Proteolysis Targeting Chimera (PROTAC) for Macrophage Migration Inhibitory Factor (MIF) Has Anti-Proliferative Activity in Lung Cancer Cells

Article abstract of DOI:10.1002/anie.202101864

Macrophage migration inhibitory factor (MIF) is involved in protein-protein interactions that play key roles in inflammation and cancer. Current strategies to develop small molecule modulators of MIF functions are mainly restricted to the MIF tautomerase active site. Here, we use this site to develop proteolysis targeting chimera (PROTAC) in order to eliminate MIF from its protein-protein interaction network. We report the first potent MIF-directed PROTAC, denoted MD13, which induced almost complete MIF degradation at low micromolar concentrations with a DC₅₀ around 100 nM in A549 cells. MD13 suppresses the proliferation of A549 cells, which can be explained by deactivation of the MAPK pathway and subsequent induction of cell cycle arrest at the G2/M phase. MD13 also exhibits antiproliferative effect in a 3D tumor spheroid model. In conclusion, we describe the first MIF-directed PROTAC (MD13) as a research tool, which also demonstrates the potential of PROTACs in cancer therapy.

Full text of DOI:10.1002/anie.202101864

A Journal of the Gesellschaft Deutscher Chemiker
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Accepted Article
Title: Proteolysis Targeting Chimera (PROTAC) for Macrophage
Migration Inhibitory Factor (MIF) Has Anti-proliferative Activity in
Lung Cancer Cells
Authors: Zhangping Xiao, Shanshan Song, Deng Chen, Ronald van
Merkerk, Petra van der Wouden, Robbert Cool, Wim Quax,
Gerrit Poelarends, Barbro Melgert, and Frank Jacobus Dekker
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.202101864
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10.1002/anie.202101864
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RESEARCH ARTICLE
Proteolysis Targeting Chimera (PROTAC) for Macrophage
Migration Inhibitory Factor (MIF) Has Anti-proliferative Activity in
Lung Cancer Cells
Zhangping Xiao,# Shanshan Song, # Deng Chen, Ronald van Merkerk, Petra E. van der Wouden,
Robbert H. Cool, Wim J. Quax, Gerrit J. Poelarends, Barbro N. Melgert, Frank J. Dekker*
[*]
Z. Xiao, S. Song, D.Chen, R. van Merkerk, P. E. van der Wouden, Dr. R. H. Cool, Prof. Dr. W. J. Quax, Prof. Dr. G. J. Poelarends, Prof. Dr. F. J. Dekker
Department Chemical and Pharmaceutical Biology, Groningen Research Institute of Pharmacy (GRIP), University of Groningen
Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands
E-mail: f.j.dekker@rug.nl
S. Song, Prof. Dr. B.N. Melgert
Molecular Pharmacology, Groningen Research Institute of Pharmacy (GRIP), University of Groningen
Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands
Prof. Dr. B.N. Melgert
University Medical Center Groningen, Groningen Research Institute of Asthma and COPD, University of Groningen
Hanzeplein 1, 9713 GZ, Groningen, The Netherlands
These authors contributed equally to this work.
[#]
Supporting information for this article is given via a link at the end of the document.
Abstract: Macrophage migration inhibitory factor (MIF) is involved in
protein-protein interactions that play key roles in inflammation and
cancer. Current strategies to develop small molecule modulators of
MIF functions are mainly restricted to MIF tautomerase active site.
Here, we use this site to develop proteolysis targeting chimera
(PROTAC) in order to eliminate MIF from its protein-protein interaction
network. We report the first potent MIF-directed PROTAC, denoted
MD13, which induced almost complete MIF degradation at low
micromolar concentrations with a DC50 around 100 nM in A549. MD13
suppresses the proliferation of A549 cells, which can be explained by
deactivation of the MAPK pathway and subsequent induction of cell
cycle arrest at G2/M phase. MD13 also exhibits antiproliferative effect
in a 3D tumor spheroid model. In conclusion, we describe the first MIF-
MIF exists as a homotrimer in which each monomer consists
of a 114-amino acids peptide.[11] Initial evidence indicated an
important role for MIF in inflammation and immune responses.
Subsequently, MIF was also discovered to function as a
hormone[12], a chemokine[13] and as a molecular chaperone[14]
.
MIF exerts its functions mainly through protein-protein
interactions with membrane-bound receptors or intracellular
signaling proteins. One of those receptors is cluster of
differentiation 74 (CD74), which is the cognate receptor for
MIF.[15,16] The interaction between MIF and CD74 triggers
activation of the mitogen-activated protein kinase (MAPK)
pathway and inhibits the p53 pathway, which results in cell
growth.[3] In addition, other non-cognate binding partners such as
CXCR4 also play key roles in cancer development.[17,18] Therefore,
the discovery of reagents that interfere with the interaction
between MIF and CD74 or other binding partners is an attractive
strategy to inhibit MIF-induced cellular signaling in relevant
disease models.
directed PROTAC (MD13) as
a research tool, which also
demonstrates the potential of PROTACs in cancer therapy.
Introduction
Apart from its function as a cytokine, MIF also harbors
enzymatic activity to catalyze keto-enol tautomerization of
substrates such as D-dopachrome and 4-hydroxylphenylpyruvate
(4-HPP).[19] MIF exerts the tautomerase activity through its
proline-1, which is a nucleophile.[20] So far, the physiological
function of the enzymatic activity remains elusive. Interestingly,
some key amino acid residues in close proximity to the
tautomerase active site are involved in binding to CD74 and
CXCR4.[21–23] This implies that small molecule inhibitors of MIF
tautomerase activity are able to interfere with the MIF-receptor
interactions. Based on this idea, several series of small-molecule
inhibitors for MIF tautomerase activity have been developed.[24,25]
One of the earliest discovered MIF inhibitors is ISO 1 (Figure 1A),
which gained wide use as a reference compound in MIF
research.[26] However, the binding potency of ISO 1 for MIF is only
in the micromolar concentration range. Research over the past
two decades yielded several MIF inhibitors with nanomolar
potency. Previously, our group and others reported structure-
activity relationships (SARs) for 7-hydroxycoumarin derivatives
Cancer treatment has improved enormously over the past
decades, but unfortunately cancer remains one of the leading
health problems worldwide. Two important reasons that limit the
success of cancer treatments are heterogeneity of the tumor and
acquired therapy resistance.[1] To address these problems, it is
imperative to discover and exploit previously unrecognized
molecular mechanisms that are involved in cell proliferation. The
protein macrophage migration inhibitory factor (MIF) has been
implicated in the pathogenesis of cancers.[2] Overexpression of
MIF has been detected in cancer types such as genitourinary
cancer,[3] melanoma,[4] neuroblastoma,[5] and lung carcinoma[6].
Remarkably, down-regulation of MIF expression by gene-
knockout[7] or gene-knockdown[8,9] not only reduced tumor
progression and metastases, but also induced antitumor immune
responses.[10] These results indicate that targeting MIF could be
a promising strategy towards development of novel cancer
therapeutics.
1
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10.1002/anie.202101864
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RESEARCH ARTICLE
based on inhibitor 2 (Figure 1A).[27,28] We also found that
compounds containing a 7-hydroxy-3,4-dihydrobenzoxazin-2-
ones backbone such as 3 can also provide potent inhibition
against MIF.[27,29] Furthermore, the Jorgensen lab discovered
potent MIF inhibitors that contain a biaryltriazole or pyrazole
scaffold.[30,31] However, the potency to inhibit MIF tautomerase
activity does not always correlate well with the potency to inhibit
the MIF-CD74 interaction or MIF induced signaling in cell-based
studies.[32,33] Altogether, this suggests that development of
molecules that merely bind to the tautomerase enzyme active site
may not be enough to effectively interfere with MIF protein-protein
interactions. In addition, proteasome-dependent MIF degradation
induced by HSP90 suppression proved to be correlated with the
inhibition of MIF activity on cell proliferation.[34] Therefore, we seek
to use proteolysis-targeting chimeras (PROTACs) as an
alternative strategy to attenuate MIF functions by depletion of MIF
protein.
1B).[35] This enables hijacking the E3 ubiquitin ligase activity to
ubiquitinate the protein of interest that is subsequently degraded
by the ubiquitin-proteasome system. After degradation of the
protein of interest the PROTAC can be recycled for a new round
of targeted degradation of the protein of interest, thus providing a
catalytic cycle. Importantly, the PROTAC strategy enables
downregulation of intercellular protein levels of the protein of
interest rather than just blocking one of its respective catalytic
activities or interaction surfaces. After its development by the
groups of Crews and Deshaies,[36] the PROTAC strategy has
progressed enormously by virtue of the identification of potent and
selective E3 ligase ligands such as pomalidomide 4 (Figure
1C).[37] Over the past years, an increasing number of proteins
have been targeted by PROTACs, including kinase, epigenetics
editors, bromodomains, nuclear receptors and others.[38] However,
PROTAC development has been largely limited to clinically
validated targets for which marketed drugs are available. The next
step to unleash the full potential of PROTAC development is
targeting the traditionally undruggable proteome, for example,
found in proteins involved in protein-protein interactions.[39]
The PROTAC strategy has emerged as a novel concept in
small-molecule drug discovery. This strategy employs
a
heterobifunctional molecule that binds both the protein of interest
and an E3 ubiquitin ligase to form a ternary complex (Figure
Figure 1. Small molecule inhibitors of MIF tautomerase activity and general mechanistic representation of PROTAC action. (A) Structure of representative MIF
tautomerase inhibitors ISO 1[40], 2 and 3.[27] (B) Mechanism representation of the action of PROTACs. (C) Chemical structure of pomalidomide. POI: protein of
interest. Ub: ubiquitin.
In the present study, we report the first MIF-directed
PROTACs by linking potent MIF binding molecules to
pomalidomide as a ligand for the cereblon Cullin RING E3
ubiquitin ligase complex. Through investigation of the structure-
activity relationship, we discovered a potent PROTAC MIF
degrader (MD13) with a DC50 < 100 nM and a Dmax > 90% in A549
cells. Control experiments were performed to demonstrate that
MD13 reduced the MIF level via cereblon ligand-induced
degradation. Moreover, MD13 inhibited the growth of cancer cells
in a 2D and a 3D cell culture system. Altogether, development of
these MIF degraders indicates a new strategy for treatment of
cancers and also provides a new class of tools to study MIF.
features of MIF tautomerase inhibitors derived from crystal
structures[28,29] and docking studies (Figure 2), we presume that
the aromatic hydroxyl functionality is deeply embedded in the
tautomerase active site, where it is involved in two key hydrogen-
bonding interactions with Asn97. Consequently, the
methoxyphenyl functionality in 2 and the ortho-dimethoxyphenyl
functionality in 3 protrude out of the pocket and are solvent-
exposed. Therefore, we replaced the methoxy-functionalities by
an amine to enable attachment of a linker by an amidation
reaction. Both MIF tautomerase inhibitor 2 and 3 were used as
MIF binding ligand in PROTACs for MIF degradation. Compound
2 was linked to the E3 ligase ligand pomalidomide to provide
PROTACs indicated as group 1 (Table S2) and compound 3 was
linked to pomalidomide to provide PROTACs indicated as group
2.
Results and Discussion
PROTAC design and synthesis. Compound 2 and 3
(Figure 1A) are inhibitors of MIF tautomerase activity with
nanomolar potency. Based on the known pharmacophoric
2
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10.1002/anie.202101864
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RESEARCH ARTICLE
of compounds with a triazole in the linker (Table 1 and Table S2).
Subsequently, the potency of the PROTACs at 2 µM was
investigated, which demonstrated the highest potency for MD9
and MD10. Based on these data MD9 was selected as the most
promising starting point to develop MIF-directed PROTACs
further.
Table 1. Optimization of linker length of MD9 and control compound with
impaired cereblon-binding ligand.
Figure 2. Design of MIF targeting PROTACs. Optimal binding poses of MIF with
2 (A, yellow, PDB 1GCZ)[28] and 3 (B, magenta, PDB 5HVT)[29]. MIF is shown as
a pale-green cartoon and the key residues forming the binding pocket are
represented as sticks. Docking studies were performed with Discovery Studio
and models were prepared with Pymol.
degradation (%)b
ID
n
R
Ki (nM)a
2 µM
--
0.2 µM
--
PROTACs retain their inhibitory potency for MIF
tautomerase activity. To verify binding of the resulting
PROTACs to MIF, we measured their ability to inhibit MIF
tautomerase activity. The candidate PROTACs inhibited MIF
enzymatic activity with nanomolar inhibition constants (Ki). The
group 1 PROTACs that contain a 7-hydroxycoumarin MIF binding
core provided Ki values between 117 to 999 nM, which is in the
same range as the Ki of MIF inhibitor 2 that was reported to be
370 nM (Table S2).[27] Interestingly, the PROTACs MD1-4 with
one carbon atom between the triazole and the amide functionality
are more potent MIF tautomerase inhibitors compared to
PROTACs with two (MD5) or three carbon atoms (MD6) in this
position. The Ki values for the PROTACs of group 2 with a 7-
hydroxy-3,4-dihydrobenzoxazin-2-one MIF binding core were all
around 100 nM. This is very well in line with the potency of their
parent inhibitor 3, which has a Ki value of 150 nM.[27] The results
demonstrated that our design strategy for linkers did not or
minimally perturb target engagement.
PROTACs induce MIF degradation. Previous studies
have demonstrated that A549 cells express a high-level of MIF.[41]
In addition, A549 cells have been successfully used for assessing
activity of cereblon ligand-based PROTACs previously.[42]
Therefore, A549 is a suitable cell line for evaluating the effect of
our putative MIF-directed PROTACs MD1 to MD12 on MIF protein
levels. The reduction in MIF levels were monitored in A549 cells
that were treated with two different PROTAC concentrations (20
and 2 µM) for 12 h in order to estimate the dose dependency,
which is important for PROTACs because of the Hook effect.[35]
The MIF levels were analyzed in cell lysates using an enzyme-
linked immunosorbent assay (ELISA).
3
--
3
4
6
7
9
7
--
H
103±10
55±3
MD7
MD8
MD9
MD13
MD14
MD15
n.s.
--
H
86±5
21±7
55±4
91±5
64±2
n.s.
--
H
51±3
24±4
71±7
51±2
n.s.
H
71±5
H
65±4
CH3
55±12
[a] Measured by MIF catalyzed 4-HPP tautomerization assay using the method
as previously reported by our group (n=3). [b] Degradation percentage is
represented as mean±SD (n = 3). Not significant (n.s.) P > 0.05.
The cellular effect of MD9 as a PROTAC was further
investigated. In comparison to the vehicle control, MD9 reduced
the MIF levels in a dose-dependent manner to provide a maximal
degradation of more than 90% with a half-maximal degradation
concentration (DC50) around 1.5 µM measured by both ELISA and
western-blot (Figure S2). The action mode of MD9 induced MIF
degradation is investigated. MIF degradation becomes visible
after 3 hours of treatment and reached its maximum effect after 6
and 9 hours with 10µM MD9 treatment. The degradation can be
rescued with pretreatment of 1, 3, 4 or proteasome inhibitor
Bortezomib[44], which indicates that the action of MD9 depends on
MIF binding as well as on CRBN E3 ligase binding, which triggers
proteasome-mediated degradation (Figure S3).
Development of a MIF-directed PROTAC with improved
potency. Although MD9 was identified as an effective MIF-
directed PROTAC, its potency remains limited to the micromolar
concentration range. The structure-activity relationship (SAR)
analysis of the PROTAC linkers of the second group suggests that
a longer aliphatic linker (MD7-9) is more favorable for MIF
degradation. To further explore the SARs and to improve the
efficacy of MIF PROTACs, we designed and synthesized MD13
and MD14, which contain longer linkers than MD9. Both new
PROTACs showed MIF binding constants (Ki) in a range similar
to the parental ligand 3 (Table 1). Subsequently, the reduction of
MIF levels upon treatment with MD13 and MD14 at concentration
of 2 and 0.2 µM was investigated. MD13 treatment resulted in
Treatment with 20 µM of any of the putative MIF PROTACs
resulted in lower MIF protein levels in A549 cells compared to
vehicle-treated controls (Table S2 and Figure S1). The only
exception is MD6 that did not trigger MIF reduction. The series of
PROTACs with 2 as warhead showed increasing potency with
increasing linker length to reach more than 50% reduction in the
MIF protein levels upon treatment with 20 µM MD4 and MD5.
However, at a concentration of 2 µM no significant degradation of
MIF was observed for this series of compounds. In the new series
of PROTACs using 3 as MIF binding ligand, treatment with 20 µM
of either of the three compounds with aliphatic linkers (MD7-9)
resulted in more than 50% lower MIF-protein levels compared to
control, whereas this was only observed for MD10 for the series
3
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91% and 71% lower MIF protein levels at 2 and 0.2 µM,
respectively. This indicates that MD13 is the most potent MIF
PROTAC in this series. In line with current knowledge, the length
of the linker appears to be critical for the potency of MIF-directed
PROTACs and the linker length of MD13 seems to be optimal.
To further confirm the action of MD13 as a MIF-degrading
PROTAC, we synthesized a control compound for MD13
containing a CRBN ligand with impaired CRBN binding. The imide
nitrogen of the piperidine-2,6-dione functionality in the CRBN
ligand is involved in a crucial hydrogen bond with CRBN.[45]
Methylation of this imide nitrogen will abolish CRBN binding.[46]
We synthesized control compound MD15 with a methylated
pomalidomide as CRBN ligand. MD15 preserved the MIF binding
potency with a Ki of 55 nM (Table 1). However, MD15 was not
capable of inducing MIF degradation at both 2 and 0.2 µM,
whereas MD13 was. Collectively, this result confirms that MD13
induced MIF degradation through binding to E3 ligase cereblon.
Characterization of MD13 as a MIF-directed PROTAC.
is in line with the result from western-blot. Interestingly, a “Hook
effect” was observed in both two assays at 20 µM of MD13.
A control experiment was performed to compare MD13 as
an active PROTAC and MD15 as an inactive PROTAC (Figure
3D). This demonstrated that MD15 was not able to reduce the MIF
levels relative to the control, thus indicating that CRBN binding is
involved in the effect of PROTAC MD13.
The ability of PROTAC MD13 to reduce the MIF levels in
A549 cells was investigated further. The kinetics of MIF
degradation proved to be relatively fast. Degradation was already
visible after 3-hour treatment, reaching > 92% degradation after 6
h (Figure 4A). Only a slight recovery of the MIF levels was
observed after 48 h. Combined treatment with PROTAC MD13
and the proteasome inhibitor Bortezomib[44] inhibited the
degradation of MIF (Figure 4B). Pretreatment of cells with either
MIF inhibitor 3 or CRBN inhibitor 4 to outcompete the formation of
ternary E3 ligase – MIF complex rescued MIF from degradation
(Figure 4C). The PROTAC MD13 also proved to be active in
Since PROTACs are relatively large heterobifunctional molecules, HEK293 cells, where it induced more than 90% MIF degradation
the efficacy of these compounds may be limited by poor cell
permeability.[47] In order to estimate the cellular uptake of
PROTAC MD13, the intrinsic fluorescence properties of the
pomalidomide part of MD13 were employed for visualization of its
subcellular localization. Clear localization of MD13 in the
cytoplasm of A549 cells was observed after one-hour incubation
(Figure S5). In parallel, the subcellular localization of MIF and its
decrease in situ upon MD13 treatment was visualized by confocal
fluorescence microscopy using a fluorescent secondary antibody.
Treatment with 1 µM MD13 significantly depleted MIF in A549
cells (Figure S5). Taken together, microscopic analysis revealed
that MD13 can enter cells to effectively induce MIF degradation.
at a concentration of 200 nM (Figure 4D). Taken together, the
results demonstrate that the activity of MD13 depends on binding
to both MIF and CRBN as well as on proteasome activity and that
near complete MIF degradation is observed in the low micromolar
range, which indicates that MD13 is a potent MIF-directed
PROTAC.
Figure 4. Characterization of the PROTAC activity of MD13 in A549. (A) Time-
dependence of MIF degradation upon treatment with MD13 in A549 cells. (B)
Rescue of MIF from degradation upon co-treatment with MD13 and Bortezomib.
(C) Rescue from MD13 mediated MIF degradation after 1 hour pretreatment
with MIF inhibitor 3 or CRBN inhibitor 4. (D) Concentration-dependent MIF
degradation was also observed in HEK293 cells. (n=2)
Anti-proliferative effect of MD13. After having identified
MD13 acts as a PROTAC that effectively reduces the MIF levels,
we employed this PROTAC to verify the role of MIF in proliferation
of A549 cells. As a first step, the toxicity of MD13 was investigated
using the MTS assay, which indicated that MD13 did not inhibit
cell viability at concentrations below 20 µM for a treatment of 24
hours (Figure S7). We next evaluated its effects on cell
proliferation, which indicated that MD13 inhibited the growth of
A549 cells in a dose-dependent manner (Figure 5A). The
inhibitory effect became visible at nanomolar concentrations and
Figure 3. The MIF-directed PROTAC MD13, but not MD15, causes depletion of
MIF protein in A549 cells. (A) A549 cells were treated with the indicated
concentrations of MD13 for 12 hours and MIF protein level was detected by
western-blot. (B) Quantification of MIF level in (B) compared with DMSO treated
control. (C) MD13 induced MIF degradation in A549 cells measured by ELISA.
R2=0.98. (D) MIF level was determined after cells were treated with the indicated
concentrations of MD13 or MD15 for 12 hours. (n=2)
The concentration dependence of PROTAC MD13-
mediated induction of MIF degradation in A549 cells was
investigated using western-blot. MD13 effectively induced MIF
degradation at nanomolar concentrations (Figure 3A). The MIF
levels were normalized to the vehicle treated control and plotted
to the respective MD13 concentrations. This provided a DC50 of
around 100 nM and a maximal degradation of around 90-95% at
concentrations higher than 1 µM (Figure 3B). The DC50 of MD13
measured by ELISA assay was about 200 nM (Figure 3C), which
reached about 50% inhibition of cell proliferation at
a
concentration of 20 µM. In contrast, the inactive control compound
MD15 showed almost no inhibition of the proliferation of A549
cells. MIF inhibitor 3 and CRBN inhibitor 4 were also included as
controls, both of which had no effect on the proliferation of cells
with concentrations up to 20 µM. Taken together, these
experiments indicates that the MIF-directed PROTAC MD13
inhibited cell proliferation of A549 cancer cells.
4
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A 3D spheroid model was employed to investigate the effect
of longer term MD13 treatment in a more complex model for tumor
growth. The 3D spheroid model was established using A549
cancer cells by a method adapted from Feng et al.[48] The
spheroids were grown over a 12-day period in absence or
presence of PROTAC MD13. Each spheroid was prepared from
about 1000 A549 cells. After three-day incubation, these
spheroids were treated with 1, 2, or 5 µM of MD13 with 72 hours
intervals over 12 days. Spheroid growth was monitored by
measuring the diameter and this was compared to day 0 of the
treatment. The tumor spheroids in MD13 treated groups were
significant smaller compared to the control group (Figure 5B and
Figure S9). With continuous exposure to 1, 2, or 5 µM of MD13
for 12 days, the growth of the spheroid tumor volume was
inhibited by 42%, 53%, and 81% compared with control group,
respectively. In contrast, 5 µM of the PROTAC-inactive control
compound MD15, 3, or 4 showed no significant influence on the
spheroid tumor growth. Collectively, our results indicate that the
MIF-directed PROTAC MD13 effectively inhibits proliferation of
A549 cancer cells in a spheroid tumor model.
the G2/M phase in A549 cells. The proportion of cells at the G2/M
phase is 12% for the control group. This percentage increases to
17%, 19%, and 23% upon treatment with 1, 2, and 5 μM MD13,
respectively. In contrast, little or no effect on the cell cycle was
observed upon treatment with 5 μM of the inactive control MD15.
These results indicate MD13 induces inhibition of cell cycle
progression, which can explain the observed inhibition of cell
proliferation.
Figure 6. Induction of cell cycle arrest by the MIF-directed PROTAC MD13 in
A549 cells. (A) A549 cells were treated with MD13, 4 or MD15 at the indicated
concentrations for 48 h. The graphs show the representative
cell cycle distribution of propidium-iodide stained cells assessed by flow
cytometry. (B) Quantification of cells in each stage of the cell cycle by Flowjo.
Data are shown as mean±SD of three replicates. *p<0.05 and **p<0.01 vs
vehicle group.
Figure 5. MD13 treatment inhibits A549 cell growth. (A) Proliferation of A549
cells were inhibited by treatment of MD13 for 72 hours. The resulting cells were
quantified by CyQUANT® assays and compared with the vehicle control. (B) The
growth of A549 cell spheroids were inhibited by treatment of MD13 for every
three days. The growth curves were obtained relative to the untreated
spheroids (day 0). Values are shown as means ± SD (n = 3 spheroids/time point,
*p<0.05, **p < 0.01 and ***p < 0.001 vs control).
MD13 inhibits ERK signaling. The effect of treatment with
MIF-directed PROTAC MD13 on MIF-related signaling pathways
was investigated by assessment of ERK phosphorylation using
western blot analysis. Treatment with 2 μM MD13 proved to inhibit
ERK phosphorylation in A549 by about 50% after 24-hour
treatment, which persisted at 48 h. In contrast, the cells exhibited
no significant decrease on the pERK levels after incubation with
the control compound 3, 4 for 24 h or MD15 for 6 h, 24 h, or 48 h
(Figure 7). Thus treatment with the MIF-directed PROTAC MD13
inhibits ERK phosphorylation as a MIF-related signaling event.
MD13 arrests cells at G2/M phase of the cell cycle. The
effect of MIF-directed PROTAC MD13 on cell cycle progression
was further analyzed using flow cytometry (Figure 6). A549 cells
were treated with MD13 at concentrations of 1, 2, or 5 μM for the
duration of 48 h before analysis using flow cytometry. Our results
showed that MD13 dose-dependently induced cell cycle arrest at
5
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Figure 7. Effect of the MIF-directed PROTAC on ERK phosphorylation in A549 cells. (A) A549 cells were treated with 2 μM of MD13, MD15, 3, 4, or DMSO for 24
h, the pERK, total ERK and GAPDH was examined by immunoblots. (B) A549 cells were treated with MD13, MD15 or DMSO for 6, 24 or 48 h, the pERK, total ERK
and GAPDH was examined. (C) Quantification of the pERK level using pERK:ERK ratio, normalized to control group at time points indicated. GAPDH was used as
a loading control on western blots. Data are shown as mean±SD of three replicates. **p<0.01 and ***p<0.001 vs vehicle group.
were conducted using 3 as a competitor for MIF binding, 4 as a
competitor of E3 ligase binding and bortezomib as a proteasome
Conclusion
inhibitor. Our results showed that 3, 4, and bortezomib were all
able to abolish the MD13 triggered degradation, thus indicating
that the activity of MD13 depends on MIF binding, CRBN-binding,
and proteasome mediated degradation. Importantly, the control
compound MD15, which contains an impaired E3 ligase ligand,
has no effect on MIF protein level. Taken together, MD13 proved
to be an effective and potent MIF-directed PROTAC.
The effect of MD13 on cell proliferation was evaluated using
cell culture assays on A549 cells. A monolayer cell culture assay
demonstrated that MD13 inhibited proliferation of A549 cells to a
maximum of about 50% at 20 μM. In contrast, the CRBN inactive
control MD15, the MIF tautomerase inhibitor 3, or the E3 ligase
ligand 4 had no or little effect on cell proliferation. Also a spheroid
cell culture assay was employed because such assays mimic the
main features of solid human tumors, such as their structural
organization, cellular layered assembling, hypoxia, and nutrient
gradients.[53] In this spheroid assay, MD13 inhibited the growth of
the spheroid volume by 53% and 81% upon treatment with 2 and
5 μM of MD13 respectively. These results indicate that depletion
of MIF using MIF-directed PROTACs provides a strong reduction
of cell proliferation, which is consistent with the results of siRNA
mediated MIF silencing.[9,54]
Overexpression of MIF was found to stimulate proliferation
of cancer cells via activation of the ERK/MAPK pathway and
inhibition of the p53 pathway.[49,50] Therefore, a number of MIF
targeting modalities have been reported as potential treatments,
including mAbs,[51] peptides,[52] small-molecule inhibitors[24,25] etc.
These modalities have been successfully applied in animal
models for MIF-related diseases.[52] However, there is no clinically
approved MIF-directed drug available yet. Use of PROTACs that
trigger degradation of the MIF protein provides novel
opportunities that might be particularly relevant for MIF.
Importantly, MIF is involved in protein-protein interactions, such
as the MIF-CD74 receptor interaction for which the interactions
site is known to be located in close proximity of the MIF
tautomerase active site.[3] However, other protein-protein
interactions might occur at different locations of the MIF protein,
thus making approaches aimed at MIF tautomerase activity
ineffective. In this perspective the value of MIF-directed
PROTACs becomes clear, because the high affinity ligands
identified for the MIF tautomerase active site can be employed to
induce degradation of the MIF protein as a whole, thus
diminishing MIF from its effector network.
The development of MIF-directed PROTACs requires the
synthesis of heterobifunctional ligands that are able to bind both
MIF and E3 ubiquitin ligase. Optimization of the linker is required
to achieve a proper orientation to trigger ubiquitination and
subsequent degradation. To synthesize MIF-targeting PROTACs,
we tethered MIF binder 2[27,28] or 3[27,29] with the cereblon E3 ligase
ligand pomalidomide by a variety of linkers constructed by click
reactions or amidation coupling reactions. Upon exploration of the
structure-activity relationships for MIF degradation, we identified
MD9 as the first MIF-directed PROTAC. Further optimization of
the linker length provides MD13 as a MIF-directed PROTAC with
improved potency, which proved to trigger almost complete (90-
95%) degradation of MIF in the low micromolar range and a DC50
of around 100 nM on A549 cells. The potency of MD13 is
comparable to PROTACs directed at other protein targets.[38]
Fluorescence microscopy demonstrated that MD13 effectively
entered the cytosol and reduced the MIF protein levels by about
80% within 3 hours. The reduction in MIF protein levels upon
treatment with 2 μM of the MIF-directed PROTAC MD13 was still
observed after 48 hours. As a bona fide MIF-targeting PROTAC,
MD13 should induce the degradation through the formation of a
ternary complex, which is followed by ubiquitination and
proteasome-mediated proteolysis. Accordingly, rescue assays
Growth of cancer cells is characterized by ordered
progression of the cell cycle.[55] MIF coordinates the cell cycle
through the association with the Jab1/CSN5 subunit of the
COP9/CSN signalosome,[56] which plays a central role in the
assembly of SCF complexes by removal of Nedd8 from Cullin.[57–
59] MIF knockout leads to DNA damage and stalled replication.[60]
Treatment of A549 cancer cells with the MIF-directed PROTAC
MD13 increased the number of cells in the G2/M phase thus
indicating inhibition of cell cycle progression. MIF as a growth
factor stimulates cell cycle progression through the MAPK
pathway.[61,62] Our results also demonstrate that MD13 treatment
attenuates the MAPK signaling by reducing ERK phosphorylation.
This result is again in line with the effect observed upon siRNA-
mediated downregulation of the MIF protein levels.[54] Collectively,
these results indicate that the MIF-directed PROTAC MD13
reduces the MIF protein levels and inhibits cell proliferation in both
2D- and 3D- cell culture, which can be explained by inhibition of
ERK phosphorylation and cell cycle progression.
In conclusion, we have developed a potent MIF-directed
PROTAC MD13 that induces MIF degradation in A549 and HEK
293 cells. MD13 effectively reduces the MIF protein level in A549
cells in a time-, cereblon-, and proteasome- dependent manner.
6
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10.1002/anie.202101864
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RESEARCH ARTICLE
Fluorescence microscopy demonstrates that MD13 enters A549
cells with concomitant reduction of the MIF levels. MD13 inhibited
proliferation by about 50% at micromolar concentrations in a 2D
cell culture assay using A549 cells. A 3D cell culture also using
A549 cells showed with 80% reduction of cell proliferation at 5 μM
MD13 an even more pronounced effect. FACS analysis
demonstrated that MD13 treatment inhibited cell cycle arrest in
the G2/M phase. MD13 treatment also inhibited ERK
phosphorylation, thus indicating that MIF degradation also inhibits
signaling pathways that respond to MIF signaling and promote cell
proliferation. In conclusion, the MIF-directed PROTAC MD13
mediates MIF degradation, which consequently results in
inhibition of cell proliferation in 2D and 3D cell cultures, which can
be explained by cell cycle arrest and inhibition of the MAPK
signaling pathway. Altogether, this study demonstrates that MIF-
directed PROTACs are novel modalities in MIF-directed drug
discovery for oncology and other MIF related diseases.
[14]
A. Israelson, D. Ditsworth, S. Sun, S. W. Song, J. Liang, M. Hruska-
Plochan, M. McAlonis-Downes, S. Abu-Hamad, G. Zoltsman, T.
Shani, M. Maldonado, A. Bui, M. Navarro, H. Zhou, M. Marsala, B.
K. Kaspar, S. DaCruz, D. W. Cleveland, Neuron 2015, 86, 218–232.
L. Leng, C. N. Metz, Y. Fang, J. Xu, S. Donnelly, J. Baugh, T.
Delohery, Y. Chen, R. A. Mitchell, R. Bucala, J. Exp. Med. 2003,
197, 1467–1476.
[15]
[16]
X. Shi, L. Leng, T. Wang, W. Wang, X. Du, J. Li, C. McDonald, Z.
Chen, J. W. Murphy, E. Lolis, P. Noble, W. Knudson, R. Bucala,
Immunity 2006, 25, 595–606.
[17]
[18]
B. Jäger, D. Klatt, L. Plappert, H. Golpon, S. Lienenklaus, P. D.
Barbosa, A. Schambach, A. Prasse, Cell. Signal. 2020, 73, 109672.
A. F. Dessein, L. Stechly, N. Jonckheere, P. Dumont, D. Monté, E.
Leteurtre, S. Truant, F. R. Pruvot, M. Figeac, M. Hebbar, C. H.
Lecellier, T. Lesuffleur, R. Dessein, G. Grard, M. J. Dejonghe, Y. De
Launoit, Y. Furuichi, G. Prévost, N. Porchet, C. Gespach, G. Huet,
Cancer Res. 2010, 70, 4644–4654.
[19]
[20]
[21]
J. B. Lubetsky, M. Swope, C. Dealwis, P. Blake, E. Lolis,
Biochemistry 1999, 38, 7346–7354.
Acknowledgements
Y. Qian, M. Schürmann, P. Janning, C. Hedberg, H. Waldmann,
Angew. Chemie. Int. Ed. 2016, 55, 7766–7771.
Z. X. and D. C. is funded by China Scholarship Council (grant no.
201706010341 for Z.X., grant no 201907720019 for D.C). S. S. is
funded by Royalties Prof. dr. H.W. Frijlink Scholarship from
University of Groningen. We thank dr. Q. Wang, Z. Wang, and B.
Zhang for inspiring discussions.
G. Pantouris, M. A. Syed, C. Fan, D. Rajasekaran, T. Y. Cho, E. M.
Rosenberg, R. Bucala, V. Bhandari, E. J. Lolis, Chem. Biol. 2015,
22, 1197–1205.
[22]
[23]
G. Pantouris, J. Ho, D. Shah, M. A. Syed, L. Leng, V. Bhandari, R.
Bucala, V. S. Batista, J. P. Loria, E. J. Lolis, Angew. Chemie. Int.
Ed. 2018, 57, 7116–7119.
Keywords: Macrophage migration inhibitory factor (MIF) •
proteolysis targeting chimera (PROTACs) • A549 cells •
Mitogen‑activated protein kinase (MAPK) pathway • cell cycle
arrest
D. Rajasekaran, S. Gröning, C. Schmitz, S. Zierow, N. Drucker, M.
Bakou, K. Kohl, A. Mertens, H. Lue, C. Weber, A. Xiao, G. Luker, A.
Kapurniotu, E. Lolis, J. Bernhagen, J. Biol. Chem. 2016, 291,
15881–15895.
[1]
[2]
[3]
I. Dagogo-Jack, A. T. Shaw, Nat. Rev. Clin. Oncol. 2018, 15, 81–94.
R. Bucala, S. C. Donnelly, Immunity 2007, 26, 281–285.
J. C. Penticuff, B. L. Woolbright, T. M. Sielecki, S. J. Weir, J. A.
Taylor, Nat. Rev. Urol. 2019, 16, 318–328.
[24]
[25]
V. C. Trivedi-Parmar, W. L. Jorgensen, J. Med. Chem. 2018, 61,
8104–8119.
T. Kok, A. A. Wasiel, R. H. Cool, B. N. Melgert, G. J. Poelarends, F.
J. Dekker, Drug Discov. Today 2018, 23, 1910–1918.
Y. Al-Abed, S. VanPatten, Future Med. Chem. 2011, 3, 45–63.
Z. Xiao, D. Chen, S. Song, R. van der Vlag, P. E. van der Wouden,
R. van Merkerk, R. H. Cool, A. K. H. Hirsch, B. N. Melgert, W. J.
Quax, G. J. Poelarends, F. J. Dekker, J. Med. Chem. 2020, 63,
11920–11933.
[4]
[5]
L. Soumoy, N. Kindt, G. Ghanem, S. Saussez, F. Journe, Cancers
(Basel). 2019, 11, 1–11.
[26]
[27]
E. Cavalli, R. Ciurleo, M. C. Petralia, P. Fagone, R. Bella, K.
Mangano, F. Nicoletti, P. Bramanti, M. S. Basile, Molecules 2020,
25, 1–19.
[6]
[7]
A. M. Coleman, B. E. Rendon, M. Zhao, M.-W. Qian, R. Bucala, D.
Xin, R. A. Mitchell, J. Immunol. 2008, 181, 2330–2337.
M. Charan, S. Das, S. Mishra, N. Chatterjee, S. Varikuti, K. Kaul, S.
Misri, D. K. Ahirwar, A. R. Satoskar, R. K. Ganju, Cell Death Dis.
2020, 11, 774.
[28]
M. Orita, S. Yamamoto, N. Katayama, M. Aoki, K. Takayama, Y.
Yamagiwa, N. Seki, H. Suzuki, H. Kurihara, H. Sakashita, M.
Takeuchi, S. Fujita, T. Yamada, A. Tanaka, J. Med. Chem. 2001,
44, 540–547.
[29]
[30]
J. A. Cisneros, M. J. Robertson, M. Valhondo, W. L. Jorgensen, J.
Am. Chem. Soc. 2016, 138, 8630–8638.
[8]
[9]
M. Zhang, L. Yan, J. A. Kim, Cancer Gene Ther. 2015, 22, 463–474.
C. S. Oliveira, C. E. de Bock, T. J. Molloy, E. Sadeqzadeh, X. Y.
Geng, P. Hersey, X. D. Zhang, R. F. Thorne, BMC Cancer 2014, 14,
1–14.
P. Dziedzic, J. A. Cisneros, M. J. Robertson, A. A. Hare, N. E.
Danford, R. H. G. Baxter, W. L. Jorgensen, J. Am. Chem. Soc.
2015, 137, 2996–3003.
[10]
[11]
K. N. Balogh, D. J. Templeton, J. V. Cross, PLoS One 2018, 13, 1–
20.
[31]
[32]
V. Trivedi-Parmar, M. J. Robertson, J. A. Cisneros, S. G. Krimmer,
W. L. Jorgensen, ChemMedChem 2018, 97, 1092–1097.
S. Balachandran, P. K. Gadekar, S. Parkale, V. N. Yadav, D.
Kamath, S. Ramaswamy, S. Sharma, R. A. Vishwakarma, N. M.
Dagia, Bioorg. Med. Chem. Lett. 2011, 21, 1508–1511.
J. Bloom, C. Metz, S. Nalawade, J. Casabar, K. F. Cheng, M. He, B.
Sherry, T. Coleman, T. Forsthuber, Y. Al-Abed, J. Biol. Chem. 2016,
291, 26502–26514.
H. W. Sun, J. Bernhagen, R. Bucala, E. Lolis, Proc. Natl. Acad. Sci.
1996, 93, 5191–5196.
[12]
[13]
R. Bucala, Cytokine Growth Factor Rev. 1996, 7, 19–24.
J. Bernhagen, R. Krohn, H. Lue, J. L. Gregory, A. Zernecke, R. R.
Koenen, M. Dewor, I. Georgiev, A. Schober, L. Leng, T. Kooistra, G.
Fingerle-Rowson, P. Ghezzi, R. Kleemann, S. R. McColl, R. Bucala,
M. J. Hickey, C. Weber, Nat. Med. 2007, 13, 587–596.
[33]
7
This article is protected by copyright. All rights reserved.

10.1002/anie.202101864
Angewandte Chemie International Edition
RESEARCH ARTICLE
[34]
R. Schulz, N. D. Marchenko, L. Holembowski, G. Fingerle-Rowson,
M. Pesic, L. Zender, M. Dobbelstein, U. M. Moll, J. Exp. Med. 2012,
209, 275–289.
[54]
X. H. Huang, W. H. Jian, Z. F. Wu, J. Zhao, H. Wang, W. Li, J. T.
Xia, Oncotarget 2014, 5, 5570–5580.
[55]
[56]
G. H. Williams, K. Stoeber, J. Pathol. 2012, 226, 352–364.
R. Kleemann, A. Hausser, G. Geiger, R. Mischke, A. Burger-
kentischer, O. Flieger, F. Johannes, T. Roger, T. Calandra, A.
Kapurniotu, Nature 2000, 408, 211–216.
[35]
[36]
[37]
[38]
M. Pettersson, C. M. Crews, Drug Discov. Today Technol. 2019, 31,
15–27.
K. M. Sakamoto, K. B. Kim, A. Kumagai, F. Mercurio, C. M. Crews,
R. J. Deshaies, Proc. Natl. Acad. Sci. U. S. A. 2001, 98, 8554–8559.
G. E. Winter, D. L. Buckley, J. Paulk, J. M. Roberts, A. Souza, S.
Dhe-Paganon, J. E. Bradner, Science. 2015, 348, 1376–1381.
X. Sun, H. Gao, Y. Yang, M. He, Y. Wu, Y. Song, Y. Tong, Y. Rao,
Signal Transduct. Target. Ther. 2019, 4, 64.
[57]
[58]
G. A. Cope, R. J. Deshaies, BMC Biochem. 2006, 7, 1–10.
Y. Asare, M. Ommer, F. A. Azombo, S. Alampour-Rajabi, M.
Sternkopf, M. Sanati, M. J. Gijbels, C. Schmitz, D. Sinitski, P. V.
Tilstam, H. Lue, A. Gessner, D. Lange, J. A. Schmid, C. Weber, M.
Dichgans, J. Jankowski, R. Pardi, M. P. J. De Winther, H. Noels, J.
Bernhagen, Proc. Natl. Acad. Sci. U. S. A. 2017, 114, E2766–
E2775.
[39]
[40]
P. M. Cromm, C. M. Crews, Cell Chem. Biol. 2017, 24, 1181–1190.
J. B. Lubetsky, A. Dios, J. Han, B. Aljabari, B. Ruzsicska, R.
Mitchell, E. Lolis, Y. Al-Abed, J. Biol. Chem. 2002, 277, 24976–
24982.
[59]
[60]
G. Fingerle-Rowson, O. Petrenko, Cell Div. 2007, 2, 1–7.
A. Nemajerova, P. Mena, G. Fingerle-Rowson, U. M. Moll, O.
Petrenko, EMBO J. 2007, 26, 987–997.
[41]
[42]
J. Li, J. Zhang, F. Xie, J. Peng, X. Wu, Int. J. Mol. Med. 2018, 41,
1062–1068.
[61]
[62]
J. C. Chambard, R. Lefloch, J. Pouysségur, P. Lenormand, Biochim.
Biophys. Acta - Mol. Cell Res. 2007, 1773, 1299–1310.
R. A. Mitchell, Cell. Signal. 2004, 16, 13–19.
J. Popow, H. Arnhof, G. Bader, H. Berger, A. Ciulli, D. Covini, C.
Dank, T. Gmaschitz, P. Greb, J. Karolyi-Özguer, M. Koegl, D. B.
McConnell, M. Pearson, M. Rieger, J. Rinnenthal, V. Roessler, A.
Schrenk, M. Spina, S. Steurer, N. Trainor, E. Traxler, C. Wieshofer,
A. Zoephel, P. Ettmayer, J. Med. Chem. 2019, 62, 2508–2520.
Z. Xiao, M. Fokkens, D. Chen, T. Kok, G. Proietti, R. van Merkerk,
G. J. Poelarends, F. J. Dekker, Eur. J. Med. Chem. 2020, 186,
111849–111862.
[43]
[44]
[45]
P. Bonvini, E. Zorzi, G. Basso, A. Rosolen, Leukemia 2007, 21,
838–842.
E. S. Fischer, K. Böhm, J. R. Lydeard, H. Yang, M. B. Stadler, S.
Cavadini, J. Nagel, F. Serluca, V. Acker, G. M. Lingaraju, R. B.
Tichkule, M. Schebesta, W. C. Forrester, M. Schirle, U. Hassiepen,
J. Ottl, M. Hild, R. E. J. Beckwith, J. W. Harper, J. L. Jenkins, N. H.
Thomä, Nature 2014, 512, 49–53.
[46]
C. Qin, Y. Hu, B. Zhou, E. Fernandez-Salas, C. Y. Yang, L. Liu, D.
McEachern, S. Przybranowski, M. Wang, J. Stuckey, J. Meagher, L.
Bai, Z. Chen, M. Lin, J. Yang, D. N. Ziazadeh, F. Xu, J. Hu, W.
Xiang, L. Huang, S. Li, B. Wen, D. Sun, S. Wang, J. Med. Chem.
2018, 61, 6685–6704.
[47]
[48]
C. A. Foley, F. Potjewyd, K. N. Lamb, L. I. James, S. V. Frye, ACS
Chem. Biol. 2020, 15, 290–295.
W. Feng, X. Y. Teo, W. Novera, P. M. Ramanujulu, D. Liang, D.
Huang, P. K. Moore, L. W. Deng, B. W. Dymock, J. Med. Chem.
2015, 58, 6456–6480.
[49]
[50]
C. O’Reilly, M. Doroudian, L. Mawhinney, S. C. Donnelly, Med. Res.
Rev. 2016, 36, 440–460.
C. C. G. Nobre, J. M. G. de Araújo, T. A. A. de M. Fernandes, R. N.
O. Cobucci, D. C. F. Lanza, V. S. Andrade, J. V. Fernandes, Pathol.
Oncol. Res. 2017, 23, 235–244.
[51]
D. Mahalingam, M. Patel, J. Sachdev, L. Hart, N. Halama, R.
Ramanathan, J. Sarantopoulos, X. Liu, S. Yazji, D. Jäger, D. Adib,
R. Kerschbaumer, M. Yoon, G. Manzur, A. Starodub, K. Sivarajan,
M. Wertheim, P. Thambi, M. Jones, S. Goel, J. Nemunaitis, A.
Tsimberidou, Ann. Oncol. 2016, 27, ii105.
[52]
[53]
D. Sinitski, C. Kontos, C. Krammer, Y. Asare, A. Kapurniotu, J.
Bernhagen, Thromb. Haemost. 2019, 119, 553–566.
M. Vinci, S. Gowan, F. Boxall, L. Patterson, M. Zimmermann, W.
Court, C. Lomas, M. Mendiola, D. Hardisson, S. A. Eccles, BMC
Biol. 2012, 10, 29–48.
8
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Angewandte Chemie International Edition
RESEARCH ARTICLE
Table of Contents
Targeting macrophage migration inhibitory factor (MIF) is challenging by small-molecule modulators, because MIF involved in
diseases via protein-protein interactions. We report the first potent MIF-directed PROTAC MD13, which eliminate MIF from its
protein-protein interaction network by inducing MIF degradation. MD13 suppresses the proliferation of A549 cells by reducing MIF
protein level and MIF-related signaling. We provide a tool for MIF study, as well as demonstrate the potential of PROTACs in cancer
therapy.
9
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