Enhancing the therapeutic value of synthetic carbohydrate polymers against chronic Escherichia coli infections in Crohn’s disease by the interplay with glycosylation dynamics on host cell surfaces – STARLET

A library of well-defined glycopolymers with homo- or heterocompositions, topologies, different architectures and a variation of densities, being either gradient densities or randomly spread, has been created from our reference building block, heptyl a-D-mannose. Other monomeric building blocks tuneable towards the most specific and highest affinity interactions with the FimH adhesin, are being conceptualized.

Our efforts are dedicated to the understanding of heptyl a-D-mannose polymer/adhesion interactions through advanced studies of the shape and form of the complexes in solution and of their antagonistic potencies in ELISA. Moreover, during the first months of the ANR project, we have developed a synthetic methodology to build anomeric heterocycles (pyrimidines and thiazoles) on mannose sugar. This chemical procedure provided access to a new class of FimH inhibitors not reachable by conventional glycosylation reactions. As other sugars can be functionalized by such protocols, this cyclization strategy may not be restricted to the design of FimH inhibitors, and is probably applicable to designing sugar antagonists of other biologically relevant lectins. In the present case, we identified a hit compound interacting strongly with FimH. A small library of analogues showing nanomolar affinity for FimH was then developed based on the co-crystallisation obtained with hit compound 1 and FimH. Lead compound 2 showed very high in vitro and ex vivo anti-adhesive effects towards the AIEC bacteria (In Press in J. Med. Chem.).

We hypothesized that the relatively low stability in acidic media was due to the anomeric amino group. Because anomerisation as a consequence may be problematic for oral treatment, the O and S analogues of 1 and 2 have been designed. The chemistry was entirely revisited and the thiazole was first functionnalized before the grafting by glycosylation to the mannoside. We recently obtained the O-analogue of the hit compound by this procedure.

By identifying factors of the host-pathogen interplay in bacterial infections through glycan structural analysis, we hope to generate original anti-bacterial agents that do not evoke resistance in the long run, but rather clear the unwanted bacteria effectively within a single shot or upon easy-manageable oral uptake. The soft approach of clearance, not killing, of the unwanted bacteria would allow the organism to recover from acute and long-standing E. coli-associated infections such as Crohn’s disease.

(2013) Thiazolylaminomannosides as potent anti-adhesives of type 1 piliated Escherichia coli isolated from Crohn’s disease patients Sami Brument, Adeline Sivignon, Tetiana I. Dumych, Nicolas Moreau, Goedele Roos, Yann Guérardel, Thibaut Chalopin, David Deniaud, Rostyslav O. Bilyy, Arlette Darfeuille-Michaud, Julie Bouckaert, Sébastien G. Gouin J. Med. Chem. See illustration below

(2013) Structural Sampling of Glycan Interaction Profiles Reveals Mucosal Receptors for Fimbrial Adhesins of Enterotoxigenic Escherichia coli Emanuela Lonardi, Kristof Moonens, Lieven Buts, Arjen R. de Boer, Johan D. M. Olsson, Manfred S. Weiss, Emeline Fabre, Yann Guérardel, André M. Deelder, Stefan Oscarson, Manfred Wuhrer and Julie Bouckaert. Biology, 2, In Press

(2013) Heptyl a-D-Mannosides Grafted on a ß-Cyclodextrin Core to Interfere with Escherichia coli Adhesion: An In Vivo Multivalent Effect. Bouckaert J, Li Z, Xavier C, Almant M, Caveliers V, Lahoutte T, Weeks SD, Kovensky J, Gouin SG. Chemistry, a European J. 19(24):7847-55.

DI MANNOSE DERIVATIVES, A PROCESS FOR PREPARING THE SAME AND THEIR USES AS A DRUG

Escherichia coli adhesion to epithelial linings in the human body and mediated by the type- fimbrial adhesin FimH can be inhibited using mannosidic substances, with affinity of up to 5 nM for a small monomeric compound heptyl a-D-mannose. Remarkably, bacterial adhesion as the first step in infection is only to specific cells of the epithelial linings and is clearly enhanced in patients with metabolic diseases such as diabetes mellitus (in the urinary tract) and inflammatory bowel diseases (in the gut).
We state that we can only make the best inhibitors of chronic E. coli infections if we understand the biological basis that optimizes the host-pathogen interactions. Concerning the “pathogenic cascade” of uropathogenic E. coli or the chronic inflammation of the intestine in ileal Crohn’s disease involving adherent-invasive E. coli (AIEC), both with intracellular survival properties, there are strong indications that the determining factor towards bacterial invasion of host cells lies in their expression of multiple high-affinity high-mannose glycan receptors. This erroneous glycosylation status of the cells is linked to common metabolic diseases states. The glycosylation pattern in the gut of Crohn’s disease patients is a major key in abnormal E. coli colonization process.
Anti-adhesives are to be further explored, because despite monovalent inhibitor such as heptyl a-D-mannose being very effective in binding the FimH adhesin, the concentrations needed to prevent infection may seem relatively high. The quasi-independence of high-mannose glycan recognition by FimH on the E. coli pathotype and on the amino acid variation within FimH, compared to the large effect of receptor glycan structure and multiplicity on the adhesion event, are indicative that refined multivalent polymannosidic compounds with the biological glycans taken as lead structures will be effective and will not be restricted to a small range of E. coli strains. We commence to apply these novel principles in our search for therapeutics to treat chronic E. coli infections and thereby we make use of the similarities in pathogenesis by UPEC in the urinary tract and by adherent-invasive E. coli (AIEC) in the gut.
The objectives are to create the most powerful, polymeric mannosidic inhibitors of type-1 fimbrial mediated adhesion of E. coli. This we envisage by carefully planning a continuous feedback between innovative chemical design, biochemical and biophysical analyses of multivalency and forces and biological studies, including validation of the therapeutic compounds. Originality and novelty lie in the chemistry, by the use of heterocycles and by creating RAFT polymers with novel architectures and gradient densities. The molecular principles behind multivalent interactions in a number of ways such as calorimetry, SAXS-DLS and AFM will be explored. The glycan receptor structures and glycosylation multiplicity, or density, as determining factors in the chronic E. coli infections will be determined, and the dynamics of the glycan structures on intestinal glycan receptors, due to inflammation or bacterial adhesion, evaluated. Finally, the polymeric mannosidic inhibitors, designed on the molecular basis of the glycan structures found, will be tested for their therapeutic value in the CEABAC10 transgenic mouse model of Crohn’s disease.