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. In this study, we combined 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. This enabled us to discover a novel binder with low nanomolar affinity and specificity for this particular Ub chain. We further chemically modified the most effective binder to generate next-generation binders. We show that our potent cyclic peptide is cell-permeable and inhibits DNA damage repair, leading to cell cycle arrest and apoptotic cell death. Concordantly, a pulldown experiment with the biotinylated analog of our lead cyclic peptide identified various proteins involved in DNA synthesis and damage repair. Collectively, we established a novel and 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 new opportunities in drug discovery areas associated with Ub signaling.