title: Design of Gram-negative selective antimicrobial peptides.
abstract: 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.
authors.affiliation: Department of Microbiology and Immunology, Vanderbilt University, A5119 MCN, Nashville, Tennessee 37232, USA.
authors.forename: S A
authors.initials: SA
authors.lastname: Muhle
authors.forename: J P
authors.initials: JP
authors.lastname: Tam
mesh_terms: D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D023181:Antimicrobial Cationic Peptides / Q000138:chemical synthesis* / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D001120:Arginine / Q000737:chemistry; D002942:Circular Dichroism; D004268:DNA-Binding Proteins / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D015195:Drug Design; D004926:Escherichia coli / Q000187:drug effects* / Q000254:growth & development; D008070:Lipopolysaccharides / Q000378:metabolism; D008239:Lysine / Q000737:chemistry; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D010456:Peptides, Cyclic / Q000138:chemical synthesis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry; D011550:Pseudomonas aeruginosa / Q000187:drug effects* / Q000254:growth & development; D020449:Repetitive Sequences, Amino Acid; D023084:alpha-Defensins
chemical_list: D023181:Antimicrobial Cationic Peptides; C429483:DEFA1A3 protein, human; D004268:DNA-Binding Proteins; D004220:Disulfides; D008070:Lipopolysaccharides; D010456:Peptides, Cyclic; D011506:Proteins; D023084:alpha-Defensins; C094880:protegrin-1; C057636:tachyplesin peptide, Tachypleus tridentatus; D001120:Arginine; D008239:Lysine; D000409:Alanine