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.

Cyclic Cell‐Penetrating Peptides with Single Hydrophobic Groups

“…In the previous study, however, the smallest ring tested was a cycloheptapeptide, which is necessary to accommodate four arginine residues, two hydrophobic residues, and a glutamine for cargo attachment. We hypothesize that a cyclohexapeptide ring might have the proper balance between conformational rigidity and spatial presentation of the arginine and hydrophobic groups for optimal interaction with the plasma and endosomal membranes and for stabilization of the negative Gaussian curvature at the budding neck during endosomal escape . Larger rings (e.g., 14 ) might be too flexible to bind tightly to the plasma and/or endosomal membranes, whereas smaller rings (e.g., 13 ) might be too rigid, locking the arginine and hydrophobic side chains out of the optimal conformation(s) for membrane binding.…”

“…Similar cellular entry efficiency and intracellular fluorescence distribution were observed when peptide 17 was labeled with positively charged tetramethylrhodamine (TMR) or a negatively charged dye, Alexa488 (Figure S1 in the Supporting Information). The rapid internalization of peptide 17 and the presence of bright fluorescent spots at the cell surface suggest that peptide 17 enters the cell primarily through direct translocation, a well‐established route of cellular entry for some CPPs, usually occurring at high CPP concentrations . The bright fluorescent spots at the cell surface have been described as “nucleation zones” .…”

Engineering Cell-Permeable Proteins through Insertion of Cell-Penetrating Motifs into Surface Loops

“…34 Amphipathic CPPs likely facilitate endosomal escape by stabilizing the budding neck structure, which features simultaneous positive and negative membrane curvatures in orthogonal directions (or negative Gaussian curvature). 41 It is hypothesized that the hydrophobic group(s) may insert into the lipid bilayer to generate positive curvature, while the arginine residues bind to and bring phospholipid head groups together, inducing negative membrane curvature. In addition, the most active cyclic CPPs [e.g., cyclo(Phephe-Nal-Arg-arg-Arg-arg-Gln), 34 where phe is D-phenylalanine, Nal is L-naphthylalanine (Nal), and arg is D-arginine] contain D-as well as L-amino acids at roughly alternating positions.…”

A gold mine for drug discovery: Strategies to develop cyclic peptides into therapies

“…Due to the common problems of linear peptides, like poor in vivo stability and low cell specificity, the clinical development of linear CPPs has always been hampered . Compared with their linear counterparts, cyclic CPPs possess many advantages, such as increased cell permeability, higher resistant to proteolysis, higher affinity to the target receptor, etc. Moreover, cyclic CPPs also exhibit efficient endosomal escape and strong ability of conjugation with therapeutic and imaging agents .…”

“…Although the application had made tremendous progress, the exact mechanism for cell‐penetration of cyclic CPPs still remains poorly defined. There are many hypotheses developed over the past two decades, including transient pores mechanism, membrane fusion mechanism, “A proton sponge” hypothesis, leaky endosomal membrane fusion and local disruption of endosomal complex . It is most likely that different kinds of cyclic CPPs can enter cells through different mechanisms, and some of them may achieve cell‐permeability through multiple pathways.…”