Temporin is an antimicrobial peptide (AMP) family discovered in the skin secretion of ranid frog that has become a promising alternative for conventional antibiotic therapy. Herein, a novel temporin peptide, Temporin-PF (TPF), was successfully identified from Pelophylax fukienensis. It exhibited potent activity against Gram-positive bacteria, but no effect on Gram-negative bacteria. Additionally, TPF exhibited aggregation effects in different solutions. Three analogs were further designed to study the relationship between the aggregation patterns and bioactivities, and the MD simulation was performed for revealing the pattern of the peptide assembly. As the results showed, all peptides were able to aggregate in the standard culture media and salt solutions, especially CaCl2 and MgCl2 buffers, where the aggregation was affected by the concentration of the salts. MD simulation reported that all peptides were able to form oligomers. The parent peptide assembly depended on the hydrophobic interaction via the residues in the middle domain of the sequence. However, the substitution of Trp/D-Trp resulted in an enhanced inter-peptide interaction in the zipper-like domain and eliminated overall biological activities. Our study suggested that introducing aromaticity at the zipper-like domain for temporin may not improve the bioactivities, which might be related to the formation of aggregates via the inter-peptide contacts at the zipper-like motif domain, and it could reduce the binding affinity to the lipid membrane of microorganisms.

Antimicrobial peptides (AMPs) are a class of molecules that play an essential role in innate immune regulation. The Brevinin-1 family are AMPs that show strong pharmacological and antimicrobial potential. A novel peptide, B1A, was designed based on the primary structure of brevinin-1PLb and brevinin-1PLc. Subsequently, a synthesised replicate was subjected to a series of bioassays and was found to display antimicrobial activity. However, it also displayed high levels of haemolysis in a horse red blood cell haemolytic assay, suggesting potential toxicity. Therefore, we rationally designed a number of B1A analogues with aim of retaining antimicrobial activity, lowering toxicity, and to explore the structure–activity relationship of its N-terminus. B1A and its analogues still retained the “Rana Box” and the FLP-motif, which is a feature of this subfamily. However, the introduction of Lys and Trp residues into the peptide sequences revealed that antimicrobial activity of these analogues remained unchanged once the hydrophobicity and the charge reached the threshold. Hence, the idea that the hydrophobicity saturation in different situations is related to antimicrobial activity can be understood via the structure–activity relationship. Meanwhile, it could also be the starting point for the generation of peptides with specific antimicrobial activity.

Antimicrobial peptides (AMPs) are considered as a promising agent to overcome the drug-resistance of bacteria. Large numbers of AMPs have been identified from the skin secretion of Rana pipiens, including brevinins, ranatuerins, temporins and esculentins. In this study, the cDNA precursor of a broad-spectrum antimicrobial peptide, ranatuerin-2Pb, was cloned and identified. Additionally, two truncated analogues, RPa and RPb, were synthesised to investigate the structure-activity relationship of ranatuerin-2Pb. RPa lost antimicrobial activity against Candida albicans, MRSA, Enterococcus faecalis and Pseudomonas aeruginosa, while RPb retained its broad-spectrum antimicrobial activity. Additionally, ranatuerin-2Pb, RPa and RPb demonstrated inhibition and eradication effects against Staphylococcus aureus biofilm. RPb showed a rapid bacterial killing manner via membrane permeabilization without damaging the cell membrane of erythrocytes. Moreover, RPb decreased the mortality of S. aureus infected Galleria mellonella larvae. Collectively, our results suggested that RPb may pave a novel way for natural antimicrobial drug design.

DMPC-10A (ALWKKLLKK-Cha-NH2) is a 10-mer peptide derivative from the N-terminal domain of Dermaseptin-PC which has shown broad-spectrum antimicrobial activity as well as a considerable hemolytic effect. In order to reduce hemolytic activity and improve stability to endogenous enzymes, a D-amino acid enantiomer (DMPC-10B) was designed by substituting all L-Lys and L-Leu with their respective D-form amino acid residues, while the Ala1 and Trp3 remained unchanged. The D-amino acid enantiomer exhibited similar antimicrobial potency to the parent peptide but exerted lower cytotoxicity and hemolytic activity. Meanwhile, DMPC-10B exhibited remarkable resistance to hydrolysis by trypsin and chymotrypsin. In addition to these advantages, DMPC-10B exhibited an outstanding antibacterial effect against Methicillin-resistant Staphylococcus aureus (MRSA) and Klebsiella pneumoniae using the Galleria mellonella larva model and displayed synergistic activities with gentamicin against carbapenem-resistant K. pneumoniae strains. This indicates that DMPC-10B would be a promising alternative for treating antibiotic-resistant pathogens.

Kunitz-like trypsin inhibitors are one of the most noteworthy research objects owing to their significance in pharmacological studies, including anticarcinogenic activity, obesity regulation and anticoagulation. In the current study, a novel Kunitz-like trypsin inhibitor, Kunitzin-AH, was isolated from the skin secretion of Amolops hainanensis. The novel peptide displayed a modest trypsin inhibitory activity with the inhibitor constant (Ki) value of 1.18 ± 0.08 µM without inducing damage to healthy horse erythrocytes. Then, a series of shortened variants of Kunitzin-AH were designed by truncating a peptide loop and site mutation inside the loop to illustrate the structure–activity relationship of the trypsin inhibition function. Among the variants, a significant decrease was observed for the Cys-Cys loop domain, while the extension of an Arg at N-terminus (RCKAAFC) retained the inhibitory activity, indicating that the -RCK-motif is essential in forming the reactive domain for exerting the inhibitory activity. Furthermore, substitutions of Ala by hydrophobic or hydrophilic residues decreased the activity, indicating suitable steric hindrance provides convenience for the combination of trypsin. Additionally, the conformational simulation of the analogues processed with Chimera and Gromacs and further combination simulations between the peptides and trypsin conducted with HDOCK offered a potential opportunity for the natural trypsin inhibitory drug design. The truncated sequence, AH-798, may be a good replacement for the full-length peptide, and can be optimized via cyclization for further study.