Synthetic glycan-based TLR4 agonists targeting caspase-4/11 for the development of adjuvants and immunotherapeutics

Gram-negative bacterial lipopolysaccharide (LPS)-induced Toll-like receptor 4 (TLR4) mediated pro-inflammatory signaling plays a key role in immunoprotection against infectious challenges and boosts adaptive immunity, whereas the activation of the cytosolic LPS receptor caspase-4/11 leads to cell death by pyroptosis and is deeply implicated in the development of sepsis. Despite tremendous advances in the understanding of the LPS-TLR4 interaction, predictably regulated TLR4 activation has not yet been achieved. The structural basis for the induction of caspase-4/11 protease activity by LPS is currently unknown. The modulation of innate and adaptive immune responses through the controlled induction of TLR4 signaling without triggering caspase-4/11 activity would open novel perspectives in the development of safe vaccine adjuvants and immunotherapeutics. We report the discovery of highly potent glycan-based immunostimulants with picomolar affinity for TLR4 which interact with caspase-4/11 and promote caspase-4/11 oligomerization while abolishing caspase-11 protease activity. The rigidity and twisted molecular shape of the alpha,alpha-(1 1')-linked disaccharide core of synthetic LPS mimicking anionic glycolipids accounted for both species-independent and adjustable TLR4-mediated NF-kappa B signaling and the modulation of caspase-4/11 activation. By the use of crystal structure based design and advanced synthetic chemistry we created a set of versatile probes for studying the structural basis of caspase-4/11 activation and established a chemical strategy for controllable TLR4 mediated cytokine release which is dissociable from the induction of caspase-11 protease activity

Creating favourable contexts for nurturing and managing innovations in organisations

This thesis gives an overview about innovations in organisations as well as the importance of a favourable organisational microclimate for the enhancement of creativity and innovation. Generating ideas is always a very fragile process for any organisation engaged in innovative activities. In order to support good and successful ideas the companies have to have a favourable organisational microclimate. This microclimate is discussed against the background of certain conditions which organisations or rather companies should obtain in order to prevail innovations and cope with them in a successful way. The main focus of this thesis is on the conditions, which can favour and support the process of emerging innovations. The conditions are teamwork, management participation, effective communication and information flow, as well as innovative culture. During the different chapters however, some interrelations can therefore be seen. Furthermore nine interviews with three different innovative Latvian companies support our theoretical framework of this thesis, how the different conditions are interrelated with each other in practice and their occurrence in the different companies. In the end a discussion about the results is started, where it can be seen whether more conditions emerged

The Amadori rearrangement as glycoconjugation method: Synthesis of non-natural C-glycosyl type glycoconjugates

The Amadori rearrangement was investigated as a potential method for the conjugation of carbohydrate moieties to suitable amino components. Starting from selected aldoheptoses, which are readily available by means of the Kiliani–Fischer C-elongation reaction of the corresponding aldohexoses, glycoconjugates presenting D-gluco, D-manno and D-galacto as well as GlcNAc motifs have been synthesised. Following this strategy, non-natural C-glycosyl type glycoconjugates, which can be utilised as building blocks for the composition of larger molecular constructions, are available by a very short synthetic approach

Tailored modulation of cellular pro-inflammatory responses with disaccharide lipid a mimetics

Pro-inflammatory signaling mediated by Toll-like receptor 4 (TLR4)/myeloid differentiation-2 (MD-2) complex plays a crucial role in the instantaneous protection against infectious challenge and largely contributes to recovery from Gram-negative infection. Activation of TLR4 also boosts the adaptive immunity which is implemented in the development of vaccine adjuvants by application of minimally toxic TLR4 activating ligands. The modulation of pro-inflammatory responses via the TLR4 signaling pathway was found beneficial for management of acute and chronic inflammatory disorders including asthma, allergy, arthritis, Alzheimer disease pathology, sepsis, and cancer. The TLR4/MD-2 complex can recognize the terminal motif of Gram-negative bacterial lipopolysaccharide (LPS)-a glycophospholipid lipid A. Although immense progress in understanding the molecular basis of LPS-induced TLR4-mediated signaling has been achieved, gradual, and predictable TLR4 activation by structurally defined ligands has not yet been attained. We report on controllable modulation of cellular pro-inflammatory responses by application of novel synthetic glycolipids-disaccharide-based lipid A mimetics (DLAMs) having picomolar affinity for TLR4/MD-2. Using crystal structure inspired design we have developed endotoxin mimetics where the inherently flexible beta(1 -> 6)-linked diglucosamine backbone of lipid A is replaced by a conformationally restricted alpha,alpha-(1 1)-linked disaccharide scaffold. The tertiary structure of the disaccharide skeleton of DLAMs mirrors the 3-dimensional shape of TLR4/MD-2 bound E. coli lipid A. Due to exceptional conformational rigidity of the sugar scaffold, the specific 3D organization of DLAM must be preserved upon interaction with proteins. These structural factors along with specific acylation and phosphorylation pattern can ensure picomolar affinity for TLR4 and permit efficient dimerization of TLR4/MD-2/DLAM complexes. Since the binding pose of lipid A in the binding pocket of MD-2 (+/- 180 degrees) is crucial for the expression of biological activity, the chemical structure of DLAMs was designed to permit a predefined binding orientation in the binding groove of MD-2, which ensured tailored and species-independent (human and mice) TLR4 activation. Manipulating phosphorylation and acylation pattern at the sugar moiety facing the secondary dimerization interface allowed for adjustable modulation of the TLR4-mediated signaling. Tailored modulation of cellular pro-inflammatory responses by distinct modifications of the molecular structure of DLAMs was attained in primary human and mouse immune cells, lung epithelial cells and TLR4 transfected HEK293 cells

Development of αGlcN(1 <-> 1)αMan-based Lipid A mimetics as a novel class of potent Toll-like Receptor 4 agonists

The endotoxic portion of lipopolysaccharide (LPS), a glycophospholipid Lipid A, initiates the activation of the Toll-like Receptor 4 (TLR4)myeloid differentiation factor 2 (MD-2) complex, which results in pro-inflammatory immune signaling. To unveil the structural requirements for TLR4 center dot MD-2-specific ligands, we have developed conformationally restricted Lipid A mimetics wherein the flexible beta GlcN(1 -> 6)GlcN backbone of Lipid A is exchanged for a rigid trehalose-like alpha GlcN(1 1)alpha Man scaffold resembling the molecular shape of TLR4 center dot MD-2-bound E. coli Lipid A disclosed in the X-ray structure. A convergent synthetic route toward orthogonally protected alpha GlcN(1 1)alpha Man disaccharide has been elaborated. The alpha,alpha-(1 1) linkage was attained by the glycosylation of 2-N-carbamate-protected alpha-GlcN-lactol with N-phenyl-trifluoroacetimidate of 2-O-methylated mannose. Regioselective acylation with (R)-3-acyloxyacyl fatty acids and successive phosphorylation followed by global deprotection afforded bis- and monophosphorylated hexaacylated Lipid A mimetics. alpha GlcN(1 1)alpha Man-based Lipid A mimetics (alpha,alpha-GM-LAM) induced potent activation of NF-kappa B signaling in hTLR4/hMD-2/CD14-transfected HEK293 cells and robust LPS-like cytokines expression in macrophages and dendritic cells. Thus, restricting the conformational flexibility of Lipid A by fixing the molecular shape of its carbohydrate backbone in the agonistic conformation attained by a rigid alpha GlcN(1 1)alpha Man scaffold represents an efficient approach toward powerful and adjustable TLR4 activation