A novel tetrabranched antimicrobial peptide that neutralizes bacterial lipopolysaccharide and prevents septic shock in vivo

Abstract

We describe the nonnatural antimicrobial peptide KKIRVRLSA (M33) and its capacity to neutralize LPS-induced cytokine release, preventing septic shock in animals infected with bacterial species of clinical interest. M33 showed strong resistance to proteolytic degradation when synthesized in tetrabranched form with 4 peptides linked by a lysine core, making it suitable for use in vivo. HPLC and mass spectrometry demonstrated its stability in serum beyond 24 h. M33 was found to be very selective for gram-negative bacteria. Minimal inhibitory concentration (MIC) ranged from 0.3 to 3 muM for multidrug resistant clinical isolates of several pathogenic species, including Pseudomonas aeruginosa, Klebsiella pneumoniae, and Acinetobacter baumannii. M33 neutralized LPS derived from P. aeruginosa and K. pneumoniae, and prevented TNF-alpha release from LPS-activated macrophages, with an EC(50) of 3.8e-8 M and 2.8e-7 M, respectively, as detected by sandwich ELISA. M33 activity was also tested in sepsis animal models. It averted septic shock symptoms due to Escherichia coli and P. aeruginosa in doses compatible with clinical use (5-25 mg/kg). These properties make tetrabranched M33 peptide a good candidate for the development of a new antibacterial drug.-Pini, A., Falciani, C., Mantengoli, E., Bindi, S., Brunetti, J., Iozzi, S., Rossolini, G. M., Bracci, L. A novel tetrabranched antimicrobial peptide that neutralizes bacterial lipopolysaccharide and prevents septic shock in vivo.

Effects of Dimerization on the Structure and Biological Activity of Antimicrobial Peptide Ctx-Ha

“…These findings are supported by recent studies that have shown that inhibition of antimicrobial activity is due to the interaction between AMPs and cell wall carbohydrates (31,37). The natural sequestration of antimicrobial agents by polysaccharides present in the cell wall also may render ineffective the activities of cAMPs, such as LL-37 and ␤-defensins (1).…”

Progressive Structuring of a Branched Antimicrobial Peptide on the Path to the Inner Membrane Target

“…Non-natural multivalent AMPs have been designed by conjugating copies of a peptide monomer to scaffold molecules via naturally occurring intermolecular disulfide bridges or unnatural scaffold linkers (7). Branched peptides have been shown to have considerable advantages over their monomeric forms, such as improved antimicrobial activity (8), maintaining high efficacy under physiological (high salt) conditions (9,10), enhanced bacterial surface binding affinity (11), decreased susceptibility to proteolytic degradation (12,13), and low cytotoxicity (14,15). However, the quantitative structure-activity relationships and physiochemical properties of branched antimicrobial peptides are still poorly understood at the atomic level.…”

Enhanced Membrane-tethered Mucin 3 (MUC3) Expression by a Tetrameric Branched Peptide with a Conserved TFLK Motif Inhibits Bacteria Adherence*

“…The monomeric and dimeric variants were inactive, and the trimeric variants were partially active (31). Pini et al (32) report that the tetrabranched peptide based on the nonnatural antimicrobial peptide KKIRVRLSA is a good candidate for the development of a new antibacterial drug. Based on the finding by Mack et al (16) showing that the M-SAA3 N-terminal 10-mer peptide induces human MUC3 transcriptional expression and inhibits EPEC adherence, we generated a synthetic tetrameric structure of branched polylysine based on the conserved TFLK-motif from M-SAA3 (GWLTFLKAAG).…”