The D enantiomers of three naturally occurring antibiotics-cecropin A, magainin 2 amide, and melittin-were synthesized. In addition, the D enantiomers of two synthetic chimeric cecropin-melittin hybrid peptides were prepared. Each D isomer was shown by circular dichroism to be a mirror image of the corresponding L isomer in several solvent mixtures. In 20% hexafluoro-2-propanol the peptides contained 43-75% a-helix. The all-D peptides were resistant to enzymatic degradation. The peptides produced single-channel conductances in planar lipid bilayers, and the D and L enantiomers caused equivalent amounts of electrical conductivity. All of the peptides were potent antibacterial agents against representative Gram-negative and Gram-positive species. The D and L enantiomers of each peptide pair were equally active, within experimental error. Sheep erythrocytes were lysed by both D-and L-melittin but not by either isomer of cecropin A, magainin 2 amide, or the hybrids cecropin A-(1-13)-melittin-(1-13)-NH2 or cecropin A-(1-8)-melittin-(1-18)-NH2. The infectivity of the bloodstream form of the malaria parasite Plasmodium falciparum was also inhibited by the D and L hybrids. It is suggested that the mode of action of these peptides on the membranes of bacteria, erythrocytes, plasmodia, and artificial lipid bilayers may be similar and involves the formation of ion-channel pores spanning the membranes, but without specific interaction with chiral receptors or enzymes.The cecropins (1, 2) and several other antibiotic peptides of the animal kingdom, including defensins (3), magainins (4), and the bee venom toxin melittin (5), are thought to function through the formation of ion channels in lipid membranes. This idea has been based on recent studies of electrical conductivity in artificial lipid bilayers (3,(6)(7)(8), where activity is a function of the structure of the peptide and the composition of the membrane lipids. The bilayer lipids and cell membranes are chiral and contain many asymmetric centers. It has been generally assumed that the chirality of the membrane would require a specific chirality ofthe peptide for it to be active, in much the same way that peptide hormones are required to fit with the conformation of their natural receptors or for a substrate and enzyme to form a tight stereospecific complex. However, we have suggested that these peptide antibiotics can exert their effect without requiring a specific target receptor on the cell membrane (7, 9).The purpose of the present study was to test this assumption by the synthesis of the all-D enantiomers of several natural, all-L peptide antibiotics and some of their active analogs. These D stereoisomers would be expected to assume equivalent, but mirror image, conformations when placed in the same environment as the all-L peptides. If a close molecular contact with the chiral components of the cell membrane is required, the D enantiomers would be expected to be inactive. However, if the interaction of the peptide with the membrane is only between achiral...

We have earlier reported two 26‐residue antibacterial peptides made up from different segments ol'cecropin A (CA) and melittin (M). We now report a substantial reduction in size at the C‐terminal section of the highly active hybrid CA(1–8)M(1–18), leading to a series of 20‐, 18‐ and 15‐residue analogs with antibiotic properties similar to the larger molecule. In particular, the 15‐residue hybrids CA(1–7)M(2–9), CA(1–7)M(4–11) and CA(1–7)M(5–12) are the shortest cecropin‐based peptide antibiotics described so far, with antibacterial activity and spectra similar or better than cecropin A and a 60% reduction in size. Their reduced size and highly α‐helical structure require an alternative mechanism for their interaction with bacterial membranes.

Solid phase synthesis was used to produce 5 hybrid peptides containing sequences from the antibacterial peptide, cecropin A, and from the bee venom toxin, melittin. Four of these chimeric peptides showed good antibacterial activity against representative Gram-negative and Gram-positive bacterial species. The best hybrid, cecropin A(l-13)-melittin(l-13) was IOO-fold more active than cecropin A against Staphylococcus uureus. It was also a lo-fold better antimalarial agent than cecropin B or magainin 2. Sheep red cells were lysed by melittin at low concentrations, but not by the hybrid molecules, even at 50 times higher concentrations.Cecropin A; Melittin; (Staphylococcus aureus, Plasmodium falciparium)

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The ability of five purified amphibian antimicrobial peptides (dermaseptin-1, temporin A, magainin I, and II, PGLa), crude peptide fractions isolated from the skin of Rana pipiens and R. catesbeiana, and four antimicrobial peptides (AMPs) from hybrid striped bass (piscidin-1N, -1H, -2, and -3) were examined for their ability to reduce the infectivity of channel catfish virus (CCV) and frog virus 3 (FV3). All compounds, with the exception of magainin I, markedly reduced the infectivity of CCV. In contrast to CCV, FV3 was 2- to 4-fold less sensitive to these agents. Similar to an earlier study employing two other amphibian peptides, the agents used here acted rapidly and over a wide, physiologically relevant, temperature range to reduce virus infectivity. These results extend our previous findings and strongly suggest that various amphibian and piscine AMPs may play important roles in protecting fish and amphibians from pathogenic viruses.

Topical antimicrobicides hold great promise in reducing human immunodeficiency virus (HIV) transmission. Amphibian skin provides a rich source of broad-spectrum antimicrobial peptides including some that have antiviral activity. We tested 14 peptides derived from diverse amphibian species for the capacity to inhibit HIV infection. Three peptides (caerin 1.1, caerin 1.9, and maculatin 1.1) completely inhibited HIV infection of T cells within minutes of exposure to virus at concentrations that were not toxic to target cells. These peptides also suppressed infection by murine leukemia virus but not by reovirus, a structurally unrelated nonenveloped virus. Preincubation with peptides prevented viral fusion to target cells and disrupted the HIV envelope. Remarkably, these amphibian peptides also were highly effective in inhibiting the transfer of HIV by dendritic cells (DCs) to T cells, even when DCs were transiently exposed to peptides 8 h after virus capture. These data suggest that amphibian-derived peptides can access DC-sequestered HIV and destroy the virus before it can be transferred to T cells. Thus, amphibian-derived antimicrobial peptides show promise as topical inhibitors of mucosal HIV transmission and provide novel tools to understand the complex biology of HIV capture by DCs.

Temporin A (TA) is a small, basic, highly hydrophobic, antimicrobial peptide amide (FLPLIGRVLSGIL-NH 2 ) found in the skin of the European red frog, Rana temporaria. It has variable antibiotic activities against a broad spectrum of microorganisms, including clinically important methicillin-sensitive and -resistant Staphylococcus aureus as well as vancomycinresistant Enterococcus faecium strains. In this investigation the antimicrobial activity and structural characteristics of TA synthetic analogs were studied. For antibacterial activity against S. aureus and enterococcal strains, the hydrophobicity of the Nterminal amino acid of TA was found to be important as well as a positive charge at amino acid position 7, and bulky hydrophobic side chains at positions 5 and 12. Replacing isoleucine with leucine at amino acid positions 5 and 12 resulted in the greatest enhancement of antibacterial activity. In addition, there was little difference between the activities of TA and its all-D enantiomer, indicating that the peptide probably exerts its effect on bacteria via non-chiral interactions with membrane lipids. ß

Many mammalian antimicrobial peptides (AMPs) have multiple effects on antimicrobial immunity. We found that temporin A (TA), a representative frog-derived AMP, induced the migration of human monocytes, neutrophils, and macrophages with a bell-shaped response curve in a pertussis toxin-sensitive manner, activated p44/42 MAPK, and stimulated Ca2+ flux in monocytes, suggesting that TA is capable of chemoattracting phagocytic leukocytes by the use of a Giα protein-coupled receptor. TA-induced Ca2+ flux in monocytes was cross-desensitized by an agonistic ligand MMK-1 specific for formyl peptide receptor-like 1 (FPRL1) and vice versa, suggesting that TA uses FPRL1 as a receptor. This conclusion was confirmed by data showing that TA selectively stimulated chemotaxis of HEK 293 cells transfected with human FPRL1 or its mouse ortholog, murine formyl peptide receptor 2. In addition, TA elicited the infiltration of neutrophils and monocytes into the injection site of mice, indicating that TA is also functionally chemotactic in vivo. Examination of two additional temporins revealed that Rana-6 was also able to attract human phagocytes using FPRL1, but temporin 1P selectively induced the migration of neutrophils using a distinct receptor. Comparison of the chemotactic and antimicrobial activities of several synthetic analogues suggested that these activities are likely to rely on different structural characteristics. Overall, the results demonstrate that certain frog-derived temporins have the capacity to chemoattract phagocytes by the use of human FPRL1 (or its orthologs in other species), providing the first evidence suggesting the potential participation of certain amphibian antimicrobial peptides in host antimicrobial immunity.