Філософія популізму як варіант сучасної філософії

We have previously reported on the functional interaction of Lipid II with human alpha-defensins, a class of antimicrobial peptides. Lipid II is an essential precursor for bacterial cell wall biosynthesis and an ideal and validated target for natural antibiotic compounds. Using a combination of structural, functional and in silico analyses, we present here the molecular basis for defensin-Lipid II binding. Based on the complex of Lipid II with Human Neutrophil peptide-1, we could identify and characterize chemically diverse low-molecular weight compounds that mimic the interactions between HNP-1 and Lipid II. Lead compound BAS00127538 was further characterized structurally and functionally; it specifically interacts with the N-acetyl muramic acid moiety and isoprenyl tail of Lipid II, targets cell wall synthesis and was protective in an in vivo model for sepsis. For the first time, we have identified and characterized low molecular weight synthetic compounds that target Lipid II with high specificity and affinity. Optimization of these compounds may allow for their development as novel, next generation therapeutic agents for the treatment of Gram-positive pathogenic infections

Turning Defense into Offense: Defensin Mimetics as Novel Antibiotics Targeting Lipid II

<div><p>We have previously reported on the functional interaction of Lipid II with human alpha-defensins, a class of antimicrobial peptides. Lipid II is an essential precursor for bacterial cell wall biosynthesis and an ideal and validated target for natural antibiotic compounds. Using a combination of structural, functional and <i>in silico</i> analyses, we present here the molecular basis for defensin-Lipid II binding. Based on the complex of Lipid II with Human Neutrophil peptide-1, we could identify and characterize chemically diverse low-molecular weight compounds that mimic the interactions between HNP-1 and Lipid II. Lead compound BAS00127538 was further characterized structurally and functionally; it specifically interacts with the N-acetyl muramic acid moiety and isoprenyl tail of Lipid II, targets cell wall synthesis and was protective in an <i>in vivo</i> model for sepsis. For the first time, we have identified and characterized low molecular weight synthetic compounds that target Lipid II with high specificity and affinity. Optimization of these compounds may allow for their development as novel, next generation therapeutic agents for the treatment of Gram-positive pathogenic infections.</p></div

Classification of lead defensin mimetics.

<p>N.D: Not Determinable by SPR. Bacterial killing: Concentration resulting in 100% killing after exposure of compound to bacteria for 30 min, determined by modified vCC assays <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003732#ppat.1003732-Ericksen1" target="_blank">[26]</a>. Binding to immobilized 3-Lipid II was analyzed by Surface Plasmon Resonance. C_50% equals compound concentration resulting in 50% cell survival measured by MTT assay following incubation for 24 h (Caco-2) or 4 h (Jurkat).</p

Lipid II antagonizes the antibacterial activity of HNP-1.

<p>Survival curves of <i>S. aureus</i> ATCC 29213 exposed to HNP-1 (at concentrations varying two-fold from 50 to 1.25 µM). Defensin peptide was pre-incubated with 3-Lipid II at varying molar ratios for 30 min prior to addition of bacteria. Bacteria were subsequently exposed to HNP-1 for 30 min. Each curve is the mean of three separate experiments (±S.D.). Points scored as zero survival could not be plotted.</p

Characterization of BAS00127538.

<p>Chemical structure (left panel), bacterial killing (middle panel) and Lipid II binding (right panel) of defensin mimetic BAS00127538. Mimetic compound was 100% bactericidal at 0.244 µM against <i>S. aureus</i> and 7.8 µM against <i>E. coli</i>. Points of zero survival could not be plotted. (right panel) Representative sensorgrams of one out of three experiments of BAS00127538 binding to immobilized 3-Lipid II.</p

Residues involved in HNP-1 Lipid II contacts.

<p>Common three letters abbreviation is used for amino acids. D: amino acid in D-configuration. MurNac: N-acetyl Muramic acid. The identified contacts are based on an analysis of the top 4 docking models.</p

Crystal structure-based model of the HNP-1-Lipid II complex obtained with HADDOCK.

<p>(A) The HNP-1 dimer is shown in surface representation. (<b>B</b>) Residue Isoleucine 20 of HNP-1 monomer A interacts with Lysine-3 of the Lipid II pentapeptide, whereas Leucine residue 25 of HNP-1 monomer A interacts with D-Ala at position 4. (<b>C</b>) Residues Arginine15, Isoleucine 20 and Leucine25 of HNP-1 monomer B interact with γD-Glu-2 and the phosphate/N-acetyl muramic acid moiety of Lipid II. Critical residues in HNP-1 for the Lipid II interactions are shown in yellow and span the two monomers indicated in green and blue.</p

Binding of HNP-1 and HNP-1 single alanine mutants on immobilized Lipid II as determined by SPR.

<p>(Upper panel) Representative sensorgrams of one out of two separate experiments of HNP-1 and analogues at 10 µM using a sensorchip with 40 RUs of soluble, 3-Lipid II. (Lower panel) Quantification of binding of HNP-1 mutants compared to binding of wild-type HNP-1, set as 100%.</p

Analysis of the BAS00127538-lipid II complex by NMR.

<p>(Upper panel) Analysis of 2D TOCSY spectra collected at 800 MHz of the aromatic region of compound BAS00127538 alone (black) overlaid with spectra of compound bound to Lipid II (red) (Lower panel). 2D natural abunance ¹³C HSQC spectrum illustrating the interaction between Lipid II and the compound BAS00127538. BAS00127538 alone (black) is overlaid with a spectrum of compound bound to Lipid II (red). Spectra were collected on a Bruker 800 MHz Avance NMR spectrometer at 25 degrees. Chemical shift changes for Lipid II upon BAS00127538 compound binding suggest that the interaction is occuring at or near the MurNAc moeity of Lipid II.</p