Exploring Phage Peptide Scaffolds: Tyrosinase-Mediated Cyclization for Selective Generation of Bioactive Cyclic Peptides

Phage display is an ideal platform for selecting peptide hits and offers a diverse array of cyclic binders with high affinity. While many recently developed phage display platforms incorporate chemical strategies, the vast majority of these are detrimental to the phage life cycle due to cross-reactivity with the capsid protein. In contrast, enzyme catalysis, which combines high efficiency and biocompatibility, offers a promising approach for phage-based cyclic peptide display. However, enzyme-mediated cyclization approaches remain underexplored. Here, we present a tyrosinase-mediated phage display platform that enables one-step cyclization via <i>o</i>-quinone-cysteine coupling, which is a simple and efficient strategy that does not compromise phage infectivity. Importantly, the catalytic property of tyrosinase is highly selective and spatially constrained, allowing it to bypass native tyrosine residues in the phage and selectively recognize only the engineered tyrosine residues. Using this platform, we constructed a macrocyclic peptide library that facilitated the discovery of macrocyclic peptide inhibitors targeting therapeutically relevant proteins. Notably, the cyclic peptide ACI1 demonstrated potent inhibition of PIP4K2A kinase activity with an IC₅₀ value of 0.93 ± 0.05 μM, while ACP1 effectively inhibited the dephosphorylation activity of PTP1B with an IC₅₀ of 1.06 ± 0.25 μM. The generality and efficiency of this strategy highlight its potential as a valuable tool for the development of bioactive cyclic peptides.