title: Scintillation proximity assay (SPA) as a new approach to determine a ligand's kinetic profile. A case in point for the adenosine A1 receptor.
abstract: Scintillation proximity assay (SPA) is a radio-isotopic technology format used to measure a wide range of biological interactions, including drug-target binding affinity studies. The assay is homogeneous in nature, as it relies on a "mix and measure" format. It does not involve a filtration step to separate bound from free ligand as is the case in a traditional receptor-binding assay. For G protein-coupled receptors (GPCRs), it has been shown that optimal binding kinetics, next to a high affinity of a ligand, can result in more desirable pharmacological profiles. However, traditional techniques to assess kinetic parameters tend to be cumbersome and laborious. We thus aimed to evaluate whether SPA can be an alternative platform for real-time receptor-binding kinetic measurements on GPCRs. To do so, we first validated the SPA technology for equilibrium binding studies on a prototypic class A GPCR, the human adenosine A1 receptor (hA1R). Differently to classic kinetic studies, the SPA technology allowed us to study binding kinetic processes almost real time, which is impossible in the filtration assay. To demonstrate the reliability of this technology for kinetic purposes, we performed the so-called competition association experiments. The association and dissociation rate constants (k on and k off) of unlabeled hA1R ligands were reliably and quickly determined and agreed very well with the same parameters from a traditional filtration assay performed simultaneously. In conclusion, SPA is a very promising technique to determine the kinetic profile of the drug-target interaction. Its robustness and potential for high-throughput may render this technology a preferred choice for further kinetic studies.
authors.affiliation: Division of Medicinal Chemistry, Leiden Academic Centre for Drug Research, Leiden University, P.O. Box 9502, 2300 RA, Leiden, The Netherlands.
authors.forename: Lizi
authors.initials: L
authors.lastname: Xia
authors.affiliation: Division of Medicinal Chemistry, Leiden Academic Centre for Drug Research, Leiden University, P.O. Box 9502, 2300 RA, Leiden, The Netherlands.
authors.forename: Henk
authors.initials: H
authors.lastname: de Vries
authors.affiliation: Division of Medicinal Chemistry, Leiden Academic Centre for Drug Research, Leiden University, P.O. Box 9502, 2300 RA, Leiden, The Netherlands. ijzerman@lacdr.leidenuniv.nl.
authors.forename: Ad P
authors.initials: AP
authors.lastname: IJzerman
authors.affiliation: Division of Medicinal Chemistry, Leiden Academic Centre for Drug Research, Leiden University, P.O. Box 9502, 2300 RA, Leiden, The Netherlands.
authors.forename: Laura H
authors.initials: LH
authors.lastname: Heitman
mesh_terms: D058916:Adenosine A1 Receptor Antagonists / Q000494:pharmacology; D000818:Animals; D001667:Binding, Competitive; D016466:CHO Cells; D002462:Cell Membrane / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D016503:Drug Delivery Systems; D005374:Filtration; D006801:Humans; D007700:Kinetics; D008024:Ligands; D011869:Radioligand Assay; D043682:Receptor, Adenosine A1 / Q000187:drug effects* / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism; D014970:Xanthines / Q000494:pharmacology
chemical_list: D058916:Adenosine A1 Receptor Antagonists; D008024:Ligands; D043682:Receptor, Adenosine A1; D043562:Receptors, G-Protein-Coupled; D014970:Xanthines; C051360:1,3-dipropyl-8-cyclopentylxanthine
keywords: Adenosine A1 receptor; Binding kinetics; G protein-coupled receptors; Residence time; Scintillation proximity assay