Generation of membrane-permeable cyclic peptides inhibiting protein–protein interaction

Small, nonpolar cyclic peptides can both bind challenging targets and cross cell membranes, making them attractive for addressing currently undruggable targets such as many protein–protein interactions (PPIs). However, developing such compounds de novo without prior information about lead structures such as natural ligands or fragments remains a notable challenge. Here we show that functional screening of structurally highly diverse cyclic peptide libraries synthesized at nanomole scale allows identification of sub-kDa inhibitors of a PPI. By screening 15,360 fully random cyclic peptides, we were able to identify an inhibitor of the E3 ligase adaptor Keap1 and its substrate Nrf2. Optimization by rapid design–build–test cycles produced a membrane-permeable compound active in live cells. This study demonstrates that large, diverse cyclic peptide libraries can enable the discovery of cell-permeable PPI inhibitors from the ground up, providing a way to harness the powerful modality of small cyclic peptides to address often difficult-to-target intracellular interactions.