Trojan vectors with clinical and therapeutic potential

What are CPPs?

Cell-penetrating peptides (CPPs), also known as protein transduction domains (PTDs), are typically 5-30 amino acid residues in length. Whether they occur naturally or are synthetic, CPPs are designed to breach cell membranes and deliver bioactive cargo to the intracellular space [1].  Like other vectors (i.e., polyplexes, liposomes, gold nanoparticles, and bioengineered viruses and bacteria), CPPs can translocate nucleic acids, small molecules, synthetic drugs, monoclonal antibodies and proteins into cells [2].  However, what sets them apart from the rest, is their unique chemical backbone and conformation, and the ease with which their side groups can be modified.  These attributes potentially allow greater cell-penetrating ability and diversity of cargo that can be delivered [3].  Cyclization and/or modification of the CPP backbone have further improved the uptake efficiency, metabolic stability and targeted delivery of CPPs and their cargo [4, 5, 6].  Notably, CPPs were the first reported vectors to ferry imaging agents into target cells, a feat that has fuelled studies into their potential use in clinical and therapeutic medicine [2].

The first CPPs to be discovered

The first naturally-occurring CPP to be identified and characterized was the 12 amino acid, Transactivator of Transcription (Tat) peptide, encoded in the Human Immunodeficiency Virus Type 1 (HIV-1) genome.  Tat was observed to cross the plasma membrane of cultured cells, and translocate to the nucleus where it trans-activated virus gene expression [7, 8].   Later, in 1991, a 16 amino acid peptide, encoded within the third helix of the Antennapedia homeodomain of Drosophila melanogaster, was found to be necessary and sufficient to translocate cargo across cell membranes, using an energy-independent mechanism that did not involve endocytosis [9].   While CPPs continue to be sourced from naturally-occurring proteins and chimeric peptides, their analogs can be synthesized in vitro.  Where there is no natural counterpart, a CPP can be designed in silico, and synthesised and tested in vitro.

Classification of CPPs

CPPs have been classified according to (i) their physicochemical properties, (ii) their origin, or (iii) their ability to target and permeate the nuclear membrane.  The so-called Nuclear Localization Sequences (NLS), are short, cationic CPPs, containing poly-lysine, -arginine, or -proline motifs. NLS enter the nucleus through the nuclear pore, a multimeric complex that contains 50-100 proteins [1].

Table 1 lists some CPPs that have been experimentally validated so far.  An extensive list can be viewed at “CPPsite 2.0 Database of Cell-Penetrating Peptides” (http://crdd.osdd.net/raghava/ cppsite/index.html) [10].  This, and other sources in the peer-reviewed literature, provide detailed information of more than 1,700 experimentally validated CPPs.

How do CPPs penetrate cell membranes?

Broadly speaking, cells internalize CPPs and their cargo by (i) Endocytosis, an energy-dependent mechanism, or (ii) Direct Translocation, an energy-independent mechanism. Which one is used, is determined by a host of factors, such as the CPP sequence, its conformation and net charge, the temperature, the chemical composition and charge of the cell’s lipid membrane, the size and chemical composition of the cargo, and how the cargo is conjugated to the CPP.

 - Endocytosis - Endocytosis of bioactive molecules occurs either by phagocytosis (uptake of large macromolecules) or pinocytosis (ingestion of fluids and solutes). Whereas phagocytosis is restricted to macrophages and leukocytes, two specialized cell types of the immune system, pinocytosis occurs in every cell, and subsumes four different types of uptake mechanisms, i.e., (i) macropinocytosis, (ii) clathrin-dependent, (ii) caveolin-dependent, or (iii) clathrin- and caveolin-independent endocytic pathways [11].

- Direct Translocation - All cells have the potential to internalize CPPs and their cargo by various energy-independent mechanisms, i.e., (i) pore formation [12], (ii) carpet-like internalization [13], (iii) membrane thinning [14], or (iv) inverted micelle formation [15]. In every case, internalization is initiated by membrane interaction, followed by membrane permeation, and ends with the release of the CPP and its cargo into the cytosol (see: http://crdd.osdd.net/raghava/cppsite1/help.php#helppage).

 

TABLE 1 – CPPs and their classification

Basis for Classification

Type of CPP

Name of CPP

Sequence

Refs

PHYSICO-CHEMICAL PROPERTIES

CATIONIC

HIV-1 TAT48-60 >

GRKKRRQRRRPQ

7, 8

Penetratin [pAntp43-58] >

RQIKIWFQNRRMKWKK

16

AMPHIPATHIC

Transportan

GWTLNSAGYLLGKINLKALAALAKKIL

17

kalata B1 (cysteine knot cyclic peptide)

CGETCVGGTCNTPGCTCSWPVCTRNGLPV

18

Pep-1

KETWWETWWTEWSQPKKKRKV

2

HYDROPHOBIC

Pept 1

PLILLRLLRGQF

2

Pept 2

PLIYLRLLRGQF

2

IVV-14

KLWMRWYSPTTRRYG

2

ORIGIN

CHIMERIC

Transportan

GWTLNSAGYLLGKINLKALAALAKKIL

17

Ig(v)

MGLGLHLLVLAAALQGAKKKRKV

2

SYNTHETIC

Amphiphilic model peptide

KLALKLALKALKAALKLA

2

PROTEIN-DERIVED

pVEC

LLIILRRRIRKQAHAHSK

2

HRSV

RRIPNRRPRR

2

HIV-1 TAT48-60 >

GRKKRRQRRRPQ

7, 8

Penetratin   [pAntp43-58] >

RQIKIWFQNRRMKWKK

16

OTHER

NUCLEAR LOCALIZATION SEQUENCES (NLS)

SV40 T-antigen NLS (Monopartite)

PKKKRKV

19

Nucleoplasmin (Bipartite)

KRPAATKKAGQAKKKL

20

NF-Kb

VQRKRQKLMP

1, 21

TFIIE-beta

SKKKKTKV

1, 21

Oct-6

GRKRKKRT

1, 21

HATF-3

ERKKRRRE

1, 21

SDC3

FKKFRKF

1, 21

 

Therapeutic applications of CPPs and current obstacles to their clinical translation

CPPs are among the most efficient and effective vectors of bioactive molecules tested to date, making them attractive candidates for use in clinical and therapeutic biomedicine.  Pre-clinical studies show they are able to cross the blood-brain barrier, intestinal mucosa, nasal mucosa and skin [22]. Elsewhere, CPPs have demonstrated therapeutic benefit in a range of experimental models, including acute cochlear injury [23], labour, multiple sclerosis, cancer, chronic pain, obesity and cardiovascular disease [18]. Pre-clinical experiments have also revealed some real and potential obstacles to their clinical translation. Some of these are listed in Table 2, together with strategies to mitigate the underlying causes.

 TABLE 2 - Potential Obstacles To The Clinical Usefulness Of CPPs And Their Solutions

OBSTACLE

SOLUTION

REFS

 

LACK OF CELL TYPE SPECIFICITY

1. Design dormant CPP that cannot penetrate cell plasma membrane (e.g. fusion peptide; side-chain modification).  2. Activate CPP by external trigger (e.g. Δ pH; Δ temp.; UV exposure; proteolysis).

6, 24, 25, 26, 27

 

 

 

 

 

 

 

 

 

 

 

POOR TRANSDUCIBILITY (Problem with CPP)

Consider physico-chemical properties of CPP:

. Guanidinium content

. Hydrophobicity

. Amphipathicity

. Charge

. Chirality (L- and D- combinations)

. Secondary structure

. Folding capacity in the presence of membranes

. Affinity of CPP for membranes

 

Possible modifications to CPP scaffold:

. Use of cell-penetrating polydisulfides

. Alpha vs beta helices; cyclization via triazole bridge; reversible bicyclization via paired S-S bonds

. N-alkylated proline spacers in-between R residues in Arginine-rich CPPs (R-X-R).

. Diketopiperazine-based oligo peptide.

. Gamma-aminoproline-based hexapeptide

. Octapeptides

. Filamentous CPPs

. Triple helical CPPs

. Supramolecular CPPs based on intermolecular interaction of repeating units

. Tryptophan-rich CPPs that assemble into spherical aggregates

6, 28, 29, 30, 31, 32

 

 

METHOD OF CONJUGATION Vs TRANSDUCTION EFFICIENCY (Problem with CPP/linker/cargo)

1. Covalent conjugation:

. Chemical (disulphide bonds, amine bonds, specific linkers).

. Expression host (E. coli or S. cerevisiae) produces the CPP-cargo conjugate.

2. Reversible, non-covalent coupling:

. Calmodulin plus calmodulin-binding motif.

. EF hand adaptor proteins (calmodulin-like protein 3 (CALML3), troponin).

2. Physical complexation

. Electrostatic and/or hydrophobic interactions between CPP and its cargo, achieved by simple bulk-mixing.

33, 34

 

 

 

ENDOSOMAL ENTRAPMENT OF CPP & CARGO

1. Direct CPP and its cargo to cytosol instead of endosomes:

. Cyclization of CPP

. Arginine-rich CPP plus fusogenic lipids, membrane-disruptive peptides, membrane-disruptive polymers, lysosomotropic agents, photochemical internalization.

. PolyArginine CPP fused to the translocation domain of Pseudomonas aeruginosa exotoxin A.

2. Incorporate subcellular targeting sequences into CPP backbone.

4, 5, 6, 35

POTENTIAL CYTOTOXICITY

 

. Affecting cell and organelle membranes

 

. Resulting from specific interactions between CPP and cell components

1. Measure cell membrane integrity as an indirect indicator of cytotoxicity:

. Trypan Blue exclusion

. MTT assay

. Fluorescein leakage

. 2-deoxyglucose-6-phosphate leakage

 

2. Test whether peptide concentration, cargo molecule or coupling strategy is the likely cause of cytotoxicity.

36, 37

 

SUSCEPTIBILITY TO PROTEOLYTIC DEGRADATION

1. Modify backbone (e.g. adjust length; test different alpha > beta peptide substitutions).

2. Introduce chirality (L- and D- combinations).

3. Cyclization (e.g. via triazole bridge).

4. Reversible bicyclization (paired S-S bonds).

28, 29, 30, 31, 32

UNFAVORABLE PHARMACOKINETICS & UNCLEAR INTERNALIZATION PATHWAYS

1. Incorporate targeting sequences into CPP sequence

2. Synthesise activatable CPPs.

3. Adjust MW.

4. Test different routes of administration (nasal, pulmonary, transdermal delivery).

5

 

 

 

METHOD OF CONJUGATION Vs TRANSDUCTION EFFICIENCY

1. Covalent conjugation:

. Chemical (disulphide bonds, amine bonds, specific linkers)

. Expression host (E. coli or S. cerevisiae) produces the CPP-cargo conjugate

2. Reversible, non-covalent coupling:

. Calmodulin plus calmodulin-binding motif

. EF hand adaptor proteins (calmodulin-like protein 3 (CALML3), troponin)

2. Physical complexation

. Electrostatic and/or hydrophobic interactions between CPP and its cargo, achieved by simple bulk-mixing

33, 34

 Concluding remarks

CPPs, by virtue of their physico-chemical properties and their structural and conformational versatility, offer promise as vehicles for the delivery of bioactive molecules to sub-cellular compartments that hitherto, could not be targeted precisely, efficiently, nor with an acceptably low level of toxicity.  Continued efforts to overcome all obstacles to their use, should pave the way for CPPs to be included in tailored treatments for a range of diseases and disorders.

 Do you require CPPs in your research?

Our experienced team at Genaxxon bioscience can provide technical and practical advice regarding:

-  Selection and purchase of CPPs

-  Our Peptide Synthesis Service > that is tailored to suit your research needs and your budget

- Here you can find more CPPs from Genaxxon:
https://www.genaxxon.com/shop/en/shop-all-products/peptides-proteins/cell-penetrating-peptides/ >

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