Discovery of Novel 5-(Piperazine-1-carbonyl)pyridin-2(1H)-one Derivatives as Orally eIF4A3-Selective Inhibitors.

Starting from our previous eIF4A3-selective inhibitor 1a, a novel series of (piperazine-1-carbonyl)pyridin-2(1H)-one derivatives was designed, synthesized, and evaluated for identification of orally bioavailable probe molecules. Compounds 1o and 1q showed improved physicochemical and ADMET profiles, while maintaining potent and subtype-selective eIF4A3 inhibitory potency. In accord with their promising PK profiles and results from initial in vivo PD studies, compounds 1o and 1q showed antitumor efficacy with T/C values of 54% and 29%, respectively, without severe body weight loss. Thus, our novel series of compounds represents promising probe molecules for the in vivo pharmacological study of selective eIF4A3 inhibition.

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 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 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 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 of eIF4A3 in RNA homeostasis.

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 53a exhibiting cellular NMD inhibitory activity. A surface plasmon resonance (SPR) biosensing assay ascertained the direct binding of 53a and its analog 52a to eIF4A3 and revealed that the binding occurs at a non-ATP binding site. Compounds 52a and 53a represent novel molecular probes for further study of eIF4A3, the EJC, and NMD.