CLIPS
Pepscan has developed a novel and broadly applicable technology termed CLIPS (Chemical LInkage of Peptides onto Scaffolds) which increases the activity and stability of a peptide by enabling its structural fixation, making use of synthetic scaffolds.
The majority of small peptides (20-30 amino acids) derived from intact proteins lack a well-defined structure in solution. Therefore, a CLIPS can be used to solve this problem by affixing the loose ends of the peptide. In this way, the scaffolded peptide may be able to adopt the same spatial structure as the corresponding sequence in the intact protein. The CLIPS technology involves the cyclization of peptide containing two or three thiol functionalities (in most cases cysteines) and a small organic molecule having the corresponding number of reactive benzyl bromide groups. The reaction can be performed on native cysteines in the peptide sequence, but also on artificially introduced (homo)cysteines at any desired position in the peptide. Hence the structure and dimensions of the CLIPS peptides can be varied at will. Pepscan has developed a toolbox of over 70 different types of CLIPS templates, varying mainly in polarity, solubility and thiol-thiol spanning distance.
A) Schematic representation of a CLIPS-reaction; B) Molecular structure of the CLIPS-linkage; C) different CLIPS-based topologies (‘bicycles’ and ‘tricycles’) for mimicry of discontinuous epitopes; D) New ‘double-loop’ CLIPS technology for functional reconstruction of discontinuous protein binding sites
The CLIPS technology is used to fix linear peptides into cyclic structures (‘single-loop’ format), and to bring together different parts of a protein binding site (‘double-loop’, ‘triple-loop’, etc. format). The CLIPS technology is highly versatile, and unique for the ease of application. The cyclization reaction lasts no longer than 30 min, runs at room temperature and does not require any sort of catalysis. Moreover, it can be applied under fully aqueous conditions and neutral pH of 7.5-8.0 and is therefore compatible with highly sensitive biological systems, like bacterial phages as used in PDL-screening. Finally, the reaction can be run at extremely dilute conditions (10-100 μM), which promotes high yields of cyclic products and avoids polymerization
Several other chemical methods are currently available for structural fixation of peptides into stable secondary structures. However, all of these are restricted to single loop peptides, are relatively complex or not compatible with all amino acids.