Optimizing HTRF Assays with Fluorescent Ligands: Time-Resolved Fluorescence in GPCR Research

What is Homogeneous Time-Resolved Fluorescence (HTRF)?

HTRF is a hybrid detection technology that combines Förster Resonance Energy Transfer (FRET) with a time-resolved measurement. FRET is a distance-dependant energy transfer between a donor and an acceptor fluorophore, and in HTRF a delay is introduced between the time of excitation of the donor and the readout of the acceptor’s emission.

The donors used in this technique have longer half-lives than other fluorophores (between 300μs–1 ms) and can be combined with the same acceptor fluorophores used in regular FRET assays.

Terbium, a second-generation donor, is brighter than Europium (10-20 times), which increases sensitivity. When the distance between donor and acceptor is close enough, energy is transferred, and a second, short-lived emission is recorded. Measuring emission at both donor (usually 620nm) and acceptor (typically 665nm) wavelengths allows for data correction, reducing variability.

Figure 1. Principle of time-resolved detection. Source: Nørskov-Lauritsen L, Thomsen AR, Bräuner-Osborne H. G protein-coupled receptor signaling analysis using homogenous time-resolved Förster resonance energy transfer (HTRF®) technology. Int J Mol Sci. 2014 Feb 13;15(2):2554-72.

The delay introduced in HTRF between excitation and emission detection lets the background signals dissipate, reducing the impact of background interference (from autofluorescence or light scattering). This makes it an ideal candidate for GPCR research, where accuracy in detecting subtle signaling changes is essential.

Homogeneous Time-Resolved Fluorescence Assays: Overcoming Common Challenges

Balancing sensitivity and scalability is one of the hardest challenges in high-throughput screening (HTS). Traditional methods like radioligand binding assays or calcium flux measurements have significant drawbacks in this context: one is limited by radioactivity-related safety and environmental concerns, while the other one has high background noise and low dynamic range.

How are these challenges overcome by HTRF?

• No washing.

• Use of 384 or 1536-well plates and compatibility with automated platforms.

• Signal interferences are kept to a minimum thanks to the timing of detection.

• The ratiometric readouts correct many inconsistencies such as pipetting errors.

• The donor lanthanum fluorophores are more stable than regular fluorophores and quite resistant to photobleaching.

• The donors act as light-harvesting antennas, capturing light from all directions, unlike the dipole-dipole alignment needed in FRET.

Enhancing HTRF Assay Performance in GPCR Research Using Fluorescent Ligands

GPCRs are involved in numerous physiological processes, making them a key target in drug development. They activate several signaling pathways, via G proteins, β-arrestins, receptor tyrosine kinases, making them a complex task to study.

GPCRs are not always the most numerous in cells. Quite often, there is a need to amplify the signal strength to detect them, which can be achieved by using fluorescent ligands. This is moreso the case when detecting partial agonism or weak receptor interactions.

This technology enhances sensitivity and assay specificity. By using two labeled ligands the transference of energy event will only happen when the adequate distance is achieved. This means that even if one of the ligands they are bound to is promiscuous, it will not compromise assay integrity the same way it would in single-label approaches. This is especially useful in GPCRs, where structural similarity happens often and thus cross-reactivity of ligands is common.

In HTRF the lanthanide-based donors with longer emission enhance signal-to-noise ratio and reduce background interference.

They can be combined with second generation acceptors like d2, as well as brighter donors, further increasing sensitivity and assay specificity. This also improves detection of low affinity or slow binding ligands. On top of that, smaller acceptors like d2 reduce steric hindrance, making them more efficient.

It can also be combined with multiplexing. By using donor-acceptor pairs with different emission spectra that don’t overlap, researchers can design assays that track multiple pathways at the same time. Terbium is compatible with both red and green acceptors. This has been done in assays tracking IP1 and cAMP to detect biased agonism in GPCR ligands

Table 1. Examples of HTRF donor/acceptor pairs

Expanding Time-Resolved Fluorescence Applications in Drug Discovery Beyond Traditional Methods

At Celtarys, we have expertise in time-resolved fluorescence applications. In a recent study, we contributed to the development of a robust HTRF assay for the discovery of new modulators for cannabinoid receptors. This assay utilized our fluorescent ligand, CELT-335, designed for hCB₁/CB₂ cannabinoid receptors, demonstrating high specificity and sensitivity in detecting ligand-receptor interactions. 

Figure 2. Saturation assays using CELT-335. Specific binding is shown, obtained from total binding and unspecific binding (a) CB1R expressing adherent HEK-293T cells and unspecific binding measurement (specific binding measured using CP55490 at 10 μM concentration) (b) CB₂R expressing adherent HEK-293T cells and unspecific binding measurement (specific binding measured using GW405833 at 10 μM concentration). Data represent the mean ± SEM (n = 3 in triplicate). Source: Navarro G, Sotelo E, Raïch I, Loza MI, Brea J, Majellaro M. A Robust and Efficient FRET-Based Assay for Cannabinoid Receptor Ligands Discovery. Molecules. 2023 Dec 15;28(24):8107.

Celtarys enhances the power of HTRF and other FRET-based technologies by providing high-performance fluorescent ligands designed specifically for pharmacological research. By combining deep expertise in GPCR biology with advanced fluorescence chemistry, Celtarys custom-developed ligands offer both high affinity and exceptional selectivity across a wide range of GPCR targets.

References

• Navarro G, Sotelo E, Raïch I, Loza MI, Brea J, Majellaro M. A Robust and Efficient FRET-Based Assay for Cannabinoid Receptor Ligands Discovery. Molecules. 2023 Dec 15;28(24):8107. doi: 10.3390/molecules28248107
  

• Nørskov-Lauritsen L, Thomsen AR, Bräuner-Osborne H. G protein-coupled receptor signaling analysis using homogenous time-resolved Förster resonance energy transfer (HTRF®) technology. Int J Mol Sci. 2014 Feb 13;15(2):2554-72. doi: 10.3390/ijms15022554.
  

• Degorce F, Card A, Soh S, Trinquet E, Knapik GP, Xie B. HTRF: A technology tailored for drug discovery - a review of theoretical aspects and recent applications. Curr Chem Genomics. 2009 May 28;3:22-32. doi: 10.2174/1875397300903010022.