Ligand-Induced Structural Dynamics Drive Allosteric Regulation of Translation Initiation Factor eIF4E

ABSTRACT The eukaryotic initiation factor 4E (eIF4E) governs cap-dependent translation, and its dysregulation contributes both to cancer and neurological disorders, making it an attractive target for therapeutic intervention. Among the few small-molecule inhibitors developed, 4EGI-1 and its analogue i4EG-BiP have shown promise in cellular and preclinical models. These compounds disrupt eIF4E’s interaction with its partner eIF4G while enhancing binding to its negative regulators, the 4E-binding proteins (4E-BPs). Despite their chemical similarity and overlapping functional effects, structural data suggest the two ligands engage different regions of eIF4E, lateral for 4EGI-1 and frontal for i4EG-BiP, raising questions about the basis of their shared activity. Here, we integrate molecular simulations, site-directed mutagenesis, and fluorescence binding assays to elucidate the mechanism of action of both ligands. Funnel metadynamics free-energy calculations reveal that the frontal binding mode is thermodynamically preferred for both compounds. Furthermore, we demonstrate that the conformational rearrangement induced by frontal binding selectively promotes 4E-BP1 over eIF4G association, explaining their common allosteric regulatory effect. These findings reconcile divergent structural observations and highlight how ligand-induced dynamics can be exploited to reprogram eIF4E interactions, offering a framework for next-generation therapeutics targeting dysregulated translation. Competing Interest Statement The authors have declared no competing interest. Footnotes ↵† Contributed to the conception and early development of the project. Some typos have been corrected. Authors, acknowledgments, contributions, and data availability have been updated.