Design of Potent, Non‐Toxic Antimicrobial Agents Based Upon the Naturally Occurring Frog Skin Peptides, Ascaphin‐8 and Peptide XT‐7

Abstract

The frog skin peptides, ascaphin-8 (GFKDLLKGAAKALVKTVLF.NH(2)) and XT-7 (GLLGPLLKIAAKVGSNLL.NH(2)), 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 < or = 25 microM) but low cytolytic activity against erythrocytes (LD(50) > 500 microM) 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 < or = 6 microM), but also increased hemolytic activities.