Asparaginyl Endopeptidase-Mediated Peptide Ligation and Cyclization for Phage Display

Genetically encoded cyclic peptide libraries are invaluable for peptide drug discovery. Here we report an enzymatic strategy for asparaginyl endopeptidase-mediated peptide ligation and cyclization, and its application in the construction of phage-displayed cyclic peptide libraries. Introduction of a low-reactive chloroacetyl group into the tripeptide recognition sequence of OaAEP1 allows intramolecular cyclization with Cys residues to generate macrocyclic peptides. By optimzing OaAEP1 activation conditions and OaAEP1-catalyzed peptide ligation, we establish an efficient OaAEP1-based enzymatic peptide ligation under acidic conditions. The OaAEP1-based enzymatic ligation is fully compatible with phage display and enables the construction of genetically encoded monocyclic and bicyclic peptide libraries. By using OaAEP1-based phage display, we identify macrocyclic peptide ligands targeting TEAD4 at the nanomolar level. One of the bicyclic peptides binds to TEAD4 with a KD value of 139 nM,16-fold lower than its linear analogue, indicating the contribution of the bicyclic scaffold to its biological activity and demonstrating the utility of the technology platform in the discovery of high-affinity cyclic peptide ligands.