Design of potent, non-toxic antimicrobial agents based upon the naturally occurring frog skin peptides, ascaphin-8 and peptide XT-7

The frog skin peptides, ascaphin-8 (GFKDLLKGAAKALVKTVLF.NH2) and XT-7 (GLLGPLLKIAAKVGSNLL.NH2), show broad-spectrum antimicrobial activity but their therapeutic potential is limited by toxicity against mammalian cells. Circular dichroism spectra demonstrate that the peptides adopt an amphipathic alpha-helical conformation in a membrane-mimetic solvent. This study has investigated the cytolytic properties of analogs containing selected amino acid substitutions that increase cationicity while maintaining amphipathicity. Substitutions at Ala(10), Val(14), and Leu(18) in ascaphin-8 by either L-Lys or D-Lys produced peptides that retained antimicrobial activity against the bacteria Escherichia coli and Staphylococcus aureus and the opportunistic yeast pathogen, Candida albicans but showed appreciably reduced toxicities (> 10-fold) against human erythrocytes, HepG2 hepatoma-derived cells, and L929 fibroblasts. The improved therapeutic index of the L-Lys(18) and D-Lys(18) analogs correlated with a decrease in % helicity and in effective hydrophobicity. Substitution of Gly(4) by L-Lys in XT-7 produced an analog with high potency against micro-organisms (MIC <= 25 mu M) but low cytolytic activity against erythrocytes (LD50 > 500 mu M) and this increase in therapeutic index also correlated with decreased helicity and hydrophobicity. Analogs of XT-7 with increased cationicity, containing multiple substitutions by L-Lys, not only displayed increased antimicrobial potencies, particularly against Candida albicans (MIC <= 6 mu M), but also increased hemolytic activities.