Modulation of CD14 and TLR4⋅MD-2 Activities by a Synthetic Lipid A Mimetic

• Published in: Chembiochem : a European journal of chemical biology 2014-01, Vol.15 (2), p.250-258
• Main Authors: Cighetti, Roberto, Ciaramelli, Carlotta, Sestito, Stefania Enza, Zanoni, Ivan, Kubik, Łukasz, Ardá-Freire, Ana, Calabrese, Valentina, Granucci, Francesca, Jerala, Roman, Martín-Santamaría, Sonsoles, Jiménez-Barbero, Jesus, Peri, Francesco
• Format: Article
• Language: English
• Subjects: Animals; Biomimetic Materials - chemistry; Biomimetic Materials - metabolism; Biomimetic Materials - pharmacology; bioorganic chemistry; carbohydrates; drug design; Endocytosis - drug effects; HEK293 Cells; Humans; Lipid A - chemistry; Lipopolysaccharide Receptors - metabolism; Lymphocyte Antigen 96 - chemistry; Lymphocyte Antigen 96 - metabolism; Macrophages - drug effects; Macrophages - metabolism; Mice; Molecular Docking Simulation; molecular modeling; Monosaccharides - chemistry; Monosaccharides - metabolism; Monosaccharides - pharmacology; NF-kappa B - metabolism; NMR; Protein Conformation; Structure-Activity Relationship; Toll-Like Receptor 4 - metabolism
• ISSN: 1439-4227; 1439-7633
• DOI: 10.1002/cbic.201300588

Monosaccharide lipid A mimetics based on a glucosamine core linked to two fatty acid chains and bearing one or two phosphate groups have been synthesized. Compounds 1 and 2, each with one phosphate group, were practically inactive in inhibiting LPS‐induced TLR4 signaling and cytokine production in HEK‐blue cells and murine macrophages, but compound 3, with two phosphate groups, was found to be active in efficiently inhibiting TLR4 signal in both cell types. The direct interaction between compound 3 and the MD‐2 coreceptor was investigated by NMR spectroscopy and molecular modeling/docking analysis. This compound also interacts directly with the CD14 receptor, stimulating its internalization by endocytosis. Experiments on macrophages show that the effect on CD14 reinforces the activity on MD‐2⋅TLR4 because compound 3's activity is higher when CD14 is important for TLR4 signaling (i.e., at low LPS concentration). The dual targeting of MD‐2 and CD14, accompanied by good solubility in water and lack of toxicity, suggests the use of monosaccharide 3 as a lead compound for the development of drugs directed against TLR4related syndromes. It takes two: A rationally designed synthetic lipid A mimetic consisting of a diphosphorylated monosaccharide with two fatty acid chains is active in inhibiting LPS‐dependent human and murine TLR4 activation in cells and stimulates CD14 internalization. The binding between the synthetic molecule and MD‐2 was studied by NMR experiments and docking calculations.