-76; EC50 = 760 ± 88 nM). We further compared
AZD1775 with 11d and 11m over a concentration range in the
medulloblastoma D458 suspension cell line, using flow
cytometry to determine cell number and percentage viability.
Cell number decreased and the percentage of nonviable cells
increased compared with DMSO control at a lower
concentration of AZD1775 (123.5 nM, p < 0.01) than 11d
Figure 1. Molecular docking of AZD1775 in the ATP-binding site of
WEE1. (A) Predicted interaction of AZD1775 (orange sticks) with
WEE1 (cyano ribbon). (B) A Connolly surface applied to the ATP-
binding site with AZD1775 (orange sticks). (C) A ligand interaction
map for AZD1775 summarizing key interactions with the ATP-binding
site of WEE1. Dashed lines indicate predicted nonbond interactions
(
green = π−π, purple = H-bond).
(
370.4 nM, p < 0.01) and 11m (1.11 μM, p < 0.001) (Figure
D). Although AZD1775 (IC50 = 18.7 ± 8 nM) exhibited
greater potency than both 11d (IC = 27.8 ± 15 nM) and 11m
4
These data validate the computational model suggesting that
the 4-methylpiperazinyl region of the molecule is solvent-
exposed when bound to the kinase and is not responsible for
key binding interactions beyond the dialkylanilino nitrogen.
However, when the pyridine ring was modified, only compound
50
(
IC50 = 30.5 ± 8 nM) in the recombinant kinase assay, the
small differences in in vitro IC50 values did not appear to
account for the disparity in single agent cellular activities,
particulary between AZD1775 and 11d.
1
1m, bearing the 2-methoxy pyridinyl substitution and
retaining the 4-methylpiperazinyl group of AZD1775, demon-
strated inhibitory potency comparable to AZD1775. These data
indicate that, although AZD1775 is amenable to modification,
small structural changes in the side chains can impact WEE1
inhibitory activity. The compounds demonstrating potent
WEE1 inhibition (11a−d, 11m, Supporting Information Figure
S2) were selected for further evaluation with AZD1775.
Effect of WEE1 Inhibitors on Medulloblastoma Cell
Growth and Viability. AZD1775 has been shown to confer
significant reduction in the growth and viability of medullo-
WEE1 Inhibitor Synergy with Cisplatin. An MTS assay
was used to determine WEE1 inhibitor synergy with the DNA-
damaging agent cisplatin, which would be an expected outcome
in response to WEE1 inhibition. Daoy cells were treated for 72
h with increasing concentrations of both cisplatin and the
WEE1 inhibitors (AZD1775, 11a−d, 11m), and the effects of
drug combinations were analyzed using the Chou−Talalay
34
equation, as previously described. The Combination Index
(CI) was determined for each potential drug combination. A CI
value less than 1 indicates a synergistic drug combination,
Figure 2. Synthesis and inhibitory activity of novel WEE1 inhibitors. (A) Synthetic scheme for the preparation of candidate WEE1 inhibitors 11a−
1n and AZD1775. (B) Inhibitor identities and in vitro inhibitory activity against WEE1 kinase. IC50 values were determined through inhibition of
recombinant WEE1 in a TR-FRET kinase assay.
1
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Figure 3. Single agent toxicity of AZD1775 shown to be significantly higher than the synthesized WEE1 inhibitors. (A) Daoy cell growth rate
represented as 1/slope (Δcell index/h) derived from a real-time cell proliferation assay (xCELLigence) between 4 and 56 h following treatment with
75 nM (blue) and 150 nM (red) WEE1 inhibitors, compared with DMSO (green). All values have p < 0.001 when compared with DMSO (n = 3,
error bars = SEM). (B) Dose−response by MTS assay for Daoy cells treated with AZD1775 (red) and the potent compounds 11d (blue) and 11m
(
7
green) for 72 h. AZD1775 (red) decreased the cellular metabolic output of Daoy cells (EC = 289 ± 22 nM) more potently than both 11d (EC =
91 ± 95 nM) and 11m (EC50 = 558 ± 34 nM; n = 3, error bars/ ± = SEM). (C) Dose−response by MTS assay for ONS-76 cells treated with
5
0
5
0
AZD1775 (red) and the potent compounds 11d (blue) and 11m (green) for 72 h. AZD1775 (red) decreased the cellular metabolic output of Daoy
cells (EC50 = 249 ± 64 nM) more potently than both 11d (EC50 = 868 ± 94 nM) and 11m (EC50 = 760 ± 88 nM; n = 3, error bars/ ± = SEM). (D)
D458 cell viability and total cell number determined by flow cytometry (Viacount) when exposed to increasing concentrations of AZD1775
(
0
orange), 11d (blue), and 11m (green) for 72 h (n = 3, error bars = SEM, compared with DMSO; total cell number; p < 0.05 = *, p < 0.01 = **, p <
.001 = ***. Nonviable cell number; p < 0.05 = +, p < 0.01 = ++, p < 0.001 = +++).
whereas a CI of greater than 1 indicates a nonsynergistic effect.
As expected, AZD1775 showed strong synergy with cisplatin
across all concentrations except the lowest cisplatin and WEE1
inhibitor concentrations (Figure 4A and B). All of our WEE1
inhibitors (11a−d, 11m) exhibited synergy with cisplatin to
some degree, especially at higher concentrations of cisplatin.
For compounds 11a−c, the degree of synergy was reduced with
respect to AZD1775 as this was only observed at the highest
concentrations of cisplatin. In contrast, the more potent WEE1
inhibitors 11d and 11m exhibited synergistic activity at lower
cisplatin concentrations and were more comparable with
AZD1775. In particular, at 600 nM cisplatin, synergy was
observed across all concentrations of 11d and all but the lowest
concentration of 11m, indicating an improvement over the
synergy profile of AZD1775. Dose−response curves from the
MTS assay were plotted for cisplatin as a single agent and when
paired with a single concentration of WEE1 inhibitor (Figure
support previous studies suggesting that WEE1 inhibition acts
in synergy with DNA damage independent of cellular p53
status.
16
Cell Permeability of WEE1 Inhibitors. Despite significant
structural similarities between AZD1775 and the series of
analogs, and the lead compounds possessing cLogP values
within acceptable limits (AZD1775; cLogP = 2.18, 11d; cLogP
= 2.35, 11m; cLogP = 2.89), it was possible that differences in
cellular permeability and retention could explain the differential
effects of each WEE1 inhibitor on cell viability. Therefore, we
determined cellular uptake for AZD1775, 11d, and 11m at
varying concentrations and incubation times (Supporting
Information Figure S5). Interestingly, there was little difference
between cellular concentrations of AZD1775 and 11d, while
11m exhibited elevated levels at all concentrations and times.
Given these favorable cell permeability and retention character-
istics, we further analyzed the effect of inhibitor treatment on
cellular WEE1 activity.
4C). A concentration of 300 nM was chosen for the WEE1
inhibitors as no effect was observed in Daoy cells treated with
WEE1 inhibitor alone at this concentration in all cases except
AZD1775. When compared with cisplatin treatment, cotreat-
ment with our inhibitors potentiated the effect of cisplatin on
cell viability. The effect was also potentiated in the presence of
AZD1775, but this was due to the extensive loss of cellular
viability in the presence of AZD1775 alone at this
concentration. Interestingly, when the treatments were
repeated using AZD1775, 11d and 11m in combination with
cisplatin in p53 wild-type ONS-76 cells, similar results were
observed (Supporting Information Figure S4). These data
Effect of WEE1 Inhibitor Treatment on Cellular CDK1
Phosphorylation. To confirm the effect of our WEE1
inhibitors on downstream signaling, we conducted immuno-
blotting analysis of phospho-CDK1 (Tyr15) levels in Daoy cell
lysates following treatment with the potent WEE1 inhibitors
(AZD1775, 11a−d and 11m; Figure 5A). Excluding 11c, all
compounds were found to reduce cellular p-CDK1 in a dose-
dependent manner, similar to AZD1775. For a more
quantitative analysis, an ELISA assay was used to determine
the relative levels of p-CDK1 (Tyr15) in Daoy cell lysates
following treatment with a broader concentration range of
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Figure 4. Identified potent inhibitors of WEE1 acting in synergy with cisplatin and potentiating the activity of cisplatin at a nontoxic concentration.
A) Combination index (CI) plots generated using an MTS assay in Daoy cells treated with WEE1 inhibitor and cisplatin combinations for 72 h (n =
). CI values determined using the Chou−Talalay equation, with combination treatments indicated as nonsynergistic (red, ≥1.05), synergistic
(
3
(
green, ≤0.95), and intermediate (yellow, 0.96−1.04). (B) Numerical representation of CI plots. (C) MTS assay in Daoy cells following treatment
with WEE1 inhibitors and cisplatin for 72 h. Dose−response for increasing cisplatin concentrations at a concentration (300 nM) of several WEE1
inhibitors, compared with cisplatin alone.
Figure 5. AZD1775 shown to decrease cellular CDK1 phosphorylation at Tyr15 at lower concentrations than potent novel WEE1 inhibitors. (A)
Immunoblotting analysis of Daoy cell lysates treated with WEE1 inhibitors and DMSO control for 24 h. Membranes were probed for p-CDK1(Y15),
total CDK1 and actin as a loading control. (B) Quantitative ELISA determination of relative p-CDK1 (Tyr15) levels in Daoy cell lysates (0.15 mg
−1
mL total protein) treated with increasing concentrations of AZD1775 (red), 11d (blue), and 11m (green) for 24 h (n = 3, error bars = SEM).
Interpolation of curves reveals that 125 nM AZD1775, 205 nM 11d, and 565 nM 11m are required to reduce cellular p-CDK1 (Tyr15) to the same
level.
AZD1775, 11d and 11m, and the relative levels between
samples were compared with untreated control. Cellular p-
CDK1 levels were decreased to lower levels in the presence of
AZD1775 versus comparable concentrations of 11d and in
particular 11m. Interpolation of the ELISA data determined
that the concentrations of 11d and 11m that result in the same
level of cellular p-CDK1 induced by 125 nM AZD1775
treatment were 205 nM and 565 nM, respectively (Figure 5B).
Inhibition of Cellular Growth at a Fixed Level of
Cellular CDK1 Phosphorylation. To evaluate the contribu-
tion of cellular p-CDK1 (Tyr15) levels, and by extension,
WEE1 activity, on the observed effects of WEE1 inhibitor
treatment, we repeated the real-time cell proliferation assay
(xCELLigence) in Daoy cells over a broad concentration range
of AZD1775 (EC50 = 120.6 ± 1.3 nM), 11d (EC50 = 302.0 ±
0.3 nM), and 11m (EC50 = 419.5 ± 1.5 nM) for 76 h (Figure
6). However, as demonstrated with ELISA determination of p-
CDK1 (Tyr15) levels (Figure 5B), AZD1775 reduces the
cellular activity of WEE1 at lower concentrations than both 11d
and 11m. To determine the contribution of cellular p-CDK1
levels toward the inhibition of Daoy cell growth, the growth
rate was plotted as a function of inhibitor concentration.
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Figure 6. AZD1775 appearing to have an increased inhibitory effect on cell growth when compared with 11d at a concentration known to result in
comparable cellular WEE1 inhibition. Real-time cell proliferation plots (xCELLigence) for Daoy cells exposed to increasing concentrations of (A)
AZD1775, (B) 11d and (C) 11m, recorded for 76 h post-treatment (drug added at 24 h; n = 3, error bars = SEM). (D) Plot for Daoy cell growth
rates represented by 1/slope (Δcell index/h) for AZD1775 (red, EC = 120.6 ± 1.3 nM), 11d (green, EC50 = 302.0 ± 0.3 nM), and 11m (blue,
50
EC = 419.5 ± 1.5 nM) across a range of inhibitor concentrations (n = 3, error bars = SEM). Growth rates were determined for the linear growth
50
phase (t = 30−80 h). Interpolation of data reveals growth rates at fixed inhibitor concentrations; 125 nM AZD1775, 1/slope = 0.033 Δcell index/h;
205 nM 11d, 1/slope = 0.063 Δcell index/h; 565 nM 11m, 1/slope = 0.012 Δcell index/h.
Incubation with 125 nM AZD1775 resulted in a significantly
reduced growth rate compared with vehicle control (0.033 vs
11d and 11m possessed some single agent activity in the cell
lines tested, the disparity in potency did not appear to be
accounted for by any differences in recombinant WEE1
inhibition. AZD1775 decreased cellular p-CDK1 levels at
lower concentrations than 11d and 11m; however, the
concentrations of 11d required to reduce p-CDK1 to a level
comparable to AZD1775 treatment were not sufficient to
account for the difference in single agent cytotoxicity. Taken
together, our data support that the potent cytotoxicity of
AZD1775 in medulloblastoma cells is uncoupled from WEE1
inhibition and that AZD1775 may interact with alternative
molecular targets that result in its potent single agent
cytotoxicity. In 2009, Hirai et al. described the small molecule
MK1775 (now known as AZD1775) as a potent and selective
WEE1 inhibitor, with an IC of 5.2 nmol/L in an in vitro kinase
0
2
.068; Figure 6D). In contrast, the equivalent concentration of
05 nM 11d resulted in a nearly 2-fold increase in the rate of
cell growth compared with AZD1775 (0.063 vs 0.033). For
compound 11m, the equivalent concentration of 565 nM
resulted in an even greater reduction in cell growth (0.012).
Taken together, these data support that WEE1 inhibition by
AZD1775, 11d, and 11m to a fixed level of cellular CDK1
phosphorylation may be uncoupled from medulloblastoma cell
growth inhibition. Therefore, the potent single agent growth
inhibitory activity of AZD1775 may occur through an
alternative molecular target, or a combination of WEE1
inhibition and an alternative molecular target.
AZD1775 was initially identified as a WEE1 inhibitor
developed from a HTS hit; however, little is known concerning
the SAR for potent WEE1 inhibition. Therefore, we designed a
series of AZD1775 analogs to determine the structural
requirements for WEE1 inhibition. The 2-propanol-pyridine
side chain of AZD1775 was poorly amenable to structural
modification as most of our attempts to modify this group
resulted in a complete loss of WEE1 inhibition, with only 11m
demonstrating potent activity against WEE1, and it retained the
50
assay and activity against eight other kinases in a panel of 223
26
kinases. These other kinase targets of AZD1775 included
Yamaguchi sarcoma viral oncogene homologue 1 (YES1) and
seven unspecified kinases that were inhibited by >80% with 1
μmol/L AZD1775. An additional study identified ABL1, LCK,
LRRK2, TNK2, and SYK as targets of AZD1775 (Pubchem ID:
3
5
24856436) with K values below 1 μM. Interstingly,
i
microarray analysis of medulloblastoma patient tumor samples
revealed that YES1, SYK, and ABL1 are all overexpressed (≥2-
fold) when compared with a normal brain (Supporting
Information Figure S6).
4-methylpiperazinyl side chain of AZD1775. However, when
the 2-propanol-pyridine side chain was unaltered, it was
possible to generate a series of compounds with increasing
complexity in the dialkylanilino substituent of AZD1775 that
retained potent WEE1 inhibition (11a−d). The WEE1
inhibitors (11a−d, 11m, Supporting Information Figure S2)
demonstrated synergy with cisplatin, with 11d and 11m
emerging as early lead compounds. Compounds 11a−d and
Given this narrow spectrum of activity within the kinome and
rapid onset of potent cell growth inhibition from real-time cell
proliferation assays (Figure 6), the alternative target(s) of
AZD1775 that contribute(s) to its potent cytotoxicity may be
outside the kinome. The rapid onset of decreased cell viability
by AZD1775 as a single agent was our initial indicator of
AZD1775 acting through alternative targets, as through WEE1
inhibition alone there would have to be a sufficient level of
unrepaired DNA damage achieved through successive rounds
11m at 300 nM potentiated the activity of cisplatin, but as
single agents they had no observable effect in the MTS assay at
this concentration. In contrast, AZD1775 significantly inhibited
cell growth in Daoy cells at this concentration, and
demonstrated potent single agent cytotoxicity in all medullo-
blastoma cell lines tested. Although the most potent inhibitors
36−40
of cell division to initiate cell death mechanisms.
In
addition, we have identified 11d as a nanomolar WEE1
F
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inhibitor that demonstrates reduced single agent toxicity and
synergy with cisplatin in medulloblastoma cells. These results
indicate that the piperazinyl N-methyl ester of 11d may have a
reduced propensity for off-target binding and confer improved
WEE1 selectivity.
METHODS
■
Molecular Modeling. All computational modeling was performed
using Schro
̈
dinger software (Release 2015−1: Maestro, version 10.1,
Schrodinger, LLC, New York, NY, 2015). The crystal structure of the
̈
WEE1 kinase domain was downloaded from the Protein Data Bank
4
4
In summary, we have examined a series of AZD1775 analogs
and evaluated their capacity to inhibit WEE1 in an in vitro
kinase assay, their effect on the phosphorylation of CDK1, their
cytotoxicity as single agents, and their ability to potentiate the
effects of cisplatin in medulloblastoma cells. We have
characterized our current lead compound 11d as a new potent
selective inhibitor of WEE1 that exhibits all the desirable
characteristics of a chemosensitizing agent targeting the G2
DNA-damage checkpoint including (1) reduced cytotoxic
effects compared with AZD1775 within its effective concen-
tration range as a single agent, (2) the capacity to potentiate the
effect of DNA-damaging agents, such as cisplatin, and (3)
favorable cell permeability and retention characteristics. In
addition, our data for AZD1775 and the series of analogs
support that the cytotoxicity of AZD1775 is uncoupled from
WEE1 inhibition and that AZD1775 interacts with alternative
molecular targets to exert its potent inhibition of cell growth.
The identification of these alternative targets may be of interest
for future work due to the rapid onset and pronounced
cytotoxicity of AZD1775 and may also help us to understand
results from clinical trials. Although clinical studies are currently
at an early stage for AZD1775, it is under investigation in
combination with various chemotherapeutic agents and for a
number of a cancer types (16 trials logged; https://clinicaltrials.
gov, accessed 09/2015). Limited clinical studies of AZD1775 as
monotherapy have been performed, but they report good
tolerance of AZD1775 with no dose-limiting toxicity up to
(www.pdb.org; PDB ID 1X8B). The protein structure was prepared
by removing water molecules and the cocrystallized ligand; bond
orders were assigned and hydrogen atoms added to the crystal
structure. Finally, a restrained minimization of the protein structure
was performed using the default constraint of 0.30 Å RMSD and the
OPLS2.1 force field. The Structure Data Format (SDF) for
AZD1775 was retrieved from the PubChem database, and prepared
using LigPrep to assign bond orders and bond angles and then
45
46
45
subjected to minimization using the OPLS2.1 force field. Then, a
Grid box was generated around the ATP-binding site of WEE1, and
docking of AZD1775 was performed using Glide XP (extra precision)
4
7
mode.
Chemistry. AZD1775 analogs were prepared as outlined in Figure
A and described in the Supporting Information. Briefly, tert-
2
butylcarbazide (2) was protected through reaction with phthalic
anhydride (1); then the carbamate nitrogen was functionalized with
allyl bromide. Removal of the phthalamide protecting group with
methyl hydrazine gave the key tert-butyl allylcarbazide (5), which was
reacted with ethyl 4-chloro-2-methylthio-5-pyrimidinecarboxylate (4)
in the presence of TFA to form the core pyrazollopyrimidinone
scaffold (6). An Ullman-type aryl amination with the relevant
functionalized pyridines (8a−c) gave the penultimate pyrazole
products (9a−c), after which activation of the thioether with m-
CPBA and reaction with anilines (10a−e) afforded the desired
AZD1775 analogs (11a−n).
Recombinant Kinase Inhibition Assay. LanthaScreen Eu time-
resolved fluorescence resonance energy transfer (TR-FRET) kinase
binding assays (Invitrogen) were performed in 384-well, low-volume
plates (Corning) using recombinant WEE1 kinase, Kinase Tracer 178
and LanthaScreen Eu-anti-GST antibody (Invitrogen). Assays were
performed at 25 °C in a reaction mixture consisting of 5 μL of serially
diluted inhibitor solution, 5 μL of Kinase Tracer 178 solution, and 5
μL of kinase/antibody solution. All reagents were prepared as
solutions in 1× kinase buffer A (Invitrogen) at 3× final desired
concentration. Inhibitor solutions were prepared such that final
DMSO concentrations did not exceed 0.5%, which was shown to have
no effect on kinase activity. Inhibitors were assayed in the final
concentration range of 0.4 nM to 100 μM. Kinase Tracer 178 was used
at a final concentration of 70 nM, and the antibody and kinase were
used at final concentrations of 2 nM and 5 nM, respectively. All
reagents were incubated together for 1 h at RT and read using a
PerkinElmer Envision 2104 Multilabel Reader enabled for TR-FRET
1300 mg; however, a concern is that when it is used in
combination therapy, the maximum tolerated dose (MTD) of
41,42
AZD1775 decreases to 200−325 mg.
In addition, the
adverse events reported for AZD1775 include hematological
events (myelosuppression), nausea/vomiting, and fatigue,
which are common for cytotoxic agents and may be masked
42
in combination therapies. Therefore, the off-target toxicities
of AZD1775 may supplement the adverse events associated
with cytotoxic chemotherapy and compromise the chemo-
sensitizing strategy of targeting WEE1 to potentiate the efficacy
of DNA-damaging agents. The use of a WEE1 inhibitor, such as
(
6
excitation = 340 nm; tracer emission = 665 nm; antibody emission =
15 nm; delay = 100 μs; integration = 200 μs). Emission ratios (665
11d, that has a minimal cytotoxicity profile may maintain a high
(
MTD) in combination with cytotoxic chemotherapy and limit
nm/615 nm) were determined for each inhibitor concentration and
the data analyzed using a nonlinear regression analysis of the log
dose−response curve to determine IC50 values.
Cell Lines and Cell Culture. Daoy, ONS-76, and D458 cells
(Medulloblastoma) were obtained from ATCC and were passaged for
the severity of additive toxicity events. Furthermore, recent
studies evaluating AZD1775 in combination with chemo-
therapy in brain tumors have reported that AZD1775 has
43
limited blood brain barrier (BBB) penetration. Although, it is
unlikely that 11d will have an improved BBB penetration
profile over AZD1775 as their calculated blood−brain partition
coefficients (qplogBB, calculated in Quikprop) are −1.8 and
<
6 months following resuscitation. Daoy and ONS-76 cells were
cultured in DMEM (Gibco) supplemented with 10% FBS (Sigma-
Aldrich), 1 mM sodium pyruvate (Gibco), 1× penicillin/streptomycin
solution (Cellgro), and 1× nonessential amino acids (Sigma-Aldrich)
−0.9, respectively (where >0.3 is excellent and >−1.0 is
at 37 °C in an incubator humidifier with 95% air and 5% CO . D458
2
considered poor), indicating that both AZD1775 and 11d have
little ability to cross the BBB; however, there may be scope to
improve BBB penetration through examining further N-
substitutions of the piperazinyl side chain. Therefore, this
initial study evaluating the SAR for WEE1 inhibition provides
valuable structural information for the development of
inhibitors with improved BBB penetration properties to achieve
optimal chemosensitizing effects in brain tumors such as
medulloblastoma.
cells were cultured in DMEM (Gibco) supplemented with 10% FBS
(Sigma-Aldrich), 1 mM sodium pyruvate (Gibco), 1× penicillin/
streptomycin solution (Cellgro), and 1× L-glutamine (Cellgro).
Seeded cells were allowed to adhere for 24 h prior to use in all
assays. Under all treatment conditions, the final DMSO concentration
did not exceed 0.5%.
Cellular Metabolic Viability Assay. Daoy and ONS-76 cells were
seeded into sterile 96-well plates (Corning Inc.) at 2000 cells/well.
Inhibitors were administered at the MTS EC50 of AZD1775 (Daoy;
EC50 = 150 nM, ONS-76; EC50 = 290 nM) and 2-fold concentrations
G
DOI: 10.1021/acschembio.5b00725
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Articles
above and below the EC . Cisplatin (Sigma-Aldrich) was diluted in
curtain gas (CUR; set at 10) and Collisionally Activated Dissociation
(CAD gas; set at 12) were nitrogen; (iv) Ion Source, gas one (GS1)
and two (GS2) were set at 30; (v) entrance potential was 10.0 V; (vi)
quadruple one (Q1) and (Q3) were Low and Unit resolution,
respectively; (vii) dwell time was 200 ms; and (viii) declustering
potential (DP), collision energy (CE), and collision cell exit potential
(CXP) are voltages (V). Compound settings were (i) AZD1775: 501.3
→ 133.9 m/z, DP = 96, CE = 31, CXP = 14; (ii) 11d: 545.4 → 527.1
m/z, DP = 96, CE = 33, CXP = 16; (iii) 11f (internal standard): 456.2
→ 415.0 m/z, DP = 81, CE = 35, CXP = 12; (iv) 11m: 473.3 → 432.1
m/z, DP = 111, CE = 35, CXP = 14. The linear ranges (0.6−900 ng/
50
media and was administered at the MTS EC50 (Daoy; EC50 = 600 nM,
ONS-76; EC50 = 60 μM) and 2-fold concentrations above and below
the EC . Cells were incubated for 72 h with 25 μL of each diluted
50
drug solution. Cell viability was measured by 2 h incubation with 30
μL CellTiter 96 AQueous One Cell Proliferation reagent (Promega)
and formazan concentration assessed through colorimetric analysis
using a BioTek Synergy H1 plate reader (absorption = 490 nm).
Real-time Cell Proliferation Assay. Daoy cells were seeded into
gold plated 96-well plates (ACEA Biosciences Inc.) at 2000 cells/well.
Cells were treated with 50 μL inhibitor solutions at relevant
concentrations or with equivalent DMSO vehicle at 37 °C with
monitoring of cell index number at 10 min intervals for an additional
2
mL) for AZD1775, 11d, 11f, and 11m had correlation R values of
0.9993, 0.9949, 0.9967, 0.9950, and 0.9962, respectively. These
standard curves were used to determine concentration in the
experimental samples. An Agilent Technologies, Zorbax extended-
C18 50 × 4.6 mm column, 5 μm particle size, equipped with a C18
column guard and heated 40 °C using a flow-rate of 0.4 mL/min was
used. The mobile phase consisted of (A) 10 mM ammonium acetate,
0.1% formic acid in water, and (B) 50:50 ACN/MeOH. Between
samples, the autosampler was washed with a 1:1:1:1 mixture of ACN/
MeOH/IPA/water containing 0.1% formic. The chromatography
solvent sequence used was 95% A for 0.5 min, ramped to 95% B at
4.5 min, and held for 3.0 min; next, it was brought back to 95% A at
8.5 min and held for 0.1 min (9.0 min total run time). Samples (10
μL) were injected onto the column.
7
6 h. Cell index number versus time and cellular growth rate for linear
growth (1/slope, Δcell index/h, t = 30−80 h) were determined for
each treatment condition.
Cell Viability Determination. D458 cells were seeded into sterile
6-well round bottomed ultralow retention plates (Corning Inc.) at
000 cells/well, and inhibitors were administered in 100 μL of media
9
5
at concentrations ranging from 10 μM to 13.7 nM or equivalent
DMSO control. Following incubation for 72 h, cells were pelleted and
media aspirated. A total of 60 μL of TrypLE Express (Gibco) was
added to each well and the plates incubated at 37 °C for 8 min
followed by mechanical resuspension. A total of 50 μL of Guava
Viacount reagent (EMD Millipore) was added to each well, and the
plates were incubated at RT for 10 min. Wells were analyzed by flow
cytometry (EMD Millipore, Guava EasyCyte Plus) with gating for
viable and nonviable cell populations, and the cell concentration and
percentage viability was recorded over 1000 events.
Microarray Analysis. Snap frozen medulloblastoma tumors (n =
39; WNT = 2, SHH = 10, group 3 = 11, group 4 = 15) were obtained
from surgeries (Children’s Hospital, Colorado), and normal brain
samples (n = 34) were collected from either autopsy, epilepsy surgery,
or commercial sources (COM-IRB 95-500 and 09-0906). Tran-
scriptomic microarray analysis (Affymetrix HG-U133plus2) was
Immunoblotting Analysis. Daoy cells were plated in sterile six-
well plates at 200 000 cells/well and treated with inhibitors at a final
concentration of 75, 150, and 300 nM. Treated cells were incubated
for 24 h, trypsinized, and resuspended in TES/SB buffer (20 mM Tris,
4
8
performed as described previously. Expression of mRNAs corre-
sponding to known AZD1775 targets (ABL1, LCK, LRRK2, SYK,
TNK2, YES1) were extracted from these microarray profiles and
compared between medulloblastoma and normal brains.
1
mM EDTA, 1 mM L-serine, 250 mM sucrose, 20 mM boric acid, pH
7.5) containing protease inhibitors (Roche, cOmplete). Lysates were
prepared by sonication, and a total protein quantity 25 μg of each
sample was loaded for SDS-PAGE. Antibodies for immunoblotting
were purchased from Cell Signaling (p-CDK1 [Y15]), Abcam
(
CDK1), Sigma-Aldrich (β-Actin), and GE Healthcare (ECL
antimouse and ECL antirabbit) and used according to recommended
protocols. The chemiluminescent signal (Thermo Scientific, Super-
Signal West Pico) was captured using X-ray film.
Quantification of p-CDK1 (Tyr15) Levels. Daoy cells were
plated in sterile six-well plates at 200 000 cells/well and treated with
inhibitors at final concentrations of 37.5, 75, 150, 300, and 600 nM
and incubated for 24 h before being trypsinized and resuspended in
TES/SB buffer containing protease inhibitors. Cells were lysed on ice
through sonication, and cell lysates were diluted with ELISA Pathscan
sample diluent to a final volume of 100 μL and protein concentration
of 0.15 mg mL prior to use. The relative concentration of p-CDK1
Tyr15 was determined using an enzyme-linked immunosorbent assay
according to the recommended protocol (Cell Signaling, ELISA
Pathscan phosphor-Cdc2 (Tyr15)).
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acschem-
bio.5b00725.
■
*
S
In vitro TR-FRET dose−response curves, chemical
structures of WEE1 inhibitors, real-time cell proliferation
plots in Daoy cells for compounds grouped by structure,
combination index plots for WEE1 inhibitor and cisplatin
treatment in ONS-76 cells, WEE1 inhibitor cell
permeability and retention data, and chemical synthesis
data (PDF)
−1
Cellular Permeability of WEE1 Inhibitors. Daoy cells were
plated in sterile 24-well plates at 40 000 cells/well and treated with
inhibitors at a final concentration of 0.1, 0.5, 1.0, and 3.0 μM for 5, 15,
30, and 60 min prior to media aspiration. The cells were immediately
washed with ice cold PBS three times, before scraping into in 500 μL
of methanol/acetonitrile/water (2:2:1) containing 20 ng/mL of 11f as
an internal standard. The resultant lysate solutions were transferred
into 96-deep-well plates and subjected to LC-MS/MS as follows: An
Applied Biosystems Sciex 4000 (Applied Biosystems) was used; it was
joined with a Shimadzu HPLC (Shimadzu Scientific Instruments, Inc.)
and Leap autosampler (LEAP Technologies). Stock DMSO solutions
AUTHOR INFORMATION
Corresponding Author
*Phone: 303-724-6431. Fax: 303-724-7266. E-mail: philip.
reigan@ucdenver.edu.
■
Author Contributions
C.J.M., D.S.B., and P.R. performed computational modeling
and inhibitor design. C.JM. carried out compound synthesis
and in vitro IC50 determination. C.J.M., S.V., P.S.H. and V.A.
performed cell testing, including synergy evaluation and single
agent toxicity studies. C.J.M. and M.F.W. characterized the
synthesized inhibitors and determined cellular uptake and
(
10.0 mM) for all compounds were prepared with (MeOH/ACN)/
H O (4):1 and used to establish LC/MS-MS conditions. Fifteen point
2
standard curves (0.6−900 ng/mL) were prepared for AZD1775, 11d,
and 11m. These compounds, and the internal standard 11f, were
monitored via electrospray ionization positive ion mode (ESI+)
settings: (i) ion-spray voltage 5500 V; (ii) temperature 450 °C; (iii)
H
DOI: 10.1021/acschembio.5b00725
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Articles
permeability. A.M.D. performed analysis of microarray data.
C.J.M. and P.R. analyzed the data and wrote the manuscript.
N.K.F., D.S.B. and R.V. analyzed data and provided review and
editorial comment of the manuscript. P.R. directed the research.
cisplatin induced ototoxicity profile may predict the need for hearing
support in children with medulloblastoma. Pediatr Blood Cancer. 60,
2
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M. H., Tang, S. H., and Chen, M. H. (2011) Medulloblastoma
presenting with pure word deafness: report of one case and review of
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Notes
The authors declare no competing financial interest.
(16) Harris, P. S., Venkataraman, S., Alimova, I., Birks, D. K.,
ACKNOWLEDGMENTS
Balakrishnan, I., Cristiano, B., Donson, A. M., Dubuc, A. M., Taylor,
M. D., Foreman, N. K., Reigan, P., and Vibhakar, R. (2014) Integrated
genomic analysis identifies the mitotic checkpoint kinase WEE1 as a
novel therapeutic target in medulloblastoma. Mol. Cancer 13, 72.
■
This study was supported by the National Institute Of
Neurological Disorders And Stroke (NINDS) of the National
Institutes of Health (NIH) under Award Number
R21NS084084, and by the University of Colorado Computa-
tional Chemistry and Biology Core Facility and the University
of Colorado Medicinal Chemistry Core Facility, which are
supported in part NIH/NCATS Colorado CTSA Grant
Number UL1 TR001082.
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DOI: 10.1021/acschembio.5b00725
ACS Chem. Biol. XXXX, XXX, XXX−XXX