Applications for Synthetic Peptides

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Numerous synthetic peptides are notable commercial or medicinal goods, including the dipeptide sugar replacement aspartame and therapeutically used hormones such as oxytocin, adrenocorticotropic hormone, and calcitonin. In 2008, the peptide therapies market attained a multibillion-dollar valuation. Over 400 peptides have commenced clinical trials so far. In addition to their medicinal value, synthetic peptides have been widely used in various fields.

Fig.1 Diverse uses utilizing peptide nanostructures. (Adler-Abramovich L., et al., 2014)

Synthetic peptides for drug delivery systems

Targeted medication delivery using peptides is on the rise, which improves the specificity of treatments. To improve treatment results while avoiding side effects, recent researches have focused on peptide-functionalized nanoparticles that can carry chemotherapeutics directly to cancer cells. Because of their biocompatibility, precise binding to target proteins and high stability, peptide-based nanostructures show great promise as drug-delivery vehicles.

(1) Stimulus-responsive release

One of the most prominent peptide-based therapies is the use of peptide-drug conjugates or PDCs. These medications have a specific peptide sequence attached to them covalently so that they can have the desired pharmacokinetic properties. Advantages of PDCs include a low drug leakage rate, a high drug loading capacity, and the formation of a precise structure by the covalent attachment of a drug molecule to a peptide fragment. The exact spatial-temporal control is provided by external stimulations through the formation of supramolecular hydrogels, which are a mix of low molecular weight medicines and self-assembled short peptides. Hydrogels may be produced when an enzyme is overexpressed in cancerous tumors, and one of these strategies is enzyme-instructed self-assembly, which provides an efficient method for forming nanofiber networks. Using this approach, a hydrogel precursor comprised of phosphorylated naphthalene, succinic acid, and taxol was produced. The activation of phosphatase in cancer cells initiates the process that transforms the precursor into self-assembled nanofibers and produces a supramolecular hydrogel. Taxol is released into an aqueous media by this hydrogel at a slow rate. It was the first demonstration of the efficacy of the prodrug's hydrogelation and enzyme-instructed self-assembly. Making use of the same brief peptide sequence (phosphorylated Nap-FFKY), a study developed a phosphatase-triggered self-assembled platinum prodrug. This drug outperforms free cisplatin in terms of antitumor growth effects against breast cancer and has less toxicity toward other important organs. DOX-KGFRWR may self-assemble into long nanofibers that stop tumors from spreading and suppress tumor development locally. In a recent development, one group has created a unique PDC called PDC-DOX2. It consists of two molecules of adriamycin (DOX) that are covalently attached to a short peptide called KIGLFRWR. In a neutral water solution, these molecules may form spherical micelles through hydrophobic interactions. Findings indicate that PDC-DOX2 is the best candidate for intravenous injection due to its spherical stability in neutral circumstances. In a neutral environment, its shape doesn't change, but as the pH drops, it starts to collect because of electrostatic interactions. Under normal physiological settings, it releases no free DOX but gradually releases the medication in phosphate-buffered saline (PBS) at a pH of 6.5. The core-shell structure of the nanomedicine HA@PDC-DOX2 is formed by coating the micelles with negatively charged hyaluronic acid (HA), a naturally occurring polysaccharide, because the micelle surface is positively charged. Because of its interaction with the overexpressed receptors in cancer cells, HA may be utilized to control the particle size and stability of HA@PDC-DOX2. This enhances the targeting of PDC-DOX2 micelles.

(2) Targeted and sustained release

A new hot subject is the fact that anticancer therapies function by means of the self-aggregation of non-toxic short peptides within or around cancer cells. The Xu lab created many different types of anti-cancer short peptides that self-assembled. One representative investigation found a hydrogelator's phosphorylate precursor (Nap-FFYp). The overproduction of phosphatases by cancer cells dephosphorylates a precursor, which in turn triggers the hydrogelator to self-assemble, resulting in the selective formation of pericellular hydrogel/nanonets around the cancer cells. Pericellular hydrogel/nanonets inhibit cellular mass exchange, leading to the death of cancer cells. With the exception of highly expressed extracellular phosphatases, the team created and manufactured tiny peptide precursors to serve as carboxylesterase substrates within cells. Intracellular carboxylesterase converted peptide precursors into self-assembled nanofibers. Precursors, when used at the right quantities, enhance cisplatin's action against drug-resistant ovarian cancer cells without damaging the cells themselves. Because of their stability, D-peptides are becoming more popular. Cellular absorption of D-peptides is inefficient due to the rarity of d-amino acid biological usage. Taurine was created artificially by and is a naturally occurring amino acid that is covalently linked to D-peptides. Taurine and D-peptide ester conjugates can enter cells via macropinocytosis and dynamin-dependent endocytosis, where they induce intracellular esterase and aid in intracellular molecular self-assembly. Biomacromolecules, aromatic carbohydrate amphiphiles, cholesterol conjugates, and short peptides are among the non-toxic therapies that can self-aggregate within or surrounding cells to produce cytotoxicity.

Fig.2 Typical applications of synthetic peptides. (Yang S., et al., 2023)

Fig.3 Application of synthetic short peptides in drug delivery. (Yang S., et al., 2023)

Synthetic peptides for diagnostics

Synthetic peptides have two main distinct roles in diagnosis. The primary function was employing peptides as pathogen recognition elements attached to the surfaces of magnetic or fluorescent nanoparticles to facilitate the capture of bacteria, followed by their separation or imaging. The alternative function involves employing peptides as thiol-rich entities to augment the adhesion of immuno-colloidal metallic nanoparticles to the surface of a mesoporous Surface-enhanced Raman scattering (SERS) template. There exists significant potential for further investigation to elucidate additional functions of peptides in nanotools for sepsis diagnostics.

The use of peptides as pathogen-capturing motifs on magnetic nanoparticles has the potential to make these nanoplatforms useful for the easy and controlled detection and separation of bacteria from human samples. One such antibiotic is vancomycin (Van), a glycopeptide that acts as a pathogen recognition molecule by hydrogen bonding with bacterial cell walls.

Superparamagnetic iron oxide nanoparticles (SPIONs) are another form of magnetic nanoparticles that can be peptide-modified to isolate human samples free of germs. SPOINs show great promise as instruments for early illness detection and diagnosis due to their non-toxicity, size controllability, huge surface area-to-volume ratio, and ability to be functionalized with targeted moieties. One example is the work of Friedrich and colleagues, who combined superparamagnetic iron oxide nanoparticles with bacterial cell wall-binding peptides and coated them with 3-aminopropyl triethoxysilane (APTES) for the purpose of separating bacterial infections from bloodstream samples. Salivary glycoprotein GP-340 is known to interact with components of bacterial cell walls; two peptide sequences, Pep1 (RKQGRVEVLYRASWGTV) and Pep2 (RKQGRVEILYRGSWGTVC) were derived from this protein. After peptide functionalization, the SPION-APTES—which had a hydrodynamic diameter of 166 nm—formed nanoparticle agglomerates with a diameter of 1679 nm. These agglomerates were created using a modified one-step coprecipitation technique. It was discovered that the nanoparticles were compatible with both cells and blood. In order to assess the separation efficiency, healthy volunteers' whole blood samples were spiked with several types of bacteria. These bacteria included Gram-negative (E. coli, S. marcescens, S. enterica, S. enteritidis, and P. aeruginosa) and Gram-positive (S. aureus). With a removal rate of over 60% for S. aureus, E. coli, and S. marcescens, and a separation rate of just 35% for P. aeruginosa, the results were mixed. In addition to its diagnostic function, the system demonstrated a highly effective control of the release of cytokines (TNF-α, IL-6, IL-1β, Il-10, and IFN-γ). Notably, this dual and straightforward theranostic strategy has the potential to expedite the identification and treatment of individuals who are suspected of having sepsis.

Fig.4 Peptides for diagnosing and drug delivery systems. (Gafar M A., et al., 2024)

Fig.5 Utilization of peptides in nanotechnology for the diagnosis and therapy of sepsis. (Gafar M A., et al., 2024)

Synthetic peptides for producing novel biomaterials

There are many fascinating uses for peptides as building blocks in biotechnology. An organized nanostructure may be self-assembled from peptides. Nanotubes and peptide nanofibers are two of the many nanoforms that have potential uses in medicine. Hydrogels may be formed using Fmoc-LD dipeptide in PBS at concentrations as low as 2 mg/mL. Dipeptide hydrogel has never been seen before. These extremely brief peptides are cheap, simple, and made entirely of synthetic materials. These biomaterials derived from peptides promote the proliferation and differentiation of several cell types without causing hemolysis or immune responses. Cell culture scaffolds, bioimaging probes, and 3D bioprinting inks are all areas where they have found useful applications.

Fig.6 Ultrashort peptide applications in bionanotechnology. (Ni M., et al., 2019)

Fig.7 Peptide-based porous materials. (Wang Y., et al., 2023)

Synthetic peptides for protein-protein interactions

Protein–protein interactions (PPIs) are acknowledged as potential therapeutic targets. As a result, interfering peptides (IPs)-synthetic peptides that can disrupt protein-protein interactions (PPIs)- are garnering heightened interest. Due to their physicochemical properties, synthetic IPs are more adept than tiny molecules at disrupting the extensive surfaces involved in protein-protein interactions (PPIs). Advancements in peptide administration, stability, biodelivery, and safety are fostering interest in peptide medication development. The notion of IPs has been substantiated for several PPIs, fostering significant anticipation for their therapeutic efficacy. This document outlines methodologies and techniques pertinent to the discovery of intellectual properties, as well as in silico, physicochemical, and biological strategies for their creation and optimization. Promising in-vivo-validated instances are delineated, and the advantages, limits, and potential of IPs as therapeutic instruments are examined.

The traditional approach to creating peptide inhibitors involves the introduction of customized oligopeptides that are changed according to critical residues at the original protein-protein interaction surfaces (e.g., hot spots) to obstruct protein binding. The creation of peptide inhibitors has made significant advancements in recent years. Ya-Qiu Long and associates developed a variety of small peptides (7-17 residues) containing essential amino acid residues vital for integrase (IN) catalytic functions or viral replication. They introduced an innovative "sequence walk" technique encompassing all 288 residues of IN to identify the "hot spots" of protein-protein interactions on IN. Two new peptides, NL-6 and NL-9, were discovered with IC50 values of 2.7 and 56 μM for strand transfer activity. Mingjie Zhang's group has created a powerful and specific GABARAP-selective peptide that targets Atg8-AnkG to suppress autophagy. The potent inhibitory peptide originating from 270/480 kDa ankyrin-G interacts with GABARAP with a dissociation constant (Kd) of approximately 2.6 nM. Despite the successful development of several peptide inhibitors by certain researchers, the pharmacokinetic characteristics of peptides continue to be significantly hindered by critical limitations, including inadequate cellular permeability and pronounced metabolic instability. These limitations significantly constrain their subsequent optimizations and clinical uses.

Fig.8 The effect of an interfering peptide (IP) targeting a PPI. (Bruzzoni-Giovanelli H., et al., 2018)

Fig.9 Peptide and peptidomimetic strategies developed to inhibit PPIs. (Arenas J L., et al., 2019)

Fig.10 Peptide-based PPI inhibitors targeting Bcl-2 Family PPIs. (Wang X., et al., 2021)

Synthetic peptides for Vaccines

Peptide vaccines consist of synthetic peptides that are highly immunogenic and provoke a targeted adaptive immune response. They exist in several forms, such as multivalent long peptide vaccines, multi-peptide vaccinations with cytotoxic T lymphocyte (CTL) and T helper epitopes, peptide cocktail vaccines, hybrid peptide vaccines, customized peptide vaccines, and peptide-pulsed dendritic cell vaccines. The effectiveness of peptide vaccines is extensively researched in relation to neurological illnesses, infectious diseases such as human immunodeficiency virus (HIV), hepatitis C virus, TB, foot and mouth disease, and cancer.

The differential expression of tumor-associated antigens (TAAs) and tumor-specific antigens (TSAs) on normal and cancer cells is utilized in the development of peptide-based cancer vaccines. Synthetic long peptides (SLPs) include 25-35 amino acids sourced from TAAs or TSAs, representing a primary category of peptide-based cancer vaccines. Trials of cancer vaccinations with SLPs shown suppression of transplanted tumor development in murine models. The Survivin-based vaccine, consisting of a combination of three SLPs with eight CD4⁺ epitopes and six CD8⁺ epitopes, has demonstrated the ability to stimulate both CD4⁺ and CD8⁺ immune responses in murine models of colorectal cancer. Fusion proteins created by amalgamating Xcl1 with Ovalbumin SLP antigen and IgG1 Fc fragment shown the ability to provoke a specific T cell response and maintain tumor control against the weakly immunogenic B16-OVA melanoma tumor. Short peptides consisting of 8–10 amino acids engage Class I major histocompatibility complex (MHC) receptors and activate CD8⁺ T cell responses. The cancer neoepitope vaccine, utilizing the MHC1-restricted short peptide Nes2LR, has been shown to elicit functional CD8⁺ T cell responses and inhibit tumor development in a mouse renal carcinoma model. Recombinant overlapping peptides (ROPs), formulated as a design technique for peptide vaccines, include a single-chain polypeptide containing several epitopes. They can elicit robust immunogenic responses in CD4⁺ and CD8⁺ T lymphocytes. Immunoinformatics methodologies were employed to develop a multi-epitope peptide vaccine targeting breast cancer, utilizing immunogenic domains of the BORIS cancer-testis antigen, which encompasses many CTL epitopes. The chosen areas were connected using a GPGPG linker, subsequently including T helper epitopes and the toll-like receptor (TLR)-4/MD-2 agonist. The resultant vaccine was reverse transcribed and subsequently integrated into pcDNA3.1 to create the DNA vaccine. Subsequent experiments shown that co-immunization with the multiepitope peptide vaccine and the corresponding DNA vaccine markedly suppressed breast tumor development, reduced tumor weight, blocked metastasis, and prolonged survival time in mouse mammary cancer.

Peptide-based vaccines possess a superiority over alternative therapeutic modalities. Metastatic tumors disseminated throughout the body can be addressed with peptide vaccines, which are non-toxic relative to alternative therapeutic modalities. In contrast to other immunotherapeutic approaches like as CAR T cell treatment, which targets cell surface antigens, peptide vaccines utilize numerous epitopes located both externally and internally within tumor cells. Creating a peptide vaccine without B cell epitopes can mitigate the danger of hypersensitivity and enable successful targeting of extremely diverse malignancies using peptide-based vaccinations. Despite the underappreciation of B cell epitopes in cancer vaccine development, current studies underscore the importance of multiepitope vaccines that include B cell epitopes with T helper and CTL epitopes for cancer prevention and treatment.

Fig.11 General features of Peptide vaccines. (Lekshmy M., et al., 2023)

Synthetic peptides in cosmetic

Synthetic peptides are gaining popularity in the cosmetic industry for their ability to enhance skin health, improve appearance, and promote anti-aging effects. Certain synthetic peptides can stimulate collagen production in the skin, helping to reduce the appearance of fine lines and wrinkles. By promoting collagen synthesis, these peptides contribute to skin elasticity and firmness. Peptides can enhance the overall texture of the skin by promoting cell turnover and encouraging the regeneration of skin cells, leading to a smoother and more youthful appearance.

Goldstein's team investigated an individual with an age spot on the dorsal aspect of the hand who applied a gel formulation comprising Leu-Lys-Lys-Thr-Glu-Thr for 28 consecutive days. They observed a considerable fading of the age spot within 7 days and a notable reduction in size after 28 days. The findings indicated that the polypeptide with the sequence Leu-Lys-Lys-Thr-Glu-Thr can mitigate or reverse skin aging and improve skin elasticity by stimulating terminal deoxynucleotidyl transferase, a non-template-dependent DNA polymerase. Lersch's team recruited 10 volunteers for a placebo-controlled clinical investigation to apply an O/W cream containing oligopeptides (Val-Glu-Ile-Pro-Glu) or a placebo twice daily on the left volar forearm for 10 days. The findings indicated that the synthesized peptide with the sequence Val-Glu-Ile-Pro-Glu may serve as a neurotransmitter inhibitor, enhancing neuronal awareness and sensitivity of the skin while facilitating prompt regeneration of injured skin cells. Dal Farra's team investigated human fibroblasts and keratinocytes incubated with the synthetic peptide (Lys-Leu-Asp-Ala-Pro-Thr) and discovered that this peptide could augment adhesion between skin cells, offer therapeutic and prophylactic benefits for aging skin manifestations (whether physiological or solar), and improve skin appearance. Nakagami's team discovered that the synthetic peptide (Glu-Leu-Lys-Leu-Ile-Phe-Leu-His-Arg-Leu-Lys-Arg-Leu-Arg-Leu-Leu-Lys-Arg-Lys) exhibited remarkable anti-aging effects by enhancing human fibroblast proliferation, stimulating hyaluronic acid synthesis, and facilitating collagen gel contraction.

Fig.12 The synthetic peptides in cosmetic for anti-aging. (Zhao X., et al., 2021)

Fig.13 Different anti-aging peptides with biological activities in cosmetic. (Zhao X., et al., 2021)

Fig.14 Patents of synthetic peptides for anti-skin aging in cosmetic. (Zhao X., et al., 2021)

Synthetic peptides for Protein identification and characterization

Synthetic peptides are essential tools in the fields of protein identification and characterization, serving various roles in proteomics and molecular biology. Synthetic peptides can be used to create a reference library for mass spectrometry-based identification. By analyzing the mass-to-charge ratios of synthetic peptides, researchers can match experimental data to known sequences, aiding in the identification of proteins. Synthetic peptides can be synthesized based on specific protein sequences to generate antibodies. These antibodies can then be used in various assays, including Western blotting and immunohistochemistry, for protein identification and quantification.

The 120-kDa surface protein antigens (SPAs) of typhus rickettsiae exhibit significant immunogenicity and are implicated in the species-specific serological responses of the typhus group rickettsiae. To investigate the immunochemistry of these proteins, overlapping decapeptides covering the whole protein were produced on modified polyethylene pins. A modified enzyme-linked immunosorbent test was employed to detect epitopes identified by rabbit hyperimmune antisera against Rickettsia prowazekii SPA. This technique identified eight unique epitopes across three areas. Four epitopes situated in the carboxyterminus of mature processed SPA were significantly blocked by native folded SPA in a competitive manner, but not by intact rickettsiae, indicating their presence on the SPA surface while remaining concealed on the rickettsial surface. Three of these epitopes were identified on both R. prowazekii and Rickettsia typhi SPAs. The immunoreactivities of five epitopes were further elucidated by the synthesis of modified peptides. Glycine substitution tests identified the essential residues inside the epitopes. The binding dependency of peptide epitopes to polyclonal antisera was delineated to individual residues. The restricted quantity and low reactivity of linear peptide epitopes identified in human and rabbit sera, likely attributable to the absence of methylated amino acids found in rickettsia-derived SPA.

References

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