Identification of Short Hydrophobic Cell-Penetrating Peptides for Cytosolic Peptide Delivery by Rational Design

Cationic CPPs (cell-penetrating peptides) have been used largely for intracellular delivery of low-molecular-mass drugs, biomolecules and particles. Most cationic CPPs bind to cell-associated glycosaminoglycans and are internalized by endocytosis, although the detailed mechanisms involved remain controversial. Sequestration and degradation in endocytic vesicles severely limits the efficiency of cytoplasmic and/or nuclear delivery of CPP-conjugated material. Re-routing the splicing machinery by using steric-block ON (oligonucleotide) analogues, such as PNAs (peptide nucleic acids) or PMOs (phosphorodiamidate morpholino oligomers), has consequently been inefficient when ONs are conjugated with standard CPPs such as Tat (transactivator of transcription), R9 (nona-arginine), K8 (octalysine) or penetratin in the absence of endosomolytic agents. New arginine-rich CPPs such as (R-Ahx-R)4 (6-aminohexanoic acid-spaced oligo-arginine) or R6 (hexa-arginine)–penetratin conjugated to PMO or PNA resulted in efficient splicing correction at non-cytotoxic doses in the absence of chloroquine. SAR (structure–activity relationship) analyses are underway to optimize these peptide delivery vectors and to understand their mechanisms of cellular internalization.

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Oligoalanine helical callipers for cell penetration

Chemical Communications ◽

10.1039/c8cc02304b ◽

2018 ◽

Vol 54 (50) ◽

pp. 6919-6922 ◽

Cited By ~ 6

Author(s):

Marta Pazo ◽

Marisa Juanes ◽

Irene Lostalé-Seijo ◽

Javier Montenegro

Keyword(s):

Amino Acids ◽

Rational Design ◽

Chemical Space ◽

Cell Penetrating Peptides ◽

Short Peptides ◽

Cell Penetration ◽

Basic Amino Acids ◽

Cell Penetrating ◽

Natural Amino Acids

Even for short peptides that are enriched in basic amino acids, the large chemical space that can be spanned by combinations of natural amino acids hinders the rational design of cell penetrating peptides.

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The rational design of cell-penetrating peptides for application in delivery systems

Peptides ◽

10.1016/j.peptides.2019.170149 ◽

2019 ◽

Vol 121 ◽

pp. 170149 ◽

Cited By ~ 10

Author(s):

Ziyao Kang ◽

Guihua Ding ◽

Zhao Meng ◽

Qingbin Meng

Keyword(s):

Rational Design ◽

Cell Penetrating Peptides ◽

Delivery Systems ◽

Cell Penetrating

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Considerations on the Rational Design of Covalently Conjugated Cell-Penetrating Peptides (CPPs) for Intracellular Delivery of Proteins: A Guide to CPP Selection Using Glucarpidase as the Model Cargo Molecule

Molecules ◽

10.3390/molecules24234318 ◽

2019 ◽

Vol 24 (23) ◽

pp. 4318 ◽

Cited By ~ 2

Author(s):

Behzadipour ◽

Hemmati

Keyword(s):

Rational Design ◽

Cellular Membrane ◽

Cell Penetrating Peptides ◽

Target Protein ◽

Folding Rate ◽

Carboxypeptidase G2 ◽

C Terminus ◽

Hydrophobic Barrier ◽

Cell Penetrating

Access of proteins to their intracellular targets is limited by a hydrophobic barrier called the cellular membrane. Conjugation with cell-penetrating peptides (CPPs) has been shown to improve protein transduction into the cells. This conjugation can be either covalent or non-covalent, each with its unique pros and cons. The CPP-protein covalent conjugation may result in undesirable structural and functional alterations in the target protein. Therefore, we propose a systematic approach to evaluate different CPPs for covalent conjugations. This guide is presented using the carboxypeptidase G2 (CPG2) enzyme as the target protein. Seventy CPPs —out of 1155— with the highest probability of uptake efficiency were selected. These peptides were then conjugated to the N- or C-terminus of CPG2. Translational efficacy of the conjugates, robustness and thermodynamic properties of the chimera, aggregation possibility, folding rate, backbone flexibility, and aspects of in vivo administration such as protease susceptibility were predicted. The effect of the position of conjugation was evaluated using unpaired t-test (p < 0.05). It was concluded that N-terminal conjugation resulted in higher quality constructs. Seventeen CPP-CPG2/CPG2-CPP constructs were identified as the most promising. Based on this study, the bioinformatics workflow that is presented may be universally applied to any CPP-protein conjugate design.

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Inhibition of IRF5 cellular activity with cell-penetrating peptides that target homodimerization

Science Advances ◽

10.1126/sciadv.aay1057 ◽

2020 ◽

Vol 6 (20) ◽

pp. eaay1057 ◽

Cited By ~ 3

Author(s):

Jaspreet Banga ◽

Dinesh Srinivasan ◽

Chia-Chi Sun ◽

Cherrie D. Thompson ◽

Francesca Milletti ◽

...

Keyword(s):

Rational Design ◽

Nuclear Translocation ◽

Susceptibility Gene ◽

Cell Penetrating Peptides ◽

Interferon Α ◽

Data Support ◽

Cell Penetrating ◽

Proinflammatory Gene ◽

Species Specific ◽

And Function

The transcription factor interferon regulatory factor 5 (IRF5) plays essential roles in pathogen-induced immunity downstream of Toll-, nucleotide-binding oligomerization domain–, and retinoic acid–inducible gene I–like receptors and is an autoimmune susceptibility gene. Normally, inactive in the cytoplasm, upon stimulation, IRF5 undergoes posttranslational modification(s), homodimerization, and nuclear translocation, where dimers mediate proinflammatory gene transcription. Here, we report the rational design of cell-penetrating peptides (CPPs) that disrupt IRF5 homodimerization. Biochemical and imaging analysis shows that IRF5-CPPs are cell permeable, noncytotoxic, and directly bind to endogenous IRF5. IRF5-CPPs were selective and afforded cell type– and species-specific inhibition. In plasmacytoid dendritic cells, inhibition of IRF5-mediated interferon-α production corresponded to a dose-dependent reduction in nuclear phosphorylated IRF5 [p(Ser462)IRF5], with no effect on pIRF5 levels. These data support that IRF5-CPPs function downstream of phosphorylation. Together, data support the utility of IRF5-CPPs as novel tools to probe IRF5 activation and function in disease.

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