Discovery of inhibitors of the bacterial lipopolysaccharide as anti-virulence agents

Résumé

Lipopolysaccharide (LPS) is a glycolipid pivotal virulence factor of Gram-negative bacteria. Its inhibition at the oligosaccharide core level generate bacteria with a truncated LPS that are much less virulent and more sensitive towards antibiotics. Thus, the inhibition of bacterial heptose, which is the first sugar unit that link Kdo2-lipid A moiety and core oligosaccharide, represents an attractive target to discover anti-virulence agents. Novel inhibitors of the enzymes that are involved in the biosynthesis of ADP-heptose, the precursor of LPS heptose, are the main topic of this thesis.
In this project, the bifunctional D-β-D-heptose phosphate kinase/D-β-D-heptose 1-phosphate adenyltransferase (HldE) and heptosyltransferase (WaaC) were successfully overexpressed and the enzymatic characterizations with the natural substrates were also carried out thanks to the chemical synthesis of these compounds in our laboratory. Then we developed robust WaaC and HldE activity assays based on spectrophotometric or HPLC technologies from the literature.
In this thesis, we mainly focused on the understanding of WaaC catalytic mechanism and the identification of novel WaaC inhibitors. Several series of compounds, such as heavily fluorinated heptoside, multivalent glycoclusters, ADP-heptose analogues, natural products and drug-like molecules were screened against WaaC. The studies of ADP-heptose analogues help to understand the binding properties of WaaC and ADP-heptose. We found that both the sugar and ADP moieties are important for WaaC catalytic process. More interestingly, novel inhibitors with nano molar inhibitory level were discovered from libraries of multivalent glucosylfullerene and drug-like molecules, an inhibition level rarely observed with glycosyltransferases. Moreover, it is the first time that natural products have been identified as WaaC inhibitors and especially flavonoids derivatives with low micro molar IC50.
It is also worth to mention that, a novel LPS biosynthesis inhibition assay was developed on Yersinia enterocolitica O:9 with a specific oligosaccharide antibody. This assay based on a flow cytometry technology and allows for the quantification of truncated LPS or whole cells. The tetrafluoro-heptosides were found to have significant inhibition of the LPS biosynthesis with dose-dependent effect. This assay was also applied to the most potent WaaC inhibitors (natural products and drug like molecules). The best inhibitor is able to inhibit 40% LPS biosynthesis at a dose of 100 μg/mL.

la date de réponse	2 oct. 2017
langue originale	Anglais
L'institution diplômante	Universite de Namur
Sponsors	China Scholarship Council
Superviseur	Stephane Vincent (Promoteur), Moreno Galleni (Jury), Xavier De Bolle (Jury), Weidong Pan (Jury) & Caroline Stévigny (Jury)