Assay Types For Gpcrs IN uHTS 1321 Radioligand Displacement Assays

The radioligand displacement assay was one of the earliest assays used to study GPCRs. For nearly 50 years, the radioligand displacement assays, also known as competition binding assays, were used by the pharmaceutical industry as a preferred screening method to identify candidate drug molecules capable of binding to GPCRs, the largest group of pharmaceutical drug targets involved in a variety of physiological disorders.

These assays, performed with purified membrane preparations from cells [13] or tissues, or on whole cells [14], require prior knowledge of the receptor ligand (agonist or antagonist) and the availability of a radiolabeled ligand. The typical filter binding assay involves the incubation of cell membranes containing GPCRs with the cognate radioligand in the presence of unlabelled compound. The radioligand bound to the GPCRs of interest on the cell membranes is then separated from the free unbound ligand by rapid filtration through glass fiber filters. The amount of radioligand bound to the target receptor is

Second messenger: i[Ca2+], IP

Second messenger: cAMP

Second messenger: i[Ca2+], IP

Second messenger: cAMP

Figure 13.2 Functional cell-based assays for GPCRs for HTS and uHTS. A schematic representation of second messenger and p-arrestin translocation assays utilizing a variety of detection technologies ranging from fluorescent dyes to reporter genes to microscopy for detection of signal.

quantitated by scintillation counting, based on the conversion of the energy from radioactive decay into light photons that can be detected using the pho-tomultiplier tubes in scintillation counters. Filtration binding assays using cell membranes can be used to quantitate GPCR expression (Bmax), as well as the affinity of the radioligand (Kd) for the GPCR. The traditional version of the radioligand binding assay requires many washes of the GPCR- containing cell membranes on the filter to wash out any nonspecifically associated signal, and is thus used primarily in traditional 96- or 384-well plate formats in HTS but is not adaptable for uHTS in 1536 or higher plate densities due to limitations with filtration technologies.

A modified version of the radioligand binding assay, called the "scintillation proximity assay" (SPA), paved the way for an automated approach to HTS for GPCRs. In SPA assays, the scintillant used to measure radioactivity is enclosed within a polystyrene bead coated with wheat germ agglutinin to capture cell membranes on the surface of the beads by nonspecific interaction between the glycosylated proteins on the cell membranes and wheat germ agglutinin. Thus, radioligand-bound GPCRs on cell membranes can be brought into close proximity to the SPA beads containing the scintillant, measured by an appropriate detector. SPA assays are homogeneous (addition only) assays and hence offer a significant advantage over the traditional filtration assays for radioligand binding in HTS laboratories. In addition, with the advent of the charge-coupled device (CCD) camera-based imagers, SPA assays are now amenable for uHTS in 1536-well plate formats. The main disadvantages of the SPA assay are related to the use of radioactive ligands and potential limitations associated with the specific activity of these radioligands. In addition, there is no straightforward method to determining Kd and/or Bmax from the SPA assays.

The advent of fluorescently labeled ligands offers an attractive alternative to radioactive binding assays for GPCRs. For example, the use of cyanine dye-labeled ligands ([Cy] ligands) with laser scanning imaging (LSI) of cells settled at the bottom of a microtiter plate enables the specific detection of the interaction of the fluorescently labeled ligand with the GPCR on the cell surface. In this approach, the need for physical separation of the GPCR-bound ligand from the "free" unbound ligand is accomplished by optical discrimination of solid-liquid phase partitioning of the fluorescent ligand (i.e., optical discrimination between the fluorescently labeled ligand on the cell surface vs. fluores-cently labeled ligand in the surrounding buffer in the well), thereby circumventing the wash step commonly used in filter binding assays [15]. This methodology is HTS and uHTS compatible in 1536-well plate formats using detectors like the FMAT (Applied Biosystems, Foster City, CA) and Acumen Explorer (TTP Labtech., Cambridge, MA).

Fluorescence polarization (FP) is another methodology that uses fluores-cently labeled ligands to measure binding to GPCRs in a homogeneous assay amenable to HTS and uHTS. FP is based on the principle that small molecules (e.g., fluorescently labeled ligands) rotate or tumble faster in solution that larger molecules (e.g., GPCRs on cell membranes) [16].

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