Cell Penetrating Peptides

What is the cell penetrating peptides?

Despite the widely held belief that hydrophilic molecules cannot pass through the plasma membrane, the discovery of cell-entering proteins was first reported in the late 80s. The results show that the HIV Trans-Activator of Transcription (Tat) protein may effectively infiltrate cells grown in a lab and boost viral gene expression. It was also discovered that the Drosophilia melanogaster transcription factor Antennapedia homeodomain could access nerve cells and control the process of neural morphogenesis. Both proteins' intriguing spontaneous entrance prompted comprehensive structure-function analyses to determine the minimal amino acid sequence required for absorption. Currently, it can be stated that CPPs are short peptides (often not surpassing 40 residues) that possess the ability to traverse biological membranes universally with little toxicity, through energy-dependent and/or independent pathways, without requiring chiral recognition by particular receptors. The most cell-penetrating peptides (CPPs) are positively charged, however a limited number of anionic or hydrophobic CPPs have also been identified. The involvement of a primary or secondary amphipathic component is suggested, albeit not absolutely necessary for internalization.

The first CPP were identified as a consequence of this process: penetratin, which corresponds to the third helix of the Antennapedia homeodomain, and Tat peptide, which corresponds to the basic domain of the HIV-1 Tat protein. This led to the discovery or rational design of more peptides with similar penetrating capabilities. The capacity to effectively transport cargo across cell membranes is a shared advantage of all CPPs.

Fig.1 Intracellular delivery of CPP-cargo complexes. (Copolovici D M., et al., 2014)

Cell penetrating peptide structure

The capacity of cell-penetrating peptides (CPPs) to penetrate cell membranes is dependent on their structural features. Despite the great variety in sequence and function of CPPs, they frequently exhibit shared structural features that allow them to penetrate membranes. In order to interact with the negatively charged cell membrane, they possess characteristics such as being cationic (positively charged) and amphipathic (having both hydrophilic and hydrophobic areas).

Linear sequences of amino acids make up the bulk of CPP structures, albeit these structures can differ substantially between different types of CPPs. Although CPPs can vary greatly, the positive charge that the peptides have is due to the high concentration of basic amino acids such as arginine and lysine. High concentrations of arginine are characteristic of CPPs, such as the HIV-1 TAT (Trans-Activator of Transcription) peptide. When bound to negatively charged components of cell membranes, such as phospholipids and proteoglycans, the guanidinium group in arginine establishes robust electrostatic connections. CPPs high in lysine likewise use their positive charge to interact with the membrane, but their translocation processes are often slightly different from those of arginine-rich CPPs.

Fig.2 Relationship between the secondary structures of Arg/dAA-based CPP, and their cell-penetrating abilities. (Yamashita, Hiroko, et al., 2016)

CPPs' capacity to interact with the cell membrane's lipid bilayer is affected by the secondary structures they can acquire. The amino acid sequence of amphipathic CPPs is helix-shaped, with one half being polar and the other half being non-polar, making them resistant to water. Because the hydrophobic side can penetrate the lipid bilayer and the hydrophilic side can interface with the water environment, this structure makes it easier to engage with the cell membrane. Amphipathic characteristics can also be displayed by some CPPs that form β-sheet structures. The formation of stable connections with the cell membrane is facilitated by this structural arrangement.

Crucial for their transport across membranes, amphipathic CPPs contain separate hydrophilic and hydrophobic areas. The hydrophobic section interacts with the fatty acyl chains of the lipid bilayer, whereas the hydrophilic section, which is usually made up of basic amino acids like arginine or lysine, interacts with the polar head groups of lipids. This two-pronged affinity makes it easier to enter and exit the cell membrane. Transportan is a hybrid peptide that incorporates both the hydrophobic and hydrophilic domains of mastoparan and galanin, respectively. Its ability to readily cross membranes is due to its amphipathic character.

Positively charged amino acids, such arginine and lysine, are abundant in CPPs. Because of their cationic charge, CPPs are able to interact with phospholipids and proteoglycans, two negatively charged components of cell membranes. To facilitate translocation, the positively charged CPP must first electrostatically contact with the negatively charged cell membrane. One important function of the guanidinium groups found in arginine side chains is to enable membrane bridging by forming bidentate hydrogen bonds with the phosphate groups in lipid bilayers.

Fig.3 Positive charges of CPPs. (Kim G C., et al., 2021)

Cell penetrating peptides classification

This categorization identifies three primary categories of CPPs: 1) peptides originating from proteins, 2) chimeric peptides formed from two natural sequences, and 3) synthetic peptides designed based on structural-activity analyses.

(1) Protein-derived CPPs: The first category contains peptides derived from the N-terminal region of capsid protein (CaP) of a plant-infecting Brome mosaic virus (BMV), such as Tat from the human immunodeficiency virus (HIV), penetratin, VP22 from the herpes simplex virus, and 22-residue peptide from the same plant.

(2) Chimeric peptides make up the second class of CPPs; these peptides inherit two or more patterns from different peptides and are partially generated from proteins or peptides found in nature. Take transportan, a 27-amino-acid peptide that shares a lysine bridge with mastoparan, a peptide produced from the venom of the Vespula lewisii wasp, and 12 functional amino acids from the neuropeptide galanin at its amino terminus. Mastoparan is the source of TP10, a peptide of 21 residues. Wasp venom provides the 14-residue peptide that is fused with the 6-residue sequence of the neuropeptide galanin by means of an additional lysine residue. In order to drive proteins into an intracellular compartment, certain chimeric CPPs, like NLS-derivatives, contain signal sequences that are recognized by acceptor proteins on the membrane of the relevant intracellular organelles.

Synthetic peptides, including MAPs and polyarginine peptides, make up the last category of CPPs. Pep-1, the first peptide with three domains and twenty-one amino acids, was synthesized. A spacer domain, a hydrophilic lysine-rich domain derived from the nuclear localization sequence (NLS) of the Simian virus 40 (SV40) big T antigen, and a hydrophobic domain with many tryptophan residues make up the three components. The third one strengthens the stability and adaptability of the first two.

Fig. 4 Protein-derived, chimeric, and synthetic cell-penetrating peptides (CPPs).
(A) Protein-derived CPPs. (B) Chimeric and synthetic CPPs. (Durzyńska J., et al., 2015)

Cell penetrating peptides mechanism

The majority of CPPs get access to cells by endocytosis, a process by which cells selectively absorb nutrients, ligands, hormones, and other compounds from the outside. Endosomes are vesicular structures that are produced by the endocytic process through the use of chemical energy and are able to budded from the plasma membrane. The vesicles are then brought into the cell's interior. Phagocytosis, macropinocytosis, receptor-mediated endocytosis, and receptor-independent endocytosis are all types of endocytosis that have been documented.

Table.1 Three mechanisms of action of cell-penetrating peptides have been reported.

Fig.5 Different mechanisms of CPPs. (Kim G C., et al., 2021)

Application of cell penetrating peptides

The capacity of cell-penetrating peptides (CPPs) to cross biological membranes and introduce various compounds into cells gives them a wide variety of potential uses. Many other areas can benefit from these, such as molecular biology research tools, gene therapy, diagnostics, and medication delivery.

Fig.6 Recent techniques based on CPPs. (Silva S., et al., 2019)

Cell penetrating peptides design and synthesis

(1) Design: While designing CPPs, it is important to prioritize their ability to traverse the cell membrane, as well as their target selectivity, biocompatibility, and cargo-binding capabilities. Many structural features are shared, including cationic residues, amphipathic structures, and the capacity to conjugate with many cargo types. The following criteria were used to design the peptide: (i) keeping the peptide sequence short so it could still penetrate cells and bind nucleic acids; (ii) including at least six arginine residues, which is the minimum number needed for cell uptake; (iii) adding tryptophan residues so it could interact hydrophobically with cell membranes; (iv) inserting histidine residues at the core so it could escape from endosomes depending on pH; and (v) adding cysteine residues at the ends to make the peptide more stable and facilitate cargo release once inside the cell.

(2) Synthesis: One common method for synthesizing CPPs is solid-phase peptide synthesis (SPPS), which involves adding amino acids in a specific order to create the desired peptide sequence. The efficiency and variety of SPPS have completely changed the game when it comes to peptide synthesis. It's now possible to create complex peptides, even ones with chemical alterations or non-natural amino acids.

Fig.7 A linear cell-penetrating peptide: a rational design. (McErlean E M., et al., 2021)

References

1. Copolovici D M., et al., Cell-penetrating peptides: design, synthesis, and applications, ACS nano, 2014, 8(3): 1972-1994.
2. Durzyńska J., et al., Viral and other cell-penetrating peptides as vectors of therapeutic agents in medicine, Journal of Pharmacology and Experimental Therapeutics, 2015, 354(1): 32-42.
3. Kim G C., et al., Challenge to overcome current limitations of cell-penetrating peptides, Biochimica et Biophysica Acta (BBA)-Proteins and Proteomics, 2021, 1869(4): 140604.
4. Guidotti G., et al., Cell-penetrating peptides: from basic research to clinics, Trends in pharmacological sciences, 2017, 38(4): 406-424.
5. Silva S., et al., Combination of cell-penetrating peptides with nanoparticles for therapeutic application: a review, Biomolecules, 2019, 9(1): 22.
6. McErlean E M., et al., Rational design and characterisation of a linear cell penetrating peptide for non-viral gene delivery, Journal of Controlled Release, 2021, 330: 1288-1299.