Recognition of the lipopolysaccharide
(LPS), a major component
of the outer membrane of Gram-negative bacteria, by the Toll-like
receptor 4 (TLR4)-myeloid differentiation factor 2 (MD-2) complex
is essential for the control of bacterial infection. A pro-inflammatory
signaling cascade is initiated upon binding of membrane-associated
portion of LPS, a glycophospholipid Lipid A, by a coreceptor protein
MD-2, which results in a protective host innate immune response. However,
activation of TLR4 signaling by LPS may lead to the dysregulated immune
response resulting in a variety of inflammatory conditions including
sepsis syndrome. Understanding of structural requirements for Lipid
A endotoxicity would ensure the development of effective anti-inflammatory
medications. Herein, we report on design, synthesis, and biological
activities of a series of conformationally confined Lipid A mimetics
based on β,α-trehalose-type scaffold. Replacement of the
flexible three-bond β(1→6) linkage in diglucosamine backbone
of Lipid A by a two-bond β,α(1↔1) glycosidic linkage
afforded novel potent TLR4 antagonists. Synthetic tetraacylated bisphosphorylated
Lipid A mimetics based on a β–GlcN(1↔1)α–GlcN
scaffold selectively block the LPS binding site on both human and
murine MD-2 and completely abolish lipopolysaccharide-induced pro-inflammatory
signaling, thereby serving as antisepsis drug candidates. In contrast
to their natural counterpart lipid IVa, conformationally constrained
Lipid A mimetics do not activate mouse TLR4. The structural basis
for high antagonistic activity of novel Lipid A mimetics was confirmed
by molecular dynamics simulation. Our findings suggest that besides
the chemical structure, also the three-dimensional arrangement of
the diglucosamine backbone of MD-2-bound Lipid A determines endotoxic
effects on TLR4.
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.
The phytosiderophore 2'-deoxymugineic acid (DMA) is exuded via the root system by all grasses (including important crop plants like rice, wheat and barley) to mobilize Fe(III) from soil and improve plant Fe nutrition, crucial for high crop yields. Elucidation of the biogeochemistry of 2'-deoxymugineic acid in the rhizosphere requires its quantification in minute amounts. To this end, (13)C4-DMA was synthesized for the first time, from cheap isotopically labeled starting materials. The synthetic route utilizes L-allyl((13)C2)glycine and L-(2-(13)C)azetidine ((13)C)carboxylic acid as versatile labeled building blocks. The title compound was recently used as an internal standard for analysis of soil and plant samples allowing the first accurate quantification of DMA in these matrices by means of LC-MS/MS. It is furthermore used in tracer experiments investigating biodegradation of DMA in soil.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.