Selective macrocyclic peptide modulators of Lys63-linked ubiquitin chains disrupt DNA damage repair.

Developing an effective binder for a specific ubiquitin (Ub) chain is a promising approach for modulating various biological processes with potential applications in drug discovery. Here, we combine the Random Non-standard Peptides Integrated Discovery (RaPID) method and chemical protein synthesis to screen an extended library of macrocyclic peptides against synthetic Lys63-linked Di-Ub to discover a specific binder for this Ub chain. Furthermore, next-generation binders are generated by chemical modifications. We show that our potent cyclic peptide is cell-permeable, and inhibits DNA damage repair, leading to apoptotic cell death. Concordantly, a pulldown experiment with the biotinylated analog of our lead cyclic peptide supports our findings. Collectively, we establish a powerful strategy for selective inhibition of protein-protein interactions associated with Lys63-linked Di-Ub using cyclic peptides. This study offers an advancement in modulating central Ub pathways and provides opportunities in drug discovery areas associated with Ub signaling.