Controlling Tricyclic Peptide Architecture in mRNA Display through Orthogonal Reactivity on Rotationally Flexible Scaffolds

Macrocyclization is a powerful strategy to enhance the conformational control and functional potential of peptide drug candidates. Embedding such architectures within genetically encoded display platforms enables the discovery of peptides predisposed toward bioactive conformations, thereby streamlining downstream development. Here, we integrate tetrafunctional scaffolds into mRNA display to enable the direct discovery of tricyclic peptides through orthogonal reactions with cysteine and azidohomoalanine residues. Scaffold rotational flexibility resolves the topological challenge by yielding a single, well-defined tricyclic architecture. Using this approach, we discover tricyclic peptides against an antibody and the Fzd5 receptor as model targets, with our best hits having low nanomolar affinity and minimal degradation in serum after 24 h, and also demonstrate target engagement in cell culture. This approach thus has the potential to accelerate peptide drug discovery through the direct generation of advanced multicyclic hits.