Optimizing HTRF Assays with Fluorescent Ligands: A Guide for Time-Resolved Fluorescence in GPCR Research
What is Homogeneous Time-Resolved Fluorescence (HTRF)?
Homogeneous Time-Resolved Fluorescence (HTRF) is a hybrid detection technology that combines Förster Resonance Energy Transfer (FRET) with time-resolved measurement. In this distance-dependent energy transfer between a donor and an acceptor fluorophore, there is a time delay between the excitation of the donor and the readout of the acceptor emission.
HTRF assays rely on long-emitting donor molecules, lanthanides like europium or terbium. These donors have long half-lives (300 μs–1 ms) compared to other fluorophores and can be used with the same acceptor molecules used in FRET.
Terbium, a second-generation donor, is 10-20 times brighter than europium, which provides higher sensitivity. When a biomolecular interaction brings it close to the acceptor, energy is transferred, and a second, short-lived emission is recorded. Measuring emission at both donor (typically 620 nm) and acceptor (usually 665 nm) wavelengths allows for ratiometric data correction, minimizing variability due to differences in well content or media composition.
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.
Unlike traditional fluorescence-based assays, which suffer from background interference due to autofluorescence or light scattering, the delay introduced in HTRF between excitation and emission detection allows the transient background signals to dissipate. The result is a cleaner, more sensitive signal. Time-resolved fluorescence is ideal for applications such as G protein-coupled receptor (GPCR) research, where accuracy in detecting subtle signaling changes is essential.
Homogeneous Time-Resolved Fluorescence Assays: Overcoming Common Challenges
One of the enduring challenges in high-throughput screening (HTS) for drug discovery has been balancing sensitivity with scalability. Traditional methods, such as radioligand binding assays or calcium flux measurements, present significant drawbacks: the former is limited by radioactivity-related safety and environmental concerns, while the latter suffers from high background noise and limited dynamic range.
HTRF solves many of these issues through its homogeneous (no-wash) format, enabling miniaturization to 384- or 1536-well plates and compatibility with automated platforms. Because of the long-lived nature of the donor fluorescence and the precise timing of detection, the method suppresses signal interference from biological matrices. In addition, the dual-wavelength ratiometric readout corrects for many inconsistencies caused by media, compound autofluorescence, or pipetting errors. The amount of false-positive or false-negative hits is reduced. Lanthanum fluorophores are not easily photobleached. It may take place after 2 minutes, a duration significantly longer than the typical timeframe applied in HTRF assays.
These molecules are also more stable and act as light-harvesting antennas, capturing light from all directions. This property eliminates the need for the precise dipole-dipole alignment, which is a great advantage of HTRF vs. FRET.
Enhancing HTRF Assay Performance in GPCR Research Using Fluorescent Ligands
GPCRs represent a vast family of receptors involved in numerous physiological processes and remain key targets in drug development. Their ability to activate multiple signaling pathways, via G proteins, β-arrestins, or receptor tyrosine kinases, makes them complex yet fascinating targets for drug discovery.
Fluorescent ligands improve assay sensitivity by amplifying signal strength and enabling the detection of low-abundance events, such as weak receptor activation or partial agonism. In HTRF, the use of lanthanide-based donors like terbium cryptate, which emits stronger and longer-lived fluorescence than earlier europium-based systems, has enhanced signal-to-noise ratios and reduced background interference. These improvements are crucial when working with receptors that are poorly expressed or only weakly coupled to downstream effectors.
The use of advanced fluorescent ligands, especially second-generation acceptors like d2 and brighter donor cryptates such as Lumi4-Tb, has further improved assay sensitivity. These ligands allow researchers to detect low-affinity or slow-binding ligands that might be missed in traditional assays. Additionally, smaller acceptor molecules such as d2 reduce steric hindrance, making the assays more efficient and compatible with a wider range of targets and experimental conditions.
This technology not only enhances detection sensitivity but also increases assay specificity. By using two labeled ligands, one with the donor (a lanthanide) and one with the acceptor fluorophore, the signal only arises when both are brought into proximity through a specific binding event. Even if one of the ligands exhibits promiscuity, it does not compromise assay integrity in the same way as in single-label approaches, since both components must participate for energy transfer to occur. This added layer of selectivity is especially important for GPCRs within the same family, where structural similarity and cross-reactivity among ligands are common.
Multiplexing is another major advantage. By using donor-acceptor pairs with non-overlapping emission spectra, researchers can design assays that track multiple pathways in the same well. Terbium is compatible with not only red acceptors but also green acceptors. This has been demonstrated, for example, in assays that simultaneously measure IP1 and cAMP to reveal pathway selectivity or biased agonism in GPCR ligands.
Some examples of HTRF donor/acceptor pairs are shown in Table 1.
Table 1. Examples of HTRF donor/acceptor pairs
Expanding Time-Resolved Fluorescence Applications in Drug Discovery Beyond Traditional Methods
While HTRF has transformed how researchers study GPCR signaling and other molecular interactions, its full potential depends heavily on the quality and specificity of the fluorescent probes used. In particular, the use of optimized fluorescent ligands is crucial for achieving reliable, reproducible, and biologically relevant readouts.
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.
We can design and implement fluorescent ligands for HTRF assays.
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.
