Cell-Permeable Bicyclic Peptide Inhibitors against Intracellular Proteins

Cyclic peptides have great potential as therapeutic agents and research tools but are generally impermeable to the cell membrane. Fusion of cyclic peptides with a cyclic cell-penetrating peptide produces bicyclic peptides that are cell-permeable and retain the ability to recognize specific intracellular targets. Application of this strategy to protein tyrosine phosphatase 1B and a peptidyl-prolyl cis−trans isomerase (Pin1) isomerase resulted in potent, selective, proteolytically stable, and biologically active inhibitors against the enzymes

Discovery of a Direct Ras Inhibitor by Screening a Combinatorial Library of Cell-Permeable Bicyclic Peptides

Cyclic peptides have great potential as therapeutic agents and research tools. However, their applications against intracellular targets have been limited, because cyclic peptides are generally impermeable to the cell membrane. It was previously shown that fusion of cyclic peptides with a cyclic cell-penetrating peptide resulted in cell-permeable bicyclic peptides that are proteolytically stable and biologically active in cellular assays. In this work, we tested the generality of the bicyclic approach by synthesizing a combinatorial library of 5.7 × 10⁶ bicyclic peptides featuring a degenerate sequence in the first ring and an invariant cell-penetrating peptide in the second ring. Screening of the library against oncoprotein K-Ras G12V followed by hit optimization produced a moderately potent and cell-permeable K-Ras inhibitor, which physically blocks the Ras-effector interactions in vitro, inhibits the signaling events downstream of Ras in cancer cells, and induces apoptosis of the cancer cells. Our approach should be generally applicable to developing cell-permeable bicyclic peptide inhibitors against other intracellular proteins

Structure-Based Optimization of a Peptidyl Inhibitor against Calcineurin-Nuclear Factor of Activated T Cell (NFAT) Interaction

Calcineurin inhibitors such as cyclosporine A and FK506 are effective immunosuppressants but produce severe side effects. Rational modification of a previously reported peptide inhibitor, GPHPVIVITGPHEE (<i>K</i>_D ∼ 500 nM), by replacing the two valine residues with <i>tert</i>-leucine and the C-terminal proline with a <i>cis</i>-proline analogue, gave an improved inhibitor ZIZIT-<i>cis</i>Pro, which binds to calcineurin with a <i>K</i>_D value of 2.6 nM and is more resistant to proteolysis

Efficient Delivery of Cyclic Peptides into Mammalian Cells with Short Sequence Motifs

Cyclic peptides hold great potential as therapeutic agents and research tools, but their broad application has been limited by poor membrane permeability. Here, we report a potentially general approach for intracellular delivery of cyclic peptides. Short peptide motifs rich in arginine and hydrophobic residues (e.g., FΦRRRR, where Φ is l-2-naphthylalanine), when embedded into small- to medium-sized cyclic peptides (7–13 amino acids), bound to the plasma membrane of mammalian cultured cells and were subsequently internalized by the cells. Confocal microscopy and a newly developed peptide internalization assay demonstrated that cyclic peptides containing these transporter motifs were translocated into the cytoplasm and nucleus at efficiencies 2–5-fold higher than that of nonaarginine (R₉). Furthermore, incorporation of the FΦRRRR motif into a cyclic peptide containing a phosphocoumaryl aminopropionic acid (pCAP) residue generated a cell permeable, fluorogenic probe for detecting intracellular protein tyrosine phosphatase activities

Early Endosomal Escape of a Cyclic Cell-Penetrating Peptide Allows Effective Cytosolic Cargo Delivery

Cyclic heptapeptide cyclo­(FΦRRRRQ) (cFΦR₄, where Φ is l-2-naphthylalanine) was recently found to be efficiently internalized by mammalian cells. In this study, its mechanism of internalization was investigated by perturbing various endocytic events through the introduction of pharmacologic agents and genetic mutations. The results show that cFΦR₄ binds directly to membrane phospholipids, is internalized into human cancer cells through endocytosis, and escapes from early endosomes into the cytoplasm. Its cargo capacity was examined with a wide variety of molecules, including small-molecule dyes, linear and cyclic peptides of various charged states, and proteins. Depending on the nature of the cargos, they may be delivered by endocyclic (insertion of cargo into the cFΦR₄ ring), exocyclic (attachment of cargo to the Gln side chain), or bicyclic approaches (fusion of cFΦR₄ and cyclic cargo rings). The overall delivery efficiency (i.e., delivery of cargo into the cytoplasm and nucleus) of cFΦR₄ was 4–12-fold higher than those of nonaarginine, HIV Tat-derived peptide, or penetratin. The higher delivery efficiency, coupled with superior serum stability, minimal toxicity, and synthetic accessibility, renders cFΦR₄ a useful transporter for intracellular cargo delivery and a suitable system for investigating the mechanism of endosomal escape