| Abstract
| - Lipopolysaccharide (LPS), a major component of Gram-negative bacteria, signals bacterialinvasion and triggers defensive host responses. However, excessive responses also lead to the seriouspathophysiological consequence of septic shock. To develop Gram-negative selective compounds thatcan inhibit the effects of LPS-induced sepsis, we have designed constrained cyclic antimicrobial peptidesbased on a cystine-stabilized β-stranded framework mimicking the putative LPS-binding sites of the LPS-binding protein family. Our prototype termed R4A, c(PACRCRAG-PARCRCAG), consists of an eightamino acid degenerated repeat constrained by a head-to-tail cyclic peptide backbone and two cross-bracingdisulfides. NMR study of K4A, an R4A analogue with four Arg → Lys replacements, confirmed theamphipathic design elements with four Lys on one face of the antiparallel β-strand and two hydrophobiccystine pairs plus two Ala on the opposite face. K4A and R4A displayed moderate microbicidal potencyand Gram-negative selectivity. However, R4A analogues with single or multiple replacements of Ala andGly 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 hydrophobicamino acids proved the best mimics of the amphipathic topology of the “active-site” β-strands of LPS-binding proteins. They displayed potent activity against Gram-negative E. coli with a minimal inhibitoryconcentration of 20 nM and a >200-fold selectivity over Gram-positive S. aureus. Our results suggestthat an LPS-targeted design may present an effective approach for preparing selective peptide antibiotics.
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