Cell selectivity and anti-inflammatory activity of a Leu/Lys-rich α-helical model antimicrobial peptide and its diastereomeric peptides

To investigate the effect of the number and distribution of d-amino acids introduced into non-cell-selective α-helical antimicrobial peptides on the cell selectivity, protease stability and anti-inflammatory activity, we synthesized an 18-meric Leu/Lys-rich α-helical model peptide (K₉L₈W) and d-amino acid-containing diastereomeric peptides. Increasing in cell selectivity of the peptides was increased in parallel with increasing in the number of d-amino acids introduced. Despite having the same number of d-amino acids, D₉-K₉L₈W-1 had better cell selectivity than D₉-K₉L₈W-2, indicating that a dispersed distribution of d-amino acids in diastereomeric peptides is more effective for cell selectivity than their segregated distribution. D₃-K₉L₈W-2, D₆-K₉L₈W, D₉-K₉L₈W-1 and D₉-K₉L₈W-2 showed complete resistance to tryptic digestion. Furthermore, K₉L₈W and all of its diastereomeric peptides significantly inhibited nitric oxide (NO) production, inducible nitric oxide synthase (iNOS) mRNA expression and tumor necrosis factor-α (TNF-α) release in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells at a lower concentration than bactericidal concentration. The order of anti-inflammatory activity for the peptides was K₉L₈W ≈ D₃-K₉L₈W-1 ≈ D₃-K₉L₈W-2 ≈ D₆-K₉L₈W ≈ D₉-K₉L₈W-2 > D₄-K₉L₈W > D₉-K₉L₈W-1. Increasing in hydrophobicity or α-helicity of the peptides was more closely correlated with increasing in hemolytic activity and anti-inflammatory activity than antimicrobial and LPS-disaggregation activities. Collectively, we successfully developed several d-amino acid-containing antimicrobial peptides (D₄-K₉L₈W, D₆-K₉L₈W and D₉-K₉L₈W-1) with good cell selectivity, protease stability and potent anti-inflammatory activity. These antimicrobial peptides could serve as templates for the development of peptide antibiotics for the treatment of sepsis, as well as microbial infection.