Radioligands vs. Fluorescent Ligands: Binding Assays

Understanding receptor-ligand interaction is key in drug discovery and biomedical research. Radioligands have been used to study GPCRs for decades, but with the advances in the fluorescence field, assays have shifted towards fluorescent-approaches, thanks to their versatility, safety and precision.

What Are the Benefits of Radioligands? 

They are ligands labeled with radioactive isotopes which can be used in binding assays to quantify other ligands’ interaction with the receptor. One of the atoms in the original molecule is replaced by a radionuclide, which emits radiation. Some examples are tritium (3H) and 125Iodine (125I), 14Carbon (14C), 35Sulfur (35S), and 32Phosphorous (32P).

Besides binding assays, they can also be used to study receptor density, binding sites and ligand kinetics in biological systems. They have several advantages, such as the minimal chemical modifications in the original ligand, high sensitivity and their extensive and successful use throughout decades.

Tritium (3H) labeled ligands are usually chemically identical to the original, because tritium will usually substitute hydrogen atoms. The problem is their long half-life, which results in lower detection efficiency. Another common radioactive atom, 125I, with a higher specific activity, but a shorter half-life (60 days), which complicates storage, besides changing the chemical structure of the ligand, as it is generally introduced into aromatic rings.

While their high specificity and extensive experience working with them, other drawbacks include radiation exposure, disposal costs and regulatory requirements for working with them. Handling of radioligands must be done by trained users in specific facilities and using specific equipment. Thus, it’s frequently outsourced to academic groups or companies who have everything set-up.

Radioligands vs. Fluorescent Probes: Differences in Binding Assays

Fluorescent ligands are a type of fluorescent probes that offer an alternative to radioligands in binding assays. They combine the desired functional activity of the original ligand, by conjugating it to a fluorescent dye (Figure 1). This can be done with agonists, antagonists, reverse agonists in the case of GPCRs.

Figure 1. General structure of a fluorescent ligand.

Strategic placement of the linker in the pharmacophore and its optimization ensure access to the binding site with minimal unspecific interactions, while also tuning the pharmacological, physicochemical and photophysical properties of the conjugated ligands.

Traditional radioligand protocols rely on filtration assay or on scintillation proximity technologies to measure ligand binding. On the other hand, fluorescence signals can be easily monitored through confocal microscopy, plate readers, fluorescence polarization and flow cytometry, proving more versatile.

Fluorescent ligands can be used at a broader range of concentration without losing accuracy, as well as detect different receptor conformation states.

Ease of handling is the other key difference, because fluorescent compounds do not require regulatory compliance, specific disposal methods and dedicated facilities, making them much easier to implement in any lab.

Fluorescent Probes and Radioligands: When should you use one or the other?

The choice between these two depends on the results needed – but in case they are similar, fluorescent probes provide a safer and more accessible alternative.

-            Quantitative cell binding studies: Radioligands have superior precision when accounting for ligand-receptor interactions, particularly in pharmacokinetic and receptor occupancy studies. However, fluorescent ligands can be used as well as a safer and more accessible alternative.

-            Live-cell imaging: Fluorescent ligands can be used to study cellular and tissue location of a receptor. They are useful tools to visualize receptors in native or transfected cells, whether living or fixed. This is complimentary with other applications, and essential in our understanding of signaling pathways.

Figure 2. Representative images of non-fixed HCTT116 colorectal tumor cells co-stained with hA2B-A3 adenosine receptor fluorescent antagonist (Celt-327) and Calcein. Images were taken 2 min after the addition of Celt-327 to the medium.

-            Fluorescent probe design for specific targets: Not all targets have available high-affinity radioligands, but with the development of potent fluorescent probes, this gap can filled in a timely and safe manner.

If you are curious about fluorescent probes, fluorescent probe design or GPCR tools, feel free to contact us!

References

Flanagan CA. GPCR-radioligand binding assays. Methods Cell Biol. 2016;132:191-215. doi: 10.1016/bs.mcb.2015.11.004. 

Soave M, Briddon SJ, Hill SJ, Stoddart LA. Fluorescent ligands: Bringing light to emerging GPCR paradigms. Br J Pharmacol. 2020 Mar;177(5):978-991. doi: 10.1111/bph.14953.

Stoddart LA, Kilpatrick LE, Briddon SJ, Hill SJ. Probing the pharmacology of G protein-coupled receptors with fluorescent ligands. Neuropharmacology. 2015 Nov;98:48-57. doi: 10.1016/j.neuropharm.2015.04.033.