Synthetic Bioactive Carbohydrates and Innate Immunity
Assoc. Professor Alla Zamyatina
The mammalian innate immune system detects the presence of pathogen associated molecular patterns (PAMPs) through pattern recognition receptors (PRRs). The outer leaflet of the outer membrane of Gram-negative bacteria is constituted by a unique antigen, a complex glycan lipopolysaccharide (LPS, also known as Endotoxin) which represents PAMP of Gram-negative bacteria.
LPS is recognized by the trans-membrane receptor protein Toll-like Receptor 4 (TLR4) and the intracellular protease caspase-4 (caspase-11 in mice) which are primarily expressed in mammalian immune cells. In response to the LPS challenge, TLR4 and caspase-4/11 trigger intracellular pro-inflammatory signaling cascades which lead to release of inflammatory mediators (chemokines and cytokines) thus contributing to the clearance of infection. When bacteria leave the located area and invade the bloodstream (bacteremia), substantial amounts of Endotoxin is released into the circulation of the host which results in the over-activation of multiple inflammatory pathways. The loss of control of inflammation and the malfunction of the regulatory mechanisms during infection may lead to a life-threatening sepsis syndrome and septic shock with an extraordinary high mortality rate (up to 60%). Besides, TLR4- and caspase-4 mediated signaling plays a key role in the pathogenesis of numerous chronic and acute inflammatory disorders such as asthma, arthritis, allergy, influenza, cardiovascular disorders and cancer, which highlights the importance of TLR4 complex and caspase-4/11 as promising therapeutic targets.
The endotoxicity of LPS resides in its terminal membrane-bound motif glycophospholipid Lipid A. Lipid A binds directly to the TLR4/MD-2 complex and to the Caspase Activation and Recruitment Domain (CARD) of caspase-4/11 which prompts immune activation. Lipid A and the mode of its interaction with the proteins of innate immune system represent an attractive target for development of anti-inflammatory (for sepsis, asthma, arthritis and influenza) and immuno-modulatory (for infection and cancer) therapeutics. Besides, TLR4 activation potentiates both the innate and adaptive immune responses which highlight an application of TLR4 agonists as adjuvants for vaccine formulations aimed at infection and cancer which require both humoral and Th1-biased immunity.Using crystal structure-based design we develop glycan-based TLR4 and caspase-4/11 ligands as potential immuno-modulators and vaccine adjuvants and versatile probes for studying LPS-protein interactions.
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Synthetic Glycans for Immunobiology and Therapeutic Immunomodulation
The major research interests are in the fields of organic chemistry (bioorganic chemistry, synthesis of complex biomolecules) and chemical biology (inflammation, infection, immune response and vaccines). We exploit the merits of advanced bioorganic chemistry and “chemical” understanding of biological processes for a breakthrough in the field of chemical biology. One of current topics focuses on the exploration of the structural and molecular basis of the host innate immune response to Gram-negative infection. Lipopolysaccharide (LPS) counts to the foremost virulence factors of Gram-negative bacteria. The endotoxic portion of LPS, a glycophospholipid Lipid A, triggers and supports the activation of the innate immune response by binding to the transmembrane Toll-like Receptor 4 (TLR4) complex and to the intracellular protease caspase-4 (caspase-11 in mice). Design and chemical synthesis of glycan-based TLR4- and caspase-4/11 specific ligands of natural and unnatural origin, exploration of their immuno-modulatory potential and specificity/affinity for diverse receptors of the innate immune system allows for advanced understanding of ligand-receptor interactions and holds potential for drug discovery and therapeutic benefit.
We are developing methodologies towards stereoselective synthesis of binary glycosyl phosphodiesters corresponding to partial structures of LPS and Lipid A from Burkholderia, Pseudomonas, Francisella and Bordetella species. Modification of the Lipid A phosphate groups by positively charged appendages is a part of survival strategy of numerous opportunistic Gram-negative bacteria. The phosphate groups of cystic fibrosis (CF)-adapted Burkholderia LPS are abundantly esterified by 4-amino-4-deoxy-β-L-arabinose (β-L-Ara4N), which imposes resistance to endogeneus cationic antimicrobial peptides (CAMPs) and to antibiotic treatment and largely contributes to bacterial virulence. To establish structural basis for the unique pro-inflammatory activity of Burkholderia LPS we synthesised Lipid A modified by β-L-Ara4N at the anomeric phosphate group and its Ara4N-free counterpart. We have shown that Ara4N modification at the glycosidically linked phosphate group of Burkholderia Lipid A contributes to significant enhancement of induction of the pro-inflammatory signaling by otherwise inactive pentaacyl Lipid A. This finding is of importance for the understanding of virulence of CF-adapted BCC species and for the development of advanced cystic fibrosis - related therapeutics. Additionally, trisaccharide-based neoglycoconjugates containing a conserved epitope βGlcN(1→6)αGlcN(1→P←1)β-L-Ara4N characteristic to CF adapted Burkholderia cepacia/cenocepacia and Pseudomonas aeruginosa species as well as an epitope βGlcN(1→6)αGlcN-(1→P←1)αGalN typical for most virulent Francisella strains were synthetically prepared. These neoglycoconjugates can be applied for generation of specific monoclonal antibodies (mAbs) which could be used in diagnostic assays for rapid antigen determination in clinical samples and for screening of not-yet-identified Ara4N producing mutants.
LPS biosynthesis pathway is a complex multistep process which involves many metabolites and small molecules. And just quite recently it became clear that several heptose derivatives swiped from the LPS biosynthesis pathway also represent PAMPs which are directly recognized by the innate immune system of the host. Using our synthetic ADP-Hep the group headed by Prof. Feng Shao identified ADP-β-D-Heptose as a substrate for bacterial autotransporter heptosyltransferase (BAHT) family and showed that ADP-L-β-D-Heptose can activate the NF-κB signalling in the cytosol of host immune cells by binding to alpha-kinase 1 (ALPK 1). D-β-D-Heptose 1,7-diphosphate gets transformed into the ADP-activated sugar by hosts adenylyltransferases and can also bind to ALPK1 though with lower affinity.
Chemical synthesis of the LPS partial structures, nucleotide-activated sugars and LPS biosynthetic precursors such as ADP-heptose and bacterial D-glycero-β-D-manno-heptose-1,7-bisphosphate (HBP) enabled a breakthrough in the exploration of LPS biosynthesis, identification of novel pathogen-associated molecular patterns involved in the host-pathogen interactions and underlying pro-inflammatory signaling pathways.
Synthetic glycolipids for studying inflammation and sepsis
Therapeutic immunomodulation through engagement of Pattern Recognition Receptors (PRR) is a promising strategy for treatment of inflammatory disorders ranging from chronic inflammation such as autoimmune disease, allergy and asthma to acute inflammatory conditions such as antibiotic-resistant infections and sepsis syndrome. Activation of the innate immune response by Gram-negative bacterial lipopolysaccharide (LPS) liberated into the blood circulation of the host results in triggering of the intracellular signaling pathways and production of the pro-inflammatory mediators which generally promotes recovery from infection. LPS-sensing receptors - transmembrane Toll-like Receptor 4/Myeloid Differentiation factor-2 complex (TLR4/MD-2) and cytosolic protease caspase-4 (caspase-11 in mice) which protect the host from bacterial assault are also responsible for the initiation and pathogenesis of sepsis syndrome. Sepsis is increasingly reputed as a common pathway to death from infection and remains the leading cause of mortality in intensive care units. In a view of the globally expanding antibiotic resistance, the incidence of sepsis is anticipated to rise. Besides, activation of TLR4 contributes to the pathogenesis of numerous inflammatory auto-immune and chronic diseases such as asthma, arthritis and cancer, which highlights the importance of TLR4 as therapeutic target.
The objective of the project is to design, synthesize and biologically evaluate a new class of TLR4- and caspase-4/11 – specific anti-inflammatory glycolipids (Disaccharide Lipid A Mimetics, DLAMs). DLAMs are rationally designed to exhibit pico- to nanomolar affinity for TLR4/MD-2 complex and to bind to Caspase Activation and Recruitment Domain (CARD) of caspase-4/11. DLAMs antagonist can displace LPS from the binding site on receptor protein, and thus block the LPS-induced endotoxicity. Specific chemical modifications of the basic antagonist structure will provide partial agonists at TLR4 and caspase-4/11. Partial agonists will compete with LPS for binding to respective receptor proteins, thereby minimizing the induction of pro-inflammatory signaling induced by LPS. At the same time, exposure of TLR4 complex to partial agonists will ensure a constant, weak level of immune activation, hampering in this way the lethal sepsis-induced immunosuppression and endotoxin tolerance. With the aim to get a straightforward access to libraries of DLAMs derived from a single disaccharide scaffold, a diversity oriented synthesis (DOS) of both antagonist and partial agonist structures having variable functional groups will be developed.
Synthetic glycans for exploration of lipopolysaccharide (LPS) recognition by the proteins of innate immune system
Activation of Toll-like Receptor 4 (TLR4) proceeds through the binding of lipopolysaccharide (LPS or Endotoxin) to the central hydrophobic pocket of the co-receptor protein Myeloid Differentiation factor 2 (MD-2), followed by dimerization of two ternary ligand-receptor complexes [TLR4·MD-2·LPS]2. LPS of different bacterial species induce dissimilar degree of the receptor complex dimerization which regulates the ensuing induction of pro-inflammatory signaling. To explore the molecular basis for LPS – induced activation of innate immune signaling pathways we design and chemically synthesize unnatural sugar-derived LPS mimetics.
Our recent findings based on the immuno-functional studies of synthetic Endotoxin mimetics derived from β(1↔1)α and α(1↔1)α linked disaccharide scaffolds disclose that the 3D-molecular shape and the ternary structure of the MD-2 – bound diglucosamine backbone of Lipid A belongs to the most decisive determinants of endotoxicity.
We perform synthetic studies toward stereoselective assembly of the nonreducing α,α- β,α- and β,β-1,1'-linked orthogonally protected disaccharides under simultaneous stereocontrol at two anomeric centers. The disaccharide scaffolds are synthetically decorated by diverse functional groups to afford biologically active Endotoxin mimetics as tools for immunobiology and valuable probes for studying LPS-protein interactions.
Prof. Holger Heine - 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
Prof. Rudi Beyaert - Department for Biomedical Molecular Biology, Unit of Molecular Signal Transduction in Inflammation, Ghent University, Center for Inflammation Research, VIB, Ghent, Belgium
Prof. Feng Shao - National Institute of Biological Sciences, Zhongguancun Life Science Park, Beijing, PR China
Prof. Roman Jerala - Department of Biotechnology, University of Ljubljana, Ljubljana, Slovenia
Prof. Chris Oostenbrink - Institute of Molecular Modelling and Simulation, University of Natural Resources and Life Sciences, Vienna, Austria
Dr. Simon Ittig - Biozentrum University of Basel, Basel, Switzerland
Prof. Johannes Stöckl - Institute of Immunology, Medical University of Vienna, Vienna, Austria
Prof. Carsten Kirschning - Institute of Medical Microbiology, University Clinic Essen, University of Duisburg-Essen, Essen, Germany
Dr. Peter van der Ley - Intravacc, Bilthoven, the Netherlands
Prof. Herbert Strobl – Medical University of Graz, Otto Loewi Research Center for Vascular Biology, Immunology and Inflammation, Graz, Austria
Prof. Pavel Kovarik - Max F. Perutz Laboratories, University of Vienna, Vienna Biocenter (VBC), Vienna, Austria
Prof. Jerrold Weiss - Inflammation Program and Department of Microbiology, University of Iowa, Iowa City, IA, USA
Dr. Jose Antonio Garate - Centro Interdisciplinario de Neurociencia de Valparaíso (CINV), Universidad de Valparaíso, Valparaíso, Chile
Prof. Jesús Jimnéz-Barbero - Chemical and Physical Biology, CIB-CSIC, Madrid, Spain
Prof. Daniel Starczynowski - Cincinnati Children's Hospital Medical Center, Experimental Hematology & Cancer Biology, Cincinnati, OH, USA
Prof. Cécile Arrieumerlou - INSERM, Institut Cochin, Paris, France
Prof. Göran Widmalm - Department of Organic Chemistry, Arrhenius Laboratory, Stockholm University, Stockholm, Sweden