Discovery and Mechanism of Highly Efficient Cyclic Cell-Penetrating Peptides

Abstract

Previous cell-penetrating peptides (CPPs) generally have low cytosolic delivery efficiencies, because of inefficient endosomal escape. In this study, a family of small, amphipathic cyclic peptides was found to be highly efficient CPPs, with cytosolic delivery efficiencies of up to 120% (compared to 2.0% for Tat). These cyclic CPPs bind directly to the plasma membrane phospholipids and enter mammalian cells via endocytosis, followed by efficient release from the endosome. Their total cellular uptake efficiency correlates positively with the binding affinity for the plasma membrane, whereas their endosomal escape efficiency increases with the endosomal membrane-binding affinity. The cyclic CPPs induce membrane curvature on giant unilamellar vesicles and budding of small vesicles, which subsequently collapse into amorphous lipid/peptide aggregates. These data suggest that cyclic CPPs exit the endosome by binding to the endosomal membrane and inducing CPP-enriched lipid domains to bud off as small vesicles. Together with their high proteolytic stability, low cytotoxicity, and oral bioavailability, these cyclic CPPs should provide a powerful system for intracellular delivery of therapeutic agents and chemical probes.

Guanidinium-Functionalized Interpolyelectrolyte Complexes Enabling RNAi in Resistant Insect Pests

“…26 pGPMA guanidinium groups provide moieties similar to arginine-rich cell penetrating peptides (CPPs), which are observed to accumulate in endomembrane vesicles, where they can cross membranes. 27−29 CPPs have also been found to enter cells through nonendocytotic routes. 30 In Sf9 cells, an RNAi-insensitive cell line derived from fall armyworms ( Spodoptera frugiperda ), naked dsRNAs are eliminated in endosomal compartments.…”

Nanoparticle mediated delivery and small molecule triggered activation of proteins in the nucleus

“…The methods often exploit pre-existing cytoplasm to endosome differences such as pH[21], enzyme content[22], access to DNA[23], redox status[24–26] or localization[27] and some utilize the physical separation of the compartments to create exploitable distinctions between the two environments[28,29]. However, despite the advantages of each of these methods, many of these techniques do not provide a real-time readout, give a non-linear output due to amplification, transcription or recombination, give no indication of post-endosome protein functionality, rely on endpoint assays on entire batches of cells, or require complex image analysis.…”

Cell-permeable bicyclic peptidyl inhibitors against T-cell protein tyrosine phosphatase from a combinatorial library

“…Additionally, substitution of Fpa for the negatively charged Asp is expected to improve the cell-permeability of the bicyclic peptide inhibitor. 10 Further modification at the pY-3 position (Table 1, peptides 28–32 ) did not improve the TCPTP/PTP1B selectivity, although substitution of D-Glu for Gln produced the most potent inhibitor of the series (IC 50 = 47 nM against TCPTP for peptide 30 ). Importantly, peptide 25 did not significantly inhibit any of the other PTPs tested, including CD45, PTPRD, SHP-1, SHP-2, and VHR (<20% inhibition at 1.5 μM; Fig.…”

“…The 12 CPP sequences consisted of different combinations of two or three aromatic hydrophobic residues (e.g., Phe and Nal) and three or four L- or D-arginine residues (Table S1). 10 The bicyclic peptide library has a theoretical diversity of 3.98 × 10 6 and was synthesized on 2 g of TentaGel microbeads (~90 μm; ~2.8 × 10 6 beads/g), as detailed in Supporting Information. Briefly, each library bead was topologically segregated into two different layers, with a unique bicyclic peptide synthesized in the surface layer and a linear peptide of identical sequence prepared in the inner core as an encoding tag.…”

“…In addition, alternating stereochemistry in the CPP sequence had previously been shown to improve the cellular uptake efficiency. 10 We also replaced the L-2-aminobutyic acid (Abu) at the pY-1 position (relative to F 2 Pmp, which is designated as position 0) with serine and other amino acids containing hydrophobic side chains (Table 1, peptides 6–12 ) and found that isoleucine gave both the highest potency against TCPTP and the best TCPTP/PTP1B selectivity (~3.4-fold). We therefore kept isoleucine as the pY-1 residue and replaced the Ala residue at the pY+1 position with Abu, Ile, Val, D-Thr, Gln, Asn, norleucine (Nle), Leu, or tert -leucine (Tle) (Table 1, peptides 13–21 ).…”