Multifunctional host-defense peptides isolated from skin secretions of the banana tree dwelling frog Boana platanera (Hylidae; Hylinae).

Five host-defense peptides (figainin 2PL, hylin PL, raniseptin PL, plasticin PL, and peptide YL) were isolated from norepinephrine-stimulated skin secretions of the banana tree dwelling frog Boana platanera (Hylidae; Hylinae) collected in Trinidad. Raniseptin PL (GVFDTVKKIGKAVGKFALGVAKNYLNS.NH2) and figainin 2PL (FLGTVLKLGKAIAKTVVPMLTNAMQPKQ. NH2) showed potent and rapid bactericidal activity against a range of clinically relevant Gram-positive and Gram-negative ESKAPE + pathogens and Clostridioides difficile. The peptides also showed potent cytotoxic activity (LC50 values < 30 µM) against A549, MDA-MB-231 and HT29 human tumor-derived cell lines but appreciably lower hemolytic activity against mouse erythrocytes (LC50 = 262 ± 14 µM for raniseptin PL and 157 ± 16 µM for figainin 2PL). Hylin PL (FLGLIPALAGAIGNLIK.NH2) showed relatively weak activity against microorganisms but was more hemolytic. The glycine-leucine-rich peptide with structural similarity to the plasticins (GLLSTVGGLVGGLLNNLGL.NH2) and the non-cytotoxic peptide YL (YVPGVIESLL.NH2) lacked antimicrobial and cytotoxic activities. Hylin PL, raniseptinPL and peptide YL stimulated the rate of release of insulin from BRIN-BD11 clonal ß-cells at concentrations ≥100 nM. Peptide YL was the most effective (2.3-fold increase compared with basal rate at 1 µM concentration) and may represent a template for the design of a new class of incretin-based anti-diabetic drugs.

Purification, conformational analysis and cytotoxic activities of host-defense peptides from the Tungara frog Engystomops pustulosus (Leptodactylidae; Leiuperinae).

The amphibian family Leptodactylidae is divided into three sub-families: Leiuperinae, Leptodactylinae, and Paratelmatobiinae. Host-defense peptides (HDPs) present in the skins of frogs belonging to the Leptodactylinae have been studied extensively, but information is limited  regarding peptides from Leiuperinae species. Peptidomic analysis of norepinephrine-stimulated skin secretions from the Tungara frog Engystomops pustulosus (Leiuperinae) collected in Trinidad led to the isolation and structural characterization of previously undescribed pustulosin-1 (FWKADVKEIG KKLAAKLAEELAKKLGEQ), [Q28E] pustulosin-1 (pustulosin-2), and pustulosin-3 (DWKETAKELLKKIGAKVAQVISDKLNPAPQ). The primary structures of these peptides do not resemble those of previously described frog skin HDPs. In addition, the secretions contained tigerinin-1EP (GCKTYLIEPPVCT) with structural similarity to the tigerinins previously identified in skin secretions from frogs from the family Dicroglossidae. Pustulosin-1 and -3 adopted extended α-helical conformations in 25% trifluoroethanol-water and in the presence of cell membrane models (sodium dodecylsulfate and dodecylphosphocholine micelles). Pustulosin-1 and -3 displayed cytotoxic activity against a range of human tumor-derived cell lines (A549, MDA-MB-231, and HT29), but their therapeutic potential for development into anti-cancer agents is limited by their comparable cytotoxic activity against non-neoplastic human umbilical vein endothelial cells. The peptides also displayed weak antimicrobial activity against Escherichia coli (MIC = 125 µM) but were inactive against Staphylococcus aureus. Tigerinin-1EP was inactive against both the tumor-derived cells and bacteria.

Purification, Conformational Analysis and Cytotoxic Activities of Host-Defense Peptides from the Giant Gladiator Treefrog Boana boans (Hylidae: Hylinae).

Frogs from the extensive amphibian family Hylidae are a rich source of peptides with therapeutic potential. Peptidomic analysis of norepinephrine-stimulated skin secretions from the Giant Gladiator Treefrog Boana boans (Hylidae: Hylinae) collected in Trinidad led to the isolation and structural characterization of five host-defense peptides with limited structural similarity to figainin 2 and picturin peptides from other frog species belonging to the genus Boana. In addition, the skin secretions contained high concentrations of tryptophyllin-BN (WRPFPFL) in both C-terminally α-amidated and non-amidated forms. Figainin 2BN (FLGVALKLGKVLG KALLPLASSLLHSQ) and picturin 1BN (GIFKDTLKKVVAAVLTTVADNIHPK) adopt α-helical conformations in trifluroethanol-water mixtures and in the presence of cell membrane models (sodium dodecylsulfate and dodecylphosphocholine micelles). The CD data also indicate contributions from turn structures. Both peptides and picturin 2BN (GLMDMLKKVGKVALT VAKSALLP) inhibited the growth of clinically relevant Gram-negative and Gram-positive bacteria with MIC values in the range 7.8-62.5 µM. Figainin 2BN was potently cytotoxic to A549, MDA-MB-231 and HT-29 human tumor-derived cells (LC50 = 7-14 µM) but displayed comparable potency against non-neoplastic HUVEC cells (LC50 = 15 µM) indicative of lack of selectivity for cancer cells.

Antimicrobial, cytotoxic, and insulin-releasing activities of the amphibian host-defense peptide ocellatin-3N and its L-lysine-substituted analogs.

The host-defense peptide ocellatin-3N (GIFDVLKNLAKGVITSLAS.NH2 ), first isolated from the Caribbean frog Leptodactylus nesiotus, inhibited growth of clinically relevant Gram-positive and Gram-negative bacteria as well as a strain of the major emerging yeast pathogen Candida parapsilosis. Increasing cationicity while maintaining amphipathicity by the substitution Asp4 →Lys increased potency against the microorganisms by between 4- and 16-fold (MIC ≤3 µM) compared with the naturally occurring peptide. The substitution Ala18 →Lys and the double substitution Asp4 →Lys and Ala18 →Lys had less effects on potency. The [D4K] analog also showed 2.5- to 4-fold greater cytotoxic potency against non-small-cell lung adenocarcinoma A549 cells, breast adenocarcinoma MDA-MB-231 cells, and colorectal adenocarcinoma HT-29 cells (LC50 values in the range of 12-20 µM) compared with ocellatin-3N but was less hemolytic to mouse erythrocytes. However, the peptide showed no selectivity for tumor-derived cells [LC50 = 20 µM for human umbilical vein endothelial cells (HUVECs)]. Ocellatin-3N and [D4K]ocellatin-3N stimulated the release of insulin from BRIN-BD11 clonal ß-cells at concentrations ≥1 nM, and [A18K]ocellatin-3N, at concentrations ≥0.1 nM. No peptide stimulated the release of lactate dehydrogenase at concentrations up to 3 µM, indicating that plasma membrane integrity had been preserved. The three peptides produced an increase in intracellular [Ca2+ ] in BRIN-BD11 cells when incubated at a concentration of 1 µM. In view of its high insulinotropic potency and relatively low hemolytic activity, the [A18K] ocellatin analog may represent a template for the design of agents with therapeutic potential for the treatment of patients with type 2 diabetes.

Immunomodulatory, insulinotropic, and cytotoxic activities of phylloseptins and plasticin-TR from the Trinidanian leaf frog Phyllomedusa trinitatis.

The aim of the study was to determine the in vitro immunomodulatory, cytotoxic, and insulin-releasing activities of seven phylloseptin-TR peptides and plasticin-TR, first isolated from the frog Phyllomedusa trinitatis. The most cationic peptides, phylloseptin-1.1TR and phylloseptin-3.1TR, showed greatest cytotoxic potency against A549, MDA-MB231, and HT-29 human tumor-derived cells and against mouse erythrocytes. Phylloseptin-4TR was the most hydrophobic and the most effective peptide at inhibiting production of the proinflammatory cytokines TNF-α and IL-1ß by mouse peritoneal cells but was without effect on production of the antiinflammatory cytokine IL-10. Phylloseptin-2.1TR and phylloseptin-3.3TR were the most effective at stimulating the production of IL-10. The noncytotoxic peptide, plasticin-TR, inhibited production of TNF-α and IL-1ß but was without effect on IL-10 production. The results of CD spectroscopy suggest that the different properties of plasticin-TR compared with the immunostimulatory activities of the previously characterized plasticin-L1 from Leptodactylus laticeps may arise from greater ability of plasticin-TR to oligomerize and adopt a stable helical conformation in a membrane-mimetic environment. All peptides stimulated release of insulin from BRIN-BD11 rat clonal ß cells with phylloseptin-3.2TR being the most potent and effective and phylloseptin-2.1TR the least effective suggesting that insulinotropic potency correlates inversely with helicity. The study has provided insight into structure-activity relationships among the phylloseptins. The combination of immunomodulatory and insulinotropic activities together with low cytotoxicity suggests that phylloseptin-3.3TR and plasticin-TR may represent templates for the development of agents for use in antiinflammatory and type 2 diabetes therapies.

Peptidomic analysis of the host-defense peptides in skin secretions of Rana graeca provides insight into phylogenetic relationships among Eurasian Rana species.

Peptidomic analysis of norepinephrine-stimulated skin secretions from the Greek stream frog Rana graeca Boulenger, 1891 led to the identification and structural characterization of a range of host-defense peptides. These comprised brevinin-1GRa, brevinin-1GRb and an N-terminally extended form of brevinin-1GRb, ranatuerin-2GR together with its oxidized form and (11-28) fragment, temporin-GRa, temporin-GRb and its non-amidated form, and a melittin-related peptide, MRP-GR and its (1-18) fragment. The most abundant peptide, MRP-GR significantly (P < 0.001) stimulated insulin release from BRIN-BD11 clonal ß-cells at concentrations ≥0.1 nM. Rana graeca (formerly Rana graeca graeca) and the morphologically similar Italian stream frog Rana italica Dubois, 1987 (formerly Rana graeca italica) were originally regarded as sub-species. However, the primary structures of the host defense peptides from both frogs support the claim based upon comparisons of the nucleotide sequences of S1 satellite DNA that R. graeca and R. italica are separate species. Cladistic analyses based upon the primary structures of the brevinin-1 and ranatuerin-2 peptides from Eurasian frogs indicate a close phylogenetic relationship between R. graeca and Rana latastei whereas R. italica is most closely related to Rana dalmatina.

Purification, Conformational Analysis, and Properties of a Family of Tigerinin Peptides from Skin Secretions of the Crowned Bullfrog Hoplobatrachus occipitalis.

Four host-defense peptides belonging to the tigerinin family (tigerinin-1O: RICTPIPFPMCY; tigerinin-2O: RTCIPIPLVMC; tigerinin-3O: RICTAIPLPMCL; and tigerinin-4O: RTCIPIPPVCF) were isolated from skin secretions of the African crowned bullfrog Hoplobatrachus occipitalis. In aqueous solution at pH 4.8, the cyclic domain of tigerinin-2O adopts a rigid amphipathic conformation that incorporates a flexible N-terminal tail. The tigerinins lacked antimicrobial (MIC > 100 µM) and hemolytic (LC50 > 500 µM) activities but, at a concentration of 20 µg/mL, significantly (P < 0.05) inhibited production of interferon-γ (IFN-γ) by peritoneal cells from C57BL/6 mice without affecting production of IL-10 and IL-17. Tigerinin-2O and -4O inhibited IFN-γ production at concentrations as low as 1 µg/mL. The tigerinins significantly (P ≤ 0.05) stimulated the rate of insulin release from BRIN-BD11 clonal ß-cells without compromising the integrity of the plasma membrane. Tigerinin-1O was the most potent (threshold concentration 1 nM) and the most effective (395% increase over basal rate at a concentration of 1 µM). Tigerinin-4O was the most potent and effective peptide in stimulating the rate of glucagon-like peptide-1 release from GLUTag enteroendocrine cells (threshold concentration 10 nM; 289% increase over basal rate at 1 µM). Tigerinin peptides have potential for development into agents for the treatment of patients with type 2 diabetes.

Host-defense and trefoil factor family peptides in skin secretions of the Mawa clawed frog Xenopus boumbaensis (Pipidae).

Peptidomic analysis of norepinephrine-stimulated skin secretions from the octoploid Mawa clawed frog Xenopus boumbaensis Loumont, 1983 led to the identification and characterization of 15 host-defense peptides belonging to the magainin (two peptides), peptide glycine-leucine-amide (PGLa; three peptides), xenopsin precursor fragment (XPF; three peptides), caerulein precursor fragment (CPF; two peptides), and caerulein precursor fragment-related peptide (CPF-RP; five peptides) families. In addition, caerulein and three peptides with structural similarity to the trefoil factor family (TFF) peptides, xP2 and xP4 from Xenopus laevis were also present in the secretions. Consistent with data from comparisons of the nucleotides sequence of mitochondrial and nuclear genes, the primary structures of the peptides suggest a close phylogenetic relationship between X. boumbaensis and the octoploid frogs Xenopus amieti and Xenopus andrei. As the three species occupy disjunct ranges within Cameroon, it is suggested that they diverged from a common ancestor by allopatric speciation.

Anti-cancer, immunoregulatory, and antimicrobial activities of the frog skin host-defense peptides pseudhymenochirin-1Pb and pseudhymenochirin-2Pa.

Pseudhymenochirin-1Pb (Ps-1Pb) and pseudhymenochirin-2Pa (Ps-2Pa) are host-defense peptides, first isolated from skin secretions of the frog Pseudhymenochirus merlini (Pipidae). Ps-1Pb and Ps-2Pa are highly cytotoxic (LC50<12 µM) against non-small cell lung adenocarcinoma A549 cells, breast adenocarcinoma MDA-MB-231 cells, and colorectal adenocarcinoma HT-29 cells but are also hemolytic against human erythrocytes (LC50=28±2 µM for Ps-1Pb and LC50=6±1 µM for Ps-2Pa). Ps-2Pa shows selective cytotoxicity for tumor cells (LC50 against non-neoplastic human umbilical vein (HUVEC) cells=68±2 µM). Ps-1Pb and Ps-2Pa (5 µg/mL) significantly inhibit production of the anti-inflammatory cytokine IL-10 and the multifunctional cytokine IL-6 from lipopolysaccharide (LPS)-stimulated peritoneal macrophages from C57BL/6 mice and enhance the production of the pro-inflammatory cytokine IL-23 from both unstimulated and LPS-stimulated macrophages. Ps-1Pb potently (MIC≤10 µM) inhibits growth of multidrug-resistant clinical isolates of the Gram-positive bacteria methicillin-resistant Staphylococcus aureus and Staphylococcus epidermidis, and the Gram-negative bacteria Acinetobacter baumannii and Stenotrophomonas maltophilia. Ps-2Pa shows the same high potency (MIC≤10 µM) against the Gram-positive bacteria but is 2-4 fold less potent against the Gram-negative isolates. Ps-1Pb at 4×MIC kills 99.9% of Escherichia coli within 30 min and 99.9% of S. aureus within 180 min. In conclusion, cytotoxicity against tumor cells, cytokine-mediated immunomodulatory properties, and broad-spectrum antimicrobial activity suggest that the Ps-1Pb and Ps-2Pa represent templates for design of non-hemolytic analogs for tumor therapy and for treatment of infections in cancer patients produced by multidrug-resistant pathogens.

Host-defense peptides from skin secretions of Fraser's clawed frog Xenopus fraseri (Pipidae): Further insight into the evolutionary history of the Xenopodinae.

Peptidomic analysis of norepinephrine-stimulated skin secretions of the tetraploid frog Xenopus fraseri Boulenger, 1905 (Pipidae) led to identification of 13 host-defense peptides. The primary structures of the peptides demonstrate that they belong to the magainin (3 peptides), peptide glycine-leucine-amide, PGLa (4 peptides), and xenopsin-precursor fragment, XPF (2 peptides) families, first identified in Xenopus laevis, together with caerulein precursor fragment-related peptides, CPF-RP (4 peptides), first identified in Silurana tropicalis. In addition, the secretions contain a molecular variant of xenopsin displaying the substitution Arg(4)→Lys compared with X. laevis xenopsin and peptide glycine-tyrosine-amide (PGYa) (GRIIPIYPEFERVFA KKVYPLY.NH2) whose function is unknown. The most potent antimicrobial peptide identified is CPF-RP-F1 (GFGSVLGKALKFGANLL.NH2) with MIC=12.5µM against Staphylococcus aureus and 50µM against Escherichia coli. On the basis of similarities in morphology and advertisement calls, X. fraseri has been placed in a species group that includes the octoploids Xenopus amieti and Xenopus andrei, and the tetraploid Xenopus pygmaeus. Cladistic analyses based upon the primary structures of magainin, PGLa, and CPF-RP peptides support a close evolutionary relationship between X. fraseri, X. amieti and X. andrei but suggest a more distant relationship with X. pygmaeus.

Conformational analysis and cytotoxic activities of the frog skin host-defense peptide, hymenochirin-1Pa.

Hymenochirin-1Pa (LKLSPKTKDTLKKVLKGAIKGAIAIASMA-NH2) is a host-defense peptide first isolated from skin secretions of the frog Pseudhymenochirus merlini (Pipidae). A nuclear magnetic resonance structural investigation demonstrates that the peptide has a random coil conformation in water but, in the membrane-mimetic solvent 50% (v/v) trifluoroethanol-water adopts a well-defined conformation characterized by two α-helical domains from residues K6 to G17 and from G21 to M28, with the N-terminal region unfolded. The presence of a GXXXG domain, the most common structural motif found at the interface between interacting trans-membrane helices, between residues 17 and 21, introduces a kink corresponding to a deviation from linearity of 93 ± 31°. Hymenochirin-1Pa shows broad spectrum anti-bacterial activity, including high potency against multidrug-resistant clinical isolates of Staphylococcus aureus, Acinetobacter baumannii, and Stenotrophomonas maltophilia. The peptide also shows high cytotoxic potency against human non-small lung adenocarcinoma A549 cells, breast adenocarcinoma MDA-MB-231 cells, and colorectal adenocarcinoma HT-29 cells but its therapeutic potential as an anti-cancer agent is limited by moderate hemolytic activity against human erythrocytes and lack of selectivity for tumor cells. Increasing cationicity of the peptide by substituting the Asp(9) residue by either L-Lys (K) or D-Lys (k) has relatively minor effects on antimicrobial and anti-tumor potencies but the [D9k] analog is non-hemolytic LC50 > 400 µM. Thus, [D9k]hymenochirin-1Pa may serve as a template for the design of non-toxic antimicrobial agents for use against multidrug-resistant pathogenic bacteria.

Host-defense peptides from skin secretions of the octoploid frogs Xenopus vestitus and Xenopus wittei (Pipidae): insights into evolutionary relationships.

The primary structures of host-defense peptides have proved useful in elucidating the evolution history of frogs. Peptidomic analysis was used to compare the diversity of host-defense peptides in norepinephrine-stimulated skin secretions from the octoploid frogs, Xenopus vestitus (Kivu clawed frog) and Xenopus wittei (De Witte's clawed frog) in the family Pipidae. Structural characterization demonstrated that the X. vestitus peptides belong to the magainin (3 peptides), peptide glycine-leucine-amide (PGLa; 4 peptides), xenopsin-precursor fragment (XPF; 1 peptide), and caerulein-precursor fragment (CPF; 5 peptides) families. The X. wittei peptides comprise magainin (4 peptides), PGLa (1 peptide), XPF (2 peptides), and CPF (7 peptides). In addition, secretions from both species contain caerulein, identical to the peptide from Xenopus laevis, but X. wittei secretions contains the novel peptide [R4K]xenopsin. The variability in the numbers of paralogs in each peptide family indicates a selective silencing of the host-defense peptide genes following the polyploidization events. The primary structures of the peptides provide insight into phylogenetic relationships among the octoploid Xenopus frogs. The data support a sister-group relationship between X. vestitus and Xenopus lenduensis, suggestive of bifurcating speciation after allopolyploidization, whereas X. wittei is more closely related to the Xenopus amieti-Xenopus andrei group suggesting a common tetraploid ancestor. Consistent with previous data, the CPF peptides showed the highest growth inhibitory activity against bacteria with CPF-W6 (GIGSLLAKAAKLAAGLV.NH2) combining high antimicrobial potency against Staphylococcus aureus (MIC=4 µM) with relatively low hemolytic activity (LC50=190 µM).

Peptides with in vitro anti-tumor activity from the venom of the Eastern green mamba, Dendroaspis angusticeps (Elapidae).

Two structurally related (48.6% amino acid sequence identity) peptides with cytotoxic activity against human non-small cell lung adenocarcinoma A549 cells were purified from the venom of the Eastern green mamba Dendroaspis angusticeps using reversed phase HPLC. The peptides were identified as members of the three-finger superfamily of snake toxins by mass fingerprinting of tryptic digests. The more potent peptide (LC50 against A549 cells = 56±4µg/ml) was identical to the previously described toxin C13S1C1 and the less active peptide (LC50 against A549 cells = 106±5µg/ml) was identical to toxin F-VIII. Toxin C13S1C1 was also cytotoxic against breast adenocarcinoma MDA-MB-231 cells (LC50 = 62±2µg/ml) and colorectal adenocarcinoma HT-29 cells (LC50 = 110±4µg/ml). Although the peptide was appreciably less hemolytic activity against human erythrocytes (LC50 >600µg/ml), it was cytotoxic to human umbilical vein endothelial HUVEC cells (57±3µg/ml) indicating no differential activity against cell lines derived from neoplastic tissues. Toxin F-VIII was not cytotoxic to MDA-MB-231, HT-29 cells, and HUVEC cells at concentrations up to 300µg/ml and was not hemolytic at concentrations up to 1mg/ml. Neither peptide inhibited growth of reference strains of Escherichia coli or Staphylococcus aureus (MIC values >200µg/ml).

Potential therapeutic applications of multifunctional host-defense peptides from frog skin as anti-cancer, anti-viral, immunomodulatory, and anti-diabetic agents.

Frog skin constitutes a rich source of peptides with a wide range of biological properties. These include host-defense peptides with cytotoxic activities against bacteria, fungi, protozoa, viruses, and mammalian cells. Several hundred such peptides from diverse species have been described. Although attention has been focused mainly on antimicrobial activity, the therapeutic potential of frog skin peptides as anti-infective agents remains to be realized and no compound based upon their structures has yet been adopted in clinical practice. Consequently, alternative applications are being explored. Certain naturally occurring frog skin peptides, and analogs with improved therapeutic properties, show selective cytotoxicity against tumor cells and viruses and so have potential for development into anti-cancer and anti-viral agents. Some peptides display complex cytokine-mediated immunomodulatory properties. Effects on the production of both pro-inflammatory and anti-inflammatory cytokines by peritoneal macrophages and peripheral blood mononuclear cells have been observed so that clinical applications as anti-inflammatory, immunosuppressive, and immunostimulatory agents are possible. Several frog skin peptides, first identified on the basis of antimicrobial activity, have been shown to stimulate insulin release both in vitro and in vivo and so show potential as incretin-based therapies for treatment of patients with Type 2 diabetes mellitus. This review assesses the therapeutic possibilities of peptides from frogs belonging to the Ascaphidae, Alytidae, Pipidae, Dicroglossidae, Leptodactylidae, Hylidae, and Ranidae families that complement their potential role as anti-infectives for use against multidrug-resistant microorganisms.

A family of antimicrobial and immunomodulatory peptides related to the frenatins from skin secretions of the Orinoco lime frog Sphaenorhynchus lacteus (Hylidae).

Peptidomic analysis of norepinephrine-stimulated skin secretions of the Orinoco lime tree frog Sphaenorhynchus lacteus (Hylidae, Hylinae) revealed the presence of three structurally related host-defense peptides with limited sequence similarity to frenatin 2 from Litoria infrafrenata (Hylidae, Pelodryadinae) and frenatin 2D from Discoglossus sardus (Alytidae). Frenatin 2.1S (GLVGTLLGHIGKAILG.NH2) and frenatin 2.2S (GLVGTLLGHIGKAILS.NH2) are C-terminally α-amidated but frenatin 2.3S (GLVGTLLGHIGKAILG) is not. Frenatin 2.1S and 2.2S show potent bactericidal activity against clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) and Staphylococcus epidermidis (MIC ≤16µM) but are less active against a range of Gram-negative bacteria. Frenatin 2.1S (LC50=80±6 µM) and 2.2S (LC50=75±5 µM) are cytotoxic against non-small cell lung adenocarcinoma A549 cells but are less hemolytic against human erythrocytes (LC50=167±8 µM for frenatin 2.1S and 169±7 µM for 2.2S). Weak antimicrobial and cytotoxic potencies of frenatin 2.3S demonstrate the importance of C-terminal α-amidation for activity. Frenatin 2.1S and 2.2S significantly (P<0.05) increased production of proinflammatory cytokines IL-1ß and IL-23 by lipopolysaccharide (LPS)-stimulated mouse peritoneal macrophages and frenatin 2.1S also enhanced production of TNF-α. Effects on IL-6 production were not significant. Frenatin 2.2S significantly downregulated production of the anti-inflammatory cytokine IL-10 by LPS-stimulated cells. The data support speculation that frenatins act on skin macrophages to produce a cytokine-mediated stimulation of the adaptive immune system in response to invasion by microorganisms. They may represent a template for the design of peptides with therapeutic applications as immunostimulatory agents.

Host-defense peptides with therapeutic potential from skin secretions of frogs from the family pipidae.

Skin secretions from frogs belonging to the genera Xenopus, Silurana, Hymenochirus, and Pseudhymenochirus in the family Pipidae are a rich source of host-defense peptides with varying degrees of antimicrobial activities and cytotoxicities to mammalian cells. Magainin, peptide glycine-leucine-amide (PGLa), caerulein-precursor fragment (CPF), and xenopsin-precursor fragment (XPF) peptides have been isolated from norepinephrine-stimulated skin secretions from several species of Xenopus and Silurana. Hymenochirins and pseudhymenochirins have been isolated from Hymenochirus boettgeri and Pseudhymenochirus merlini. A major obstacle to the development of these peptides as anti-infective agents is their hemolytic activities against human erythrocytes. Analogs of the magainins, CPF peptides and hymenochirin-1B with increased antimicrobial potencies and low cytotoxicities have been developed that are active (MIC < 5 µM) against multidrug-resistant clinical isolates of Staphylococcus aureus, Escherichia coli, Acinetobacter baumannii, Stenotrophomonas maltophilia and Klebsiella pneumoniae. Despite this, the therapeutic potential of frog skin peptides as anti-infective agents has not been realized so that alternative clinical applications as anti-cancer, anti-viral, anti-diabetic, or immunomodulatory drugs are being explored.

Anti-tumor activities of the host-defense peptide hymenochirin-1B.

The hymenochirins are a family of cationic, amphipathic, α-helical host-defense peptides, first isolated from skin secretions of the Congo clawed frog Hymenochirus boettgeri (Pipidae). Of the four hymenochirins tested, hymenochirin-1B (IKLSPETKDNLKKVLKGAIKGAIVAKMV.NH2) shows the greatest cytotoxic potency against non-small cell lung adenocarcinoma A549 cells (LC50=2.5±0.2 µM), breast adenocarcinoma MDA-MB-231 cells (LC50=9.0±0.3 µM), colorectal adenocarcinoma HT-29 cells (LC50=9.7±0.2 µM), and hepatocarcinoma HepG2 cells (LC50=22.5±1.4 µM) with appreciably less hemolytic activity against human erythrocytes (LC50=213±18µM). Structure-activity relationships were investigated by synthesizing analogs of hymenochirin-1B in which Pro(5), Glu(6) and Asp(9)on the hydrophilic face of the helix were replaced by one or more L-lysine or D-lysine residues. The [D9K] analog displays the greatest increase in potency against all four cell lines (up to 6 fold) but hemolytic activity also increases (LC50=174±12 µM). The [D9k] and [E6k,D9k] analogs retain relatively high cytotoxic potency against the tumor cells (LC50 in the range 2.1-21 µM) but show reduced hemolytic activity (LC50>300 µM). The data suggest that hymenochirin-1B has therapeutic potential as a template to generate potent, non-toxic anti-cancer agents.

Cytotoxic activities of [Ser49]phospholipase A2 from the venom of the saw-scaled vipers Echis ocellatus, Echis pyramidum leakeyi, Echis carinatus sochureki, and Echis coloratus.

Fractionation by reversed-phase HPLC of venom from four species of saw-scaled viper: Echis ocellatus, Echis pyramidum leakeyi, Echis carinatus sochureki, and Echis coloratus led to identification in each sample of an abundant protein with cytotoxic activity against human non-small cell lung adenocarcinoma A549 cells. The active component in each case was identified by MALDI-TOF mass fingerprinting of tryptic digests as [Ser49]phospholipase A2 ([Ser49]PLA2). An isoform of [Ser49]PLA2 containing the single Ala¹8→ Val substitution and a partially characterized [Asp49]PLA2 were also present in the E. coloratus venom. LC50 values against A549 cells for the purified [Ser49]PLA2 proteins from the four species are in the range 2.9-8.5 µM. This range is not significantly different from the range of LC50 values against human umbilical vein endothelial HUVEC cells (2.5-12.2 µM) indicating that the [Ser49]PLA2 proteins show no differential anti-tumor activity. The LC50 value for [Ser49]PLA2 from E. ocellatus against human erythrocytes is >100 µM and the MIC values against Escherichia coli and Staphylococcus aureus are >100 µM. It is suggested that the [Ser49]PLA2 proteins play a major role in producing local tissue necrosis and hemorrhage at the site of envenomation.

Transformation of the naturally occurring frog skin peptide, alyteserin-2a into a potent, non-toxic anti-cancer agent.

Alyteserin-2a (ILGKLLSTAAGLLSNL.NH(2)) is a cationic, amphipathic α-helical cell-penetrating peptide, first isolated from skin secretions of the midwife toad Alytes obstetricans. Structure-activity relationships were investigated by synthesizing analogs of alyteserin-2a in which amino acids on the hydrophobic face of the helix were replaced by L-tryptophan and amino acids on the hydrophilic face were replaced by one or more L-lysine or D-lysine residues. The Trp-containing peptides display increased cytotoxic activity against non-small cell lung adenocarcinoma A549 cells (up to 11-fold), but hemolytic activity against human erythrocytes increases in parallel. The potency of the N15K analog against A549 cells (LC(50) = 13 µM) increases sixfold relative to alyteserin-2a and the therapeutic index (ratio of LC(50) for erythrocytes and tumor cells) increases twofold. Incorporation of a D-Lys(11) residue into the N15K analog generates a peptide that retains potency against A549 cells (LC(50) = 15 µM) but whose therapeutic index is 13-fold elevated relative to the native peptide. [G11k, N15K] alyteserin-2a is also active against human hepatocarcinoma HepG2 cells (LC(50) = 26 µM), breast adenocarcinoma MDA-MB-231 cells (LC(50) = 20 µM), and colorectal adenocarcinoma HT-29 cells (LC(50) = 28 µM). [G11k, N15K] alyteserin-2a, in concentrations as low as 1 µg/mL, significantly (P < 0.05) inhibits the release of the immune-suppressive cytokines IL-10 and TGF-ß from unstimulated and concanavalin A-stimulated peripheral blood mononuclear cells. The data suggest a strategy of increasing the cationicity while reducing the helicity of naturally occurring amphipathic α-helical peptides to generate analogs with improved cytotoxicity against tumor cells but decreased activity against non-neoplastic cells.

An immunomodulatory peptide related to frenatin 2 from skin secretions of the Tyrrhenian painted frog Discoglossus sardus (Alytidae).

Norepinephrine-stimulated skin secretions of the Tyrrhenian painted frog Discoglossus sardus Tschudi, 1837 (Alytidae) did not contain any peptide with antimicrobial or hemolytic activity. However, peptidomic analysis of the secretions revealed the presence of an abundant peptide with structural similarity to frenatin 2, previously isolated from the Australian frog Litoria infrafrenata (Hylidae). The primary structure of the peptide, termed frenatin 2D, was established as DLLGTLGNLPLPFI.NH2 by automated Edman degradation and mass spectrometry with electron-transfer dissociation (ETD)-based fragmentation and confirmed by chemical synthesis. The structure of a second frenatin 2-related peptide, termed frenatin 2.1D, that was present in much lower abundance was established as GTLGNLPAPFPG. Frenatin 2D (20 µg/ml) significantly stimulated production of the proinflammatory cytokines TNF-α (P<0.05) and IL-1ß (P<0.01) by mouse peritoneal macrophages but the peptide did not potentiate the stimulation produced by lipopolysaccharide (LPS). The peptide increased IL-12 production in both unstimulated (P<0.01) and LPS-stimulated (P<0.05) cells but stimulatory effects on IL-6 production were not significant. The biological role of frenatin 2D is unknown but it is speculated that the peptide acts on skin macrophages to produce a cytokine-mediated stimulation of the adaptive immune system in response to invasion by microorganisms.