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High-throughput confocal microscopy for beta-arrestin-green fluorescent protein translocation G protein-coupled
Ralph J Garippa1, Ann F Hoffman, Gabriele Gradl
1Roche Discovery Technologies, Roche Inc., Nutley, NJ, USA.
Abstract:
Ligand-activated G protein-coupled receptors (GPCRs) are known to regulate a myriad of homeostatic functions. Inappropriate signaling is associated with several pathophysiological states. GPCRs belong to a approximately 800 member superfamily of seven transmembrane-spanning receptor proteins that respond to a diversity of ligands. As such, they present themselves as potential points of therapeutic intervention. Furthermore, orphan GPCRs, which are GPCRs without a known cognate ligand, offer new opportunities as drug development targets. This chapter describes a systems-based biological approach, one that combines in silico bioinformatics, genomics, high-throughput screening, and high-content cell-based confocal microscopy strategies to (1) identify a relevant subset of protein family targets, (2) within the therapeutic area of energy metabolism/obesity, (3) and to identify small molecule leads as tractable combinatorial and medicinal chemistry starting points. Our choice of screening platform was the Transfluor beta-arrestin-green fluorescent protein translocation assay in which full-length human orphan GPCRs were stably expressed in a U-2 OS cell background. These cells lend themselves to high-speed confocal imaging techniques using the Evotec Technologies Opera automated microscope system. The basic assay system can be implemented in any laboratory using a fluorescent probe, a stably expressed GPCR of interest, automation-assisted plate and liquid-handling techniques, an optimized image analysis algorithm, and a high-speed confocal microscope with sophisticated data analysis tools.
Insights
Researchers developed a systems-based approach to discover drug leads for orphan G protein-coupled receptors (GPCRs) involved in metabolism and obesity. This method combines bioinformatics, genomics, and high-throughput screening for novel therapeutic targets.
Area of Science:
- Pharmacology
- Biotechnology
- Genomics
Background:
- G protein-coupled receptors (GPCRs) regulate vital homeostatic functions, and dysregulation is linked to diseases.
- Orphan GPCRs, lacking known ligands, represent promising targets for drug discovery.
- GPCRs are a large superfamily of transmembrane receptors crucial for cellular signaling.
Purpose of the Study:
- To identify therapeutic targets within the energy metabolism/obesity domain.
- To discover small molecule leads for drug development against orphan GPCRs.
- To apply a systems-based biological strategy for target and lead identification.
Main Methods:
- Utilized in silico bioinformatics and genomics for target selection.
- Employed high-throughput screening with a Transfluor beta-arrestin-green fluorescent protein translocation assay.
- Integrated high-content cell-based confocal microscopy using U-2 OS cells and automated imaging systems.
Main Results:
- Identified a subset of relevant protein family targets in energy metabolism.
- Discovered small molecule leads serving as starting points for medicinal chemistry.
- Validated the utility of the Transfluor assay and automated microscopy for orphan GPCR screening.
Conclusions:
- The described systems-based approach is effective for identifying orphan GPCR targets and small molecule leads.
- This strategy facilitates drug discovery in therapeutic areas like obesity and metabolic disorders.
- The assay system is adaptable for various laboratories and GPCR targets.
