Radioligands vs. Fluorescent Ligands: Binding Assays

Studying receptor-ligand interaction is a crucial approach in drug discovery and biomedical research. Radioligands have been the gold standard for binding assays since their first use to study G-protein coupled receptors (GPCRs) in 1970. While radioligands have been widely used for decades, advances in fluorescent probe design have shifted towards fluorescence-based approaches due to their versatility, safety, and precision.

What Are the Benefits of Radioligands?

Radioligands are molecules labeled with radioactive isotopes commonly used in radioligand binding assays to quantify receptor-ligand interactions. A radionuclide replaces a stable atom in the ligand, emitting detectable radiation. Common radioisotopes used to generate radioligands are tritium (³H) and ¹²⁵Iodine (¹²⁵I), although ¹⁴Carbon (¹⁴C), ³⁵Sulfur (³⁵S), and ³²Phosphorous (³²P) may also be used. 

Radioligands are used to assess ligand-binding measuring compound affinities, to study receptor density, binding sites, and ligand kinetics in various biological systems. Some benefits of using radioligands for receptor binding assays are the minimal chemical modification required of the ligand, their sensitivity, and extensive validation during the last decades, making it a robust and reliable assay. ³H-labeled ligands are usually chemically identical to the equivalent nonradioactive ligand (because hydrogen atoms are substituted with ³H). They also show a slow radioactive decay due to the long half-life (12.4 years), which results in a low detection efficiency. Nevertheless, ¹²⁵I presents a high specific activity but exhibits a short half-life (60 days). It is a large atom generally introduced into an aromatic ring of the ligand, changing the chemical structure which may affect interaction with the receptor in the radioligand binding assay. 

Despite these specific advantages, radioligands come with significant general drawbacks, including radiation exposure risks, high disposal costs, and stringent regulatory requirements. Handling radioactivity requires specific facilities and equipment, as well as safety training and specific certification for the users. Due to this, experiments involving radioactivity usually get outsourced, losing control over the protocol. Radioligands have been the gold standard in binding assays since they were long discovered 50 years ago, but the advances in synthesizing high-affinity fluorescent ligands offer an alternative to bypass these limitations.

Radioligands vs. Fluorescent Probes: Differences in Binding Assays

Fluorescent ligands are a specific type of fluorescence probes that offer a powerful alternative to radioligands in binding assays. 

Fluorescent ligands combine a ligand with the desired functional activity (e.g. agonist, neutral antagonist or reverse agonist for receptors such as GPCRs) for the target of interest conjugated to a fluorescent dye. The pharmacophore or ligand, the molecule that interacts with the target, and the fluorophore, the fluorescent dye, are conjugated by a third component, the linker (Figure 1).

Figure 1. Fluorescent ligand general structure

Strategic pharmacophore labeling and linker optimization ensure access of the pharmacophore to the receptor binding site and minimal unspecific interactions, tuning finely pharmacological, physicochemical, and photophysical properties of the final probes. 

Radioligand binding assay protocols rely on a filtration assay or scintillation proximity technologies to measure ligand binding. On the other hand, fluorescent ligand binding can be easily monitored through confocal microscopy, plate readers, or fluorescence polarization and flow cytometry, making them valuable alternatives to radioligand binding assays in receptor studies and drug screening.

Fluorescent ligands can be used at a broader range of concentrations compared to radioligands without losing accuracy. Additionally, fluorescent ligands can help detect different receptor conformation states. 

Convenience and ease of handling in the laboratory are other major differences in binding assays using fluorescent ligands vs. radioligands. Unlike radioligands, which require strict regulatory compliance, controlled storage, and specialized disposal methods, fluorescent ligands are much simpler to implement. They can be stored at standard laboratory conditions; there are no supply shortages, they are safer to use, and they do not require additional licensing or radiation protection measures. This makes fluorescence-based approaches more accessible and cost-effective, gaining complete control over the binding assay.

When to Use Radioligands or Fluorescent Probes in Receptor Studies?

While radioligands remain valuable in certain applications, fluorescent ligands are increasingly preferred due to their safety, flexibility, and ability to enable live-cell imaging. The choice between radioligands and fluorescence probes depends on the specific research needs:

• Quantitative binding studies: Radioligand binding assays excel in precise quantification of ligand-receptor interactions, particularly in pharmacokinetic and receptor occupancy studies. However, fluorescent ligands offer quantitative fluorescence-based alternatives that can provide sensitivity without radiation risks.
• Live-cell imaging: Fluorescent ligand applications extend to advanced imaging techniques to study the cellular and tissue localization of a receptor. Fluorescent ligands have been validated as useful tools to visualize receptors in native and transfected cells, both in living cells and in cells after fixation (Figure 2). This capability is complementary to other applications, such as competition binding microscopy, and it is essential in understanding receptor dynamics and intracellular 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: High-affinity radioligands are not available for all the targets. Modern advancements in fluorescent probe design enable the creation of highly selective probes tailored to specific receptors. This allows to fill the gap related to the lack of available optimal fluorescent probes for targeted imaging and binding assays for many proteins with pharmacological interest. 

At Celtarys, we have developed a new rational, convergent, and efficient synthetic strategy that allows the development of customized fluorescent probes. Celtarys technology allows the generation of new fluorescent ligands for any therapeutical target. Thanks to our proprietary conjugation technology, we can generate hundreds of different combinations of pharmacophore, linker architecture, and fluorophore at the same time. This allows us to develop fluorescent probes with high affinity and selectivity for any target of interest in a time and cost-effective manner. 

Our expertise in ligand-receptor interactions and fluorescence techniques allows us to manage all the process phases, from the design of the new fluorescent probes to their validation as optimal tools for your assays. 

Contact us and tell us about your idea, we are sure we can design the right fluorescent ligand for your research. 

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.