Identification of a selective DDX3X inhibitor with newly developed quantitative high-throughput RNA helicase assays

Biochemical and Biophysical Research Communications xxx (xxxx) xxx

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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 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 b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 16 December 2019 Accepted 17 December 2019 Available online xxx

The DEAD-box family of RNA helicases plays essential roles in both transcriptional and translational mRNA degradation; they unwind short double-stranded RNA by breaking the RNAeRNA interactions. Two DEAD-box RNA helicases, eukaryotic translation initiation factor 4A3 (eIF4A3) and DEAD-box 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 inhibit cancer cell growth; however, more potent compounds are required for cancer therapeutics, and there is a critical need for high-throughput assays to screen for RNA helicase inhibitors. In this study, we developed novel fluorescence resonance energy transfer-based high-throughput RNA helicase assays for eIF4A3 and DDX3X. Using these assays, we identified several eIF4A3 allosteric inhibitors whose inhibitory 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/).

Keywords: RNA helicase eIF4A3 DDX3X RNA helicase inhibitor

1. Introduction The Asp-Glu-Ala-Asp (DEAD) box RNA helicases play key physiological roles in cellular RNA metabolism, including in transcription, 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: [email protected] (S. Araki).

ATP and RNA, and they unwind double-stranded RNA in the presence 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/).

Please cite this article as: S. Nakao et al., Identification of a selective DDX3X inhibitor with newly developed quantitative high-throughput RNA helicase assays, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.094

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entry sites [11,12]. It has been shown that translational changes regulated by DDX3X upregulate genes associated with tumor progression [5]. Recurrent mutations of DDX3X broadly inhibit translation 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 assays 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 highthroughput RNA helicase assays using a double-stranded RNA substrate. We then showed that, for several eIF4A3 allosteric inhibitors, 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 Information, 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-imidazol-2-yl)phenyl]prop-2-enamide}; and C3, N-[4-{3-[(2aminoethyl)carbamoyl]phenyl}-6-(4-chloro-2-hydroxyphenyl)-3cyanopyridin-2-yl]-2-methoxybenzamide. In addition, we purchased human DDX3X recombinant protein (MBS1093676; MyBioSource, San Diego, CA), rabbit anti-DDX3X antibody (A300474A; Bethyl Laboratories, Inc., Montgomery, TX), and goat antirabbit 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 NiNTA Superflow affinity column (QIAGEN, Valencia, CA) and Superdex 200 gel filtration column (GE Healthcare). The His-SUMO or His tags were cleaved with SUMO protease or tobacco etch virus protease, 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 fluorophore TAMRA (carboxytetramethylrhodamine) and the compatible quencher Black Hole Quencher 2 (BHQ2), as follows (50 to 30 ): GGG GAG AAA AAC AAA ACA AAA CUA GCA CCG UAA AGC ACG CBHQ2 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 oligonucleotides 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:

Helicase activity

  Fs  Fb  100; % ¼ 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 halfmaximal 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).

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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 reannealing 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

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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 quantification. 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 according 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 independent 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 concentrations (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).

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compounds that target eIF4A3, we measured eIF4A3 RNA helicase activity in the presence of the non-hydrolyzable ATP analog AMPPNP, 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 reported 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], using 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 activity correlated well with those for ATPase activity (Fig. 2D),

suggesting that allosteric eIF4A3 inhibitor compounds show wellcorrelated ATPase and helicase inhibitory activity. 3.3. Compound screening using the quantitative DDX3X RNA helicase assay Fig. 3A schematically illustrates the RNA helicase assay developed 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 significantly 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% homology. 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).

Please cite this article as: S. Nakao et al., Identification of a selective DDX3X inhibitor with newly developed quantitative high-throughput RNA helicase assays, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.094

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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 nonspecifically. 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 critical for the identification of helicase inhibitors. The difference between allosteric inhibition and ATPase inhibition for eIF4A3 could

Please cite this article as: S. Nakao et al., Identification of a selective DDX3X inhibitor with newly developed quantitative high-throughput RNA helicase assays, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.094

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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; however, 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. Highthroughput 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 significant 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]. Tumors 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 helicase inhibitor C1 may be a valuable tool for gaining a better understanding 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 publish, or preparation of the manuscript. Author contributions SN, MN, and SAr conceived and designed the research and

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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 discussions. 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. References [1] E. Jankowsky, RNA helicases at work: binding and rearranging, Trends Biochem. Sci. 36 (2011) 19e29. [2] P. Linder, E. Jankowsky, From unwinding to clamping - the DEAD box RNA helicase family, Nat. Rev. Mol. Cell Biol. 12 (2011) 505e516. [3] J. Zhang, J.L. Manley, Misregulation of pre-mRNA alternative splicing in cancer, Cancer Discov. 3 (2013) 1228e1237. [4] G.M. Bol, F. Vesuna, M. Xie, J. Zeng, K. Aziz, N. Gandhi, A. Levine, A. Irving, D. Korz, S. Tantravedi, M.R. Heerma van Voss, K. Gabrielson, E.A. Bordt, B.M. Polster, L. Cope, P. van der Groep, A. Kondaskar, M.A. Rudek, R.S. Hosmane, E. van der Wall, P.J. van Diest, P.T. Tran, V. Raman, Targeting DDX3 with a small molecule inhibitor for lung cancer therapy, EMBO Mol. Med. 7 (2015) 648e669. [5] B. Phung, M. Ciesla, A. Sanna, N. Guzzi, G. Beneventi, P. Cao Thi Ngoc, M. Lauss, R. Cabrita, E. Cordero, A. Bosch, F. Rosengren, J. Hakkinen, K. Griewank, A. Paschen, K. Harbst, H. Olsson, C. Ingvar, A. Carneiro, H. Tsao, D. Schadendorf, K. Pietras, C. Bellodi, G. Jonsson, The X-linked DDX3X RNA helicase dictates translation reprogramming and metastasis in melanoma, Cell Rep. 27 (2019) 3573e3586, e3577. [6] M. Iwatani-Yoshihara, M. Ito, Y. Ishibashi, H. Oki, T. Tanaka, D. Morishita, T. Ito, H. Kimura, Y. Imaeda, S. Aparicio, A. Nakanishi, T. Kawamoto, Discovery and characterization of a eukaryotic initiation factor 4A-3-selective inhibitor that suppresses nonsense-mediated mRNA decay, ACS Chem. Biol. 12 (2017) 1760e1768. [7] H. Le Hir, J. Sauliere, Z. Wang, The exon junction complex as a node of posttranscriptional networks, Nat. Rev. Mol. Cell Biol. 17 (2016) 41e54. [8] T. Kurosaki, M.W. Popp, L.E. Maquat, Quality and quantity control of gene expression by nonsense-mediated mRNA decay, Nat. Rev. Mol. Cell Biol. 20 (2019) 406e420. [9] F. Pastor, D. Kolonias, P.H. Giangrande, E. Gilboa, Induction of tumour immunity by targeted inhibition of nonsense-mediated mRNA decay, Nature 465 (2010) 227e230. [10] M.W. Popp, L.E. Maquat, Attenuation of nonsense-mediated mRNA decay facilitates the response to chemotherapeutics, Nat. Commun. 6 (2015), 6632. [11] R. Soto-Rifo, T. Ohlmann, The role of the DEAD-box RNA helicase DDX3 in mRNA metabolism, Wiley interdisciplinary reviews. RNA. 4 (2013) 369e385.

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Please cite this article as: S. Nakao et al., Identification of a selective DDX3X inhibitor with newly developed quantitative high-throughput RNA helicase assays, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.094