ABSTRACT
HYPOTHESIS: Release of lipopolysaccharides (LPS) from bacteria into bloodstream may cause serious unwanted stimulation of the host immune system. P-113 is a clinically active histidine-rich antimicrobial peptide. Nal-P-113, a ß-naphthylalanine-substituted P-113, is salt-resistant but has limited LPS neutralizing activity. We suspected the size and shape of the non-natural bulky amino acid may affect its LPS neutralizing activity. Herein, antimicrobial, LPS neutralizing, and antiproteolytic effects of phenylalanine- (Phe-P-113), ß-naphthylalanine- (Nal-P-113), ß-diphenylalanine- (Dip-P-113), and ß-(4,4'-biphenyl)alanine- (Bip-P-113) substituted P-113 were studied. EXPERIMENTS: Structure-activity relationships of P-113, Phe-P-113, Nal-P-113, Dip-P-113, and Bip-P-113 were evaluated using antimicrobial activity assays, serum proteolytic assays, peptide-induced permeabilization of large unilamellar vesicles, zeta potential measurements, dynamic light scattering measurement of LPS aggregation, and Limulus amebocyte lysate assays for measuring LPS neutralization. In vitro and in vivo LPS neutralizing activities were further confirmed by LPS-induced inflammation inhibition in an endotoxemia mouse model. FINDINGS: Bip-P-113 and Dip-P-113 had the longest and widest non-nature amino acids, respectively. Bip-P-113 enhanced salt resistance, serum proteolytic stability, peptide-induced permeabilization, zeta potential measurements, LPS aggregation, and in vitro and in vivo LPS neutralizing activities. These results could help design novel antimicrobial peptides that have enhanced stability in vivo and that can have potential therapeutic applications.
Subject(s)
Amino Acids/chemistry , Anti-Bacterial Agents/pharmacology , Antimicrobial Cationic Peptides/pharmacology , Endotoxemia/drug therapy , Inflammation/drug therapy , Lipopolysaccharides/antagonists & inhibitors , Animals , Anti-Bacterial Agents/blood , Anti-Bacterial Agents/chemistry , Antimicrobial Cationic Peptides/blood , Antimicrobial Cationic Peptides/chemistry , Disease Models, Animal , Dose-Response Relationship, Drug , Dynamic Light Scattering , Endotoxemia/chemically induced , Endotoxins , Escherichia coli/drug effects , Fibroblasts , Hemolytic Plaque Technique , Humans , Inflammation/chemically induced , Lipopolysaccharides/chemistry , Lipopolysaccharides/pharmacology , Male , Mice , Mice, Inbred C57BL , Microbial Sensitivity Tests , Particle Size , Pseudomonas aeruginosa/drug effects , Staphylococcus aureus/drug effects , Structure-Activity Relationship , Surface PropertiesABSTRACT
Lipopolysaccharide (LPS, endotoxin) is the major component of Gram-negative bacterial outer surface membrane. LPS released from bacteria into bloodstream during infection may cause serious unwanted stimulation of host's immune system and lead to septic shock of the patient. Recently, we have developed a strategy to increase salt resistance and LPS neutralization of short antimicrobial peptides by adding ß-naphthylalanine end-tags to their termini. Herein, correlations between membrane immersion depth, orientation, and antiendotoxin activities of the antimicrobial peptides S1 and S1-Nal-Nal have been investigated via solution structure, paramagnetic resonance enhancement, and saturation transfer difference NMR studies. Unlike the parent peptide S1, S1-Nal-Nal rotated its two terminal ß-naphthylalanine residues into the hydrophobic lipid A motif of LPS micelles. The LPS-induced inflammation may then be prohibited by the blocked lipid A motif.
