Scintillation proximity assay (SPA) as a new approach to determine a ligand’s kinetic profile. A case in point for the adenosine A1 receptor
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- 作者：Lizi Xia ; Henk de Vries ; Ad P. IJzerman ; Laura H. Heitman
- 关键词：G protein ; coupled receptors ; Scintillation proximity assay ; Binding kinetics ; Adenosine A1 receptor ; Residence time
- 刊名：Purinergic Signalling
- 出版年：2016
- 出版时间：March 2016
- 年：2016
- 卷：12
- 期：1
- 页码：115-126
- 全文大小：1,033 KB
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Henk de Vries (1)
Ad P. IJzerman (1)
Laura H. Heitman (1)
1. Division of Medicinal Chemistry, Leiden Academic Centre for Drug Research, Leiden University, P.O. Box 9502, 2300 RA, Leiden, The Netherlands - 刊物类别：Biomedical and Life Sciences
- 刊物主题：Biomedicine
Biomedicine
Pharmacology and Toxicology
Human Physiology
Neurosciences
Cancer Research - 出版者：Springer Netherlands
- ISSN：1573-9546
文摘
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. Keywords G protein-coupled receptors Scintillation proximity assay Binding kinetics Adenosine A1 receptor Residence time