Search

Hymenochirin-1B (IKLSPETKDN(10)LKKVLKGAIK(20)GAIAVAKMV.NH2) is a cationic, amphipathic, α-helical, host-defense peptide, first isolated from skin secretions of the Congo clawed frog Hymenochirus boettgeri (Pipidae). Structure-activity relationships were investigated by synthesizing analogs in which the Pro(5), Glu(6) and Asp(9) on the hydrophilic face of the α-helix are substituted by one or more l-lysine or d-lysine residues. Although replacement with l-lysine generates analogs with increased antimicrobial potency against a range of Gram-positive and Gram-negative bacteria (up to 8-fold), the peptides are more hemolytic. Increasing the cationicity of hymenochirin-1B while reducing the helicity by substitutions with d-lysine generates analogs that are between 2 and 8 fold more potent than the native peptide and are equally or less hemolytic. [E6k,D9k]hymenochirin-1B represents a candidate for drug development as it shows high potency against clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) and a range of Gram-negative bacteria, including multidrug-resistant strains of Acinetobacter baumannii and Stenotrophomonas maltophilia (MIC in the range 0.8-3.1 μM) and NDM-1 carbapenemase-producing clinical isolates of Klebsiella pneumoniae, Escherichia coli, Enterobacter cloacae and Citrobacter freundii (MIC in the range 3.1-6.25 μM), and low hemolytic activity (LC50=302 μM). [E6k,D9k]hymenochirin-1B, at a concentration of 2.5 μM, significantly (P

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 (P0.05) inhibits the release of the immune-suppressive cytokines IL-10 and TGF-β 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.

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).

International audience; Peptidomic analysis was used to compare the diversity of host-defense peptides in norepinephrine-stimulated skin secretions from laboratory-generated female F1 hybrids of Xenopus laevis and Xenopus borealis (Pipidae). Skin secretions of hybrids with maternal X. laevis (XLB) contained 12 antimicrobial peptides (AMPs), comprising 8 from X. laevis and 4 from X. borealis. Magainin-B1, XPF-B1, PGLa-B1 CPF-B2, CPF-B3 and CPF-B4 from X. borealis and XPF-1, XPF-2, and CPF-6 from X. laevis were not detected and CPF-1 and CPF-7 were present in low concentration. The secretions contained caerulein and caerulein-B1 derived from both parents but lacked X. laevis xenopsin and X. borealis caerulein-B2. Skin secretions of hybrids with maternal X. borealis (XBL) contained 14 AMPs comprising 6 from X. borealis and 8 from X. laevis. Magainin-B1, XPF-B1, PGLa-B1, CPF-B2, XPF-1, CPF-5, and CPF-7 were absent and CPF-B3, CPF-B4, CPF-1 and CPF-6 were present only in low concentration. Xenopsin and caerulein were identified in the secretions but caerulein-B2 was absent and caerulein-B1 was present in low concentration. No peptides were identified in secretions of either XLB or XBL hybrids that were not present in the parental species. The data indicate that hybridization between X. laevis and X. borealis results in increased diversity of host-defense peptides in skin secretions but point to extensive AMP gene silencing compared with previously studied female X. laevis×X. muelleri F1 hybrids and no novel peptide expression.

Population declines due to amphibian chytridiomycosis among selected species of ranid frogs from western North America have been severe, but there is evidence that the Oregon spotted frog, Rana pretiosa Baird and Girard, 1853, displays resistance to the disease. Norepinephrine-stimulated skin secretions were collected from a non-declining population of R. pretiosa that had been exposed to the causative agent Batrachochytrium dendrobatidis. Peptidomic analysis led to identification and isolation, in pure form, of a total of 18 host-defense peptides that were characterized structurally. Brevinin-1PRa, -1PRb, -1PRc, and -1PRd, esculentin-2PRa and -PRb, ranatuerin-2PRa, -2PRb, -2PRc, and -2PRe, temporin-PRb and -PRc were identified in an earlier study of skin secretions of frogs from a different population of R. pretiosa known to be declining. Ranatuerin-2PRf, -2PRg, -2PRh, temporin-PRd, -PRe, and -PRf were not identified in skin secretions from frogs from the declining population, whereas temporin-PRa and ranatuerin-2PRd, present in skin secretions from the declining population, were not detected in the current study. All purified peptides inhibited the growth of B. dendrobatidis zoospores. Peptides of the brevinin-1 and esculentin-2 families displayed the highest potency (minimum inhibitory concentration = 6.25–12.5 μM). The study provides support for the hypothesis that the multiplicity and diversity of the antimicrobial peptide repertoire in R. pretiosa and the high growth-inhibitory potency of certain peptides against B. dendrobatidis are important in conferring a measure of resistance to fatal chytridiomycosis.

International audience; Skin secretions of frogs from the subfamily Xenopodinae (Xenopus+Silurana) within the family Pipidae are a rich source of antimicrobial peptides with therapeutic potential but species from the sister taxon Hymenochirus in the subfamily Pipinae (Hymenochirus+Pseudhymenochirus+Pipa) have not been investigated. Peptidomic analysis of norepinephrine-stimulated skin secretions from two distinct populations of the Congo dwarf clawed frog Hymenochirus boettgeri (Tornier, 1896) has led to identification of five structurally related peptides with broad-spectrum antimicrobial activity. Hymenochirin-1B (IKLSPETKDNLKKVLKGAIKGAIAVAKMV.NH(2)) is C-terminally α-amidated whereas hymenochirins-2B-5B have the general structure XKIPX(2)VKDTLKKVAKGX(2)SX(2)AGAX(3).COOH. Hymenochirin-3B (IKIPAVVKDTLKKVAKGVLSAVAGALTQ) was the most abundant peptide in the secretions. The hymenochirins show very low structural similarity with the antimicrobial peptides isolated from skin secretions of Silurana tropicalis and Xenopus laevis consistent with the proposed ancient divergence of the Pipinae and Xenopodinae. Synthetic replicates of hymenochirin-1B-4B inhibit the growth of Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Staphylococcus aureus (MIC in the range 10-40 μM) and Candida albicans (MIC=80 μM). The peptides display relatively weak hemolytic activity against human erythrocytes (LC(50) in the range 160 to >300 μM).