Anti-endotoxic activity and structural basis for human MD-2·TLR4 antagonism of tetraacylated lipid A mimetics based on βGlcN(1↔1)αGlcN scaffold

• Published in: Innate immunity (London, England) England), 2015-07, Vol.21 (5), p.490-503
• Main Authors: Garate, Jose Antonio, Stöckl, Johannes, del Carmen Fernández-Alonso, María, Artner, Daniel, Haegman, Mira, Oostenbrink, Chris, Jiménez-Barbero, Jesús, Beyaert, Rudi, Heine, Holger, Kosma, Paul, Zamyatina, Alla
• Format: Article
• Language: English
• Subjects: Binding Sites; Cell Line; Cytokines - biosynthesis; Dendritic Cells - drug effects; Dendritic Cells - metabolism; Endotoxins - antagonists & inhibitors; Epithelial Cells - drug effects; Escherichia coli - chemistry; Glucosamine - chemistry; Glucosamine - pharmacology; Humans; Immunity, Innate - immunology; Lipid A - analogs & derivatives; Lipid A - chemistry; Lipid A - pharmacology; Lipopolysaccharides - metabolism; Lymphocyte Antigen 96 - antagonists & inhibitors; Lymphocyte Antigen 96 - chemistry; Lymphocyte Antigen 96 - metabolism; Molecular Conformation; Original; Signal Transduction - physiology; Toll-Like Receptor 4 - antagonists & inhibitors; Toll-Like Receptor 4 - chemistry; Toll-Like Receptor 4 - metabolism
• ISSN: 1753-4259; 1753-4267
• DOI: 10.1177/1753425914550426

Interfering with LPS binding by the co-receptor protein myeloid differentiation factor 2 (MD-2) represents a useful approach for down-regulation of MD-2·TLR4-mediated innate immune signaling, which is implicated in the pathogenesis of a variety of human diseases, including sepsis syndrome. The antagonistic activity of a series of novel synthetic tetraacylated bis-phosphorylated glycolipids based on the βGlcN(1↔1)αGlcN scaffold was assessed in human monocytic macrophage-like cell line THP-1, dendritic cells and human epithelial cells. Two compounds were shown to inhibit efficiently the LPS-induced inflammatory signaling by down-regulation of the expression of TNF-α, IL-6, IL-8, IL-10 and IL-12 to background levels. The binding of the tetraacylated by (R)-3-hydroxy-fatty acids (2 × C12, 2 × C14), 4,4′-bisphosphorylated βGlcN(1↔1)αGlcN-based lipid A mimetic DA193 to human MD-2 was calculated to be 20-fold stronger than that of Escherichia coli lipid A. Potent antagonistic activity was related to a specific molecular shape induced by the β,α(1↔1)-diglucosamine backbone. ‘Co-planar’ relative arrangement of the GlcN rings was inflicted by the double exo-anomeric conformation around both glycosidic torsions in the rigid β,α(1↔1) linkage, which was ascertained using NOESY NMR experiments and confirmed by molecular dynamics simulation. In contrast to the native lipid A ligands, the binding affinity of βGlcN(1↔1)αGlcN-based lipid A mimetics to human MD-2 was independent on the orientation of the diglucosamine backbone of the synthetic antagonist within the binding pocket of hMD-2 (rotation by 180°) allowing for two equally efficient binding modes as shown by molecular dynamics simulation.