Development of a Cell-Permeable Cyclic Peptidyl Inhibitor against the Keap1-Nrf2 Interaction.

Macrocyclic peptides have proven to be highly effective inhibitors of protein-protein interactions but generally lack cell permeability to access intracellular targets. We show herein that macrocyclic peptides may be rendered highly cell-permeable and biologically active by conjugating them with a cyclic cell-penetrating peptide (CPP). A previously reported cyclic peptidyl inhibitor against the Kelch-like ECH-associated protein 1 (Keap1)-nuclear factor erythroid-2 (Nrf2) interaction ( = 18 nM) was covalently attached to a cyclic CPP through a flexible linker. The resulting bicyclic peptide retained the Keap1-binding activity, resisted proteolytic degradation, readily entered mammalian cells, and activated the transcriptional activity of Nrf2 at nanomolar to low micromolar concentrations in cell culture. The inhibitor provides a useful tool for investigating the biological function of Keap1-Nrf2 and a potential lead for further development into a novel class of anti-inflammatory and anticancer agents. Our data suggest that other membrane-impermeable cyclic peptides may be similarly rendered cell-permeable by conjugation with a cyclic CPP.