Sortase-Mediated Peptide Ligation and Cyclization for Phage Display

Macrocyclic peptide library has proven to be an invaluable source for discovering high-affinity ligands for disease-associated proteins. Here we propose a sortase-mediated peptide ligation and one-pot cyclization strategy to generate monocyclic and bicyclic peptide libraries. Employing the promiscuity of sortase on the X site of the LPXTG motif, we introduce a low-reactive electrophile to the X residue for side-chain cyclization with the cysteine residue of an N-terminal glycine peptide. We combine the SrtA-mediated peptide cyclization strategy with phage display to construct a large-scale genetically encoded cyclopeptide library. After three-round in vitro selection, we identify a potent bicyclic peptide that binds TEAD4 with a nanomolar KD value (63.9 nM) and effectively blocks the TEAD۰YAP interaction. Importantly, the binding affinity of the bicyclic peptide ligand is 102 times lower than that of the noncyclized analogue. This SrtA-based phage display platform will provide a versatile technique for the discovery of macrocyclic peptide ligands.