Celtarys – High-Throughput Screening of GPCRs for Drug Discovery.

High-throughput GPCR screening has revolutionized the drug discovery process through the automated testing of millions of biological/chemical compounds for specific biological targets over a relatively short time period. It establishes the affinity rate of these molecules with target GPCR ligands, providing the starting point for identifying potential drug candidates in pharmacological assays.

What are GPCRs & Why are they Important in Novel Drug Development?

G protein-coupled receptors (GPCRs) represent the largest and most diverse family of cell membrane receptors in eukaryotes. They are responsible for the transduction of extracellular stimuli into intracellular responses. Activated by many stimulants, such as light, peptides, hormones, growth factors and lipids, they control numerous physiological processes, including sensory transduction, cell–cell communication, neuronal transmission and hormonal signaling 1.

During ligand binding GPCRs couple to heterotrimeric G proteins, activating Gα subunits and causing an influx in secondary messenger molecules and subsequential downstream signaling events. GPCR kinases then phosphorylate activated GPCRs, triggering the recruitment of β-arrestin proteins which desensitize G protein signaling 2.

GPCRs are vital to many biological processes, bind to a diverse range of ligands and play a central role in numerous pathological conditions, making them popular targets for drug development 3. These ligand binding receptors are the target of over 50% of pharmaceutical drugs, however, over 200 of the 360 non-sensory GPCRs still remain orphan (unidentified), representing potential targets for novel drug discovery 1, 4. GPCR screening is a frontline tool in the drug discovery process and has resulted in the identification of new and repurposing of existing drugs.

High-Throughput GPCR Screening Methods

High-throughput GPCR screening automatedly tests many chemical/biological compounds to identify potential target ligands through measuring the secondary messenger response of the cell, generated by GPCR ligand binding. Currently several GPCR screening methods exist and here are some of the most utilised.

Cell-based GPCR screening of secondary messenger cAMP (cyclic adenosine monophosphate)

This GPCR screening approach measures changes in the concentration of cellular cAMP, induced by Gαs, Gαi subunits during activation from GPCR ligand binding. The activation of Gαs results in increased cAMP accumulation, while the activation of Gαi causes a decrease in cAMP. The activation of a GPCR ligand can also be linked to RNA barcode reporters, using fluorescent labels, allowing different GPCRs to be pooled together and tested against a particular molecule for the isolation of the highest affinity ligand binders 4.

Calcium-based GPCR screening

The activation of Gαq subunit, from GPCR ligand binding, results in an accumulation of calcium within the target cell, which is documented by high-throughput GPCR screening using calcium dyes, such as Fluo-4. Two drawbacks of this GPCR screening method are that these dyes are costly and that the additional assay time limits the high-throughput screening process. However, recent developments of genetically encoded calcium biosensors (such as GCaMP) reduce the expense of calcium-based GPCR screening 4.

β-arrestin recruitment-based GPCR screening

After activation, GPCRs finally become desensitized through the recruitment of β-arrestin, linked to a protease. When a GPCR is activated by a ligand, it undergoes a conformational change that allows it to interact with and activate a G protein, which in turn triggers downstream signaling pathways. However, prolonged or repeated receptor activation can lead to excessive or sustained signaling, which can be harmful to the cell or organism. To prevent this, the cell recruits β-arrestin to the activated receptor, which acts as an adaptor protein and binds to the receptor, effectively "arresting" its signaling activity. This desensitizes the receptor, making it less responsive to further stimulation. The recruitment of β-arrestin is measured using reports like luciferase, GFP, split luciferase, b-lactamase and RNA barcodes to quantify GPCR activation 4.

In Vitro High-Throughput GPCR screening

The screening of GPCRs outside of cell membrane systems is particularly challenging as they are highly unstable. In vitro GPCR screening utilizes GPCRs engineered for stability and uses fluorescence polarization and affinity mass spectrometry for the identification of potential ligand drug candidates. The main limitation of this GPCR screening technique has been the availability of fluorescently labeled substrates, yet current optimization of fluorescently labelled, high affinity ligands is combatting this limitation.

The application of high-throughput GPCR screening is shedding light on the different cell types that are activated by different ligands, key to the initial target identification of drug candidates. Developments in cell-based GPCR screening techniques are providing us with a more accurate understanding of GPCR targeting compounds. The use of fluorescently marked ligands in GPCR screening, as well as being safer than traditional radioligand binding assays, offer a more powerful and versatile analysis. All in all, high-throughput GPCR screening is constantly improving the efficiency of identifying potential new drug candidates in pharmacological assays.


  1. Zhang, R. and Xie, X. 2012. Tools for GPCR drug discovery. Acta Pharmacologica Sinica. 33(3), pp.372-384.
  2. Kumari, P., Ghosh, E. and Shukla, A.K. 2015. Emerging approaches to GPCR ligand screening for drug discovery. Trends in Molecular Medicine. 21(11), pp.687-701.
  3. Chen, L., Jin, L. and Zhou, N. 2012. An update of novel screening methods for GPCR in drug discovery. Expert opinion on drug discovery. 7(9), pp.791-806.
  4. Yasi, E.A., Kruyer, N.S. and Peralta-Yahya, P. 2020. Advances in G protein-coupled receptor high-throughput screening. Current opinion in biotechnology. 64, pp.210-217.
  5. Sittadjody, S., Thangasamy, T., NickKolgh, B., & Balaji, K. C. (2016). Non-androgen signaling pathways in castration-resistant prostate cancer. Managing metastatic prostate cancer in your urological oncology practice, 35-63.