Discovery of Cbl-b Targeting Cyclic Peptide Ligands by Phage Display

Cbl-b is a RING-type E3 ubiquitin ligase and intracellular immune checkpoint that negatively regulates immune-cell activation. However, ligand discovery for Cbl-b remains limited, largely because the RING-type E3 ubiquitin ligase is regulated by shallow and extended protein-protein interaction interfaces that are difficult to target with conventional small molecules. Here, we employed a genetically encoded, macrocyclization-enabled phage-display library incorporating the cysteine-reactive noncanonical amino acid O2beY to identify cyclic peptide ligands for Cbl-b. Affinity selection followed by biolayer interferometry revealed a macrocyclic peptide binder with a dissociation constant of Kd = 798 ± 50 nM. These results highlight the ability of conformationally constrained macrocyclic scaffolds to engage challenging Cbl-b surfaces and provide a peptide-based starting point for developing chemical probes targeting Cbl-b-regulated ubiquitin signaling.