A Scintillation Proximity Assay for Real-Time Kinetic Analysis of Chemokine–Chemokine Receptor Interactions

Abstract

Chemokine receptors are extensively involved in a broad range of physiological and pathological processes, making them attractive drug targets. However, despite considerable efforts, there are very few approved drugs targeting this class of seven transmembrane domain receptors to date. In recent years, the importance of including binding kinetics in drug discovery campaigns was emphasized. Therefore, kinetic insight into chemokine–chemokine receptor interactions could help to address this issue. Moreover, it could additionally deepen our understanding of the selectivity and promiscuity of the chemokine–chemokine receptor network. Here, we describe the application, optimization and validation of a homogenous Scintillation Proximity Assay (SPA) for real-time kinetic profiling of chemokine–chemokine receptor interactions on the example of ACKR3 and CXCL12. The principle of the SPA is the detection of radioligand binding to receptors reconstituted into nanodiscs by scintillation light. No receptor modifications are required. The nanodiscs provide a native-like environment for receptors and allow for full control over bilayer composition and size. The continuous assay format enables the monitoring of binding reactions in real-time, and directly accounts for non-specific binding and potential artefacts. Minor adaptations additionally facilitate the determination of equilibrium binding metrics, making the assay a versatile tool for the study of receptor–ligand interactions.

Distinct Activation Mechanisms of CXCR4 and ACKR3 Revealed by Single-Molecule Analysis of their Conformational Landscapes

“…1A). To promote monomeric receptor incorporation in each nanodisc, we utilized MSP1E3D1, which forms nanodiscs with a diameter of approximately 13 nm (46, 47).…”

“…1A). To promote monomeric receptor incorporation in each nanodisc, we utilized MSP1E3D1, which forms nanodiscs with a diameter of approximately 13 nm (46,47). Crystal and cryo-electron microscopy (cryo-EM) structures of homologous class A GPCRs, such as β 2 AR, in inactive and active conformations, reveal that TM6 moves outwards from the TM helical bundle during activation, whereas the position of TM4 remains relatively fixed (48).…”

“…1). To restrict receptor incorporation to one per nanodisc, we utilized MSP1E3D1, which forms nanodiscs with a diameter of approximately 13 nm 42,43 . ACKR3 exhibits greater conformational dynamics than CXCR4.…”