Figure 1.
Crystal structure-based model of the HNP-1-Lipid II complex obtained with HADDOCK.
(A) The HNP-1 dimer is shown in surface representation. (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. (C) 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.
Table 1.
Residues involved in HNP-1 Lipid II contacts.
Figure 2.
Binding of HNP-1 and HNP-1 single alanine mutants on immobilized Lipid II as determined by SPR.
(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%.
Figure 3.
Lipid II antagonizes the antibacterial activity of HNP-1.
Survival curves of S. aureus 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.
Figure 4.
Characterization of BAS00127538.
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 S. aureus and 7.8 µM against E. coli. 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.
Table 2.
Classification of lead defensin mimetics.
Table 3.
Broth microdilution susceptibility testing for lead defensin mimetics and comparators.
Figure 5.
Mechanism of action studies of BAS00127538.
Exponentially growing S. aureus 29213 cells were exposed to compound and comparators in triplicate using 2.5% DMSO as “no drug” control. Cells were added to Mueller-Hinton Broth or M9 medium for protein synthesis and further incubated in the presence of [14C]N-acetyl glucosamine (cell wall), [3H]glycerol (lipid), [3H]Thymidine (DNA), or [3H]Leucine (protein). Following incubation, reactions were stopped by adding TCA (DNA, protein), 8% SDS (cell wall) or chloroform/methanol (lipid) and analyzed by scintillation counting.
Figure 6.
Membrane activity of BAS00127538 and 1499-1221.
(A) Compounds were diluted in sextuplet in phosphate-buffered saline solution and incubated for 30 min at RT with DPPC LUVs with or without Lipid II as indicated. Release of fluorophore was expressed as percentage of release compared to controls containing Triton X-100. Results represent average of three experiments, each carried out in sextuplet (B) Defibrinated human blood was washed three times with buffer (10 mM Tris-HCl, pH 7.4, 0.9% NaCl) and was resuspended to a final concentration of 3% RBCs immediately prior to performing the assay. Incubation of compounds and RBCs was for 30 minutes at room temperature, followed by centrifugation at 1,300× g for 5 minutes to pellet the RBCs. 300 µl of the supernatant was removed to a 96-well plate and the released hemoglobin was measured at A540 using a SpectraMax (Molecular Dynamics) plate reader.
Figure 7.
Analysis of the BAS00127538-lipid II complex by NMR.
(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.
Figure 8.
Model of the BAS00127538-lipid II complex obtained with CADD in conjunction with the NMR data.
Upper panel includes BAS00127538 shown in CPK atom-colored representation, Lipid II in a licorice, atom-colored representation, with the exception of the N-MurNac moeity, which is green, and water molecules included in the simulation are shown in stick format. Lower panel is the same as upper panel except Lipid II is shown in van der Waals representation. Images created with VMD [63].
Figure 9.
Efficacy of BAS00127538 in vivo.
Blood samples were collected from vehicle-treated animals at 20 h or at 50 h post-infection from vancomycin and BAS00127538-treated animals. * One animal treated with compound did not survive beyond 28 h.