Topomimetics of amphipathic beta-sheet and helix-forming bactericidal peptides neutralize lipopolysaccharide endotoxins

Abstract

Release of lipopolysaccharide (LPS) endotoxin from Gram negative bacterial membranes triggers macrophages to produce large quantities of cytokines that can lead to septic shock and eventual death. Agents that bind to and neutralize LPS may provide a means to clinically prevent septic shock upon bacterial infection. Previously, we reported the design of antibacterial helix peptide SC4 and beta-sheet-forming betapep peptides that neutralize LPS in vitro. We hypothesized that the ability of these and other such peptides to neutralize LPS rested in the common denominator of positively charged amphipathic structure. Here, we describe the design and synthesis of nonpeptide, calixarene-based helix/sheet topomimetics that mimic the folded conformations of these peptides in their molecular dimensions, amphipathic surface topology, and compositional properties. From a small library of topomimetics, we identified several compounds that neutralize LPS in the 10-8 M range, making them as effective as bactericidal/permeability increasing protein and polymyxin B. In an endotoxemia mouse model, three of the most in vitro effective topomimetics are shown to be at least partially protective against challenges of LPS from different bacterial species. NMR studies provide mechanistic insight by suggesting the site of molecular interaction between topomimetics and the lipid A component of LPS, with binding being mediated by electrostatic and hydrophobic interactions. This research contributes to the development of pharmaceutical agents against endotoxemia and septic shock.

Figure 1

LPS structure. A generic structure of lipopolysaccharide (LPS) is shown. The phospholipid “lipid A” moiety is common in LPS from all species of Gram negative bacteria, whereas the polysaccharide moiety is highly variable in LPS from different bacteria.

Figure 2

Molecular design approach. (A) The topological design features influencing our choice of a calixarene scaffold for arraying hydrophobic and hydrophilic substituents, are shown. The folded structures of βpep-25 and SC4 peptides are illustrated to scale with the calix[4]arene scaffold. Chemical structures for calixarene analogs in the topomimetic library are displayed (B–E). These are grouped as (B) tertiary amines, (C) guanidinium, (D) triazole, primary amines, and negatively charged groups, and (E) generic partial cone conformer.

Figure 3

Examples of dose response curves for endotoxin neutralization. Several dose response curves for LPS neutralization are shown for some of the sheet/helix mimetics listed in Table 1. Symbols indicate actual data points, and lines are the best fit of a sigmoidal function to the data points. For all compounds listed in Table 1, IC₅₀ values have been derived from dose response curves fitted similarly.

Figure 4

Helix/sheet topomimetics protect mice from LPS endotoxin. Three helix/sheet topomimetics (5, 13 and 17) were used in mouse endotoxemia models to assess in vivo efficacy. In three separate studies, these compounds were tested against LPS derived from E. coli serotype 0111 :B4, E. coli serotype 055:B5 LPS, and Salmonella. The compounds (in a final concentration of 2 % DMSO v/v), were first mixed individually with LPS, and incubated for 30 minutes prior to i.p. injection into C57/BL6 mice (n = 4–8/group). The control mice were treated with DMSO (2% v/v) alone. Each mouse received a lethal dose of LPS, with or without one of the compounds. (A) Survival of mice after being challenged with 600 μ1 LPS form E. coli serotype 055:B5 with or without one of the compounds. The survival percentage of treatment with compound 5 and 13 are significantly significant (p = 0.03 and 0.006 respectively). (B) Survival of mice after being challenged with 500 μl LPS form E. coli serotype 0111:B4 with or without one of the compounds. The survival percentage of treatment with compound 13 is significantly increased (p = 1.3 10⁻⁵). (C) Survival of mice after being challenged with 600 μ1 LPS form Salmonella with or without one of the compounds. The survival percentage of treatment with compound 17, 5 and 13 are significantly increased (p = 9×l0⁻⁸, 0.008 and 0.002 respectively). In all panels, symbols are defined as: control (□), 5 (•), 17 (π), 13(θ).

Figure 5

TOCSY spectra and chemical shift changes upon titration of 5 and 13 with lipid A. Spectra were collected on Inova 800 MHz NMR-spectrometer at 25 °C. Lipid A was dissolved in chloroform/methanol/water 74/23/3 mixture at the concentration of 1 mg/ml. (A) Expanded regions of two-dimensional proton TOCSY spectra are displayed (overlaid) in the absence (blue) and presence (red) of compound 13 at 1:2 lipid A:compound molar ratio. Assignments for anomeric protons are indicated. (B–E) Chemical shift changes for some proton resonances of lipid A and compounds 13 and 5 are shown as a function of the lipid A:compound molar ratio. These changes were calculated by subtraction of the chemical shift of a given proton resonance from pure lipid A from the chemical shift of the same proton resonance from lipid A to which the calixarene compound was added. Resonances for protons 1 to 4 (blue) and 1′ to 4′ (red) are shown using different colors for clarity. (F) Relative chemical shift changes are illustrated for resonances from lipid A, and compounds 13 and 5, induced by addition of compounds to lipid A at different ratios. Each point was obtained by dividing the chemical shift change shown in panels B–E by the chemical shift change between pure lipid A and after addition of compound at the ratio 1:1. The arithmetic averages of relative chemical shifts are plotted. Error bars represent the standard deviation of chemical shifts calculated for different proton resonances.

Figure 6

Structures of lipid A and compounds 5 and 13 highlighting probable binding sites. The chemical structure (A left) and calculated Connoly surface (A right) of hexaacyl lipid A from E. coli are shown. In panel A left, proton resonances affected by the binding of compounds 5 and 13 are labeled using solid circles. Large red circles correspond to the largest chemical shift changes (>0.03 ppm), whereas orange and yellow circles indicate intermediate (0.02–0.03 ppm) and small (<0.02 ppm) chemical shift changes. The same color codes are used in A right to indicate the probably site of interaction with 5 and 13. (B) Energy minimized structures of compounds 5 and 13 are displayed. Chemical groups whose resonances are most affected by interaction with lipid A are color coded as described above.

Scheme 1

(A) synthesis of tertiary amine calixarene derivatives 1 and 5. (B) synthesis of guanidine calixarene derivatives 13 and 14 and primary amine calixarene derivative 16. (C) synthesis of triazole linked primary amine calixarene derivatives 17 and 18. Reaction conditions: a) AlCl₃, PhOH, toluene, rt; b) ethyl bromoacetate, K₂CO₃, acetone, reflux; c) N,N-dimethylethylenediamine, toluene, reflux; d) i. NaH, 3-chloro-2-methylpropene, THF, DMF, 80 °C; ii. N,N-dimethylaniline, 200 °C; e) Pd/C, H₂, 1atm, EtOAc, rt; f) 4-bromobutyronitrile, K₂CO₃, acetone, reflux; g) 4-bromobutyronitrile, NaH, DMF, 75 °C; h) NaBH₄, CoCl₂, MeOH; i) 1,3-bis(tert-butoxycarbonyl)-2-methyl-2-thiopseudourea, HgCl₂, Et₃N, CH₂Cl₂; j) TEA, 5% anisole in CH₂Cl₂, rt; k) NaH, propargyl bromide, THF, DMF, reflux; 1) N₃(CH₂)₂NHBoc, ascorbic acid, NaOAc, CuSO₄, t-BuOH, H₂O, THF; m) TFA, 5% anisole in CH₂Cl₂, 0 °C to rt.