Final Activity Report Summary - GANGLIOMIM (Design and synthesis of glycosidase-stable ganglioside mimics as cholera toxin ligands)
Specific binding between bacterial enterotoxins and oligosaccharides on the host cell membrane is a paradigm for protein-sugar interaction. One of the best characterized recognition pairs is formed by ganglioside GM1 and the cholera toxin (CT). Glycosidase stable and synthetically accessible ganglioside mimics that behave as high-affinity ligands for CT may lead to drugs capable of blocking the interaction between the toxin and target cells, thus preventing the onset of the disease.
Previously reported artificial CT ligands suffer of complex synthesis and/or expected instability towards hydrolytic enzymes (glycosidases), which make them poor candidates for potential medicinal applications. The present project was aimed to the design and synthesis of glycosidase-stable mimics of the GM1 ganglioside as efficient and selective CT inhibitors.
As a general strategy, the two pharmacophoric fragments of GM1, the monosaccharides sialic acid and galactose, were connected to each other through specially designed linkers using non-glycosidic bonds. The design of the linker was computer-aided and typically all linker candidates were submitted to initial docking tests to assess their applicability. The advantage of using non-glycosidic bonds is two-fold: it allows to simplify the synthetic sequence and it yields molecules which are resistant to enzyme-catalysed hydrolysis.
A first set of molecules built from three families of linear mimics varying in the galactose part of the ligand were analysed by Weak Affinity Chromatography (WAC) and allowed to draw some interesting indications about structure-activity relationships. This part of the work afforded an initial scaffold which was optimized by introduction of an additional (lypophilic) sidechain onto the linker backbone, which could enhance affinity through complementary (hydrophobic) interactions inside the CT binding pocket.
The latter series of compounds demonstrated higher activity, with some ligands arriving at retention times (measured by WAC) that corresponded to Kd values 2.3-2.8 times lower than that of the reference compound (O-meta-nitrophenyl-galactoside, MNPG), thus pointing to the lower end of micromolar affinity scale (<100 microM).
Hence, the approach has already led to compounds significantly more active than reference MNPG, which are at the same time likely to be stable in vivo (devoid from O-glycosidic bonds) and much easier to synthesise then previously reported high affinity mimics closely resembling natural ligand GM1 structure.
Finally, pursuing the long-term goal of deeper understanding of the CT-inhibitor binding process, a collaboration with Prof. Ute Krengel from the University of Oslo was established recently through COST D34 workgroup meetings, with the objective to run co-crystallisation experiments of the most active ligands with CT. The experiments are in progress at the moment.
Previously reported artificial CT ligands suffer of complex synthesis and/or expected instability towards hydrolytic enzymes (glycosidases), which make them poor candidates for potential medicinal applications. The present project was aimed to the design and synthesis of glycosidase-stable mimics of the GM1 ganglioside as efficient and selective CT inhibitors.
As a general strategy, the two pharmacophoric fragments of GM1, the monosaccharides sialic acid and galactose, were connected to each other through specially designed linkers using non-glycosidic bonds. The design of the linker was computer-aided and typically all linker candidates were submitted to initial docking tests to assess their applicability. The advantage of using non-glycosidic bonds is two-fold: it allows to simplify the synthetic sequence and it yields molecules which are resistant to enzyme-catalysed hydrolysis.
A first set of molecules built from three families of linear mimics varying in the galactose part of the ligand were analysed by Weak Affinity Chromatography (WAC) and allowed to draw some interesting indications about structure-activity relationships. This part of the work afforded an initial scaffold which was optimized by introduction of an additional (lypophilic) sidechain onto the linker backbone, which could enhance affinity through complementary (hydrophobic) interactions inside the CT binding pocket.
The latter series of compounds demonstrated higher activity, with some ligands arriving at retention times (measured by WAC) that corresponded to Kd values 2.3-2.8 times lower than that of the reference compound (O-meta-nitrophenyl-galactoside, MNPG), thus pointing to the lower end of micromolar affinity scale (<100 microM).
Hence, the approach has already led to compounds significantly more active than reference MNPG, which are at the same time likely to be stable in vivo (devoid from O-glycosidic bonds) and much easier to synthesise then previously reported high affinity mimics closely resembling natural ligand GM1 structure.
Finally, pursuing the long-term goal of deeper understanding of the CT-inhibitor binding process, a collaboration with Prof. Ute Krengel from the University of Oslo was established recently through COST D34 workgroup meetings, with the objective to run co-crystallisation experiments of the most active ligands with CT. The experiments are in progress at the moment.