Dual-point competition association assay: a fast and high-throughput kinetic screening method for assessing ligand-receptor binding kinetics

• Published in: Journal of biomolecular screening Vol. 18; no. 3; pp. 309 - 320
• Main Authors: Guo, Dong, van Dorp, Erika J H, Mulder-Krieger, Thea, van Veldhoven, Jacobus P D, Brussee, Johannes, Ijzerman, Adriaan P, Heitman, Laura H
• Format: Journal Article
• Language: English
• Published: United States 01.03.2013
• Subjects: Animals; Binding, Competitive; Cell Line; CHO Cells; Cricetinae; Cricetulus; High-Throughput Screening Assays - methods; Kinetics; Ligands; Protein Binding; Radioligand Assay - methods; Receptors, Cell Surface - chemistry; Receptors, Cell Surface - metabolism; Reproducibility of Results
• ISSN: 2472-5552; 1552-454X
• DOI: 10.1177/1087057112464776

The concept of ligand-receptor binding kinetics is emerging as an important parameter in the early phase of drug discovery. Since the currently used kinetic assays are laborious and low throughput, we developed a method that enables fast and large format screening. It is a so-called dual-point competition association assay, which measures radioligand binding at two different time points in the absence or presence of unlabeled competitors. Specifically, this assay yields the kinetic rate index (KRI), which is a measure for the binding kinetics of the unlabeled ligands screened. As a prototypical drug target, the adenosine A(1) receptor (A(1)R) was chosen for assay validation and optimization. A screen with 35 high-affinity A(1)R antagonists yielded seven compounds with a KRI value above 1.0, which indicated a relatively slow dissociation from the target. All other compounds had a KRI value below or equal to 1.0, predicting a relatively fast dissociation rate. Several compounds were selected for follow-up kinetic quantifications in classical kinetic assays and were shown to have kinetic rates that corresponded to their KRI values. The dual-point assay and KRI value may have general applicability at other G-protein-coupled receptors, as well as at drug targets from other protein families.