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Developing tools for the study of protein carbohydrate interactions is an important goal in glycobiology. Cholera toxin inhibition is an interesting target in this context, as its inhibition may help the fight against cholera. For the study of novel ligands an affinity capillary electrophoresis (ACE) method was optimised and applied. The method uses unlabeled cholera toxin B-subunit (CTB) and unlabeled carbohydrate ligands based on ganglioside GM1-oligosaccharides (GM1os). In an optimized method at pH 4, adsorption of the protein to the capillary walls was prevented by a polybrene-dextran sulfate-polybrene (PB-DS-PB) coating. Different concentrations of the ligands were added to the background electrolyte. CTB binding was observed by a mobility shift that could be used for dissociation constant (Kd) determination. The Kd values of two GM1 derivatives differed by close to an order of magnitude (600 ± 20 nM and 90 ± 50 nM) which was in good agreement with the differences in their reported nanomolar IC50 values of an ELISA-type assay. Moreover, the selectivity of GM1os towards CTB was demonstrated using Influenza hemagglutinin (H5) as a binding competitor. The developed method can be an important platform for preclinical development of drugs targeting pathogen-induced secretory diarrhea. This article is protected by copyright. All rights reserved

Ligand‐based NMR techniques to study protein–ligand interactions are potent tools in drug design. Saturation transfer difference (STD) NMR spectroscopy stands out as one of the most versatile techniques, allowing screening of fragments libraries and providing structural information on binding modes. Recently, it has been shown that a multi‐frequency STD NMR approach, differential epitope mapping (DEEP)‐STD NMR, can provide additional information on the orientation of small ligands within the binding pocket. Here, the approach is extended to a so‐called DEEP‐STD NMR fingerprinting technique to explore the binding subsites of cholera toxin subunit B (CTB). To that aim, the synthesis of a set of new ligands is presented, which have been subject to a thorough study of their interactions with CTB by weak affinity chromatography (WAC) and NMR spectroscopy. Remarkably, the combination of DEEP‐STD NMR fingerprinting and Hamiltonian replica exchange molecular dynamics has proved to be an excellent approach to explore the geometry, flexibility, and ligand occupancy of multi‐subsite binding pockets. In the particular case of CTB, it allowed the existence of a hitherto unknown binding subsite adjacent to the GM1 binding pocket to be revealed, paving the way to the design of novel leads for inhibition of this relevant toxin., Fingerprinting: A powerful protocol by using NMR spectroscopy and molecular dynamics to study multi‐subsite binding pockets has been developed. For cholera toxin subunit B (CTB), the combination of differential epitope mapping saturation transfer difference (DEEP‐STD) NMR, STD competition experiments, transfer‐NOESY, and Hamiltonian replica exchange molecular dynamics (HREMD) unveiled the presence of a novel subsite adjacent to the known subsites of GM1 on CTB.