iPEP: peptides designed and selected for interfering with protein interaction and function

Abstract

Semi-rational design is combined with PCAs (protein-fragment complementation assays) and phage-display screening techniques to generate a range of iPEPs (interfering peptides) that target therapeutically relevant proteins with much higher interaction stability than their native complexes. PCA selection has been improved to impose a competitive and negative design initiative on the library screen, thus simultaneously improving the specificity of assay 'winners'. The folding pathways of designed pairs imply that early events are dominated by hydrophobic collapse and helix formation, whereas later events account for the consolidation of more intricate intermolecular electrostatic interactions.

Thermodynamic analysis of Jun-Fos coiled coil peptide antagonists

“…The antagonists (see Table 1 for sequences and Fig. 2 for example ITC profiles) have previously been shown to be capable of sequestering cJun or cFos using a variety of techniques, including CD thermal denaturation studies [11,12,20], kinetic folding studies [15,21] and native gel analysis [12,15]. We observe that the enthalpic component is strongly favoured for our antagonist–target complexes and that the change in entropy is unfavourable.…”

“…In order to increase the specificity of PCA‐generated PPIs, we incorporated a competitive and negative design initiative (CANDI) into the screen. CANDI is used to ensure that the energy gap between desired and nondesired complexes is maximized and works by including sequences competing for an interaction with either the target and/or the library member in the bacterial selection [12,13]. Library members that bind to the competitor, are promiscuous in their binding selection or cannot compete with the competitor–target complex are subsequently removed from the bacterial pool.…”

Design and Development of Peptides and Peptide Mimetics as Antagonists for Therapeutic Intervention

“…This technique is termed a 'competitive and negative design initiative' (CANDI) and has been used by the author and co-workers in the Jun-Fos system to create a peptide that has been derived to bind to cFos without binding to cJun. This CANDI-PCA technique generated a more specific antagonist relative to conventional PCA; by using cJun directly in the assay as a competitor the energy gap between the nondesired antagonist-cJun competing complex and the desired antagonist-cFos complex was maximized [19,20].…”

Electrostatic contacts in the activator protein‐1 coiled coil enhance stability predominantly by decreasing the unfolding rate

“…Protein–protein interactions capable of sequestering oncogenic Jun–Fos AP‐1 leucine zipper proteins were previously generated using genetic libraries containing partially randomized oligonucleotides [20–22]. These libraries retained the vast majority of wild‐type parent residues, with electrostatic options at e / g positions and hydrophobic options at a positions, known to conform to coiled coil structures (Fig.…”