Cationic antimicrobial peptides (CAMPs) are a promising alternative to treat multidrug-resistant bacteria, which have developed resistance to all the commonly used antimicrobial, and therefore represent a serious threat to human health. One of the major drawbacks of CAMPs is their sensitivity to proteases, which drastically limits their half-life. Here we describe the design and synthesis of three nine-residue CAMPs, which showed high stability in serum and broad spectrum antimicrobial activity. As for all peptides a very low selectivity between bacterial and eukaryotic cells was observed, we performed a detailed biophysical characterization of the interaction of one of these peptides with liposomes mimicking bacterial and eukaryotic membranes. Our results show a surface binding on the DPPC/DPPG vesicles, coupled with lipid domain formation, and, above a threshold concentration, a deep insertion into the bilayer hydrophobic core. On the contrary, mainly surface binding of the peptide on the DPPC bilayer was observed. These observed differences in the peptide interaction with the two model membranes suggest a divergence in the mechanisms responsible for the antimicrobial activity and for the observed high toxicity toward mammalian cell lines. These results could represent an important contribution to unravel some open and unresolved issues in the development of synthetic CAMPs.

Antimicrobial peptides are promising candidates as future therapeutics in order to face the problem of antibiotic resistance caused by pathogenic bacteria. Myxinidin is a peptide derived from the hagfish mucus displaying activity against a broad range of bacteria. We have focused our studies on the physico-chemical characterization of the interaction of myxinidin and its mutant WMR, which contains a tryptophan residue at the N-terminus and four additional positive charges, with two model biological membranes (DOPE/DOPG 80/20 and DOPE/DOPG/CL 65/23/12), mimicking respectively Escherichia coli and Pseudomonas aeruginosa membrane bilayers. All our results have coherently shown that, although both myxinidin and WMR interact with the two membranes, their effect on membrane microstructure and stability are different. We further have shown that the presence of cardiolipin plays a key role in the WMR-membrane interaction. Particularly, WMR drastically perturbs the DOPE/DOPG/CL membrane stability inducing a segregation of anionic lipids. On the contrary, myxinidin is not able to significantly perturb the DOPE/DOPG/CL bilayer whereas interacts better with the DOPE/DOPG bilayer causing a significant perturbing effect of the lipid acyl chains. These findings are fully consistent with the reported greater antimicrobial activity of WMR against P. aeruginosa compared with myxinidin.

The G-quadruplex-forming VEGF-binding aptamer V7t1 was previously found to be highly polymorphic in a K⁺-containing solution and, to restrict its conformational preferences to a unique, well-defined form, modified nucleotides (LNA and/or UNA) were inserted in its sequence. We here report an in-depth biophysical characterization of V7t1 in a Na⁺-rich medium, mimicking the extracellular environment in which VEGF targeting should occur, carried out combining several techniques to analyse the conformational behaviour of the aptamer and its binding to the protein. Our results demonstrate that, in the presence of high Na⁺ concentrations, V7t1 behaves in a very different way if subjected or not to annealing procedures, as evidenced by native gel electrophoresis, size exclusion chromatography and dynamic light scattering analysis. Indeed, not-annealed V7t1 forms both monomeric and dimeric G-quadruplexes, while the annealed oligonucleotide is a monomeric species. Remarkably, only the dimeric aptamer efficiently binds VEGF, showing higher affinity for the protein compared to the monomeric species. These findings provide new precious information for the development of improved V7t1 analogues, allowing more efficient binding to the cancer-related protein and the design of effective biosensors or theranostic devices based on VEGF targeting.

Antimicrobial peptides (AMPs) are membrane-active peptides with a broad spectrum of activity against different pathogenic organisms and they represent promising new drugs to overcome the emergence of resistance to antibiotics in bacteria. (P)GKY20 is an antimicrobial peptide with a low hemolytic effect on eukaryotic cells and a strong antimicrobial activity especially against Gram-negative bacteria. However, its mechanism of action is still unknown. Here, we use fluorescence spectroscopy and differential scanning calorimetry combined with atomic force microscopy to characterise the binding of (P)GKY20 with model biomembranes and its effect on the membrane's microstructure and thermotropic properties. We found that (P)GKY20 selectively perturbs the bacterial-like membrane via a carpet-like mechanism employing peptide conformational changes, lipid segregation and domain formation as key steps in promoting membrane disruption. These results shed a first light on the action mechanism of (P)GKY20 and could represent an important contribution to the development of new peptides serving as antimicrobial agents.

The designed peptide dH3w (dansyl-HPHGHW-NH ₂ ) showed a selective fluorescence turn-on response to Zn ²⁺ and a turn-off response to Cu ²⁺ .