Understanding Cell Penetration of Cyclic Peptides

Abstract

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.

Figure 1.

Publications in the cyclic peptide field by year (left Y-axis) and cumulative total (right Y-axis).

Figure 2.

Examples of cyclic peptides that enter cells by passive diffusion.

Figure 3.

Examples of N^α-methylated cyclic peptides, with N^α-methylated positions highlighted in red.

Figure 4.

Dynamic conformations of CsA when transitioning between high and low ε environments. CsA exists in an open comformation inside the aqueous extra- and intracellular environments, but adopts a closed conformation containing three intramolecular hydrogen bonds upon entering the lipid bilayer. Nitrogen atoms are shown in blue color, oxygen atoms in red, and hydrogen bonds shown as dotted lines.

Figure 5.

Steric occlusion of backbone amides in sanguinamide A and danamide D, represented as solvent-accessible surface colored in tan. Solvent-accessible amide bonds are colored in blue and the occluding side-chain is colored in red.

Figure 6.

Cyclic cell-penetrating peptides. Disulfide bonds in MCoTI-II (15) are shown in yellow, while the grafted arginines in ZF5.3 (16) are highlighted in pink. ZF5.3, although not a cyclic peptide, is included here because it shares the structural constraints and high cellular entry efficiency of cyclic CPPs.

Figure 7.

Time-lapse confocal microscopic images of HeLa cells upon treatment with 3 μM CPP9^FITC (11) for 0 (A), 30 (B), 60 (C), or 120 min (D) at 37 °C. Reproduced from ref 114. Copyright 2019 American Chemical Society.

Figure 8.

Proposed mechanisms of endosomal escape. (A) Proton sponge effect and osmotic lysis of the endosome/lysosome. (B) Membrane fusion mechanism for liposome-based delivery systems. (C) Barrel-stave pore formation. (D) Toroidal pore formation. (E) Membrane destabilization mechanism for polymer-based delivery systems; and (F) Vesicle budding and collapse mechanism.

Figure 9.

Structures of dfTat (17) and non-peptidic CPM3 (18).

Figure 10.

Effect of negative Gaussian curvature at the budding neck on endosomal escape efficiency. (A) Selective binding of CPPs (indicated by small red cricles) to the saddle-shaped membrane surface at the budding neck and the associated negative Gaussian curvature (as shown, positive and negative curvatures in the vertical and horizontal directions, respectively, on the intraluminal leaflet). (B) Energy diagrams for the vesicle budding event in the absence and presence of CPP. (C) Scheme showing the induction of positive membrane curvature by insertion of hydrophobic groups in between phospholipids, whereas hydrogen bonding interactions between a guanidinium group of a CPP and two adjacent lipid molecules generate negative curvature by bringing the lipid head groups together.

Figure 11.

Cellular entry of CPPs by direct translocation. (A) Scheme showing the formation of nucleation zones on the plasma membrane of a cell, followed by direct translocation across the membrane. (B) Confocal microscopic images of Hela cells upon treatment with 25 μM FITC-labeled cyclic CPP12 for 0, 5, and 10 min. CPP12 forms nucleation zones (bright fluorescent spots) on the cell surface prior to direct translocation.

Figure 12.

Examples of cyclic peptides that likely enter cells by active transport.

Figure 13.

Examples of cell-permeable cyclic peptide natural products that have been subjected to analog synthesis, with structural modifications highlighted in red.

Figure 14.

Examples of synthetic, membrane-permeable cyclic peptides by the formation of intramolecular hydrogen bonds (highlighted in red).

Figure 15.

Examples of synthetic, membrane-permeable cyclic peptidomimetics.

Figure 16.

Different cargo delivery modes of cyclic CPPs. (A) Cargo conjugation to a cyclic CPP using the exocyclic strategy. (B) Incorporation of a cell-penetrating motif into a macrocycle for endocyclic delivery. (C) Incorproation of a second transport ring using the bicyclic delivery strategy. (D) Reversible cyclization of CPP-cargo fusion peptides into mono- or bicyclic peptides via disulfide bonds.

Figure 17.

Examples of exocyclic CPP-cargo conjugates.

Figure 18.

Examples of endocyclic CPP-cargo conjugates.

Figure 19.

Examples of bicyclic CPP-cargo conjugates.

Figure 20.

Reversibly cyclized bicyclic CPP-cargo conjugates.

Figure 21.

Examples of stapled peptides.

Figure 22.

Overview of pathways utilized by cyclic CPPs to enter the cell. Left, some hydrophobic cyclic peptides have demonstrated ability to cross the plasma membrane by passive diffusion. Middle, other peptides can directly cross the plasma membrane by yet poorly defined mechanisms. A number of putative mechanisms including pore formation, carpet model, and inverted micelle have been put forward in the literature. Right, cyclic peptides are brough into the early endosome by various endocytic and pinocytic mechanisms and some of them efficiently escape the endosomal/lysosomal pathway into the cytosol.