Crafting Attractive Non-Covalent Interactions for the Study of β-Hairpins with Long Loops

In this study, we developed a new peptide motif called β-strap (strap = strand + cap) used to fold β-hairpins of varying length. β-Straps are mean to be short sequences (4 to 8 a-amino acids) forming β-sheets using a judicious combination of non-covalent interactions (NCI) to overcome the entropic penalty inherent to long loop closure. Among those, we proved that a couple of CH-π / NH-π interactions from a tryptophan zipper motif were critical to create a stable packing of the structure. To optimize these interactions, we incorporated unnatural tryptophan derivatives having functionalized indole side chains. Finally, the innate ability of the β-strap to bring β-stand in close contact was exploited to promote macrocyclization of long coiled peptides (up to 16 residues).
Then, we studied a more complex β-hairpin loop mimics found at the apex of monoclonal antibodies (mAb) complementary determining region 3 (CDR-H3). Using a set of bioinformatics tools, a search of PDB crystal structures revealed that a large set of mAb crystals possess a β-bulge, located at the edge of CDR-H3 loops. A cluster analysis revealed it has an impressive adaptability towards different H3-loop sizes and conformations. In order to evaluate its function in antibodies, we synthesized several β-hairpin models bearing a prototypical β-bulge. By combining short β-straps and the β-bulge, we were able to design β-hairpin peptides mimic of mAb with a variety of lengths and rigidity while retaining a high degree of folding. Starting from pembrolizumab, the most outstanding blocker of the PD-1/PD-L1 checkpoint currently available in clinic, we scoped ~30 CDR-H3 mAb mimics (H3 loop). As a result, several novel β-hairpin peptide inhibitors of the PD-1/PD-L1 pathway were identified (IC50 <0.3 μM).