Rational Optimization of Conformational Effects Induced By Hydrocarbon Staples in Peptides and their Binding Interfaces

Abstract

eIF4E is frequently over-expressed in different cancers and causes increased translation of oncogenic proteins via deregulated cap-dependent translation. Inhibitors of the eIF4E:eIF4G interactions represent an approach that would normalize cap-dependent translation. Stapled peptides represent an emerging class of therapeutics that can target protein: protein interactions. We present here molecular dynamics simulations for a set of rationally designed stapled peptides in solution and in complex with eIF4E, supported with biophysical and crystallographic data. Clustering of the simulated structures revealed the favoured conformational states of the stapled peptides in their bound or free forms in solution. Identifying these populations has allowed us to design peptides with improved affinities by introducing mutations into the peptide sequence to alter their conformational distributions. These studies emphasise the effects that engineered mutations have on the conformations of free and bound peptides, and illustrate that both states must be considered in efforts to attain high affinity binding.m RNAs are hypothesized to compete with one another for binding to the eIF4F protein complex and in turn their subsequent translation. The eIF4F complex consists of eIF4E, eIF4A and eIF4G proteins. eIF4E initiates cap-dependent translation by binding to the cap structure (m 7 GTP) found at the 59 end of mRNA. eIF4F forms a complex with the 40S ribosomal subunit and eIF3. This complex shuttles along the 59-untranslated region (59-UTR) of the mRNA until it arrives at the AUG initiation codon 1 . The short, unstructured 59-UTRs of most cellular mRNAs enable the eIF4E containing complex to scan efficiently for the translation initiation codon (AUG). In comparison, the lengthy, G 1 C-rich, highly structured 59-UTRs typical of protooncogenic mRNAs (e.g. cyclin D1, VEGF) hinder recognition of the AUG start codon by the initiation complex, leading to mRNAs being poorly translated 2 . eIF4E contributes to malignancy by increasing the translation of mRNAs with structured 59UTRs, either when over-expressed or when the eIF4F complex is not regulated correctly 2-4 . Failure to regulate the eIF4F complex frequently occurs in cancers when the 4E-binding proteins (4E-BPs), which regulate eIF4F activity by displacing eIF4G from eIF4E, are hyperphosphorylated by mTOR 5 .Inhibiting the protein interface between eIF4E and eIF4G is attractive for the design of anti-cancer therapeutics. Peptides derived from eIF4G1 and 4E-BP1 containing the residues responsible for their interactions with eIF4E (YXXXXLW motif, where W signifies any hydrophobic residue), are structural mimics of each other 6 . The tyrosine (Y4) (Figure 1) is engaged in multiple van der Waal contacts with eIF4E and an h-bond between its side chain hydroxyl and the carbonyl backbone of P38 in eIF4E. The leucine (L9) exploits a shallow cavity on the surface of eIF4E and interacts with W73 of eIF4E via a hydrogen bond between its backbone and the indole of the tryptophan. The conserve...

Stapled peptide design: principles and roles of computation

“…One of the stapled peptides generated had two interface residues replaced by a staple and displayed a drastic reduction in cellular activity. Another study observed no improvement in eIF4E binding affinity when two interacting phenylalanine residues in the eIF4EG peptide were replaced by an i, i + 4 hydrocarbon staple [56].…”

“…REMD also proved useful for guiding the design of a set of stapled eIF4G peptides, for which binding affinity was found to correlate with the extent of conformational similarity between the unbound and bound peptides [56]. Besides analysing simulations of the unbound peptide, simulations of the protein-peptide complex can also yield valuable information that helps in stapled peptide design.…”

“…The results also indicated that a new staple position that was untried in the experiments is likely to provide a bigger affinity boost than the current optimal position. Simulations of the protein-peptide complex were also used in conjunction with REMD simulations of the unbound peptides to design stapled eIF4G peptides with improved binding affinities [56]. As mentioned above, MD simulations of the protein-peptide complex were also instrumental in revealing the interaction of the hydrocarbon staple of a stapled p53 peptide with the surface of MDM2 [57], which was subsequently validated by X-ray crystallography [59].…”

“…Several studies have shown that staples that replace important binding residues invariably attenuate the binding affinity of the stapled peptides [55,56]. Staple scanning, which involves the sampling of all possible staple positions along the peptide helix, was carried out on the BIM BH3 sequence [55].…”

Macrocyclic α-Helical Peptide Drug Discovery

“…[19][20][21][22][23][24][27][28][29] and 62 for reviews) for the modulation a plethora of intracellular targets (i.e. Bcl2-family, 19,[22][23][24]43,47,48,58,[67][68][69][70][71]76,77,83,92,93,98 p53:MDM2/X, 42,57,65,66,72,79,81,82,87,89,94,95 MAML:Notch, 49 eIF4E, 158 ERa/ERb, 50 IRS1, 84 HIF-1:p300, 56,90 RAS:SOS, 59 Rab-GTPase, 96 b-catenin, 64,78,80 protein kinase-A, 91 RPA, 97 HIV-1 integrase, …”