Free thiol group of MD-2 as the target for inhibition of the lipopolysaccharide-induced cell activation

Abstract

MD-2 is a part of the Toll-like 4 signaling complex with an indispensable role in activation of the lipopolysaccharide (LPS) signaling pathway and thus a suitable target for the therapeutic inhibition of TLR4 signaling. Elucidation of MD-2 structure provides a foundation for rational design of inhibitors that bind to MD-2 and inhibit LPS signaling. Since the hydrophobic binding pocket of MD-2 provides little specificity for inhibitors, we have investigated targeting the solvent-accessible cysteine residue within the hydrophobic binding pocket of MD-2. Compounds with affinity for the hydrophobic pocket that contain a thiol-reactive group, which mediates covalent bond formation with the free cysteine residue of MD-2, were tested. Fluorescent compounds 2-(4'-(iodoacetamido)anilino)naphthalene-6-sulfonic acid and N-pyrene maleimide formed a covalent bond with MD-2 through Cys(133) and inhibited LPS signaling. Cell activation was also inhibited by thiol-reactive compounds JTT-705 originally targeted against cholesterol ester transfer protein and antirheumatic compound auranofin. Oral intake of JTT-705 significantly inhibited endotoxin-triggered tumor necrosis factor alpha production in mice. The thiol group of MD-2 also represents the target of environmental or endogenous thiol-reactive compounds that are produced in inflammation.

FIGURE 1.

Free cysteine residue is positioned within the binding pocket of MD-2, which can react with thiol-reactive compounds. Left, MD-2 structure reveals an internal hydrophobic cavity with a cysteine residue 133 (van der Waals radii shown in yellow), which is located inside the cavity near the bound lipid IVa antagonist and presents a target for compounds with a thiol-reactive group. Bottom, surface representation of MD-2 with docked pyrene-maleimide molecules. Right image, MD-2 with covalently bound pyrene-maleimide, indicating steric overlap with bound lipid IVa. Right, tested compounds comprising a hydrophobic moiety and a thiol-reactive group. N-Pyrene maleimide and IAANS were used as fluorescent probes, whereas JTT-705 and auranofin are drugs already used in therapy or in clinical trials.

FIGURE 2.

Compounds interact with MD-2 in solution. The addition of 1 μm MD-2 to 5 μm tested compound solution increases the fluorescence emission of hydrophobic probes IAANS (A) and N-pyrene maleimide (B). C, replacement of a fluorescent probe bis-ANS bound to MD-2 with nonfluorescent auranofin is reflected in a decrease of bis-ANS fluorescence emission. A–C, interaction between MD-2 and IAANS or N-pyrene maleimide or auranofin is impaired in mutant MD-2C133F. Fluorescence intensities are presented in relative units (r. u.) or a percentage (%) of maximal fluorescence intensity.

FIGURE 3.

N-Pyrene maleimide covalently binds to MD-2 WT. Extraction of N-pyrene maleimide from the aqueous phase into the organic phase determines binding of N-pyrene maleimide to MD-2. A, N-pyrene maleimide was completely extracted from the buffer to chloroform. Preincubation of MD-2, either WT or WT treated with iodoacetamide or mutant C133F, with N-pyrene maleimide and subsequent extraction retained some N-pyrene maleimide in the aqueous phase as a result of tight binding. B, disruption of tertiary structure of MD-2 by the addition of a chemical denaturant guanidine HCl (6 m) before the extraction caused the removal of noncovalently bound N-pyrene maleimide from the aqueous phase. Fluorescence intensities are presented in relative units (r. u.).

FIGURE 4.

Binding site of tested compounds on MD-2 overlaps with the LPS-binding site. Sandwich ELISA shows competition of compound and LPS for the same binding site on MD-2. Recombinant hMD-2 WT and IAANS, N-pyrene maleimide, JTT-705, or auranofin was preincubated to allow the formation of complex and added to the chicken anti-MD-2 polyclonal antibody-coated plate. Detection of complex bound to polyclonal antibody was achieved by anti-MD-2 9B4 monoclonal antibody, which recognizes only LPS-free MD-2. Compound bound to MD-2 prevented 9B4 binding in a concentration-dependent manner.

FIGURE 5.

Compounds inhibit LPS induced NO production. RAW264.7 cells were seeded and preincubated with increasing concentrations of N-pyrene maleimide (top left), IAANS (top right), JTT-705 (bottom left), or auranofin (bottom right) for 1 h. After LPS stimulation for 16 h, release of NO into the medium was determined using Griess reagent, and the NO amount was calculated from the standard curve.

FIGURE 6.

Auranofin and JTT-705 inhibit the MyD88-dependent signaling pathway. HEK293T cells were transfected with MyD88 and NF-κB reporter plasmids. Afterward, cells were treated with auranofin and JTT-705 for 16 h. Inhibition of MyD88 overexpression was determined by measuring relative luciferase activity in cell lysate. *, significantly different from vehicle (p < 0.1); ***, significantly different from vehicle (p < 0.01).

FIGURE 7.

JTT-705 inhibition of LPS signaling depends on the reactivity with free cysteine group of MD-2. A, ELISA confirmed the importance of free Cys¹³³. JTT-705 was preincubated with MD-2 WT or MD-2C133F. JTT-705 bound to MD-2C133F to a lesser extent. Binding of MD-2 without a compound was defined as 100%. B, preincubation of recombinant human MD-2 WT with JTT-705 inhibits LPS-stimulated cell signaling, monitored by the NF-κB-responsive luciferase reporter of TLR4-transfected HEK293. The addition of preincubated MD-2C133F protein with JTT-705 to TLR4-expressing HEK293 cells had only minor influence on LPS signaling. **, significantly different from vehicle (p < 0.05); ****, significantly different from vehicle (p < 0.005).

FIGURE 8.

JTT-705 inhibits TNFα production in endotoxin-treated mice. Mice were treated intraperitoneally with 50 ng of LPS and 18 mg of galactosamine. As a positive control of protection against endotoxic shock, mice from one of the groups fed regular mouse pellets received 150 μg of PMB immediately after LPS challenge. Mice were treated with JTT705 by adding it to the food pellets 6 days before the LPS treatment. Serum levels of TNFα were determined in the animal groups (n = 4) at 60 min postinoculation, coinciding with the cytokine peak production as established in previous experiments.