Design of synthetic antimicrobial peptides based on sequence analogy and amphipathicity

Novel α-helical antimicrobial peptides have been devised by comparing the N-terminal sequences of many of these peptides from insect, frog and mammalian families, extracting common features, and creating sequence templates with which to design active peptides. Determination of the most frequent amino acids in the first 20 positions for over 80 different natural sequences allowed the design of one peptide, while a further three were based on the comparison of the sequences of α-helical antimicrobial peptides derived from the mammalian cathelicidin family of precursors. These peptides were predicted to assume a highly amphipathic α-helical conformation, as indicated by high mean hydrophobic moments. In fact, circular dichroism experiments showed clear transitions from random coli in aqueous solution to an α-helical conformation on addition of trifluoroethanol. All four peptides displayed a potent antibacterial activity against: selected gram-positive and gram-negative bacteria (minimum inhibitory concentrations in the range 1-8 μM), including some antibiotic resistant strains. Permeabilization of both the outer and cytoplasmic membranes of the gram-negative bacterium, Escherichia coli, by selected peptides was quite rapid and a dramatic drop in colony forming units was observed within 5 min in time-killing experiments. Permeabitization of the cytoplasmic membrane of the gram-positive bacterium, Staphylococcus aureus, was instead initially quite slow, gathering speed after 45 min, which corresponds to the time required for significant inactivation in time-killing studies. The cytotoxic activity of the peptides, determined on several normal and transformed cell lines, was generally low at values within the minimum inhibitory concentration range.