Asparaginyl Endopeptidase-Mediated Peptide Ligation and Cyclization for Phage Display

07 February 2024, Version 1
This content is a preprint and has not undergone peer review at the time of posting.


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.


phage display
cyclic peptide library
enzyme-mediated cyclization
bicyclic peptide

Supplementary materials

Supporting Information
Detailed description of the experiments, analysis of the compounds mentioned in the study, and characterisation and activity testing of macrocyclic peptides.


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