Search Results (20)

Background/Objectives: The clinical potential of antimicrobial peptides (AMPs) against dual threats like antimicrobial resistance (AMR) and cancer is often limited by their high host cell toxicity. Here, we focused on brevinin-2OS (B2OS), a novel peptide from the skin of Odorrana schmackeri with potent haemolytic activity. The objective was to study the structure–activity relationship and optimise the safety via targeted modifications. Methods: A dual-modification strategy involving C-terminal truncation and subsequent N-terminal D-amino acid substitution was employed. The bioactivities and safety profiles of the resulting analogues were evaluated using antimicrobial, haemolysis, and cytotoxicity assays. Result: Removal of the rana box in B2OS(1-22)-NH₂ substantially reduced haemolysis while maintaining bioactivities. Remarkably, the D-leucine substitution in [D-Leu²]B2OS(1-22)-NH₂ displayed a superior HC₅₀ value of 118.1 µM, representing a more than ten-fold improvement compared to its parent peptide (HC₅₀ of 10.44 µM). This optimised analogue also demonstrated faster bactericidal kinetics and enhanced membrane permeabilisation, leading to a greater than 22-fold improvement in its therapeutic index against Gram-positive bacteria. Conclusions: The C-terminal rana box is a primary determinant of toxicity rather than a requirement for activity in the B2OS scaffold. The engineered peptide [D-Leu²]B2OS(1-22)-NH₂ emerges as a promising lead compound, and this dual-modification strategy provides a powerful design principle for developing safer, more effective peptide-based therapeutics. Full article

Amphibians use their skin as an effective defense mechanism against predators and microorganisms. Specialized glands produce antimicrobial peptides (AMPs) that possess antioxidant properties, effectively reducing reactive oxygen species (ROS) levels. These peptides are promising candidates for treating diseases associated with oxidative stress (OS) and redox imbalance, including neurodegenerative disorders such as Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease (HD), and amyotrophic lateral sclerosis (ALS), as well as age-related conditions, like cardiovascular diseases and cancer. This review highlights the multifaceted roles of AMPs and antioxidant peptides (AOPs) in amphibians, emphasizing their protective capabilities against oxidative damage. They scavenge ROS, activate antioxidant enzyme systems, and inhibit cellular damage. AOPs often share structural characteristics with AMPs, suggesting a potential evolutionary connection and similar biosynthetic pathways. Peptides such as brevinin-1FL and Cath-KP demonstrate neuroprotective effects, indicating their therapeutic potential in managing oxidative stress-related diseases. The antioxidant properties of amphibian-derived peptides pave the way for novel therapeutic developments. However, a deeper understanding of the molecular mechanisms underlying these peptides and their interactions with oxidative stress is essential to addressing ROS-related diseases and advancing therapeutic strategies in clinical practice. Full article

Background/Objective: Amphibian skin is a valuable source of host defense peptides (HDPs). This study aimed to identify HDPs with novel amino acid sequences from the skin of Rana tagoi yakushimensis and analyze their functions. Methods: cDNAs encoding HDP precursors were cloned and sequenced using RT-PCR and 3′-RACE. The novel HDPs were synthesized to evaluate their antimicrobial activity, antioxidant activity, and cytotoxicity. Antimicrobial activity was evaluated by way of broth microdilution and endotoxin- and β-glucan-binding capacity using an enzyme-linked endotoxin binding assay (ELEBA) and a modified ELEBA, respectively. Results: Nine cDNAs encoding precursors for various HDP families, including temporin, ranatuerin-2, brevinin-1, amurin-9, and a novel yakushimin peptide, were identified. Brevinin-1TYa exhibited antibacterial activity against Staphylococcus aureus, and brevinin-1TYa and amurin-9TYa induced morphological changes in Escherichia coli and S. aureus. Yakushimin-TYa, amurin-9TYa, and brevinin-1TYa showed concentration-dependent antibacterial effects against the plant pathogens Xanthomonas oryzae pv. oryzae and Clavibacter michiganensis subsp. michiganensis. Amurin-9TYa demonstrated strong binding affinity to lipopolysaccharide, lipoteichoic acid, and β-glucan, exhibited antioxidant activity, and lacked cytotoxicity, making it a promising therapeutic candidate. Moreover, brevinin-1TYa showed strong cytotoxicity, whereas yakushimin-TYa exhibited weak cytotoxicity. Conclusions: These findings highlight the potential of these peptides, particularly amurin-9TYa, for future applications as antimicrobial and therapeutic agents. Full article

Antibiotic resistance has emerged as a grave threat to global public health, leading to an increasing number of treatment failures. Antimicrobial peptides (AMPs) are widely regarded as potential substitutes for traditional antibiotics since they are less likely to induce resistance when used. A novel AMP named Brevinin-1BW (FLPLLAGLAASFLPTIFCKISRKC) was obtained by the Research Center of Molecular Medicine of Yunnan Province from the skin of the Pelophylax nigromaculatus. Brevinia-1BW had effective inhibitory effects on Gram-positive bacteria, with a minimum inhibitory concentration (MIC) of 3.125 μg/mL against Enterococcus faecalis (ATCC 29212) and 6.25 μg/mL against both Staphylococcus aureus (ATCC 25923) and multidrug-resistant Staphylococcus aureus (ATCC 29213) but had weaker inhibitory effects on Gram-negative bacteria, with a MIC of ≥100 μg/mL. Studies using scanning electron microscopy (SEM) and flow cytometry have revealed that it exerts its antibacterial activity by disrupting bacterial membranes. Additionally, it possesses strong biofilm inhibitory and eradication activities as well as significant lipopolysaccharide (LPS)-binding activity. Furthermore, Brevinin-1BW has shown a significant anti-inflammatory effect in LPS-treated RAW264.7 cells. In conclusion, Brevinin-1BW is anticipated to be a promising clinical agent with potent anti-Gram-positive bacterial and anti-inflammatory properties. Full article

Peptides released on frogs’ skin in a stress situation represent their only weapon against micro-organisms and predators. Every species and even population of frog possesses its own peptidome being appropriate for their habitat. Skin peptides are considered potential pharmaceuticals, while the whole peptidome may be treated as a taxonomic characteristic of each particular population. Continuing the studies on frog peptides, here we report the peptidome composition of the Central Slovenian agile frog Rana dalmatina population. The detection and top-down de novo sequencing of the corresponding peptides was conducted exclusively by tandem mass spectrometry without using any chemical derivatization procedures. Collision-induced dissociation (CID), higher energy collision-induced dissociation (HCD), electron transfer dissociation (ETD) and combined MS³ method EThcD with stepwise increase of HCD energy were used for that purpose. MS/MS revealed the whole sequence of the detected peptides including differentiation between isomeric Leu/Ile, and the sequence portion hidden in the disulfide cycle. The array of the discovered peptide families (brevinins 1 and 2, melittin-related peptides (MRPs), temporins and bradykinin-related peptides (BRPs)) is quite similar to that of R. temporaria. Since the genome of this frog remains unknown, the obtained results were compared with the recently published transcriptome of R. dalmatina. Full article

Amphibians have been consumed as an alternative protein source all around the world due to their delicacy. The skin of edible amphibians, particularly frogs and giant salamanders, always goes to waste without further utilization. However, these wastes can be utilized to extract protein and bioactive peptides (BPs). Various BPs have been extracted and reported for numerous biological activities such as antioxidant, antimicrobial, anticancer, antidiabetic, etc. The main BPs identified were brevinins, bombesins, dermaseptins, esculentins, magainin, temporins, tigerinins, and salamandrins. This review provides a comprehensive discussion on various BPs isolated and identified from different amphibian skins or skin secretion and their biological activities. The general nutritional composition and production statues of amphibians were described. Additionally, multiple constraints against the utilization of amphibian skin and secretions are reported. Finally, the prospective applications of BPs in food and biomedical industries are presented such as multifunctional food additives and/or supplements as well as drug delivery agents. Full article

Antimicrobial peptides (AMPs) are a diverse class of short, often cationic biological molecules that present promising opportunities in the development of new therapeutics to combat antimicrobial resistance. Newly developed in silico methods offer the ability to rapidly discover numerous novel AMPs with a variety of physiochemical properties. Herein, using the rAMPage AMP discovery pipeline, we bioinformatically identified 51 AMP candidates from amphibia and insect RNA-seq data and present their in-depth characterization. The studied AMPs demonstrate activity against a panel of bacterial pathogens and have undetected or low toxicity to red blood cells and human cultured cells. Amino acid sequence analysis revealed that 30 of these bioactive peptides belong to either the Brevinin-1, Brevinin-2, Nigrocin-2, or Apidaecin AMP families. Prediction of three-dimensional structures using ColabFold indicated an association between peptides predicted to adopt a helical structure and broad-spectrum antibacterial activity against the Gram-negative and Gram-positive species tested in our panel. These findings highlight the utility of associating the diverse sequences of novel AMPs with their estimated peptide structures in categorizing AMPs and predicting their antimicrobial activity. Full article

Several years have passed since the Zika virus (ZIKV) pandemic reoccurred in 2015–2016. However, there is still a lack of proved protective vaccines or effective drugs against ZIKV. The peptide brevinin-2GHk (BR2GK), pertaining to the brevinin-2 family of antimicrobial peptides, has been reported to exhibit only weak antibacterial activity, and its antiviral effects have not been investigated. Thus, we analyzed the effect of BR2GK on ZIKV infection. BR2GK showed significant inhibitory activity in the early and middle stages of ZIKV infection, with negligible cytotoxicity. Furthermore, BR2GK was suggested to bind with ZIKV E protein and disrupt the integrity of the envelope, thus directly inactivating ZIKV. In addition, BR2GK can also penetrate the cell membrane, which may contribute to inhibition of the middle stage of ZIKV infection. BR2GK blocked ZIKV E protein expression with an IC₅₀ of 3.408 ± 0.738 μΜ. In summary, BR2GK was found to be a multi-functional candidate and a potential lead compound for further development of anti-ZIKV drugs. Full article

Brevinins are a well-characterised, frog-skin-derived, antimicrobial peptide (AMP) family, but their applications are limited by high cytotoxicity. In this study, a wild-type des-Leu2 brevinin peptide, named brevinin-1OS (B1OS), was identified from Odorrana schmackeri. To explore the significant role of the leucine residue at the second position, two variants, B1OS-L and B1OS-D-L, were designed by adding L-leucine and D-leucine residues at this site, respectively. The antibacterial and anticancer activities of B1OS-L and B1OS-D-L were around ten times stronger than the parent peptide. The activity of B1OS against the growth of Gram-positive bacteria was markedly enhanced after modification. Moreover, the leucine-modified products exerted in vivo therapeutic potential in an methicillin-resistant Staphylococcus aureus (MRSA)-infected waxworm model. Notably, the single substitution of D-leucine significantly increased the killing speed on lung cancer cells, where no viable H838 cells survived after 2 h of treatment with B1OS-D-L at 10 μM with low cytotoxicity on normal cells. Overall, our study suggested that the conserved leucine residue at the second position from the N-terminus is vital for optimising the dual antibacterial and anticancer activities of B1OS and proposed B1OS-D-L as an appealing therapeutic candidate for development. Full article

Antimicrobial peptides (AMPs) are considered potential alternatives to antibiotics due to their advantages in solving antibiotic resistance. Brevinin-2GUb, which was extracted from the skin secretion of Hylarana guentheri, is a peptide with modest antimicrobial activity. Several analogues were designed to explore the structure–activity relationship and enhance its activity. In general, the Rana box is not an indispensable motif for the bioactivity of Brevinin-2GUb, and the first to the 19th amino acids at the N-terminal end are active fragments, such that shortening the peptide while maintaining its bioactivity is a promising strategy for the optimisation of peptides. Keeping a complete hydrophobic face and increasing the net charges are key factors for antimicrobial activity. With the increase of cationic charges, α-helical proportion, and amphipathicity, the activity of t-Brevinin-2GUb-6K (tB2U-6K), in combatting bacteria, drastically improved, especially against Gram-negative bacteria, and the peptide attained the capacity to kill clinical isolates and fungi as well, which made it possible to address some aspects of antibiotic resistance. Thus, peptide tB2U-6K, with potent antimicrobial activity against antibiotic-resistant bacteria, the capacity to inhibit the growth of biofilm, and low toxicity against normal cells, is of value to be further developed into an antimicrobial agent. Full article

Cancer has always been one of the most common malignant diseases in the world. Therefore, there is an urgent need to find potent agents with selective antitumor activity against cancer cells. It has been reported that antimicrobial peptides (AMPs) can selectively target tumor cells. In this study, we focused on the anti-tumor activity and mechanism of Brevinin-1RL1, a cationic α-helical AMP isolated from frog Rana limnocharis skin secretions. We found that Brevinin-1RL1 preferentially inhibits tumor cells rather than non-tumor cells with slight hemolytic activity. Cell viability assay demonstrated the intermolecular disulfide bridge contributes to the inhibitory activity of the peptide as the antitumor activity was abolished when the disulfide bridge reduced. Further mechanism studies revealed that both necrosis and apoptosis are involved in Brevinin-1RL1 mediated tumor cells death. Moreover, Brevinin-1RL1 induced extrinsic and mitochondria intrinsic apoptosis is caspases dependent, as the pan-caspase inhibitor z-VAD-FMK rescued Brevinin-1RL1 induced tumor cell proliferative inhibition. Immunohistology staining showed Brevinin-1RL1 mainly aggregated on the surface of the tumor cells. These results together suggested that Brevinin-1RL1 preferentially converges on the cancer cells to trigger necrosis and caspase-dependent apoptosis and Brevinin-1RL1 could be considered as a pharmacological candidate for further development as anti-cancer agent. Full article

Amphibians are widely distributed on different continents, except for the polar regions. They are important sources for the isolation, purification and characterization of natural compounds, including peptides with various functions. Innate immune antimicrobial peptides (AMPs) play a critical role in warding off invading pathogens, such as bacteria, fungi, parasites, and viruses. They may also have other biological functions such as endotoxin neutralization, chemotaxis, anti-inflammation, and wound healing. This article documents a bioinformatic analysis of over 1000 amphibian antimicrobial peptides registered in the Antimicrobial Peptide Database (APD) in the past 18 years. These anuran peptides were discovered in Africa, Asia, Australia, Europe, and America from 1985 to 2019. Genomic and peptidomic studies accelerated the discovery pace and underscored the necessity in establishing criteria for peptide entry into the APD. A total of 99.9% of the anuran antimicrobial peptides are less than 50 amino acids with an average length of 24 and a net charge of +2.5. Interestingly, the various amphibian peptide families (e.g., temporins, brevinins, esculentins) can be connected through multiple length-dependent relationships. With an increase in length, peptide net charge increases, while the hydrophobic content decreases. In addition, glycine, leucine, lysine, and proline all show linear correlations with peptide length. These correlations improve our understanding of amphibian peptides and may be useful for prediction and design of new linear peptides with potential applications in treating infectious diseases, cancer and diabetes. Full article

Antimicrobial peptides (AMPs) are a class of molecules that play an essential role in innate immune regulation. The Brevinin-1 family are AMPs that show strong pharmacological and antimicrobial potential. A novel peptide, B1A, was designed based on the primary structure of brevinin-1PLb and brevinin-1PLc. Subsequently, a synthesised replicate was subjected to a series of bioassays and was found to display antimicrobial activity. However, it also displayed high levels of haemolysis in a horse red blood cell haemolytic assay, suggesting potential toxicity. Therefore, we rationally designed a number of B1A analogues with aim of retaining antimicrobial activity, lowering toxicity, and to explore the structure–activity relationship of its N-terminus. B1A and its analogues still retained the “Rana Box” and the FLP-motif, which is a feature of this subfamily. However, the introduction of Lys and Trp residues into the peptide sequences revealed that antimicrobial activity of these analogues remained unchanged once the hydrophobicity and the charge reached the threshold. Hence, the idea that the hydrophobicity saturation in different situations is related to antimicrobial activity can be understood via the structure–activity relationship. Meanwhile, it could also be the starting point for the generation of peptides with specific antimicrobial activity. Full article

Antimicrobial peptides (AMPs) are promising therapeutic alternatives compared to conventional antibiotics for the treatment of drug-resistant bacterial infections. However, the application of the overwhelming majority of AMPs is limited because of the high toxicity and high manufacturing costs. Amphibian skin secretion has been proven to be a promising source for the discovery and development of novel AMPs. Herein, we discovered a novel AMP from the skin secretion of Odorrana schmackeri, and designed the analogues by altering the key factors, including conformation, net charge and amphipathicity, to generate short AMPs with enhanced therapeutic efficacy. All the peptides were chemically synthesised, followed by evaluating their biological activity, stability and cytotoxicity. OSd, OSe and OSf exhibited broad-spectrum antibacterial effects, especially OSf, which presented the highest therapeutic index for the tested bacteria. Moreover, these peptides displayed good stability. The results from scanning electron microscopy and transmission electron microscopy studies, indicated that brevinin-OS, OSd, OSe and OSf possessed rapid bactericidal ability by disturbing membrane permeability and causing the release of cytoplasmic contents. In addition, OSd, OSe and OSf dramatically decreased the mortality of waxworms acutely infected with MRSA. Taken together, these data suggested that a balance between positive charge, degrees of α-helicity and hydrophobicity, is necessary for maintaining antimicrobial activity, and these data successfully contributed to the design of short AMPs with significant bactericidal activity and cell selectivity. Full article

The Sado wrinkled frog Glandirana susurra has recently been classified as a new frog species endemic to Sado Island, Japan. In this study, we cloned 12 cDNAs encoding the biosynthetic precursors for brevinin-2SSa–2SSd, esculentin-2SSa, ranatuerin-2SSa, brevinin-1SSa–1SSd, granuliberin-SSa, and bradykinin-SSa from the skin of G. susurra. Among these antimicrobial peptides, we focused on brevinin-2SSb, ranatuerin-2SSa, and granuliberin-SSa, using their synthetic replicates to examine their activities against different reference strains of pathogenic microorganisms that infect animals and plants. In broth microdilution assays, brevinin-2SSb displayed antimicrobial activities against animal pathogens Escherichia coli, Salmonella enterica, Pseudomonas aeruginosa, and Candida albicans and plant pathogens Xanthomonas oryzae pv. oryzae, Clavibacter michiganensis subsp. michiganensis, and Pyricularia oryzae. Ranatuerin-2SSa and granuliberin-SSa were active against C. albicans and C. michiganensis subsp. michiganensis, and granuliberin-SSa also was active against the other plant pathogenic microbes. Scanning electron microscopic observations demonstrated that brevinin-2SSb, ranatuerin-2SSa, and granuliberin-SSa induced morphological abnormalities on the cell surface in a wide range of the reference pathogens. To assess the bacterial-endotoxin-binding ability of the peptides, we developed an enzyme-linked endotoxin-binding assay system and demonstrated that brevinin-2SSb and ranatuerin-2SSa both exhibited high affinity to lipopolysaccharide and moderate affinity to lipoteichoic acid. Full article