Approximately 75% of all disease-relevant human proteins, including those involved in intracellular protein−protein interactions (PPIs), are undruggable with the current drug modalities (i.e., small molecules and biologics). Macrocyclic peptides provide a potential solution to these undruggable targets because their larger sizes (relative to conventional small molecules) endow them the capability of binding to flat PPI interfaces with antibody-like affinity and specificity. Powerful combinatorial library technologies have been developed to routinely identify cyclic peptides as potent, specific inhibitors against proteins including PPI targets. However, with the exception of a very small set of sequences, the vast majority of cyclic peptides are impermeable to the cell membrane, preventing their application against intracellular targets. This Review examines common structural features that render most cyclic peptides membrane impermeable, as well as the unique features that allow the minority of sequences to enter the cell interior by passive diffusion, endocytosis/endosomal escape, or other mechanisms. We also present the current state of knowledge about the molecular mechanisms of cell penetration, the various strategies for designing cell-permeable, biologically active cyclic peptides against intracellular targets, and the assay methods available to quantify their cell-permeability.

Cell-penetrating peptides (CPPs) are capable of delivering membrane-impermeable cargoes (including small molecules, peptides, proteins, nucleic acids, and nanoparticles) into the cytosol of mammalian cells and have the potential to revolutionize biomedical research and drug discovery. However, the mechanism of action of CPPs has remained poorly understood, especially how they escape from the endosome into the cytosol following endocytic uptake. We show herein that CPPs exit the endosome by inducing budding and collapse of CPP-enriched vesicles from the endosomal membrane. This mechanism provides a theoretical basis for designing CPPs and other delivery vehicles of improved efficiencies.

Stapled peptides recapitulate the binding affinity and specificity of α-helices in proteins, resist proteolytic degradation, and may provide a novel modality against challenging drug targets such as protein–protein interactions. However, most of the stapled peptides have limited cell permeability or are impermeable to the cell membrane. We show herein that stapled peptides can be rendered highly cell-permeable by conjugating a cyclic cell-penetrating peptide to their N-terminus, C-terminus, or stapling unit. Application of this strategy to two previously reported membrane-impermeable peptidyl inhibitors against the MDM2/p53 and β-catenin/TCF interactions resulted in the generation of potent proof-of-concept antiproliferative agents against key therapeutic targets.

A new family of cyclic cell‐penetrating peptides (CPPs) has been discovered; they differ from previously reported cyclic CPPs by containing only a single hydrophobic residue. The optimal CPP structure consists of four arginine residues and a hydrophobic residue with a long alkyl chain (e.g., a decyl group) in a cyclohexapeptide ring. The most active member of this family, CPP 17, has an intrinsic cellular entry efficiency similar to that of cyclic CPP12, the most active CPP reported to date. However, CPP 17 is 2.8 times more active than CPP12 under high serum protein concentrations, presumably because of the lower protein binding. CPP 17 enters the cell primarily by direct translocation at a relatively low concentration (≥5 μm).

Mitochondrial dysfunction is linked to a variety of human illnesses, but selective delivery of therapeutics into the mitochondrion has been challenging. We report herein a family of amphipathic cell-penetrating motifs (CPMs) consisting of four guanidinium groups and one or two aromatic hydrophobic groups (e.g., naphthalene) assembled through a central scaffold (e.g., a benzene ring). The CPMs and CPM-cargo conjugates efficiently enter the interior of cultured mammalian cells and are specifically localized into the mitochondrial matrix, as revealed by high-resolution confocal microscopy. With a membrane-impermeable peptide as cargo, the CPMs exhibited ≥170-fold higher delivery efficiency than previously reported mitochondrial delivery vehicles. Conjugation of a small-molecule inhibitor of heat shock protein 90 to a CPM resulted in accumulation of the inhibitor inside the mitochondrial matrix and greatly enhanced its anticancer activity. The CPMs showed minimal effect on the viability or the mitochondrial membrane potential of mammalian cells.