A2A Fluorescent Competitive Binding: Advancing NanoBRET® Target Engagement for GPCR Drug Discovery

The A2A adenosine receptor (A2AAR) is one of four adenosine receptor subtypes expressed in the human body (A1, A2A, A2B, and A3). It plays a key role in immune system downregulation, making it an attractive target for conditions in which immune reactivation is desired. A2AAR-targeted therapies have advanced to phase II clinical trials for various cancers, particularly in combination with other immune checkpoint inhibitors.1

In a shared effort to develop robust screening approaches that can serve as practical alternatives to radioligand binding assays, Celtarys Research and PROMEGA combined their respective technologies to support GPCR drug discovery. A new NanoBRET® competitive binding assay² was developed in collaboration with Professor Kevin Pfleger’s laboratory (University of Western Australia), using a new Celtarys fluorescent ligand.

Figure 1. Scheme of a competitive NanoBRET® assay.

GPCRs are part of Celtarys’ expertise fields. Using a similar pharmacophore to the one present in the A_2AAR probe CELT-300, new fluorescent ligands adapted to the NanoBRET® technology were designed and synthesized. The Nanobret® 590 Dye commercialized by PROMEGA was used as the fluorophore tag. The competitive assay design and optimization were performed in the University of Western Australia.

GPCRs are part of Celtarys’ expertise fields. Using a similar pharmacophore to the one present in the A_2AAR probe CELT-300, new fluorescent ligands adapted to the NanoBRET® technology were designed and synthesized. The Nanobret® 590 Dye commercialized by PROMEGA was used as the fluorophore tag. The competitive assay design and optimization were performed in the University of Western Australia.

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Combining Two Technologies Into One to Measure A2A Fluorescent Competitive Binding

Bioluminescence resonance energy transfer (BRET) serves as the basis for the NanoBRET® Target Engagement (TE) Technology. In this proximity-based approach, a NanoLuc®luciferase genetically fused to the target protein transfers bioluminescence to a fluorescent tracer binding to the target protein. In the competitive assay, A_2AAR ligands are set to compete with the probe, and the interactions between the ligands and protein are quantified in real time by measuring the probe’s emission in intact cells.

To synthesize the probe, Celtarys has applied its proprietary conjugation technology. First the fluorescent ligand is properly functionalized to keep its activity intact. Then, the linker composition is optimized, by using different hinges and spacers. Afterwards, the fluorophore is attached and the final probes evaluated. 

Celtarys’ technology significantly reduces the time it takes to obtain fluorescent tags and test varying linker structures and lengths, and it was used here to ensure optimal performance in NanoBRET® TE assays.

Results

The combination of both technologies, and the expertise of Prof. Pfleger’s group in developing assays, led to two new A_2AAR tracers (CELT-463 and CELT-464), bearing the same pharmacophore and fluorophore, but with different linkers. They can be used to verify target engagement and calculate ligand affinity in a NanoBRET®-based competitive binding assay. 

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Saturation binding experiments on NanoLuc®-tagged A2A receptors.

First, saturation binding assays were performed. They are needed to identify the best concentrations for each tool. The curves obtained were consistent, and high specific binding and optimal signal to noise ratio were observed.

Figure 2. Saturation Binding Experiment for CELT-463 (KD=33±4nM) and CELT-464 (KD=44±5nM) using HEK293FT cells transiently transfected with signal peptide nanoluc®-A2AAR expression vector. Transfected cells were treated with increasing concentrations of CELT-463 or CELT-464 in the presence (non-specific binding) or absence (total binding) of SCH 442416. Specific binding was calculated by subtracting non-specific binding from total binding (mean±SEM, n=6).

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NanoBRET® competitive binding assays on A2AAR with CELT-463 and CELT-464

Using the previous data as reference, 50nM was chosen as the tracer concentration for the assays, as it produces a sufficiently large window to perform the experiment. Increasing concentrations of the competitor compounds were added and the signal measured. As seen in figure 2, a heterogenous set of compounds (agonists and antagonists, different structures) was measured using both CELT-463 and CELT-464.

Figure 3. Measurement of competitive ligand binding to A2AAR using tracers CELT-463 and CELT-464 in NanoBRET® assays for a set of reference compounds. Cells expressing signal peptide-nanoluc®-A2AAR were treated with 50nM CELT-463 or CELT-464 in the presence of increasing concentrations of various competitor compounds (mean±SEM, n=6).

Selective or promiscuous, agonists or antagonists were for all 4 Adenosine Receptors were included in this set. the data obtained were compared with those reported in literature, which were obtained employing radioligand binding assays.

Table 1. Set of reference compounds tested for assay validation, together with the reported and experimental binding data. PKI values were derived from pIC50 values using the Cheng-Prusoff equation.³

The pKi values display a similar order of affinity to the reported values, guaranteeing the NanoBRET® competitive binding assay on A_2AAR, meaning CELT-463 and CELT-464 are a valid alternative to radioligand binding and other traditional methodologies.

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Conclusions

This collaboration between PROMEGA, Celtarys Research, and the University of Western Australia led to the identification of two fluorescent ligands, CELT-463 and CELT-464, optimized for NanoBRET®-based A2A fluorescent competitive binding affinity screening. Both are effective as NanoBRET® TE tracers, leading to similar results to those present in literature using traditional screening methods.

As a proof of concept, the study shows that Celtarys’ chemistry can be translated into NanoBRET® TE GPCR assays compatible with 384-well screening formats. For research teams, this provides a practical framework for integrating fluorescent ligand design with live-cell target engagement assays. The next step will be to determine how broadly this approach can be extended across GPCR families and how predictive these measurements are in downstream discovery workflows.

Check the method to perform this assay and other case studies we have done on our website: https://www.celtarys.com/case-studies 

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References

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