Fluorescent Probes for High-Throughput Screening: A Cost-Effective Alternative to Radioligands in Preclinical Research

What Is High-Throughput Screening?

High-throughput screening (HTS) is a starting point to discover a new drug candidate. This method is used primarily in the early stages of drug development to evaluate large libraries of chemical compounds for potential biological activity. HTS involves exposing biological targets, such as G protein-coupled receptors (GPCRs), to a wide range of candidates and measuring their effect using a variety of technologies and readouts. The goal is to identify new “hits”, which means compounds that show a moderate activity against the target of interest, laying the foundation for further optimization, which will lead to the final therapeutic agents (also called ”leads”).

Biochemical screening in HTS measures the binding of ligands or the inhibition of enzymaticactivity in vitro. Competitive binding is a common biochemical screening method used to determine if the tested compounds can displace a known ligand from its binding site in the  target. These assays are compatible with 384-well and 1536-well plates, automation, and read out using optical methods such as absorbance, fluorescence, or luminescence. These are essential requirements for HTS, allowing tens of thousands of experiments in a matter of days.

As the volume of testing increases thanks to HTS assays, so do the demands for safer, more robust, and economically sustainable assay components. This is where fluorescent probes are becoming game-changers.

Why Fluorescent Probes are Transforming High-Throughput Screening in Drug Discovery

Fluorophores are molecules that emit light upon excitation and can be employed to generate fluorescent ligands that bind specifically to the desired biological targets. In high-throughput screening assays, they are used to detect interactions between ligands and their targets or to monitor downstream biological effects. Fluorescent methods have largely replaced traditional radiolabeled ligand assays due to their non-radioactive, stable, and safer use, significantly reducing regulatory and operational burdens.

One of the most compelling advantages of fluorescent ligands lies in their versatility. They are compatible with various assay formats, including:

• Fluorescence Polarization (FP): Ideal for studying molecular interactions, fluorescence polarization assay detects changes in the rotation of fluorescent molecules upon binding, making it highly useful for detecting small-molecule–protein interactions. The fluorescence polarization protocol is compatible with 384-well plates.
  
• Förster Resonance Energy Transfer (FRET): This technique detects energy transfer between a donor and an acceptor fluorophore nearby. It allows real-time monitoring of conformational changes or protein-protein interactions.
  
• Homogeneous Time-Resolved Fluorescence (HTRF): It is a variation of FRET combined with time-resolved (TR) measurement. It introduces a delay between excitation and emission measurement using lanthanides as donors to reduce background noise and enhance sensitivity. 

Figure 1 shows an example of the HTRF competition binding between hCB1/CB2 cannabinoid receptor fluorescent ligand CELT-335 and three natural cannabinoids (THC, THCA, and THCV).

Figure 1. Competition experiments of binding of fluorophore-conjugated CELT-335 to living HEK-293 T cells expressing the CB₁R. Source: Raïch I, Rivas-Santisteban R, Lillo A, Lillo J, Reyes-Resina I, Nadal X, Ferreiro-Vera C, de Medina VS, Majellaro M, Sotelo E, Navarro G, Franco R. Similarities and differences upon binding of naturally occurring Δ9-tetrahydrocannabinol-derivatives to cannabinoid CB1 and CB2 receptors. Pharmacol Res. 2021 Dec;174:105970.

From a cost-effective perspective, fluorescent assays offer significant advantages. Fluorescent ligands can be developed in a conventional laboratory and their use is not only restricted to binding assays. They are also compatible with the visualization and quantification of receptors by imaging techniques, such as fluorescence microscopy or flow cytometry. 

Experiments involving radioligands usually require outsourcing due to stringent safety protocols and regulations and the lack of specialized radioactive waste disposal. Fluorescent assays can be performed in the laboratory using common equipment. Fluorimeters are standard equipment in most biology laboratories, while scintillators typically need to be specially acquired for working with radioactivity. Besides, fluorescent ligands can be stored and often have longer shelf lives, reducing repeat orders and minimizing assay variability due to reagent degradation.Moreover, advances in probe design and detection technologies have dramatically improved the sensitivity and specificity of fluorescent assays. Signal-to-background ratios have increased, thanks to techniques such as homogeneous time-resolved fluorescence or the use of confocal microscopes, allowing for reliable detection even in complex biological matrices. This makes fluorescence-based HTS a competitive and, in many cases, superior alternative to radioligand approaches.

Assay Development and Optimization: How Fluorescent Probes Improve HTS Workflow Efficiency

Developing a reliable high-throughput assay involves multiple steps, from target selection and probe validation to signal optimization and data analysis. Fluorescent probes streamline several of these stages, ultimately improving workflow efficiency and data quality:

• Fluorescent assays are generally easier to miniaturize and automate. Since they do not involve the same safety restrictions as radioligands, they can be integrated into high-density plate formats and robotic systems without additional infrastructure or protocols. 
• Fluorescent probes maintain stability under proper storage conditions, ensuring consistent performance over time. Some radioligands, such as ¹²⁵Iodine, suffer from relatively short half-lives and require careful handling to avoid decay-related variability. 
• Fluorescence allows for real-time and kinetic measurements. A separate assay point is required for each time point using radioligands. Fluorescent ligands allow direct measurement in real time using microplate readers or confocal microscopes with frequent measurements. 
• The multiplexing capability of fluorescence readouts using different fluorescent probes emitting at distinct wavelengths enables researchers to monitor multiple targets or cellular structures simultaneously within the same assay well, increasing throughput without additional resources.

Advances in imaging and plate-reader technologies, along with the increasing availability of commercial fluorescent ligands, are making fluorescent probe uses more accessible to labs of all sizes. When suitable commercial ligands are not available,  Celtarys, applying its proprietary can generate custom fluorescent ligands tailored to specific targets, designing the most appropriate fluorescent probes according to each project’s requirements. Academic research centers, biotech startups, and large pharmaceutical companies alike can now access affordable, scalable tools to develop their own fluorescent-based high-throughput screening assays, democratizing the early stages of drug discovery.

References

Blay V, Tolani B, Ho SP, Arkin MR. High-Throughput Screening: today’s biochemical and cell-based approaches. Drug Discov Today. 2020 Oct;25(10):1807-1821. doi: 10.1016/j.drudis.2020.07.024.

Fang X, Zheng Y, Duan Y, Liu Y, Zhong W. Recent Advances in Design of Fluorescence-Based Assays for High-Throughput Screening. Anal Chem. 2019 Jan 2;91(1):482-504. doi: 10.1021/acs.analchem.8b05303.

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

Raïch I, Rivas-Santisteban R, Lillo A, Lillo J, Reyes-Resina I, Nadal X, Ferreiro-Vera C, de Medina VS, Majellaro M, Sotelo E, Navarro G, Franco R. Similarities and differences upon binding of naturally occurring Δ9-tetrahydrocannabinol-derivatives to cannabinoid CB1 and CB2 receptors. Pharmacol Res. 2021 Dec;174:105970. doi: 10.1016/j.phrs.2021.105970. 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.