TY - JOUR

T1 - A Scintillation Proximity Assay for Real-Time Kinetic Analysis of Chemokine-Chemokine Receptor Interactions

AU - Eberle, Stefanie Alexandra

AU - Gustavsson, Martin

PY - 2022

Y1 - 2022

N2 - 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.

AB - 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.

KW - 7TM receptor

KW - ACKR3

KW - CXCL12

KW - SDF-1

KW - chemokine

KW - chemokine receptor

KW - kinetics

KW - association

KW - dissociation

KW - Scintillation Proximity Assay (SPA)

KW - PROTEIN-COUPLED RECEPTORS

KW - TARGET BINDING-KINETICS

KW - IN-VIVO

KW - MEMBRANE-PROTEIN

KW - DRUG DISCOVERY

KW - K-I

KW - LIGAND

KW - CXCR4

KW - ANTAGONISTS

KW - TC14012

U2 - 10.3390/cells11081317

DO - 10.3390/cells11081317

M3 - Journal article

C2 - 35455996

VL - 11

JO - Cells

JF - Cells

SN - 2073-4409

IS - 8

M1 - 1317

ER -