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The C-type lectin receptor DC-SIGN is a pattern recognition receptor expressed on macrophages and dendritic cells. It has been identified as a promiscuous entry receptor for many pathogens, including epidemic and pandemic viruses such as SARS-CoV-2, Ebola virus, and HIV-1. In the context of the recent SARS-CoV-2 pandemic, DC-SIGN-mediated virus dissemination and stimulation of innate immune responses has been implicated as a potential factor in the development of severe COVID-19. Inhibition of virus binding to DC-SIGN, thus, represents an attractive host-directed strategy to attenuate overshooting innate immune responses and prevent the progression of the disease. In this study, we report on the discovery of a new class of potent glycomimetic DC-SIGN antagonists from a focused library of triazole-based mannose analogues. Structure-based optimization of an initial screening hit yielded a glycomimetic ligand with a more than 100-fold improved binding affinity compared to methyl ��-d-mannopyranoside. Analysis of binding thermodynamics revealed an enthalpy-driven improvement of binding affinity that was enabled by hydrophobic interactions with a loop region adjacent to the binding site and displacement of a conserved water molecule. The identified ligand was employed for the synthesis of multivalent glycopolymers that were able to inhibit SARS-CoV-2 spike glycoprotein binding to DC-SIGN-expressing cells, as well as DC-SIGN-mediated trans-infection of ACE2+ cells by SARS-CoV-2 spike protein-expressing viruses, in nanomolar concentrations. The identified glycomimetic ligands reported here open promising perspectives for the development of highly potent and fully selective DC-SIGN-targeted therapeutics for a broad spectrum of viral infections.

Siglec-8 is a promising target for the treatment of eosinophil- and mast cell-associated diseases such as asthma. Based on pharmacophore analysis of 6’-sulfo-sialyl Lewisx, a specific Siglec-8 ligand identified in glycan array screening, potent glycomimetics were developed. While maintaining the neuraminic acid core, a carbocyclic replacement of the Gal moiety and the introduction of a sulfonamide led to a 20-fold improvement of affinity.

Antibiotic resistance is a major threat for our society and finding novel antibacterial therapies is of great importance. In this thesis, we investigate bacterial sialic acid uptake inhibitors as a novel antibacterial approach. Bacteria do not biosynthesise sialic acid and therefore harvest it from the host. Sialic acid plays a crucial role for pathogenic bacteria, since it is used as a source of carbon and in a immuno-evasive mechanism called “molecular mimicry”. Disrupting genes involved in bacterial sialic acid catabolism and transport has been proven to alter the bacterial growth and infectivity in vivo. We identified the SiaT transporter, from the sodium solute transporter (SSS) family, from Proteus mirabilis (PmSiaT) and Staphylococcus aureus (SaSiaT), as starting targets for our investigations. Firstly, a library of sialic acid derivatives with single modifications at O4, N5 and C9 was designed, synthesised and tested on the two targets with a thermal shift assay called nano differential scanning fluorimetry (nanoDSF). Subsequentially, the most promising hits were investigated with isothermal titration calorimetry (ITC), and proteoliposome and bacterial growth assays. Our best compound, with a 3,5-dibromobenzyl substituent at O4, showed mid-nanomolar affinity, a 185-fold increase for PmSiaT compared to the natural substrate. The best compounds block sialic acid uptake with a competitive mode of action and delay bacterial growth in the case of S. aureus. With these initial promising results, we focussed on targeting multiple bacterial sialic acid transporter families, in particular the substrate binding proteins (SBP) from the ATP-binding cassette (ABC) and tripartite ATP-indipendent periplasmic (TRAP) transporter families. NanoDSF and ITC were again employed as methods to evaluate the previously developed compound library and establish structure activity relationships for the new targets. We identified C9 and, potentially, O4 as promising sites of derivatisation for broad spectrum bacterial sialic acid uptake inhibitors. To follow on the promising leads obtained from the 4-O-benzyl series, we designed a new library of derivatives bearing 4-N-piperidine and piperazine as spacers between C4 and the aromatic moiety. Clear trends were observed when adding either electron-withdrawing or donating groups, with the former significantly enhancing affinity. Lastly, we developed a new methodology to functionalise C4 of sialic acid, starting from methyl (5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-D-galacto-2-nonulopyranosid)onate. The procedure allows for the introduction of nitrogen, sulfur and carbon nucleophiles in a single step, with retention of the configuration, without the requirement for special conditions. With this procedure, the scope of C4 functionalisations is greatly expanded to sustain future sialic acid centered drug discovery and chemical biology investigations. The results of this thesis represent the first examples of bacterial sialic acid uptake inhibition. Our efforts enabled the identification of promising leads and methodologies to be used in the future to develop this class of compounds as novel antibacterial drugs.