Modulation of the hydrophobic domain of polymyxin B nonapeptide: Effect on outer-membrane permeabilization and lipopolysaccharide neutralization

Polymyxin B nonapeptide (PMBN), a cationic cyclic peptide derived from the antibacterial peptide polymyxin B, is capable of specifically increasing the permeability of the outer membrane (OM) of Gram-negative bacteria toward hydrophobic antibiotics. In this study, we evaluated the contribution of the hydrophobic segment of PMBN (i.e., D-Phe⁵-Leu⁶) to this activity. Accordingly, we synthesized four analogs of PMBN by replacing D-Phe⁵ with either with D-Trp or D-Tyr and Leu⁶ with Phe or Ala and evaluated their ability to bind cell-free lipopolysaccharide (LPS) and increase bacterial OM permeability. Compared with PMBN, [D-Tyr⁵]PMBN and [Ala⁶]PMBN possessed reduced LPS affinity (IC₅₀ = 2.5, 25, and 12 μM, respectively) and significantly reduced OM permeability and LPS neutralization activity. [Phe⁶]PMBN exhibited rather similar affinity to cell-free LPS (IC₅₀ = 5 μM) and the same OM permeability capacity as PMBN. However, [D-Trp⁵]PMBN, despite its similar affinity to cell-free LPS (IC₅₀ = 4 μM), had moderately reduced OM permeability capacity. These results demonstrate the significant role of the PMBN hydrophobic segment in promoting biological activity.