Homogeneous time-resolved G protein-coupled receptor–ligand binding assay based on fluorescence cross-correlation spectroscopy

Abstract

G protein-coupled receptors (GPCRs) mediate many important physiological functions and are considered as one of the most successful therapeutic target classes for a wide spectrum of diseases. Drug discovery projects generally benefit from a broad range of experimental approaches for screening compound libraries and for the characterization of binding modes of drug candidates. Owing to the difficulties in solubilizing and purifying GPCRs, assay formats have been so far mainly limited to cell-based functional assays and radioligand binding assays. In this study, we used fluorescence cross-correlation spectroscopy (FCCS) to analyze the interaction of detergent-solubilized receptors to various types of GPCR ligands: endogenous peptides, small molecules, and a large surrogate antagonist represented by a blocking monoclonal antibody. Our work demonstrates the suitability of the homogeneous and time-resolved FCCS assay format for a robust, high-throughput determination of receptor-ligand binding affinities and kinetic rate constants for various therapeutically relevant GPCRs.

Considerations for improved performance of competition association assays analysed with the Motulsky–Mahan's “kinetics of competitive binding” model

“…Not surprisingly, this parameter is nowadays monitored in drug discovery programmes all the way from the hit identification and lead optimisation phases up to the candidate evaluation and selection (Georgi et al, ). To this end, the Motulsky–Mahan “kinetics of competitive binding” model is widely applied, with recent efforts aiming at screening assay formats with increased robustness and throughput (Antoine et al, ; Guo et al, ; Schiele et al, ; Stoddard et al, ).…”

“…The growing interest in BK has promoted the development of assay technologies dedicated to measure association (k on ) and dissociation (k off ) rate constants, as well as binding affinities, ( K D = k off /k on ; Antoine et al, ; de Witte et al, ; Guo, Mulder‐Krieger, IJzerman, & Heitman, ; Schiele, Ayaz, & Fernández‐Montalván, ; Stoddard et al, ; Stoddart et al, ; Swinney et al, ; Sykes et al, ; Xia, de Vries, IJzerman, & Heitman, ). Among them, competition association assays, monitoring the simultaneous binding of a labelled tracer (we have referred to the labelled compound as tracer, although any type of fluorescent or luminescent probes and radioactive tracers can be used for competition association assays) and an unlabelled compound to the target molecule, have gained popularity due to their relatively high throughput and ability to deal with both soluble and membrane‐bound proteins (Schiele et al, ; Stoddart et al, ; Xia et al, ).…”

High-Affinity Binding of Chemokine Analogs that Display Ligand Bias at the HIV-1 Co-receptor CCR5

“…Antoine et al performed similar ligand binding measurements by FCCS using several green fluorescent protein (GFP) receptor fusion proteins with fluorescently-labeled small molecules and antibodies. (29) Our approach differs in two major ways. First, we used highly purified receptor samples for ligand-binding measurements, instead of GPCRs solubilized in crude cell lysates without any purification.…”

XPO1 target occupancy measurements confirm the selinexor recommended phase 2 dose

“…Assays to measure target occupancy have been developed to verify drug-target engagement for a variety of indications using many different methods such as microscopic detection of fluorescently labeled probes [ 20 , 21 ], positron emission tomography of tracers [ 22 , 23 ], or flow cytometry [reviewed in 3, 4]. Fluorescently labeled molecules can be used as probes to measure target occupancy for the Bruton tyrosine kinase inhibitor ibrutinib in PBMCs [ 21 ], or when combined with fluorescent polarized microscopy to quantitate drug-target engagement in single cells [ 24 ].…”