Cationic Amphipathic Peptides Research Articles

N-terminal modification of an LAH4-derived peptide increases mRNA delivery in the presence of serum.

The recently developed mRNA-based coronavirus SARS-CoV-2 vaccines highlighted the great therapeutic potential of the mRNA technology. Although the lipid nanoparticles used for the delivery of the mRNA are very efficient, they showed, in some cases, the induction of side effects as well as the production of antibodies directed against particle components. Thus, the development of alternative delivery systems is of great interest in the pursuit of more effective mRNA treatments. In the present work, we evaluated the mRNA transfection capacities of a series of cationic histidine-rich amphipathic peptides derived from LAH4. We found that while the LAH4-A1 peptide was an efficient carrier for mRNA, its activity was highly serum sensitive. Interestingly, modification of this cell penetrating peptide at the N-terminus with two tyrosines or with salicylic acid allowed to confer serum resistance to the carrier.

Killing of Plasmodium Sporozoites by Basic Amphipathic α-Helical Fusion Peptides.

Membranolytic molecules constitute the first line of innate immune defense against pathogenic microorganisms. Plasmodium sporozoites are potentially exposed to these cytotoxic molecules in the hemolymph and salivary glands of mosquitoes, as well as in the skin, blood, and liver of the mammalian host. Here, we show that sporozoites are resistant to bacteriolytic concentration of cecropin B, a cationic amphipathic antimicrobial insect peptide. Intriguingly, anti-tumoral cell-penetrating peptides derived from the anti-apoptotic protein AAC11 killed P. berghei and P. falciparum sporozoites. Using dynamic imaging, we demonstrated that the most cytotoxic peptide, called RT39, did not significantly inhibit the sporozoite motility until the occurrence of a fast permeabilization of the parasite membrane by the peptide. Concomitantly, the cytosolic fluorescent protein constitutively expressed by sporozoites leaked from the treated parasite body while To-Pro 3 and FITC-labeled RT39 internalized, respectively, binding to the nucleic acids and membranes of sporozoites. This led to an increase in the parasite granularity as assessed by flow cytometry. Most permeabilization events started at the parasite's posterior end, resulting in the appearance of a fluorescent dot in the anterior part of sporozoites. Understanding and exploiting the susceptibility of sporozoites and other plasmodial stages to membranolytic molecules might foster strategies to eliminate the parasite and block its transmission.

Materials derived from the human elastin-like polypeptide fusion with an antimicrobial peptide strongly promote cell adhesion.

Protein and peptide materials have attracted great interest in recent years, especially for biological applications, in light of their possibility to easily encode bioactivity whilst maintaining cytocompatibility and biodegradability. Heterologous recombinant expression to produce antimicrobial peptides is increasingly considered a convenient alternative for the transition from conventional methods to more sustainable production systems. The human elastin-like polypeptide (HELP) has proven to be a valuable fusion carrier, and due to its cutting-edge properties, biomimetic materials with antimicrobial capacity have been successfully developed. In this work, we have taken advantage of this platform to produce a difficult-to-synthesise sequence as that of the human β-defensin 1 (hBD1), an amphipathic cationic peptide with structural folding constraints relevant to its bioactivity. In the design of the gene, highly specific endoproteinases recognition sites were introduced to release the active forms of hBD1. After the expression and purification of the new fusion construct, its biological activity was evaluated. It was found that both the fusion biopolymer and the released active forms can inhibit the growth of Escherichia coli in redox environments. Remarkably, 2D and 3D materials derived from the biopolymer showed a strong cell adhesion-promoting activity. These results suggest that HELP represents a multitasking platform that not only facilitates the production of bioactive domains and derived materials but could also pave the way for the development of new approaches to study biological interactions at the molecular level.

D- and N-Methyl Amino Acids for Modulating the Therapeutic Properties of Antimicrobial Peptides and Lipopeptides.

Here we designed and synthesized analogs of two antimicrobial peptides, namely C10:0-A2, a lipopeptide, and TA4, a cationic α-helical amphipathic peptide, and used non-proteinogenic amino acids to improve their therapeutic properties. The physicochemical properties of these analogs were analyzed, including their retention time, hydrophobicity, and critical micelle concentration, as well as their antimicrobial activity against gram-positive and gram-negative bacteria and yeast. Our results showed that substitution with D- and N-methyl amino acids could be a useful strategy to modulate the therapeutic properties of antimicrobial peptides and lipopeptides, including enhancing stability against enzymatic degradation. The study provides insights into the design and optimization of antimicrobial peptides to achieve improved stability and therapeutic efficacy. TA4(dK), C10:0-A2(6-NMeLys), and C10:0-A2(9-NMeLys) were identified as the most promising molecules for further studies.

Evolutionary trend of bovine β-defensin proteins toward functionality prediction: A domain-based bioinformatics study

Defensins are small cationic cysteine-rich and amphipathic peptides that form of three-dimensional β-strand structure connected by disulfide bonds. Defensins form key elements of the innate immune system of multicellular organisms. They not only possess broad-spectrum antimicrobial activity but also have diverse roles, including cell signaling, ion channel agitation, toxic functions, and enzyme inhibitor activities in various animals. Although the role of β-defensins in immune responses against infectious agents and reproduction could be significant, inadequate genomic information is available to explain the whole β-defensin repertoire in cattle. No domain or motif-based functional analyses have been previously reported. In addition, how do defensins possess this magnitude of functions in the immune system is still not clear. Our present study, therefore, investigated the sequence divergence and evolutionary relations of bovine defensin proteins with those of humans. Our domain-based evolutionary analysis revealed four major clusters with significant domain variation while reserving a main antimicrobial activity. Our study revealed the β-defensin domain as the ancestor domain, and it is preserved in the first group of defensin protein with no α-helix in its structure. Due to natural selection, some domains have evolved independently within clusters II and III, while some proteins have lost their domain characteristics. Cluster IV contains the most recently evolved domains. Some proteins of all but cluster I might have adopted the functional characteristics of α-defensins which is largely absent in cattle. The proteins show different patterns of disulfide bridges and multiple signature patterns which might render them specialized functions in different tissue to combat against various pathogens.

Synthesis and antimicrobial activity of aminoalkyl resveratrol derivatives inspired by cationic peptides

Antimicrobial resistance is a global concern, far from being resolved. The need of new drugs against new targets is imminent. In this work, we present a family of aminoalkyl resveratrol derivatives with antibacterial activity inspired by the properties of cationic amphipathic antimicrobial peptides. Surprisingly, the newly designed molecules display modest activity against aerobically growing bacteria but show surprisingly good antimicrobial activity against anaerobic bacteria (Gram-negative and Gram-positive) suggesting specificity towards this bacterial group. Preliminary studies into the action mechanism suggest that activity takes place at the membrane level, while no cross-resistance with traditional antibiotics is observed. Actually, some good synergistic relations with existing antibiotics were found against Gram-negative pathogens. However, some cytotoxicity was observed, despite their low haemolytic activity. Our results show the importance of the balance between positively charged moieties and hydrophobicity to improve antimicrobial activity, setting the stage for the design of new drugs based on these molecules.

Structure-Based Rational Design of Small α-Helical Peptides with Broad-Spectrum Activity against Multidrug-Resistant Pathogens.

A series of small (7-12 mer) amphipathic cationic peptides were designed and synthesized to create short helical peptides with broad-range bactericidal activity and selectivity toward the bacterial cells. The analysis identified a lead 12-mer peptide 8b with broad-spectrum activity against Gram-positive (MIC = 3.1-6.2 μg/mL) and Gram-negative (MIC = 6.2-12.5 μg/mL) bacteria and selectivity toward prokaryotic versus eukaryotic cells (HC50 = 280 μg/mL, >75% cell viability at 150 μg/mL). The rapid membranolytic action of 8b was demonstrated by a calcein dye leakage assay and confirmed using scanning electron microscopy. According to circular dichroism and NMR spectroscopy, the peptides have an irregular spatial structure in water. A lipid bilayer induced an amphipathic helix only in 12-mer peptides, including 8b. Molecular dynamics simulations provided detailed information about the interaction of 8b and its closest analogues with bacterial and mammalian membranes and revealed the roles of particular amino acids in the activity and selectivity of peptides.

A new triphenylphosphonium-conjugated amphipathic cationic peptide with improved cell-penetrating and ROS-targeting properties

We study for the first time whether triphenylphosphonium (TPP) moiety can improve cellular delivery and redox properties of amphipathic cationic peptides based on YRFK/YrFK cell-penetrating and cytoprotective motif. TPP moiety was found to increase reducing activity of both stereoisomeric peptides in solution and on electrode surface in association with TPP-mediated intramolecular interactions. Among TPP-conjugated peptides, newly synthesized TPP3-YrFK featured both increased antioxidant efficacy and proteolytic resistance. TPP-conjugated peptides preferably mitigated endogenic ROS in mitochondria and cytoplasm of model glioblastoma cells with increased oxidative status. This anti-ROS effect was accompanied by mild reversible decrease of reduced glutathione level in the cells with relatively weak change in glutathione redox forms ratio. Such low interference with cell redox status is in accordance with non-cytotoxic nature of the compounds. Intracellular concentrations of label-free peptides were analyzed by LC–MS/MS, which showed substantial TPP-promoted penetration of YrFK motif across cell plasma membrane. However, according to ΔΨm analysis, TPP moiety did not profoundly enhance peptide interaction with mitochondrial inner membrane. Our study clarifies the role of TPP moiety in cellular delivery of amphipathic cationic oligopeptides. The results suggest TPP moiety as a multi-functional modifier for the oligopeptides which is capable of improving cellular pharmacokinetics and antioxidant activity as well as targeting increased ROS levels. The results encourage further investigation of TPP3-YrFK as a peptide antioxidant with multiple benefits.

Plasma metabolites-based design of long-acting peptides and their anticancer evaluation

Antimicrobial peptides (AMPs) are generally small cationic amphipathic peptides, which are thought to be ideal antineoplastic agents, owing to their favorable selectivity to cancer cells and the ability to overcome drug-resistance. In this study, an anticancer AMP (Mastoparan (INLKALAALAKKIL-NH2)) was selected as the lead compound and a series of Mastoparan derivatives were designed. Preliminary studies verified that an analogue of Mastoparan, KM8 (KLLKINLKALAALAKKIL-NH2), exhibited prominent selective antitumor effects. Instead, it presents a significant defect of metabolic instability, with a half-life in plasma of only about 0.5 h. Metabolite profiling of KM8 was performed and indicated the structure 9AL10 in peptide sequence could be the fragile site for KM8. Thus, the Aib (unnatural amnio acid) was employed to substitute the 9Ala residue in KM8, and generating a long-acting KM8 derivative, namely KM8-Aib. Further investigations revealed KM8-Aib possessed higher metabolic stability, more potent anticancer activity in vitro &in vivo, and lower toxicity. Therefore, KM8-Aib is suggested be a potential antimalignant agent that worthy of more in-depth study.

Abstract 13518: Augmenting SOD2 Levels in Apoe -/- Mice With Peptide-mRNA Nanoparticles Preserves Atherosclerotic Plaque Stability in Advanced Atherosclerosis

Erosion or rupture of the advanced atherosclerotic plaque is the leading cause of atherosclerosis complications such as acute coronary syndrome and stroke. Atherosclerotic plaques in aging hypercholesterolemic mice have decreased SOD2 levels and increased mitochondrial oxidative stress, which leads to cell apoptosis, necrotic core expansion, and fibrous cap thinning and rupture. We hypothesized that increasing SOD2 expression in advanced atherosclerotic plaque cells in mice using mRNA polyplex would protect mitochondria from oxidative damage and preserve plaque stability. Macrophages isolated from Sod2 +/- mice treated with amphipathic cationic peptide p5RHH: Sod2 mRNA polyplexes had markedly increased levels of Sod2 mRNA and mitochondrial SOD2 (p<0.01), higher SOD activity, which resulted in lower levels of mitochondrial and cellular reactive oxygen species (ROS) and improved mitochondrial function. RNA-Seq analysis showed that macrophages treated with Sod2 nanoplexes had broadly decreased inflammatory cytokines and immune activation pathways. In Apoe -/- mice, atherosclerotic lesions with vulnerable plaque features were induced in the carotid artery by placing a perivascular collar over the carotid artery and 4-months Western diet. Mice were treated for 4 weeks with saline or with 0.5 mg/kg p5RHH: Sod2 mRNA polyplexes which selectively accumulated in atherosclerotic lesion macrophages. The carotid plaques of the mice treated with Sod2 mRNA differed significantly from the plaques of the control Apoe -/- mice by higher SOD2 expression, lower mitochondrial ROS and oxidative DNA damage, and reduced expression of CCL2, IL1, IL6, MMP 2 and 9 (p<0.01). Sod2 polyplexes-treated mouse plaques exhibited 25% fewer inflammatory macrophages (p<0.01) and 98% more smooth muscle cells (p<0.001) with a reduced lipid core, preserved fibromuscular cap, and decreased plaque vulnerability index (p<0.05). The results suggest that selective overexpression of Sod2 in plaque macrophages using nanoplexes reduces mitochondrial stress and preserves plaque stability without adverse effects. Using nanoparticle-based mRNA therapeutics to modulate plaque morphology has great potential in the prevention and treatment of atherosclerosis complications.

In silico assessment of missense point mutations on human cathelicidin LL-37

Cathelicidin antimicrobial peptides are a diverse family of cationic amphipathic peptides with multiple activities. In humans, cathelicidin LL-37 is one of the main host defense peptides with a remarkable medical and biotechnological potential. Deregulation of LL-37 expression has been associated with inflammatory diseases. However the effects of point mutations driven by single nucleotide polymorphisms (SNPs) on LL-37 are unknown. Here we applied an array of computational tools to investigate the effects of such mutations on LL-37 structure and activity. Due to the fact that, on cathelicidins, the prodomain is more conserved than the mature peptide, the SNP effect predictions were biased and, overall, resulted in neutral effects; and due to the slight changes in physicochemical properties, the antimicrobial predictions indicated the maintenance of such activity. Nonetheless, R07P, R07W, R29Q, R29W mutations reduced the peptide net charge, which in turn could result in less active LL-37 variants. Molecular dynamics data indicated that R07Q and N30Y mutations altered the LL-37 structure, leading to potential deleterious effects. In addition, the helix dipole is altered in G03A, R07P, R07W and L31P mutations, which could also alter the antimicrobial activity. Our results indicated that despite the mutations did not alter the residues from LL-37 active core, they could influence the antimicrobial activity and consequently, could be involved in inflammatory diseases.

Роль дефензинів у неспецифічному захисті макроорганізму від інфекційних агентів при запальних захворюваннях порожнини рота і ротоглотки (огляд літератури)

The purpose of the study was to analyze literature sources containing information about defensins, cysteine-rich cationic amphipathic peptides produced by circulating white blood cells and tissue cells. This review describes the antimicrobial, antiviral, anti-inflammatory and immunomodulatory properties of defensins, as well as their molecular and cellular interactions. These substances, which are present on the epithelium and body fluids, are active against bacteria, fungi and viruses, as well as produced by immune and epithelial cells. These natural antimicrobial cationic peptides play an important role in innate and adaptive immunity. Defensins are divided into alpha and beta families. Alpha-defensins (α-defensins) are found in neutrophils, macrophages and Paneth cells in the intestine. Beta-defensins (β-defensins) are secreted by most leukocytes and epithelial cells. Extensive antimicrobial activity and multifaceted immunomodulatory functions of defensins confirm their role in innate immunity as the main protective component of the human body against bacterial, viral and fungal infections. Thus, they are key effector molecules in protecting the organism from infection due to their broad-spectrum antimicrobial activity. Their common antimicrobial function is the formation of destructive pores in the membranes of pathogens, including enveloped viruses. Antiviral activity includes the direct effect of defensin on viral envelopes, glycoproteins and capsids. Binding and modulation of host cell surface receptors and disruption of intracellular signaling by defensins may also inhibit virus replication. These peptides block infection with enveloped and non-enveloped viruses by aggregating particles, blocking receptor binding, inhibiting virus penetration or depletion of particles, inhibiting stem cell signaling, or viral gene expression. In addition, defensins may function as chemokines to enhance and alter adaptive immune responses by exhibiting an indirect antiviral mechanism. Conclusion. However, sources of scientific information have shown that defensins attract immune cells and modulate adaptive immune responses. It has also been shown that defensins can both induce inflammation and suppress inflammatory responses by acting on certain cells through various mechanisms. Due to this, they can be used as one of the markers in the development of inflammatory diseases of the mouth and oropharynx. The main drugs that activate the production of defensins are probiotics, vitamin D and leukotriene B4. This expands the possibility of their use as a new class of non-toxic antimicrobials and immunomodulators

Short peptide analogs of LfcinB synthesized by Solid Phase Peptide Synthesis as an alternative to global microbial resistance

Abstract Antibiotics are the main therapy in the treatment of infections caused by pathogenic bacteria. Their frequent and prolonged use leads to the development and spread of resistant microorganisms. According to the WHO, there is a worrying increase in global resistance to conventional antimicrobial therapy. This in turn poses a serious risk to public health and requires the search for an alternative to traditional pathogen therapy. Antimicrobial peptides (AMPs) are of great interest in this regard. They are produced naturally in all living organisms and are the first line of defense against fungi, viruses and bacteria. An example ofAMP with proven antimicrobial action are lactoferricins. They are cationic amphipathic peptides and have been found in both humans (LfcinH) and other mammals (cattle (LfcinB), mice (LfcinM) and goats (LfcinC). The most powerful antibacterial effect among them shows LfcinB. Using SPPS methods, short peptide molecules were obtained, analogs of LfcinB, which show a stronger antibacterial effect compared to the native peptide. In recent years, lactoferricins have been the subject of a number of scientific studies and represent a potential alternative for the development of new therapies to combat antimicrobial drug resistance.

In Vitro Biophysical Characterization of Candidalysin: A Fungal Peptide Toxin.

In 2016, the first peptide toxin in any human fungal pathogen was identified. It was discovered in Candida albicans and was named candidalysin. Candidalysin is an amphipathic cationic peptide that damages cell membranes. Like most lytic peptides, candidalysin shows alpha-helical secondary structure. As the helicity and the membrane lytic activity of candidalysin are key factors for pathogenicity, here we describe in vitro approaches to monitor both its membrane-lytic function and the secondary structure. First, membrane permeabilization activity of candidalysin is measured in real time by direct electrical recording. Second, the secondary structure and helicity of candidalysin are determined by circular dichroism spectroscopy. These biophysical methods provide a means to characterize the activity and physical properties of candidalysin in vitro and will be useful in determining the structural and functional features of candidalysin and other similar cationic membrane-active peptides.

Hydrophobicity-tuned anion responsiveness underlies endosomolytic cargo delivery mediated by amphipathic vehicle peptides

Peptide conformation can change subject to environment cues. This concept also applies to many cationic amphipathic peptides (CAPs) known to have cell membrane lytic or penetrative activities. Well-conditioned CAPs can match the properties of the target membrane to support their intended biological functions, e.g., intracellular cargo delivery; however, the intricacy in such conditioning surpasses our current understanding. Here we focused on hydrophobicity, a key biophysical property that dictates the membrane activity of CAPs, and applied a structure–function strategy to evolve a template peptide for endosomolytic cargo delivery. The template was subjected to iterative adjustment to balance hydrophobicity between its N-terminal linear and C-terminal helical domains. We demonstrate that the obtained peptide, LP6, could dramatically promote cargo cell entry and facilitate cytosolic delivery of biomacromolecules such as FITC-dextran, saporin, and human IgG. Among the evolved peptide series, LP6 has low cytotoxicity and moderate hydrophobicity, exhibits maximum change in helical conformation in response to negatively charged phospholipids, and also shows an apparent aggregational behavior in response to sialic acid enrichment. These attributes of LP6 collectively indicate that its anion-responsive conformational change is a critical underlining of its endosomolytic cargo delivery capability. Our results also suggest that modulation of hydrophobicity serves as a key to the precise tuning of CAP's membrane activity for future biomedical applications.

Role of antimicrobial peptides in atopic dermatitis.

Host defense peptides (HDPs) or antimicrobial peptides (AMPs) are short cationic amphipathic peptides of divergent sequences, which are part of the innate immune system and produced by various types of cells and tissues. The predominant role of HDPs is to respond to and protect humans against infection and inflammation. Common human HDPs include defensins, cathelicidin, psoriasin, dermcidin, and ribonucleases, but these peptides may be dysregulated in the skin of patients with atopic dermatitis (AD). Current evidence suggests that the antimicrobial properties and immunomodulatory effects of HDPs are involved in AD pathogenesis, making HDPs research a promising area for predicting disease severity and developing novel treatments for AD. In this review, we describe a potential role for human HDPs in the development, exacerbation, and progression of AD and propose their potential therapeutic benefits.

Bacterial-Specific Aggregation and Killing of Immunomodulatory Host Defense Peptides.

This study involves the design and development of disulfide bridge-linked antimicrobial peptides using the host defense protein Angiogenin 4 (chAng4) as a template. The mini peptides derived from chAng4 (mCA4s) were evaluated for their antibacterial efficacies in various pathogenic bacterial strains, and the role of the oxidation state of thiols in the peptide sequence and its implication on antibacterial properties were explored. A remarkable property of these synthetic mCA4 peptides is their capability to flocculate bacteria and mediate bacterial-specific killing, in the absence of any other external stimulus. mCA4s were further evaluated for their cellular uptake, hemolytic activities, toxicities, and immunomodulatory activities in different eukaryotic cell lines. The results indicate that disulfide bridge-containing cationic amphipathic peptides show superior antibacterial efficacies, are nontoxic and nonhemolytic, and mediate bacterial flocculation and killing, in the absence of external stimuli.

The Role of Antimicrobial Peptides in Preterm Birth.

Antimicrobial peptides (AMPs) are short cationic amphipathic peptides with a wide range of antimicrobial properties and play an important role in the maintenance of immune homeostasis by modulating immune responses in the reproductive tract. As intra-amniotic infection and microbial dysbiosis emerge as common causes of preterm births (PTBs), a better understanding of the AMPs involved in the development of PTB is essential. The altered expression of AMPs has been reported in PTB-related clinical presentations, such as preterm labor, intra-amniotic infection/inflammation, premature rupture of membranes, and cervical insufficiency. Moreover, it was previously reported that dysregulation of AMPs may affect the pregnancy prognosis. This review aims to describe the expression of AMPs associated with PTBs and to provide new perspectives on the role of AMPs in PTB.

Self-assembled miRNA-switch nanoparticles target denuded regions and prevent restenosis

Cardiovascular disease is the leading cause of death and disability worldwide. Effective delivery of cell-selective therapies that target atherosclerotic plaques and neointimal growth while sparing the endothelium remains the Achilles heel of percutaneous interventions. The current study utilizes synthetic microRNA switch therapy that self-assembles to form a compacted, nuclease-resistant nanoparticle <200nM in size when mixed with cationic amphipathic cell-penetrating peptide (p5RHH). These nanoparticles possess intrinsic endosomolytic activity that requires endosomal acidification. When administered in a femoral artery wire injury mouse model invivo, the mRNA-p5RHH nanoparticles deliver their payload specifically to the regions of endothelial denudation and not to the lungs, liver, kidney, or spleen. Moreover, repeated administration of nanoparticles containing a microRNA switch, consisting of synthetically modified mRNA encoding for the cyclin-dependent kinase inhibitor p27Kip1 that contains one complementary target sequence of the endothelial cell-specific miR-126 at its 5' UTR, drastically reduced neointima formation after wire injury and allowed for vessel reendothelialization. This cell-selective nanotherapy is a valuable tool that has the potential to advance the fight against neointimal hyperplasia and atherosclerosis.

Rational design and characterisation of an amphipathic cell penetrating peptide for non-viral gene delivery

RALA is a cationic amphipathic peptide which has shown great promise as an efficient, multifunctional delivery system for the delivery of nucleic acids. Rational peptide design was utilised in this study to understand the essential amino acids required for delivery and if any improvements to the RALA peptide could be made. Six amphipathic peptides were synthesised with strategic sequences and amino acid substitutions to reduce peptide sequence, while maintaining the functional characteristics of RALA including amphipathicity, alpha-helicity and pH responsiveness for endosomal escape. Data demonstrated that all six peptides complexed pEGFP-N1 to produce cationic nanoparticles <200 nm in diameter, but not all peptides resulted in successful transfection; indicating the influence of peptide design for cellular uptake and endosomal escape. Pep2, produced nanoparticles with similar characteristics and transfection efficiency to the parent peptide, RALA. However, Pep2 had issues with toxicity and a lack of pH-responsive alpha-helcity. Therefore, RALA remains the superior sequence for non-toxic gene delivery.