Design of Gram-negative selective antimicrobial peptides

Biochemistry. 2001 May 15;40(19):5777-85. doi: 10.1021/bi0100384.


Lipopolysaccharide (LPS), a major component of Gram-negative bacteria, signals bacterial invasion and triggers defensive host responses. However, excessive responses also lead to the serious pathophysiological consequence of septic shock. To develop Gram-negative selective compounds that can inhibit the effects of LPS-induced sepsis, we have designed constrained cyclic antimicrobial peptides based on a cystine-stabilized beta-stranded framework mimicking the putative LPS-binding sites of the LPS-binding protein family. Our prototype termed R4A, c(PACRCRAG-PARCRCAG), consists of an eight amino acid degenerated repeat constrained by a head-to-tail cyclic peptide backbone and two cross-bracing disulfides. NMR study of K4A, an R4A analogue with four Arg --> Lys replacements, confirmed the amphipathic design elements with four Lys on one face of the antiparallel beta-strand and two hydrophobic cystine pairs plus two Ala on the opposite face. K4A and R4A displayed moderate microbicidal potency and Gram-negative selectivity. However, R4A analogues with single or multiple replacements of Ala and Gly with Arg or bulky hydrophobic amino acids displayed increased potency and selectivity in both low- and high-salt conditions. Analogues R5L and R6Y containing additional cationic and bulky hydrophobic amino acids proved the best mimics of the amphipathic topology of the "active-site" beta-strands of LPS-binding proteins. They displayed potent activity against Gram-negative E. coli with a minimal inhibitory concentration of 20 nM and a >200-fold selectivity over Gram-positive S. aureus. Our results suggest that an LPS-targeted design may present an effective approach for preparing selective peptide antibiotics.

Publication types

  • Comparative Study
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Alanine / chemistry
  • Amino Acid Sequence
  • Amino Acid Substitution / genetics
  • Antimicrobial Cationic Peptides / chemical synthesis*
  • Antimicrobial Cationic Peptides / genetics
  • Antimicrobial Cationic Peptides / metabolism
  • Antimicrobial Cationic Peptides / pharmacology*
  • Arginine / chemistry
  • Circular Dichroism
  • DNA-Binding Proteins / chemistry
  • Disulfides / chemistry
  • Drug Design
  • Escherichia coli / drug effects*
  • Escherichia coli / growth & development
  • Lipopolysaccharides / metabolism
  • Lysine / chemistry
  • Microbial Sensitivity Tests
  • Molecular Sequence Data
  • Nuclear Magnetic Resonance, Biomolecular
  • Peptides, Cyclic / chemical synthesis*
  • Peptides, Cyclic / chemistry
  • Peptides, Cyclic / genetics
  • Peptides, Cyclic / metabolism
  • Peptides, Cyclic / pharmacology*
  • Protein Binding
  • Proteins / chemistry
  • Pseudomonas aeruginosa / drug effects*
  • Pseudomonas aeruginosa / growth & development
  • Repetitive Sequences, Amino Acid
  • alpha-Defensins


  • Antimicrobial Cationic Peptides
  • DEFA1A3 protein, human
  • DNA-Binding Proteins
  • Disulfides
  • Lipopolysaccharides
  • Peptides, Cyclic
  • Proteins
  • alpha-Defensins
  • protegrin-1
  • tachyplesin peptide, Tachypleus tridentatus
  • Arginine
  • Lysine
  • Alanine