Chemical Biology of Carbohydrates

Zoonotic viruses can rapidly emerge from animals and transmit to humans as has been well documented for various avian/porcine influenza and bat corona viruses. It has now been observed repeatedly that these animal-to-human transmissions can result in a pandemic causing millions of deaths and a massive economic burden for humanity. All those pandemic viruses and numerous other classes of human viruses exploit animal and human sialic acid receptors for transmission and infection. Therefore, viral entry inhibitors interfering with this process are of broad interest. In this early discovery project, the development of novel sialomimetic therapeutics will be established. After initial validation using known viruses with known and distinct sialic acid binding specificity, the platform will then be applied onto viruses with reported but unexplored sialic acid binding, i.e. Parainfluenza, Zika and BK Polyoma Viruses, all of which currently lack drugs or vaccines. This pipeline will therefore yield sialomimetic entry inhibitors for the treatment of viral infections with clinical need, and importantly, the established workflow will serve as an important platform in the scope of Pandemic Preparedness.

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The increase of antimicrobial resistance is a major threat for individual and public health. Consequently, new weapons to fight the emerging multidrug-resistant bacteria are quickly required. The ESKAPE pathogens are at the frontline of these problematic life-threatening drug resistant nosocomial pathogens. In this project, we aim to overcome antimicrobial resistance by hijacking energy-driven carbohydrate uptake systems (PTS and ABC transporters) to actively pump carbohydrate-antibiotic conjugates into bacteria which will be liberated by cellular glycosidases. These transporters are able to accumulate mM intracellular concentrations of their substrates. Exploring if they will boost intracellular concentrations of the carbohydrate-antibiotic conjugates as well is the main objective of the CO-TEAM project. Our preliminary work shows that the expression of the transporters of these carbohydrates is significantly induced under infection conditions. The project is divided into 4 tasks. 4 different oligosaccharide conjugates carrying 6 different antibiotics from 4 different classes will be chemically synthesized. These conjugates will then be studied for antimicrobial efficacy and resistance development against a panel of strains with a focus on the most urgent ESKAPE pathogens. In addition, mechanistic analyses to identify the transporters importing the conjugates will be conducted in 2 Gram-positive and 2 Gram-negative pathogens. Finally, we will evaluate the most promising conjugates in an insect and mouse model. Boosting intracellular antibiotic concentrations through active import is expected to overcome antimicrobial resistance mechanisms, both, target modifications lowering the antibiotic's efficacy, also permeability affecting resistance, the major problem in Gram-negative bacteria.

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Bacterial infections are now a global threat demanding novel treatments due to the appearance of resistances against antibiotics at a high pace. The ESKAPE pathogens are those with highest importance in the EU and chronic infections due to biofilm formation are a particular task. Noninvasive pathogen-specific imaging of the infected tissue is not clinically available. Its successful implementation will enable the choice of appropriate therapy and boost efficacy. Furthermore,
Gram-negative bacteria have a highly protective cellular envelope as an important resistance mechanism for drugs acting intracellularly, resulting in an alarmingly empty drug-pipeline.
To overcome this gap, I will establish Lectin-directed Theranostics targeting pathogens via their extracellular carbohydrate-binding proteins at the site of infection for specific imaging and treatment. This will be implemented for the highly resistant ESKAPE pathogen Pseudomonas aeruginosa through 3 different work packages.
WP1 Sweet Imaging: Design & conjugation of lectin-directed ligands to imaging probes, Optimization of ligand/linker, in vivo proof-of-concept imaging study.
WP2 Sweet Targeting: Delivery of antibiotics to the infection through covalent linking of lectindirecting groups. Employing different antibiotics, assessment of bactericidal potency and targeting efficiency. Manufacturing of nano-carriers with surface exposed lectin-directed ligands, noncovalent charging with antibiotics. In vitro and in vivo targeting.
WP3 Sweet SMART Targeting: Conjugates as SMART drugs: specific release of anti-biofilm lectin inhibitor and drug cargo upon contact with pathogen, development of linkers cleavable by pathogenic enzymes.
SWEETBULLETS will establish fundamentally novel lectin-directed theranostics to fight these deleterious infections and provide relief to nosocomially infected and cystic fibrosis patients. It is rapidly extendable towards other ESKAPE pathogens, e.g. Klebsiella spp..

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Protein-carbohydrate interactions play a key role in the first step of numerous biological processes, e.g. fertilization and tissue homing of immune cells, but also in infection, inflammation, migration of tumor cells and other pathologies. Pathogenic microorganisms (viruses, bacteria, fungi and parasites) have developed strategies for utilizing glycan epitopes on human tissues for specific recognition, for adhesion and sometimes for cellular internalization. The proteins involved can be viral capsid domains, adhesins on top of pili, soluble lectins or carbohydrate binding domains of enzymes or toxins. Their carbohydrate-binding sites are specific for glycans located on human epithelia such as the histo/blood group oligosaccharides of ABO and Lewis systems. Molecules that could interfere with such lectin/glycan interaction are therefore of high interest as anti-infectious agents. Similarly human lectins are involved in chronic diseases related to inflammation and cancer, and consequently the search for lectin inhibitors to interfere with these pathological conditions is of outstanding interest. The project aims at the development of the underexplored area of non-carbohydrate drug-like lectin inhibitors. Such glycomimetics could overcome current limitations of carbohydrate-based therapeutics such as pharmacokinetic and pharmacodynamic limitations as well as synthetic tractability. Targeting three lectins from opportunistic bacteria, the consortium of two French and two German laboratories proposes to combine virtual and in vitro screening of chemical libraries using functional assays and NMR approaches, followed by structure (X-ray, NMR), thermodynamics (ITC) and kinetics (SPR, MD) guided lead optimization. Assembling of lead structures into oligomeric compounds in order to use natural lectin multivalency to gain very high affinity will be also envisaged. This project aims at the establishment of a broadly applicable methodology for the development of non-carbohydrate lectin inhibitors. A set of three diverse and representative bacterial lectins was chosen to explore the potential of the discovery pipeline. However, this approach will not be limited to bacterial targets and other viral, fungal, or human lectins of high therapeutic significance can be targeted as well. While pipeline development is our major concern, generating novel anti-infectives is foreseen (i.e. a problem-solving endeavor). Moreover, this endeavor will contribute to our fundamental understanding of biological processes involving multivalent receptors such as membrane dynamics and lipid raft-mediated internalization (i.e. a curiosity driven research).

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