title: Development of a fluorescence polarization binding assay for asialoglycoprotein receptor.
abstract: Asialoglycoprotein receptor (ASGP-R) has been actively investigated for targeted delivery of therapeutic agents into hepatocytes because this receptor is selectively and highly expressed in liver and has a high internalization rate. Synthetic cluster glycopeptides (e.g., triGalNAc) bind with high affinity to ASGP-R and, when conjugated to a therapeutic agent, can drive receptor-mediated uptake in liver. We developed a novel fluorescent polarization (FP) ASGP-R binding assay to determine the binding affinities of ASGP-R-targeted molecules. The assay was performed in 96-well microplates using membrane preparations from rat liver as a source of ASGP-R and Cy5 fluorophore-labeled triGalNAc synthetic ligand as a tracer. This high-throughput homogeneous assay demonstrates advantages over existing multistep methods in that it minimizes both time and resources spent in determining binding affinities to ASGP-R. At the optimized conditions, a Z' factor of 0.73 was achieved in a 96-well format.
authors.affiliation: In Vitro Pharmacology department, Merck, West Point, PA 19486, USA. anna_kornilova@merck.com
authors.forename: Anna Y
authors.initials: AY
authors.lastname: Kornilova
authors.forename: Bethany
authors.initials: B
authors.lastname: Algayer
authors.forename: Michael
authors.initials: M
authors.lastname: Breslin
authors.forename: Victor
authors.initials: V
authors.lastname: Uebele
mesh_terms: D000818:Animals; D037263:Asialoglycoprotein Receptor / Q000032:analysis / Q000737:chemistry*; D001665:Binding Sites; D005453:Fluorescence; D005454:Fluorescence Polarization / Q000379:methods*; D007700:Kinetics; D008099:Liver / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D037263:Asialoglycoprotein Receptor