Selective degradation-inducing probes for studying cereblon (CRBN) biology

Article abstract of DOI:10.1039/d0md00382d

Targeted protein degradation represents a rapidly growing area in drug discovery and development. Moreover, small molecules that induce the targeted degradation of a given protein also represent an important addition to the chemical probes toolbox as these compounds can achieve selective protein knockdown, thus providing an approach that is orthogonal to genetic knockdowns. In order to develop degradation-inducing chemical probes for studying cereblon (CRBN) biology, we generated six CRBN-CRBN (homo-PROTAC) degraders and six CRBN-VHL (hetero-PROTAC) degraders. From these compounds we identified two potent and selective CRBN degraders (ZXH-4-130 and ZXH-4-137), both of which are CRBN-VHL compounds. We characterized these lead degraders by quantitative proteomics in five cell lines (MM1.S, Kelly, SK-N-DZ, HEK293T, and MOLT-4) and observed high selectivity for CRBN in all cell lines. Furthermore, we directly compared our compounds to current lead CRBN degraders and demonstrated how these probes can be used as chemical knockdown reagents for studying CRBN-dependent processes. Overall, our work provides a roadmap for thorough degrader characterization by combination western and proteomic analysis, as illustrated by the identification of ZXH-4-130 and ZXH-4-137 as CRBN-knockdown tool compounds suitable for cell-based studies.

Full text of DOI:10.1039/d0md00382d

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RESEARCH ARTICLE
Selective degradation-inducing probes for
studying cereblon (CRBN) biology†
Cite this: DOI: 10.1039/
d0md00382d
ab
Chelsea E. Powell,
‡
Guangyan Du,‡ab Jonathan W. Bushman,‡ab Zhixiang He,ab
Tinghu Zhang,ab Eric S. Fischerab and Nathanael S. Gray
ab
*
Targeted protein degradation represents a rapidly growing area in drug discovery and development.
Moreover, small molecules that induce the targeted degradation of a given protein also represent an
important addition to the chemical probes toolbox as these compounds can achieve selective protein
knockdown, thus providing an approach that is orthogonal to genetic knockdowns. In order to develop
degradation-inducing chemical probes for studying cereblon (CRBN) biology, we generated six CRBN–
CRBN (homo-PROTAC) degraders and six CRBN–VHL (hetero-PROTAC) degraders. From these
compounds we identified two potent and selective CRBN degraders (ZXH-4-130 and ZXH-4-137), both of
which are CRBN–VHL compounds. We characterized these lead degraders by quantitative proteomics in
five cell lines (MM1.S, Kelly, SK-N-DZ, HEK293T, and MOLT-4) and observed high selectivity for CRBN in all
cell lines. Furthermore, we directly compared our compounds to current lead CRBN degraders and
demonstrated how these probes can be used as chemical knockdown reagents for studying CRBN-
dependent processes. Overall, our work provides a roadmap for thorough degrader characterization by
combination western and proteomic analysis, as illustrated by the identification of ZXH-4-130 and ZXH-4-
137 as CRBN-knockdown tool compounds suitable for cell-based studies.
Received 9th November 2020,
Accepted 15th March 2021
DOI: 10.1039/d0md00382d
rsc.li/medchem
small molecule degraders, compounds that bind and recruit
an E3 ubiquitin ligase to a target protein, resulting in target
Introduction
A large amount of knowledge about the function of gene
products has been derived through genetic modifications
using tools such as RNA interference,1 transcription activator-
like effector nucleases (TALENs),2,3 and the clustered regularly
interspaced short palindromic repeats (CRISPR)-Cas9
system.4,5 However, there are limitations to these genetic
knockdown strategies, including the high expense and time
costs of developing animal models, particularly in nonhuman
primates, and the potential for genetic compensations or
spontaneous mutations in animal models which can obscure
the biological consequences of the genetic perturbation.6,7
Therefore, orthogonal methods are needed to overcome some
of the pitfalls of genetic knockdown strategies, and small
molecule tool compounds have emerged as useful in this
context. A recent addition to the chemical probe tool box are
ubiquitination and proteasomal degradation. These degrader
molecules lead to chemically induced protein-knockdown and
thus represent
processes.
a valuable tool for studying biological
Cereblon (CRBN) is a substrate receptor subunit of the E3
ubiquitin ligase complex CRL4, which also includes damaged
DNA binding protein 1 (DDB1), cullin-4A (CUL4A), and RING-
box protein 1 (RBX1 or ROC1). Although CRBN was identified
more than 15 years ago,8 critical details about its endogenous
biological function remain unknown. Individual studies have
suggested that CRBN may play a role in metabolic regulation
or a protective role against DNA damage-induced apoptosis,
but these hypotheses have not been fully tested.9,10
More recently CRBN has gained popularity as an E3
ubiquitin ligase that can be hijacked by small molecule
degraders and used for targeted protein degradation. The
utility of CRBN in this context stems from the discovery that
it binds immunomodulatory drugs (IMiDs), such as
thalidomide, lenalidomide, and pomalidomide,11 resulting in
recruitment of the zinc finger transcription factors (TFs)
Ikaros (IKZF1) and Aiolos (IKZF3), and their subsequent
a Department of Biological Chemistry & Molecular Pharmacology, Harvard Medical
School, Boston, Massachusetts 02115, USA
b Department of Cancer Biology, Dana-Farber Cancer Institute, Boston,
Massachusetts 02215, USA. E-mail: Nathanael_Gray@dfci.harvard.edu
† Electronic supplementary information (ESI) available: Chemical synthesis
methods, compound characterization, and Fig. S1–S3. See DOI: 10.1039/
d0md00382d
ubiquitination
and
proteasomal
degradation.12–14
Additionally, IMiDs can be incorporated into bifunctional
degrader molecules (also called PROTACs), which can induce
‡ These authors contributed equally.
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the degradation of target proteins beyond TFs by bringing
them into proximity of an E3 ligase.15–17 These compounds
are composed of an E3 ligase recruiting element, such as
IMiDs that recruit CRBN, and a binding motif for a target of
interest. Bifunctional degraders function in the same way as
IMiDs, by facilitating formation of the ubiquitination-
competent ternary complex. In addition to CRBN,
bifunctional degrader molecules that hijack other ligases,
such as the von-Hippel Lindau (VHL) E3 ligase, have been
reported. Both the CRBN and VHL E3 ligases have been
successfully recruited by small molecule degraders for a
range of targets, including BRD4, BRD9, TRIM24, BCR-ABL,
activity (hetero-PROTACs).27,28,30 Additionally, we observe that
degraders that use IMiD scaffolds as both the E3 ligase
recruiting moiety and the target binding arm (homo-
PROTACs) are not as effective as CRBN–VHL compounds.
Using a workflow of western blot analysis to expression
proteomics, we thoroughly characterize the selectivity profiles
of two highly selective CRBN degraders, ZXH-4-130 and ZXH-
4-137, including proteomic analysis of ZXH-4-130 in five
different cell lines. These compounds may represent useful
tools for achieving chemical knockdown of CRBN as a
strategy for studying its biology.
CDK9, Aurora A, FAK, FER, BTK, ALK, and other Results and discussion
kinases.15,18–25
In
addition
to
offering
a
novel
The chemical synthesis of all compounds discussed is
presented in full in the ESI.†
pharmacological strategy with translational implications, the
use of selective degraders has yielded important insights into
the biology of these targets, such as the identification of
TRIM24 as a novel dependency in acute leukemia.24
CRBN–CRBN degraders (homo-PROTACs)
Previous work has shown that VHL may be recruited to
degrade itself,26 and that CRBN degradation may be induced
through recruitment of itself or VHL.27–30 To enable further
studies of CRBN, here we aimed to develop selective small
molecule degraders of CRBN as tool compounds to probe
CRBN biology. We generated two different sets of degraders
(CRBN–CRBN and CRBN–VHL) and examined their ability to
induce selective CRBN degradation. This led to the
identification of two CRBN–VHL degraders as potent and
selective inducers of CRBN degradation. Our work is in
agreement with the recently reported observation that CRBN
degradation can be achieved via VHL E3 ubiquitin ligase
We designed three CRBN–CRBN degraders that linked two
pomalidomide groups together with all carbon linkers of
varying lengths (Fig. 1A). All three compounds induced
potent CRBN degradation at a dose range of 0.01–0.1 μM
after 4 h treatments in the multiple myeloma cell line MM1.S
(Fig. 1B). At a higher dose range of 1–10 μM the degraders
displayed the “hook effect” that has been previously reported
as a trait of small molecule degraders, where at higher doses
degrader potency is lost.31,32 In Fig. 1B the cause of the hook
effect is well illustrated, with CRBN degradation decreasing
as Ikaros degradation increases, indicating that the degraders
are out-competing themselves for binding and behaving
Fig. 1 Pomalidomide CRBN–CRBN degrader behavior in MM1.S cells. (A) Structures of pomalidomide derived CRBN–CRBN degraders. (B)
Immunoblot after 4 h of treatment with dose titrations of degraders. Pom. is pomalidomide. Dotted white lines provided only as visual aids and do
not indicate cuts. (C) Immunoblot after treatment with 50 nM of compound for the indicated amount of time. Quantification shown as percentage
of DMSO control normalized to β-actin or vinculin.
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selectivity profile of the CRBN–CRBN degraders, we generated
three CRBN–CRBN (homo-PROTAC) compounds that use a
tricyclic analog of thalidomide that has been previously
reported by our group as a strategy to dial out degradation of
IMiD neo-substrates, such as IKZF1, while gaining superior
affinity for CRBN compared to pomalidomide (Fig. 3A).35
Although these compounds were selective for CRBN, they
induced less potent degradation than the pomalidomide–
pomalidomide compounds (Fig. 3B and C).
CRBN–VHL degraders (hetero-PROTACs)
Our next effort towards developing a selective CRBN degrader
focused on examining whether degraders that recruit the VHL
E3 ligase show improvement. We generated six pomalidomide–
VHL ligand based CRBN–VHL degraders using varying linkers
(Fig. 4A). As seen from 4 h dose titration treatments in MM1.S
cells, ZXH-4-130 and ZXH-4-137 were highly potent and selective
degraders of CRBN (Fig. 4B). These two compounds use the
same 11-carbon linker, with the VHL ligand used in ZXH-4-137
being a methylated analog of the VHL ligand in ZXH-4-130.
Interestingly, ZXH-4-133 and ZXH-4-136 also share a linker
(4-carbon) with ZXH-4-136 containing a methylated VHL ligand.
For the ZXH-4-133/ZXH-4-136 pair the methylated VHL ligand
improves potency, with ZXH-4-136 being the more potent
degrader. However, for the ZXH-4-130/ZXH-4-137 pair the
potencies are equivalent. ZXH-4-132 which has a 2-carbon linker
induced weak CRBN degradation, likely due to the short linker
Fig. 2 Expression proteomics in MOLT-4 cells after 6 h treatment with
50 nM of ZXH-3-159 or DMSO (singlicate analysis of compound;
triplicate analysis of DMSO control).
similar to single agent pomalidomide. These pomalidomide
derived CRBN–CRBN degraders were able to induce
degradation as early as 1 h and as late as 24 h after 50 nM
treatment, with the most potent degradation seen roughly
between 4–8 h (Fig. 1C).
We used expression proteomics to examine the selectivity
profile of ZXH-3-159 in the leukemia cell line MOLT-4 (50 nM
dose for 6 h) and observed that this compound induced
down regulation of IKZF1 (Ikaros), ZFP91, and ZNF653 in
addition to CRBN (Fig. 2). These off-targets are all known
IMiD neo-substrates.12–14,33,34 In an effort to increase the
Fig. 3 Tricyclic thalidomide analog CRBN–CRBN degrader behavior. (A) Structures of tricyclic thalidomide analog derived CRBN–CRBN degraders.
(B) Immunoblot after 4 h of treatment with dose titrations of degraders in MM1.S cells. Vinculin representative of 3 blots. Quantification shown as
percentage of DMSO control normalized to vinculin. Pom. is pomalidomide. Dotted white lines provided only as visual aids and do not indicate
cuts. (C) Expression proteomics in MOLT-4 cells after 6 h treatment with 100 nM of DGY-8-127 or DMSO (singlicate analysis of compound;
triplicate analysis of DMSO control).
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Fig. 4 Thalidomide–VHL ligand based CRBN–VHL degrader behavior in MM1.S cells. (A) Structures of thalidomide and VHL ligand derived CRBN–
VHL degraders. (B) Immunoblots after 4 h of treatment with dose titrations of degraders. Vinculin representative of 3 blots. Pom. is pomalidomide.
Dotted white lines provided only as visual aids and do not indicate cuts. (C) Immunoblots after treatment with 50 nM of compound for the
indicated amount of time. Vinculin representative of 2 blots. Quantification shown as percentage of DMSO control normalized to vinculin.
being less amenable to formation of the ternary complex. While
slightly more potent than ZXH-4-132, ZXH-4-135 which contains
a 4-polyethylene glycol (PEG) linker also induced weak CRBN
degradation. It is possible that since both the CRBN and VHL
ligands are fairly hydrophilic, the longer alkyl linker used in
ZXH-4-130 and ZXH-4-137 increases the hydrophobicity and
clog P of the whole compound, improving cell permeability.
A time course in MM1.S cells with a 50 nM dose
demonstrated that ZXH-4-130 was the most potent between
2–8 h, while ZXH-4-137 was the most potent between 2–16 h
(Fig. 4C).
these degraders are highly selective for CRBN, with CRBN
being the only significantly downregulated target (Fig. 5). The
high degree of selectivity for ZXH-4-130 was also confirmed
by expression proteomics in Kelly, SK-N-DZ, HEK293T, and
MOLT-4 cell lines, indicating that this compound may be
useful as a tool compound in many cell lines (ESI:† Fig. S1).
Additionally, we confirmed by western in MM1.S cells and
expression proteomics in MOLT-4 cells that ZXH-4-130 and
ZXH-4-137 did not affect the expression of other members of
the CRBN E3 ligase complex: DDB1, CUL4A, and ROC1 (Fig.
S2 and S3†). This indicates that both of these compounds are
promising lead probes for chemically inducing CRBN
degradation.
Expression proteomics in MM1.S cells after 6 h treatments
with 50 nM of ZXH-4-130 or ZXH-4-137 further validated that
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Fig. 5 Expression proteomics in MM1.S cells after 6 h treatment with 50 nM of ZXH-4-130, 50 nM of ZXH-4-137, or DMSO (triplicate analysis).
We further assessed the selectivity profile of ZXH-4-130 over
time by using expression proteomics in the neuroblastoma cell
line SK-N-DZ after 6 h, 12 h, and 24 h treatments of 100 nM
compound or DMSO (Fig. 6). ZXH-4-130 remained highly
selective for CRBN across these time points (note that proteins
F2 and F5 seen at the 6 h time point were not significantly up-
or down-regulated at any other time points or in any other cell
lines; these secreted proteins are occasionally seen as false
positives in cellular proteomics experiments).36
of CRBN at a dose range of 10–500 nM (Fig. 7B). ZXH-4-130 and
ZXH-4-137 induced CRBN degradation to a similar extent as
CRBN-6-5-5-VHL at a dose range of 10–500 nM (Fig. 7C). All four
CRBN degraders prevented pomalidomide cytotoxicity to a
significant extent, with St-15a having the least statistically
significant amount of prevention and ZXH-4-130, ZXH-4-137,
and CRBN-6-5-5-VHL having equally statistically significant
amounts of prevention (Fig. 7D).
In order to compare the selectivity profiles of St-15a
(CRBN–CRBN), CRBN-6-5-5-VHL (CRBN–VHL), and ZXH-4-130
(CRBN–VHL) we used expression proteomics in MOLT-4 cells
(Fig. 8). After 6 h treatments with 50 nM compound or
DMSO, all three compounds were highly selective for CRBN.
Degradation of CRBN could potentially lead to upregulation
of its endogenous substrates. However, known CRBN
substrates were not among the proteins upregulated with
ZXH-4-130, and we were unable to replicate upregulation of
these proteins in an independent MOLT-4 experiment or any
other cell lines. Upregulated protein C3 is a member of the
complement system, of which several other components (F2,
F5, FN1) occasionally appear as false positives in our cellular
proteomics experiments; ECM1 also occasionally appears as a
false positive.36
Comparison to previously reported CRBN degraders
We compared our lead CRBN degraders to the pomalidomide
based CRBN–CRBN degrader St-15a as well as CRBN-6-5-5-
VHL, a potent CRBN targeting thalidomide–VHL ligand based
degrader, both of which have been previously reported by
Steinebach et al. (Fig. 7A).29,30 CRBN-6-5-5-VHL was selected
as the CRBN–VHL compound for comparison to our lead
degraders due to its higher potency compared to the lead
degrader 14a reported by Girardini et al.27 and the lead
degrader TD-165 reported by Kim et al.28 Importantly, both
ZXH-4-130 and ZXH-4-137 appear more potent against CRBN
than 14a and TD-165, which have reported DC50s of >100 nM
in HeLa cells and 20.4 nM in HEK293T cells, respectively.
While St-15a appeared similarly selective for CRBN over
Ikaros by western, ZXH-4-130 was a much more potent degrader
Interestingly, ZXH-4-130 differs only by an exit vector to
the compound TD-165 that was reported by Kim et al.; TD-
165 uses an amide exit vector from the pomalidomide group
Fig. 6 Expression proteomics in SK-N-DZ cells after 100 nM of ZXH-4-130 for various treatment times compared to 24 h DMSO control (duplicate
analysis of compound; triplicate analysis of control).
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Fig. 7 Comparison of lead CRBN degraders to previously reported compounds by Steinebach et al. (A) Structure of pomalidomide based CRBN–
CRBN degrader St-15a and thalidomide–VHL ligand based CRBN–VHL degrader CRBN-6-5-5-VHL. (B and C) Immunoblot after 4 h of treatment with
dose titrations of degraders in MM1.S cells. Vinculin representative of 4 blots. Quantification shown as percentage of DMSO control normalized to
vinculin. Pom. is pomalidomide. The VHL targeting homo-PROTAC CM11 is shown for comparison.26 Dotted white lines provided only as visual aids
and do not indicate cuts. (D) MM1.S cell viability after 2 h pre-treatments with 100 nM of ZXH-4-130, ZXH-4-137, St-15a, or CRBN-6-5-5-VHL
followed by 96-hour treatment with 1 μM of pomalidomide (four biological replicates; Graphpad Prism 8 software; Welch's t test).
Fig. 8 Expression proteomics in MOLT-4 cells after 6 h of 50 nM treatment with St-15a, CRBN-6-5-5-VHL, ZXH-4-130, or DMSO (singlicate
analysis of compound; triplicate analysis of DMSO control).
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Fig. 9 Immunoblots of ZXH-4-130 and ZXH-4-137 as tool compounds for studying CRBN related biology. (A) Immunoblot after 2 h pre-
treatments with DMSO, carfilzomib (Car.), ZXH-4-130 (130), ZXH-4-137 (137), or pomalidomide (Pom.) followed by 4 h treatment with CC-885 in
MM1.S cells. (B) Immunoblot after 2 h pre-treatments with DMSO, carfilzomib (Car.), ZXH-4-130 (130), ZXH-4-137 (137), or pomalidomide (Pom.)
followed by 6 h treatment with THAL-SNS-032 in MOLT-4 cells.
instead of an ether.28 Despite this high degree of similarity,
TD-165 was reported to be less potent than ZXH-4-130. While
TD-165 has a DC50 of 20.4 nM in HEK293T cells, ZXH-4-130
induces ∼80% CRBN degradation at 10 nM in MM1.S cells (n
= 3, Fig. 4B, 7B, and C); however, direct cell line comparison
is needed. The selectivity profile of TD-165 was not assessed
by proteomics, but Kim et al. did report proteomic analysis of
a 7-carbon linker analog of TD-165 after 1 μM treatment for
PROTAC by testing the activity of the cyclin-dependent kinase
9 (CDK9) degrader THAL-SNS-032 in MOLT-4 cells after 2 h
pre-treatment with 0.1 μM of the CRBN degraders (Fig. 9B).19
ZXH-4-130 and ZXH-4-137 were indeed able to partially
prevent CDK9 degradation compared to the DMSO control
and 0.1 μM pomalidomide treatment, but to a lesser extent
than carfilzomib treatment or 10 μM pomalidomide
competition. Of note, both ZXH-4-130 and ZXH-4-137
induced nearly complete CRBN degradation, but THAL-SNS-
032's activity against CDK9 was only partially prevented; this
speaks to the catalytic power of small molecule degraders.
12
h in Jurkat cells. This demonstrated statistically
significant downregulation of two proteins in addition to
CRBN: CNIH1 and LMBRD2.28
Conclusions
Use of lead CRBN degraders as tool compounds
We examined the potential of using ZXH-4-130 and ZXH-4-
137 as probes for studying CRBN biology. We pre-treated
MM1.S cells for 2 h with 50 nM of ZXH-4-130 or ZXH-4-137,
before exposing them to CC-885, a compound that induces
G1 to S phase transition protein 1 (GSPT1) degradation via
CRBN E3 ligase recruitment.37 We observed that this pre-
treatment with 50 nM of ZXH-4-130 or ZXH-4-137 rescued
GSPT1 degradation (Fig. 9A). This prevention of induced
degradation was comparable to that observed after pre-
treatment with the proteasome inhibitor carfilzomib. Pre-
treatment with 50 nM or 10 μM of pomalidomide did not
prevent GSPT1 degradation to a similar extent as pre-
treatment with ZXH-4-130 or ZXH-4-137, indicating that the
ability to prevent induced GSPT1 degradation was due to the
CRBN degradative abilities of ZXH-4-130 and ZXH-4-137, not
just competition for CRBN binding. We also examined
whether or not the chemical knockdown of CRBN by ZXH-4-
130 or ZXH-4-137 could prevent the degradative activity of a
Here we describe the development and characterization of
two potent and highly selective degraders of CRBN, ZXH-4-
130 and ZXH-4-137. These compounds feature both a CRBN
recruiting ligand and a VHL E3 ligase binding ligand, and
therefore represent an example of bifunctional degrader
molecules that bring two E3 ubiquitin ligases into close
proximity. In our studies, this strategy was more successful in
achieving CRBN degradation than degraders that featured
two CRBN recruiting ligands (homo-PROTACs). These
observations were consistent with the literature.30 One
possible explanation for the poor performance of homo-
PROTACs is that as CRBN is degraded by these compounds,
it rapidly reaches a point where the amount of cellular CRBN
is lowered to a level such that all remaining CRBN is
occupied by one end of a degrader compound, and thus there
is no free protein to engage with the second CRBN-recruiting
group. This may be considered a variation of the hook effect
where lowered protein levels prevent CRBN from being
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completely degraded by CRBN–CRBN degraders. Hetero-
PROTACs can avoid this by recruiting two different E3
ligases, allowing for more complete CRBN degradation.
Interestingly, the CRBN–VHL degraders only induced
degradation of CRBN and not VHL, which is also consistent
with previously published studies on CRBN–VHL
degraders.27,28,30 A possible explanation is that the ternary
complex formed by the small molecule degraders positions
the E3 ligases in a manner where CRBN is more amenable to
degradation than VHL. Additionally, it has been suggested by
the use of VHL–VHL degraders that VHL in the CRL2-VHL
ubiquitin ligase complex may be protected from
ubiquitination as selective degradation of pVHL30, a form of
VHL that is thought to have ligase independent functions, is
seen over degradation of pVHL19, a form of VHL that may
preferentially act in the E3 ligase complex.26
and phosphatase inhibitor cocktails (Roche) at 4 °C for 15
minutes. The cell lysate was vortexed before being centrifuged
at 14 000 × g for 20 min at 4 °C. Protein in cell lysate was
quantified by BCA assay (Pierce). Primary antibodies used in
this study include β-actin (Cell Signaling Technology, 3700S),
CDK9 (Cell Signaling Technology, 2316S), CK1α (Abcam,
ab206652), CRBN (Novus Biologicals, NBP1-91810), CUL4A (Cell
Signaling Technology, 2699S), DDB1 (Cell Signaling Technology,
5428S), eRF3/GSPT1 (Abcam, ab49878), IKZF1 (Ikaros) (Cell
Signaling Technology, 5443S), IKZF3 (Aiolos) (Cell Signaling
Technology, 15103S), RBX1 (ROC1) (Cell Signaling Technology,
11922S), VHL (Cell Signaling Technology, 68547S), and vinculin
(Abcam, ab130007). Blot quantification was performed using
Image Studio 4.0 software, normalizing to loading controls.
Sample preparation for TMT LC-MS3 mass spectrometry
Our lead CRBN degraders ZXH-4-130 and ZXH-4-137
performed as expected in our cell-based studies that tested
their ability to block the activity of other degraders developed
to hijack the CRBN E3 ligase. These results show that CRBN
knockdown due to ZXH-4-130 and ZXH-4-137 pre-treatment
rescues small molecule-mediated degradation of two different
targets (GSPT1 and CDK9). Overall, ZXH-4-130 and ZXH-4-137
are potent and selective CRBN degraders that may assist with
new lines of inquiry into CRBN biology.
Kelly, MOLT-4, MM1.S, SK-N-DZ, or HEK293T cells were
treated for 6, 12, or 24 hours with DMSO in biological
triplicates or compound in biological singlicate, duplicate, or
triplicate as indicated. Treatment concentrations were as
follows: 0.1% DMSO, 50 or 100 nM ZXH-4-130, 50 nM ZXH-4-
137, 50 nM ZXH-3-159, 50 nM St-15a, 50 nM CRBN-6-5-5-
VHL, 100 nM DGY-8-127, or 50 nM CM11. Cells were
harvested by centrifugation and prepared for mass
spectrometry as previously described.33,38
Experimental
LC-MS data analysis
Chemical synthesis
Data were collected using an Orbitrap Fusion Lumos mass
spectrometer (Thermo Fisher Scientific) using an MS3-based
TMT method.39 Proteome Discoverer 2.2 (Thermo Fisher
Scientific) was used for .RAW file processing as previously
described.33,38 Reporter ion intensities were normalised and
scaled using in-house scripts in the R framework.40 Statistical
analysis was carried out using the limma package within the
R framework.41
Chemical synthesis and compound characterization are
presented in full in the ESI.†
Cell culture
MM1.S (CRL-2974), MOLT-4 (CRL-1582), SK-N-DZ (CRL-2149),
and HEK293T cells (CRL-3216) were purchased from ATCC.
Kelly cells (ECACC 92110411) were purchased from Millipore
Sigma.
All media was supplemented with 10% fetal bovine serum
(FBS) and 1% penicillin/streptomycin. MM1.S and MOLT-4
cells were cultured in RPMI-1640 media containing
L-glutamine. Kelly cells were cultured in RPMI-1640 media
without L-glutamine. SK-N-DZ cells were cultured in DMEM
media containing L-glutamine. HEK293T cells were cultured
in DMEM media without L-glutamine. Mycoplasma testing
was performed on a monthly basis using the MycoAlert
mycoplasma detection kit (Lonza) and all lines were negative.
Notes
The mass spectrometry datasets generated during this study
are available in the Proteomics Identification Database
(PRIDE) archive under the project accessions PXD022021,
PXD022022, PXD022058, PXD022059, and PXD022060. All
other data presented are available in the main text and ESI.†
Conflicts of interest
Nathanael S. Gray is a founder, science advisory board
member (SAB) and equity holder in Gatekeeper, Syros, Petra,
C4, B2S, Aduro, Jengu and Soltego (board member). The Gray
lab receives or has received research funding from Novartis,
Takeda, Astellas, Taiho, Janssen, Kinogen, Voronoi, Her2llc,
Deerfield and Sanofi. Eric S. Fischer is a founder, science
advisory board member and equity holder in Civetta, Jengu
(board member), and Neomorph, an equity holder in C4
Cell viability assays
Cell viability was evaluated using the CellTiter-Glo
luminescent cell viability assay (Promega) following the
manufacturer's standards.
Immunoblotting
Cells were washed with PBS before being lysed with cell lysis
buffer (Cell Signaling Technology) supplemented with protease
Therapeutics, and
Deerfield, and EcoR1 capital. The Fischer lab receives or has
a
consultant to Astellas, Novartis,
RSC Med. Chem.
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RSC Medicinal Chemistry
Research Article
received research funding from Novartis, Astellas, and
Deerfield.
17 D. P. Bondeson, A. Mares, I. E. D. Smith, E. Ko, S. Campos,
A. H. Miah, K. E. Mulholland, N. Routly, D. L. Buckley, J. L.
Gustafson, N. Zinn, P. Grandi, S. Shimamura, G. Bergamini,
M. Faelth-Savitski, M. Bantscheff, C. Cox, D. A. Gordon, R. R.
Willard, J. J. Flanagan, L. N. Casillas, B. J. Votta, W. den
Besten, K. Famm, L. Kruidenier, P. S. Carter, J. D. Harling, I.
Churcher and C. M. Crews, Nat. Chem. Biol., 2015, 11,
611–617.
18 A. C. Lai, M. Toure, D. Hellerschmied, J. Salami, S. Jaime-
Figueroa, E. Ko, J. Hines and C. M. Crews, Angew. Chem.,
2015, 128, 818–821.
19 C. M. Olson, B. Jiang, M. A. Erb, Y. Liang, Z. M. Doctor, Z.
Zhang, T. Zhang, N. Kwiatkowski, M. Boukhali, J. L. Green,
W. Haas, T. Nomanbhoy, E. S. Fischer, R. A. Young, J. E.
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Acknowledgements
Funding for this work was received from the National
Institutes of Health (NIH): Grant R01 CA214608 (E. S. F.) and
5 F31 CA210619-02 (C. E. P.). We would like to thank Nicholas
A Eleuteri and Katherine A Donovan (Dana-Farber Cancer
Institute) for help with proteomics experiments. We would
also like to thank Milka Kostic (Dana-Farber Cancer Institute)
for helpful feedback on the manuscript.
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