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.