Strategy for the Identification of Host-Defense Peptides in Frog Skin Secretions with Therapeutic Potential as Antidiabetic Agents.

Several amphibian peptides that were first identified on the basis of their antimicrobial or cytotoxic properties have subsequently shown potential for development into agents for the treatment of patients with Type 2 diabetes. A strategy is presented for the isolation and characterization of such peptides that are present in norepinephrine-stimulated skin secretions from a range of frog species. The methodology involves (1) fractionation of the secretions by reversed-phase HPLC, (2) identification of fractions containing components that stimulate the rate of release of insulin from BRIN-BD11 clonal ß-cells without simultaneously stimulating the release of lactate dehydrogenase, (3) identification of active peptides in the fractions in the mass range 1-6 kDa by MALDI-ToF mass spectrometry, (4) purification of the peptides to near homogeneity by further reversed-phase HPLC on various column matrices, and (5) structural characterization by automated Edman degradation. The effect of synthetic replicates of the active peptides on glucose homeostasis in vivo may be evaluated in appropriate animal models of Type 2 diabetes such as db/db mice and mice fed a high fat diet to produce obesity, glucose intolerance, and insulin resistance.

Amphibian Skin and Skin Secretion: An Exotic Source of Bioactive Peptides and Its Application.

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.

Peptides Isolated from Amphibian Skin Secretions with Emphasis on Antimicrobial Peptides.

The skin of amphibians is a tissue with biological functions, such as defense, respiration, and excretion. In recent years, researchers have discovered a large number of peptides in the skin secretions of amphibians, including antimicrobial peptides, antioxidant peptides, bradykinins, insulin-releasing peptides, and other peptides. This review focuses on the origin, primary structure, secondary structure, length, and functions of peptides secreted from amphibians' skin. We hope that this review will provide further information and promote the further study of amphibian skin secretions, in order to provide reference for expanding the research and application of amphibian bioactive peptides.

Engineering and Structural Insights of a Novel BBI-like Protease Inhibitor Livisin from the Frog Skin Secretion.

The Bowman-Birk protease inhibitor (BBI) family is a prototype group found mainly in plants, particularly grasses and legumes, which have been subjected to decades of study. Recently, the discovery of attenuated peptides containing the canonical Bowman-Birk protease inhibitory motif has been detected in the skin secretions of amphibians, mainly from Ranidae family members. The roles of these peptides in amphibian defense have been proposed to work cooperatively with antimicrobial peptides and reduce peptide degradation. A novel trypsin inhibitory peptide, named livisin, was found in the skin secretion of the green cascade frog, Odorrana livida. The cDNA encoding the precursor of livisin was cloned, and the predicted mature peptide was characterized. The mature peptide was found to act as a potent inhibitor against several serine proteases. A comparative activity study among the native peptide and its engineered analogs was performed, and the influence of the P1 and P2' positions, as well as the C-terminal amidation on the structure-activity relationship for livisin, was illustrated. The findings demonstrated that livisin might serve as a potential drug discovery/development tool.

The beneficial roles of poisonous skin secretions in survival strategies of the odorous frog Odorrana andersonii.

The evolution of predatory, anti-predatory, and defensive strategies regarding environmental adaptation in animals is of significant research interest. In particular, amphibians, who represent a transition between aquatic and terrestrial vertebrates, play an important role in animal evolution. The bioactive skin secretions of amphibians are of specific interest due to their involvement in the crucial physiological functions of amphibian skin. We previously isolated and identified several bioactive peptides, including those showing antioxidant, antimicrobial, and wound-healing properties, from the skin secretions of the odorous frog species Odorrana andersonii. Currently, however, the biological significance of skin secretions in O. andersonii survival remains unclear. Here, we studied the biological significance of skin glands and secretions in regard to environmental adaptations of O. andersonii. Our research found that O. andersonii may secrete and excrete bioactive secretions through many glands (peptides and proteins as the main components in glands) distributed in the skin. The skin secretions not only displayed toxicity but also showed antioxidant, antibacterial, and repair promoting activities, suggesting that they play a protective role in O. andersonii when facing environmental threats. These bioactive skin secretions appear to act as a chemical survival strategy in O. andersonii, allowing the species to gain advantages in survival behavior.

Host defense or parasite cue: Skin secretions mediate interactions between amphibians and their parasites.

Amphibian skin secretions (substances produced by the amphibian plus microbiota) plausibly act as a first line of defense against parasite/pathogen attack, but may also provide chemical cues for pathogens. To clarify the role of skin secretions in host-parasite interactions, we conducted experiments using cane toads (Rhinella marina) and their lungworms (Rhabdias pseudosphaerocephala) from the range-core and invasion-front of the introduced anurans' range in Australia. Depending on the geographical area, toad skin secretions can reduce the longevity and infection success of parasite larvae, or attract lungworm larvae and enhance their infection success. These striking differences between the two regions were due both to differential responses of the larvae, and differential effects of the skin secretions. Our data suggest that skin secretions play an important role in host-parasite interactions in anurans, and that the arms race between a host and parasite can rapidly generate spatial variation in critical features of that interaction.

A novel peptide from the skin of amphibian Rana limnocharis with potency to promote skin wound repair.

In clinical trials, the healing of wounds remains a substantial physiological and financial incumbrance on patients. Therefore, the development of new drugs that can accelerate wound healing is vital. Based on genomic methods, we identified a new peptide (RL-RL10) with the amino acid sequence 'RLFKCWKKDS' from the skin of an amphibian frog species (Rana limnocharis). RL-RL10 promoted wound healing of human keratinocytes (HaCaT) in a concentration-dependent manner. RL-RL10 also had an effect on the migration and proliferation of HaCaT cells and promoted healing of a full-thickness wound in mice in a dose-dependent manner. In conclusion, we discovered RL-RL10 that promoted healing activity of cellular and animal wounds, thus providing a new peptide template for the development of novel wound-repairing drugs.

Peptidomic Analysis of Skin Secretions of the Caribbean Frogs Leptodactylus insularum and Leptodactylus nesiotus (Leptodactylidae) Identifies an Ocellatin with Broad Spectrum Antimicrobial Activity.

Ocellatins are peptides produced in the skins of frogs belonging to the genus Leptodactylus that generally display weak antimicrobial activity against Gram-negative bacteria only. Peptidomic analysis of norepinephrine-stimulated skin secretions from Leptodactylus insularum Barbour 1906 and Leptodactylus nesiotus Heyer 1994, collected in the Icacos Peninsula, Trinidad, led to the purification and structural characterization of five ocellatin-related peptides from L. insularum (ocellatin-1I together with its (1-16) fragment, ocellatin-2I and its (1-16) fragment, and ocellatin-3I) and four ocellatins from L. nesiotus (ocellatin-1N, -2N, -3N, and -4N). While ocellatins-1I, -2I, and -1N showed a typically low antimicrobial potency against Gram-negative bacteria, ocellatin-3N (GIFDVLKNLAKGVITSLAS.NH2) was active against an antibiotic-resistant strain of Klebsiella pneumoniae and reference strains of Escherichia coli, K. pneumoniae, Pseudomonas aeruginosa, and Salmonella typhimurium (minimum inhibitory concentrations (MICs) in the range 31.25-62.5 µM), and was the only peptide active against Gram-positive Staphylococcus aureus (MIC = 31.25 µM) and Enterococcus faecium (MIC = 62.5 µM). The therapeutic potential of ocellatin-3N is limited by its moderate hemolytic activity (LC50 = 98 µM) against mouse erythrocytes. The peptide represents a template for the design of long-acting, non-toxic, and broad-spectrum antimicrobial agents for targeting multidrug-resistant pathogens.

Accelerated Wound Healing Induced by a Novel Amphibian Peptide (OA-FF10).

BACKGROUND: Despite the continued development of modern medicine, chronic wounds are still a critical issue in clinical treatment, placing a great physiological, psychological, and financial burden on patients. Researchers have investigated many methods to solve this problem, with bioactive peptides gaining increasing attention due to their considerable advantages and diverse functions, as well as low cost, simple storage, and easy transportation. METHODS: In this research, a novel peptide (named OA-FF10) was identified from the skin secretions of the odorous frog species Odorrana andersonii. The sequence of mature OA-FF10 was "FFTTSCRSGC", which was produced by the post-translational processing of a 61-residue prepropeptide. RESULTS: Similar to most frog peptides, OA-FF10 showed an intramolecular disulfide bridge at the C-terminus. OA-FF10 demonstrated no antibacterial, antioxidant, hemolytic, or acute toxic activity, but promoted wound healing and proliferation of human keratinocytes (HaCaT) both time- and dose-dependently. Furthermore, while OA-FF10 had no effect on wound healing of Human Skin Fibroblasts (HSF), it did accelerate healing in a full-thickness skin-wound mouse model. CONCLUSION: Our research revealed the strong wound-healing activity of OA-FF10 in vivo and in vitro, thus providing a new candidate for the development of novel wound-healing drugs.

Biological Properties, Current Applications and Potential Therapeautic Applications of Brevinin Peptide Superfamily.

The Brevinin peptides are antimicrobial agents obtained from frog skin secretions. Brevinin-2R has attracted many attentions due to its very low hemolytic activity, cationic property, and high affinity to cancer cells. Moreover, it has shown little toxicity against normal mammalian cells, while having killed several tumor cell lines by activation of lysosome-mitochondrial death pathway. In this review, we introduced the Brevinin superfamily with a focus on its therapeutic applications. Next, some unique properties of Brevinins were briefly discussed, including their ability to stimulate insulin secretion, dendritic cell maturation, and wound healing. In this context, we also provide information about the decoration of nanoparticles, such as cerium nano-oxide, by Brevinins. Finally, we addressed their potential for anti-tumor and drug design applications.

Identification of Arenin, a Novel Kunitz-Like Polypeptide from the Skin Secretions of Dryophytes arenicolor.

Amphibian skin secretions are enriched with complex cocktails of bioactive molecules such as proteins, peptides, biogenic amines, alkaloids guanidine derivatives, steroids and other minor components spanning a wide spectrum of pharmacological actions exploited for centuries in folk medicine. This study presents evidence on the protein profile of the skin secretions of the canyon tree frog, Dryophytes arenicolor. At the same time, it presents the reverse-phase liquid chromatography isolation, mass spectrometry characterization and identification at mRNA level of a novel 58 amino acids Kunitz-like polypeptide from the skin secretions of Dryophytes arenicolor, arenin. Cell viability assays performed on HDFa, CaCo2 and MCF7 cells cultured with different concentrations of arenin showed a discrete effect at low concentrations (2, 4, 8 and 16 µg/mL) suggesting a multi-target interaction in a hormetic-like dose-response. Further work is required to investigate the mechanisms underlying the variable effect on cell viability produced by different concentrations of arenin.

A novel antimicrobial peptide, Ranatuerin-2PLx, showing therapeutic potential in inhibiting proliferation of cancer cells.

Antimicrobial peptides are a promising resource for developing novel antibiotic and even anticancer drugs. Here, a 28-mer polypeptide, Ranatuerin-2PLx (R2PLx), was identified from lyophilised skin secretions. The chemically synthetic replicates exhibited moderate and broadspectrum antimicrobial effect against various microorganisms including methicillin-resistant Staphylococcus aureus (MRSA, minimal inhibitory concentration = 256 µM). In addition, R2PLx was found to inhibit the proliferation of several tumour cells, especially showing more potent effect on prostate cancer cell, PC-3. The early cell apoptosis was observed in 6 h by Annexin V-FITC/propidium iodide staining, as well as the activation of Caspase-3 at 5 µM peptide concentration. R2PLx may therefore be promising for developing new therapeutic approach for cancer treatment. Moreover, the artificial deficiency of conserved rana-box loop or net positive charge in C-terminal domain notably reduced the biological activities of the truncated and substituted isoforms, respectively, suggesting for maintaining their biological potency of ranatuerin family requires both cysteine-bridged segment and cationincity within the loop domain in C-terminus.

Unveiling the Multifaceted Mechanisms of Antibacterial Activity of Buforin II and Frenatin 2.3S Peptides from Skin Micro-Organs of the Orinoco Lime Treefrog (Sphaenorhynchus lacteus).

Amphibian skin is a rich source of natural compounds with diverse antimicrobial and immune defense properties. Our previous studies showed that the frog skin secretions obtained by skin micro-organs from various species of Colombian anurans have antimicrobial activities against bacteria and viruses. We purified for the first time two antimicrobial peptides from the skin micro-organs of the Orinoco lime treefrog (Sphaenorhynchus lacteus) that correspond to Buforin II (BF2) and Frenatin 2.3S (F2.3S). Here, we have synthesized the two peptides and tested them against Gram-negative and Gram-positive bacteria, observing an effective bactericidal activity at micromolar concentrations. Evaluation of BF2 and F2.3S membrane destabilization activity on bacterial cell cultures and synthetic lipid bilayers reveals a distinct membrane interaction mechanism. BF2 agglutinates E. coli cells and synthetic vesicles, whereas F2.3S shows a high depolarization and membrane destabilization activities. Interestingly, we found that F2.3S is able to internalize within bacterial cells and can bind nucleic acids, as previously reported for BF2. Moreover, bacterial exposure to both peptides alters the expression profile of genes related to stress and resistance response. Overall, these results show the multifaceted mechanism of action of both antimicrobial peptides that can provide alternative tools in the fight against bacterial resistance.

OA-GL21, a novel bioactive peptide from Odorrana andersonii, accelerated the healing of skin wounds.

Nowadays, the number of chronic trauma cases caused by a variety of factors such as the world's population-ageing and chronic diseases is increasing steadily, and thus effective treatment for chronic wounds has become a severe clinical challenge, which also burdens the patient both physically and financially. Therefore, it is urgent to develop new drugs to accelerate the healing of wounds. Bioactive peptides, which are relatively low cost, easy to produce, store and transport, have become an excellent choice. In this research, we identified a novel peptide OA-GL21, with an amino acid sequence of 'GLLSGHYGRVVSTQSGHYGRG', from the skin secretions of Odorrana andersonii Our results showed that OA-GL21 exerted the ability to promote wound healing of human keratinocytes (HaCaT) and human fibroblasts in a dose- and time-denpendent manner. However, OA-GL21 had no significant effect on the proliferation of these two cells. Significantly, OA-GL21 showed obvious ability to promote wound healing in the full-thickness skin wound model in dose- and scar-free manners. Further studies showed that OA-GL21 had no direct antibacterial, hemolytic, and acute toxic activity; it had weak antioxidant activities but high stability. In conclusion, this research proved the promoting effects of OA-GL21 on cellular and animal wounds, and thus provided a new peptide template for the development of wound-repairing drugs.

Identification of Components in Frog Skin Secretions with Therapeutic Potential as Antidiabetic Agents.

Several peptides that were first identified on the basis of their antimicrobial or immunomodulatory properties have subsequently shown potential for development into agents for the treatment of patients with Type 2 diabetes. A strategy is presented for the isolation and characterization of such peptides in norepinephrine-stimulated skin secretions from a range of frog species. The methodology involves fractionation of the secretions by reversed-phase HPLC, identification of fractions containing components that stimulate the rate of release of insulin from BRIN-BD11 clonal ß-cells without simultaneously stimulating the release of lactate dehydrogenase, identification of active peptides in the mass range 1-6 kDa by MALDI-TOF mass spectrometry, purification of the peptides to near homogeneity by further HPLC, and structural characterization by automated Edman degradation. The effect of synthetic replicates of the active peptides on glucose homeostasis in vivo may be evaluated in mice fed a high fat diet to produce obesity, glucose intolerance, and insulin resistance.

Characterization of an insulinotropic peptide from skin secretions of Odorrana andersonii.

Insulinotropic peptide agents are regarded as potential candidates for anti-diabetic treatment. In the present study, a novel insulinotropic peptide, termed OA-A1, was purified from frog skin secretions of Odorrana andersonii. Mature OA-A1 was determined to be a 1965.049 Da peptide with an amino acid sequence of LVGKLLKGAVGDVCGLLPIC, in which an intramolecular disulfide bridge was formed by two cysteine residues. At the cellular level, OA-A1 exhibited potent proliferation promoting effects on mouse-derived pancreatic ß-TC-6 cells and significantly stimulated insulin release in ß-TC-6 cells at a minimum concentration of 1 nM. In the animal model, OA-A1 also showed a dose-dependent insulin-releasing role in mice. At concentrations ranging from 1 nmol/kg to 1 µmol/kg, OA-A1 had a significant acute hypoglycemic effect on streptozotocin (STZ)-induced diabetic mice. The pancreatic islet areas of diabetic mice increased dose-dependently after 21 days of OA-A1 treatment (1-100 nmol/kg) compared with those of the saline control group. Moreover, OA-A1 significantly improved the oral glucose tolerance of STZ-induced diabetic mice. Taken together, these results suggest that OA-A1 provides an excellent template for the development of novel anti-diabetic therapeutic agents. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

Peptidomic approach identifies cruzioseptins, a new family of potent antimicrobial peptides in the splendid leaf frog, Cruziohyla calcarifer.

UNLABELLED: Phyllomedusine frogs are an extraordinary source of biologically active peptides. At least 8 families of antimicrobial peptides have been reported in this frog clade, the dermaseptins being the most diverse. By a peptidomic approach, integrating molecular cloning, Edman degradation sequencing and tandem mass spectrometry, a new family of antimicrobial peptides has been identified in Cruziohyla calcarifer. These 15 novel antimicrobial peptides of 20-32 residues in length are named cruzioseptins. They are characterized by having a unique shared N-terminal sequence GFLD- and the sequence motifs -VALGAVSK- or -GKAAL(N/G/S) (V/A)V- in the middle of the peptide. Cruzioseptins have a broad spectrum of antimicrobial activity and low haemolytic effect. The most potent cruzioseptin was CZS-1 that had a MIC of 3.77µM against the Gram positive bacterium, Staphylococcus aureus and the yeast Candida albicans. In contrast, CZS-1 was 3-fold less potent against the Gram negative bacterium, Escherichia coli (MIC 15.11µM). CZS-1 reached 100% haemolysis at 120.87µM. Skin secretions from unexplored species such as C. calcarifer continue to demonstrate the enormous molecular diversity hidden in the amphibian skin. Some of these novel peptides may provide lead structures for the development of a new class of antibiotics and antifungals of therapeutic use. BIOLOGICAL SIGNIFICANCE: Through the combination of molecular cloning, Edman degradation sequencing, tandem mass spectrometry and MALDI-TOF MS we have identified a new family of 15 antimicrobial peptides in the skin secretion of Cruziohyla calcarifer. The novel family is named "Cruzioseptins" and contains cationic amphipathic peptides of 20-32 residues. They have a broad range of antimicrobial activity that also includes effective antifungals with low haemolytic activity. Therefore, C. calcarifer has proven to be a rich source of novel peptides, which could become leading structures for the development of novel antibiotics and antifungals of clinical application.

Characteristics of hemolytic activity induced by skin secretions of the frog Kaloula pulchra hainana

Background: Previous works had shown that scorpion venom induced neurotransmitter elevation and an inflammatory response associated with various anatomo-pathological modifications. The most dangerous scorpions species in Algeria responsible for these effects are Androctonus australis hector (Aah) and Androctonus amoreuxi (Aam). Results: Comparison of the physiopathological effects induced by the two venoms showed differences in the kinetic of cytokine release and in lung injury. The lung edema was only observed in response to Aah venom and it was correlated with cell infiltration. In order to better understand the involved mechanism in inflammatory response, we used two antagonists, atropine (non-selective muscarinic antagonist) and propranolol (ß adrenergic antagonist), which lead to a decrease of cell infiltration but has no effect on edema forming. Conclusion: These results suggest another pathway in the development of lung injury following envenomation with Aam or Aah venom.(AU)

Characteristics of hemolytic activity induced by skin secretions of the frog Kaloula pulchra hainana

The hemolytic activity of skin secretions obtained by stimulating the frog Kaloula pulchra hainana with diethyl ether was tested using human, cattle, rabbit, and chicken erythrocytes. The skin secretions had a significant concentration-dependent hemolytic effect on erythrocytes. The hemolytic activity of the skin secretions was studied in the presence of osmotic protectants (polyethylene glycols and carbohydrates), cations (Mg2+, Ca2+, Ba2+, Cu2+, and K+), or antioxidants (ascorbic acid, reduced glutathione, and cysteine). Results Depending on their molecular mass, osmotic protectants effectively inhibited hemolysis. The inhibition of skin hemolysis was observed after treatment with polyethylene glycols (1000, 3400, and 6000 Da). Among divalent cations, only 1 mM Cu2+ markedly inhibited hemolytic activity. Antioxidant compounds slightly reduced the hemolytic activity. Conclusions The results suggested that skin secretions of K. pulchra hainana induce a pore-forming mechanism to form pores with a diameter of 1.36-2.0 nm rather than causing oxidative damage to the erythrocyte membrane.