Design of Potent, Non-Toxic Antimicrobial Agents Derived from the Frog Skin Peptide Raniseptin-PL Active Against Gram-Negative ESKAPE+ Pathogens

Background/Objectives: The frog skin peptide raniseptin-PL (GVFDTVKKIGKAVGKFALGVAKNYLNS.NH2) is active against a range of antibiotic-resistant bacteria including ESKAPE+ pathogens, but its therapeutic potential is limited by high cytotoxicity against mammalian cells. The study aimed to determine the effects of selective amino acid substitutions by L- and D-lysine residues on the biological activity of the peptide. Methods: Conformational analysis of raniseptin-PL and its analogs was accomplished using circular dichroism spectroscopy. Antimicrobial activity was determined using reference strains and antibiotic-resistant strains of ESKAPE+ bacteria, and cytotoxicity against human neoplastic and non-neoplastic cells was evaluated. Results: Raniseptin-PL adopts an extended helical conformation in membrane-mimetic environments. Replacement of the Asp-4 residue by either L-Lys or D-Lys or the Leu-18 residue by L-Lys produced only modest changes in antimicrobial potency but high cytotoxicity against human-derived cells was retained. In contrast, substitution at Leu-18 by D-Lys attenuated potency against the Gram-positive bacteria Staphylococcus aureus and Staphylococcus epidermidis (MIC ≥ 50 µg.mL−1), but high potency against the antibiotic-resistant Gram-negative ESKAPE+ pathogens Acinetobacter baumannii, Klebsiella pneumoniae, Pseudomonas aeruginosa and Escherichia coli was either maintained or increased (MIC 3.1–12.5 µg.mL−1; approx.1–4 µM). Importantly, [L18k]raniseptin-PL and [D4k,L18k]raniseptin-PL lacked cytotoxic activity against human-derived HaCaT keratinocytes, A549 non-small cell lung adenocarcinoma cells, PC-3 prostate cancer cells and erythrocytes at concentrations up to 100 µM. Conclusions: [L18k]raniseptin-PL and [D4k,L18k]raniseptin-PL show therapeutic potential as templates for development of a non-toxic antimicrobial agent for use against systemic infections by Gram-negative bacteria, particularly ESKAPE+ pathogens.