Discovery of Novel 1,4-Diacylpiperazines as Selective and Cell-Active eIF4A3 Inhibitors

Eukaryotic initiation factor 4A3 (eIF4A3), a member of the DEAD-box RNA helicase family, is 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). In order to identify molecular probes to investigate the functions and therapeutic relevance of eIF4A3, a search for selective eIF4A3 inhibitors was conducted. Through the chemical optimization of 1,4-diacylpiperazine derivatives identified via high-throughput screening (HTS), we discovered the first reported selective eIF4A3 inhibitor <b>53a</b> exhibiting cellular NMD inhibitory activity. A surface plasmon resonance (SPR) biosensing assay ascertained the direct binding of <b>53a</b> and its analog <b>52a</b> to eIF4A3 and revealed that the binding occurs at a non-ATP binding site. Compounds <b>52a</b> and <b>53a</b> represent novel molecular probes for further study of eIF4A3, the EJC, and NMD

Discovery of Novel 1,4-Diacylpiperazines as Selective and Cell-Active eIF4A3 Inhibitors

Eukaryotic initiation factor 4A3 (eIF4A3), a member of the DEAD-box RNA helicase family, is 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). In order to identify molecular probes to investigate the functions and therapeutic relevance of eIF4A3, a search for selective eIF4A3 inhibitors was conducted. Through the chemical optimization of 1,4-diacylpiperazine derivatives identified via high-throughput screening (HTS), we discovered the first reported selective eIF4A3 inhibitor <b>53a</b> exhibiting cellular NMD inhibitory activity. A surface plasmon resonance (SPR) biosensing assay ascertained the direct binding of <b>53a</b> and its analog <b>52a</b> to eIF4A3 and revealed that the binding occurs at a non-ATP binding site. Compounds <b>52a</b> and <b>53a</b> represent novel molecular probes for further study of eIF4A3, the EJC, and NMD

Discovery of Novel 1,4-Diacylpiperazines as Selective and Cell-Active eIF4A3 Inhibitors

Eukaryotic initiation factor 4A3 (eIF4A3), a member of the DEAD-box RNA helicase family, is 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). In order to identify molecular probes to investigate the functions and therapeutic relevance of eIF4A3, a search for selective eIF4A3 inhibitors was conducted. Through the chemical optimization of 1,4-diacylpiperazine derivatives identified via high-throughput screening (HTS), we discovered the first reported selective eIF4A3 inhibitor <b>53a</b> exhibiting cellular NMD inhibitory activity. A surface plasmon resonance (SPR) biosensing assay ascertained the direct binding of <b>53a</b> and its analog <b>52a</b> to eIF4A3 and revealed that the binding occurs at a non-ATP binding site. Compounds <b>52a</b> and <b>53a</b> represent novel molecular probes for further study of eIF4A3, the EJC, and NMD

Discovery and Characterization of a Eukaryotic Initiation Factor 4A-3-Selective Inhibitor That Suppresses Nonsense-Mediated mRNA Decay

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 <i>via</i> 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 <b>2</b>, which exhibited noncompetitive 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 the deuterium-incorporation pattern of compound <b>2</b> overlapped with that of an allosteric pan-eIF4A inhibitor, hippuristanol, suggesting that compound <b>2</b> 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 of eIF4A3 in RNA homeostasis