Modulating the eIF4E-eIF4G Protein-Protein Interaction in Human Disease

Abstract

Dysregulation of the mTORC1 pathway has been linked to several human diseases, particularly cancer; but attempts to target mTORC1 directly have been mostly unsuccessful due to observed drug resistance. eIF4E is a downstream effector of mTORC1 signaling and the rate-limiting factor in cap-dependent translation. Cellular eIF4E activity is regulated by the 4E-BPs, which act as gatekeepers of eIF4E by binding and sequestering the protein to prevent cap-dependent translation initiation. The aim of this work is to develop an inhibitor of the eIF4E–eIF4G PPI using eIF4G and 4E-BP1 peptides as models. These peptides are intrinsically disordered and share the same binding site on eIF4E’s surface. We analyzed the linear peptides by CD and SPR and found that their structures in solution and their kinetics of binding are quite different. The 4E-BP peptide is somewhat helical in solution, demonstrates little temperature dependence of its binding constant (KD), and has a slower off-rate (kd) and stronger KD with increasing ionic strength. Conversely, the eIF4G peptide is not helical in solution, has a faster kd with increasing temperature, and a weaker Kd with increasing ionic strength. The ka of eIF4G was faster under all circumstances than the ka of 4E-BP, but the 4E-BP peptide had a much slower kd and therefore a stronger KD. While eIF4G associates much more quickly with eIF4E, its binding motif appears to be less stable. We hypothesize that the differences are due to different binding mechanisms adopted by each peptide, in which eIF4G favors an induced fit binding mode, but 4E-BP likely adopts a combination of induced fit and conformational selection. We next made stapled version of each peptide. As expected, the 4E-BP stapled peptide (HCS 4E-BP1) was more helical than the linear peptide and had an improved binding constant (26 nM to 4 nM). However, the eIF4G stapled peptide (HCS eIF4G) was barely helical in solution and lost activity (29 nM to 90 nM). The constraint of the staple appears to be preventing its preferred association mechanism, and its loss in affinity is entirely due to a decreased ka. We hypothesize that this is because HCS eIF4G still favors an induced fit binding mechanism. HCS 4E-BP1 suffered from poor solubility and a tendency to aggregate. This problem was solved by changing the staple type from hydrocarbon to a lactam staple, which significantly improved solubility and behavior while maintaining activity. This new lead peptide is 40% helical in solution and binds eIF4E with a KD of 2 nM. Additionally, the lactam stapled peptide is cell permeable, as demonstrated with CAPA performed by the Kritzer lab. We solved the NMR structure of this peptide in solution and found that it forms a compact structure in phosphate buffer and hypothesize that this structure is critical for cell penetration. Future directions on this project will focus on optimizing this peptide for use as a probe for the eIF4E–eIF4G PPI. Optimizations will be made to improve the chemical and metabolic stability of the peptide. Additionally, we will further investigate the effects of staple length, type, and orientation on the activity and cell penetrability of the peptide. Finally, we will explore options for good models to use the optimized peptide to validate the eIF4E–eIF4G PPI in vivo.