Effect of Double Replacement of L-Pro, D-Pro, D-Leu or Nleu in Hydrophobic Face of Amphipathic α-Helical Model Antimicrobial Peptide on Structure, Cell Selectivity and Mechanism of Action

In order to investigate the effects of the double replacement of L-Pro, D-Pro, D-Leu or Nleu (the peptoid residue for Leu) in the hydrophobic face (positions 9 and 13) of amphipathic a-helical non-cell-selective antimicrobial peptide L8K9W1 on the structure, cell selectivity and mechanism of action, we synthesized a series of L8K9W1 analogs with double replacement of L-Pro, D-Pro, D-Leu or Nleu in the hydrophobic face of L8K9W1. In this study, we have confirmed that the double replacement of L-Pro, D-Pro, or Nleu in the hydrophobic face of L8K9W1 let to a great increase in the selectivity toward bacterial cells and a complete destruction of a-helical structure. Interestingly, L8K9W1-L-Pro, L8K9W1-D-Pro and L8K9W1-Nleu preferentially interacted with negatively charged phospholipids, but unlike L8K9W1 and L8K9W1-D-Leu, they did not disrupt the integrity of lipid bilayers and depolarize the bacterial cytoplasmic membrane. These results suggested that the mode of action of L8K9W1-L-Pro, L8K9W1-D-Pro and L8K9W1-Nleu involves the intracellular target other than the bacterial membrane. In particular, L8K9W1-L-Pro, L8K9W1-D-Pro and L8K9W1-Nleu had powerful antimicrobial activity (MIC range, 1 to 4 mu M) against methicillin-resistant Staphylococcus aureus (MRSA) and multidrug-resistant Pseudomonas aeruginosa (MDRPA). Taken together, our results suggested that L8K9W1-L-Pro, L8K9W1-D-Pro and L8K9W1-Nleu with great cell selectivity may be promising candidates for novel therapeutic agents, complementing conventional antibiotic therapies to combat pathogenic microorganisms.