Anti-endotoxic activity and structural basis for human MD-2·TLR4 antagonism of tetraacylated lipid A mimetics based on βGlcN(1↔1)αGlcN scaffold.
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.
Institute of Molecular Modelling and Simulation, University of Natural Resources and Life Sciences, Vienna, Austria.
Jose Antonio
JA
Garate
Institute of Immunology, Medical University of Vienna, Vienna, Austria.
Johannes
J
Stöckl
Chemical and Physical Biology, CIB-CSIC, Madrid, Spain.
María del Carmen
Mdel C
Fernández-Alonso
Department of Chemistry, University of Natural Resources and Life Sciences, Vienna, Austria.
Daniel
D
Artner
Department for Biomedical Molecular Biology, Unit of Molecular Signal Transduction in Inflammation, Ghent University, Inflammation Research Center, VIB, Ghent, Belgium.
Mira
M
Haegman
Institute of Molecular Modelling and Simulation, University of Natural Resources and Life Sciences, Vienna, Austria.
Chris
C
Oostenbrink
Chemical and Physical Biology, CIB-CSIC, Madrid, Spain.
Jesús
J
Jiménez-Barbero
Department for Biomedical Molecular Biology, Unit of Molecular Signal Transduction in Inflammation, Ghent University, Inflammation Research Center, VIB, Ghent, Belgium.
Rudi
R
Beyaert
Research Group Innate Immunity, Research Center Borstel, Leibniz-Center for Medicine and Biosciences, Airway Research Center North (ARCN), Member of the German Center for Lung Research (DZL), Borstel, Germany.
Holger
H
Heine
Department of Chemistry, University of Natural Resources and Life Sciences, Vienna, Austria.
Paul
P
Kosma
Department of Chemistry, University of Natural Resources and Life Sciences, Vienna, Austria alla.zamyatina@boku.ac.at.
Alla
A
Zamyatina
D001665:Binding Sites; D002460:Cell Line; D016207:Cytokines / Q000096:biosynthesis; D003713:Dendritic Cells / Q000187:drug effects / Q000378:metabolism; D004731:Endotoxins / Q000037:antagonists & inhibitors*; D004847:Epithelial Cells / Q000187:drug effects; D004926:Escherichia coli / Q000737:chemistry; D005944:Glucosamine / Q000737:chemistry / Q000494:pharmacology; D006801:Humans; D007113:Immunity, Innate / Q000276:immunology; D008050:Lipid A / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology*; D008070:Lipopolysaccharides / Q000378:metabolism; D051218:Lymphocyte Antigen 96 / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D008968:Molecular Conformation; D015398:Signal Transduction / Q000502:physiology; D051197:Toll-Like Receptor 4 / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism
D016207:Cytokines; D004731:Endotoxins; C495377:LY96 protein, human; D008050:Lipid A; D008070:Lipopolysaccharides; D051218:Lymphocyte Antigen 96; D051197:Toll-Like Receptor 4; D005944:Glucosamine
Antagonist; MD-2; NMR; Toll-like receptor 4; glycolipids; lipid A; lipopolysaccharide; molecular dynamics simulation