Ascaphin-8

Ascaphin-8 is an α-helical anti-tumor and antimicrobial peptide containing 19 residues, which was isolated from norepinephrine-stimulated skin secretions of the North American tailed frog Ascaphus truei. To improve both its stability and biological activities, a series of hydrocarbon-stapled analogs of Ascaphin-8 were synthesized and investigated for their potential antiproliferative activities. The activity studies were evaluated using the CCK-8 method and colony formation assay on human cancer cell lines. Ascaphin-8-3, as the most active peptide, showed a stronger inhibition effect when compared with the parent peptide for the tested cell lines. In addition, the effect of Ascaphin-8-3 on inhibiting the metastatic capabilities of A549 cells was more powerful than that of the parent peptide. This peptide derivative showed potentiality for further optimization in antitumor drugs.

Extended-spectrum beta-lactamase (ESBL)-producing Gram-negative bacteria are becoming increasingly prevalent and their antibiotic resistance necessitates novel therapeutic intervention. Ascaphin-8 is a cationic alpha-helical peptide that shows broad-spectrum antibacterial activity but is also toxic to human erythrocytes (LC(50)= 55 microM). This study assesses the activity of ascaphin-8, and a series of l-lysine-substituted analogs, against a range of clinical isolates of ESBL-producing bacteria. All ESBL-producing Escherichia coli (MIC=1.5-6 microM) and Klebsiella pneumoniae (MIC=12.5-25 microM) strains tested were susceptible to ascaphin-8, as well as a group of miscellaneous ESBL-producing strains (Citrobacter, Salmonella, Serratia, Shigella spp.) (MIC< or = 25 microM). The Lys4- and Lys8-substituted analogs were generally the most potent against bacteria but showed the highest hemolytic activity. However, the Lys10, Lys14, and Lys18 analogs also displayed potent antibacterial activity while showing very low hemolytic activity (LC50> 500 microM). An unexpected finding was the susceptibility of ESBL-producing Proteus mirabilis strains to ascaphin-8 (MIC=12.5-25 microM) and its Lys4-substituted analog (MIC= 6 microM), although non-ESBL isolates of this organism were resistant to these peptides (MIC> 100 microM).

Ascaphins are cationic antimicrobial peptides that have been shown to have potential in the treatment of infectious diseases caused by multidrug-resistant pathogens (MDR). However, to date, their principal molecular target and mechanism of action are unknown. Results from peptide prediction software and molecular dynamics simulations confirmed that ascaphin-8 is an alpha-helical peptide. For the first time, the peptide was described as membranotrophic using biophysical approaches including calcein liposome leakage, Laurdan general polarization, and dynamic light scattering. Ascaphin-8's activity and selectivity were modulated by rearranging the spatial distribution of lysine (Var-K5), aspartic acid (Var-D4) residues, or substitution of phenylalanine with tyrosine (Var-Y). The parental peptide and its variants presented high affinity toward the bacterial membrane model (≤2 μM), but lost activity in sterol-enriched membranes (mammal and fungal models, with cholesterol and ergosterol, respectively). The peptide-induced pore size was estimated to be >20 nm in the bacterial model, with no difference among peptides. The same pattern was observed in membrane fluidity (general polarization) assays, where all peptides reduced membrane fluidity of the bacterial model but not in the models containing sterols. The peptides also showed high activity toward MDR bacteria. Moreover, peptide sensitivity of the artificial membrane models compared with pathogenic bacterial isolates were in good agreement.