Rational optimization of conformational effects induced by hydrocarbon staples in peptides and their binding interfaces.

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.

Stapled peptides with improved potency and specificity that activate p53.

By using a phage display derived peptide as an initial template, compounds have been developed that are highly specific against Mdm2/Mdm4. These compounds exhibit greater potency in p53 activation and protein-protein interaction assays than a compound derived from the p53 wild-type sequence. Unlike Nutlin, a small molecule inhibitor of Mdm2/Mdm4, the phage derived compounds can arrest cells resistant to p53 induced apoptosis over a wide concentration range without cellular toxicity, suggesting they are highly suitable for cyclotherapy.

Improved eIF4E binding peptides by phage display guided design: plasticity of interacting surfaces yield collective effects.

Eukaryotic initiation factor (eIF)4E is over-expressed in many types of cancer such as breast, head and neck, and lung. A consequence of increased levels of eIF4E is the preferential translation of pro-tumorigenic proteins (e.g. c-Myc and vascular endothelial growth factor) and as a result is regarded as a potential therapeutic target. In this work a novel phage display peptide has been isolated against eIF4E. From the phage sequence two amino acids were delineated which improved binding when substituted into the eIF4G1 sequence. Neither of these substitutions were involved in direct interactions with eIF4E and acted either via optimization of the helical capping motif or restricting the conformational flexibility of the peptide. In contrast, substitutions of the remaining phage derived amino acids into the eIF4G1 sequence disrupted binding of the peptide to eIF4E. Interestingly when some of these disruptive substitutions were combined with key mutations from the phage peptide, they lead to improved affinities. Atomistic computer simulations revealed that the phage and the eIF4G1 derivative peptide sequences differ subtly in their interaction sites on eIF4E. This raises the issue, especially in the context of planar interaction sites such as those exhibited by eIF4E, that given the intricate plasticity of protein surfaces, the construction of structure-activity relationships should account for the possibility of significant movement in the spatial positioning of the peptide binding interface, including significant librational motions of the peptide.

C-terminal substitution of MDM2 interacting peptides modulates binding affinity by distinctive mechanisms.

The complex between the proteins MDM2 and p53 is a promising drug target for cancer therapy. The residues 19-26 of p53 have been biochemically and structurally demonstrated to be a most critical region to maintain the association of MDM2 and p53. Variation of the amino acid sequence in this range obviously alters the binding affinity. Surprisingly, suitable substitutions contiguous to this region of the p53 peptides can yield tightly binding peptides. The peptide variants may differ by a single residue that vary little in their structural conformations and yet are characterized by large differences in their binding affinities. In this study a systematic analysis into the role of single C-terminal mutations of a 12 residue fragment of the p53 transactivation domain (TD) and an equivalent phage optimized peptide (12/1) were undertaken to elucidate their mechanistic and thermodynamic differences in interacting with the N-terminal of MDM2. The experimental results together with atomistically detailed dynamics simulations provide insight into the principles that govern peptide design protocols with regard to protein-protein interactions and peptidomimetic design.