Synthetic TLR4-MD-2-dependent endotoxin mimetics

Endotoxins (LPS, lipopolysaccharides) are unique, highly abundant surface glycolipids of Gram-negative bacteria, which are capable to potently induce proinflammatory responses in mammalian host by TLR4-MD-2 -dependent cell activation. Homodimerization of TLR4, which leads to the activation of the intracellular signaling cascade, is triggered directly by MD-2 - bound LPS, with electrostatic forces and hydrophobic interactions governing the structural adjustment of endotoxin-receptor complex and the geometry of the endotoxically active conformation of lipid A. It remains unclear which conformational rearrangements occur to lipid A upon its binding to TLR4-MD-2 complex, followed by receptor dimerization, wherein acyl chains of lipid A directly participate in the cross-linking of the neighbouring TLR4 and receptor clustering. The spatial arrangement of acyl chains in lipid A significantly depends on the relative orientation of GlcN sugar rings of the lipid A backbone, which, in turn, is reflected by the dihedral angles of hydroxymethyl fragment of reducing GlcN engaged in the 1 6 O-glycosidic linkage. To assess the influence of hydroxymethyl dihedrals of diglucosamine backbone of lipid A on its endotoxic potential by TLR4-MD-2-dependent cell activation, a series of conformationally constrained endotoxin mimetics based on crystal structures of a - a; ß - ß and a - ß trehalose will be synthesised. The trehalose type backbone serves as a scaffold mimicking the spatial arrangement of the carbohydrate backbone of lipid A as seen in the crystal structures of TLR4-MD-2-bound agonistic and antagonistic LPS. For the stereoselective preparation of nonreducing disaccharides the problem of simultaneously controlling the stereochemistry at two anomeric centres while forming a glycosidic linkage will be addressed. Along with conformationally constrained endotoxin mimetics of trehalose type, a number of analogues having the same acylation and phosphorylation pattern but equipped with the flexible C-glycosidic methyloxy linkage will be prepared. This would allow straight comparison of the endotoxic properties of conformationally constrained mimetics vs. flexible analogues mimicking natural lipid A, and, thus, would make possible a direct correlation of hydroxymethyl conformation and relative orientation of GlcN rings, which determines the direction of lipid chains in endotoxically active conformation, to agonistic activity. The project will be carried out within national and international multidisciplinary cooperations. Differential induction of the TLR4-MD-2 MyD88-dependent and -independent signaling pathways by synthetic endotoxin mimetics in relation to chemical structure will be studied in TLR4-MD-2 transfected HEK293 cells and mouse macrophages (cooperations S. Knapp, P. Kovarik). Agonistic lipid A analogues will be further tested for the ability to modulate LPS-induced inflammation as well as induction of LPS tolerance in rodents in vivo (S. Knapp). In- vitro imaging of the TLR4-MD-2-endotoxin complex will be examined by FRET using a set of fluorescently labelled proteins (TLR4, MD-2, MyD88) and azido-functionalized endotoxin mimetics which will be fluorescently labelled in vitro by means of "click chemistry" (cooperation R. Jerala). These data will provide a new insight in the structural determinants of the lipid A endotoxicity and, concurrently, in the mechanisms of LPS - triggered TLR4 activation, which, in turn, will permit improvements in the design of future TLR4-dependent adjuvants and therapeutics.

Lipopolysaccharide (LPS, Endotoxin) is a large 20 kDa molecule situated in the outer membrane of the cell wall of Gram-negative bacteria. LPS represents a pathogen associated molecular pattern (PAMP) that can be detected by the mammalian innate immune system. Immune cells such as macrophages or dendritic cells possess specific receptor proteins (Toll-like Receptors) which are responsible for the recognition of PAMPs and ensuing initiation of the innate immune response. Minor amounts of LPS are detected by the Toll-like Receptor 4 (TLR4) which, after binding of LPS, triggers a cascade of immune reactions resulting in the release of pro-inflammatory mediators (cytokines). The latter contributes to the establishment of an immediate immune response to infection and eradication of the invading pathogens. TLR4 can recognize and bind only a small amphiphilic part of LPS which is called Lipid A and consists of a carbohydrate backbone substituted by the two anionic phosphate groups and by up to six hydrophobic lipid tails. The degree of TLR4 activation depends mainly on the chemical structure of Lipid A. In this project we explored the structure-function relationships in the ternary Lipid AMD-2TLR4 complex wherein we focused on the three-dimensional molecular shape of the protein-bound ligand as the major structural determinant of endotoxicity. We directed our research on restricting the inherent flexibility of the ß(1?6)-linked diglucosamine backbone of Lipid A by exchanging a flexible three-bond ß(1?6)-glycosidic linkage for a rigid two-bond 1,1-anomerically linked trehalose-type connection. Rational X-ray structure based design and chemical synthesis of a series of Lipid A mimetics (LAMs) based on the 1,1-linked disaccharide backbones having variable anomeric configuration afforded both antagonistic (anti-endotoxic) and agonistic (TLR4 stimulating) TLR4 ligands. The immunostimulating activity of LAMs was dependent on the chemical structure (e.g. number of lipid tails and phosphate groups attached to the carbohydrate backbone) and, most importantly, on the topology and three-dimensional arrangement of the sugar backbone. Lipid A mimetics based on the disaccharide scaffold having planar oriented sugar rings displayed antagonistic properties, whereas LAMs based on the disaccharide scaffold with twisted relative arrangement of sugar rings were highly agonistic. Our data contribute to the comprehension of structure activity relationships in the LPS-TLR4 ligand-receptor complex and provide new insights on the structural requirements to TLR4 specific ligands. Additionally, within a cooperation framework with research groups in Germany, Switzerland, Belgium and Slovenia, the immuno-biological examination of novel synthetic TLR4 ligands was accomplished. Therapeutic potential of novel synthetic TLR4 antagonists and agonists was demonstrated in a variety of immune and non-immune cells, highlighting several compounds from antagonistic series as suitable lead structures for the development of antisepsis drug candidates and the compounds of the agonistic series as immuno-modulators and potential vaccine adjuvants, respectively.