A proximity-driven and regioselective peptide bicyclization (PReP-Bicyc) approach for phage display libraries

Peptide macrocyclization is a powerful strategy to enhance the stability, rigidity, binding affinity, and cellular permeability of therapeutic candidates. Peptide bicycles provide superior conformational constraint but remain difficult to generate in phage-displayed libraries because of limited phage-compatible chemistries. Here, we report Proximity-Driven, Regioselective Peptide Bicyclization (PReP-Bicyc), a general, additive-free, phage-compatible strategy for site-selective bicyclization of peptide libraries. PReP-Bicyc uses a rationally designed linker, 4,6-dichloropyrimidine-2-carbonitrile (4,6-DCCPm), that rapidly reacts with an N-terminal cysteine through nitrile-mediated thiazoline formation, followed by proximity-driven intramolecular thiol arylation of two internal cysteines. The resulting bicycles form efficiently under mild conditions while preserving phage viability. Screening a PReP-Bicyc-modified CX4CX4C library against programmed cell death protein 1 (PD-1) identifies sub-micromolar bicyclic ligands (best Kd ≈ 400 nM) that selectively label PD-1-expressing cells and inhibit the PD-1/PD-L1 interaction. PReP-Bicyc provides a robust platform for evolving high-affinity bicyclic ligands and expands the chemical toolkit for macrocyclic therapeutic discovery. Peptide bicycles offer superior conformational constraint but remain challenging to construct within phage-displayed peptide libraries due to the necessity for phage-compatible chemistry. Here, the authors report Proximity-Driven, Regioselective Peptide Bicyclization (PReP-Bicyc), a general, additive-free, and phage-compatible strategy for site-selective bicyclization of phage-displayed peptide libraries.