G protein-coupled receptor internalization assays in the high-content screening format.

High-content screening (HCS), a combination of fluorescence microscopic imaging and automated image analysis, has become a frequently applied tool to study test compound effects in cellular disease-modeling systems. This chapter describes the measurement of G protein-coupled receptor (GPCR) internalization in the HCS format using a high-throughput, confocal cellular imaging device. GPCRs are the most successful group of therapeutic targets on the pharmaceutical market. Accordingly, the search for compounds that interfere with GPCR function in a specific and selective way is a major focus of the pharmaceutical industry today. This chapter describes methods for the ligand-induced internalization of GPCRs labeled previously with either a fluorophore-conjugated ligand or an antibody directed against an N-terminal tag of the GPCR. Both labeling techniques produce robust assay formats. Complementary to other functional GPCR drug discovery assays, internalization assays enable a pharmacological analysis of test compounds. We conclude that GPCR internalization assays represent a valuable medium/high-throughput screening format to determine the cellular activity of GPCR ligands.

Comparison of G-protein coupled receptor desensitization-related beta-arrestin redistribution using confocal and non-confocal imaging.

High Content Screening (HCS), a combination of fluorescence microscopic imaging and automated image analysis, has become a frequently applied tool to study test compound effects in cellular disease-modelling systems. In this work, we compared a confocal and a non-confocal cellular HCS system, the IN Cell Analyzers(1) 3,000 and 1,000, respectively. As a cellular model system we used the Transfluor technology in the 384-well microtiter plate (MTP) format. The Transfluor HCS assay for G-protein coupled receptor (GPCR) activation is based on the recruitment of a green fluorescent protein-labelled arrestin (ArrGFP) from the cytosol to the plasma membrane. We investigated two GPCRs, the wild-type (wt) beta2 adrenergic receptor (beta2AR) and the beta2AR-enhanced (E), a C-terminally mutated receptor with a higher affinity to arrestin. Upon agonist stimulation, the beta2AR-wt induced the redistribution of ArrGFP to coated pits, the beta2AR-E maintained the interaction with ArrGFP down to the formation of endocytic vesicles. Our findings reveal that the assay is feasible on both instruments, with sufficiently robust Z' statistics. Improved Z' statistics, though, are achieved with the confocal system, particularly in case of weak signals. Moreover, throughput is dramatically higher for the IN Cell Analyzer 3,000. We conclude that, depending on the needs for throughput and assay biology, either instrument may fulfil a successful role in the drug discovery process. Confocal optics, however, provide a better basis for the detection of smaller subcellular structures with lower fluorescence intensity.

Confocal fluorescence microscopy for high-throughput screening of G-protein coupled receptors.

In the pharmaceutical industry, G-protein coupled receptors (GPCRs) are the most successful group of therapeutic targets. Finding compounds that interfere with the ligand-GPCR interaction in a specific and selective way is a major focus of pharmaceutical research today. As compound libraries of large pharmaceutical companies have increased to hundreds of thousands of test compounds, there is a growing need for miniaturization of drug discovery assays to save bioreagents and to reduce the consumption of test compounds. Due to its high sensitivity combined with a femtoliter-sized measurement volume, confocal fluorescence microscopy enables designs for GPCR binding assays with tiny sample volumes. The GPCRs are prepared in the form of plasma membrane fragments from GPCR-overexpressing cells or may be integrated into virus-like particles (VLiPs). One technique to extract binding data from confocal fluorescence experiments is the so-called fluorescence intensity distribution analysis (FIDA). In this review article, we describe the applicability of FIDA to GPCR-focussed high-throughput screening (HTS) and compare FIDA to two other GPCR-adaptable drug discovery techniques for ligand binding studies, the scintillation proximity assay (SPA) and macroscopic fluorescence polarization (FP) measurements. FIDA measures the absolute concentrations of both GPCR-bound and unbound ligand, thereby providing an internal control to the drug screening data. FIDA is amenable to work with relatively low amounts of GPCRs so that the assay may be carried out with biomembranes of a low GPCR density. Moreover, the fluorescence intensity readout of the FIDA technique may be combined with other confocal fluorescence readouts such as fluorescence anisotropy or lifetime. The combination of a low sample volume with an information-rich measurement means that confocal fluorescence spectroscopy can bring substantial benefits as a bioassay platform to pharmaceutical GPCR-directed research.

Evaluation of the InteraX system technology in a high-throughput screening environment.

The authors have developed a cell-based high-throughput screening (HTS)-compatible assay to measure EGFR dimerization using the InteraX enzyme complementation technology of Applied Biosystems. The cells contain 2 chimeric proteins with complementing deletion mutants of the beta galactosidase enzyme, each fused to the extracellular and transmembrane part of EGFR. On binding of EGF, EGF receptor dimerizes and an active beta galactosidase is built. The authors used this homogeneous 384-well assay to screen about 20,000 diverse compounds. From 2 independent primary screen runs 239 hits were identified. For run 1, a mean S/B ratio of 4.26 and a mean Z' factor of 0.74 were obtained, for run 2 a mean S/B ratio of 3.88 and a mean Z' factor of 0.71 were obtained. After hit confirmation, repeated 4 times, 112 hits remained with a confirmation rate of 48.9%. Thirty of the 112 could be identified as cytotoxic. Fifty-one of the remaining 82 compounds could be shown to be inhibitors of the beta galactosidase enzyme itself. In summary, 31 compounds remained as potential EGFR dimerization or EGF stimulation inhibitors. The authors conclude that the InteraX system technology is HTS capable and can detect small molecule inhibitors capable of inhibiting protein-protein interactions.

High-throughput screening of interactions between G protein-coupled receptors and ligands using confocal optics microscopy.

Interactions of extracellular ligands with proteins in the cellular plasma membrane are the starting point for various intracellular signaling cascades. In the pharmaceutical industry, particular attention has been paid to G protein- coupled receptors (GPCRs), which are involved in various disease processes. In so-called high-throughput screening (HTS) campaigns, large medicinal chemistry compound libraries were searched for bioactive molecules that would either induce or inhibit the activity of a specific disease-relevant GPCR. In the respective drug discovery assays, the test compound typically competes with the physiological ligand for a binding site on the receptor. The transmembrane receptor is prepared in the form of membrane fragments or, as described here, in so-called virus-like particles (VLiPs). As hundreds of thousands of test compounds must be analyzed, there is a strict need for low volume binding assays to save the expensive bioreagents, and to reduce the consumption of the test compounds. In this chapter, we describe the application of confocal optics microscopy to measure GPCR ligand interactions in low microliter assay volumes.

Peptide protein binding assay using ImageFlashPlates or Imaging Beads.

Imaging devices used for the measurement of radioligand-receptor binding assays are typically based on charge-coupled device (CCD) cameras, which are more sensitive for red-shifted scintillation. In the past, red-shifted scintillants had only been integrated into microspheres, referred to as scintillation proximity assay (SPA) Imaging Beads. More recently, ImageFlashPlates have been developed that emit light at 615 nm when exposed to beta-radiation. In this article, we report the establishment of peptide-protein binding assays using either streptavidin-coated ImageFlashPlates or Imaging Beads in a low volume 384-well format. In these assays, we employed a biotinylated peptide X and a [33P]-phosphorylated protein Y as the binding partner. The FlashPlates required a washing step, the bead-filled microtiter plates (MTPs) needed a centrifugation step for optimal performance in the scintillation measurements. Both the peptide X-loaded FlashPlates and the beads displayed saturable binding of [33P]-phosphorylated protein Y with a similar scintillation efficiency. A KD value of about 30 nmol/l was measured using the bead-based assay. Due to the washing step in the FlashPlate experiment, approximately two-thirds of the [33P]-phosphorylated protein Y were withdrawn from equilibrium binding. This resulted in correspondingly lower scintillation signals for the FlashPlate experiment. For this reason, the FlashPlate produced a Z' value of 0.64 that was lower than the Z' value of 0.87 for the beads. Using a reference inhibitor in a competition assay produced similar IC50 values for the bead-based assay as for the FlashPlate. Depending on the local automation environment either the centrifugation step for the beads or the washing step for the FlashPlates may be considered more or less of a challenge. Low volume 384-well high-throughput screening (HTS) applicable assay formats are achievable using either the ImageFlashPlates or the Imaging Beads.

Miniaturization and validation of a high-throughput serine kinase assay using the AlphaScreen platform.

Reducing costs while maintaining the highest readout quality is a precept of modern high-throughput screening. Given the trend toward nonradiometric screening platforms, this has been a big challenge for some kinase target classes. Common issues include low sensitivity, susceptibility to nonspecific interference, or the need for costly reagents. In this study, the authors describe the feasibility of miniaturization of a serine kinase assay using generic reagents in the AlphaScreen format. They have validated the robustness of this assay in the course of miniaturization from a 35-to 4.375-microL final assay volume in 384-and 1536-well formats. Within this volume range, they consistently obtained Z' values above 0.5 and have investigated the suitability of these assay formats for measuring compound effects by testing a set of 25 previously identified active compounds. These active compounds were also reliably identified in the miniaturized assay formats. The results presented here show that the AlphaScreen technology permits robust and cost-efficient miniaturization of serine/threonine kinase assays.

Confocal optics microscopy for biochemical and cellular high-throughput screening.

In recent years, both academia and pharmaceutical industry have produced significant advances in confocal detection and spectroscopy by laser-induced fluorescence. Confocal fluorescence studies provide information on identity, size, diffusion coefficient and concentration of the fluorescently labeled entity. This enables the establishment of sophisticated biochemical drug screening assays using the multitude of fluorescence parameters that can be observed (e.g. molecular brightness, fluorescence lifetime, anisotropy, resonance energy transfer). In cellular screening assays, confocality introduces spatial resolution in the vertical direction and reduces background fluorescence from outside the focal plane. Confocal HTS systems focusing on femtoliter-sized observation volumes allow for assay volumes far beyond current limits.