High-content assays for ligand regulation of G-protein-coupled receptors

doi:10.1016/S1359-6446(03)02738-7

Drug Discovery Today

Volume 8, Issue 13, 1 July 2003, Pages 579-585

https://doi.org/10.1016/S1359-6446(03)02738-7 Get rights and content

Abstract

High-content assays rely on the imaging of cellular events. They can be used to monitor the activation of G-protein-coupled receptors (or other receptors), their internalization into the cell, or alterations in their amount. In addition, multiplexed assays can provide further information about the characteristics of the receptor. Recent improvements in throughput using high-content screening platforms means that such assays are now an integral element of functional analysis in the drug discovery process.

Section snippets

Assays based on protein translocation

Variants of the autofluorescent green protein from the jellyfish Aequoria victoria have been developed that have both altered emission spectra and enhanced fluorescence properties, and this has revolutionized many aspects of cell biology in recent years 12., 13.. In addition, it opened up possibilities for the development of ligand-screening assays for GPCRs based on cell imaging. That the vast majority of GPCRs internalize from the cell surface into acidic endosomes in response to agonist

Assays based on altered protein concentration

All GPCRs display some level of constitutive activity. That is, they are able to transduce signals in the absence of an agonist ligand. The extent of constitutive activity varies significantly between different GPCRs and can often be increased substantially by judicious mutagenesis [30]. Many of these mutations also result in destabilization of the GPCR such that it denatures more easily and displays a reduced half-life in cells 31., 32., 33., 34.. Importantly, binding of ligands to such

Assays based on altered spectral properties

The ideal assay for a high-content screen for GPCR activation would involve either a GPCR or an associated protein whose spectral properties change in response to activation. Potentially, assays based on resonance energy transfer techniques can offer this. Indeed, as GPCRs appear to exist and potentially function as dimeric or oligomeric species 41., 42., there was considerable interest in several reports indicating that agonist ligands could induce or inhibit GPCR dimerization [43].

In either

Concluding remarks

High-content screening assays have become of increasing importance in the drug discovery process. This reflects both improvements in speed and analysis of samples, and the capacity to multiplex assays. Several assay formats are now available and this is expected to increase markedly in the future.

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Label-free optical biosensor for ligand-directed functional selectivity acting on β<inf>2</inf> adrenoceptor in living cells
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Recent realization of ligand-directed functional selectivity demands high-resolution tools for studying receptor biology and ligand pharmacology. Here we use label-free optical biosensor to examine the dynamic mass redistribution of human epidermoid A431 cells in response to diverse β₂-adrenoceptor ligands. Multi-parameter analysis reveals distinct patterns in activation and signaling of the receptor induced by different agonists. Sequential and co-stimulation assays categorize various ligands for their ability to modulate signaling induced by catechol, a structural component of catecholamines. This study documents multiple ligand-specific states of the β₂-adrenoceptor and highlights the power of the biosensor assays for screening pathway-biased ligands.
Pharmacological profiling of chemokine receptor-directed compounds using high-content screening
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High-content screening, typically defined as automated fluorescence microscopy combined with image analysis, is now well established as a means to study test compound effects in cellular disease-modeling systems. In this work, the authors establish several high-content screening assays in the 384-well format to measure the activation of the CC-type chemokine receptors 2B and 3 (CCR2B, CCR3). As a cellular model system, the authors use Chinese hamster ovary cells, stably transfected with 1 of the respective receptors. They characterize receptor stimulation by human monocyte chemoattractant protein-1 for CCR2B and by human eotaxin-1 for CCR3: Receptor internalization and receptor-induced phosphorylation of ERK1/2 (pERK) were quantified using fluorescence imaging and image analysis. The 4 assay formats were robust, displayed little day-to-day variability, and delivered good Z′ statistics for both CCRs. For each of the 2 receptors, the authors evaluated the potency of inhibitory compounds in the internalization format and the pERK assay and compared the results with those from other assays (ligand displacement binding, Ca²⁺ mobilization, guanosine triphosphate exchange, chemotaxis). Both physiological agonists and test compounds differed significantly with respect to potencies and efficacies in the various profiling assays. The diverse assay formats delivered partially overlapping and partially complementary information, enabling the authors to reduce the probability of test compound–related technology artifacts and to specify the mode of action for individual test compounds. Transfer of the high-content screening format to a fully automated medium-throughput screening platform for CCR3 enabled the profiling of large compound numbers with respect to G protein signaling and possible tolerance-inducing liabilities.
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ReviewHigh-content assays for ligand regulation of G-protein-coupled receptors

Abstract

Section snippets

Assays based on protein translocation

Assays based on altered protein concentration

Assays based on altered spectral properties

Concluding remarks

Drug Discov. Today

J. Biomol. Screen.

Curr. Opin. Pharmacol.

Methods Enzymol.

Pharmacol. Ther.

Trends Pharmacol. Sci.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Trends Pharmacol. Sci.

J. Biol. Chem.

J. Biol. Chem.

Trends Pharmacol. Sci.

Trends Endocrinol. and Metabol.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Methods

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

The sequence of the human genome

Science

Initial sequencing and analysis of the human genome

Nature

The G protein-coupled receptor repertoires of human and mouse

Proc. Natl. Acad. Sci. U. S. A.

Orphan G-protein coupled receptors: novel drug targets for the pharmaceutical industry

Drug Des. Discov.

Strategies to identify ligands for orphan G-protein-coupled receptors

Biochem. Soc. Trans.

Functional assays for identifying ligands at orphan G protein-coupled receptors

Recept. Channels

Aequorin-based functional assays for G-protein-coupled receptors, ion channels and tyrosine kinase receptors

Recept. Channels

Review
High-content assays for ligand regulation of G-protein-coupled receptors