title: Development of an AlphaScreen assay for discovery of inhibitors of low-affinity glycan-lectin interactions.
abstract: The development of high-throughput screening (HTS) assays with increased sensitivity for the identification of potent and selective inhibitors of galectins has been hampered by the weak binding affinities between galectins and their carbohydrate ligands. To circumvent this obstacle, we have developed an AlphaScreen assay for a 384-well plate format in a competitive binding configuration for discovery of new inhibitors of galectin-3. His-tagged galectin-3 was bound to nickel chelate acceptor beads, whereas biotinylated asialofetuin (biotin-ASF), a galectin-3 nanomolar binding partner, was bound to streptavidin-coated donor beads. Inhibitors of the carbohydrate-galectin interaction lead to a reduction of the AlphaScreen signal by competing with the biotin-ASF. The obtained IC50 values for known carbohydrate ligands of galectin-3 are in good agreement with the Kd values reported and measured for galectin-3 by isothermal titration calorimetry (ITC). Thus, the developed AlphaScreen assay in a competitive binding configuration offers several advantages over the existing screening assays for inhibitors of glycan-lectin interactions. In addition, the assay format for the galectin-3/ASF pair could be easily applied in screening for glycan- and/or small molecule-based inhibitors of other members of the galectin family.
authors.affiliation: Torrey Pines Institute for Molecular Studies, Port St. Lucie, FL 34987, USA.
authors.forename: Svetlana
authors.initials: S
authors.lastname: Yegorova
authors.forename: Anais E
authors.initials: AE
authors.lastname: Chavaroche
authors.forename: Maria C
authors.initials: MC
authors.lastname: Rodriguez
authors.forename: Dmitriy
authors.initials: D
authors.lastname: Minond
authors.forename: Mare
authors.initials: M
authors.lastname: Cudic
mesh_terms: D002236:Carbohydrate Conformation; D004926:Escherichia coli; D037502:Galectin 3 / Q000037:antagonists & inhibitors* / Q000378:metabolism; D005786:Gene Expression Regulation; D006801:Humans; D055767:Immobilized Proteins / Q000737:chemistry; D020128:Inhibitory Concentration 50; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D011134:Polysaccharides / Q000737:chemistry*; D011485:Protein Binding; D012680:Sensitivity and Specificity
chemical_list: D037502:Galectin 3; D055767:Immobilized Proteins; D011134:Polysaccharides
keywords: AlphaScreen technology; Competitive binding assay; Galectin-3; Glycan–lectin interactions; High-throughput screening