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Protein toxins produced by bacteria are the cause of many life-threatening diarrheal diseases. Many of these toxins, including cholera toxin (CT), enter the cell by first binding to glycolipids in the cell membrane. Inhibiting these multivalent protein/carbohydrate interactions would prevent the toxin from entering cells and causing diarrhea. Here we demonstrate that the site-specific modification of a protein scaffold, which is perfectly matched in both size and valency to the target toxin, provides a convenient route to an effective multivalent inhibitor. The resulting pentavalent neoglycoprotein displays an inhibition potency (IC50) of 104 pM for the CT B-subunit (CTB), which is the most potent pentavalent inhibitor for this target reported thus far. Complexation of the inhibitor and CTB resulted in a protein heterodimer. This inhibition strategy can potentially be applied to many multivalent receptors and also opens up new possibilities for protein assembly strategies., (© 2014 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.)

Cholera toxin (CT), the causative agent of cholera, displays a pentavalent binding domain that targets the oligosaccharide of ganglioside GM1 (GM1os) on the periphery of human abdominal epithelial cells. Here, we report the first GM1os-based CT inhibitor that matches the valency of the CT binding domain (CTB). This pentavalent inhibitor contains five GM1os moieties linked to a calix[5]arene scaffold. When evaluated by an inhibition assay, it achieved a picomolar inhibition potency (IC50 = 450 pM) for CTB. This represents a significant multivalency effect, with a relative inhibitory potency of 100,000 compared to a monovalent GM1os derivative, making GM1os-calix[5]arene one of the most potent known CTB inhibitors.

Eight symmetric and pentavalent corannulene derivatives were functionalized with galactose and the ganglioside GM1-oligosaccharide (GM1os) via copper-catalyzed alkyne-azide cycloaddition (CuAAC) reactions. The compounds were evaluated for their ability to inhibit the binding of the pentavalent cholera toxin to its natural ligand, ganglioside GM1. In this assay, all ganglioside GM1os-sym-corannulenes proved to be highly potent nanomolar inhibitors of cholera toxin.