Cellular molecule through peptide design

Organisms fold and assemble proteins into higher spatial conformational tissues (secondary, tertiary, and quaternary structures)
according to their primary structure, i.
e.
amino acid sequences.
Based on the same principle, short peptides are also capable of taking on spatial secondary structures such as α helixes and β folds
.

These conformations are often driven by weak non-covalent interactions, such as electrostatic interactions, hydrogen bonding, hydrophobic interactions, or van der Waals forces
.
In addition, biparental short peptides are prone to self-assembly
due to weak intermolecular interactions between peptide monomers.
Advances in chemistry provide an environment for programming and control of molecular design: this article will focus on recent advances
in the molecular design of Cell Penetrating Peptides (CPPs).

Cell penetrating peptide is a class of short peptides about 5~30 amino acids long, which can carry peptides, nucleic acids, small molecule drugs and virus particles through the cell membrane into cells
.
It is used as a vehicle to transport carriers into cells
.
Past studies have demonstrated efficacy in treating cancer and inflammatory diseases with cell-penetrating peptides carrying proteins and peptides
.
Based on animal studies, it is thought that it will be possible
for cells to penetrate peptides to carry DNA or SiRNA to treat diseases.

Cell-penetrating peptides can also improve the efficiency of
viral transfection.
Therefore, for this double-edged sword, which can help treat diseases, but also help to cause abuse, and accelerate the spread of the virus, people need to increase research efforts to seek benefits and avoid harm
.
In addition, cell-penetrating peptides can carry fluorescent or radiometric reagents for imaging applications
.
In short, cell penetrating peptides carrying therapeutic genes or drug molecules into cells will have a very broad clinical application prospect.

The Cell Penetrating Peptide Database was established in 2012 by Indraprastha Institue of Information Technology, India, and has been updated to version 2.
0 and has a strong guiding effect
on the molecular design of cell penetrating peptides.
The Cell Penetrating Peptide Database shows 1699 unique cell penetrating peptide sequences, most of which are linear polypeptides (94.
5%)
.
The carrying molecule aspect is mainly used for the transport of fluorophores (54%), and the main cargo molecule of biomedical relevance is nucleic acids (15%)
.
The remaining cargo molecules mainly include proteins (9%), biotin (8%), nanoparticles (7%), and peptide drugs (4%)
.
The main body of cell penetrating peptide research focuses on synthetic polypeptides composed of whole L-amino acids (84.
3%) (54.
8%)
.
In addition, the internalization efficiency and stability of cell-penetrating peptides composed of all D-amino acids, and cell-penetrating peptides composed of L- and D-amino acid mixtures, have also been reported
in the literature.
(Figure 1)

Conventional molecular design of cell-penetrating peptides does not bypass the following three key parameters: guanidine content (or cationic amino acid content), hydrophobicity, and biphilia
.
Arginine-rich cell penetrating peptides (arginine side chains with guanidine groups with a pKa of about 12, and protonated guanidine cations at physiological pH) have undergone in-depth research
in the field of primary structure-efficacy.

Membrane models, membrane extracts, and in vivo studies play a key role in studying the amino acid sequence of cell-penetrating peptides and the contribution of individual amino acid residues (e.
g.
, positive charge), all of which guide the molecular design of cell-penetrating peptides
.
Despite these theoretical guidelines, it is still very difficult
to predict membrane penetration from peptide sequences alone.

In addition to
the sequence.
Multiple chemical and physicochemical properties, such as charge, chirality, aromatics, and hydrophobicity, and their interactions are often important drivers of cell penetrating peptide internalization
.
It can be said that the peptide sequence is the foundation, and the interaction between molecules on this basis determines the efficacy
of cells to penetrate peptides.
To design efficient cell-penetrating peptides, numerous parameters including charge, guanidine group, chirality, hydrophobicity, and aromatics, and their interactions require further study
.

Based on the above considerations, the structure-activity relationship of cell-penetrating peptides and the spatial conformation of biparental polypeptides have been fully studied
.
Charge and biphilia are two major factors
to consider when designing cell-penetrating peptide molecules.
The internalization effect of cell-penetrating peptides varies depending on the overall properties of biphiles and polycations
.
In addition, peptide sequence length and spatial conformation are as important for peptide internalization as
polycation.

Peptide sequences rich in side-chain cationic amino acids are critical
in the field of cell-penetrating peptides.
The discovery of the Tat peptide (Figure 2) was a landmark event
in the field of cell penetrating peptides.
Tat (Transactivating transcriptional activator) peptides are peptide fragments
of residues 48 to 60 in the original transcriptional activator of the human immunodeficiency virus (HIV-1).
The gene delivery system modified by Tat shows enhanced transport function
across multiple biomembranes, such as cell, endosomal and nuclear membranes.

Tat peptide contains six arginine and two lysine residues, both of which are basic amino acids, and the side chains are guanidine and amino groups, all of which are positively charged
due to protonation under physiological pH conditions.
Given the significant guiding significance of Tat, it is not surprising that the simplest cell-penetrating peptide mimics are designed as oligoarginine
.

The correlation between the internalization effect of cell-penetrating peptides and cationic residues has been demonstrated
.
Inspired by Tat (GRKKRRQRRRPQ) and permosin (RQIKIWFQNRRMKWKK), the molecular design of cell-penetrating peptides mainly focuses on small cationic peptides (carrying 5 or so positive charges), such as polyarginine and polylysine peptides
.
The most in-depth cell penetrating peptide models are oligoarginine-based R8 and R9 (8 or 9 continuous arginine residue peptides, Figure 3).

The absorption efficiency of polycationic cell penetrating peptides depends on the sequence length and the location
of arginine residues in the peptide sequence.
Cyclization has been reported to maximize the interaction between arginine and cell membranes, resulting in higher absorption efficiency
.
In addition to the guanidine group of the arginine side chain, the positive charge carried by the peptide can also be introduced through other amino acid side chains, such as lysine (side chain amino) and histidine (side chain imidazolyl).

However, the internalization of polyarginine outperforms polylysine and polyhistidine
.
Therefore, polyarginine is also the most deeply studied cell-penetrating polypeptide
in terms of internalization and introduction mechanisms.
The internalization efficiency of linear polyarginine depends on the sequence length and the number and location
of arginine residues.
Guanidine groups have the ability to form double-tooth hydrogen bonds with membranes, so their peptide internalization should be associated with membrane translocation (Figure 4).

In addition to the cationic moiety, the lipophilic moiety is also important
for cellular uptake.
In this regard, the researchers proposed a mechanism that believes that guanidine-rich cells penetrate peptides, and hydrophobic anti (anion) ions are formed near the main chain, which play a role
in the internalization of cell penetrating peptides.
The counterion effect is first manifested by the interaction of hydrogen bonds between arginine residues and membrane components to form complexes
that can cross the membrane.
In contrast, this strong antiion binding was not observed in lysine-rich polypeptides, a finding that reversely demonstrates the relevance
of guanidine counterions to promote cell-penetrating peptide internalization.
This ability of cell-penetrating peptides to bind to hydrophobic counterions is called the self-activating properties
of these peptides.
Therefore, while paying attention to the positive charge of peptides, the study of the hydrophobicity of cell-penetrating peptides and the contribution of
single fat and aromatic functional groups cannot be ignored.

Hydrophobicity can be achieved
by adding aliphatic or aromatic structures.
Some studies of cell-penetrating peptide mimics have confirmed that aromatic activation is superior to aliphatics
.
Lipidation is achieved by attaching hydrocarbon chains (alkyl groups) of different lengths to the N-terminus of known cell penetrating peptides or other suitable functional groups
.
This alkylation modification enhances the internalization of
cell-penetrating peptides by enhancing hydrophobic interaction with membranes.
In addition to integrating fat chains, the improvement of the hydrophobicity of peptide molecules can also be achieved
by introducing hydrophobic amino acids (e.
g.
, leucine, isoleucine, alanine, valine, phenylalanine).
It has been reported that after replacing methyl groups with more hydrophobic butyls, the activity of one cell-penetrating peptide increased threefold
.
8 Correspondingly, in the same study, when γ-dimethylsilaproline replaced Proline to construct a cell-penetrating peptide with a polyproline PPII helical structure (Figure 5), its internalization efficiency was improved
.
This result may be due to the increased
overall hydrophobicity of the peptide.

A hydrophobic viral peptide gH 625 with cell-penetrating properties (sequence: HGLASTLTRWAHYNALIRAF, Figure 6)
was recently reported.
This cell-penetrating peptide is derived from herpes simplex virus type I and is used to carry liposomes, quantum dots, dendritic macromolecules, disordered proteins, and SPIONS (superparamagnetic iron oxide nanoparticles).

The biphility of its chemical structure is also evident
.

Peptide hydrophobicity can also be enhanced
by the introduction of aromatic amino acid residues (tryptophan, phenylalanine and tyrosine).
According to Wimley and White, aromatic residues have advantageous free energy
during the insertion of peptides into the bilayer interface of cell membranes.
Pyrene, halobenzene, and fullerenes have been shown to stimulate the activation of guanidine cations, allowing arginine-rich cells to penetrate peptides across cell membranes
.

Through a series of experiments, the researchers verified whether aromatic functional groups provide better internalization efficiency
than aliphatic functional groups under the premise of maintaining the same hydrophobicity of polypeptides.
These studies have found that aromatic functional groups are more conducive to the internalization
of cell-penetrating peptides than to fat chains.
Because aromatic polypeptides can interact with aromatic amino acids on membrane proteins π-π, they may help promote or stabilize peptide-membrane interactions and aid internalization
.
8 The concept is extended to include the effects
of various π interactions (π-π, π-cation, π-anion, and π-dipole) on peptide molecule internalization.

In the molecular design of cell-permeable peptides, cationic and hydrophobic characteristics need to be considered
comprehensively.
Penetratin peptides are such examples (Figure 7).

Permothin is obtained from a Drosophila homologous protein
.
The non-electrostatic interaction of permothin with the non-polar portion of the plasma membrane is important for internalization (which also explains the basis of the biparental molecular design, which contains non-polar amino acid residues such as leucine, tryptophan, and phenylalanine).

The number of cationic residues, their spacing on the primary structure, their relative position on the secondary structure, and the integration containing non-peptide elements such as hydrophobic lipid moieties are critical
for the molecular design of cell penetrating peptides.

Another example is PEP-1, which includes 5 tryptophan (hydrophobic amino acids to enhance the hydrophobic effect of the peptide with cell membranes) and a positively charged lysine-rich fragment (KKKRKV, nuclear localization sequence from the nuclear localization sequence on the viral SV-40 T-antigen), and a proline interval to increase the flexibility
of the peptide.

Similarly, the MPG peptide (GALFLGFLGAAGSTMGAWSQPKKKRKV) is designed to contain the same hydrophilic nuclear localization sequence and couple this sequence to a hydrophobic sequence of the HIV-gp-41 viral fragment
.
By integrating cationic amino acid residues, hydrophobic fragments, and biparental structures in the same peptide molecule in different ways, more efficient cell-penetrating peptides
can be constructed and derived.

Faced with various challenges in the realization of cell penetrating peptide function, such as internalization efficiency, endosomal escape efficiency, cycle time, and specificity and selectivity (for cells, tissues, diseases), cell penetrating peptide design has made corresponding progress
.
One example of this is cysteine-rich (CYS)-rich cell-penetrating peptides
.

The design of this molecule is inspired by Crotamine, a toxin found in snake venom, which contains two nuclear localization domains (CROT(2-18) and CROT(27-39)
).
By examining Crot(27-39) (sequence: KMDCRWRWKCCKK), researchers designed a cysteine-rich decapeptide (CRWRWKCCKK) molecule using systematic substitution and/or omission of amino acid residues, as well as in-depth structure-activity studies (Figure 9).

This potential cell-penetrating peptide has enhanced internalization efficiency
.

Cell-penetrating peptides have become a hot area of research
for intracellular therapeutics.
Because of the natural limitations of linear cell penetrating peptides, such as endosomal embedding, toxicity, poor cell specificity, poor stability and strong degradability, as well as imperfect cell penetration, modified cell penetrating cyclic peptides came into being
.
Compared to its straight-chain precursors, cell penetrating cyclic peptides have enhanced cell penetration and improved physicochemical properties, as well as stability
against hydrolytic degradation.
Some cell penetrating cyclic peptides can exhibit endosomal uptake-independent properties, and some cell penetrating cyclic peptides have been reported to have nuclear targeting properties
.

Figure 10 shows some cell penetrating cyclic peptides with monocyclic
, bicyclic and tricyclic structures with arginine and other amino acid residues.

[WR]4 and [WR]5, two cell penetrating cyclic peptide building blocks, are characterized by the presence
of alternating positive charges (arginine) and hydrophobic amino acids (tryptophan) in the sequence.
Monocyclic cell penetrating cyclic peptides containing tryptophan and arginine residues can also be conjugated
to potential therapeutic agents.
For example, monocyclic peptides are coupled with doxorubicin, paclitaxel, and camptothecin, wherein the adduct of doxorubicin-cyclic peptide demonstrates the effect
of internalization.

In addition, several monocyclic peptides containing cysteine and arginine residues also significantly enhance cellular uptake
of impermeable phosphopeptide (F)-Gly-(pTyr)-Glu-Glu-Ile (F-GpYEEI).
Cyclic decapeptides containing tryptophan and histidine effectively increase intracellular delivery
of cell-impermeable phosphopeptides to the anti-HIV drug emtricitabine.
[WR]4-[WR]4-[WR]4 tricyclic peptide contains alternating arginine and tryptophan residues, which improves cellular uptake
of the breast cancer cell line MDA-MB-231 from F-GpYEEI and fluorescently labeled anti-HIV drugs (lamivudine (3TC), emtricitabine (FTC), and siRNA).

In the above cell-penetrating cyclic peptide, the continuous arrangement and combination of tryptophan and arginine residues have derived different types of cell-penetrating cyclic peptides
with different cell transport characteristics.
These data reveal the molecular transport potential of monocyclic , bicyclic and tricyclic cell penetrating peptides and provide a good basis
for designing the next generation of drug delivery peptides.

It should be noted that when discussing the molecular design of cell-penetrating peptides, this article only introduces the effect
of peptide primary structure, that is, amino acid sequence.
Due to space reasons, the activity
of secondary structures for cell-penetrating peptides has not been introduced for the time being.

Although the molecular design of cell-penetrating peptides currently reported in the literature is very detailed, translating them into a clinical setting remains a formidable challenge
.
The application field of cell-penetrating peptides is broad and evolving, and deeper mechanistic exploration has led to increased
molecular design complexity.
This includes the development of stable and multi-domain circular or self-assembling nanostructures
.
In addition, selectivity, targeting, high efficiency and other aspects need to be further developed
.
Many researchers have proposed to improve the stability and activity of cell penetrating peptides by controlled spatial folding, cyclization, dimerization, staple, self-assembly, and even peptide mimetics with different backbones
.

However, due to the development of various complex internalization mechanisms, the heterogeneity of diseases, and the wide variety of cell lines available for in vitro studies, there is still no uniform standard to evaluate internalization efficiency improvements
in one molecular design relative to another.
In addition, the large heterogeneity of cargo molecules means that more parameters
need to be considered in assessing the efficiency and activity of cells in penetrating peptides.
In this regard, standardized harmonization and more in-depth comparative studies are needed to measure the performance
of different types of cell-penetrating peptides.
Although great progress has been made in the design of cell-penetrating peptides, there is undoubtedly still a lot of room
for development in this field.

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