Author: Oleg Melnyk

Ten novel sNPF analogs were designed and synthesized based on the structure of lead I-3 by molecular docking and peptidomimetic methods. Compound A-1 exhibits better binding affinity with the receptor, possesses excellent aphidicidal activity, and shows low toxicity to bees, making it a promising candidate for environmentally friendly insecticides.

ABSTRACT

Short neuropeptide F (sNPF) is a peptide unique to insects, characterized by a C-terminal phenylalanine and a conserved RLRFa motif, and plays key roles in controlling feeding behavior, growth, circadian rhythms, and water–salt homeostasis. We previously identified an sNPF analog, I-3, with aphidicidal activity. In this study, 10 sNPF analogs with aromatic or nonaromatic modifications at the N-terminus were designed based on I-3, using molecular docking and peptidomimetic strategies to investigate the role of N-terminal residues. Aphicidal activity showed that A-1 has stronger activity than I-3 and pymetrozine. Structure–activity analysis indicated that a benzene ring with electronegative and lipophilic groups at the N-terminus is key for aphicidal activity. Molecular docking and molecular dynamics simulations showed A-1 binds more stably to the receptor than I-3. Toxicity tests on honeybees (Apis mellifera) confirm that compound A-1, which exhibits strong aphidicidal activity, is safe for nontarget organisms. Additionally, Admetsar3 evaluations indicate low toxicity risks for all compounds. Therefore, A-1 represents a promising, selective, and eco-friendly insecticide for controlling pea aphids, and this study validates the feasibility of developing novel green pesticides based on sNPF.

No abstract is available for this article.

This review explores various computational methods based on structure and ligand-based approaches along with advanced AI models in transforming peptide design. We also discuss the impact of peptide databases in accelerating peptide research.

ABSTRACT

Peptides play essential roles in biological systems and serve as key agents in therapeutics, biomaterials, and drug delivery. Despite their broad utility, peptide design is limited by rapid degradation, low oral bioavailability, and the inefficiency of conventional synthesis and screening methods. This review provides a comprehensive overview of computational approaches that have emerged as effective alternatives, enabling the exploration of a large chemical space and the virtual screening of thousands of peptides. We detail the critical role of specialized peptide databases, computational tools, and advanced methodologies, including structure-based design, molecular dynamics (MD) simulations, and ligand-based approaches. A particular focus is placed on the transformative impact of machine learning (ML), deep learning (DL), and generative AI models, which are accelerating the discovery of novel peptides. While these methods offer promising solutions, we also address key challenges like data inconsistency, model interpretability, and the need for better forcefields. By highlighting these advancements and limitations, this review aims to provide a roadmap for leveraging computational design in peptide research.

No abstract is available for this article.

The theoretical diversity of peptide libraries is astronomical. We define a practical lower bound as Y ≥ 1/(s × a × ∏k=1mfk$$ {prod}_{k=1}^m{f}_k $$). A literature survey estimates that a diversity of ≈1.6 × 10⁵ (m ≈ 4, a ≈ 1, s ≈ 1) is required to include one specific binder.

ABSTRACT

Molecular library display systems utilizing phage, bacteria, and genetic material are powerful tools for identifying target-specific peptides. Libraries of sufficient diversity are required to isolate target-specific peptides. Although the evolution of molecular display techniques—from phage display to mRNA display—has substantially expanded achievable library diversities, the minimum diversity required to reliably isolate target-specific peptides remains unclear. Here, we propose a straightforward equation to estimate the minimum diversity (Y). Y is defined by three experimentally accessible parameters: (i) the number of important amino acids (m, consensus motif residues whose substitution markedly reduces binding), (ii) the number of independent binding sites (s) on the target molecule and (iii) the arrangement factor (a) that counts the possible positional permutations of the motif within the random region. By analyzing 35 target-specific peptides reported previously, we found that the average value of m was ≈4, whereas a and s were most frequently ≈1. These representative values imply a practical lower-bound benchmark of diversity, Y ≥ 1.6 × 10⁵ for random peptide libraries in screening. Our findings will aid researchers in rationalizing the design and construction of peptide libraries, facilitating efficient identification of high-affinity, target-specific peptides.

Short fiber-forming peptides were modified by histidine substitution and mannose or galactose attachment and combined with different metal ions. Although no generalizable trends were observed, Lys to His substitution at position 25 with galactose in the presence of copper produced a species with highly favorable viscoelastic properties.

ABSTRACT

Mucus is the biological hydrogel that lines the mucosal surfaces of mammals and acts as a protective barrier. Its main proteinaceous component is mucin, the high molecular weight, degree of glycosylation, and hardly uniquely defined nature of which hamper precise structures/property investigations based on biological samples. In contrast, chemically precisely defined peptide model systems inspired by such natural glycoproteins represent synthetically readily obtainable tools with excellent properties for both fundamental research and biomedical applications. Herein, we report the design and characterization of a library of histidine- and monosaccharide-containing coiled coil peptides that form hydrogels to different degrees in the presence of divalent metal ions Cu²⁺, Zn²⁺, Ca²⁺, and Fe²⁺. Using rheology, circular dichroism, and transmission electron microscopy, we determined the viscoelastic properties and global structures of these glycopeptide materials. This study reflects the interplay between glycan identity, histidine position, and divalent metal ion on the mechanical strength of these hydrogels.

In vitro dose–response tests reveal that moricin peptide retards the growth of Streptococcus pneumoniae. Molecular docking and MD simulation tests show that moricin binds strongly to Pneumococcal Surface Adhesin A (PsaA). ADMET profile shows that moricin can be safely absorbed, has minimal toxicity and could be used as a treatment. Moricin disrupts membrane potential and causes membrane leakage of bacteria. It is biocompatible, with no significant toxicity to BV2 microglial cells.

ABSTRACT

The rise of multidrug-resistant Streptococcus pneumoniae poses a major public health threat, necessitating novel therapeutic targets. Pneumococcal Surface Adhesin A (PsaA), a conserved surface lipoprotein, plays a key role in manganese acquisition, colonization and virulence. Immunization with PsaA elicits protective immunity, while fragment-based drug design has identified inhibitors disrupting its function. PsaA emerges as a promising molecular target for innovative therapeutic strategies against pneumococcal diseases. Antimicrobial peptides (AMPs) are crucial components of the innate immune system, providing a potent defence mechanism against a broad spectrum of pathogens. Moricin, an AMP initially identified in Bombyx mori, exhibits robust antimicrobial activity against Gram-positive bacteria. This study explores the inhibitory effects of moricin on S. pneumoniae, a significant human pathogen responsible for severe infections such as pneumonia, meningitis and sepsis. In silico analyses, including molecular docking and molecular dynamics simulations, revealed a strong interaction between moricin and the PsaA. In vitro studies corroborated the computational findings, demonstrating a dose-dependent inhibition of S. pneumoniae growth. Moricin induced bacterial membrane disruption, evidenced by increased membrane permeability, release of intracellular contents and altered membrane potential, highlighting the bactericidal mode of action. Furthermore, time-kill kinetics revealed rapid bacterial eradication, underscoring moricin’s efficacy. Additionally, toxicity assays on the macrophage BV2 cell line demonstrated that moricin caused no significant structural or organelle damage, emphasizing its biocompatibility and safety. The integration of in silico and in vitro approaches provides comprehensive mechanistic insights into moricin’s antimicrobial action and establishes its potential as a safe and effective therapeutic agent against S. pneumoniae.

Highly selective chemical modification of peptides. This review describes peptide tags that direct covalent chemical modification of proteins.

ABSTRACT

Bioconjugation chemistry is an important tool for studying proteins, developing pharmaceutical agents, and for many other applications. Conventional methods for protein functionalization rely on chemoselective reactions but often have poor regioselectivity. Peptide tags facilitating site-selective chemical covalent modification of proteins are of great value in the synthesis of protein conjugates. Ideally, a protein would only have to be minimally mutated prior to chemical modification to avoid interfering with native protein folding, trafficking, and function. This short review summarizes the advances in the developments and applications of peptide tags for covalent modifications that proceed without enzymatic assistance.

Gelsolin peptides form amyloids, and aggregation is accelerated by a single point mutation. The gelsolin amyloids can be inhibited using plant hormones.

ABSTRACT

Amyloidosis, a self-assembly of proteins or peptides, is associated with numerous degenerative diseases, such as gelsolin amyloidosis, which remain without a cure. Gelsolin protein is an actin-binding protein, but when aggregated in a diseased state, it is a potential drug target. Specifically, gelsolin mutations, N184K and D187Y, have been linked to renal amyloidosis and systemic progressive deposition of amyloids, respectively. Understanding how such mutations mitigate gelsolin aggregation and how this process can be prevented through small molecule inhibitors is of interest. Herein, we explored the efficacies of plant-based naturally occurring cytokinin (CK) molecules as aggregation modulators in vitro. Using various biophysical methods, such as spectroscopy and microscopy, the aggregation of wild-type gelsolin peptide 184NNGDCFILDL193 and its mutants (N184K, D187Y) was investigated. The mutations significantly promoted aggregation, which is of biological significance. The CK trans-zeatin (tZ) was a more effective disaggregation promoter compared with kinetin (Kin). The experimentally determined IC₅₀ values were in the 9–20 μM range. The mode of inhibition was identified as direct non-covalent complexation between the CK and the peptides by using mass spectrometry and molecular docking studies. Data show that CKs are promising amyloid modulators, which can be easily translatable to other amyloid systems.

A peptide library from nonbloodsucking leech Whitmania pigra was established. Through molecular docking and dynamics (MD) simulations, a thrombin-targeting PEP^WP (LRELEDALEQER) was screened out, showing significant in vitro/vivo anticoagulant activity. Thrombin’s Arg233 and Arg101 in Exosite II were revealed as the key electrostatic interaction site, distinguishing its mechanism from hirudin.

ABSTRACT

Targeting thrombin to screen safe thrombin inhibitors from natural plants and animals is a critical direction in anticoagulant drug development. This study aimed to screen thrombin inhibitors from the nonbloodsucking leech Whitmania pigra (WP) and elucidate the mechanism of anticoagulation through a “computation-guided experimentation” strategy. A peptide library was constructed from WP hydrolysates, and virtual screening was performed using molecular docking and dynamics simulations. A novel thrombin-targeting anticoagulant peptide PEP^WP (LRELEDALEQER) was screened out from the peptide library and validated through in vitro/in vivo experiments. PEP^WP significantly prolonged thrombin time (TT) and prothrombin time (PT) in a dose-dependent manner in vitro, indicating its role in the common and extrinsic coagulation pathways. Surface plasmon resonance (SPR) analysis then confirmed strong thrombin binding (K

_d
 = 7.242 × 10⁻⁶ mol/L). Furthermore, PEP^WP prolonged TT while reducing blood viscosity in acute blood stasis rats. Finally, structural analysis revealed that PEP^WP bound to Exosite II of thrombin. Arg233 and Arg101 were the key residues for the binding. In conclusion, PEP^WP exhibited good anticoagulant activity and significant application potential.

This study demonstrates that novel octenyl-alanine–modified hPep peptides efficiently encapsulate siRNA into well-defined nanoparticles. Our findings highlight the potential of these hPep/siRNA nanoparticles to induce RNA interference, effectively silencing therapeutically important CD45 expression.

ABSTRACT

The development of therapeutic small interfering RNAs (siRNAs) has lately gained significant momentum due to their ability to silence genes in a highly specific manner. The main obstacle withholding the wider translation of siRNA-based drug modalities is their limited half-life and poor bioavailability, especially in extra-hepatic tissues. Consequently, various drug delivery systems (DDSs) have been developed to improve the delivery of siRNAs, including short delivery peptides called cell-penetrating peptides (CPPs). In this study, we explore the potential of using alkenyl-alanine modifications to enhance the siRNA delivery efficacy with CPPs. We demonstrate on hPep peptides that incorporation of alkenyl-alanines enhances the encapsulation of siRNAs into stable nanoparticles and contributes to increased cellular uptake. Furthermore, we demonstrate that the lead peptide, hPep3, induces effective RNAi-mediated gene silencing in a reporter cell model as well as on the disease-implicated endogenous CD45 gene target. The biodistribution studies in mice show that the alkenyl-alanines are systemically well tolerated, and employing such modifications in the peptide backbone improves siRNA delivery in several tissues, including extra-hepatic sites. As demonstrated on hPep peptides, alkenyl-alanines offer a simple yet robust way to enhance the delivery efficacy of CPPs and have the potential to advance siRNA therapeutics beyond the liver targets.

Stapled Panurgines peptides were developed to enhance stability and permeability. PNG-5 showed improved helicity, protease resistance, and anti-breast cancer activity, highlighting the potential of hydrocarbon stapling for creating effective peptide-based therapeutics.

ABSTRACT

The development of novel candidate molecules for breast cancer treatment holds significant clinical value. Panurgines (PNG), derived from the venom of the wild bee Panurgus calcaratus, are particularly noteworthy for their anti-breast cancer activity and antibacterial properties. However, linear peptides are often hindered by poor stability and limited cell membrane permeability, making them highly susceptible to protease degradation. To tackle this challenge, the current study focused on synthesizing a range of stapled Panurgines peptides through hydrocarbon stapling modifications, followed by a comprehensive evaluation of their chemical and biological properties. Remarkably, PNG-5 demonstrated notable improvements in helicity, cell membrane permeability, proteolytic stability, and antitumor activity. This study examines how the hydrocarbon stapling technique significantly affects the secondary structure, hydrolase stability, and biological activity of PNG, revealing its potential as a transformative and powerful anti-breast cancer therapeutic. These findings lay a strong foundation for the development of innovative and highly effective peptide-based anti-tumor drugs.

The mystery of PTH-cysteine and diPTH-cystine in the analysis of disulfide bonds is solved. Both derivatives were synthesized and applied to Edman sequencing. A time-efficient combined workflow applying Edman and MS/MS sequencing for a comprehensive analysis of the complex disulfide connectivity in peptides and proteins was developed.

ABSTRACT

The annotation of disulfide bridges in peptides and proteins can be an elaborate process and requires careful revision of multiple data sets to avoid wrong assignment in the structural analysis. Herein, we provide additional support to elucidate the cysteine connectivity by re-implementation of Edman sequencing for the analysis of this specific structural feature. By synthesizing diPTH-cystine and PTH-cysteine for comparison, we were able to identify the respective derivative during Edman sequencing when a disulfide bond is detected in a peptide. Application of Edman sequencing to selected peptides with two or three disulfide bridges provides further insight into the differentiation of cysteines that form a disulfide bridge for both half-cystines in the same cycle and in separated cycles. A combined approach for the implementation of automated Edman sequencing in the process of disulfide bond assignment is described to alleviate structural elucidation in the future analysis of cysteine-rich peptides and proteins.

No abstract is available for this article.

The use of a non-swelling resin for solid-phase peptide synthesis allows the reduction of solvent consumption by up to 50%.

ABSTRACT

Non-swelling silica-based resin was used for peptide synthesis. The strategy used is similar to that of solid-phase peptide synthesis (SPPS), referred to as silica-assisted solid-phase peptide synthesis (SiPPS). A 2-h coupling seemed to favor the coupling compared to that of 1-h standard coupling. The use of this non-swelling resin allows a 50% reduction in the consumption of solvents. The strategy was well demonstrated for the synthesis of H-YSSFL-NH₂, linear oxytocin, angiotensin II, and afamelanotide using a silica-based support (Fmoc-Rink amide SiliaBond manufactured from SiliCycle Inc.). The peptides were found to have more than acceptable purity, although there was a loss in overall yields.

A novel antioxidant peptide WQK was identified from Silkworm pupa by virtual digestion, ADMET prediction, and molecular docking. WQK can reduce ABTS·⁺ and ferric-tripyridyltriazine, in vitro. Besides, WQK can promote the expression of antioxidant genes and eliminate reactive oxygen species in oxalic acid-treated human proximal tubular epithelial cells (HK-2).

ABSTRACT

Nrf2-Keap1 is an important defense system against oxidative stress damage and enhances the body’s antioxidant capacity. Targeting the Keap1-Nrf2 signaling pathway and activating Nrf2 has become an effective strategy for treating oxidative stress and related diseases. In this study, virtual digestion, ADMET prediction, and molecular docking were used to screen antioxidant peptide from silkworm pupa protein. Then, a novel tripeptide (WQK) was identified, exhibiting good water solubility, nontoxicity, high intestinal absorption, and the ability to cross the blood–brain barrier. Molecular docking showed that WQK established six H-bond interactions with some key sites of Keap1 (Arg380, Arg415, Gln530, Ser555, Ile416, and Leu365), which is similar to the identified antioxidant molecule 12e. Additionally, WQK has the function to reduce ABTS·⁺ and ferric-tripyridyltriazine (Fe³⁺-TPTZ) in vitro. Furthermore, WQK can promote the expression of antioxidant genes and eliminate reactive oxygen species (ROS) in oxalic acid-treated human proximal tubular epithelial cells (HK-2). The results suggested that WQK may activate the Keap1-Nrf2 signaling pathway by binding to Keap1 and activating the antioxidant system.

Amorphous nanostructures from polyunsaturated peptide amphiphiles such as eicosapentaenoyl-VVVAAAKKK-NH₂ promote gene delivery by viral vectors.

ABSTRACT

Peptide amphiphiles can form fibrillar and amorphous structures. While fibrillar assemblies have previously been shown to enhance viral infectivity or retroviral transduction for gene delivery, we now elucidate the mechanism behind amorphous peptide amphiphiles that promote virus–cell interactions. Using electron microscopy, we reveal that amorphous fragments of polyunsaturated peptide amphiphiles allow for more VLPs to bind directly to the plasma membrane, explaining previously observed efficient viral entry and superior biodegradation compared to state-of-the-art adjuvants. We believe our work highlights the potential of unsaturated fatty acid peptide hybrid materials for clinical applications.

Thermal and photolytic stability of liraglutide was studied, as it is essential to understand the effects of potential in-use mishandling and environmental stress conditions to understand its stability and degradation. Techniques, such as RP-HPLC and LC-HRMS (Orbitrap) were utilized to identify and characterize the generated impurities.

ABSTRACT

Liraglutide (LGT), a synthetic glucagon-like peptide-1 (GLP-1) analogue, is widely used in the treatment of Type 2 diabetes and obesity. Due to its peptide-based nature, it is prone to degradation, particularly under improper storage or handling conditions. Environmental stressors, such as heat and light, can lead to the formation of impurities that may compromise the quality and safety of the drug. In this study, we have investigated the impact of thermal and photolytic stress on LGT stability using reverse-phase liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS). This method enabled the separation, identification, and characterization of intact LGT and its degradation products (DPs). Ten impurities were identified, including four DPs that have not been reported so far. Further, MS/MS fragmentation analysis was employed to elucidate the sequences of LGT and its impurities, pinpointing the modifications and their respective sites. The formation mechanisms of these impurities were also proposed, providing critical insights into the degradation pathways. This study offers a comprehensive analytical approach for assessing the stability of peptide drugs, contributing to enhanced quality control and safer pharmaceutical formulations.

The introduction of an isopeptide bond at the lysine residues in the backbone of Esc(1-21) led to the generation of five analogs. Among these, Esc(1-21)ε20 showed the most promising features, having antimicrobial activity comparable with those of the parent peptide, a lower cytotoxicity, and a higher stability to proteolytic degradation.

ABSTRACT

Antimicrobial peptides (AMPs) represent valid alternatives to conventional antibiotics primarily due to their mechanism of action, which consists of cytoplasmic membrane disruption. However, their clinical application is often limited by cytotoxicity at high concentrations and low intrinsic biostability. To address these limitations, various biochemical approaches have been explored. In recent years, the frog-skin derived AMP Esc(1-21) has been extensively characterized for its potent antimicrobial activity, especially against Gram-negative bacteria, both in vitro and in vivo. In this study, we designed and synthesized novel Esc(1-21) analogs in which a single isopeptide bond was introduced in place of a conventional peptide bond at specific positions within the sequence. The resulting five analogs were evaluated for their (i) chemical and structural properties, (ii) resistance to proteolytic degradation, (iii) antimicrobial and antibiofilm activities, (iv) hemolytic and cytotoxic effects, and (v) ability to perturb bacterial cytoplasmic membranes. Among these, Esc(1-21)ε20 showed the most promising features, maintaining antimicrobial and antibiofilm activities comparable to those of the parent peptide while exhibiting lower cytotoxicity towards eukaryotic cells at higher concentrations and greater resistance to enzymatic degradation. These findings highlight Esc(1-21)ε20 as an attractive lead candidate for the development of new antibiotic therapeutics.

Regarding the acquisition of antibacterial activity of BKBA-20 derivatives against Gram-negative bacteria by histidine incorporation, the suitable introduction position depends on pH.

ABSTRACT

Cationic antimicrobial peptides (CAMPs) exhibit potent antibacterial activity by disrupting bacterial membranes. We investigated the effect of histidine incorporation on BKBA-20, a designed amphiphilic helical peptide composed of alternating 2-aminoisobutyric acid (Aib) and lysine. Substitution at lysine sites (1a–1e series) reduced net charge and antimicrobial activity, though certain analogues (1c, 1d) demonstrated minimal antibacterial activity against Escherichia coli. In contrast, substitution at Aib sites (2a–2c series) preserved some extent of helical structure and improved activity under acidic conditions. Notably, substitutions at the terminal of the peptide were more effective at acidic pH, while the slightly medial side of the peptide favored activity at neutral pH. Hemolysis assays confirmed low cytotoxicity of the modified peptides. These results suggest histidine incorporation as a promising strategy to broaden the spectrum of CAMPs, particularly against Gram-negative bacteria, without increasing toxicity.

Journal of Peptide Science, Volume 31, Issue 9, September 2025.

The three main approaches commonly employed for peptide synthesis are solution synthesis or also called classical solution-phase peptide synthesis (CSPS), solid-phase peptide synthesis (SPPS), and liquid-phase peptide synthesis (LPPS).

ABSTRACT

Various approaches to make peptides have been adopted globally owing to their high demand. The three main approaches commonly used for this purpose are solution synthesis, also called classical solution-phase peptide synthesis (CSPS), solid-phase peptide synthesis (SPPS), and liquid-phase peptide synthesis (LPPS). Each method offers unique advantages: CSPS for scalability, SPPS for automation and efficiency, and LPPS for combining solution-phase simplicity with iterative synthesis using soluble tags.

Synthesis and biologicals activities of the natural cyclic peptide stylissatin A and its various analogues arereported. The findings suggest that specific amino acid residues such as Ile at position 2, Pro at 4 and 6, and Tyr at position 1 are key contributors to the peptide’s therapeutic potential.

ABSTRACT

Marine sponges are sessile invertebrates found in moderate, arctic, and tropical regions, serving as a valuable reservoir of bioactive compounds, particularly Pro-rich peptides. Among these, cyclic peptides have attracted significant interest due to their diverse therapeutic properties. One notable example is Stylissatin A (SA), a Pro-rich cyclic peptide reported from the marine sponge Stylissa massa. SA and its analogues have shown promising biological activities, including anti-inflammatory, anticancer, and anti-obesity effects. Despite the vast potential of marine-derived peptides, only a small number have progressed to the pharmaceutical market. Cyclic peptides like SA offer unique opportunities for molecular modifications and total synthesis, enabling the enhancement of potency, improvement of physicochemical properties, and optimization of synthetic yields. This review highlights the synthetic strategies developed for the total synthesis of SA, explores its structural features and related analogues, and discusses their therapeutic potential, underscoring the promise of SA-based scaffolds as novel peptide-based drug candidates.

No abstract is available for this article.

The reported high-yield synthesis of Medin, the most common human amyloid, uses automated solid-phase peptide synthesis (SPPS) with pseudoproline dipeptides and a removable aggregation-suppressing linker. After linker removal, synthetic Medin exhibits identical ThT kinetics and amyloid fibrils as natural Medin. This method enables high-purity production of Medin and analogs, aiding research into its role in Alzheimer’s disease. (Figure created in BioRender.com) (Institute and/or researcher Twitter usernames: https://x.com/VIB_Switch_Lab
)

ABSTRACT

Medin, a 50-amino acid fragment derived from the protein MFG-E8 (lactadherin), is the most prevalent amyloid found in humans, present in the vasculature of nearly all individuals over the age of 50. Its biological relevance is highlighted by its co-localization with amyloid-β (Aβ) deposits in both Alzheimer’s disease patients and transgenic mice models. Notably, Medin promotes amyloid-β aggregation, forming mixed fibrils with Aβ and enhancing its deposition in blood vessels. Here we report a new and efficient strategy to chemically access this compound. Our approach employs a solubilizing linker that not only ensures high solubility but also suppresses aggregation, allowing efficient purification of the product. The linker can be removed without a trace, after which the product behaves identically to wild-type Medin and forms amyloid fibrils. The synthesis route allows opening up a new chemical space, including nonnatural modifications like biotinylation. Together with the control over the aggregation properties, this is a powerful tool for amyloid protein studies.

Peptide-drug conjugates (PDCs) are emerging cancer therapeutics inspired by antibody-drug conjugates, offering targeted delivery via peptide ligands. Despite structural versatility, challenges like instability and rapid clearance hinder clinical progress. Innovations in linker design and targeting strategies drive ongoing development, with several candidates advancing through late-stage clinical trials.

ABSTRACT

Peptide-drug conjugates (PDCs) are advancing as targeted cancer therapies, leveraging lessons from antibody-drug conjugates (ADCs) to improve tumour specificity. These molecules combine a homing peptide with a cytotoxic payload via a linker, enabling precise drug delivery while sparing healthy tissue. Despite their potential, PDCs face challenges including metabolic instability, premature payload release and rapid clearance, limiting clinical success. Only Lutathera remains FDA-approved after Pepaxto’s withdrawal, though Pepaxto retains EMA and MHRA approval—highlighting regulatory and technical complexities. Most PDCs target overexpressed receptors (e.g., somatostatin and GnRH), though novel designs like CBX-12 employ alternative strategies. Currently, six PDCs are in Phase III trials, with ~96 in development, signalling growing interest. This review explores how ADC research has guided PDC optimisation, particularly in linker chemistry and payload selection. We analyse key structural features governing PDC efficacy, including peptide-receptor binding and intracellular trafficking. Innovations in stable linkers and tumour-selective activation mechanisms are critical to overcoming pharmacokinetic hurdles. Promising candidates in late-stage trials are highlighted, emphasising their potential to address unmet needs in oncology. By refining targeting precision and payload delivery, next-generation PDCs may expand treatment options for resistant cancers, bridging the gap between biologics and small-molecule therapies.

When tetrahydrothiophene (THT) was added to the solvent in solid phase peptide synthesis (SPPS), the oxidation of Met to Met(O) was significantly decreased, and the yields of Met-containing peptides improved. Using this method, we could synthesize an insulin-like peptide identified in the kuruma shrimp Marsupenaeus japonicus.

ABSTRACT

The oxidation of Met residue(s) in peptides and proteins is sometimes found in solid phase peptide synthesis (SPPS). In this study, in order to develop a method to prevent the oxidation of Met during SPPS, various sulfide compounds were added to the solvent and the oxidation rate was measured. As a result, it was found that tetrahydrothiophene (THT) was most efficient for reducing the extent of Met oxidation. THT tended to prevent the oxidation of Met in a concentration-dependent manner, although the oxidation of Met could not be completely prevented even at a concentration of 20% (v/v). On the other hand, when the SPPS in the presence of THT and then reduction of Met(O) to Met with NH₄I were performed, the yield was much improved. These results indicate that the combination of preventing oxidation with THT and reducing Met with NH₄I is effective for the synthesis of peptides containing Met residue(s). Using the method established here, we could synthesize an insulin-like peptide from the kuruma shrimp. This method is likely to be applicable to the synthesis of various Met-containing peptides.

Two orthogonal stability-indicating reverse-phase high-performance liquid chromatography (RP-HPLC) methods were developed and validated to analyze semaglutide and its degradation products. Stability assessments were conducted under varying temperature conditions (5°C–80°C), and the effects of pH and buffer strength were systematically evaluated at 25°C and 40°C. These experiments were designed to simulate conditions relevant to formulation development for both oral and long-acting injectable delivery.

ABSTRACT

Therapeutic peptides’ physical and chemical stability is of significant interest to the pharmaceutical industry. This study examines the effects of pH, temperature, and buffer strength on semaglutide (SEMA) stability using two orthogonal stability-indicating reverse-phase high-performance liquid chromatography (RP-HPLC) methods. The findings aid in designing novel oral and long-acting injectable (LAI) SEMA formulations. Two RP-HPLC methods were developed and validated to separate SEMA and its degradants. Stability studies were conducted at 25°C, 40°C, and 55°C for 24 h in water, with extended studies at 5°C, 25°C, 40°C, 60°C, and 80°C. pH and buffer strength effects were assessed at 25°C and 40°C. SEMA remained stable for 3 h at 80°C. The results obtained from pH-dependent stability studies indicated that SEMA was relatively stable at pH 1.2 at 25°C and 40°C for a day. A higher extent of degradation was observed between the pH of 4.5–5.5; this is due to the isoelectric point of SEMA (pH 5.4), and hence, the finished product pH should be > 7.0. These findings highlight the critical influence of buffer, temperature, and pH on SEMA stability. The results obtained by this study would help develop both oral and LAI SEMA formulations.

Chemoselective photocatalytic diselenide contraction (PDC) enables site-selective biomimetic ubiquitylation of substrate peptides. The resulting selenalysine linkage undergoes efficient cleavage by deubiquitylating enzymes.

ABSTRACT

Ubiquitylation is a highly conserved post-translational modification (PTM) in eukaryotes, which serves as a critical regulatory mechanism for protein homeostasis, cellular transport, signal transduction pathways, and numerous other functions. The biological function of ubiquitylation is dictated predominantly by the topology of its linkage. Deciphering ubiquitin’s complex biochemistry necessitates novel synthetic methods that deliver well-defined, biosimilar ubiquitylation. To this end, a semisynthetic strategy relying on the recombinant expression of ubiquitin combined with chemoselective photocatalytic diselenide contraction (PDC) was established to enable site-selective biomimetic selenalysine-linked ubiquitylation. The modification of ubiquitin with a C-terminal selenol was fine-tuned to avoid hydrolysis. The conditions of the PDC reaction, such as solvent composition, phosphine concentration, and irradiation, were optimized for efficient ubiquitylation of a Tau F derived peptide. Furthermore, it was demonstrated that the selenalysine linkage undergoes efficient cleavage by deubiquitylating enzymes, comparable to the native isopeptide linkage. The presented method expands the toolbox of site-selective ubiquitylation techniques. It is tolerant to many functional groups and will help to further elucidate the complexities of ubiquitylation.

A novel polymeric mesoporous support (pDVB) has been proposed with the aim of improving the efficiency and sustainability of SPPS. This new resin enables the use of a wider range of green solvents.

ABSTRACT

This study explores the use of a novel polymeric mesoporous support (pDVB) for solid-phase peptide synthesis (SPPS), with the aim of improving the efficiency and sustainability of the process. The pDVB support, functionalized with the Fmoc-Rink amide linker, offers advantages over conventional supports based on gel-type, lightly crosslinked polymer skeletons, particularly with regard to reduced reliance on swelling capacity, which allows the use of a wider range of solvents. The work focuses on greener and eco-friendly solvents such as TEP, ACN, IPA, and their mixtures with DMSO to replace toxic solvents such as DMF. The synthesis of two model peptide sequences, Fmoc-LLVF-NH₂ and ACP(65–74), showed that pDVB-Rink performs better than a conventional-type Rink Amide MBHA support, especially when using environmentally friendly solvents. These results suggest that mesoporous pDVB-Rink is a promising solid support for SPPS to reduce the use of toxic solvents and to improve sustainability.

No abstract is available for this article.

Triple-negative breast cancer has a high potential for metastasis, resulting in a poor 5-year overall survival rate. Our research demonstrated that PepH3-vCPP2319 can inhibit cell migration and proliferation, which prevents cancer metastasis to distant organs, including the brain.

ABSTRACT

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype affecting mostly younger women with a poor 5-year overall survival. It is characterized by a high metastization rate, particularly to the brain, where the blood–brain barrier (BBB) hinders the pharmaceuticals delivery. New anticancer drugs able to inhibit cell migration are required to effectively prevent the development of metastasis. PepH3-vCPP2319 (AGILKRW(Ahx)WRRRYRRWRRRRRQRRRPRR-amide), consisting of the conjugation of the BBB peptide shuttle (BBBpS) PepH3 (AGILKRW-amide) to the anticancer peptide (ACP) vCPP2319 (WRRRYRRWRRRRRQRRRPRR-amide), was reported to have high anticancer activity (IC₅₀ = 5.0 μM) toward highly aggressive TNBC cells (MDA-MB-231) paired with 2-fold increased accumulation in the brain when compared to unconjugated vCPP2319. Herein, we demonstrate that PepH3-vCPP2319 inhibits cell migration and proliferation in wound healing assays, outperforming the gold standard small chemical inhibitor, iCRT-3. The concentration required to inhibit cell migration is 10-fold lower for PepH3-vCPP2319 (0.5 μM) when compared with iCRT-3 (50 μM). Likewise, PepH3-vCPP2319 at 2.5 μM was more efficient in preventing cell proliferation when compared with 50 μM iCRT-3, with 45% reduction in spheroid diameter. This study sheds light on the antimetastatic potential of PepH3-vCPP2319 through abrogation of cell migration to distant sites, including the brain.

A schematic representation illustrating phage display technology and the application of phage display–derived peptides in research and clinical applications for cancer imaging. These peptides are employed in advanced imaging modalities, including PET/SPECT, MRI, in vivo fluorescence imaging, ultrasound, and photoacoustic imaging.

ABSTRACT

Phage display has emerged as a groundbreaking technique for discovering novel biomolecules with significant applications in cancer diagnosis and therapy. This technique employs genetically engineered bacteriophages to display diverse libraries of peptides on their coat proteins, enabling the identification of candidates through a biopanning process targeting specific cancer markers. Biomolecules identified via phage display are widely used as molecular tools, often labeled with imaging agents or conjugated to nanoparticles for noninvasive tumor imaging. This review provides a comprehensive overview of recent advancements in phage display applications in cancer research over the past 5 years and prominent examples of clinical studies. The analysis underscores the potential of phage display to deliver diagnostic and therapeutic biomolecules, highlighting its promise for future clinical implementation in cancer imaging.

The results of an analysis on the presence of π-turns, characterized by an i ← i + 5 C=O···H–N intramolecular hydrogen bond, in the X-ray diffraction structures of peptides are discussed. π-Turns are often found at the C-end of incipient or fully developed α-helices, 3₁₀-helices, and mixed α-/3₁₀-helices, thus acting as a C-capping motif.

ABSTRACT

The results of an analysis on the presence of π-turns, characterized by an i ← i + 5 C=O···H–N intramolecular hydrogen bond, in the X-ray diffraction structures of peptides are discussed. The survey returned a total of 55 π-turn occurrences in linear and cyclic peptides. π-Turns characterized by a helical conformation for residue i + 4, but with a screw sense opposite to that of the three preceding residues, are largely prevailing. They are often found at the C-end of incipient or fully developed α-helices, 3₁₀-helices, and mixed α-/3₁₀-helices, thus acting as a C-capping motif. However, the structures of two linear peptides and 15 cyclopeptides indicate that these types of π-turns can exist in isolation, without the support of a preceding helix. The frequent presence of additional intramolecular hydrogen bonds internal to the π-turn is also investigated. Cyclopeptides offered examples of two types of π-turns that have no parallel in the structures of proteins. Differently from proteins, π-turns characterized by helical ϕ, ψ sets of the same screw sense for all internal residues are hitherto unreported in the X-ray diffraction structures of peptides. A suggestion for the rational design in peptides/peptidomimetics of a π-turn featuring the screw-sense reversal of residue i + 4 is proposed.

   

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Scanning a protein using overlapping synthetic peptides is a valuable strategy for the identification of therapeutic peptide candidates. This review provides a brief overview of the various types of peptide libraries that can be prepared—either as soluble peptides or in the form of peptide arrays—and discusses their strengths and limitations in mapping protein–protein interaction (PPI) interfaces, identifying immunogenic epitopes and discovering bioactive peptides.

ABSTRACT

Therapeutic peptides targeted at various diseases are becoming increasingly relevant for the pharmaceutical industry. Several of these drugs were originally designed by mimicking a segment of a protein of interest. As such, protein mimicry represents a promising strategy both in immunology, for the identification of B- and T-cell epitopes, as well as for the modulation of protein activity, including the disruption of protein–protein interactions (PPIs) and the interference with biological or pathological cellular functions. Several methods have been developed to pinpoint the (binding) epitopes of a protein or the regions responsible for biological activity. One of such strategies is the scanning of the protein or selected domains with synthetic overlapping peptides. As the mechanism of action of a mimetic peptide can be similar to that of the whole protein, this method offers a powerful tool for the investigation of protein function, along with providing a solid basis for the development of therapeutic candidates. This review gives a general overview of different applications of the peptide scanning methodology, describing a comparison of the preparation and use of solid-phase libraries (peptide arrays) with isolated peptide libraries and highlighting their strengths and most common applications.

Ubiquitination mediates the turnover of proteins in eukaryotic cells. The coadministration of endosomolytic peptides enables the traceless entry of exogenous ubiquitin into human cells and its incorporation into cellular proteins. This strategy can enable the illumination of underexplored aspects of ubiquitin biology.

ABSTRACT

The proteostasis network involves complex protein signaling cascades. The tagging of proteins with ubiquitin is central to the degradation of cellular proteins, but understanding its exact role in processing proteins is complicated by the complexity and extent of its utilization within cells. Here, we describe the application of a traceless protein delivery strategy to effect the uptake of exogenous ubiquitin into the cytosol of human cells. We find that coadministration of the endosomolytic peptides L17E and, especially, L17ER₄ provides not only cytosolic access to ubiquitin but also its functional incorporation into endogenous proteins. By enabling the study of semisynthetic ubiquitin variants in the human cytosol, this strategy could advance the field of ubiquitin biology.

Stylissamide B was successfully synthesized for the first time using Fmoc-SPPS monitored using ReD-A method followed by cyclization in solution phase. The linear peptide, Pro-Pro-Ile-Tyr-Pro-Phe-Pro, was cyclized to form stylissamide B. It is noted that position of the Pro-Pro-Pro motif affects cyclization efficiency.

ABSTRACT

Stylissamide B was successfully synthesized for the first time using Fmoc solid-phase peptide synthesis (Fmoc-SPPS) followed by cyclization in solution phase. The Fmoc-SPPS process was monitored using the reversible detection method for amino groups (ReD-A method). The linear peptide, Pro-Pro-Ile-Tyr-Pro-Phe-Pro, was cyclized to form stylissamide B. The spectral data obtained were in agreement with previously reported literature, thereby confirming the reliability of the synthetic method.

Peptide-based vaccines, formulated with an appropriate adjuvant, offer a versatile platform for targeted cancer immunotherapy. Herein, we explore the synthesis of two tissue-restricted cancer-testis antigens; (NY-ESO-1 and BAGE4) covalently linked to the toll-like receptor agonist, Pam₂Cys. These constructs were evaluated in vivo along with a lipid nanoparticle formulation of BAGE4.

ABSTRACT

Peptide-based vaccines, formulated with an appropriate adjuvant, offer a versatile platform for targeted cancer immunotherapy. While adjuvants are usually coadministered for nucleic acid and protein vaccines, synthetic peptide antigens afford a more effective opportunity to covalently and regioselectively graft immunostimulatory motifs directly onto the antigen scaffold to yield self-adjuvanting vaccines. Herein, we explore the synthesis of two tissue-restricted cancer-testis antigens (CTAs); New York oesophageal cell carcinoma 1 (NY-ESO-1) and B melanoma antigen 4 (BAGE4), both carrying the toll-like receptor (TLR) agonist, Pam₂Cys. These constructs were evaluated in vivo along with a lipid nanoparticle (LNP) preparation of the underexplored BAGE4 melanoma antigen.

KF19 and LR16 showed potent activity against Staphylococcus aureus with low toxicity. They killed S. aureus within 2 h and significantly disrupted biofilms. SEM confirmed membrane destruction.

ABSTRACT

The antimicrobial resistance (AMR) crisis represents a significant global threat. Unlike traditional antibiotics, antimicrobial peptides offer a promising pathway because of their primary mechanisms. This study aimed to evaluate and rationally design novel AMPs based on tobacco nectar’s AMP (Pep 6) to combat antibiotic resistance issues. Substitution and truncation of some amino acids were applied. Four peptides, KF19, KF16, LK16, and LR16, were designed with enhanced net charge hydrophobicity. They were evaluated for their in vitro antibacterial activity. However, only promising AMPs were further evaluated for their hemolytic activity, time-killing kinetics, mode of action, and anti-biofilm properties. The results showed that only KF19 and LR16 have potent activity against Staphylococcus aureus ATCC25923 and resistant isolates with MIC values from 7.81 to 15.62 μg/mL. Hemolysis ratios were 2.38% and 2.24% at 125 μg/mL for KF19 and LR16, respectively. Both peptides were able to kill S. aureus ATCC25923 within 2 h. SEM results showed their ability to target the cell membrane. Both peptides destroyed the S. aureus biofilms significantly at 62.5 and 125 μg/mL (**p < 0.01, ***p < 0.001, ****p < 0.0001). This study supported rational design in developing new antibacterial agents and demonstrated the therapeutic potency of novel peptides that could solve the resistance issues.

The advent of epimerization-free methods for coupling in N- to C-direction synthesis offers improved atom economy for sustainable peptide synthesis.

ABSTRACT

A revolution in peptide production arrived from the innovation of carboxylate to amine C– to N-direction solid-phase synthesis. This cornerstone of modern peptide science has enabled multiple academic and industrial applications; however, the process of C– to N-solid phase peptide synthesis (C-N-SPPS) has extreme process mass intensity and poor atom economy. Notably, C-N-SPPS relies upon the use of atom-intensive protecting groups, such as the fluorenylmethyloxycarbonyl (Fmoc) protection and wasteful excess of protected amino acids and coupling agents. On the other hand, peptide synthesis in the amine to carboxylate N– to C-direction offers potential to minimize protection and may arguably enable more efficient means for manufacturing peptides. For example, efficient amide bond formation in the N– to C-direction has been accomplished using methods employing thioesters, vinyl esters, and transamidation to achieve peptide synthesis with minimal epimerization. This review aims to provide an overview of N– to C-peptide synthesis indicating advantages in taking this avenue for sustainable peptide production.

De novo sequencing can combine with parallel SPOT synthesis to highly accelerate the biological peptides screening from natural origin. Seahorse was utilized as an example, for which peptides possessing synergistic antioxidant and α-glucosidase inhibition activities were efficiently screened out for potential hyperglycemia management.

ABSTRACT

In this research, de novo sequencing was innovatively combined with parallel SPOT synthesis for the efficient screening of biological peptides from TCM or seafood: seahorse with synergistic antioxidant and α-glucosidase inhibitory activities, which is promising for postprandial hyperglycemia management. Gastrointestinal digestion mimic and de novo sequencing were sequentially carried out to predict new peptides from seahorse. After bioinformatic analysis using Peptide Ranker, 82 peptides were eventually synthesized by efficient parallel SPOT technique, and Ser-Val-Try-Leu-Gly-Gly-Ser-Leu-Leu (SVWLGGSLL) was screened out as the most efficient peptide with synergistic antioxidant (DPPH radical scavenging activity of 77%) and α-glucosidase inhibitory activity (IC₅₀ = 0.36 mM). Molecular docking was further carried out to illustrate the favorable ligand-receptor interactions formed such as hydrogen bonding and van der Waals force with low binding free energy of −7.8 kcal/mol. Moreover, pharmacokinetic analysis indicated that SVWLGGSLL was unrelated to toxicity with the advantage of gastrointestinal stability.

Analogues of the heavy chain of the antimicrobial peptide EeCentrocin 1, originated from the marine sea urchin, Echinus esculentus, maintained high potency even in its truncated form.

ABSTRACT

We have synthesised a series of 12-residue analogues of a previously reported lead peptide (P6) developed from the heavy chain of the marine peptide EeCentrocin 1, isolated from the sea urchin Echinus esculentus. We optimised the lead peptide by increasing its net positive charge, its lipophilicity through N-terminal fatty acid acylation or incorporation of a Trp residue, and by synthesising head-to-tail cyclic peptides under pseudo–high-dilution conditions. All peptides were screened for antimicrobial and antifungal activity, and toxicity was determined against human red blood cells. The two most potent peptide analogues were the linear peptide P6-W6R8 and its head-to-tail cyclic analogue cP6-W6R8 displaying minimum inhibitory concentrations of 0.4–6.6 μM against Gram-positive and Gram-negative bacteria and 6.2–13 μM against fungi. All peptides showed low haemolytic activity except for two of the lipopeptides, in which haemolytic activity correlated with increasing acyl chain length. Mode of action studies using bacterial biosensor strains revealed a membrane disruptive effect of both the linear and the cyclic peptide. Overall, the results of our study demonstrated that relatively simple structural modifications could be successfully employed in the development of potent antimicrobial lead peptides derived from marine natural products.

This review highlights the synergistic integration of peptides with peptide-functionalized poly(lactic-co-glycolic acid) (PLGA) and addresses key challenges of peptide-based therapeutics.

ABSTRACT

Peptide-based therapeutics have gained attention in cancer treatment because of their good specificity, low toxicity, and ability to modulate immune responses. However, challenges such as enzymatic degradation and poor bioavailability limit their clinical application. Peptide-functionalized poly(lactic-co-glycolic acid) (PLGA) systems have emerged as a transformative platform in cancer therapy that offers unique advantages, including enhanced stability, sustained release, and precise delivery of therapeutic agents. This review highlights the synergistic integration of peptides with PLGA and addresses key challenges of peptide-based therapeutics. The application of peptide-functionalized PLGA systems encompasses a diverse range of strategies for cancer therapy. In chemotherapy, peptides disrupt critical tumor pathways, induce apoptosis, and inhibit angiogenesis, demonstrating their versatility in targeting various aspects of tumor progression. In immunotherapy, peptides act as antigens to stimulate robust immune responses or as immune checkpoint inhibitors to restore T cell activity, overcoming tumor immune evasion. These systems also harness the enhanced permeability and retention effect, facilitating preferential accumulation in tumor tissues while leveraging tumor microenvironment (TME)-responsive mechanisms, such as pH-sensitive or enzyme-triggered drug release, to achieve controlled, localized delivery. Collectively, peptide-functionalized PLGA systems represent a promising, versatile approach for precise cancer therapy that integrates innovative delivery strategies with highly specific, potent therapeutic agents.

Peptides are increasingly used in diagnosis and treatment due to their selectivity and low side effects. Over 11% of FDA-approved drugs from 2016 to 2024 were synthetic peptides. However, immunogenicity—an adverse immune response—can limit their safety and efficacy. This may result from the peptide itself or impurities, triggering antidrug antibodies. Regulatory guidelines now require immunogenicity risk assessment. Developing robust immunogenicity assays reflecting immune complexity and population variability is crucial and remains a priority as greener synthesis methods are introduced.

ABSTRACT

Peptides are gaining remarkable popularity in clinical diagnosis and treatment due to their high selectivity and minimal side effects. Over 11% of all new pharmaceutical chemical entities authorised by the FDA between 2016 and 2024 were synthetically manufactured peptides. A critical factor that can potentially limit the efficacy and safety of peptide-based therapeutics or biologics is immunogenicity, defined as an unintended or adverse immune response to a protein or peptide therapy. This response may be triggered by the peptide itself or by impurities in the production or formulation steps, leading to the production of antidrug antibodies (ADAs). To address this, current regulatory guidelines require the assessment of risks in market authorization applications, which include identifying drug impurity levels and immunogenicity. The development and critical evaluation of appropriate immunogenicity assays is therefore highly warranted. Such assays must consider the fine complexities of the immune response, as well as its variation within the human population. Moreover, immunogenicity testing is expected to remain a priority as the shift toward greener chemistries in peptide synthesis may require reassessment of novel impurities in peptide formulations.

This study cyclized conotoxin Vc1.1 using polyethylene glycol (PEG) linkers of different lengths and demonstrates that linker length modulates the peptide’s helicity, thereby influencing its biological activity and stability.

ABSTRACT

α-Conotoxin Vc1.1 is a disulfide-rich peptide and a promising drug candidate for treating neuropathic and chronic pain. Backbone cyclization was applied to enhance its drug-like properties, resulting in improved serum stability and oral bioavailability. However, this modification also adversely affected its stability and activity in simulated intestinal fluid (SIF). To address these adverse effects, we explored the use of polyethylene glycol (PEG) linkers as substitutes for peptide backbone cyclization linkers. PEG linkers are smaller, more flexible, and more stable than peptide linkers. Furthermore, previous studies have demonstrated that PEG backbone linkers can enhance the activity of conotoxins. In this study, we synthesized four PEG-backboned cyclic Vc1.1 (cVc1.1) analogues with varying lengths of PEG linkers and used a chemo-enzymatic method to cyclize these analogues. Their structure, stability, and activity were subsequently evaluated. Although the results revealed that PEG linkers preserved the SIF stability and activity of cVc1.1, they highlighted the crucial role of the peptide’s helical structure in maintaining its stability and activity. Additionally, this work introduces a novel approach for synthesizing cyclic conotoxins.

The solid-phase synthesis of natural product phakellistatin 21/22 afforded an unnatural conformer 1, with all trans-proline residues. The synthetic peptide 1 lacked cytotoxicity, unlike the natural product. However, peptide 1 and its analogs 2, 3 and 5 were found to have neuroprotective effects in terms of supporting growth of the primary cortical cells.

ABSTRACT

The phakellistatins are a class of cyclic peptide natural products among which phakellistatin 21 and 22 isolated from the marine sponge Stylissa flabelliformis are cyclo(Pro¹-Pro²-Met(O)³-Phe⁴-Glu⁵-Leu⁶-Pro⁷-Pro⁸-Tyr⁹-Ile¹⁰) epimeric at the methionine sulfoxide residue. The natural product contains two cis and two trans proline residues and is reported to have significant cytotoxic activities. We attempted the total synthesis of phakellistatin 21/22 via on-resin macrocyclization using methionine as a building block. The final product contained methionine sulfoxide, suggesting that aerial oxidation took place during the synthesis and during the original isolation of the natural product. Our synthetic peptide cyclo(trans-Pro

^1,2,7,8

)-Pha21 (1) was identified as an unnatural conformer of natural product phakellistatin 21/22 with all Pro residues present as trans amides. The Peptide 1 was inactive against human cancer cell lines, unlike the natural product. We additionally synthesized alanine scanning Analogs 2–5 in which a Pro residue was replaced by Ala and Analog 6, where all four Pro residues were substituted by Ala. Peptides 1, 2, 3, and 5 were found to have neuroprotective effects on primary cortex cells and are potential leads for the treatment of neurodegenerative disorders.

Ultra-short antimicrobial peptides containing acyl groups at the N-terminus and the NCAA His* were prepared. The conformational analysis demonstrated that the peptides exhibiting the highest degree of structure were also those with the greatest antimicrobial activity. A promising candidate (P8) resistant to proteolysis with enhanced biological activity was identified.

ABSTRACT

Antimicrobial resistance represents a significant global health threat, prompting the exploration of alternative therapeutic strategies. Antimicrobial peptides (AMPs) and lipopeptides are promising candidates due to their unique ability to disrupt bacterial cell membranes through mechanisms distinct from conventional antibiotics. These peptides are typically enhanced by motifs involving cationic amino acids, positive charge, and aromatic residues. Additionally, the conjugation of acyl chains to the N-terminus of AMPs has been shown to improve their antimicrobial activity and selectivity. However, the susceptibility of peptides to enzymatic degradation presents a major limitation. To address this, we investigated the incorporation of non-coded amino acids (NCAAs) to enhance peptide stability. Specifically, we synthesized the NCAA 2-amino-3-(1H-imidazol-1-yl)propanoic acid [His*], producing both enantiomers with high yield and optical purity. We then designed various analogs of ultra-short AMPs by inserting His* at specific positions, evaluating their antimicrobial properties with different acyl chain lengths (C16 and C12) at the N-terminus and the C-terminus. We were able to identify a very promising candidate for applications (P8) characterized by resistance to proteolysis and enhanced biological effectiveness.

A pentaALFA peptide may be reacted with a SNAP- or HaloTag and allows for signal enhancement using fluorescently labelled nanobodies. Tagged and peptide/nanobody treated cell surface receptors give generally higher signal intensity using fluorescence microscopy and are amenable for stimulated emission by depletion (STED) super-resolution imaging.

ABSTRACT

Self-labelling proteins like SNAP- and HaloTag have advanced imaging in life sciences by enabling live-cell labeling with fluorophore-conjugated substrates. However, the typical one-fluorophore-per-protein system limits signal intensity. To address this, we developed a strategy using the ALFA-tag system, a 13-amino acid peptide recognized by a bio-orthogonal and fluorescently labelled nanobody, for signal amplification. We synthesized a pentavalent ALFA₅ peptide and used an azidolysine for conjugation with a Cy5-modified SNAP- or HaloTag ligand through strain-promoted click chemistry. In vitro measurements on SDS-PAGE showed labelling, and the peptides covalently reacted with their respective tag. HEK293 cells expressing SNAP- and HaloTag-mGluR2 fusion proteins were labeled with ALFA₅-Cy5 substrates, and confocal microscopy revealed a significant enhancement in the far-red signal intensity upon nanobody addition, as quantified by integrated signal density ratios. Comparisons between SNAP- and HaloTag substrates showed superior performance for the latter, achieving better signal-to-noise and signal-to-background ratios, as well as overall signal intensity in plasma membrane-localized regions. Our results demonstrate the potential of ALFA-tag-based systems to amplify SLP fluorescent signals. This strategy combines the photostability of synthetic fluorophores with multivalent labeling, providing a powerful tool for advanced imaging applications including super-resolution in cells. Its versatility is expandable across diverse protein systems and colors.

Proposed green chemical synthesis scheme for efficient preparation of a (hypothetical) 20 residue peptide. Aqueous SPPS employing N-carboxyanhydrides (NCAs) minimal side chain protection, sample displacement mode HPLC purification and convergent condensation of unprotected peptide segments by native chemical ligation in aqueous solution.

ABSTRACT

This perspective essay will briefly recount fundamental physicochemical properties of the peptide-resin that have led to the almost universal use of stepwise solid phase peptide synthesis (SPPS) for the chemical synthesis of peptides. The essay discusses multiple aspects that must be addressed if we are to develop truly green chemical peptide synthesis. An optimal SPPS approach that retains the advantages inherent to polymer-supported chemical synthesis, combined with convergent synthesis based on modern chemical ligation methods for the condensation of unprotected peptide segments, will be described as a path to green synthesis of peptides and their efficient manufacture. Only the most pertinent primary literature is cited.