Fluorescence Polarization in GPCR Research

The ability to identify compounds that interact with molecular targets involved in disease pathways is where the development of new therapeutics lies. Target-based screening is now a fundamental pillar of drug development, and GPCRs are key targets for disease treatment. Using this method, binding affinity, kinetics and selectivity can all be measured and used to establish the best compounds.

To efficiently run these screening campaigns high throughput screening assays are used. They provide rapid and scalable assessment of pharmacological parameters. There are several fluorescent techniques used in HTS, among them Fluorescence Polarization (FP) is especially useful since it allows real time detection and there is no need for washing steps.

How Fluorescence Polarization Assays Work: Principles and Applications in GPCR Research

FP assays work on the principle that a fluorescent ligand bound to protein rotates slower than a free fluorescent ligand. This combined with the use of polarized light leads to a signal that can only be detected when the fluorescent ligand is bound to the protein.

When polarized light hits a free fluorescent ligand, its rotation speed is so fast that the emission is depolarized, which is not read by the FP filters. On the other hand, when the polarized light hits the bound fluorescent ligand, the slower rotation speed means a polarized emission which is detected by the filter. This means no washing steps are needed to reduce background signal.

Figure 1. Mechanism of fluorescence polarization. Adapted from: Zhang Y, Tang H, Chen W, Zhang J. Nanomaterials Used in Fluorescence Polarization Based Biosensors. Int J Mol Sci. 2022 Aug 3;23(15):8625

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This technique is most effective at studying interactions between large proteins and small ligands thanks to this change in rotation speed. This makes them very useful for GPCR drug discovery, since receptors are a lot bigger than their small molecule ligands.

Optimizing GPCR Drug Discovery with Fluorescence Polarization: Key Advantages and Future Perspectives

1.       FP assays are convenient and easy to manipulate.

They are more accessible than radioligand assays, since they do not require complex equipment for analysis and plate readers are usually available in regular laboratories, although not all have FP filters, they are usually included.

In this article by Miranda-Pastoriza et al. we have demonstrated that both radioligands and FP assays using CELT-228 A3AR fluorescent antagonist provide similar binding affinity values. These results support the use of fluorescence-based screening methods as a reliable alternative to classical radioligand binding assays.

Table 1. Comparison of hA3 binding affinities or percentage of displacement at 1 µM measured for different compounds in human cell lines. hA₁, hA₂A, hA₂B, and hA₃ (radioligand assays): Displacement of specific radiolabeled ligands in CHO, HeLa, or HEK-293 cells, expressed as K_i (nM ± SEM, n=3) or percentage displacement at 1 µM (n=2). hA₃ (fluorescence polarization assay): Displacement of CELT-228 detected by FP measurements (n=3). Reference compounds XAC, ISVY-130, and MRS 1220 were included as standard A₃AR antagonists. 

2.       Compatible with various sources of GPCRs.

Conventional membrane preparations or baculoviruses can be used among others. We highlight this article by Tahk et al. where they used CELT-419 for D3 dopamine receptor binding assays in baculoviruses.

3.       Not time-sensitive

Once equilibrium has been reached the assay remains stable for extended periods (at least 60 to 90minutes), being limited by the stability of the fluorescent ligand or receptor preparations in the assay media.

4.       No energy transfer between two fluorophores.

Unlike other techniques (FRET-based), only one fluorophore is needed. In this case only the distance between fluorophore and protein matters (for the rotation to be slowed), not the distance between the protein, donor and acceptor.

Advancing Fluorescence Polarization Assays with Custom Fluorescent Ligands

Looking ahead, fluorescence polarization technology is expected to expand its role in GPCR research through the development of novel fluorescent probes and advanced imaging techniques. New generations of fluorophores with improved photostability and brightness will enhance assay sensitivity, while integration with artificial intelligence-driven data analysis will accelerate hit identification and lead optimization.

The future of fluorescence polarization assays is closely tied to fluorescent ligands. Contact us to drive further advancements in GPCR drug discovery!

References

Kumar V, Chunchagatta Lakshman PK, Prasad TK, Manjunath K, Bairy S, Vasu AS, Ganavi B, Jasti S, Kamariah N. Target-based drug discovery: Applications of fluorescence techniques in high throughput and fragment-based screening. Heliyon. 2023 Dec 19;10(1):e23864. doi: 10.1016/j.heliyon.2023.e23864. 

Miranda-Pastoriza D, Bernárdez R, Azuaje J, Prieto-Díaz R, Majellaro M, Tamhankar AV, Koenekoop L, González A, Gioé-Gallo C, Mallo-Abreu A, Brea J, Loza MI, García-Rey A, García-Mera X, Gutiérrez-de-Terán H, Sotelo E. Exploring Non-orthosteric Interactions with a Series of Potent and Selective A3 Antagonists. ACS Med Chem Lett. 2022 Jan 10;13(2):243-249. doi: 10.1021/acsmedchemlett.1c00598.