Design, synthesis and evaluation of bacterial sialic acid uptake inhibitors

Antibiotic resistance is a major threat for our society and finding novel antibacterial therapies is of greatimportance. 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 forpathogenic bacteria, since it is used as a source of carbon and in a immuno-evasive mechanism called “molecularmimicry”. Disrupting genes involved in bacterial sialic acid catabolism and transport has been proven to alter thebacterial 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 ourinvestigations. 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 bestcompounds 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, inparticular the substrate binding proteins (SBP) from the ATP-binding cassette (ABC) and tripartite ATPindipendentperiplasmic (TRAP) transporter families. NanoDSF and ITC were again employed as methods toevaluate the previously developed compound library and establish structure activity relationships for the newtargets. We identified C9 and, potentially, O4 as promising sites of derivatisation for broad spectrum bacterialsialic acid uptake inhibitors.To follow on the promising leads obtained from the 4-O-benzyl series, we designed a new library of derivativesbearing 4-N-piperidine and piperazine as spacers between C4 and the aromatic moiety. Clear trends wereobserved when adding either electron-withdrawing or donating groups, with the former significantly enhancingaffinity.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 theintroduction of nitrogen, sulfur and carbon nucleophiles in a single step, with retention of the configuration, withoutthe requirement for special conditions. With this procedure, the scope of C4 functionalisations is greatly expandedto 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 enabledthe identification of promising leads and methodologies to be used in the future to develop this class ofcompounds as novel antibacterial drugs