The dermaseptin antimicrobial peptide family contains members of 27–34 amino acids in length that have been predominantly isolated from the skins/skin secretions of phyllomedusine leaf frogs. By use of a degenerate primer in Rapid amplification of cDNA ends (RACE) PCR designed to a common conserved domain within the 5′-untranslated regions of previously-characterized dermaseptin encoding cDNAs, two novel members of this peptide family, named dermaseptin-PD-1 and dermaseptin-PD-2, were identified in the skin secretion of the phyllomedusine frog, Pachymedusa dacnicolor. The primary structures of both peptides were predicted from cloned cDNAs, as well as being confirmed by mass spectral analysis of crude skin secretion fractions resulted from reversed-phase high-performance liquid chromatography. Chemically-synthesized replicates of dermaseptin-PD-1 and dermaseptin-PD-2 were investigated for antimicrobial activity using standard model microorganisms (Gram-positive bacteria, Gram-negative bacteria and a yeast) and for cytotoxicity using mammalian red blood cells. The possibility of synergistic effects between the two peptides and their anti-cancer cell proliferation activities were assessed. The peptides exhibited moderate to high inhibition against the growth of the tested microorganisms and cancer cell lines with low haemolytic activity. Synergistic interaction between the two peptides in inhibiting the proliferation of Escherichia coli and human neuronal glioblastoma cell line, U251MG was also manifested.

Antimicrobial peptides (AMPs) in the skin secretions of amphibians are fundamental components of a unique defence system that has evolved to protect these hosts from microbial invasion. Medusins constitute a recently-discovered AMP family from phyllomedusine leaf frog skin and exhibit highly-conserved structural characteristics. Here, we report a novel medusin, medusin-PT, from the skin secretion of the Tarsier Leaf Frog, Phyllomedusa tarsius. The mature peptide was initially identified from its cloned biosynthetic precursor-encoding cDNA as obtained by the rapid amplification of cDNA ends (RACE) method. Reverse-phase HPLC and tandem mass spectrometry confirmed both the presence of medusin-PT in the skin secretion and its primary structure. In a range of bioassays, medusin-PT exhibited antimicrobial activity against only the Gram-positive bacterium Staphylococcus aureus at 64 μg/ml. However, after directed changes to enhance the cationicity and amphipathicity of the peptide structure, three analogues showed more potent antimicrobial activity against several additional bacteria including the antibiotic-resistant bacterium MRSA. In addition, these analogues exhibited activity against microbial biofilm (minimum biofilm inhibitory and eradication concentrations of 32 μg/ml and over 64 μg/ml, respectively). These data provide evidence that medusins might be promising candidates as novel antibiotic leads and that the targeted modification of a natural AMP can both improve its efficacy so as to provide new insights into antibiotic design and development.

Antimicrobial peptides belonging to the phylloseptin family are mainly found in phyllomedusine frogs. These peptides not only possess potent antimicrobial activity but exhibit low toxicity against eukaryotic cells. Therefore, they are considered as promising drug candidates for a number of diseases. In a recent study, potent antimicrobial activity was correlated with the conserved structures and cationic amphiphilic characteristics of members of this peptide family. A phylloseptin peptide precursor was discovered here in the skin secretion of Phyllomedusa tarsius and the mature peptide was validated by MS/MS sequencing, and was subsequently named phylloseptin-PT. The chemically-synthesized and purified phylloseptin-PT displayed activity against Staphylococcus aureus and Candida albicans. Nevertheless, a range of cationicity-enhanced peptide analogues of phylloseptin-PT, which contained amino acid substitutions at specific sites, exhibited significant increases in antimicrobial activity compared to native phylloseptin-PT. In addition, alternative conformers which were designed and chemically-synthesized with d-lysine, showed potent antimicrobial activity and enhanced bioavailability. These data indicate that phylloseptins may represent potential candidates for next-generation antibiotics. Thus, rational design through modification of natural antimicrobial peptide templates could provide an accelerated path to overcoming obstacles en-route to their possible clinical applications.