Cholera toxin inhibitors studied with high-performance liquid affinity chromatography: a robust method to evaluate receptor-ligand interactions.
Anti-adhesion drugs may be an alternative to antibiotics to control infection of micro-organisms. The well-characterized interaction between cholera toxin and the cellular glycolipid GM1 makes it an attractive model for inhibition studies in general. In this report, we demonstrate a high-performance liquid affinity chromatography approach called weak affinity chromatography to evaluate cholera toxin inhibitors. The cholera toxin B-subunit was covalently coupled to porous silica and a (weak) affinity column was produced. The K(D) values of galactose and meta-nitrophenyl alpha-D-galactoside were determined with weak affinity chromatography to be 52 and 1 mM, respectively, which agree well with IC(50) values previously reported. To increase inhibition potency multivalent inhibitors have been developed and the interaction with multivalent glycopolypeptides was also evaluated. The affinity of these compounds was found to correlate with the galactoside content but K(D) values were not obtained because of the inhomogeneous response and slow off-rate from multivalent interactions. Despite the limitations in obtaining direct K(D) values of the multivalent galactopolypeptides, weak affinity chromatography represents an additional and valuable tool in the evaluation of monovalent as well as multivalent cholera toxin inhibitors. It offers multiple advantages, such as a low sample consumption, high reproducibility and short analysis time, which are often not observed in other methods of analysis.
Bergström
Maria
M
School of Pure and Applied Natural Sciences, University of Kalmar, SE-391 82 Kalmar, Sweden. maria.bergstrom@hik.se
Liu
Shuang
S
Kiick
Kristi L
KL
Ohlson
Sten
S
D000595:Amino Acid Sequence; D000818:Animals; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002772:Cholera Toxin / Q000037:antagonists & inhibitors* / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005677:G(M1) Ganglioside / Q000737:chemistry / Q000378:metabolism; D008024:Ligands; D008969:Molecular Sequence Data; D015394:Molecular Structure; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism
D008024:Ligands; D021122:Protein Subunits; D005677:G(M1) Ganglioside; D002772:Cholera Toxin