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 (kon and koff) 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.

A two‐state model for the kinetics of competitive radioligand binding

“…Additionally, the method remains laborious as it requires a sufficiently high number of data points for reliable kinetic estimation. To further adapt the assay into a high‐throughput format, one may consider combining the two‐state model with homogenous binding assay techniques, such as the scintillation proximity assay (Xia et al ., ) or the time‐resolved fluorescence energy transfer assay (Schiele et al ., ; Nederpelt et al ., ), which allow continuous readout of ligand–receptor binding without physical separation between free and bound ligands.…”

Public–Private Partnerships in Lead Discovery: Overview and Case Studies

“…Drug–target kinetics, in contrast, has lacked assays with sufficient throughput and robustness, preventing an optimal integration of binding kinetics analyses in the drug discovery and development process. In this arena, efforts by members of the K4DD consortium have already resulted in the development of a new scintillation proximity assay , as well as a universally applicable kinetic probe competition assay (kPCA) which allows for significantly higher throughput in determining binding kinetics with different classes of pharmacologically relevant target enzymes, protein–protein interactions, and G protein‐coupled receptors (GPCRs) . In the past, variable assay conditions and different protocols made it difficult to compare drug–target kinetics between laboratories.…”

Dynamics of ligand binding to GPCR: Residence time of melanocortins and its modulation

“…This method has been found to be very useful for detection of ligand binding to particular receptors [6] or for high throughput screening [7], but it has not found wider usage in radioligand binding kinetic studies. In one study, SPA worked well for on-line 4 monitoring of the radioligand binding to adenosine A1 receptors, but the system did not sufficiently characterize the competitive ligands' kinetics [8]. The use of Dual-Point IC50/Ki value determination for characterization of a competitor's residence time has been suggested [9], but this approach can be used only for ligands with significantly longer residence time.…”

“…As MC4 receptors exist in complexes, of which the minimal size is a homodimer [39], it has been proposed that binding occurs on separate receptor subunits within the complex. The minimal 8 functional model describing these data is presented as Figure 1. The receptors exist as preformed dimers RR and the binding of a ligand may occur to only one of the receptor subunits at a time.…”