Phage Display of Cyclized CLIPSᵀᴹ Peptides

We have developed a unique technology to discover new highly constrained peptides. Our proprietary CLIPSTM technology generates highly constrained redox-stable peptides with enhanced affinity, selectivity and proteolytic stability suited for diagnostic and therapeutic applications.

Phage display is a technology that enables the de novo discovery of peptides against any target of interest by exploring DNA-encoded peptide libraries. Phages are a type of virus that infect bacterial cells. By mimicking the process of evolution, we can identify the peptide binders for a given target. This powerful technology can screen libraries of billions of different peptides in less than four weeks.

The ability to screen such large libraries, however, does not guarantee the identification of a lead peptide for a protein of interest. This is because the search must also entail the right chemical space, which is known to be highly target-dependent. This is why we focus on monocyclic peptides in the context of a large portfolio of CLIPSTM crosslinking molecules. We can thus probe different parts of the conformational peptide space, since binding to different proteins requires a large variety of complementary shapes on the peptide level.

CLIPSTM Peptide Phage Display Technology

Our monocyclic phage display library encodes for 10 random positions (X) flanked by two cysteines (ACXXXXXXXXXXCG). The phage display library was generated with trinucleotides, ensuring an equal distribution of all 19 natural amino acids (excluding Cys) over all randomized positions and possessing a diversity exceeding 4E+10 variants.

Subset of proprietary CLIPSTM scaffolds

Linking the two cysteines can be accomplished with a subset of proprietary CLIPSTM scaffolds, with over 50 variants.

Structure of the various different T-2 scaffolds and corresponding (anticipated) peptide conformations.

Combining the monocyclic libraries with different types of CLIPSTM scaffolds serves two purposes:

1. Additional contacts

Combining monocyclic libraries with different types of CLIPSTM scaffolds helps us create additional contacts between the CLIPSTM scaffolds and hydrophobic/aromatic/cationic residues on the target’s surface. Crosslinking moieties significantly contribute to binding energies.¹

2. Different conformational shapes

Combining monocyclic libraries with different types of CLIPSTM aids us in exploring different conformational shapes in the peptide structure space. Prof. Heinis’ lab (EPFL, Lausanne, Switzerland) published results showing that combining the same library with different scaffolds can yield unique solutions for recognizing the target protein.² Swapping scaffolds after the selection often abolishes peptide binding. Prof. Suga’s lab (Tokyo, Japan) combines one encoded library but varies the N- terminal amino acid that is used for crosslinking, e.g. N-chloroacetyl-L-Tyr to D-Tyr.³ Such variation usually leads to unique candidates for each selection, where the enantiomer used is essential for target recognition.

1. Glas, A. et al. Constrained Peptides with Target-Adapted Cross-Links as Inhibitors of a Pathogenic Protein–Protein Interaction. Angew. Chem. Int. Ed. 53, 2489–2493 (2014).
2. Chen, S., Bertoldo, D., Angelini, A., Pojer, F. & Heinis, C. Peptide ligands stabilized by small molecules. Angew. Chem. Int. Ed. Engl. 53, 1602–1606 (2014).
3. Kawamura, A. et al. Highly selective inhibition of histone demethylases by de novo macrocyclic peptides. Nature Communications 8, 14773 (2017).