A cysteine-free 15-residue host-defense peptide analogs combines low MICs against Gram-negative bacteria with no detectable keratinocyte cytotoxicity

Cathelicidins are host-defense peptides with activity against multidrug-resistant bacteria, but peptide length and host cell toxicity can limit their therapeutic use. Cathelicidin-PP, a 32-residue peptide, inhibits both Gram-negative and Gram-positive bacteria and is cytotoxic to human keratinocytes. We sought to develop shorter analogs that retain activity against WHO critical-priority bacteria, while reducing cytotoxicity. Twelve 15-residue analogs (Pep1–Pep12) were designed and synthesized by Fmoc solid-phase synthesis. Minimum inhibitory concentrations (MICs) were determined by broth microdilution against nine bacterial strains and Candida albicans over a concentration range of 1–128 µM. Hemolysis was assessed using human erythrocytes, and HaCaT keratinocyte viability was measured using the MTT assay. Concentrations causing 50% hemolysis (HC₅₀) or a 50% reduction in HaCaT cell viability (IC₅₀) were estimated by four-parameter logistic regression when supported by the concentration–response data. All 12 analogs inhibited carbapenem-resistant Acinetobacter baumannii at 1–64 µM, compared with 2 µM for the wild-type (WT) peptide. Pep12 was the only analog to inhibit Staphylococcus aureus, with an MIC of 64 µM compared with 4 µM for the WT peptide. Pep2, Pep3, Pep8, Pep11, and Pep12 inhibited both Escherichia coli and Pseudomonas aeruginosa, with MICs of 2–8 µM. The corresponding MICs of the WT peptide were 2 and 4 µM, respectively. Eight analogs inhibited C. albicans at 32–64 µM, whereas the MIC of the WT peptide was >64 µM. Pep8 inhibited A. baumannii, E. coli, P. aeruginosa, and Klebsiella pneumoniae at 1, 2, 4, and 8 µM, respectively, compared with 2, 2, 4, and 2 µM for the WT peptide. None of the analogs caused 50% hemolysis or reduced HaCaT cell viability by 50% at concentrations up to 128 µM. Both HC₅₀ and HaCaT IC₅₀ therefore exceeded 128 µM for all analogs. The WT peptide had a HaCaT IC₅₀ of 4.8 µM (SE, 0.3 µM). These findings show that the toxicity of cathelicidin-PP can be separated from its Gram-negative activity in a short sequence, giving a new, promising candidate scaffold against carbapenem-resistant infections, as shown by Pep8.