Peptide amphiphiles are composed of hydrophobic alkyl tails and peptide regions designed to self-assemble into cylindrical supramolecular nanofibers in solution. While hydrogen bonds form some β-sheets between short β-strands (2 or 3 residues), others are formed by extended-strands.

Smaller Ions Stabilize β-sheets

The strongly-hydrated ions (F- and Cl-) are more attracted to the positively charged lysine residues on the surface of the peptide nanofiber. When peptide residues form β-sheets, an F- or Cl- ion forms a salt bridge between the side chains of lysine residues from two neighboring peptide amphiphile chains. The salt bridge stabilizes the peptide by bringing the backbones closer, resulting in a transition from random coil to extended β-sheets structures. The smaller ions (F- and Cl-, 50mM NaF and NaCl solutions) tend to stabilize β-sheets slightly better compared to the larger ions (I-, Br-).

So, the self-assembly of peptide amphiphiles into supramolecular nanofibers can be regulated by modifying the salt solution.

Reference: doi.org/10.1021/acs.jpcb.9b05532

Hydrogels with a capacity to absorb and hold water within a porous, swelled structure make it a great candidate as a drug delivery system due to their broad range of physical properties as well as chemical adaptability. For example, a positively charged polypeptide (poly-l-lysine, PLL) coupling to a self-assembling dipeptide (Fmoc-FF) leads to the formation of hydrogels with rheological properties suitable for injection.

Self-Assembling Peptide Hydrogels As a Drug Delivery System

Hydrogenation via self-assembly is a hierarchical process. The hydrogels can be easily prepared via simple mixing and incorporated with various stoichiometric ratios of peptides without any additional synthetic processes. Peptides can form hydrogels by multiple non-covalent interactions. It was found that PTZ-Gly-Phe-Phe-Tyr can form gels at ultra-low concentrations of 0.01 wt.%. The π-π stacking may be another driving forces in the self-assembly to form the final hydrogel structure. The N-cadherin mimic peptide (CLRAHAVDIN) and TGF-β1 mimic peptide (CESPLKRQ) synthesized by LifeTein were used to form an injectable 3D hydrogel. Increased retention of stem cells by using an injectable hydrogel has resulted in successful tissue engineering outcomes.

Another efficient method to facilitate hydrogel formation is based on the electrostatic attraction of oppositely charged peptides. The order of amino acids is of great importance for hydrogenation. Self-assembling beta-hairpin peptides, with high arginine content, exhibited extremely good performance in killing bacteria. The peptide hydrogels also have been demonstrated to be used as wound healing agents and other therapeutic instruments under different conditions. Curcumin could be encapsulated into hairpin hydrogels as an injectable agent for localized delivery.

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Smaller Ions Stabilize β-sheets

Synthetic Scorpion Toxin Peptides for Chronic Pain

LifeTein’s Braftide & Cancer Therapy

Blocking the interaction between Programmed Death Ligand 1 (PD-L1) and its receptor, PD-1, is an effective method of treating many types of cancers.

1. Identifying the PD-L1 binding peptide and mock peptide: Select from a library of peptides for stability and high affinity.
2. Fluorescent target peptide-Cy5 as Marker for Flow Cytometry.
3. Detection of PD-L1 in circulating tumor cells using the Cy5 or Cy7 (infrared) labeled peptides.
4. Biotinylated peptides to detect PD-L1 in sample tissues. The streptavidin-HRP are used.
5. Fluorescent Cy5-labeled peptides detects PD-L1 in sample tissues.

By utilizing a peptide-based approach, it is possible to detect all levels of PD-L1 with high sensitivity and specificity.

Reference:

https://www.nature.com/articles/s41598-017-10946-2

1. Design a biotin-labeled peptide library: overlapping or Alanine scan
2. The peptide library is coated on the avidin-coated 96 well plates.
3. The Peptide library is used to evaluate the binding of each peptide to different cell lines using CyQUANT Cell Proliferation Assays.
4. Flow cytometry: Determine the cellular uptake of selected FITC-labeled peptides.
5. Competitive assay: The cells are incubated with FITC-peptide with or without excess unlabeled peptide (100-fold)
6. Finetune the peptide sequence: structure analysis, cyclization, and D amino acid modification.
7. Find the promising receptor-specific cancer cell binding peptide that can be conjugated directly to a chemotherapeutic drug or to nanoparticles for targeted drug delivery to enhance the efficacy of chemotherapy for cancer treatment.

Reference:

https://www.nature.com/articles/s41598-019-38574-y

Biochemists are excited by the possibilities presented by peptides and proteins as pharmaceuticals because they so often mimic exactly the behavior of a natural ligand – the substance that interacts with the receptor on an enzyme or cell to cause a biological process.

Peptides are used for:
1. Self-Assembling Peptide Epitopes as Novel Platform for Anticancer Vaccination: OVA 250−264 and HPV16 E7 43−57.

2.Incorporation of DSPE-PEG and cRGD-modified DSPE-PEG molecules improves the biocompatibility and cellular uptake of the nanoprodrug platform: Click chemistry conjugation of peptides to PEGs. 

3. Designing Tracers for Imaging from Cyclic Peptides: ATTO, FITC, FAM, Cy3, Cy5, infrared C7. 

4. Co-delivery of tumor antigen and dual toll-like receptor ligands into dendritic cell: Cell Penetrating Peptides, HIV-TAT proteins, R8.

There are many ways to design improved peptides: site-directed mutagenesis, computational design approaches, synthetic libraries, template-assisted methodologies, and mechanism-based strategies.

1. Site-directed mutagenesis:
   Adding, deleting, or replacing one or more amino acid residues are useful approaches to re-engineer a peptide. The alanine or lysine scanning are examples of valuable strategies, as they allow for coverage of all positions within a peptide chain, thus making it possible to analyze the effect of all amino acid side chains on structure and function.
2. De novo design:
   Peptides present both basic and hydrophobic residues in a given sequence. The antimicrobial peptides favor an amphipathic structure. The helix-stabilizing residues such as leucine, alanine, valine, isoleucine, lysine, and arginine, or prototypical destabilizing residues such as proline and glycine are frequently introduced to manipulate the peptide structure. The design can also base on the native bioactive peptides as the template-assisted approach.
3. Synthetic libraries:
   A synthetic combinatorial library is practical to scan for the active binding site. The positional scanning or iterative approaches are costly and time-consuming.
4. Computational methods:
   The antimicrobial databases are widely available. The design can be based on the bioinformatics tools, statistical modeling, SAR studies, genetic algorithms or deep learning. The sequence motifs, structure, net charge, hydrophobicity, amphipathicity, and unnatural modifications are essential guidelines for the peptide design.

There are many methods to modify cysteine, which include alkylation, conjugate addition, oxidation, and reduction.

Recently, it was found that it is possible to arylate cysteine in the context of bioconjugation using sufficiently activated reagents. The cysteine perfluoroarylation can be used for mild functionalization of cysteine thiolate moieties.

This is a new and mild synthetic platform for cysteine arylation. The developed method operates at room temperature in polar organic solvents and shows excellent selectivity and functional group tolerance. As a result, one can utilize this approach towards stapling unprotected peptides, thereby positively altering their biological properties. It was demonstrated incorporation of a perfluoroaryl staple within the small tri-helical affibody protein.

Perfluoroarene-based peptide macrocycle is a stapling modification performed on a peptide sequence showed enhancement in binding, cell permeability, and proteolytic stability properties, as compared to the unstapled analog.

It was found that the utility of peptide macrocyclization through perfluoroaryl-cysteine chemistry to improve the ability of peptides to cross the blood−brain barrier. Multiple macrocyclic analogues of the peptide transportan-10 were investigated that displayed increased uptake in two different cell lines and improved proteolytic stability. The abiotic peptide macrocycles can exhibit significantly enhanced penetration of the brain.

Cell-penetrating peptides (CPPs) are a promising class of short peptides with the ability to translocate across the cell membrane.

In general, CPPs can be divided into three classes: Cationic, amphipathic and hydrophobic. Cationic peptides are a class of peptides that contain a high positive charge such as arginine-based peptides R8. Amphipathic CPPs are chimeric peptides such as SV40 NLS PKKRKV. Hydrophobic CPPs are derived from signal peptide sequences and contain only apolar residues such as transportan and stapled peptides.

CPPs have been widely used as a delivery vector due to their high transduction efficiency and capacity for delivering large molecules into a cell. CPPs have the capability to deliver various cargoes without causing any cellular injury. Thus, a wide range of CPP applications are being developed, such as imaging agents and vehicles to deliver therapeutic drugs, small interfering RNA (siRNA), nucleotides, proteins, and peptides.

Application of cell-penetrating peptides:

1. Imaging: CPPs can function as vectors to carry fluorescent particles into cells due to their internalization properties and have become promising tools for delivering imaging agents, contrast agents, and quantum dots in the field of imaging. The advantage of such imaging technology is the ability to visualize and quantify biomarkers or biochemical and cellular processes, detect the stage of diseases, identify the extent of disease, and measure the effect of treatment.
2. Anti-inflammation therapy: Antisense peptide nucleic acids (PNAs) have been shown to specifically inhibit gene expression and growth of E.coli, and are a promising anti-inflammation agent.
3. Tumor therapy: CPP-delivered anticancer therapeutics can increase the cellular membrane permeability of anticancer drugs
   to target tumor cells, expanding the broad application of CPPs in tumor therapy.
4. Nucleic acid and protein delivery: CPPs can facilitate the cellular uptake of large molecules and have been developed as a delivery tool for nucleic acids and proteins. siRNA has been widely used for gene silencing and used to treat diseases such as cancer, infectious diseases, and genetic disorders.
5. Viral delivery: CPPs can also be applied to enhance the efficiency of viral transduction. CPP‑modified Adv as a delivery vector is an attractive tool for transducing cells and gene therapy.