1. Affinity capillary electrophoresis for the assessment of binding affinity of carbohydrate-based cholera toxin inhibitors
- Author
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Aizpurua-Olaizola, Oier, Sastre Torano, Javier, Pukin, Aliaksei, Fu, Ou, Boons, Geert Jan, de Jong, Gerhardus J, Pieters, Roland J, Afd Chemical Biology and Drug Discovery, Sub Algemeen Scheikunde, Chemical Biology and Drug Discovery, Afd Chemical Biology and Drug Discovery, Sub Algemeen Scheikunde, and Chemical Biology and Drug Discovery
- Subjects
0301 basic medicine ,Cholera Toxin ,Clinical Biochemistry ,Oligosaccharides ,Context (language use) ,G(M1) Ganglioside ,medicine.disease_cause ,01 natural sciences ,Biochemistry ,Protein-carbohydrateinteraction ,Analytical Chemistry ,03 medical and health sciences ,Capillary electrophoresis ,medicine ,Affinity capillary electrophoresis ,Protein–carbohydrate interactions ,Cholera toxin inhibitors ,Secretory diarrhea ,Enzyme Inhibitors ,Ganglioside ,Formamides ,Glycobiology ,Chemistry ,010401 analytical chemistry ,Cholera toxin ,Electrophoresis, Capillary ,Hemagglutinin ,0104 chemical sciences ,Dissociation constant ,030104 developmental biology ,Protein Binding - Abstract
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
- Published
- 2017
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