Single-molecule detection technologies in miniaturized high throughput screening: Binding assays for G protein-coupled receptors using fluorescence intensity distribution analysis and fluorescence anisotropy

G Protein-coupled receptors (GPCRs) represent one of the most important tar get classes for drug discovery. Various assay formats are currently applied to screen large compound libraries for agonists or antagonists, However, t he development of nonradioactive, miniaturizable assays that are compatible with the requirements of ultra-high throughput screening (uHTS) has so far been slow, In this report we describe homogeneous fluorescence-based bindi ng assays that are highly amenable to miniaturization. Fluorescence intensi ty distribution analysis (FIDA) is a single-molecule detection method that is sensitive to brightness changes of individual particles, such as those i nduced by binding of fluorescent ligands to membrane particles with multipl e receptor sites, As a confocal detection technology, FIDA inherently allow s reduction of the assay volume to the microliter range and below without a ny loss of signal, Binding and displacement experiments are demonstrated fo r various types of GPCRs, such as chemokine, peptide hormone, or small-mole cule ligand receptors, demonstrating the broad applicability of this method , The results correlate quantitatively with radioligand binding data, We co mpare FIDA with fluorescence anisotropy (FA), which is based on changes of molecular rotation rates upon binding of fluorescent ligands to membranes, While FA requires a higher degree of binding, FIDA is sensitive down to low er levels of receptor expression, Both methods are, within these boundary c onditions, applicable to uHTS.