De Novo Discovery of Nonstandard Thioisoindole‐Bridged Bicyclic Peptides Targeting Traf2‐ and NCK‐Interacting Kinase

ABSTRACT Macrocyclization strategies that generate conformationally constrained peptide scaffolds within nucleotide‐encoded library screening platforms have significantly advanced the discovery of de novo bioactive peptides. We previously reported ribosomal synthesis of topologically defined thioisoindole‐bridged bicyclic (TiB) peptides, but complete conversion required overnight incubation. Here, we reengineer the key ribosomally incorporated substrate to accelerate TiB formation to full conversion within 2 h. This was followed by a streptavidin‐based pulldown step to efficiently remove linear species and purify the desired TiB peptides. The optimized chemistry was incorporated into the RaPID (Random nonstandard peptides integrated discovery) system to enable de novo identification of TiB peptide ligands. As a proof of concept, two complementary TiB libraries were screened against Traf2‐ and NCK‐interacting kinase (TNIK). Of the five TiB candidates identified, four exhibited nanomolar affinity for TNIK (best K D = 12.3 nM), and the most potent ligand, TK7, inhibited TNIK kinase activity with an IC 50 of 60 nM. X‐ray crystallography revealed that TK7 adopts a unique binding mode on the TNIK surface. Collectively, these findings establish a practical platform for TiB peptide discovery, highlighting their potential in early‐stage peptide drug development.