Isothiocyanate-mediated cyclization of phage-displayed peptides enables discovery of macrocyclic binders.

Cyclic peptides exhibit advantages in binding protein targets with high affinity and competency in inhibiting protein-protein interactions. Cyclic peptide phage display with more than a billion variants is an invaluable tool in drug discovery. However, achieving efficient peptide cyclization on phages remains a challenge because of the limited availability of reaction sites, which also restrict scaffold diversity. Here, we report an isothiocyanate-derived cross-linker featuring dual reactive groups: a bromide that covalently attaches to cysteine thiols and a thiocyanogen that selectively forms a thiourea bridge with either the N-terminal amino group or ε-amines of lysine, depending on pH. This strategy enables pH-modulated cyclization. At pH 6.5, head-to-side chain cyclization occurs, and at pH 9.5, side chain-to-side chain ligation is performed. Both processes simultaneously generate thiourea scaffolds. To demonstrate the versatility and biocompatibility of this approach, we constructed cyclic peptide libraries using both cyclization methods and successfully selected binders for several targets, including cyclophilin D, murine double minute 2, and Keap1, with dissociation constants ranging from micromolar to nanomolar. Given the broad pharmacological potential of the thiourea moiety, this phage display library opens previously unidentified chemical space with high scaffold diversity and the integration of a proven pharmacophore for the development of cyclic peptide therapeutics.