Discovery and Characterization of a Eukaryotic Initiation Factor 4A-3

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Discovery and characterization of a eukaryotic initiation factor 4A-3selective inhibitor that suppresses nonsense-mediated mRNA decay Misa Iwatani-Yoshihara, Masahiro Ito, Yoshihiro Ishibashi, Hideyuki Oki, Toshio Tanaka, Daisuke Morishita, Takashi Ito, Hiromichi Kimura, Yasuhiro Imaeda, Samuel A. Aparicio, Atsushi Nakanishi, and Tomohiro Kawamoto ACS Chem. Biol., Just Accepted Manuscript • Publication Date (Web): 25 Apr 2017 Downloaded from http://pubs.acs.org on April 27, 2017

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ACS Chemical Biology

Discovery and characterization of a eukaryotic initiation factor 4A-3-selective inhibitor that suppresses nonsense-mediated mRNA decay Misa Iwatani-Yoshihara1*, Masahiro Ito2, Yoshihiro Ishibashi1, Hideyuki Oki1, Toshio Tanaka2, Daisuke Morishita3, Takashi Ito1, Hiromichi Kimura3, Yasuhiro Imaeda2, Samuel Aparicio4,5, Atsushi Nakanishi6, Tomohiro Kawamoto1 1

Biomolecular Research Laboratories., 2Medicinal Chemistry Research Laboratories.,

3

Oncology Drug Discovery Unit,6Shonan Incubation Laboratories., Takeda Pharmaceutical

Company Ltd., Pharmaceutical Research Division, 26-1, Muraoka-Higashi 2-chome, Fujisawa, Kanagawa, 251-8555, Japan, 4BC Cancer Agency, Department of Molecular Oncology, Vancouver, BC, V5Z 1L3, Canada, 5University of British Columbia, Department of Pathology and Laboratory Medicine, Vancouver, BC, V6T 2B5, Canada *

Corresponding author

Email: [email protected] Abstract Eukaryotic initiation factor 4A-3 (eIF4A3) is an Asp-Glu-Ala-Asp (DEAD) box-family adenosine triphosphate (ATP)-dependent RNA helicase. Subtypes eIF4A1 and eIF4A2 are required for translation initiation, but eIF4A3 participates in the exon junction complex (EJC) and functions in RNA metabolism including nonsense-mediated RNA decay (NMD). No small molecules for NMD inhibition via selective inhibition of eIF4A3 have been discovered. Here we identified allosteric eIF4A3 inhibitors from a high-throughput screening campaign. Chemical optimization of the lead compounds based on ATPase activity yielded Compound 2, which exhibited non-competitive inhibition with ATP or RNA and high selectivity for eIF4A3 over other helicases. The optimized compounds suppressed the helicase activity of eIF4A3 in an ATPase-dependent manner. Hydrogen/deuterium exchange mass spectrometry demonstrated that deuterium-incorporation pattern of Compound 2 overlapped with that of an allosteric pan-eIF4A inhibitor, hippuristanol, suggesting that Compound 2 binds to an allosteric region on eIF4A3. We examined NMD activity using a luciferase-based cellular reporter system and a quantitative real-time polymerase chain reaction-based cellular system to monitor levels of endogenous NMD substrates. NMD suppression by the compounds correlated positively with their ATPase-inhibitory activity. In conclusion, we developed a novel eIF4A3 inhibitor that targets the EJC. The optimized chemical probes represent useful tools for understanding the functions 1

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of eIF4A3 in RNA homeostasis. Key words: eukaryotic initiation factor 4A-3 (eIF4A3), RNA helicase, allosteric, nonsense-mediated mRNA decay

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Introduction Eukaryotic initiation factor 4A (eIF4A) is an adenosine triphosphate (ATP)-dependent RNA helicase of the Asp-Glu-Ala-Asp (DEAD) box family in superfamily (SF) 2.1 Three isoforms of eIF4A exist: eIF4A1, eIF4A2, and eIF4A3. Although eIF4A1 and 2, abundant proteins in many cell types, are required for translation initiation,2 eIF4A3 is mainly localized in the nucleus and functionally distinct.3 eIF4A3 participates in the exon junction complex (EJC), a multiprotein that assembles 20–24 nucleotides upstream of exon-exon boundaries in a sequence-independent manner.4 The EJC is a marker for splice-site positions until its disassembly during translation.5 One of the critical cellular functions of the EJC is an RNA surveillance mechanism termed nonsense-mediated RNA decay (NMD) .6, 7 NMD is a quality control system that degrades mRNAs containing premature termination codons (PTC), thereby preventing the accumulation of dysfunctional RNAs and proteins, such as stalled RNA-protein complex in protein translation and C-terminally truncated proteins. Mammalian NMD occurs only if a PTC resides over 50–55 nucleotides upstream of an exon-exon junction, and it is triggered by a ribosome stalled at the PTC.8 Approximately one-third of alternative spliced mRNAs are candidates for NMD, indicating its essential role in the regulation of mRNA homeostasis.9, 10 The EJC core comprises four components, eIF4A3, MLN51, MAGOH and Y14.11 Crystal-structure analysis revealed two conformations of eIF4A3: open and closed.12 eIF4A3 adopts the open conformation when it is in the apo form or complex with MLN51, and the closed form when it is in a stable EJC with MLN51, MAGOH, Y14, RNA and ATP. During EJC formation or disassembly, the ATP hydrolysis and RNA-binding capabilities of eIF4A3 changed. MLN51 stimulates RNA-dependent ATPase activity of eIF4A3 by increasing its RNA-binding affinity.13 The interaction of MAGOH and Y14 with eIF4A3-MLN51 inhibits the ATPase activity and enhances the RNA-binding affinity of eIF4A3, generating a stable formation of EJC.11 eIF4A3 functions as an RNA clamp by binding to single-stranded RNAs in an ATP-dependent manner, but how its helicase activity affects EJC formation or NMD activity remains unclear. eIF4A3 has multiple cellular functions. It functions as a translation initiation factor for nuclear cap-binding complex-dependent translation by unwinding secondary structures in 5'untranslated region.14 EJC components including eIF4A3 regulate alternative splicing linked with RNA polymerase II elongation rates.15 Genome-wide transcriptome mapping of eIF4A3 coupled to high-throughput sequencing (CLIP-seq) revealed that eIF4A3 localizes in the canonical EJC region, ~24 nucleotides upstream of exon junction and in non-canonical regions within the exons, implying that the EJC has multiple functions.16 The identification of chemical probes for eIF4A3 is essential to clarify the diverse cellular functions of eIF4A and the EJC. 3



Three natural products bind to eIF4A: hippuristanol, pateamine A and silvestrol. Hippuristanol is a selective inhibitor of eIF4A1 and eIF4A2 with less potency toward eIF4A3.17 Nuclear magnetic resonance (NMR) analysis indicates that hippuristanol binds to an allosteric site on eIF4A1.17 Pateamine A stabilizes the interaction between eIF4A and RNA, preventing formation of the eIF4F complex necessary for translation initiation.18 Pateamine A also stimulates the ATPase activity of eIF4A3 and inhibits NMD through stabilization of UPF1 and the EJC complex.19 Silvestrol appears to be similar to pateamine A in inhibitory mechanism of eIF4A1 and eIF4A2.20 However, no selective small-molecule eIF4A3 inhibitors have been identified to date. We report the discovery and characterization of a novel a series of selective eIF4A3 inhibitors. Hydrogen/Deuterium Exchange Mass Spectrometry (HDX-MS) analysis revealed that a representative inhibitor, Compound 2, binds to an allosteric region on eIF4A3. The optimized eIF4A3 inhibitors suppress cellular NMD in correlation with their ATPase-inhibitory activity. Our findings indicate that Compound 2 is a selective and cell-active chemical probe for eIF4A3.

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Results and Discussion Discovery of a small-molecule eIF4A3-selective inhibitor. To identify a small-molecule eIF4A3 inhibitor, we established an RNA-dependent ATPase assay for eIF4A3. The ATPase activity for eIF4A3 is enhanced by MLN51, its cofactor.13 We constructed a high-throughput screening (HTS) system that monitored the RNA-dependent ATPase activity of the eIF4A3– MLN51 complex. We conducted HTS of approximately 500,000 compounds from our internal chemical library. Following a secondary assay to evaluate the compounds’ selectivity for eIF4A3 over eIF4A1, we identified a 1,4-diacylpiperazine derivative with a half-maximal inhibitory concentrations (IC50) value against eIF4A3 in the micro molar range. Chemical optimization of prototypic 1,4-diacylpiperazine derivatives, represented by Compound 1 (IC50=2.2 µM), led to the discovery of Compound 2 (S form, IC50=0.11 µM) (Figure 1A). Compound 2 exhibited ~550-fold stronger activity than its distomer, Compound 3 (R form, IC50=60 µM). We conducted substrate-competition assay to analyze their mechanism of action. The inhibitory potency of Compound 2 was unchanged when the ATP concentration was increased to 10 times the Michaelis constant (Km) or the RNA concentration was increased to 40 times the Km, suggesting that it inhibits eIF4A3 non-competitively with both substrates (Figure 1B). Compound 2 was demonstrated to be highly selective: it exerted no inhibitory activity against the subfamily members eIF4A1 and eIF4A2 or the SF2 helicase family members DHX29 and Brr2, even at 100 µM (Figure 1C). The potency and selectivity of Compound 2 were higher than those of hippuristanol, with IC50 values of 70 µM against eIF4A3 and 3.1 µM against eIF4A1,21 which was consistent with the reported values. 17 Thus, Compound 2 is a highly selective, non-competitive eIF4A3 inhibitor. Inhibition of RNA unwinding activity by eIF4A3 inhibitors. eIF4A3 exerts RNA-unwinding activity in vitro.13 Although eIF4A3 is an ATP-dependent RNA helicase, the correlation between ATPase and RNA-unwinding capabilities remain unclear. As for inhibitors of other DEAD-box family, such as HCV NS3 or DDX3, there is no clear relationship between ATPase and helicase activities. The ATP analogs RTP and RDP inhibit both the ATPase and helicase activities of NS3, whereas paclitaxel inhibits ATPase but not helicase activity, even at high concentrations.22 The quinoline-based chemical QU663 inhibits helicase activity without inhibiting ATPase activity.23 To evaluate the effects of eIF4A3 inhibitors on RNA unwinding, we established a helicase activity based assay to monitor single- or double-stranded fluorescence-labeled RNAs. We observed no unwinding activity by eIF4A3 without ATP, indicating that it is ATP-dependent (Figure 2). We examined the potency of the eIF4A3 inhibitors (Figure 1). The most potent inhibitor, Compound 2 inhibited helicase activity at 3 µM, whereas its enantiomer, Compound 3 did not even at 100 µM (Figure 2). Compound 1 inhibited helicase activity at 10 µM, but it was 5



less potent than Compound 2. Thus, the helicase activity of eIF4A3 correlates with its ATPase activity. Therefore, ATPase activity based HTS is a suitable strategy for the discovery of eIF4A3 inhibitors: an ATPase activity based assay is more feasible and sensitive than a helicase activity based assay. Chemical optimization of eIF4A3 can also be achieved based on ATPase activity. Investigation of the Compound 2-binding site on eIF4A3 using differential HDX-MS. Elucidating the structure of protein–ligand complex is essential to understanding the mechanism of inhibition. Since we could not obtain co-crystal structures of eIF4A3 with the inhibitors, we employed differential HDX-MS, a recently matured technology to probe protein–ligand interactions.24, 25 Firstly, we analyzed interactions between eIF4A3 and the pan-eIF4A inhibitor hippuristanol. Differential HDX showed that most affected regions were in the C-terminal domain (CTD; Figure 3A and supplementary Figure S1). Almost 80% of the affected regions were consistent with the reported data derived from NMR experiments using eIF4A1 with hippuristanol (green bars in Figure 3A, highlighted in blue in supplementary Figures S1 and S2). 17

Then, interactions between eIF4A3 and Compound 2 were also analyzed by HDX-MS. The

perturbations elicited by Compound 2 were mapped on the primary sequence (Figure 3A and supplementary Figure S2) and crystal structure (Figure 3B). The HDX regions were divided into three parts based on the structure of eIF4A3: regions adjacent to the ATP-binding site; regions adjacent to the RNA binding site; and the interface between the CTD of eIF4A3 and MAGOH protein. In regions adjacent to the ATP binding site, Compound 2 caused 15% decrease in HDX in the N terminal ends of β-strands S8 and S13, and 15% decrease in C terminal end of α-helix H13 and the loop connecting β-strand S13 to α-helix H13 (Figure 3A). In regions adjacent to the RNA-binding site, Compound 2 decreased the HDX by 20% in β-strand S10 and by 25% in a segment connecting α-helix H12 to β-strand S11. HDX in the interface with MAGOH was increased by 5% around the C terminal side of α-helix H14 by Compound 2. The deuterium-incorporation pattern of Compound 2 for eIF4A3 overlapped with that of hippuristanol as shown in Figure 3A. The common HDX region includes the C-terminal end of β-strand S13, which corresponds to the residues in eIF4A1 where NOE contacts (

Discovery and Characterization of a Eukaryotic Initiation Factor 4A-3

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