Direct Selection of Functional De Novo Macrocycles for Activation of <i>On‐Cellulo</i> Insulin Receptor

ABSTRACT The integration of mRNA display with genetic reprogramming, known as RaPID (Random non‐standard Peptides Integrated Discovery) system, has emerged as a promising platform for nonstandard peptide screening. The RaPID system relies on mRNA display of macrocyclic peptides which employs affinity‐based in vitro selection against an immobilized target protein on magnetic beads, followed by amplification of active species for repetitive rounds, enabling the enrichment of potent binders. Although the binding ability of such macrocyclic peptides is essential for its downstream functional activity, not all binders represent functionally active molecules. Here we have upgraded the RaPID system to directly discover functionally active species by ex tending to cell ular s creening, referred to as RaPID‐ExCells system. Using this system, we have discovered a potent de novo macrocycle (HL4) that specifically agonizes the insulin receptor (IR) on cells. Based on its sequence, we further performed the RaPID‐ExCells for deep mutational scanning of non‐proteinogenic amino acids without compromising the parental agonist activity and elucidated essential roles of residues in HL4. This work underscores the RaPID‐ExCells system as a unique platform for functional screening of macrocycles.