Biochemical and Biophysical Research Communications 523 (2020) 795e801

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Biochemical and Biophysical Research Communications

journal homepage: www.elsevier.com/locate/ybbrc

Identification of a selective DDX3X inhibitor with newly developed

quantitative high-throughput RNA helicase assays
Shoichi Nakao a, Masahiro Nogami a, Misa Iwatani a, Toshihiro Imaeda a, Masahiro Ito a,
Toshio Tanaka a, Michiko Tawada a, Satoshi Endo a, Douglas R. Cary a, Momoko Ohori a,
Yasuhiro Imaeda a, Tomohiro Kawamoto a, Samuel Aparicio b, c, Atsushi Nakanishi a,
Shinsuke Araki a, *
a
Research, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-Higashi 2-chome, Fujisawa, Kanagawa, 251-8555, Japan
b
Department of Molecular Oncology, BC Cancer Agency, Vancouver, BC, V5Z 1L3, Canada
c
Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, BC, V6T 2B5, Canada

a r t i c l e i n f o a b s t r a c t

Article history: The DEAD-box family of RNA helicases plays essential roles in both transcriptional and translational
Received 16 December 2019 mRNA degradation; they unwind short double-stranded RNA by breaking the RNAeRNA interactions.
Accepted 17 December 2019 Two DEAD-box RNA helicases, eukaryotic translation initiation factor 4A3 (eIF4A3) and DEAD-box
Available online 15 January 2020
helicase 3 (DDX3X), show high homology in the ATP-binding region and are considered key molecules
for cancer progression. Several small molecules that target eIF4A3 and DDX3X have been reported to
Keywords:
inhibit cancer cell growth; however, more potent compounds are required for cancer therapeutics, and
RNA helicase
there is a critical need for high-throughput assays to screen for RNA helicase inhibitors. In this study, we
eIF4A3
DDX3X
developed novel fluorescence resonance energy transfer-based high-throughput RNA helicase assays for
RNA helicase inhibitor eIF4A3 and DDX3X. Using these assays, we identified several eIF4A3 allosteric inhibitors whose inhibi-
tory effect on eIF4A3 ATPase showed a strong correlation with inhibitory effect on helicase activity. From
102 compounds that exhibited eIF4A3 ATPase inhibition, we identified a selective DDX3X inhibitor, C1,
which showed stronger inhibition of DDX3X than of eIF4A3. Small-molecule helicase inhibitors can be
valuable for clarifying the molecular machinery of DEAD-box RNA helicases. The high-throughput
quantitative assays established here should facilitate the evaluation of the helicase inhibitory activity
of compounds.
© 2020 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND
license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction
The Asp-Glu-Ala-Asp (DEAD) box RNA helicases play key phys-
iological roles in cellular RNA metabolism, including in transcrip-
tion, RNA splicing, mRNA export, the initiation of translation, and
mRNA degradation [1,2]. Members of the DEAD-box family have a
highly conserved helicase core, which harbors the binding sites for
Abbreviations: AMP-PNP, adenylyl-imidodiphosphate; BHQ2, Black Hole
Quencher 2; DDX3X, DEAD box helicase 3; ds, double-stranded; eIF4A3, eukaryotic
translation initiation factor 4A3; FRET, fluorescence resonance energy transfer; His,
histidine; IC50, half-maximal inhibitory concentration; NMD, nonsense-mediated
mRNA decay; ss, single-stranded; TAMRA, carboxytetramethylrhodamine (a
fluorophore).
* Corresponding author.
E-mail address: shinsuke.araki@takeda.com (S. Araki).
ATP and RNA, and they unwind double-stranded RNA in the pres-
ence of ATP. Several DEAD-box RNA helicases have been linked to
diseases, especially cancers; these include eukaryotic translation
initiation factor 4A3 (eIF4A3) and DEAD-box helicase 3 (DDX3X)
[3e6].
eIF4A3 is a core component of the exon junction complex (EJC),
together with the proteins MLN51, MAGOH, and Y14. It is involved
in several post-transcriptional steps, including mRNA export,
translation, and nonsense-mediated mRNA decay (NMD) [7,8].
NMD is a critical surveillance mechanism that prevents the
expression of mRNAs that contain a premature termination codon.
Recent studies have shown that the inhibition of NMD is associated
with the induction of tumor immunity and with the enhancement
of chemotherapeutics in cancer [9,10].
DDX3X plays a role in global protein synthesis, initiating the
translation of 50 -untranslated regions, including internal ribosome

https://doi.org/10.1016/j.bbrc.2019.12.094
0006-291X/© 2020 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.
0/).
796 S. Nakao et al. / Biochemical and Biophysical Research Communications 523 (2020) 795e801
entry sites [11,12]. It has been shown that translational changes
regulated by DDX3X upregulate genes associated with tumor pro-
gression [5]. Recurrent mutations of DDX3X broadly inhibit trans-
lation and result in the hyperassembly of stress granules [13].
These findings suggest that targeting eIF4A3 or DDX3X may
have therapeutic potential for cancer treatment. Recent studies
have therefore investigated the therapeutic potential of various
small-molecule compounds that target eIF4A3 or DDX3X
[4,6,14,15]. These reported eIF4A3 inhibitors that inhibited NMD
and induced G2/M cell cycle arrest and apoptosis in cancer cells
[6,15], and a DDX3X inhibitor that induced G1 cell cycle arrest and
apoptosis [4]. These findings shed light on the potential for cancer
therapeutics; however, there is a need for more potent compounds
with good pharmacokinetics.
Screening and evaluating compounds to identify eIF4A3 and
DDX3X helicase inhibitors requires suitable high-throughput as-
says that can measure helicase activity. However, no such assays for
eIF4A3 and DDX3X were available. A further issue was the lack of
data about the relationship between ATPase activity and helicase
activity. For instance, eIF4A3 acts ATP-dependently as an RNA
clamp by binding to single-stranded RNAs, but it is unclear how its
helicase activity affects the formation of the EJC and NMD activity.
The aims of this study were to develop high-throughput RNA
helicase assays for eIF4A3 and DDX3X and to use these to identify
inhibitors that may have therapeutic potential. We developed novel
fluorescence resonance energy transfer (FRET)-based high-
throughput RNA helicase assays using a double-stranded RNA
substrate. We then showed that, for several eIF4A3 allosteric in-
hibitors, there was a positive correlation between ATPase inhibitory
activity and helicase inhibitory activity. There is a high degree of
homology between the ATP-binding site of eIF4A3 and DDX3X; we
therefore assessed DDX3X ATPase and helicase inhibitory activity
by using a compound series for eIF4A3 ATPase inhibitor. In this way,
we identified a novel selective DDX3X helicase inhibitor, C1, which
showed stronger helicase inhibitory activity for DDX3X than for
eIF4A3.
2. Materials and methods
2.1. Materials
Adenylyl-imidodiphosphate (AMP-PNP) was obtained from
Sigma-Aldrich (St. Louis, MO) and hippuristanol from Centaurus
Biopharma Co. (Beijing, China). The series of eIF4A3 inhibitors had
been synthesized as previously reported [6,14]. The synthetic route
for the production of C1 is described in the Supplemental Infor-
mation, C2 was obtained from Cayman Chemical Co. (Ann Arbor,
MI), and C3 was synthesized and purified as described in a previous
patent application [16]. The full descriptions of these compounds
are as follows: C1, N-(6-aminohexyl)-2-[6-(4-chlorophenyl)-2-[2-
(4-methoxyphenyl)ethyl]-4-oxoquinazolin-3(4H)-yl]acetamide;
C2, (2E,20 E)-3,3’-(1,4-phenylene)bis{N-[4-(4,5-dihydro-1H-imida-
zol-2-yl)phenyl]prop-2-enamide}; and C3, N-[4-{3-[(2-
aminoethyl)carbamoyl]phenyl}-6-(4-chloro-2-hydroxyphenyl)-3-
cyanopyridin-2-yl]-2-methoxybenzamide. In addition, we pur-
chased human DDX3X recombinant protein (MBS1093676;
MyBioSource, San Diego, CA), rabbit anti-DDX3X antibody (A300-
474A; Bethyl Laboratories, Inc., Montgomery, TX), and goat anti-
rabbit IgG-HRP (sc-2030; Santa Cruz Biotechnology, Inc., Dallas,
TX).
2.2. Preparation of enzymes
The human recombinant proteins eIF4A3 and MLN51 (residues
137e283) were expressed in Escherichia coli BL21 (DE3) as fusion
proteins with 6  histidine (His)-small ubiquitin-like modifier
(SUMO) or His tags, followed by a tobacco etch virus protease
cleavage site at the N-terminus. These were then purified with a Ni-
NTA Superflow affinity column (QIAGEN, Valencia, CA) and Super-
dex 200 gel filtration column (GE Healthcare). The His-SUMO or His
tags were cleaved with SUMO protease or tobacco etch virus pro-
tease, and the protein concentrations were determined using a BCA
Protein Assay Kit (Thermo Fisher Scientific Inc.).
2.3. RNA oligonucleotides and double-stranded RNA for the helicase
assay
Oligonucleotides for the helicase assay were purchased from
Gene Design (Osaka, Japan). The single-stranded RNAs (ssRNAs)
used in the EIF4A3 helicase assays were designed according to
previously described sequences [17], using the reporter fluo-
rophore TAMRA (carboxytetramethylrhodamine) and the compat-
ible quencher Black Hole Quencher 2 (BHQ2), as follows (50 to 30 ):
GGG GAG AAA AAC AAA ACA AAA CUA GCA CCG UAA AGC ACG C-
BHQ2 and TAMRA-GCU UUA CGG UGC. The underscoring indicates
the sequence in BHQ2 complementary to TAMRA. The capture
ssRNA was GCU UUA CGG UGC (50 to 30 ). Similarly, the oligonucle-
otides used in the DDX3X helicase assays were designed based on
previously described sequences [18], as follows (50 to 30 ): ACC AGC
UUU GUU CCU UGG GUU CUU GGG AGC AGC AGG-BHQ2 and
TAMRA-CCC AAG AAC CCA AGG AAC. The capture ssRNA was CCC
AAG AAC CCA AGG AAC (50 to 30 ). The TAMRA- and BHQ2-labeled
ssRNAs were prepared at 270 nM and mixed into a reaction
buffer containing 40 mM Tris-HCl (pH 7.5), 3 mM dithiothreitol
(DTT), 50 mM NaCl, 0.01% Tween-20, and 2 mM MgCl2. For the
production of double-stranded RNA (dsRNA), the RNA mixtures
were heated at 90  C for 5 min and then cooled to 4  C for 90 min.
2.4. RNA helicase assays for eIF4A3 and DDX3X
eIF4A3 helicase activity was evaluated with 1 mM MLN51, 2 mM
eIF4A3, 30 nM dsRNA, 1.5 mM capture RNA, and 2 mM ATP in a
reaction buffer containing 40 mM Tris-HCl (pH 7.5), 3 mM DTT,
50 mM NaCl, 0.01% Tween-20, and 2 mM MgCl2. DDX3X helicase
activity was evaluated with 200 nM DDX3X, 20 nM dsRNA, 1 mM
capture RNA, and 500 mM ATP in a reaction buffer containing
50 mM Tris-HCl (pH 7.5), 1 mM DTT, 0.2 mg/ml BSA, 5% glycerol, and
10 mM MgCl2.
These mixtures were incubated at 37  C for 1 h and the reaction
was terminated with a mixture of 10 mM EDTA, 0.8% sodium
dodecyl sulfate, and 10% glycerol. Samples were diluted four-fold
with the reaction buffer and their fluorescence intensity was
quantified with an MF20 (Olympus, Tokyo, Japan) or Envision
(PerkinElmer, Waltham, MA, USA) detection system. The helicase
activity was calculated from the following formula:
 
Fs  Fb
Helicase activity % ¼  100;
Fn  Fb
where Fs, Fn, and Fb are the fluorescence intensities of the sample,
negative control (dimethyl sulfoxide), and background sample,
respectively. Helicase assays using gel electrophoresis were
analyzed using a 15% native polyacrylamide gel with a native buffer
containing 2.5 mM Tris and 19.2 mM glycine. The gel was visualized
with a LAS-4000 imaging system (FujiFilm, Tokyo, Japan). The half-
maximal inhibitory concentrations (IC50) of compounds in the RNA
helicase assays were calculated from a sigmoidal doseeresponse
(variable slope) curve with the top and bottom constrained to be
100% and 0%, respectively, using GraphPad Prism v.6.04 software
(GraphPad Inc., La Jolla, CA, USA).
 S. Nakao et al. / Biochemical and Biophysical Research Communications 523 (2020) 795e801 797

3. Results
3.1. The quantitative RNA helicase assay system for eIF4A3
Fig. 1A schematically illustrates the quantitative RNA helicase
assay developed for eIF4A3, modified from a previously described
electrophoresis-based approach [17]. The RNA used comprised a
12-base pair double-stranded (ds) region with a 24-nucleotide 50 -
single-stranded (ss) region and a 40 -nucleotide 30 -ss region. The
short strand was end-labeled with the fluorophore TAMRA and the
long strand with the quencher BHQ2. The presence of any excess
non-labeled capture oligonucleotide would prevent the re-
annealing of the labeled ssRNAs. Gel electrophoresis analysis
confirmed that the TAMRA fluorescence was detected in the ssRNA
but absorbed by BHQ2 in the dsRNA (Fig. 1B). No fluorescence was
detected in eIF4A3 alone, but eIF4A3 dose-dependent helicase
activity was observed after co-incubation with MLN51 (Fig. 1B). This
was consistent with previous results [17]. The fluorescence was
then detected using a single-molecule fluorescence spectroscopy
system, MF-20, which enabled high-throughput, accurate quanti-
fication. Consistent with the gel electrophoresis analysis, strong
fluorescent intensity was detected in the ssRNA sample and a
sample that had been heat denatured at 95  C for 5 min, but not in
dsRNA sample (Fig. 1C). Fluorescence was hardly detected in the
eIF4A3 alone, the intensity was approximately seven-fold stronger
in the sample of MLN51 alone, and the intensity increased ac-
cording to the eIF4A3 dose when co-incubated with 1 mM MLN51
(Fig. 1C). It has been reported that MLN51 interacts with RNA in the
absence of the other EJC components (eIF4A3, Y14, and MAGOH)
[17], suggesting that MLN51 alone unwinds dsRNA, which is inde-
pendent of EJC.
To investigate whether the helicase assay system could evaluate

Fig. 1. The RNA helicase assay for eIF4A3.

A, Schematic illustration of the principles underlying the eIF4A3 RNA helicase assay. In the double-stranded RNA (dsRNA) form, the fluorescence of TAMRA is quenched by BHQ2;
however, the fluorescence can be detected and quantified for unwound TAMRA-labeled single-stranded RNA (ssRNA). Any excess non-labeled capture RNA oligonucleotide binds to
the ssRNA labeled with BHQ2, preventing re-annealing of the labeled ssRNAs. B, The dsRNA substrate was incubated with eIF4A3 and MLN51 proteins at the indicated concen-
trations (lanes 3e10) and the products were separated by gel electrophoresis. The dsRNA substrate in the absence of proteins was used as the input (lane 1) and the ssRNA labeled
with TAMRA and the heat-denatured dsRNA substrate were used as positive controls (lanes 2 and 11). C, eIF4A3 helicase activity, quantified using TAMRA fluorescence intensity
measured by an MF20 detection system. The treatment and positive controls were as in B, with the dsRNA substrate used as a negative control. D, eIF4A3 helicase activity, quantified
by TAMRA fluorescence intensity. The dsRNA substrate in the presence of eIF4A3 and MLN51 proteins was incubated with AMP-PNP at the indicated concentrations. C and D, The
data represent the mean ± SD of triplicate samples. ***P  0.0005 (Williams’ test).
798 S. Nakao et al. / Biochemical and Biophysical Research Communications 523 (2020) 795e801

compounds that target eIF4A3, we measured eIF4A3 RNA helicase
activity in the presence of the non-hydrolyzable ATP analog AMP-
PNP, a helicase inhibitor [19,20]. This showed a dose-dependent
decrease in activity (Fig. 1D), suggesting that our eIF4A3 RNA
helicase assay could be used for quantitative, high-throughput
compound screening.
3.2. Correlation between helicase activity and ATPase activity using
eIF4A3 inhibitors
For further validation, we examined helicase activity in the
presence of various eIF4A3 inhibitors. The first was hippuristanol,
which binds to the carboxy terminal domain of eIF4A1, blocking
both ATPase and helicase activity [21,22]; however, the carboxy
terminal domains of eIF4A1 and eIF4A3 have low homology, and
hippuristanol has less potency for eIF4A3 than for eIF4A1 [6,21]. At
a concentration of 300 mM, hippuristanol inhibited 16% of the
eIF4A3 helicase activity (Fig. 2A), consistent with previously re-
ported ATPase activity [6].
We then tested the allosteric eIF4A3 inhibitors T-595 and T-202,
which have shown potent, selective inhibition of eIF4A3 in ATPase
and gel electrophoresis-based helicase unwinding assays [14], us-
ing T-598, an inactive distomer compound, as a negative control
(Fig. 2B). In the presence of 3 mM T-595 or T-202, eIF4A3 helicase
activity was inhibited by more than 50%, whereas there was no
inhibition with T-598. This was consistent with the IC50 values for
eIF4A3 ATPase activity (T-595, 0.11 mM; T-202, 0.58 mM; and T-598,
60 mM) (Fig. 2C).
We compared eIF4A3 helicase activity to ATPase activity by
synthesizing eight analogs of T-202 and using these in both ATPase
and helicase analyses for eIF4A3. The IC50 values for helicase ac-
tivity correlated well with those for ATPase activity (Fig. 2D),
suggesting that allosteric eIF4A3 inhibitor compounds show well-
correlated ATPase and helicase inhibitory activity.
3.3. Compound screening using the quantitative DDX3X RNA
helicase assay
Fig. 3A schematically illustrates the RNA helicase assay devel-
oped for DDX3X. The RNA used comprised a 12-base pair ds region
with a 24-nucleotide 50 -ss region and a 40 -nucleotide 30 -ss region.
The short strand was end-labeled with TAMRA and the long strand
with BHQ2. TAMRA fluorescence in the presence of DDX3X
depended on ATP concentration (Fig. 3B), and AMP-PNP signifi-
cantly inhibited helicase fluorescence activity in a dose-dependent
manner (Fig. 3C). The signal-to-background ratio was high (3.1) and
the Z0 factor was at least 0.6, indicating that the assay was highly
robust and sufficiently reliable to be used for high-throughput
screening.
The ATP-binding pockets of DDX3X and eIF4A3 show 71% ho-
mology. We therefore selected 102 compounds for DDX3X RNA
helicase analysis based on their ATPase inhibitory activity of
eIF4A3. Of these, three compounds (C1, C2, and C3) inhibited more
than 70% of DDX3X RNA helicase activity (Fig. 3D and E).
3.4. Further evaluation of the compounds showing helicase
inhibitory activity for DDX3X and eIF4A3
We further evaluated the selectivity of the four compounds (C1,
C2, C3, and T-595) shown to inhibit helicase activity for DDX3X and
eIF4A3. At high concentrations (1e10 mM), C1, C2, and C3 inhibited
DDX3X RNA helicase activity, whereas the eIF4A3 inhibitor T-595
did not (Fig. 4A and C). C1 and T-595 reproducibly induced DDX3X
helicase activity at low concentrations (0.1e1 mM). Interestingly,

Fig. 2. Correlations between eIF4A3 ATPase activity and helicase activity for eIF4A3 inhibitors
A, eIF4A3 helicase activity, quantified by TAMRA fluorescence intensity. The dsRNA substrate in the presence of eIF4A3 and MLN51 proteins was incubated with hippuristanol at the
indicated concentrations. B, Chemical structures of T-595, T-598, and T-202. C, eIF4A3 helicase activity, quantified by TAMRA fluorescence intensity. The dsRNA substrate was
incubated with T-595, T-598, or T-202. D, Scatter plot comparing eIF4A3 ATPase inhibitory activity with eIF4A3 helicase inhibitory activity after incubation with a series of eIF4A3
allosteric inhibitors. The axes show the log10(IC50) values for eIF4A3 ATPase and helicase inhibition. A and C, The data represent the mean ± SD of triplicate samples. *P  0.025,
***P  0.0005 (Williams’ test).
 S. Nakao et al. / Biochemical and Biophysical Research Communications 523 (2020) 795e801 799

Fig. 3. The DDX3X RNA helicase assay and compound screening

A, Schematic of the DDX3X fluorescence-based RNA helicase assay. This is explained in the legend to Fig. 1A. B, DDX3X helicase activity, quantified by TAMRA fluorescence intensity.
The dsRNA substrate in the presence of DDX3X protein was incubated in the absence or presence of 500 mM ATP. C, DDX3X helicase activity, quantified by TAMRA fluorescence
intensity. The dsRNA substrate in the presence of DDX3X protein was incubated with AMP-PNP at the indicated concentrations. B and C, The data represent the mean ± SD of
triplicate samples; ***P  0.0005 (William’s test). D, DDX3X helicase activity for 108 compounds, quantified by TAMRA fluorescence intensity. E, Chemical structures of the
compounds that showed inhibition of DDX3X RNA helicase activity (C1, C2, and C3).

eIF4A3 helicase activity was inhibited by C2, C3, and T-595 but not
by C1 (Fig. 4B and C). This was confirmed by gel electrophoresis
helicase analysis. The unwinding activity of DDX3X was inhibited
by 10 mM C1; in contrast, C1 did not inhibit the unwinding activity
of eIF4A3 (Fig. 4D). These results suggest that C1 is more selective
for DDX3X than for eIF4A3, and that it does not bind RNA non-
specifically.
4. Discussion
In this study, we developed RNA helicase assays for eIF4A3 and
DDX3X to facilitate compound screening. We also showed a good
correlation between ATPase and helicase inhibitory activity with
allosteric eIF4A3 inhibitors. Interestingly, however, there was a
discrepancy between ATPase and helicase inhibitory activity for
eIF4A3 with the compounds that inhibited DDX3X helicase activity.
It has been reported that eIF4A remains able to unwind dsRNA even
in the presence of a non-hydrolyzable ATP analog [2,23,24]. ATP
binding, but not ATPase activity, is required for RNA unwinding,
whereas ATPase activity is required for the release of eIF4A protein
from the RNA [23]. These findings suggest that the measurement of
RNA helicase activity that can affect downstream signaling is crit-
ical for the identification of helicase inhibitors. The difference be-
tween allosteric inhibition and ATPase inhibition for eIF4A3 could
800 S. Nakao et al. / Biochemical and Biophysical Research Communications 523 (2020) 795e801

Fig. 4. Analyses of the four inhibitors of DDX3X and eIF4A3 helicase activity.
A, DDX3X helicase activity in the presence of C1, C2, C3, and T-595. B, eIF4A3 helicase activity in the presence of the indicated compounds. A and B, The y-axes show the helicase
activity normalized to dimethyl sulfoxide. The data points represent the means ± SD of triplicate samples. C, IC50 of eIF4A3 ATPase and helicase activity, and DDX3X helicase activity,
for C1, C2, C3, and T-595. D, Electrophoresis-based helicase assays of DDX3X (upper panel) and eIF4A3 (lower panel), showing that C1 selectively inhibits DDX3X. Each dsRNA,
generated from TAMRA-labeled ssRNA and non-fluorescent ssRNA, was incubated with C1 at the indicated concentrations and the products were separated by gel electrophoresis.
The dsRNA substrate in the absence of proteins or ATP was used as negative controls (lanes 1 and 3) and the ssRNAs labeled with TAMRA and the heat-denatured dsRNA substrate
were used as controls for the ssRNAs (lanes 2 and 11). AMP-PNP and T-202 were used as positive controls for DDX3X and eIF4A3, respectively (lane 10).

potentially result in different pharmacological phenotypes; how-
ever, this requires confirmation using multiple compounds in cells.
In the FRET-based DDX3X helicase assays, C1 and T-595 induced
helicase activity at low concentrations and inhibited it at high
concentrations. However, gel electrophoresis assays confirmed this
only at high concentrations and not at low concentrations. High-
throughput systems detect false-positive or -negative compounds
at a constant rate, so this suggests that a secondary confirmation
assay system may be needed.
Recent anti-tumor molecular targeting drugs have shown sig-
nificant anti-tumor effects for specific patients, with the emergence
of predictive biomarkers to help increase response rates among
cancer patients. Recently, a mutation of DDX3X has been reported
in medulloblastoma and chronic lymphatic leukemia [25e27]. Tu-
mors with the DDX3X mutation contain a concurrent mutation of
b-catenin and activate the Wnt signaling pathway [25]. A DDX3X
inhibitor may therefore provide effective treatment for patients
with the DDX3X mutation.
In conclusion, the newly developed RNA helicase assays for
eIF4A3 and DDX3X enable high-throughput compound screening
and the characterization of compounds that show RNA helicase or
ATPase inhibition. In addition, the newly discovered DDX3X heli-
case inhibitor C1 may be a valuable tool for gaining a better un-
derstanding of molecular mechanisms involving DDX3X and might
serve as a basis for future cancer therapeutics.
Funding
Takeda Pharmaceutical Company Limited provided support in
the form of salaries for authors but did not have any additional role
in the study design, data collection and analysis, decision to pub-
lish, or preparation of the manuscript.
Author contributions
SN, MN, and SAr conceived and designed the research and
 S. Nakao et al. / Biochemical and Biophysical Research Communications 523 (2020) 795e801
experiments. SN, MI, MO performed the experiments. TI, MIto, TT,
DC, and YI synthesized the compounds. MT and SE performed
chemo informatics analysis. SA, AN, TK, and YI supervised the study.
SN, TI, and SAr wrote the manuscript.
Declaration of competing interest
In accordance with the journal’s policy regarding conflicts of
interest: SN, MN, MI, TI, MIto, TT, MT, SE, DC, MO, YI, TK, AN, and SAr
are/were employees of Takeda Pharmaceutical Co. Ltd.
Acknowledgements
We are grateful to our laboratory members for insightful dis-
cussions. We also thank H. Kato and T. Ogasawara for technical
assistance.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.bbrc.2019.12.094.
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