Ligand binding affinities at G-protein coupled receptors (GPCRs) have historically been determined using a radioligand that competes for receptor binding sites against an unlabeled drug-like compound. However, the potential hazards of open-source radioisotope handling, and the environmental impact of radioisotope disposal, make this a less desirable, costly technology.
Development of fluorescent ligands for GPCRs provides a safer method for determining ligand binding affinities. However, quantification of fluorescent ligand binding tends to rely on high-resolution fluorescence image capture. This approach can be lengthy and tedious, resulting in 96-well plate read times of approximately 30 minutes or more. Complex post-capture image analysis algorithms also extend data-processing time. This results in high content analysis (HCA) assays that are too slow to be useful for high throughput screening (HTS).
In this study we use the adjustable focal height feature and advanced bottom reading of the PHERAstar FS plate reader from BMG LABTECH, in a live-cell 96-well plate-based assay, to quantify fluorescent ligand binding associated with the adherent cell layer. Depending on several variables, “read to IC50 pKi curve plot” times for a 96-well plate were between 2.5 and 10 minutes. This is in stark contrast to HCA assays, which can take around 60 minutes.
Here the PHERAstar FS performs fluorescent ligand binding assays using two fluorescent ligands from CellAura for adenosine (Figure 1A) and dopamine (not shown) receptors. Furthermore, the suitability of such a combination of plate reader and fluorescent ligand for higher throughput receptor binding assays and screening is postulated.