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In this study, we present in vitro actions of pure commercial preparations of oxidized and/or dehydrated metabolites of cholesterol (OS) identified in the lipid fraction of Fraction B (FB) prepared from a catfish skin preparation on calcium transients and on the formation of human neutrophil extracellular traps (NETs). These investigations are part of an ongoing effort to understand the important roles these compounds play as components of FB when FB is applied to accelerate the healing of wounds and the healing of highly infected non-healing diabetic foot ulcers, without the use of antibiotics. Our aim was to determine potential therapeutic interventions for various disease states. Our results reveal interesting findings, demonstrating specific actions of the individual compounds. Compounds 7α-hydroxy-cholesterol (S3), Cholestane-3,5,6-triol (S5), 5-cholesten-3β-ol-7-one (S8) and Cholesta-3,5 dien-7-one (S10) are inhibitory, while Cholesterol 5β,6β-epoxide (S4) and 5α-cholestane-3,6-dione (S11) activate the response for calcium influx in human neutrophils. A somewhat similar response is observed in dHL60 cell lines, where S3, S5, S7, S8, and cholesta-2,4-diene (S14) inhibit the calcium influx, although S4, S10, and S11 activate the response in this cell line. Furthermore, we observed a relationship between actions against NETosis and calcium transients. Interestingly, relative to the vehicle control, S3, Cholesta-3,5 diene (S9), and S14 appeared to significantly stimulate DNA release (NETosis), while S2, 7α-hydroxy-cholesterol (S6) and cholesta-3,5 dien-7-one (S10) caused lesser stimulation. We provide the IC50 activities for each compound tested in each assay. Calcium influx and NETs formation (NETosis) correlate with diseases exacerbation. These findings offer valuable insights into the potential therapeutic applications of individual OS for various diseases, highlighting their importance in future interventions.

The number of multidrug-resistant pathogenic microorganisms has been growing in recent years, most of which is due to the inappropriate use of the commercial antibiotics that are currently available. The dissemination of antimicrobial resistance represents a serious global public health problem. Thus, it is necessary to search for and develop new drugs that can act as antimicrobial agents. Antimicrobial peptides are a promising alternative for the development of new therapeutic drugs. Anurans' skin glands are a rich source of broad-spectrum antimicrobial compounds and hylids, a large and diverse family of tree frogs, are known as an important source of antimicrobial peptides. In the present study, two novel antimicrobial peptides, named Raniseptins-3 and -6, were isolated from Boana raniceps skin secretion and their structural and biological properties were evaluated. Raniseptins-3 and -6 are cationic, rich in hydrophobic residues, and adopt an α-helix conformation in the presence of SDS (35 mM). Both peptides are active against Gram-negative bacteria and Gram-positive pathogens, with low hemolytic activity at therapeutic concentrations. No activity was observed for yeasts, but the peptides are highly cytotoxic against B16F10 murine melanoma cells and NIH3T3 mouse fibroblast cells. None of the tested compounds showed improvement trends in the MTT and LDH parameters of MHV-3 infected cells at the concentrations tested.

Skin secretions of toads are a complex mixture of molecules. The substances secreted comprise more than 80 different compounds that show diverse pharmacological activities. The compounds secreted through skin pores and parotid glands are of particular interest because they help toads to endure in habitats full of pathogenic microbes, i.e., bacteria, fungi, viruses, and protozoa, due to their content of components such as bufadienolides, alkaloids, and antimicrobial peptides. We carried out an extensive literature review of relevant articles published until November 2022 in ACS Publications, Google Scholar, PubMed, and ScienceDirect. It was centered on research addressing the biological characterization of the compounds identified in the species of genera Atelopus , Bufo , Duttaphrynus , Melanophryniscus , Peltopryne , Phrynoidis , Rhaebo , and Rhinella , with antibacterial, antifungal, antiviral, and antiparasitic activities; as well as studies performed with analogous compounds and skin secretions of toads that also showed these activities. This review shows that the compounds in the secretions of toads could be candidates for new drugs to treat infectious diseases or be used to develop new molecules with better properties from existing ones. Some compounds in this review showed activity against microorganisms of medical interest such as Staphylococcus aureus , Escherichia coli , Bacillus subtilis , Coronavirus varieties, HIV, Trypanosoma cruzi , Leishmania chagasi , Plasmodium falciparum , and against different kinds of fungi that affect plants of economic interest.

The skin secretion of tree frogs contains a vast array of bioactive chemicals for repelling predators, but their structural and functional diversity is not fully understood. Paxilline (PAX), a compound synthesized by Penicillium paxilli, has been known as a specific antagonist of large conductance Ca 2+ -activated K + Channels (BK Ca ). Here, we report the presence of PAX in the secretions of tree frogs ( Hyla japonica ) and that this compound has a novel function of inhibiting the potassium channel subfamily K member 18 (KCNK18) channels of their predators. The PAX-induced KCNK18 inhibition is sufficient to evoke Ca 2+ influx in charybdotoxin-insensitive DRG neurons of rats. By forming π-π stacking interactions, four phenylalanines located in the central pore of KCNK18 stabilize PAX to block the ion permeation. For PAX-mediated toxicity, our results from animal assays suggest that the inhibition of KCNK18 likely acts synergistically with that of BK Ca to elicit tingling and buzzing sensations in predators or competitors. These results not only show the molecular mechanism of PAX-KCNK18 interaction, but also provide insights into the defensive effects of the enriched PAX.

Among the vast repertoire of animal toxins and venoms selected by nature and evolution, mankind opted to devote its scientific attention-during the last century-to a restricted group of animals, leaving a myriad of toxic creatures aside. There are several underlying and justifiable reasons for this, which include dealing with the public health problems caused by envenoming by such animals. However, these studies became saturated and gave rise to a whole group of animals that become neglected regarding their venoms and secretions. This repertoire of unexplored toxins and venoms bears biotechnological potential, including the development of new technologies, therapeutic agents and diagnostic tools and must, therefore, be assessed. In this review, we will approach such topics through an interconnected historical and scientific perspective that will bring up the major discoveries and innovations in toxinology, achieved by researchers from the Butantan Institute and others, and describe some of the major research outcomes from the study of these neglected animals.

Species of the family Bufonidae, better known as true toads, are widespread and produce bioactive substances in the secretions obtained from specialized skin macroglands. Some true toads have been employed as a folk remedy to treat infectious diseases caused by microbial pathogens. Recent publications based on in silico analysis highlighted the Bufonidae as promising sources of antimicrobial peptides. A review of the literature reveals that Bufonidae skin secretion extracts show inhibitory activity in vitro against clinical isolates of bacteria, resistant and standard strains of bacterial, and fungal and parasitic human pathogens. Secondary metabolites belonging to the classes of alkaloids, bufadienolides, and peptides with antimicrobial activity have been isolated from species of the genera Bufo , Bufotes , Duttaphrynus, and Rhinella . Additionally, some antimicrobial extracts and purified compounds display low cytotoxicity against mammal cells.

Amphibian skin secretions are abundant in bioactive compounds, especially antimicrobial peptides. These molecules are generally cationic and rich in hydrophobic amino acids, have an amphipathic structure and adopt an α-helical conformation when in contact with microorganisms membranes. In this work, we purified and characterized Figainin 1, a novel antimicrobial and antiproliferative peptide from the cutaneous secretion of the frog Boana raniceps . Figainin 1 is a cationic peptide with eighteen amino acid residues-rich in leucine and isoleucine, with an amidated C-terminus-and adopts an α-helical conformation in the presence of trifluoroethanol (TFE). It displayed activity against Gram-negative and especially Gram-positive bacteria, with MIC values ranging from 2 to 16 µM, and showed an IC 50 value of 15.9 µM against epimastigote forms of T. cruzi ; however, Figanin 1 did not show activity against Candida species. This peptide also showed cytolytic effects against human erythrocytes with an HC 50 of 10 µM, in addition to antiproliferative activity against cancer cells and murine fibroblasts, with IC 50 values ranging from 10.5 to 13.7 µM. Despite its adverse effects on noncancerous cells, Figainin 1 exhibits interesting properties for the development of new anticancer agents and anti-infective drugs against pathogenic microorganisms.

Pain is a distressful experience that can have a major impact on an individual's quality of life. The need for new and better analgesics has been further intensified in light of the current opioid epidemic. Substances obtained from amphibians have been shown to contain bioactive peptides that exert analgesic effects. The genus Phyllomedusa represents an important source of peptides and bioactive components. The aim of this study was to investigate the antinociceptive effects of the skin secretion of Phyllomedusa rohdei in rodent models of pain. The crude skin extract of P. rohdei was tested in different pain models: acetic acid-induced writhing test (mice), formalin test (rats), Von Frey electronic test for hypernociception induced by PGE 2 (rats), and hot plate test (mice). Motor-impairing effects were tested using the rota-rod test. The results showed that the skin extract of P. rohdei exerted antinociceptive effects in all pain models tested. Particularly, the highest dose tested of the skin extract decreased acetic acid-induced writhing by 93%, completely blocked formalin-induced nociception both during the acute and inflammatory phases of the test, PGE 2 -induced hypernociception by 73% and increased latency to paw withdrawal in the hot plate test by 300%. The effects observed in the hot plate test were reversed by pretreatment with selective µ and κ, but not δ, opioid receptor antagonists, indicating a mechanism of action dependent on µ and κ opioid receptors. The results were not influenced by sedative effects. Further studies remain necessary to reveal the specific compounds involved in the antinociceptive effects of P. rohdei skin extract as a new therapeutic tool in pain management.

Bradykinin (BK) and bradykinin-related peptides (BRPs), which were identified from a diversity of amphibian skin secretions, exerted contractile and relaxing effects on non-vascular and vascular smooth muscle, respectively. Here, we report a novel bradykinin-related peptide with a molecular mass of 1890.2 Da, RVAGPDKPARISGLSPLR, which was isolated and identified from Ordorrana hejiangensis skin secretions, followed by a C-terminal extension sequence VAPQIV. The biosynthetic precursor-encoding cDNA was cloned by the "shotgun" cloning method, and the novel RR-18 was identified and structurally confirmed by high-performance liquid chromatography (HPLC) and tandem mass spectrometry (MS/MS). Subsequently, the myotropic activity of the synthetic replicate of RR-18 was investigated on the rat bladder, uterus, tail artery and ileum smooth muscle. The peptide was named RR-18 in accordance (R = N-terminal arginine, R = C-terminal arginine, 18 = number of residues). In this study, the synthetic replicates of RR-18 showed no agonist/antagonism of BK-induced rat bladder and uterus smooth muscle contraction. However, it displayed an antagonism of bradykinin-induced rat ileum contraction and arterial smooth muscle relaxation. The EC 50 values of BK for ileum and artery, were 214.7 nM and 18.3 nM, respectively. When the tissue was pretreated with the novel peptide, RR-18, at the maximally effective concentration of bradykinin (1 × 10 -6 M), bradykinin-induced contraction of the ileum and relaxation of the arterial smooth muscle was reduced by 50-60% and 30-40%, respectively. In conclusion, RR-18 represents novel bradykinin antagonising peptide from amphibian skin secretions. It may provide new insight into possible treatment options for chronic pain and chronic inflammation.

A novel Kunitz-type neurotoxin peptide that inhibited voltage-gated sodium channel was purified and characterized from the skin secretions of rufous-spotted torrent frog, Amolops loloensis. It has a 240-bp cDNA encoding an 79-amino acid residue (aa) precursor protein containing 6 half-cysteines. The precursor was proven to release a 57-aa mature peptide with amino acid sequence, DRNPICNLPPKEGFCLWMMRRSFFNPSKGRCDTFGYRGCGGNKNNFETPRACKEACG. The mature was named amotoxin. Amotoxin shares sequence homology with other Kunitz-type toxins and also has three cysteine bridges. Amotoxin showed an inhibitory ability against trypsin with an inhibitory constant (Ki) of 0.087 μM. To the best of our knowledge, this is the first gene-encoded neurotoxin found in Amolops loloensis. Recombinant amotoxin showed similar functional properties as the native amotoxin. The functional properties of amotoxin may provide insights into the ecological adaptation of amphibians and deepen our understanding about the biological function spectrum of amphibian skin peptides., Competing Interests: Declaration of competing interest The authors claim no conflicts of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

In recent years, the number of new antimicrobial drugs launched on the market has decreased considerably even though there has been an increase in the number of resistant microbial strains. Thus, antimicrobial resistance has become a serious public health problem. Amphibian skin secretions are a rich source of host defense peptides, which generally are cationic and hydrophobic molecules, with a broad-spectrum of activity. In this study, one novel multifunctional defense peptide was isolated from the skin secretion of the Chaco tree frog, Boana raniceps. Figainin 2 ( 1 FLGAILKIGHALAKTVLPMVTNAFKPKQ 28 ) is cationic and hydrophobic, adopts an α-helical structure in 50% ( v/v ) trifluoroethanol (TFE), and is thermally stable. This peptide exhibited activity against Gram-negative and Gram-positive pathogenic bacteria arboviruses, T. cruzi epimastigotes; however, it did not show activity against yeasts. Figainin 2 also showed antiproliferative activity on cancer cells, is moderately active on human erythrocytes, and activates the oxidative burst in human neutrophils.

Background: Argenteohyla siemersi (red-spotted Argentina frog) is a casque-headed tree frog species belonging to the Hylidae family. This species has a complex combination of anti-predator defense mechanisms that include a highly lethal skin secretion. However, biochemical composition and biological effects of this secretion have not yet been studied., Methods: The A. siemersi skin secretion samples were analyzed by mass spectrometry and chromatographic analysis (MALDI-TOF/MS, RP-HPLC and GC-MS). Proteins were also studied by SDS-PAGE. Among the biological activities evaluated, several enzymatic activities (hemolytic, phospholipase A 2 , clotting, proteolytic and amidolytic) were assessed. Furthermore, the cytotoxic activity (cytolysis and fluorescence staining) was evaluated on myoblasts of the C2C12 cell line., Results: The MALDI-TOF/MS analysis identified polypeptides and proteins in the aqueous solution of A. siemersi skin secretion. SDS-PAGE revealed the presence of proteins with molecular masses from 15 to 55 kDa. Steroids, but no alkaloids or peptides (less than 5 KDa), were detected using mass spectrometry. Skin secretion revealed the presence of lipids in methanolic extract, as analyzed by CG-MS. This secretion showed hemolytic and phospholipase A 2 activities, but was devoid of amidolytic, proteolytic or clotting activities. Moreover, dose-dependent cytotoxicity in cultured C2C12 myoblasts of the skin secretion was demonstrated. Morphological analysis, quantification of lactate dehydrogenase release and fluorescence staining indicated that the cell death triggered by this secretion involved necrosis., Conclusions: Results presented herein evidence the biochemical composition and biological effects of A. siemersi skin secretion and contribute to the knowledge on the defense mechanisms of casque-headed frogs., Competing Interests: Competing interests: The authors declare that they have no competing interests.

Traditional sources of mRNA for molecular cloning on amphibian skin secretion studies have been the frog's skin and skin secretions. Here, we demonstrate that mRNA isolated from chromatographic fractions of skin secretions is amenable for molecular cloning assays. We identified precursor sequences of the Arg 0 , Trp 5 , Leu 8 -bradykinin and six antimicrobial peptides of Pelophylax esculentus (Ranidae). These results show that both transcriptomic and peptidomic analyses can be performed with a single sample reducing in half the amount of starting skin secretion required. This is a significant advantage when working with endangered or very rare amphibian species, where minimal samples are available., (Copyright © 2018. Published by Elsevier Inc.)

Background: Animal poisons and venoms are sources of biomolecules naturally selected. Rhinella schneideri toads are widespread in the whole Brazilian territory and they have poison glands and mucous gland. Recently, protein from toads' secretion has gaining attention. Frog skin is widely known to present great number of host defense peptides and we hypothesize toads present them as well. In this study, we used a RNA-seq analysis from R. schneideri skin and biochemical tests with the gland secretion to unravel its protein molecules., Methods: Total RNA from the toad skin was extracted using TRizol reagent, sequenced in duplicate using Illumina Hiseq2500 in paired end analysis. The raw reads were trimmed and de novo assembled using Trinity. The resulting sequences were submitted to functional annotation against non-redundant NCBI database and Database of Anuran Defense Peptide. Furthermore, we performed caseinolytic activity test to assess the presence of serine and metalloproteases in skin secretion and it was fractionated by fast liquid protein chromatography using a reverse-phase column. The fractions were partially sequenced by Edman's degradation., Results: We were able to identify several classes of antimicrobial peptides, such as buforins, peroniins and brevinins, as well as PLA 2 , lectins and galectins, combining protein sequencing and RNA-seq analysis for the first time. In addition, we could isolate a PLA 2 from the skin secretion and infer the presence of serine proteases in cutaneous secretion., Conclusions: We identified novel toxins and proteins from R. schneideri mucous glands. Besides, this is a pioneer study that presented the in depth characterization of protein molecules richness from this toad secretion. The results obtained herein showed evidence of novel AMP and enzymes that need to be further explored., Competing Interests: Not applicable.Not applicable.The authors declare there are no competing interests.Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Two novel peptides belonging to the dermaseptin family, namely DRS-CA-1 and DRS-DU-1, were encoded from cDNA libraries derived from the skin secretions of Phyllomedusa camba and Callimedusa (Phyllomedusa) duellmani . Both natural peptides are highly-conserved and exhibited high potency against wild-type Gram-positive, Gram-negative bacteria, yeast and antibiotic-resistant bacteria (MRSA and Pseudomonas aeruginosa ) (MICs 4-8 µM) with no obvious hemolytic activity. Collectively these results suggest that both peptides may have potential as novel antibiotics. Additionally, DRS-DU-1 exhibited selective cytotoxicity to tumor cells. The truncated analogue, DP-1 and TAT-fused DP-1 (namely DP-2) were subsequently synthesised. It showed that DP-1 had low antimicrobial activity, no hemolytic and cytotoxicity to tumor cells. However, DP-2 possessed strong antimicrobial activity and the similar selective, no obvious hemolytic activity and cytotoxicity on normal human cells, but enhanced cytotoxicity to tumor cells of DRS-DU-1. These findings indicate that the N-terminus of the dermaseptins may contribute to their bioactivity, and that addition of the TAT peptide can improve biological activity. The results provide a new insight for designing novel peptide-based antimicrobial or anticancer agents with low hemolytic activity and cytotoxicity., Competing Interests: The authors declare there are no competing interests.

In efforts to find new antimicrobial peptides (AMPs), we studied the skin secretion of the endemic Colombian frog Dendropsophus columbianus belonging to a genus that has not been investigated previously. From HPLC-fractionated secretion, we identified one peptide with slightly antibacterial activity. Its peptide sequence showed no sequence similarity to current annotated peptides. We named this novel peptide dendropsophin 1 (Dc1). Afterward, two analogues were designed (Dc1.1 and Dc1.2) to improve the cationic and amphipathic features. Then, their antiproliferative and cytotoxic properties were evaluated against several pathogens including bacteria, fungi, protozoa and also mammalian cells. Dc1 and its two analogues exhibited moderate antibacterial activities and no hemolytic and cytotoxic effects on mammalian cells. Analogue Dc1.2 showed slightly improved antibacterial properties. Their secondary structures were characterised using CD spectroscopy and Dc1.2 displayed a higher α-helix content and thermal stability compared to Dc1 and Dc1.1 in hydrophobic experimental conditions.

The amphibian skin plays an important role protecting the organism from external harmful factors such as microorganisms or UV radiation. Based on biorational strategies, many studies have investigated the cutaneous secretion of anurans as a source of bioactive molecules. By a peptidomic approach, a novel antioxidant peptide (AOP) with in vitro free radical scavenging ability was isolated from Physalaemus nattereri. The AOP, named antioxidin-I, has a molecular weight [M+H] + = 1543.69Da and a TWYFITPYIPDK primary amino acid sequence. The gene encoding the antioxidin-I precursor was expressed in the skin tissue of three other Tropical frog species: Phyllomedusa tarsius, P. distincta and Pithecopus rohdei. cDNA sequencing revealed highly homologous regions (signal peptide and acidic region). Mature antioxidin-I has a novel primary sequence with low similarity compared with previously described amphibian's AOPs. Antioxidin-I adopts a random structure even at high concentrations of hydrophobic solvent, it has poor antimicrobial activity and poor performance in free radical scavenging assays in vitro, with the exception of the ORAC assay. However, antioxidin-I presented a low cytotoxicity and suppressed menadione-induced redox imbalance when tested with fibroblast in culture. In addition, it had the capacity to substantially attenuate the hypoxia-induced production of reactive oxygen species when tested in hypoxia exposed living microglial cells, suggesting a potential neuroprotective role for this peptide., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Bombesin-like peptides, which were identified from a diversity of amphibian skin secretions, have been demonstrated to possess several biological functions such as stimulation of smooth muscle contraction and regulation of food intake. Here, we report two novel bombesin-like peptides, bombesin-OS and bombesin-PE, which were isolated from Odorrana schmackeri and Pelophylax kl. esculentus, respectively. The mature peptides were identified and structurally confirmed by high performance Scliquid chromatography (HPLC) and tandem mass spectrometry (MS/MS). Subsequently, the effects of these purified chemically-synthetic peptides on smooth muscle were determined in bladder, uterus, and ileum. The synthetic replications were revealed to have significant pharmacological effects on these tissues. The EC 50 values of bombesin-OS for bladder, uterus and ileum, were 10.8 nM, 33.64 nM, and 12.29 nM, respectively. Furthermore, compared with bombesin-OS, bombesin-PE showed similar contractile activity on ileum smooth muscle and uterus smooth muscle, but had a higher potency on bladder smooth muscle. The EC 50 value of bombesin-OS for bladder was around 1000-fold less than that of bombesin-PE. This suggests that bombesin-OS and bombesin-PE have unique binding properties to their receptors. The precursor of bombesin-OS was homologous with that of a bombesin-like peptide, odorranain-BLP-5, and bombesin-PE belongs to the ranatensin subfamily. We identified the structure of bombesin-OS and bombesin-PE, two homologues peptides whose actions may provide a further clue in the classification of ranid frogs, also in the provision of new drugs for human health., Competing Interests: The authors declare no conflict of interest.

Bombinin and bombinin H are two antimicrobial peptide (AMP) families initially discovered from the skin secretion of Bombina that share the same biosynthetic precursor-encoding cDNAs, but have different structures and physicochemical properties. Insight into their possible existing relationship lead us to perform the combination investigations into their anti-infectious activities. In this work, we report the molecular cloning and functional characterization of two novel AMPs belonging to bombinin and bombinin H families from secretions of Bombina orientalis Their mature peptides (BHL-bombinin and bombinin HL), coded by single ORF, were chemically synthesized along with an analogue peptide that replaced L-leucine with D-leucine from the second position of the N-terminus (bombinin HD). CD analysis revealed that all of them displayed well-defined α-helical structures in membrane mimicking environments. Furthermore, BHL-bombinin displayed broad-spectrum bactericidal activities on a wide range of microorganisms, while bombinin H only exhibited a mildly bacteriostatic effect on the Gram-positive bacteria Staphylococcus aureus The combination potency of BHL-bombinin with either bombinin HL or bombinin HD showed the synergistic inhibition activities against S. aureus (fractional inhibitory concentration index (FICI): 0.375). A synergistic effect has also been observed between bombinin H and ampicillin, which was further systematically evaluated and confirmed by in vitro time-killing investigations. Haemolytic and cytotoxic examinations exhibited a highly synergistic selectivity and low cytotoxicity on mammalian cells of these three peptides. Taken together, the discovery of the potent synergistic effect of AMPs in a single biosynthetic precursor with superior functional selectivity provides a promising strategy to combat multidrug-resistant pathogens in clinical therapy., (© 2017 The Author(s).)

The defensive skin secretions of amphibians continue to be an excellent source of novel biologically-active peptides. Here we report the identification and pharmacological activity of a novel C-terminally amided myotropic octapeptide from the skin secretion of the African hyperoliid frog, Kassina senegalensis . The 8-amino acid peptide has the following primary structure: WMSLGWSL-amide and has a molecular mass of 978 Da. The primary structure and organisation of the biosynthetic precursor of WL-8 amide was successfully deduced from cloned skin secretion-derived cDNA. The open-reading frame encoded a single copy of WL-8, located at the C-terminus. Synthetic WL-8 amide was found to cause relaxation of rat tail artery smooth muscle with an EC 50 of 25.98 nM. This peptide is unique in terms of its primary structure and is unlike any other peptide previously isolated from an amphibian source which has been archived in the NCBI database. WL-8 amide thus represents the prototype of a novel family of myotropic peptide from amphibian defensive skin secretions., Competing Interests: The authors declare no conflict of interest.

Frog skin secretions contain complex peptidomes and peptidic protease inhibitors that are one of the biologically and structurally described groups of components. In the present study, by use of molecular 'shotgun' cloning and LC MS/MS fractionation sequencing, a novel Bowman-Birk-type heptadecapeptide (AALKGCWTKSIPPKPCF-amide), named O dorrana s chmackeriT rypsin I nhibitor (OSTI), with a canonical Cys 6 -Cys 16 disulfide bridge, was isolated and identified in piebald odorous frog ( O. schmackeri ) skin secretion. A synthetic replicate of OSTI-exhibited trypsin inhibitory activity with a K i value of 0.3 ± 0.04 nM and also a tryptase inhibitory effect with a K i of 2.5 ± 0.6 μM. This is the first time that this property has been reported for a peptide originating from amphibian sources. In addition, substituting lysine (K) with phenylalanine (F) at the presumed P1 position, completely abrogated the trypsin and tryptase inhibition, but produced a strong chymotrypsin inhibition with a K i of 1.0 ± 0.1 μM. Thus, the specificity of this peptidic protease inhibitor could be optimized through modifying the amino acid residue at the presumed P1 position and this novel native OSTI, along with its analogue, [Phe 9 ]-OSTI, have expanded the potential drug discovery and development pipeline directed towards alleviation of serine protease-mediated pathologies., (© 2017 The Author(s).)

A novel peptide was isolated from the skin secretion of Chinese large odorous frog, Odorrana livida, and was named as Rana-BI. The cDNA sequencing was obtained by 'shotgun' cloning. The amino acid sequence of the mature peptide was identified as Gly-Leu-Leu-Ser-Gly-Lys-Ser-Val-Lys-Gly-Ser-Ile-OH by automated Edman degradation, and the molecular weight of the peptide was confirmed to be 1144.68 Da by MALDI-TOF and liquid chromatography/MS. Subsequently, the bioactivity of synthetic peptide was evaluated by smooth muscle assay using isolated rat bladder preparation. It was demonstrated that Rana-BI inhibited the contraction of rat bladder induced by bradykinin. Comparing with other peptides by searching from database, the primary structure of Rana-BI showed high similarity with that of an antimicrobial peptide of Rana family (12/12 residues). These data revealed a novel biological function of this peptide., (Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.)

Defensins are one of the major families of antimicrobial peptides (AMPs), and have been reported from prokaryotic to eukaryotic kingdoms. But defensins are seldom found in amphibian which is a major resource of novel AMPs. A novel defensin-like AMP (defensin-TK) was isolated and characterized from skin secretions of the tree frog Theloderma kwangsiensis. The cDNA encoding defensin-TK precursor was cloned from the skin cDNA library of T. kwangsiensis. The deduced precursor of defensin-TK was composed of three domains, a signal peptide of 16 residues, a spacer peptide of 1 residues and a mature peptide of 42 residues. The mature peptide of defensin-TK shared the highest identity with the salamander (Cynops fudingensis) defensin CFBD-1. The six conserved cysteines which formed intramolecular disulfide bonds of defensins also exist in defensin-TK. Phylogenetic analysis indicated that defensin-TK was closely related to fish β-defensins. Defensin-TK showed potent and broad-spectrum antimicrobial activity. In addition, defensin-TK exerted a low hemolytic activity on human red cells. These results suggested defensin-TK might play an important role in defense the skin pathogenic microbes of tree frog T. kwangsiensis, and might be a promising candidate for development of novel antimicrobial agents., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Albeit many bioactive peptides have been reported from amphibian skins, no anti-platelet peptide has been identified till to date. Here, an anti-platelet peptide, namely Zongdian platelet inhibitor (ZDPI), with the molecular weight of 1798.6 Da, was purified and characterized from skin secretions of the frog, Amolops loloensis. The amino acid sequence of ZDPI was determined as FRGCWLKNYSPRGCL-NH2 by combination methods of Edman degradation, mass spectrometry analysis and carboxypeptidase Y treatment revealing that it is composed of 15 amino acid residues with two cysteines formed an intra-molecular disulfide bridge and C-terminal amidation. cDNA encoding ZDPI precursor was cloned from skin cDNA library of A. loloensis. The precursor is composed of 63 amino acid (aa) residues including the predicted signal peptide (22 aa), an acidic spacer peptide (19 aa), and mature ZDPI. BLAST search indicates that ZDPI belongs to antimicrobial peptide family of ranacyclin, peptide leucine arginine or odorranain. It was found to inhibit ADP-induced platelet aggregation in a dose-dependent manner. At the concentration of 32 μg/ml, ZDPI completely inhibited platelet aggregation induced by ADP. To the best of our knowledge, this is the first report about an anti-platelet peptide from amphibian skin secretions. Considering its strong inhibitory ability on platelets and simple structure, ZDPI might be an excellent candidate or template to develop anti-thrombosis agent. In addition, the discovery of anti-platelet peptide in the frog skin increases biological function spectrum of amphibian skin peptides.

Anuran secretions are rich sources of bioactive molecules, including antimicrobial and antitumoral compounds. The aims of this study were to investigate the therapeutic potential of Physalaemus nattereri skin secretion against skin cancer cells, and to assess its cytotoxic action mechanisms on the murine melanoma cell line B16F10. Our results demonstrated that the crude secretion reduced the viability of B16F10 cells, causing changes in cell morphology (e.g., round shape and structure shrinkage), reduction in mitochondrial membrane potential, increase in phosphatidylserine exposure, and cell cycle arrest in S-phase. Together, these changes suggest that tumor cells die by apoptosis. This skin secretion was also subjected to chromatographic fractioning using RP-HPLC, and eluted fractions were assayed for antiproliferative and antibacterial activities. Three active fractions showed molecular mass components in a range compatible with peptides. Although the specific mechanisms causing the reduced cell viability and cytotoxicity after the treatment with crude secretion are still unknown, it may be considered that molecules, such as the peptides found in the secretion, are effective against B16F10 tumor cells. Considering the growing need for new anticancer drugs, data presented in this study strongly reinforce the validity of P. nattereri crude secretion as a rich source of new anticancer molecules.

Amphibians exhibit various, characteristic adaptations related to their "incomplete" shift from the aquatic to the terrestrial habitat. In particular, the integument was subject to a number of specialized modifications during the evolution of these animals. In this review, we place special emphasis on endogenous host-defence skin peptides from the cuteanous granular glands anuran amphibians (frogs and toads). The overview on the two broad groups of neuroactive and antimicrobial peptides (AMPs) goes beyond a simple itemization in that we provide a new perspective into the evolution and function of anuran AMPs. Briefly, these cationic, amphipathic and α-helical peptides are traditionally viewed as being part of the innate immune system, protecting the moist skin against invading microorganisms through their cytolytic action. However, the complete record of anuran species investigated to date suggests that AMPs are distributed sporadically (i.e., non-universally) across Anura. Together with the intriguing observation that virtually all anurans known to produce neuropeptides in their granular glands also co-secrete cytolytic peptides, we call the traditional role for AMPs as being purely antimicrobial into question and present an alternative scenario. We hypothesize AMPs to assist neuroactive peptides in their antipredator role through their cytolytic action increasing the delivery of the latter to the endocrine and nervous system of the predator. Thus, AMPs are more accurately viewed as cytolysins and their contribution to the immune system is better regarded as an accessory benefit., (Copyright © 2014 Elsevier Inc. All rights reserved.)