Author: Oleg Melnyk

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.

Cytosolic acquisition of various cell-penetrating peptides through translocation across the plasma membrane (direct translocation) is inhibited by plasma membrane depolarization in all tested cell lines.

ABSTRACT

Cell-penetrating peptides (CPPs) are small peptides that can carry bioactive cargoes into cells. CPPs access the cell’s cytosol via direct translocation across the plasma membrane. We and others have shown that direct translocation of CPPs occurs through water pores that are formed upon hyperpolarization of the cell’s membrane. Direct translocation through water pores can therefore be blocked by depolarizing the plasma membrane. Other direct translocation mechanisms have been proposed that would not rely on membrane hyperpolarization. It has been reported, for example, that in HEK cells, CPP translocation occurs in a plasma membrane potential-independent manner, in contrast to HeLa cells, where CPP access to the cytosol required plasma membrane hyperpolarization. To address these apparent discrepant data, we have tested the requirement of plasma membrane hyperpolarization in a series of cell lines, including HEK and HeLa cells, for CPP direct translocation. Our data, obtained from a wide range of CPP concentrations, show that efficient direct translocation always requires plasma membrane hyperpolarization. We discuss the possible reasons why earlier studies have not evidenced the importance of the plasma membrane potential in the cytosolic uptake of CPPs in some cell lines.

No abstract is available for this article.

Keynote speakers Florine Cavelier Team Stereoselective synthesis & modified amino acids IBMM, CNRS, Univ. Montpellier, France website Two decades of Neurotensin research for therapeutic advances Anna Maria Papini PeptLab Università di Firenze, Italy website From peptide-based drugs and diagnostics to cosmetics to inspire next generation EU researchers to transfer academic results into the market Invited …

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This work examines the interaction between farnesyltransferase (FTase) and cell-permeable CaaX peptides. Particularly, peptide CaaX-1 is presented as efficient binder to FTase capable to inhibit further interaction of other CaaX motif–bearing peptides. Moreover, biological effects require an intact CaaX motif. Thus, we suggest CaaX peptides as versatile tools to study protein prenylation.

ABSTRACT

Recently, we presented cell-permeable CaaX peptides as versatile tools to study intracellular prenylation of proteins. These peptides consist of a cell-penetrating peptide (CPP) and a C-terminal CaaX motif derived from Ras proteins and demonstrated high cellular accumulation and the ability to influence Ras signaling in cancerous cells. Here, we aimed to gain a deeper insight into how such cell-permeable CaaX peptides, particularly the KRas4B-derived CaaX-1 peptide, interact with farnesyltransferase (FTase) and likely influence further intracellular processes. We show that CaaX-1 is farnesylated by FTase ex cellulo and that an intact CaaX motif is required for modification. A competition experiment revealed a slower farnesylation of CaaX-1 by FTase compared to a CaaX motif-containing control peptide. CaaX-1 inhibited farnesylation of this control peptide at considerably lower concentrations; thus, a higher affinity for FTase is hypothesized. Notably, AlphaFold3 not only predicted interactions between CaaX-1 and FTase but also suggested interactions between the peptide and geranylgeranyltransferase type I. This finding encourages further investigation, as cross-prenylation is a well-known drawback of FTase inhibitors. Our results are further evidence for the usefulness of CaaX peptides as tools to study and manipulate the prenylation of proteins. They offer real potential for the development of novel inhibitors targeting the prenylation pathway.

The synthesis of peptide hydrazides on hydrazine 2CT and hydrazone resin was optimized using CMR-free reagents and solvents. The suggested protocols allow for reduced reagent consumption and the avoidance of DMF and DCM. The obtained results can simplify the synthesis of peptide building blocks for native chemical ligation.

ABSTRACT

Peptide hydrazides are widely applied as precursors of peptide thioesters, valuable building blocks for the synthesis of proteins by native chemical ligation. In addition, they can be applied for the selective modification of cargo or carrier molecules using hydrazone ligation technique. In this work, we describe key aspects of solid phase synthesis of peptide hydrazides on hydrazine 2CT and hydrazone resin. Special attention is paid to the optimization of synthetic procedures using “preferred” and “usable” organic solvents. Thus, optimization of 2-CTC resin loading with Fmoc-hydrazine permits to reduce reagents consumption and avoid DMF and DCM application. The final products can be released from the polymer support with simultaneous BOC removal with 5% HCl (aq) in acetone. Although this protocol demands subsequent peptide deprotection to remove other protecting groups, it benefits of significantly reduced TFA consumption. Because of improved stability in acidic conditions and the possibility of selective Mtt removal and peptide cleavage in green solvents, hydrazone resin can be considered as a useful alternative for peptide hydrazides synthesis. Obtained results can simplify the synthesis of peptide building blocks for native chemical ligation using CMR-free reagents and solvents.

We have attached a wide range of blocking groups to glucagon with the intention of interfering with the quaternary interactions that drive self-association. In so doing, we have identified multiple groups that are able to significantly block fibril formation.

ABSTRACT

Protein self-interaction leading to aggregation is a major challenge facing protein pharmaceuticals. It leads to a range of problems, including increases in immunogenicity and loss of activity. In this work, we describe an approach for blocking or antagonizing the quaternary interactions that drive self-association. We applied the approach to glucagon, a therapeutic peptide known for its propensity to form fibrils due to self-interaction. We synthesized a regio-pure common feedstock that allowed easy modification with potential blocking peptides that represented a range of chemical types (anionic, cationic, polar, and nonpolar). From these synthesized materials, we identified two modified glucagons that showed significant stabilization against fibril formation compared with unmodified glucagon. This was confirmed by three complementary biophysical techniques. Both successful modifications introduced excess net charge to glucagon, consistent with overall electrostatic repulsion being at the root of the observed fibrillation resistance. This approach can potentially be applied to other therapeutic proteins that suffer from the problems associated with self-association.

No abstract is available for this article.

A novel GAPDH-derived peptide JFP-2826, which showed selective antimicrobial activity against marine-specific Vibrio bacteria, was identified from the transcriptome of Rhopilema esculentum. Jellyfish GAPDH may have a newly discovered antimicrobial-related function conducted by releasing JFP-2826-like cryptic peptides.

ABSTRACT

Marine organisms serve as a rich source of bioactive natural compounds, including antimicrobial agents. Jellyfish, which are ancient marine invertebrates with hundreds of millions of years of evolutionary history, have been in continuous contact with a diverse array of pathogenic microorganisms from seawater, which may give rise to a distinctive innate immune system and related defensive molecules. However, it is difficult and inefficient to isolate active ingredients directly from jellyfish for enrichment, though few jellyfish-sourced antimicrobial peptides (AMPs) have been reported. In this study, we utilized transcriptomic big data with bioinformatic tools to dig deeper into potential antimicrobial components in jellyfish, and identified a new AMP JFP-2826 from Rhopilema esculentum. The 20-mer peptide exhibited an alpha-helix structure and showed antimicrobial activity against selected bacterial strains; more importantly, JFP-2826 demonstrated good selectivity for marine-specific Vibrio including Vibrio vulnificus. Sequence analysis of the full-length protein of JFP-2826 revealed that it is derived from the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which is probably produced through enzymatic cleavage of the N-terminal fragment. This suggests that GAPDH of jellyfish might have a newly discovered antimicrobial-related function that is conducted by releasing JFP-2826-like cryptic peptides. JFP-2826 can be subjected to further structural modifications and optimizations to potentially become a potent lead peptide for the development of novel antimicrobial drugs treating infections of marine pathogens.

The integration of a polyarginine cell penetrating peptide with a GRP78-targeting peptide provides selective and effective peptide-based siRNA transfection directly within the GRP78 overexpressing prostate cancer (DU145) cells.

ABSTRACT

This study investigated a peptide-based GRP78-targeting strategy for short-interfering (si) RNA delivery in cancer cells. Synthetic fluorescein-labeled amphiphilic peptides composed of the hydrophobic cell surface (cs) GRP78-targeting and hydrophilic, polycationic arginine-rich cell penetrating peptides demonstrated GRP78-dependent cell uptake in the DU145 prostate cancer cells, and to a lesser extent in the non-cancerous human lung fibroblast WI-38 cell line. Mechanistic studies revealed energy-dependent GRP78 receptor-mediated endocytosis of the GRP78-targeting peptide with polyarginine (W1-R9). The cytosolic accumulation of this peptide underscored its potential utility in siRNA delivery. Peptide:siRNA complexes formed stably condensed nanoparticles, with calcium functioning as an ionic stabilizer and additive promoting endosomal siRNA escape for RNA interference (RNAi) activity. Preliminary peptide-based siRNA transfections in the DU145 cells demonstrated that GRP78 knockdown led to an interplay in between pro-survival and cell death outcomes under ER stress induction. Thus, the GRP78-targeting polyarginine peptides enables efficient cell uptake for specific siRNA delivery in the DU145 cells. This class of bio-active synthetic peptides is important for the investigation of cancer biology, leading to the innovation of cancer-targeted gene delivery and therapy approaches.

Interaction analysis of the antibody mimetic peptide H1H3s with its target protein through cross-linking mass spectrometry confirmed the proposed binding site. Binding and virus neutralizing properties were preserved in a proteolytically stable D-amino acid peptide variant. K➔D exchanges yielded a peptide that assembles into nanofibrils and enhances retroviral infection.

ABSTRACT

Antibody mimetic peptides have evolved as versatile tools for biomedical applications, based on their ability to interfere with protein–protein interactions. We had previously designed a functional mimic of the broadly neutralizing HIV-1 antibody b12 that recognizes the CD4 binding site of the HIV-1 envelope glycoprotein gp120. The molecular details of the interaction of a linear variant of this peptide (H1H3s) with gp120 have now been characterized through cross-linking mass spectrometry, confirming the proposed involvement of the CD4 binding site of gp120 in the interaction. In addition, a variant of the b12 mimetic peptide composed mostly of D-amino acids was shown to be stable towards proteolytic degradation, while the binding and HIV-1 neutralizing properties were largely preserved. Furthermore, a peptide variant in which aspartate residues were replaced with lysine was shown to strongly enhance infection of cells with HIV-1 and GALV glycoprotein pseudotyped viral vectors, respectively, introducing this peptide as a tool to facilitate retroviral gene transfer. Collectively, the presented results highlight the versatile potential therapeutic and gene transfer applications of H1H3s and its variants in particular, as well as antibody mimetic peptides in general.

The Insect Antimicrobial Peptide Database (IAMPDB) represents a specialized database of insect-derived antimicrobial peptides (IAMPs). The data are sourced from UniProt and its associated literature from PubMed. Currently, the database holds 438 entries of IAMPs from 148 insects across 10 taxonomical insect orders. The data can be used to target 181 microorganisms and study 33 activities. Researchers, scientists, and pharmaceutical industries can utilize this database for drug development, disease-resistant crop development, and more.

ABSTRACT

Insects, a majority of animal species, rely on innate immunity and antimicrobial peptides (AMPs), which are a part of their innate immunity, to combat diverse parasites and pathogens. These peptides have applications ranging from agriculture to antimicrobial resistance (AMR). However, there is a lack of a specialized database, prompting the development of the Insect Antimicrobial Peptide Database (IAMPDB) as a pioneering comprehensive Knowledgebase dedicated to

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nsect-derived antimicrobial peptides (IAMPs), serving as a resource for researchers and industry professionals. Curated from UniProt and associated literature(s), IAMPDB currently houses 438 curated entries of IAMPs from various insect species, spanning 10 taxonomical orders of insects. Each entry is meticulously annotated with details on peptide sequence, source organism, activities, physicochemical properties, and more. IAMPDB offers a user-friendly interface with diverse search options, interactive visualizations, and links to external databases; advanced tools, including a peptide sequence alignment toolbox and a peptide feature calculation toolbox, facilitating sequence alignment, physicochemical property calculation, and in-depth analysis. The knowledgebase is accessible online (at URL https://bblserver.org.in/iampdb/).

Guidelines from regulatory agencies support the analysis and quality control of peptide and protein drugs. Key aspects of the analysis process are summarized here.

ABSTRACT

Peptides and proteins have become increasingly important in the treatment of various diseases, including infections, metabolic disorders, and cancers. Over the past decades, the number of approved peptide- and protein-based drugs has grown significantly, now accounting for about 25% of the global pharmaceutical market. This increase has been recorded since the introduction of the first therapeutic peptide, insulin, in 1921. Therapeutic peptides and proteins offer several advantages over small molecule drugs, including high specificity, potency, and safety; however, they also face challenges related to instability in liquid formulations. To address this issue, numerous formulation techniques have been developed to enhance their stability. In either state, physical and chemical characterization of the peptide or protein of interest is crucial for ensuring the identity, purity, and activity of these therapeutic agents. Regulatory bodies such as the FDA, ICH, and EMA have established guidelines for the analysis, stability testing, and quality control of peptides and biologics to ensure the safety and effectiveness of these drugs. In the present review, these guidelines and the consequences thereof are summarized and provided to support the notion of developing tailored bioanalytical workflows for each peptide or protein drug.

A divergent synthesis strategy was developed for producing various anabaenopeptins (AP) for harmful algal bloom monitoring. The synthesis involved on-resin stepwise pentapeptide assembly on a MeDbz linker, then N-α-ureido amino acid attachment and cyclization. This approach proved effective as a general platform for anabaenopeptin synthesis, allowing rapid access to anabaenopeptins A, B, F, and oscillamide Y.

ABSTRACT

A divergent synthesis strategy was developed for producing various anabaenopeptins (AP) for harmful algal bloom monitoring. The synthesis involved on-resin stepwise pentapeptide assembly on a MeDbz linker then N-α-ureido amino acid attachment and cyclization. To manage N-methylated amino acids, modified coupling conditions were employed. Lysine’s ε-amino group reacted with the activated MeDbz linker in a self-cleaving head-to-side chain cyclization. Cyclization conditions were optimized by screening different pH levels to control lysine α-amine cyclization and prevent hydrolysis. Global cleavage and purification afforded the pure anabaenopeptins. This approach proved effective as a general platform for anabaenopeptin synthesis, allowing rapid access to anabaenopeptins A, B, F, and oscillamide Y.

The self-assembly and bioactivity of the lipopeptide lipoyl-KTTKS which comprises lipoic acid conjugated to a collagen-stimulating pentapeptide are examined. Self-assembly into curly fibrils (wormlike micelles) is observed, these structures being dissociated in the presence of a chemical reductant. The lipopeptide also undergoes photo-degradation in the presence of UV radiation. Lipoyl-KTTKS has excellent cytocompatibility, stimulates collagen production and enhances the rate of cell repopulation in a simple in vitro scratch assay.

ABSTRACT

Lipoic acid is a biocompatible compound with antioxidant activity that is of considerable interest in cosmetic formulations, and the disulfide group in the N-terminal ring confers redox activity. Here, we study the self-assembly and aspects of the bioactivity of a lipopeptide (peptide amphiphile) comprising the KTTKS collagen-stimulating pentapeptide sequence conjugated to an N-terminal lipoic acid chain, lipoyl-KTTKS. Using SAXS, SANS and cryo-TEM, lipoyl-KTTKS is found to form a population of curly fibrils (wormlike micelles) above a critical aggregation concentration. Upon chemical reduction, the fibrils (and β-sheet structure) are disrupted because of the breaking of the disulfide bond, which produces dihydrolipoic acid. Lipoyl-KTTKS also undergoes photo-degradation in the presence of UV radiation. Through cell assays using fibroblasts, we found that lipoyl-KTTKS has excellent cytocompatibility across a wide concentration range, stimulates collagen production, and enhances the rate of cell coverage in a simple in vitro scratch assay of ‘wound healing’. Lipoyl-KTTKS thus has several notable properties that may be useful for the development of cosmetics, cell scaffolds or tissue engineering materials.

A post-docking analysis was used to explore the binding modes formed between 44 peptides identified by phage display and nine proteins overexpressed in HeLa and MDA-MB-231 cell lines, facilitating the selection of the best peptide candidates.

ABSTRACT

Peptides binding overexpressed breast and cervical cancer cell surface proteins can be isolated by phage display technology, and their affinity to their potential receptors can be assessed by molecular docking. We isolated 44 phage clones displaying dodecapeptides with high affinity to HeLa cervical cancer and MDA-MB-231 (MDA) breast cancer cells by repeated biopanning of an MK13 phage library and explored their affinity to specific proteins by molecular docking. Six peptides appeared repeatedly during biopanning: two with affinity to HeLa (H5/H21), and four with affinity to MDA cells (M3/M7/M15/M17). Peptide pairs M3/H5 and H1/M17 had affinity to both cell lines. A systematic review identified Annexin A2, EGFR, CD44, CD146, and Integrin alpha V as potential protein targets in HeLa cells, and Vimentin, Galectin-1, and Annexins A1 and A5 in MDA cells. Via virtual screening, we selected six peptides with the highest total docking scores: H1 (−916.32), H6 (−979.21), H19 (−1093.24), M6 (−732.21), M16 (−745.5), and M19 (−739.64), and identified that docking scores were strengthened by the protein type, the interacting amino acid side chains, and the polarity of peptides. This approach facilitates the selection of relevant peptides that could be further explored for active targeting in cancer diagnosis and treatment.

L’article Peptide mimetics, peptoids and peptide nucleic acids est apparu en premier sur Groupe Français des Peptides et Protéines.

YdgR transporter from E. coli selectively transports the fluorescence reporter substrate β-Ala-Lys(AMCA), but modifications such as reducing the lysine chain or substituting or altering the AMCA fluorophore block transport.

ABSTRACT

Fluorescent probes are widely used in cellular imaging and disease diagnosis. Acting as substitute carriers, fluorescent probes can also be used to help transport drugs within cells. In this study, commonly used fluorophores, TAMRA (5-carboxytetramethylrhodamine), PBA (1-pyrenebutyric acid), NBD (nitrobenzoxadiazole), OG (Oregon Green), and CF (5-carboxyfluorescein) were conjugated with the dipeptide β-Ala-Lys, the peptide moiety of the well-established peptide transporter substrate β-Ala-Lys(AMCA) (AMCA: 7-amino-4-methyl-coumarin-3-acetic acid) by modifying it with respect to side-chain length and functional end groups. The analogs were tested for transport through or inhibition of YdgR, a prototypical peptide transporter from E. coli and apparently homologous to the human PEPT1. Strikingly, none of the dipeptide-fluorophore conjugates nor minor modifications in the reporter substrate were tolerated by YdgR, indicating discrepancies to PEPT1. These findings underscore intricate substrate recognition mechanisms governing substrate recognition by YdgR.

Multiepitope DNA constructs harboring the immunogenic and conserved epitopes of the L1, L2 and E7 proteins of HPV16/18 were delivered using KALA and REV cell-penetrating peptides. The stability of the REV/DNA and KALA/DNA complexes against DNase I and serum protease was investigated and their entry into HEK-293T eukaryotic cells was analyzed qualitatively and quantitatively. The anti-tumor effects of the peptide/DNA complexes were investigated in the C57BL/6 mouse model.

ABSTRACT

Developing human papillomavirus (HPV) therapeutic DNA vaccines requires an effective delivery system, such as cell-penetrating peptides (CPPs). In the current study, the multiepitope DNA constructs harboring the immunogenic and conserved epitopes of the L1, L2, and E7 proteins of HPV16/18 (pcDNA-L1-L2-E7 and pEGFP-L1-L2-E7) were delivered using KALA and REV CPPs with different properties in vitro and in vivo. Herein, after confirmation of the REV/DNA and KALA/DNA complexes, their stability was investigated against DNase I and serum protease. Then, their entry into HEK-293T eukaryotic cells was analyzed by qualitative and quantitative methods. Finally, anti-tumor effects of the peptide/DNA complexes were investigated in the C57BL/6 mouse model. Based on the obtained data, the REV/DNA and KALA/DNA complexes at the N/P ratio of 5:1 demonstrated successful penetration into HEK-293T cells. Furthermore, in vivo studies represented that the REV/DNA (survival rate: 75%) and KALA/DNA (survival rate: 50%) complexes provided significant protection against C3 tumors in mice. Indeed, REV CPP exhibited a higher survival rate and lower tumor volume than KALA CPP, 50 days after the C3 challenge. These findings represented the potential of KALA and REV CPPs, especially REV, as promising gene delivery systems for developing HPV therapeutic DNA vaccine candidates.

No abstract is available for this article.

The natural Short Neuropeptide F (sNPF) from the pea aphid (Acyrthosiphon pisum) was used as lead compound to design five novel sNPF analogs, altering the N-terminal amino acid to Ser, Thr, Tyr, Leu, or Gln. Aphid bioassays showed that the analog I-3 was more active than the natural Acypi-sNPF-1 and pymetrozine. I-3 can be utilized as a selective and environmentally friendly insecticide to manage pea aphids.

ABSTRACT

Short neuropeptide F (sNPF) is an insect-specific neuropeptide named for its C-terminal phenylalanine. It consists of 6–19 amino acids with a conserved RLRFa structure, regulating feeding, growth, circadian rhythms, and water-salt balance in insects. Its receptor belongs to GPCR-As and binds sNPF to regulate the insect nervous system. Many research groups are evaluating sNPF for plant protection and pest control. In this study, the natural sNPF from the pea aphid (Acyrthosiphon pisum) was used as a lead compound. Five novel sNPF analogs were designed and synthesized through molecular docking and peptidomimetics, altering the N-terminal amino acid to Ser, Thr, Tyr, Leu, or Gln. Aphid bioassays showed that the analog I-3 (YLRLRFa, LC₅₀ = 1.820 mg/L) was more active than the natural Acypi-sNPF-1 and pymetrozine. The structure–activity relationship analysis indicated that N-terminal tyrosine incorporation, combined with increased ClogP and TPSA, enhanced aphidicidal activity. Furthermore, Toxtree’s toxicity predictions suggest a low risk for all compounds, and a toxicity assay conducted on the honeybee (Apis mellifera) for I-3, which exhibits high aphidicidal activity, indicates that I-3 does not pose a toxicity risk to non-target organisms. Thus, I-3 can be utilized as a selective and environmentally friendly insecticide to manage pea aphids.

L’article test-FH est apparu en premier sur Groupe Français des Peptides et Protéines.

PepFuNN is a new open-source version of the PepFun package with functions to study the chemical space of peptide libraries and perform structure–activity relationship analyses. It is a Python package comprising five modules to study peptides with natural amino acids and, in some cases, sequences with non-natural amino acids based on the availability of a public monomer dictionary.

ABSTRACT

We present PepFuNN, a new open-source version of the PepFun package with functions to study the chemical space of peptide libraries and perform structure–activity relationship analyses. PepFuNN is a Python package comprising five modules to study peptides with natural amino acids and, in some cases, sequences with non-natural amino acids based on the availability of a public monomer dictionary. The modules allow calculating physicochemical properties, performing similarity analysis using different peptide representations, clustering peptides using molecular fingerprints or calculated descriptors, designing peptide libraries based on specific requirements, and a module dedicated to extracting matched pairs from experimental campaigns to guide the selection of the most relevant mutations in design new rounds. The code and tutorials are available at https://github.com/novonordisk-research/pepfunn.

Peptides were comprehensively summarized in the aspects of sources, function, the use of peptides in cosmetics and skin care, and indications for the delivery of cosmetic peptides. Attention was also given to the development of cosmetic peptides in the future.

ABSTRACT

Peptides are molecules that consist of at least two amino acids linked by peptide bonds. The difference between peptides and proteins is primarily based on size and structure. Typically, oligopeptides consist of fewer than about 10–20 amino acids, and polypeptides consist of more than 20 amino acids, whereas proteins usually are made up more than 50 amino acids and often contain multiple peptide subunits as stated in the International Union of Pure and Applied Chemistry rules. Beyond the nutritional properties, peptides are also structural components of hormones, enzymes, toxins, and antibiotics and play several fundamental physiological roles in the body. Since the introduction of the first commercial peptide drug, insulin, peptide-based drugs have gained increased interest. So far, more than 80 peptide-based drugs have reached the market for a wide range of conditions, such as diabetes, cardiovascular diseases, and urological disorders. Meanwhile, peptides have also gained significant attention in the cosmetic industry because of their potential in boosting skin health. In this review, peptides were comprehensively summarized in the aspects of sources, function, the use of peptides in cosmetics and skin care, and indications for the delivery of cosmetic peptides.