CLIPS technology
Pepscan has developed a novel and broadly applicable technology termed CLIPS (Chemical LInkage of Peptides onto Scaffolds) which often improves the performance (increase binding activity and/or proteolytic stability) of a peptide by enabling its structural fixation. The technology makes use of synthetic scaffolds.
The majority of small peptides (20-30 amino acids) derived from intact proteins lack a well-defined secondary structure in solution. CLIPS-scaffolds can be used to affix the loose ends of the peptide. The CLIPSed peptide may be able to adopt the same spatial structure as the corresponding sequence in the intact protein. CLIPS technology involves cyclization of linear peptides with two or three (homo)cysteines via reaction with a small synthetic entity carrying reactive benzyl bromides. Cyclization can be performed on native cysteines, but also on artificially introduced (homo)cysteines at any desired position in the sequence. Hence, the structure and dimensions of the CLIPSed peptides can be varied at will. Pepscan has developed a toolbox of over 70 different types of CLIPS scaffolds, varying mainly in polarity, solubility and thiol-thiol spanning distance.
Schematic representation of a CLIPS-reaction (left panel), different CLIPS-based topologies (double and triple loops) for mimicry of discontinuous epitope (right panel)
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’, 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 (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 high-dilution 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, and not compatible with sidechain-unprotected amino acids.