A Tag‐Free Platform for Synthesis and Screening of Cyclic Peptide Libraries

In the realm of high‐throughput screening (HTS), macrocyclic peptide libraries traditionally necessitate decoding tags, essential for both library synthesis and identifying hit peptide sequences post‐screening. Our innovation introduces a tag‐free technology platform for synthesizing cyclic peptide libraries in solution and facilitates screening against biological targets to identify peptide binders through unconventional intramolecular CyClick and DeClick chemistries (CCDC) discovered through our research. This combination allows for the synthesis of diverse cyclic peptide libraries, the incorporation of various amino acids, and facile linearization and decoding of cyclic peptide binder sequences. Our sensitivity‐enhancing derivatization method, utilized in tandem with nano LC‐MS/MS, enables the sequencing of peptides even at exceedingly low picomolar concentrations. Employing our technology platform, we have successfully unearthed novel cyclic peptide binders against a monoclonal antibody and the first cyclic peptide binder of HIV capsid protein responsible for viral infections as validated by microscale thermal shift assays (TSA), biolayer interferometry (BLI) and functional assays. The platform PEPTIC for cyclic peptide drug discovery offers a tag‐free workflow for the synthesis, sequencing, and identification of cyclic peptide binders. This innovative approach enables the generation of diverse cyclic peptide libraries, as well as facile linearization and decoding of cyclic peptide binder sequences, and is expected to expedite the discovery of novel cyclic peptide binders of biological targets.