In vitro selection of multiple libraries created by genetic code reprogramming to discover macrocyclic peptides that antagonize VEGFR2 activity in living cells.

We report the in vitro selection of thioether-macrocyclized peptides against vascular endothelial growth factor receptor 2 (VEGFR2) from multiple, highly diverse peptide libraries constructed utilizing genetic code reprogramming. The macrocyclic peptide libraries consisted of combinations of four types of amino acid linkers for cyclization and two types of elongator amino acid compositions, including four backbone-modified non-proteinogenic amino acids. Affinity selection from these libraries, using our recently developed TRAP (Transcription-translation coupled with Association of Puromycin-linker) display, yielded multiple anti-VEGFR2 macrocyclic peptide leads. Further antagonizing activity-based screening of the chemically synthesized lead peptides identified a potent macrocyclic peptide that inhibited VEGF-induced VEGFR2 autophosphorylation, proliferation, and angiogenesis of living vascular endothelial cells. The TRAP display-based selection from multiple, highly diverse peptide libraries followed by activity-based screening of selected peptides is a powerful strategy for discovering biologically active peptides targeted to various biomolecules.