Advantages of Fluorescent Probes in GPCR Assays

GPCRs pose a critical challenge in drug discovery: a vast therapeutic potential with many targets yet to be explored.  35% of FDA-approved drugs target GPCRs, showing their importance in numerous physiological processes. However, over half of non-sensory GPCRs do not have drugs available in the clinic, opening the door to groundbreaking treatments for various diseases.

In order to fast-track these advancements, an improvement in tools for their study must happen. Thus we propose the use of fluorescent probes in GPCR screening assays due to their numerous advantages over traditional techniques like radioligands.

The advantages of Fluorescent Probes in GPCR assays over other methods

Fluorescent ligands are made by choosing an adequate pharmacophore with the desired affinity and/or functional activity, that is modified to introduce a linked and then conjugated with a fluorescent ligand. These probes facilitate localization of receptors, tracking of their internalization and are also safer alternatives to radioligand binding assays. They can also further pharmacological and structural studies, as they can be used to determine receptor activation dynamics, ligand binding geometry and receptor interactions.

The traditional binding assays for GPCRs use radioligands, whose limitations, especially safety concerns, low temporal resolution and the need for specialized facilities including disposal, mean their use should be reduced in favor of more accessible and safer alternatives, such as fluorescent ligands. On the other hand, fluorescent ligands have a superior safety and environmental profile. They are easier to handle and store, which also lowers outsourcing.

Fluorescent ligands provide real-time data on receptor activation, ligand binding and downstream signaling using live-imaging modalities. This means fluorescent probes enable a dynamic observation of GPCRs. For example, in Figure 1A, the dual A2B/A3 adenosine receptor fluorescent antagonist (CELT-327) was added to HCT116 cells (colon cancer cell line), detectable fluorescence starting at 1minute after addition. The fluorescence plateaus after approximately 10minutes. It can be appreciated how fast the ligand diffuses and is distributed homogeneously within monolayers, staining both apicobasal and lateral membranes (Figure 1B).

Figure 1. Real-time dynamic labeling of A_2BAR. A) Representative confocal images of HCT116 cells pre-stained with CellTracker Green (lower panels) and labeled in real-time with CELT-327 250 nM, added at timepoint 0 (upper panels). Scale bars: 50 μm. B) Orthogonal projections on a selected area of images in A. Scale bar: 20 μm. C) Quantification of CELT-327 average fluorescence intensity over time.

The Challenges Fluorescent Probes Face

There are several challenges fluorescent ligands face for their use as tools.

1.        Photobleaching. This happens when prolonged exposure to light in the excitation range causes the emission signal to decrease over time. This can lower the quality of long-term imaging studies but can be fixed by using stable fluorescent tags or specialized imaging techniques. The combination of the correct linker and dye can offer better photostability, maintaining signal integrity for longer.

2.        Spectral Overlap. The cells’ autofluorescence and the background noise from other dyes present in the experiment can interfere with measurements. This can be fixed by using fluorescent dyes that emit at longer wavelengths, where fluorescence from the cell components is minimal, as well as expanding the working range for imaging in vivo. Confocal microscopy is a technique that can be used to mitigate this issue.

At Celtarys, we focus on developing fluorescent tools that keep minimal background signal even without washing before cell visualization (Figure 2). Our portfolio also includes all kinds of dyes, such as pharmacophores conjugated to near-infrared fluorophores (CELT-075 hD2 dopamine receptor fluorescent antagonist and CELT-095 hM1/M2 muscarinic receptor fluorescent antagonist). Our proprietary conjugation technology also facilitates the synthesis of different linker sizes and types, making it easier to find the adequate probe for a certain technique and reduce these issues.

Another issue, that is present in all probes but can sometimes be stronger in fluorescent ligands, is non-specific binding. The dyes might have charges or be so hydrophilic/hydrophobic that they bind to intracellular structures. Thus, rational design using efficient synthetic strategies is needed to make these probes as specific as possible.

Figure 2. Representative confocal images of co-cultures of HCT116 cells and human primary CAFs. The dashed line represents the frontier between both cell types. CAFs are labeled with CellTracker (Green), and cocultures are labeled with CELT-327 250 nM. Scale bar 100 μm.

Protocols for GPCR assays using Fluorescent Probes

The preparation of the cellular system is the most common starting point for employing fluorescent probes in GPCR assays. Cells expressing or over-expressing the receptor of interest are cultured, preserving their physiological characteristics. Cell fragments (such as membranes) can also be used for some assays, reducing the possibility of non-specific binding.

Once the cells are ready, the fluorescent ligand is added, and the cells are incubated with it for a short time. For visualization purposes, cells can be co-stained with a green live-cell dye (Calcein) or Hoechst. For fixed cell preparations, DAPI (4′,6-diamidino-2-phenylindole), a DNA-specific fluorescent probe, can be used. After incubation, cells are observed using a confocal microscope.

In competition binding assays, another ligand is introduced in the medium to displace the fluorescent ligand, and the decrease in fluorescence as the concentration of the competitor is increased is the one used to obtain an affinity constant.

Celtarys’ protocol section includes detailed protocols for diverse applications and fluorescent ligands.

Contact us if you have any questions about our fluorescent ligands. Our scientific team can guide you choose or design the right fluorescent ligand for your research.

References

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Ciruela F, Jacobson KA, Fernández-Dueñas V. Portraying G protein-coupled receptors with fluorescent ligands. ACS Chem Biol. 2014 Sep 19;9(9):1918-28. doi: 10.1021/cb5004042. 

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.

Sridharan R, Zuber J, Connelly SM, Mathew E, Dumont ME. Fluorescent approaches for understanding interactions of ligands with G protein coupled receptors. Biochim Biophys Acta. 2014 Jan;1838(1 Pt A):15-33. doi: 10.1016/j.bbamem.2013.09.005.