Abstract 2802: Discovery of the first selective and cell-active eIF4A3 inhibitors

Background: Eukaryotic initiation factor 4A3 (eIF4A3) is a member of the Asp-Glu-Ala-Asp (DEAD) box RNA helicase family. There are three subtypes of eIF4A, eIF4A1, 2, and 3. eIF4A1 and eIF4A2 are required for translation initiation as their name suggest, however eIF4A3 is functionally distinct and one of the core components of the exon junction complex (EJC). The EJC is known to be involved in a variety of RNA metabolic processes typified by nonsense-mediated RNA decay (NMD), which is the surveillance mechanism that recognizes mRNAs containing premature termination codons to prevent the accumulation of truncated proteins. It is known that eIF4A3 is an ATP-dependent RNA clamp that can serve as a nucleation center to recruit other EJC components, and siRNA-mediated knockdown of eIF4A3 leads to a defect in NMD. In order to investigate the functions of eIF4A3 further and evaluate the therapeutic potential, we conducted a search for molecular probes of eIF4A3. Methods: Using an RNA dependent ATPase assay as a guide, intensive structure activity relationship (SAR) study and chemical optimization of high-throughput screening hit were conducted. Thereafter, NMD inhibitory activity, selectivity, helicase inhibitory activity, and physicochemical properties of optimized compounds were confirmed. Additionally, direct binding of inhibitors to eIF4A3 and their binding mode were analyzed using biophysical methods. Results: Optimized compounds showed high selectivity for eIF4A3 and exhibited significant NMD inhibitory activity in HEK293T cells. The drastic difference in eIF4A3 inhibitory activity between eutomers and distomer revealed the importance of stereochemistry at the 3-position of the piperazine ring for eIF4A3 inhibition. The lack of significant NMD inhibition by distomers suggests that NMD inhibition of eutomers is mediated by eIF4A3 inhibition. A surface plasmon resonance (SPR) biosensing assay and hydrogen/deuterium exchange mass spectrometry (HDX MS) provided strong evidence for the direct binding of the inhibitors to eIF4A3 and implications for their binding site. Conclusion: We discovered the first selective eIF4A3 inhibitors exhibiting cellular NMD inhibitory activity. These novel inhibitors not only represent novel molecular probes for investigation of RNA biology but also could be promising lead compounds for drug discovery research. References: [1] Ito, M.; Tanaka, T.; Cary, D. R.; Iwatani-Y, M.; Kamada, Y.; Kawamoto, T.; Aparicio, S.; Nakanishi, A.; Imaeda, Y. Discovery of novel 1,4-diacylpiperazines as selective and cell-active eIF4A3 inhibitors. J. Med. Chem. 2017, 60, 3335-3351. [2] Iwatani-Y, M.; Ito, M.; Ishibashi, Y.; Oki, H.; Tanaka, T.; Morishita, D.; Ito, T.; Kimura, H.; Imaeda, Y.; Aparicio, S.; Nakanishi, A.; Kawamoto, T., Discovery and characterization of a eukaryotic initiation factor 4A-3-selective inhibitor that suppresses nonsense-mediated mRNA decay. ACS Chem. Biol. 2017, 12, 1760-1768. Citation Format: Masahiro Ito, Misa Iwatani-Yoshihara, Toshio Tanaka, Douglas R. Cary, Yusuke Kamada, Yoshihiro Ishibashi, Hideyuki Oki, Satoshi Sogabe, Shoichi Nakao, Daisuke Morishita, Takashi Ito, Hiromichi Kimura, Tomohiro Kawamoto, Samuel Aparicio, Atsushi Nakanishi, Yasuhiro Imaeda. Discovery of the first selective and cell-active eIF4A3 inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2802.