Development and Characterization of a Wee1 Kinase Degrader

Article abstract of DOI:10.1016/j.chembiol.2019.10.013

The G1/S cell cycle checkpoint is frequently dysregulated in cancer, leaving cancer cells reliant on a functional G2/M checkpoint to prevent excessive DNA damage. Wee1 regulates the G2/M checkpoint by phosphorylating CDK1 at Tyr15 to prevent mitotic entry. Previous drug development efforts targeting Wee1 resulted in the clinical-grade inhibitor, AZD1775. However, AZD1775 is burdened by dose-limiting adverse events, and has off-target PLK1 activity. In an attempt to overcome these limitations, we developed Wee1 degraders by conjugating AZD1775 to the cereblon (CRBN)-binding ligand, pomalidomide. The resulting lead compound, ZNL-02-096, degrades Wee1 while sparing PLK1, induces G2/M accumulation at 10-fold lower doses than AZD1775, and synergizes with Olaparib in ovarian cancer cells. We demonstrate that ZNL-02-096 has CRBN-dependent pharmacology that is distinct from AZD1775, which justifies further evaluation of selective Wee1 degraders. Li, Pinch et al. develop and characterize the first selective Wee1 degrader, ZNL-02-096, by linking the clinical candidate inhibitor, AZD1775, to the cereblon-binding ligand, pomalidomide. They demonstrate that ZNL-02-096 exhibits cereblon-dependent pharmacology, induces G2/M phase accumulation and apoptosis at lower doses than AZD1775, and synergizes with Olaparib.

Full text of DOI:10.1016/j.chembiol.2019.10.013

Brief Communication
Development and Characterization of a Wee1 Kinase
Degrader
Graphical Abstract
Authors
Zhengnian Li, Benika J. Pinch,
Calla M. Olson, ..., Peter K. Sorger,
Eric S. Fischer, Nathanael S. Gray
Correspondence
nathanael_gray@dfci.harvard.edu
In Brief
Li, Pinch et al. develop and characterize
the first selective Wee1 degrader, ZNL-
02-096, by linking the clinical candidate
inhibitor, AZD1775, to the cereblon-
binding ligand, pomalidomide. They
demonstrate that ZNL-02-096 exhibits
cereblon-dependent pharmacology,
induces G2/M phase accumulation and
apoptosis at lower doses than AZD1775,
and synergizes with Olaparib.
Highlights
d
d
d
A selective Wee1 degrader was generated by conjugating
pomalidomide to AZD1775
Wee1 degradation induced G2/M accumulation at lower
doses than Wee1 inhibition
Wee1 degradation synergized with Olaparib in ovarian
cancer cells
Li et al., 2020, Cell Chemical Biology 27, 1–9
January 16, 2020 ª 2019 Elsevier Ltd.
https://doi.org/10.1016/j.chembiol.2019.10.013

Please cite this article in press as: Li et al., Development and Characterization of a Wee1 Kinase Degrader, Cell Chemical Biology (2019), https://
doi.org/10.1016/j.chembiol.2019.10.013
Cell Chemical Biology
Brief Communication
Development and Characterization
of a Wee1 Kinase Degrader
Zhengnian Li,1,2,7 Benika J. Pinch,1,3,4,7 Calla M. Olson,1,2 Katherine A. Donovan,1,2 Rados1aw P. Nowak,1,2 Caitlin E. Mills,5
David A. Scott,1,2 Zainab M. Doctor,1,6 Nicholas A. Eleuteri,1,2 Mirra Chung,5 Peter K. Sorger,5 Eric S. Fischer,1,2
and Nathanael S. Gray1,2,8,
*
1Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA, USA
2Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA
3Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, USA
4Department of Chemical Biology, Harvard University, Cambridge, MA, USA
5Laboratory of Systems Pharmacology, Department of Systems Biology, Harvard Medical School, Boston, MA, USA
6Department of Biological and Biomedical Sciences, Harvard Medical School, Boston, MA, USA
7These authors contributed equally
8Lead Contact
*Correspondence: nathanael_gray@dfci.harvard.edu
https://doi.org/10.1016/j.chembiol.2019.10.013
SUMMARY
induces premature mitosis, sensitizes cancer cells to
DNA-damaging agents, and causes tumor regression in preclin-
The G1/S cell cycle checkpoint is frequently dysregu- ical cancer models (Fu et al., 2018; Guertin et al., 2013; Hirai
et al., 2009). Numerous clinical trials are underway to evaluate
AZD1775 in combination with DNA-damaging agents in
lated in cancer, leaving cancer cells reliant on a
functional G2/M checkpoint to prevent excessive
DNA damage. Wee1 regulates the G2/M checkpoint
by phosphorylating CDK1 at Tyr15 to prevent mitotic
entry. Previous drug development efforts targeting
Wee1 resulted in the clinical-grade inhibitor,
AZD1775. However, AZD1775 is burdened by dose-
limiting adverse events, and has off-target PLK1 ac-
advanced solid tumors and leukemia. Several trials are also
testing AZD1775 monotherapy, for example, in ovarian cancer
patients with germline BRCA1/2 mutations (Leijen et al., 2016).
Although AZD1775 shows promising efficacy in the clinic, it
has been associated with dose-limiting toxicities, including neu-
tropenia, thrombocytopenia, anemia, and nausea (Do et al.,
2015; Guertin et al., 2013). Furthermore, AZD1775 has off-target
activity against other kinases; for example, it is equipotent
against recombinant Wee1 and the Ser/Thr kinase, PLK1 (Wright
tivity. In an attempt to overcome these limitations, we
developed Wee1 degraders by conjugating AZD1775
to the cereblon (CRBN)-binding ligand, pomalido- et al., 2017), and analogs of AZD1775 have been reported that
maintain potency against Wee1 but with reduced single-agent
cellular cytotoxicity (Matheson et al., 2016b, 2018). These limita-
mide. The resulting lead compound, ZNL-02-096, de-
grades Wee1 while sparing PLK1, induces G2/M
accumulation at 10-fold lower doses than AZD1775,
and synergizes with Olaparib in ovarian cancer cells.
We demonstrate that ZNL-02-096 has CRBN-depen-
dent pharmacology that is distinct from AZD1775,
which justifies further evaluation of selective Wee1
degraders.
tions highlight the need for an agent that selectively eliminates
Wee1 activity and that offers the potential for lower dosing.
Bifunctional degraders are small molecules composed of two
protein-binding ligands, one that recruits an E3 ligase and the
other that binds a protein of interest (POI). The resulting ternary
complex enables the ubiquitination and proteasomal degrada-
tion of the POI. Degraders can offer advantages over traditional
inhibitors, including catalytic target engagement to give a sub-
stoichiometric drug requirement, and the ability to achieve selec-
INTRODUCTION
tive degradation with a non-selective ligand (Toure and Crews,
2016; Winter et al., 2015). For example, selective degradation
Wee1, a tyrosine kinase, regulates the G2/M cell cycle check- of CDK4 or CDK6 can be achieved with dual CDK4/6 inhibitor-
point by phosphorylating and inactivating CDK1 in response to based degraders (Jiang et al., 2019), and selective degradation
extrinsic DNA damage and errors in DNA synthesis, thereby of CDK9 is possible with degraders based on a non-selective
preventing mitotic entry (Schmidt et al., 2017). Cancer cells CDK inhibitor (Olson et al., 2017). In a study that probed the
often have a deficient G1/S checkpoint, frequently via mutation degradable kinome, Wee1 was among the kinases degraded
of p53, which leaves them reliant on the G2/M checkpoint to by a promiscuous multi-kinase degrader (Huang et al., 2018),
avoid mitotic catastrophe. Therefore, abrogation of the G2/M suggesting that Wee1 is a suitable target for small-molecule-
checkpoint by inhibiting Wee1 can sensitize tumors to DNA- induced degradation.
damaging therapies (Carrassa and Damia, 2017; Matheson
et al., 2016a).
Here, we describe the design and characterization of a selec-
tive Wee1 degrader. Through docking and structure-guided
AZD1775 (Figure 1A) is an ATP-competitive Wee1 inhibitor design (Nowak et al., 2018), we developed a series of degraders
that decreases downstream phosphorylation of CDK1 Tyr15, by conjugating AZD1775 to pomalidomide, which binds the
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Figure 1. Design and Characterization of Wee1-Targeted Degraders
(A) Chemical structure of AZD1775, with linker attachment point highlighted in blue.
(B) Chemical structures of Wee1 degraders and the negative control, ZNL-02-178.
(C) Histogram of the shortest pairwise distances between lenalidomide and AZD1775, with the red arrow corresponding to the docking pose in (D) (Rosetta).
(D) Shortest pairwise distance docking pose between lenalidomide and AZD1775 (Rosetta).
ubiquitously expressed E3 ligase, CRBN. Using cell-based as- binant Wee1, with half maximal inhibitory concentration (IC50
)
says, we demonstrate that the resulting lead compound, ZNL- values of 1–10 nM, as evaluated using the fluorescence
02-096, induces potent and rapid Wee1 degradation, while resonance energy transfer (FRET)-based LanthaScreen assay
sparing degradation of AZD1775’s secondary target, PLK1. We (Invitrogen) (Table S1). Both compounds also induced Wee1
further show that ZNL-02-096 induces G2/M accumulation, in- degradation following a 5-h treatment in MOLT4 cells, an acute
creases unrepaired DNA damage and apoptosis, and synergizes lymphoblastic leukemia cell line that is sensitive to AZD1775
with the PARP inhibitor, Olaparib, in ovarian cancer cells.
treatment (Di Rora et al., 2018). Although a 1 mM treatment of
ZNL-02-012 was required to achieve maximal Wee1 degrada-
tion, 100 nM of ZNL-02-047 was sufficient to degrade Wee1,
likely due to enhanced cell permeability (Figure S1). We also
confirmed that treatment with either AZD1775 or pomalidomide
RESULTS
Development and Optimization of Wee1 Degraders
To develop a Wee1-targeted degrader, we modified AZD1775 by alone was insufficient to induce Wee1 degradation (Figure S1).
conjugating it to the CRBN-binding ligand, pomalidomide That ZNL-02-012 and ZNL-02-047 successfully engaged recom-
(Chamberlain et al., 2014; Chanan-Khan et al., 2013). The avail- binant Wee1 and mediated its cellular degradation validated the
able co-crystal structure of Wee1 with AZD1775 (PDB: 5V5Y) N-methyl piperazine of AZD1775 as a suitable linker attach-
suggests that the N-methyl piperazine is solvent exposed (Fig- ment site.
ure 1A) and we therefore selected this site for the attachment
of linkers to pomalidomide.
To optimize these initial compounds, we determined the min-
imum required linker length for ternary complex formation using
Compounds were alkylated from the 4-nitrogen of AZD1775’s Rosetta docking to predict degrader-mediated protein-protein
piperazine, and extended with a polyethylene glycol chain interactions (Nowak et al., 2018). We performed docking with
conjugated to pomalidomide. By this approach, we synthesized 40,000 models based on the X-ray crystal structures of lenalido-
ZNL-02-012, as well as a derivative without the amide bond mide bound to CRBN (PDB: 5FQD) and apo Wee1 (PDB: 3CR0)
(ZNL-02-047) to reduce the number of hydrogen bond donors, to identify the top 200 low-energy minima conformations. We
with the goal of enhancing cell penetration (Figure 1B). ZNL- then aligned the structure of AZD1775 bound to Wee1 (PDB:
02-012 and ZNL-02-047 were similarly potent binders of recom- 5V5Y) to the docked poses of apo Wee1, which allowed us to
2
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calculate the shortest possible distances between the N-methyl mide prevented ZNL-02-096-induced Wee1 degradation,
piperazine of AZD1775 and lenalidomide (Figure S1). Use of the demonstrating a requirement for both Wee1 and CRBN
apo structure to generate a docking model, followed by addition engagement (Figure 2B), although pomalidomide pretreatment
of the AZD1775 ligand post-docking, made possible the explora- gave only partial rescue. Notably, the decrease in pCDK Y15
tion of binding conformations that would otherwise have been induced by 100 nM of ZNL-02-096 was similarly dependent
excluded due to steric clashes. This approach thereby enables on proteasome function and CRBN binding, as indicated by
the design of degraders with short linkers, including molecules the rescue of this downstream phenotype upon pretreatment
in which the two ligands are directly fused. Accordingly, we iden- with carfilzomib, MLN4924, or pomalidomide. As expected,
˚
tified a cluster of linkers 3–7 A in length, corresponding to two to pretreatment with 1 mM of AZD1775 decreased pCDK1 Y15
six linker atoms, as sufficient to connect the two moieties (Fig- by inhibiting Wee1, independent of ZNL-02-096 co-treatment
ures 1C and 1D).
To test this docking prediction, we synthesized two deriva-
(Figure 2B).
To evaluate the kinetics of Wee1 degradation, we performed a
tives with short saturated carbon chain linkers, either six time course in MOLT4 cells using 100 nM of ZNL-02-096. Degra-
(ZNL-02-040) or three (ZNL-02-096) atoms in length (Figure 1B). dation occurred rapidly, with over 50% degradation achieved
Although both compounds potently engaged recombinant within 30 min, and complete degradation reached by 3 h.
Wee1 (LanthaScreen) (Table S1) and induced Wee1 degrada- Wee1 degradation was accompanied by a similarly rapid
tion following a 5-h exposure in MOLT4 cells, degradation reduction in pCDK1 Y15 (Figure 2C). In addition to being rapid,
was most pronounced for ZNL-02-096, which we selected as Wee1 degradation was prolonged, with a single 100 nM treat-
our lead compound (Figure S1). We also synthesized a corre- ment of ZNL-02-096 resulting in sustained Wee1 loss for at least
sponding negative control (ZNL-02-178) by methylating the ni- 48 h. By contrast, the negative control, ZNL-02-178, had no
trogen of the glutarimide ring to prevent CRBN binding effect on Wee1 protein levels out to 48 h after treatment
(Figure 1B). ZNL-02-178 inhibits but does not degrade Wee1, (Figure 2D).
enabling comparisons with ZNL-02-096 to distinguish between
To compare their biochemical selectivity, we submitted ZNL-
inhibition- and degradation-dependent pharmacology (Fig- 02-096 and AZD1775 for KINOMEscan profiling (DiscoverX) at
ure S1). Ultimately, we used Rosetta docking to inform linker 1 mM. As expected, both compounds displayed a similar off-
development, which yielded ZNL-02-096 as a potent, nanomo- target profile, for example, exhibiting strong engagement of
lar Wee1 degrader.
both Wee1 and PLK1 (Figure S1; Data S1). This result was
confirmed in a FRET-based Z0-LYTE kinase assay (Invitrogen),
in which ZNL-02-096 potently inhibited PLK1 (IC50 = 102 nM;
Table S1).
ZNL-02-096 Is a Rapid and Selective CRBN-Mediated
Wee1 Degrader
To assess the mechanism of ZNL-02-096, we compared its ac-
Given the precedent that selective degraders can be gener-
tivity in parental versus CRBNꢀ/ꢀ MOLT4 cells. In parental ated from non-selective ligands (Jiang et al., 2019; Olson et al.,
MOLT4 cells, ZNL-02-096 induced potent Wee1 degradation af- 2017), we next assessed the selectivity of degradation by per-
ter 5 h, with maximal degradation observed at 100 nM and a forming multiplexed mass spectrometry-based proteomics
‘‘hook effect’’ apparent at the top concentration of 10 mM, where (Donovan et al., 2018) in MOLT4 cells treated with 100 nM of
ZNL-02-096-CRBN and ZNL-02-096-Wee1 binary complexes ZNL-02-096 for 2 h. Wee1 was the only protein significantly
inhibit the formation of a productive ternary complex (Pettersson (p = 8.22 3 10ꢀ9) downregulated (log2 fold change = ꢀ1.97)
and Crews, 2019; Toure and Crews, 2016). By contrast, treat- across the proteome, thus establishing ZNL-02-096 as a Wee1
ment with ZNL-02-096 in CRBNꢀ/ꢀ MOLT4 cells did not affect degradation selective molecule. No downregulation of PLK1
Wee1 abundance, demonstrating that ZNL-02-096-mediated was evident by proteomics, or by immunoblot analysis (Figures
Wee1 degradation was CRBN dependent (Figure 2A). Further- 2D and 2E).
more, treatment with ZNL-02-096 in the parental MOLT4 cells
Zinc-finger transcription factors such as Ikaros (IKZF1), Aiolos
reduced phosphorylation of CDK1 at Tyr15 (pCDK1 Y15) starting (IKZF3), and ZFP91 are established off-targets of IMiD-based
¨
at a 100 nM dose, while decreased pCDK1 Y15 was observed in degraders (Donovan et al., 2018; Kronke et al., 2014). Although
the CRBNꢀ/ꢀ MOLT4 cells starting at a 1 mM dose. Therefore, low levels of ZFP91 degradation was evident in the proteomics
low concentrations of ZNL-02-096 (100 nM) yielded CRBN- experiment (Figure 2E), no degradation of either IKZF1 or
dependent effects on pCDK1 Y15, while high concentrations IKZF3 by ZNL-02-096 was observed at short time points. How-
(1 mM or above) resulted in the combined effects of inhibition ever, degradation of both IKZF1 and IKZF3 was apparent
and degradation.
following a 24 h treatment, as assayed by immunoblotting
We next evaluated the necessity of proteasome function and (Figure S1).
target engagement for ZNL-02-096 activity. Degradation of
These data show that ZNL-02-096 induces potent degrada-
Wee1 by ZNL-02-096 was rescued upon pretreatment tion of Wee1 in a CRBN- and proteasome-dependent manner,
with the proteasome inhibitor, carfilzomib, establishing a with maximal degradation achieved at 100 nM. Although ZNL-
requirement for proteasome function (Figure 2B). Wee1 degra- 02-096 inhibits PLK1 in vitro, it does not degrade PLK1 in cells.
dation was also prevented following pretreatment with the Finally, while ZNL-02-096 induces selective Wee1 degradation
NEDD8-activating enzyme inhibitor, MLN4924, indicating a at short time points (2–5 h), it degrades the common IMiD targets
dependence on Cullin-RING ligase activity given that CUL4 (IKZF1/3) by 24 h post-treatment. Therefore, it is critical to
neddylation is required for E3 ligase activity (Figure 2B). Finally, include an IMiD control in cell-based assays to distinguish be-
pretreatment with either an excess of AZD1775 or pomalido- tween the phenotypes of Wee1 versus IKZF1/3 degradation.
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Figure 2. ZNL-02-096 Induces Potent, Rapid, Selective, and CRBN-Dependent Wee1 Degradation
(A) Immunoblot analysis of MOLT4 parental or CRBNꢀ/ꢀ cells treated with ZNL-02-096 for 5 h.
(B) Immunoblot analysis of MOLT4 cells pre-treated with DMSO, Carfilzomib (Carf.), MLN4924 (MLN), pomalidomide (Pom.), or AZD1775 for 2 h, and then co-
treated with ZNL-02-096 for 5 h.
(C) Immunoblot analysis of MOLT4 cells treated with ZNL-02-096 for the indicated time points.
(D) Immunoblot analysis of MOLT4 cells treated with ZNL-02-096 or ZNL-02-178 for the indicated time points.
(E) Log2 fold change in abundance of proteins as measured using multiplexed quantitative-mass spectrometry-based proteomics of MOLT4 cells treated with
ZNL-02-096 (100 nM) for 2 h versus p value. n = 3 biological replicates.
ZNL-02-096 Induces DNA Damage, Apoptosis, and
Deregulation of the G2/M Checkpoint
data suggest that ZNL-02-096 induces the same downstream
effects as AZD1775, but at 10-fold lower doses, likely because
We next evaluated the effects of targeted Wee1 degradation on the degrader’s catalytic turnover enables efficacy at lower doses
the cell cycle. Following loss of Wee1 activity, cancer cells are than the inhibitor.
expected to enter mitosis before completing DNA repair, result-
Treatment with 100 nM of ZNL-02-096 increased the propor-
ing in unrepaired DNA damage and mitotic catastrophe (Beck tion of MOLT4 cells in G2/M in a time-dependent manner,
et al., 2012). Accordingly, a 24 h treatment of ZNL-02-096 measured by propidium iodide staining, while an equivalent
(100 nM) in MOLT4 cells reduced pCDK1 Y15, increased dose of ZNL-02-178 or AZD1775 did not appreciably affect
apoptosis, as monitored by PARP cleavage, and increased unre- the cell cycle (Figure 3B). Mirroring the concentration depen-
paired DNA, as indicated by the expression of the DNA double- dence of the downstream signaling effects (Figure 3A), a 10-
strand break marker, gH2AX. By contrast, treatment with an fold higher dose of AZD1775 (1 mM) was needed to elicit the
equivalent dose of ZNL-02-178 or AZD1775 did not produce same cell cycle changes as 100 nM of ZNL-02-096. Furthermore,
these downstream changes. Rather, a 1 mM treatment of the G2/M accumulation mediated by 100 nM of ZNL-02-096 was
AZD1775 was necessary to produce the same downstream absent in CRBNꢀ/ꢀ MOLT4 cells, highlighting a requirement for
changes as 100 nM of ZNL-02-096. Serving as the IMiD control, CRBN-dependent degradation (Figure 3C).
1 mM of pomalidomide did not affect phosphorylation of CDK1 To assess whether this cell cycle phenotype was extensible to
Y15, apoptosis, or levels of DNA damage (Figure 3A). These other cancer cell lines, we tested ZNL-02-096, ZNL-02-178, and
4
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Figure 3. ZNL-02-096 Induces DNA Damage, Apoptosis, and Premature Mitosis
(A) Immunoblot analysis of MOLT4 cells treated with the ZNL-02-096, ZNL-02-178, AZD1775, or pomalidomide for 24 h.
(B) MOLT4 cells treated with ZNL-02-096, ZNL-02-178, or AZD1775 for the indicated time points and stained with propidium iodide. Reported as mean SEM for
n = 3 biological replicates.
(C) Parental or CRBNꢀ/ꢀ MOLT4 cells treated with ZNL-02-096, ZNL-02-178, AZD1775, or lenalidomide for 24 h and stained with propidium iodide. Reported as
mean SEM for n = 3 biological replicates.
AZD1775 in triple-negative breast cancer (TNBC) and ovarian of ZNL-02-096 in MOLT4 CRBNꢀ/ꢀ cells. AZD1775 treatment
cancer cell lines using an image-based method to assay induced the same downstream phenotypes as ZNL-02-096,
changes in cell-cycle state (Hafner et al., 2016). In the TNBC but only at higher doses.
(BT549, HCC1806) and ovarian cancer (COV362, Kuramochi,
OVCAR8) cell lines tested, ZNL-02-096 induced G2/M phase ZNL-02-096 Induces CRBN-Dependent Antiproliferative
accumulation at lower doses than either ZNL-02-178 or Effects and Synergizes with Olaparib
AZD1775 (Figure S2). Strikingly, neither ZNL-02-096 nor We next profiled the antiproliferative activity of ZNL-02-096. In
AZD1775 induced significant cell cycle changes in the non- MOLT4 cells, ZNL-02-096 exhibited slightly enhanced cytotox-
transformed mammary epithelial cell line, MCF10A, indicating a icity as compared with AZD1775 (ZNL-02-096 IC50 = 390 nM;
cancer cell line-dependent sensitivity to Wee1 loss (Figure S2). AZD1775 IC50 = 800 nM), while ZNL-02-178 was significantly
This agrees with previous reports that normal cells are protected less potent (IC50 = 4,557 nM). This shift in the IC50 values of
from AZD1775-induced DNA damage and toxicity due to low ZNL-02-096 versus ZNL-02-178 is the CRBN-dependent contri-
basal replication stress (Fang et al., 2019).
bution to the antiproliferative activity. The CRBN-dependence
Collectively, these data reveal that ZNL-02-096 (100 nM) treat- was further indicated by the approximately 6-fold shift in the
ment leads to loss of pCDK1 Y15, increased apoptosis and un- IC50 of ZNL-02-096 in MOLT4 CRBNꢀ/ꢀ cells (IC50 = 2,220 nM)
repaired DNA damage, and G2/M phase accumulation in a versus in the parental cells (Figure 4A). Pomalidomide alone
CRBN-dependent manner. The requirement for CRBN engage- had no detectable cytotoxicity (Figure S3), suggesting that the
ment was shown by (1) rescue upon treatment with ZNL-02- CRBN-dependent antiproliferative activity of ZNL-02-096 is
178, which cannot engage CRBN, and (2) rescue upon treatment attributable to Wee1 degradation.
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Figure 4. ZNL-02-096 Induces Antiproliferative Effects and Synergizes with Olaparib
(A) Cell viability of parental or CRBNꢀ/ꢀ MOLT4 cells treated with AZD1775, ZNL-02-096, or ZNL-02-178 for 72 h, using CellTiter-Glo. Plotted as mean SEM for
n = 4 replicates.
(B) Cell viability of OVCAR8 cells treated with AZD1775, ZNL-02-096, or ZNL-02-178 for 72 h, using CellTiter-Glo. Plotted as mean SEM for n = 4 replicates.
(C) Cell viability of OVCAR8 cells co-treated with ZNL-02-096 and Olaparib, or AZD1775 and Olaparib for 72 h, using CellTiter-Glo. Plotted as mean SEM for
n = 4 replicates.
To more broadly profile the antiproliferative activity of ZNL-02- evaluated in a phase 1b clinical trial (ClinicalTrials.gov). There-
096, we submitted it to the PRISM platform (Broad Institute), a fore, we profiled ZNL-02-096 in a panel of ovarian cell lines.
pooled cytotoxicity assay, to evaluate its potency against 300 Notably, while ZNL-02-096 induced potent cell death in the three
suspension and hematopoietic cancer cell lines (Yu et al., ovarian cancer cell lines tested (OVCAR8, COV283, and Kuramo-
2016). We also profiled ZNL-02-178 to distinguish between chi), it showed little cytotoxicity in the non-malignant ovarian line,
CRBN-dependent and CRBN-independent effects. ZNL-02- OCE1, therefore showing cancer cell line-dependent antiprolifer-
096 exhibited sub-1 mM IC50 values in almost all of the lines ative activity (Figure S3). ZNL-02-096 demonstrated the most
tested, while ZNL-02-178 was uniformly less potent. The largest potent antiproliferative activity in OVCAR8 cells, as well as the
shift in potency between ZNL-02-096 and ZNL-02-178 was largest difference in potency relative to ZNL-02-178, so we
observed in multiple myeloma (MM) cell lines, which may be selected this cell line for further study. After confirming that
partially attributable to IKZF1/3 degradation given the therapeu- ZNL-02-096 degraded Wee1 in OVCAR8 cells, with maximal
tic efficacy of lenalidomide in MM patients (Data S2). In the cell degradation observed at 100 nM (Figure S4), we next evaluated
lines profiled, sensitivity to ZNL-02-096 was not dependent on the efficacy of ZNL-02-096 and Olaparib combination treat-
p53 mutational status (Figure S3) (Chen et al., 2018; Guertin ments. ZNL-02-096 synergized with Olaparib in OVCAR8 cells
et al., 2013). These data highlight that ZNL-02-096 demonstrates following a 72 h treatment, with similar synergy observed upon
single-agent antiproliferative activity against a broad range of AZD1775 and Olaparib co-treatment (Figures 4E and S4). By
cancer cell lines, and that the CRBN-dependent contribution, contrast, ZNL-02-178 displayed less synergistic activity with
as assessed by the shift in potency relative to ZNL-02-178, var- Olaparib than ZNL-02-096 at low doses. Pomalidomide and Ola-
ied considerably between cell lines, which may be explained by parib co-treatment was not synergistic, nor was pomalidomide
varying expression of CRBN. Moreover, cell lines dependent on and AZD1775 co-treatment (Figure S4). These data highlight
IKZF1/3 exhibited striking sensitivity to ZNL-02-096, likely due to that ZNL-02-096 demonstrates single-agent cytotoxicity and
the additional effects of IMiD-dependent IKZF1/3 degradation.
displays CRBN-dependent synergy with Olaparib in OVCAR8
To avoid any confounding phenotypes from IKZF1/3 degrada- cells, suggesting a potential therapeutic value to combination
tion, we chose to perform follow-up studies in ovarian cancer cell therapies of PARP inhibitors and Wee1 degraders.
lines, which are insensitive to IKZF1/3 degradation. Furthermore,
AZD1775 has demonstrated efficacy in ovarian cancer in both DISCUSSION
preclinical models and in the clinic, making ovarian cancer a ther-
apeutically relevant system in which to evaluate ZNL-02-096 Despite the proven utility of Wee1 inhibition in preclinical models
(Guertin et al., 2013; Zhang et al., 2017). In particular, AZD1775 and the clinic, to our knowledge small molecules that selectively
is reported to enhance the sensitivity of ovarian cancer cells to induce Wee1 degradation have not been investigated previously.
the PARP inhibitor, Olaparib (Kim et al., 2015; Meng et al., Given that a previous study implicated Wee1 as a degradable ki-
2018), and AZD1775/Olaparib combination therapy is being nase (Huang et al., 2018), and since bifunctional degraders offer
6
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Please cite this article in press as: Li et al., Development and Characterization of a Wee1 Kinase Degrader, Cell Chemical Biology (2019), https://
doi.org/10.1016/j.chembiol.2019.10.013
d KEY RESOURCES TABLE
d LEAD CONTACT AND MATERIALS AVAILABILITY
d EXPERIMENTAL MODEL AND SUBJECT DETAILS
d METHOD DETAILS
catalytic target engagement and the potential for lower
dosing, we were motivated to develop Wee1 degraders. ZNL-
02-096 exhibited Wee1 degradation at sub-mM doses, induced
G2/M accumulation, increased unrepaired DNA damage and
apoptosis, and exhibited antiproliferative activity across a panel
of 300 cancer cell lines. Comparing ZNL-02-096 directly with
AZD1775 showed that the degrader induced downstream
changes at 10-fold lower doses than the inhibitor. Moreover,
the negative control, ZNL-02-178, which inhibits Wee1 but
cannot engage CRBN, enabled us to distinguish between the
CRBN-dependent and CRBN-independent phenotypes of
ZNL-02-096. These data suggest that targeted Wee1 degraders
could be efficacious at lower doses than Wee1 inhibitors.
Selective protein degradation can be achieved with degraders
that incorporate a non-selective inhibitor into their design (Jiang
et al., 2019; Olson et al., 2017). Accordingly, although ZNL-02-
096 maintained the same biochemical selectivity profile as
AZD1775 and inhibited recombinant PLK1, cell-based assays
and proteomics demonstrated that PLK1 was not degraded by
ZNL-02-096 (Bondeson et al., 2018). As observed for other de-
graders that hijack CRBN, ZNL-02-096 degraded IKZF1/3,
which warrants additional medicinal chemistry efforts to elimi-
nate this effect. However, incorporating an IMiD control in our
cell-based assays enabled us to distinguish between the pheno-
types of Wee1 versus IKZF1/3 degradation. Furthermore, this
off-target IMiD activity could be leveraged in cancers that are
sensitive to IMiDs, such as MM, which is supported by the po-
tency of ZNL-02-096 in MM cell lines. By analogy, a degrader
of BTK and IKZF1/3 showed efficacy in B cell malignancies
(Dobrovolsky et al., 2019).
B Immunoblotting
B Cell Viability Assays
B Cell Cycle Analysis (PI Staining)
B In Vitro Kinase Assays
B Inhibitor Treatment and Western Blotting Experiments
B Synergy
B Proteomics
B Image-based Measurement of Cell Viability and Cell
Cycle Perturbation
B Rosetta Docking
B Chemistry Experimental Procedures
d QUANTIFICATION AND STATISTICAL ANALYSIS
B Antiproliferation Assay
B FACS
d DATA AND CODE AVAILABILITY
SUPPLEMENTAL INFORMATION
Supplemental Information can be found online at https://doi.org/10.1016/j.
chembiol.2019.10.013.
ACKNOWLEDGMENTS
We thank the Gray lab for helpful discussions, and M. Kostic and B. Nabet for
providing feedback on the manuscript. This work was supported by the NIH
F31 Fellowship CA225066 (to B.J.P.), NIH training grants 5 T32 GM007306-
41 and 5 T32 GM095450-04 (to B.J.P.), the Chleck Family Foundation (to
B.J.P. and Z.M.D.), NIH/NCI Grant HL127365 (to P.S. and N.S.G.), and NIH
R01 CA218278 01A1 (to N.S.G. and E.S.F.).
Collectively, the data presented in this study justify the evalu-
ation of Wee1 degradation in ovarian cancer as a monotherapy
or combined with Olaparib. We envision that ZNL-02-096 and
future analogs may serve as starting points for developing ther-
apeutic Wee1-targeted degraders, which could be broadly
applicable in cancer.
AUTHOR CONTRIBUTIONS
Z.L. designed and synthesized the compounds and B.J.P. performed their bio-
logical characterization and wrote the manuscript. C.M.O. supported the
degrader profiling, K.A.D. and N.A.E. performed proteomics, and R.P.N. per-
formed the docking. D.A.S. and E.S.F. provided input on project directions,
and Z.M.D. performed the PRISM/p53 data analysis. C.E.M., M.C., and
P.K.S. developed and performed the DeepDyeDrop experiment. N.S.G.
conceived of and oversaw the project.
SIGNIFICANCE
Wee1 kinase regulates the G2/M cell cycle checkpoint via
phosphorylation and inactivation of CDK1. Because cancer
cells often have a deficient G1/S checkpoint, Wee1 inhibition
can sensitize tumors to PARP inhibition and DNA-damaging
therapies. Here, we describe the development and charac-
terization of the selective Wee1 degrader, ZNL-02-096,
generated by linking the clinical-candidate Wee1 inhibitor,
AZD1775, to the CRBN-binding ligand, pomalidomide. The
CRBN-dependent pharmacology of ZNL-02-096 can be eval-
uated when compared with its negative control (ZNL-02-
178), which cannot engage CRBN. Ultimately, we show
that ZNL-02-096 induces apoptosis and G2/M accumulation
at up to 10-fold lower doses than AZD1775, demonstrating
that Wee1 degradation can enable efficacy at lower doses
than Wee1 inhibition.
DECLARATION OF INTERESTS
N.S.G. is a Scientific Founder and member of the Scientific Advisory Board
(SAB) of C4 Therapeutics, Syros, Soltego, B2S, Gatekeeper, and Petra Phar-
maceuticals, and has received research funding from Novartis, Astellas,
Taiho, and Deerfield. E.S.F. is a founder, SAB, and/or equity holder of C4
and Civetta and consults for Novartis, AbbVie, and Deerfield. The Fischer
lab receives/has received funding from Novartis, Deerfield, and Astellas.
P.S. is a founder, SAB member, and equity holder in Merrimack Pharmaceu-
tical and Glencoe Software; member of Board of Directors of Applied Biomath
and SAB of RareCyte Inc. P.S. declares that none of these relationships are
related to the content of this manuscript. B.J.P, Z.L., D.A.S., E.S.F., C.M.O.,
R.P.N., K.A.D., and N.S.G are inventors on a patent application related to
the Wee1 degraders described here.
STAR+METHODS
Received: June 28, 2019
Revised: September 23, 2019
Accepted: October 28, 2019
Published: November 14, 2019
Detailed methods are provided in the online version of this paper
and include the following:
Cell Chemical Biology 27, 1–9, January 16, 2020
7

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doi.org/10.1016/j.chembiol.2019.10.013
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doi.org/10.1016/j.chembiol.2019.10.013
STAR+METHODS
KEY RESOURCES TABLE
REAGENT or RESOURCE
SOURCE
IDENTIFIER
Antibodies
Rabbit polyclonal anti Wee1
Mouse monoclonal anti b-Actin
Rabbit monoclonal anti phospho-cdc2 (Tyr15)
Mouse monoclonal anti PLK1
Cell Signaling Technology
Cell Signaling Technology
Cell Signaling Technology
Abcam
Cat # 4936; RRID: AB_2288509
Cat # 3700; RRID: AB_2242334
Cat # 4539; RRID: AB_560953
Cat # ab17056; RRID: AB_443612
Cat # 9718; RRID: AB_2118009
Rabbit monoclonal anti phospho-histone H2A.X
(Ser139)
Cell Signaling Technology
Rabbit polyclonal anti PARP
Rabbit monoclonal anti cleaved PARP (Asp214)
Rabbit monoclonal anti Ikaros (D10E5)
Rabbit monoclonal anti Aiolos (D1C1E)
Rabbit polyclonal anti CDK1
Rabbit monoclonal anti CRBN
Chemicals, Peptides, and Recombinant Proteins
CellTiter Glo
Cell Signaling Technology
Cell Signaling Technology
Cell Signaling Technology
Cell Signaling Technology
Abcam
Cat # 9542; RRID: AB_2160739
Cat # 5625; RRID: AB_10699459
Cat # 9034; RRID: AB_2797691
Cat # 15103; RRID: AB_2744524
Cat # ab131450; RRID: AB_11155872
Cat # 71810; RRID: AB_2799810
Cell Signaling Technology
Promega
Cat # G7571
Cat # S1060
Cat # 15140122
Cat # 11965118
Cat # 11875119
Cat # 901558
Cat # P0018
Cat # S2853
Cat # 50-547-70001
Cat # BP231-100
Cat # S1525
N/A
Olaparib
Selleck Chemical LLC
Thermo Fisher Scientific
Thermo Fisher Scientific
Thermo Fisher Scientific
Sigma Aldrich
Sigma Aldrich
Selleck Chemicals
Thermo Fisher Scientific
Fisher
Penicillin Streptomycin
DMEM Medium
RPMI 1640 Medium
Lenalidomide
Pomalidomide
Carfilzomib
MLN4924
DMSO
AZD1775
Selleckchem
ZNL-02-012
This study
ZNL-02-040
This study
N/A
ZNL-02-047
This study
N/A
ZNL-02-096
This study
N/A
ZNL-02-178
This study
N/A
Protease Inhibitor Cocktail
Phosphatase Inhibitor Cocktail (PhosSTOP)
EdU
Roche
Cat # 11836170001
Cat # PHOSS-RO
Cat # 10540
Cat # L34974
Cat # 927-40000
Roche
Lumiprobe
LIVE/DEAD Far Red
Odyssey Blocking Buffer
Critical Commercial Assays
BCA Protein Assay Kit
Lanthascreen Binding (Wee1)
Z’ Lyte (PLK1)
Thermo Fisher Scientific
LI-COR Biosciences
Thermo Fisher Scientific
Invitrogen
Cat # 23227
Assay ID 1223
Assay ID 706
Cat # A34807
Invitrogen
Tandem mass tag (TMT) reagents
Deposited Data
Thermo Fisher Scientific
ZNL-02-096 Proteomics in MOLT4 cells
PRIDE PXD015513
(Continued on next page)
e1 Cell Chemical Biology 27, 1–9.e1–e9, January 16, 2020

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doi.org/10.1016/j.chembiol.2019.10.013
Continued
REAGENT or RESOURCE
Experimental Models: Cell Lines
OVCAR8
SOURCE
IDENTIFIER
Panagiotis A. Konstantinopoulos’s laboratory
Panagiotis A. Konstantinopoulos’s laboratory
Panagiotis A. Konstantinopoulos’s laboratory
N/A
N/A
N/A
N/A
Kuramochi
COV362
OCE1
Live Tumor Culture Core, University of Miami,
item # SC8
MOLT4
MOLT4 CRBN-/-
ATCC
CRL-1582
Generated by Winter et al.
(Winter et al., 2017)
CRL-10317
CRL-2335
MCF10A
ATCC
ATCC
ATCC
HCC1806
BT549
HTB-122
Software and Algorithms
Prism
GraphPad
N/A
FlowJo
FlowJo, LLC
N/A
Adobe Illustrator
Proteome Discoverer 2.2
R framework
Adobe Creative Cloud
Thermo Fisher Scientific
N/A
Cat # 30795
Team RCR: A Language and Environment for
Statistical Computing
R Version 3.5.1
– Feather Spray
Statistical Analysis, Limma Package (R framework)
Bioconductor
(Ritchie et al., 2015)
LEAD CONTACT AND MATERIALS AVAILABILITY
Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Nathanael
S. Gray (Nathanael_Gray@dfci.harvard.edu). Requested compounds will be provided following completion of an MTA.
EXPERIMENTAL MODEL AND SUBJECT DETAILS
HCC1806 (donor sex: female), BT549 (donor sex: female), MOLT4 (donor sex: male), OVCAR8 (donor sex: female) and Kuramochi
(donor sex: female) cells were maintained in RPMI (Corning, Corning, NY) supplemented with 10% fetal bovine serum (FBS) (Life
Technologies, Carlsbad, CA) and 1% penicillin/streptomycin (Corning, Corning, NY). MCF10A (donor sex: female) cells were main-
tained in a 1:1 mix of DMEM:F12 supplemented with 5% horse serum (Life Technologies, Carlsbad, CA), 1% penicillin/strepto-
mycin (Corning, Corning, NY), 20 ng/ml EGF (Peprotech, Rocky Hill, NJ), 500 ng/ml hydrocortisone (Sigma Aldrich, St. Louis,
MO), 10 mg/ml insulin (Sigma Aldrich, St. Louis, MO), and 100 ng/ml cholera toxin (Sigma Aldrich, St. Louis, MO). COV362 (donor
sex: female) cells were maintained in DMEM (Corning, Corning, NY) supplemented with 10% FBS (Life Technologies, Carlsbad,
CA), 1% penicillin/streptomycin (Corning, Corning, NY) and 2mM L-glutamine (Life Technologies, Carlsbad, CA). OCE1 (donor
sex: female) cells were maintained in FOMI (Wit-Fo) (PMID: 24303006) supplemented with 25 ng/ml cholera toxin (Sigma Aldrich,
St. Louis, MO). All cell lines were cultured at 37ꢁC and 5% CO2 and were tested for the absence of Mycoplasma infection on a
monthly basis.
METHOD DETAILS
Immunoblotting
Whole cell lysates for immunoblotting were prepared by pelleting cells from each cell line at 4ꢁC (300 g) for 5 minutes. The resulting
cell pellets were washed 1x with ice-cold PBS and then resuspended in RIPA lysis buffer (Sigma, cat#R0278) supplemented with
protease and phosphatase inhibitor tablets (Roche cat#4906845001). Lysates were clarified at 14,000 rpm for 15 minutes at 4ꢁC prior
to quantification by BCA assay (Pierce, cat#23225). Whole cell lysates were loaded into Bolt 4-12% Bis-Tris Gels (Thermo Fisher,
cat#NW04120BOX) and separated by electrophoreses at 95 V for 1.5 hours. The gels were transferred to a nitrocellulose membrane
using the iBlot Gel Transfer at P3 for 7 minutes (Thermo Fisher, cat#IB23001) and then blocked for 1 hr at room temperature in Od-
yssey blocking buffer (LICOR Biosciences, cat#927-50010). Membranes were probed using antibodies against the relevant proteins
at 4ꢁC overnight in 20% Odyssey Blocking Buffer in 1x TBST. Membranes were then washed three times with 1x TBST (at least
Cell Chemical Biology 27, 1–9.e1–e9, January 16, 2020 e2

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doi.org/10.1016/j.chembiol.2019.10.013
5 minutes per wash) followed by incubation with the IRDye goat anti-mouse (LICOR, cat#926-32210) or goat anti-rabbit (LICOR, cat
#926-32211) secondary antibody (diluted 1:10,000) in 20% Odyssey Blocking Buffer in 1x TBST for 1 hr at room temperature. After
three washes with 1x TBST (at least 5 minutes per wash), the immunoblots were visualized using the ODYSSEY Infrared Imaging Sys-
tem (LICOR).
Cell Viability Assays
The indicated cell lines were plated in flat bottom 384-well plates (Corning cat#3570). MOLT4 cells were plated at a density of 3,000
cells per well in 50 mL media, while OVCAR8, COV362, and Kuramochi cells were plated at a density of 750 cells per well in 50 mL
media. Cells were treated the next day (except for MOLT4 cells, which were treated the same day as plating) with 0.1 mL of the
indicated compounds in a four-fold dilution series using the Janus pinner. Cells were incubated with compounds for 72 hours
at 37ꢁC 5% CO2. Anti-proliferative effects were then assessed by CellTiter-Glo Luminescent Cell Viability Assay (Promega
cat#G7570) according to the manufacturer’s standards, measuring luminescence using an Envision plate-reader. IC50 values
were determined using a non-linear regression curve fit in GraphPad Prism 8. N=4 biological replicates were used for each treat-
ment condition.
Cell Cycle Analysis (PI Staining)
MOLT4 cells were plated at 1.5 million cells per well in a 6-well plate, with 3 mL of media per well. Immediately after plating, cells were
treated with the indicated concentrations of the indicated compound. At the indicated time points, cells were collected by transferring
each well to a 15 mL conical, centrifuging (1000 rpm for 3 min), and then washing the cell pellet 3x with cold PBS. Cell pellets were
then fixed with 1 mL cold 80% ethanol in PBS at 4ꢁC overnight. The cell pellets were then washed by adding 1 mL of PBS and then
centrifuging (1500 rpm for 5 minutes), followed by aspiration of the PBS/ethanol mix. Cells were resuspended in cell cycle staining
solution (100 mg/mL of RNAse A (Roche, 10109169001) and 50 mg/mL propidium iodide (Life Technologies, P1304MP), diluted in
PBS, and incubated for 10 minutes at room temperature. Cell cycle data was acquired by flow cytometry on a Guava easyCyte
flow cytometer (Millipore) using the InCyte software. Data was analyzed using FlowJo. N=3 biological replicates were used for
each treatment condition.
In Vitro Kinase Assays
LanthaScreen assays were conducted for Wee1 in a 10-point dose response as performed in the commercial assay service by Life
Technologies. Z’-LYTE assays were conducted for PLK1 as performed in the commercial assay service by Life Technologies in a
10-point dose response using Km ATP concentrations.
Inhibitor Treatment and Western Blotting Experiments
MOLT4 cells were plated at 1.5 million cells per well in a 6-well plate, with 3 mL of media per well. Cells were then immediately treated
with the indicated concentration of the indicated compound and incubated for the indicated amount of time. Cells were collected by
transferring each well to a 15 mL conical, centrifuging (1000 rpm for 3 min), then washing the cell pellet 2x with cold PBS. Each cell
pellet was then lysed in 100 mL of RIPA lysis buffer (+protease/phosphatase inhibitors). Samples were normalized and prepped in 4x
LDS + 10% b-mercaptoethanol and boiled for 5 min at 95ꢁC. Lysates were probed for specified proteins by western blotting using the
Bolt system (Life Technologies).
Synergy
Plate Cells
OVCAR8 cells were plated at a density of 750 cells per well in 50 mL media in a white 384-well plate (Corning cat#3570).
Prepare Compound Plates and Treat Cells
Each compound was plated in a 384-well plate in a 5-point, four-fold dilution curve as illustrated below (where blue is ZNL-02-
096 (A, below), yellow is Olaparib (B, below), gray and white is DMSO; dark blue or yellow is the highest compound concen-
tration, with dilutions represented by the gradations to light blue or yellow). Cells were treated the day after plating with
0.1 mL from each compound plate using the Janus pinner (ZNL-02-096 plate pinned with orientation A1 to back left (A, below);
Olaparib plate pinned with orientation A1 to front right (B, below), to give the co-treatment pattern (C, below) in which green
represents co-treatment regions).
Incubate Cells with Compound
Cells were incubated with compounds for 72 hours at 37ꢁC 5% CO2. Anti-proliferative effects were then assessed by CellTiter-Glo
Luminescent Cell Viability Assay (Promega cat#G7570) according to the manufacturer’s standards, measuring luminescence using
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an Envision plate-reader. IC50 values were determined using a non-linear regression curve fit in GraphPad Prism 8. N=4 biological
replicates were used for each treatment condition.
Proteomics
Sample Preparation TMT LC-MS3 Mass Spectrometry
MOLT4 cells were treated with DMSO or 100 nM of ZNL-02-096 for 2 or 4 hours in biological triplicates and lenalidomide in biological
duplicates for 4 hours and cells were harvested by centrifugation. Lysis buffer (8 M Urea, 50 mM NaCl, 50 mM 4-(2hydroxyethyl)-1-
piperazineethanesulfonic acid (EPPS) pH 8.5, Protease and Phosphatase inhibitors from Roche) was added to the cell pellets and
homogenized by 20 passes through a 21 gauge (1.25 in. long) needle to achieve a cell lysate with a protein concentration between
1 – 4 mg mL-1. A micro-BCA assay (Pierce) was used to determine the final protein concentration of protein in the cell lysate. 200 mg of
protein for each sample were reduced and alkylated as previously described(Donovan et al., 2018).
Proteins were precipitated using methanol/chloroform. In brief, four volumes of methanol were added to the cell lysate, followed by
one volume of chloroform, and finally three volumes of water. The mixture was vortexed and centrifuged to separate the chloroform
phase from the aqueous phase. The precipitated protein was washed with three volumes of methanol, centrifuged and the resulting
washed precipitated protein was allowed to air dry. Precipitated protein was resuspended in 4 M Urea, 50 mM HEPES pH 7.4, fol-
lowed by dilution to 1 M urea with the addition of 200 mM EPPS, pH 8. Proteins were first digested with LysC (1:50; enzyme: protein)
for 12 hours at room temperature. The LysC digestion was diluted down to 0.5 M Urea with 200 mM EPPS pH 8 and then digested with
trypsin (1:50; enzyme:protein) for 6 hours at 37ꢁC. Tandem mass tag (TMT) reagents (Thermo Fisher Scientific) were dissolved in
anhydrous acetonitrile (ACN) according to manufacturer instructions. Anhydrous ACN was added to each peptide sample to a final
concentration of 30% v/v, and labeling was induced with the addition of TMT reagent to each sample at a ratio of 1:4 peptide:TMT
label. The 10-plex labeling reactions were performed for 1.5 hours at room temperature and the reaction quenched by the addition of
hydroxylamine to a final concentration of 0.3% for 15 minutes at room temperature. The sample channels were combined at a
1:1:1:1:1:1:1:1:1:1:1 ratio, desalted using C18 solid phase extraction cartridges (Waters) and analyzed by LC-MS for channel ratio
comparison. Samples were then combined using the adjusted volumes determined in the channel ratio analysis and dried down
in a speed vacuum. The combined sample was then resuspended in 1% formic acid and acidified (pH 2ꢀ3) before being subjected
to desalting with C18 SPE (Sep-Pak, Waters). Samples were then offline fractionated into 96 fractions by high pH reverse-phase
HPLC (Agilent LC1260) through an aeris peptide xb-c18 column (phenomenex) with mobile phase A containing 5% acetonitrile
and 10 mM NH4HCO3 in LC-MS grade H2O, and mobile phase B containing 90% acetonitrile and 10 mM NH4HCO3 in LC-MS grade
H2O (both pH 8.0). The 96 resulting fractions were then pooled in a non-continuous manner into 24 fractions and these fractions were
used for subsequent mass spectrometry analysis.
Data were collected using an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) coupled
with a Proxeon EASY-nLC 1200 LC pump (Thermo Fisher Scientific). Peptides were separated on an EasySpray ES803 75 mm inner
diameter microcapillary column (ThermoFisher Scientific). Peptides were separated using a 190 min gradient of 6–27% acetonitrile in
1.0% formic acid with a flow rate of 350 nL/min.
Each analysis used an MS3-based TMT method as described previously(McAlister et al., 2014) The data were acquired using a
mass range of m/z 340 – 1350, resolution 120,000, AGC target 5 x 105, maximum injection time 100 ms, dynamic exclusion of
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doi.org/10.1016/j.chembiol.2019.10.013
120 seconds for the peptide measurements in the Orbitrap. Data dependent MS2 spectra were acquired in the ion trap with a normal-
ized collision energy (NCE) set at 35%, AGC target set to 1.8 x 104 and a maximum injection time of 120 ms. MS3 scans were ac-
quired in the Orbitrap with a HCD collision energy set to 55%, AGC target set to 2 x 105, maximum injection time of 150 ms, resolution
at 50,000 and with a maximum synchronous precursor selection (SPS) precursors set to 10.
LC-MS Data Analysis
Proteome Discoverer 2.2 (Thermo Fisher) was used to for .RAW file processing and controlling peptide and protein level false dis-
covery rates, assembling proteins from peptides, and protein quantification from peptides. MS/MS spectra were searched against
a Uniprot human database (September 2016) with both the forward and reverse sequences. Database search criteria are as follows:
tryptic with two missed cleavages, a precursor mass tolerance of 20 ppm, fragment ion mass tolerance of 0.6 Da, static alkylation of
cysteine (57.02146 Da), static TMT labelling of lysine residues and N-termini of peptides (229.16293 Da), and variable oxidation of
methionine (15.99491 Da). TMT reporter ion intensities were measured using a 0.003 Da window around the theoretical m/z for
each reporter ion in the MS3 scan. Peptide spectral matches with poor quality MS3 spectra were excluded from quantitation
(summed signal-to-noise across 11 channels < 200 and precursor isolation specificity < 0.5).
Reporter ion intensities were normalized and scaled using in-house scripts in the R framework (R Core Team, 2014). Statistical
analysis was carried out using the limma package within the R framework(Ritchie et al., 2015).
Image-based Measurement of Cell Viability and Cell Cycle Perturbation
These measurements are related to Figures S2 and S3. Breast cancer cell lines, BT549, HCC1806, and MCF10A cells were plated at a
density of 1000 cells per well in 384-well Cell Carrier plates (Perkin Elmer, Waltham, MA) using a Multidrop Combi Reagent Dispenser
(Thermo Fisher Scientific, Waltham, MA) and allowed to adhere to for 24 hours prior to drug treatment. Ovarian cancer cell were
platted similarly at the following densities: COV362 and OVCAR8 500 cells/well, Kuramochi 1500 cells per well and, OCE1 1000 cells
per well. Cells were treated with a dilution series of the indicated drugs using a D300 Digital Dispenser (Hewlett-Packard, Palo Alto,
CA). Cells were stained and fixed for analysis at the time of drug delivery and after 24, 48 or 72 hrs of incubation. Cells were pulsed for
one hour with EdU (Lumiprobe, Hunt Valley, MD) and stained with 1:2000 LIVE/DEAD Far Red Dead Cell Stain (LDR) (Thermo Fisher
Scientific, Waltham, MA). Cells were then fixed with 3.7% formaldehyde (Sigma Aldrich, St. Louis, MO) for 30 minutes and permea-
bilized with 0.5% Triton X-100 in PBS. The EdU was labeled with cy3-azide (Lumiprobe, Hunt Valley, MD) for 30 min. The cells were
then blocked for one hour with Odyssey blocking buffer (LI-COR, Lincoln, NE), and stained overnight at 4ꢁC with 2 mg/ml Hoechst
33342 (Sigma Aldrich, St. Louis, MO) and a 1:1000 dilution of anti-phospho-histone H3 (pHH3) Alexa 488 (Ser10, clone D2C8) con-
jugated antibody (Cell Signaling Technologies, Danvers, MA). Fixed cells were imaged with a 10x objective using an Operetta micro-
scope and analyzed using the Columbus image data storage and analysis system (Perkin Elmer, Waltham, MA). Nuclei were
segmented using Columbus software (Perkin Elmer, Waltham, MA) based on their Hoechst signal. DNA content was defined by
the total Hoechst intensity within the nuclear mask to identify cells in the G1 and G2 phases of the cell cycle. The average LDR,
EdU and phospho-histone H3 intensities within the nuclear masks were determined. The LDR signal was used to classify cells as
live or dead, the EdU and pHH3 signals to identify S and M phase cells respectively. Cells with intermediate DNA content and no
EdU signal were classified as S phase dropout cells. Live cell counts were normalized to DMSO-treated controls on the same plates
to yield normalized growth rate inhibition (GR) values as described previously (Hafner et al., 2016). Experiments in breast cancer cell
lines were performed as technical triplicates in biological duplicate, and as technical and biological triplicates in ovarian cancer
cell lines.
Rosetta Docking
All protein docking was carried out using Rosetta 3.8 provided through SBGrid(Morin et al., 2013).Input models were downloaded
from the PDB (CRBN bound lenalidomide pdb: 5fqd, chain B; Wee1 pdb: 3cr0, chain A with inhibitor, glycerol and Cl removed).
Ligand parameters for lenalidomide were generated using OpenEye Omega (OpenEye Scientific) and parameter files generated us-
ing Rosetta ‘molfile_to_params.py’. Pdb structure coordinates were combined into a single file and prepared for docking using the
Rosetta ‘docking_prepack_protocol’ program. Initial global docking was performed by running Rosetta ‘docking_protocol’ 80 times
(total 40,000 poses) with the following command line options:
-partners A_B -dock_pert 5 25 -randomize2 -ex1 -ex2aro -nstruct 500 -beta.
providing the combined pdb, lenalidomide specific parameter files and database file as input.
To assess the landscape of possible low energy binding modes for Wee1, the top 200 lowest I_sc scoring docking decoys were
selected. X-ray crystal structure model of Wee1 bound AZD1775 (pdb: 5v5y) was superimposed to each of the docked poses of
¨
Wee1 using Pymol ‘align’ command (The PyMOL Molecular Graphics System, Version 1.8.6.0 Schrodinger, LLC). The shortest pair-
wise distance between selected atoms on lenalidomide and AZD1775 (see highlighted atoms in Figure S1) was calculated in Pymol
as Euclidean distance for each of the top 200 poses. The histogram was obtained in GraphPad Prism 7 using Column Analysis - Fre-
quency Distribution. Data analysis and statistics for all steps were performed using GraphPad Prism 7.
Chemistry Experimental Procedures
Chemical Synthesis and Structure Validation
Starting materials, reagents and solvents were purchased from commercial suppliers and were used without further purification
unless otherwise noted. All reactions were monitored using a Waters Acquity UPLC/MS system (Waters PDA el Detector, QDa
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doi.org/10.1016/j.chembiol.2019.10.013
Detector, Sample manager - FL, Binary Solvent Manager) using Acquity UPLCꢀ BEH C18 column (2.1 x 50 mm, 1.7mm particle size):
solvent gradient = 85% A at 0 min, 1% A at 1.7 min; solvent A = 0.1% formic acid in Water; solvent B = 0.1% formic acid in Acetonitrile;
flow rate: 0.6mL/min. Reaction products were purified by flash column chromatography using CombiFlashꢀRf with Teledyne Isco
RediSepꢀ normal-phase silica flash columns (4 g, 12 g, 24 g, 40 g or 80 g) and Waters HPLC system using SunFireTM Prep C18
column (19 x 100 mm, 5 mm particle size): solvent gradient = 80% A at 0 min, 10% A at 25 min; solvent A = 0.035% TFA in Water;
solvent B = 0.035% TFA in MeOH; flow rate: 25 mL/min. 1H NMR spectra were recorded on 500 MHz Bruker Avance III spectrometers
and 13C NMR spectra were recorded on 125 MHz Bruker Avance III spectrometer. Chemical shifts are reported in parts per million
(ppm, d) downfield from tetramethylsilane (TMS). Coupling constants (J) are reported in Hz. Spin multiplicities are described as br
(broad), s (singlet), d (doublet), t (triplet), q (quartet) and m (multiplet).
Scheme 1: 2-Allyl-1-(6-(2-hydroxypropan-2-yl)pyridin-2-yl)-6-((4-(piperazin-1-yl)phenyl)amino)-1,2-dihydro-3H-pyrazolo[3,4-d]
pyrimidin-3-one (4).
To a solution of known intermediate 1(Matheson et al., 2016b) (220 mg, 0.6 mmol) in toluene was added m-chloroperoxybenzoic
acid (145 mg, 0.65 mmol) and the reaction mixture was stirred for 1 hour. N,N-Diisopropylethylamine and aniline 2 (205 mg,
0.74 mmol) were added and the reaction mixture was stirred overnight. Sat. NaHCO3 (10 mL) was added and the mixture was ex-
tracted with EtOAc (15 mL32). The organic phase was washed with brine, dried over MgSO4, filtered, and concentrated in vacuo.
The crude material was purified via silica gel chromatography (20:1 DCM:MeOH) to afford intermediate 3 (255mg, 73%). LC-MS:
m/z 587.34 [M+1]+. TFA (1 mL) was added to a solution of (3) (255 mg, 0.43 mmol) in DCM (4 mL). The reaction was stirred for 2 hours.
The solvent was removed under reduced pressure to obtain the TFA salt of the Boc-deprotected amine (4). LC-MS: m/z
487.31 [M+1]+.
1H NMR (500 MHz, DMSO) d 10.20 (s, 1H), 8.87-8.84 (m, 3H), 8.03 (t, J= 7.8 Hz, 1H), 7.73 (d, J = 7.8 Hz, 1H), 7.61 (d, J = 7.5 Hz, 3H),
6.99 (d, J = 9.0 Hz, 2H), 5.66 (ddt, J = 16.3, 10.3, 6.0 Hz, 1H), 4.99 (dd, J = 10.2, 1.2 Hz, 1H), 4.82 (dd, J = 17.1, 1.3 Hz, 1H), 4.68 (d,
J = 5.4 Hz, 2H), 3.36-3.18 (m, 8H), 1.46 (s, 6H).
Scheme 2: Synthesis of ZNL-02-040, ZNL-02-047 and ZNL-02-096.
4-((2-(2-(2-(2-(4-(4-((2-Allyl-1-(6-(2-hydroxypropan-2-yl)pyridin-2-yl)-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)
phenyl)piperazin-1-yl)ethoxy)ethoxy)ethoxy)ethyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (ZNL-02-047). To a solution
of Intermediate (4) (35 mg, 0.073 mmol) in DMF(1.5mL) was added tert-Butyl (2-(2-(2-(2-bromoethoxy)ethoxy)ethoxy)ethyl)carbamate
(28 mg, 0.08 mmol) and K2CO3 (20 mg, 0.15 mmol). The mixture was heated at 60ꢁC for 6 h. Then allowing the reaction to cool to
room temperature, the solution was dissolved in water and ethyl acetate. The mixture extracted with EtOAc (3 x 10 ml). The organic
extract was washed with brine (20 ml), dried over MgSO4, and concentrated under reduced pressure. The residue was purified via silica
gel chromatography (10:1 DCM: MeOH) to give compound (5) (44 mg, 80 % yield). LC-MS: m/z 762.46 [M+1]+. Intermediate (5) (22 mg,
0.03 mmol) was dissolved in DCM (2 mL). TFA (0.5 mL) was added and the mixture was stirred for 30 minutes. The solvent was removed
under reduced pressure and the residue was dissolved in DMSO (1 mL). 2-(2,6-dioxopiperidin-3-yl)-4-fluoroisoindoline-1,3-dione (8)
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(10 mg, 0.035 mmol) and N, N-diisopropylethylamine (15 mL, 0.09 mmol) were added, and the mixture was stirred at 90ꢁC for 3 h. The
mixture was purified by HPLC (10-90% MeOH in H2O) to give TFA salt ZNL-02-047 (8 mg, 30%) as a yellow solid. LC-MS: m/z
918.46 [M+1]+.
1H NMR (500 MHz, DMSO) d 11.11 (s, 1H), 10.20 (s, 1H), 9.75 (s, 1H), 8.85 (s, 1H), 8.03 (t, J = 7.8 Hz, 1H), 7.76 (d, J = 7.9 Hz, 1H), 7.62
(d, J = 7.6 Hz, 3H), 7.57 (dd, J = 8.4, 7.2 Hz, 1H), 7.13 (d, J = 8.6 Hz, 1H), 7.04 (d, J = 7.0 Hz, 1H), 6.99 (d, J = 9.0 Hz, 2H), 6.60 (s, 1H),
5.67 (ddt, J = 16.3, 10.3, 6.0 Hz, 1H), 5.06 (dd, J = 12.8, 5.4 Hz, 1H), 5.00 (dd, J = 10.2, 1.1 Hz, 1H), 4.83 (dd, J = 17.1, 1.2 Hz, 1H), 4.69
(d, J = 4.8 Hz, 2H), 3.80 – 3.75 (m, 5H), 3.64 –3.57 (m, 12H), 3.49 – 3.44 (m, 2H), 3.39 (s, 2H), 3.22 (d, J = 9.8 Hz, 2H), 3.01 (t, J = 11.9 Hz,
2H), 2.89 (ddd, J = 17.1, 14.0, 5.4 Hz, 1H), 2.65 – 2.53 (m, 1H), 2.06 – 1.98 (m, 1H), 1.47 (s, 6H).
13C NMR (125 MHz, DMSO) d 173.29, 171.19, 170.57, 169.45, 168.12, 167.75, 161.54, 160.90, 158.64, 158.37, 156.56, 147.54,
146.87, 145.90, 139.26, 136.73, 132.66, 132.57, 121.71, 118.73, 117.92, 116.75, 111.20, 109.73, 72.79, 70.15, 69.99, 69.36,
67.29, 53.92, 51.38, 49.04, 47.04, 46.61, 42.17, 40.59, 36.59, 36.10, 31.46, 30.93, 24.45, 22.61.
4-((6-(4-(4-((2-Allyl-1-(6-(2-hydroxypropan-2-yl)pyridin-2-yl)-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)phenyl)
piperazin-1-yl)hexyl)amino)-2-(2,6- dioxopiperidin-3-yl)isoindoline-1,3-dione (ZNL-02-040). To a solution of Intermediate (4)
(35 mg, 0.073 mmol) in DMF (1.5mL) was added tert-Butyl (6-bromohexyl) carbamate (24 mg, 0.08 mmol) and K2CO3 (20 mg,
0.15 mmol). The mixture was heated at 60ꢁC for 6 h. Then allowing the reaction to cool to room temperature, the solution was
dissolved in water and ethyl acetate. The mixture extracted with EtOAc (3 x 10 ml). The organic extract was washed with brine
(20 ml), dried over MgSO4, and concentrated under reduced pressure. The residue was purified via silica gel chromatography
(10:1 DCM: MeOH) to give compound (6) (37 mg, 75 % yield). LC-MS: m/z 686.47 [M+1]+. Intermediate (6) (20 mg, 0.03 mmol)
was dissolved in DCM (2 mL). TFA (0.5 mL) was added and the mixture was stirred for 30 minutes. The solvent was removed under
reduced pressure and the residue was dissolved in DMSO (1 mL). 2-(2,6-dioxopiperidin-3-yl)-4-fluoroisoindoline-1,3-dione (8)
(10 mg, 0.035 mmol) and N, N-diisopropylethylamine (15 mL, 0.09 mmol) were added, and the mixture was stirred at 90ꢁC for
3 h. The mixture was purified by HPLC (10-90% MeOH in H2O) to give TFA salt ZNL-02-040 (10 mg, 39%) as a yellow solid.
LC-MS: m/z 842.52 [M+1]+.
1H NMR (500 MHz, DMSO) d 11.08 (s, 1H), 10.13 (s, 1H), 8.82 (s, 1H), 8.04 (s, 1H), 7.75 (s, 1H), 7.59 (dd, J = 14.7, 7.4 Hz, 4H), 7.09 (d,
J = 8.5 Hz, 1H), 7.02 (d, J = 7.0 Hz, 1H), 6.90 (d, J = 8.5 Hz, 2H), 6.53 (s, 1H), 5.66 (dd, J = 16.6, 10.4 Hz, 1H), 5.33 (s, 1H), 5.04 (dd,
J = 12.7, 5.2 Hz, 1H), 4.99 (d, J = 10.1 Hz, 1H), 4.82 (d, J = 17.2 Hz, 1H), 4.67 (s, 2H), 3.07 (s, 5H), 2.87 (dd, J = 21.4, 8.8 Hz, 1H), 2.60 –
2.53 (m, 1H), 2.29 (d, J = 6.5 Hz, 2H), 2.11 – 1.95 (m, 1H), 1.59 (s, 2H), 1.46 (s, 6H), 1.42 – 1.21 (m, 6H).
13C NMR (125 MHz, DMSO) d 173.28, 170.57, 169.44, 167.78, 161.64, 160.98, 156.47, 147.71, 146.92, 139.29, 136.77, 132.68,
131.25, 121.62, 118.73, 117.68, 116.75, 115.92, 110.86, 109.49, 72.79, 58.30, 53.28, 49.16, 49.02, 47.07, 42.29, 40.59, 40.43,
40.26, 40.09, 31.46, 30.93, 29.12, 27.16, 26.70, 22.64.
4-((3-(4-(4-((2-Allyl-1-(6-(2-hydroxypropan-2-yl)pyridin-2-yl)-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)phenyl)
piperazin-1-yl)propyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (ZNL-02-096). To a solution of Intermediate (4)
(100 mg, 0.2 mmol) in DMF (2 mL) was added tert-Butyl (3-bromopropyl) carbamate (60 mg, 0.24 mmol) and K2CO3 (55 mg,
0.4 mmol). The mixture was heated at 60ꢁC for 6 h. Then allowing the reaction to cool to room temperature, the solution
was dissolved in water and ethyl acetate. The mixture extracted with EtOAc (3 x 10 ml). The organic extract was washed
with brine (20 ml), dried over MgSO4, and concentrated under reduced pressure. The residue was purified via silica gel
chromatography (10:1 DCM: MeOH) to give compound (7) (110 mg, 85 % yield). LC-MS: m/z 644.44 [M+1]+. Intermediate (7)
(30 mg, 0.04 mmol) was dissolved in DCM (2 mL). TFA (0.5 mL) was added and the mixture was stirred for 30 minutes. The
solvent was removed under reduced pressure and the residue was dissolved in DMSO (1 mL). 2-(2,6-dioxopiperidin-3-yl)-4-
fluoroisoindoline-1,3-dione (8) (15 mg, 0.055 mmol) and N, N-diisopropylethylamine (20 mL, 0.12 mmol) were added, and the
mixture was stirred at 90ꢁC for 3 h. The mixture was purified by HPLC (10-90% MeOH in H2O) to give TFA salt ZNL-02-096
(11 mg, 35%) as a yellow solid. LC-MS: m/z 800.52 [M+1]+.
1H NMR (500 MHz, DMSO) d 11.10 (s, 1H), 10.72 (s, 1H), 10.21 (s, 1H), 8.84 (s, 1H), 8.04 (t, J = 7.6 Hz, 1H), 7.75 (d, J = 8.0 Hz, 1H),
7.67 – 7.57 (m, 4H), 7.18 (d, J = 8.6 Hz, 1H), 7.06 (d, J = 7.0 Hz, 1H), 7.00 (d, J = 9.0 Hz, 2H), 6.78 (s, 1H), 5.66 (ddt, J = 16.4, 10.3,
6.0 Hz, 1H), 5.06 (dd, J = 12.7, 5.4 Hz, 1H), 5.02 – 4.96 (m, 1H), 4.82 (dd, J = 17.1, 1.1 Hz, 1H), 4.73 – 4.61 (m, 2H), 3.82 – 3.64 (m, 3H),
3.62 – 3.52 (m, 2H), 3.52 – 3.36 (m, 3H), 3.22 – 3.08 (m, 6H), 2.89 (ddd, J = 16.9, 13.9, 5.4 Hz, 1H), 2.66 – 2.53 (m, 2H), 2.15 – 1.98 (m,
3H), 1.46 (s, 6H).
13C NMR (125 MHz, DMSO) d 173.29, 170.57, 169.21, 168.11, 167.76, 163.49, 161.53, 161.40, 160.88, 156.51, 147.51, 146.55,
145.96, 139.28, 136.78, 132.77, 132.66, 132.44, 121.69, 118.73, 117.81, 116.80, 116.70, 111.17, 109.94, 72.88, 72.79, 72.63,
70.99, 70.39, 68.08, 65.64, 63.27, 63.20, 60.65, 60.61, 53.72, 51.16, 49.03, 47.03, 46.36, 31.46, 30.93, 23.63, 22.65.
Scheme 3: Synthesis of 3-(2-(2-((2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy) propanoic acid (11).
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tert-Butyl3-(2-(2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-ylamino)ethoxy)ethoxy) propanoate (10). To a solution of
2-(2,6-dioxopiperidin-3-yl)-4-fluoroisoindoline-1,3-dione (8) (0.4 g, 4.3 mmol) and NH2-PEG2-t-Bu (9) (0.25 g, 4.3 mmol) in
DMF (10 mL) was added N, N-Diisopropylethylamine (2.5 mL). The solution was heated at 90ꢁC for 2 hours, then cooled, diluted
with water (15 mL) and acidified to pH 4-5 with 10% KHSO4. The mixture was extracted with ethyl acetate (3 x 20 mL) and
the combined organic extracts washed with brine, dried over anhydrous sodium sulfate and concentrated in vacuo. The
residue was purified by silica chromatography (2:1 petroleum ether: EtOAc) to give compound 10 (120 mg, 23% yield) as a yel-
low solid.
1H NMR (400 MHz, CDCl3): d 8.09 (s, 1H), 7.49 (m, 1H), 7.10 (d, 1H), 6.92 (d, 1H), 4.92 (m, 1H), 3.72 (m, 4H), 3.64 (m, 4H), 3.46 (t, 2H),
2.67ꢂ2.94 (m, 3H), 2.51 (t, 2H), 2.13 (m, 1H), 1.44 (s, 9H). LC-MS: m/z 512.17 [M+23]+.
3-(2-(2-((2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy) propanoic acid (11). To a solution of tert-butyl
3-(2-(2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-ylamino)ethoxy)ethoxy)propanoate (185 mg, 0.38 mmol) in DCM (5 mL)
was added TFA (1.5 mL) and the solution was stirred for 2 hours. The solvent was removed under reduced pressure to give compound
11 (150 mg, 94% yield) as a yellow solid.
1H NMR (400 MHz, CDCl3): d 8.62 (s, 1H), 7.48 (m, 1H), 7.10 (d, 1H), 6.91 (d, 1H), 6.54 (br s, 1H), 4.93 (m, 1H), 3.77 (t, 2H), 3.73 (t, 2H),
3.67 (s, 4H), 3.46 (t, 2H), 2.86 (m, 1H), 2.77 (m, 2H), 2.65 (t, 2H), 2.13 (m, 1H). LC-MS: m/z 434.25 [M+1]+.
Scheme 4: Synthesis of N-(3-(4-(4-((2-allyl-1-(6-(2-hydroxypropan-2-yl)pyridin-2-yl)-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-
yl)amino)phenyl)piperazin-1-yl)propyl)-3-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)propenamide
(ZNL-02-012).
To a solution of intermediate 7 (20mg, 0.03 mmol) in DCM (2 mL) was added TFA (0.5 mL). The reaction was stirred for 1 hour.
The solvent was removed to give the title compound as the TFA salt. HATU (23 mg, 0.06 mmol) 3-(2-(2-((2-(2,6-dioxopiperidin-3-
yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy) propanoic acid 11 (13 mg, 0.03 mmol) and diisopropylethylamine (28 mL,
0.15 mmol) were added to a solution of the TFA salt in DMF (1.5 mL). The reaction was stirred for 1 h and the mixture was
purified by HPLC (10-90% MeOH in H2O) to give TFA salt ZNL-02-012 (23 mg, 82%) as a yellow solid. LC-MS: m/z
959.70 [M+1]+.
1H NMR (500 MHz, DMSO) d 11.02 (s, 1H), 10.13 (s, 1H), 9.44 (s, 1H), 8.78 (s, 1H), 8.04 – 7.85 (m, 2H), 7.68 (d, J = 8.1 Hz, 1H), 7.51
(ddd, J = 23.5, 13.9, 8.4 Hz, 3H), 7.07 (d, J = 8.6 Hz, 1H), 7.04 – 6.88 (m, 3H), 6.53 (s, 1H), 5.60 (ddt, J = 16.3, 10.3, 6.0 Hz, 1H), 5.04 –
4.87 (m, 2H), 4.76 (dd, J = 17.1, 1.3 Hz, 1H), 4.61 (d, J = 5.2 Hz, 2H), 3.72 (d, J = 12.4 Hz, 2H), 3.59 – 3.35 (m, 12H), 3.08 (m, 6H), 2.95 –
2.73 (m, 3H), 2.58 – 2.46 (m, 2H), 2.27 (t, J = 6.4 Hz, 2H), 2.01 – 1.88 (m, 1H), 1.82 – 1.71 (m, 2H), 1.39 (s, 6H).
13C NMR (125 MHz, DMSO) d 173.29, 171.19, 170.57, 169.45, 168.12, 167.75, 161.54, 160.90, 158.64, 158.37, 156.56, 147.54,
146.87, 145.90, 139.26, 136.73, 132.66, 132.57, 121.71, 118.73, 117.92, 116.75, 111.20, 109.73, 72.79, 70.15, 69.99, 69.36,
67.29, 53.92, 51.38, 49.04, 47.04, 46.61, 42.17, 40.59, 40.43, 40.26, 40.09, 36.59, 36.10, 31.46, 30.93, 24.45, 22.61.
Scheme 5: Synthesis of 4-Fluoro-2-(1-methyl-2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (12).
To DMF (3mL) was added NaH 60% (21mg, 0.54mmol), then 2-(2,6-dioxopiperidin-3-yl)-4-fluoroisoindoline-1,3-dione 8
(100mg, 0.36mmol) was dissolved in DMF (1 mL) was dropped at 0ꢁC, the mixture was stirred for 15 minutes, MeI (35uL,
0.54mmol) was added and the mixture was stirred for 8 h warming to room temperature. The mixture was diluted with EtOAc
(20 mL) and water (30mL), and extracted with EtOAc (3 x 10 mL). The organic extract was washed with brine (20 ml), dried
(MgSO4) and concentrated under reduced pressure, and the residue was purified via silica gel chromatography (DCM:
MeOH =10:1) to give 4-fluoro-2-(1-methyl-2,6-dioxopiperidin-3-yl) isoindoline-1,3-dione (80 mg, 76 % yield). LC-MS: m/z
291.09 [M+1]+.
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Please cite this article in press as: Li et al., Development and Characterization of a Wee1 Kinase Degrader, Cell Chemical Biology (2019), https://
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Scheme 6: Synthesis of 4-((3-(4-(4-((2-allyl-1-(6-(2-hydroxypropan-2-yl)pyridin-2-yl)-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-
6-yl)amino)phenyl)piperazin-1-yl)propyl)amino)-2-(1-methyl-2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (ZNL-02-178).
To a solution of Intermediate (7) (40 mg, 0.05 mmol) was in DCM (2 mL) was added TFA (0.5 mL) and the resulting solution was
stirred for 30min at room temperature. The solvent was removed under reduced pressure and the residue was dissolved in
DMSO (1 mL). 4-Fluoro-2-(1-methyl-2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (12) (28 mg, 0.078 mmol) and N, N-diisopropyle-
thylamine (40 mL, 0.22 mmol) were added, and the mixture was stirred at 90ꢁC for 3 h. The mixture was purified by HPLC
(10-90% MeOH in H2O) to give TFA salt ZNL-02-178 (15 mg, 37%) as a yellow solid. LC-MS: m/z 814.46 [M+1]+.
1H NMR (500 MHz, DMSO) d 10.22 (s, 1H), 9.68 (s, 1H), 8.84 (s, 1H), 8.03 (t, J = 7.8 Hz, 1H), 7.75 (d, J = 8.0 Hz, 1H), 7.70 – 7.57 (m,
4H), 7.17 (d, J = 8.6 Hz, 1H), 7.08 (d, J = 7.0 Hz, 1H), 7.00 (d, J = 8.9 Hz, 2H), 6.80 (d, J = 5.7 Hz, 1H), 5.66 (ddd, J = 16.4, 11.0, 6.0 Hz,
1H), 5.14 (dd, J = 13.0, 5.4 Hz, 1H), 4.99 (d, J = 10.2 Hz, 1H), 4.82 (d, J = 17.1 Hz, 1H), 4.68 (d, J = 4.8 Hz, 2H), 3.80 (d, J = 12.5 Hz, 2H),
3.62 (d, J = 11.3 Hz, 2H), 3.43 (d, J = 6.0 Hz, 2H), 3.31 – 3.09 (m, 4H), 3.02 (s, 3H), 3.00 – 2.87 (m, 3H), 2.77 (dd, J = 13.4, 2.9 Hz, 1H),
2.62 – 2.53 (m, 1H), 2.11 – 1.93 (m, 3H), 1.46 (s, 6H).
13C NMR (125 MHz, DMSO) d 172.29, 170.33, 169.21, 168.12, 167.71, 161.54, 161.45, 160.89, 158.60, 158.32, 156.55, 147.54,
146.55, 145.91, 139.25, 136.82, 132.78, 132.66, 132.52, 118.73, 117.76, 116.72, 111.24, 109.95, 72.79, 53.82, 51.36, 49.60,
47.04, 40.59, 40.42, 40.25, 40.17, 40.09, 31.59, 30.93, 27.08, 23.79, 21.89.
QUANTIFICATION AND STATISTICAL ANALYSIS
For all experiments, the number of replicates and error bars are described in the respective figure legends. All biological experiments
were performed at least twice.
Antiproliferation Assay
IC50 values were determined using a non-linear regression curve fit in GraphPad Prism. N=4 biological replicates were used for each
treatment condition. Values are reported as mean +/- SEM, as also indicated in the figure legends
FACS
Data were analyzed using FlowJo. N=3 biological replicates were used for each treatment condition. Bar height represents mean
percentage of cells in each cell cycle phase, and error bars represent SEM, as also indicated in figure legends.
DATA AND CODE AVAILABILITY
The mass spectrometry raw data files for multiplexed proteomics have been deposited in the PRIDE Archive under the accession
number PXD015513.
e9 Cell Chemical Biology 27, 1–9.e1–e9, January 16, 2020