A robust and Efficient FRET-Based Assay for Cannabinoid Receptor Ligands Discovery.

1.     Introduction

1.1 The Endocannabinoid System

The Endocannabinoid System (ECS) is composed of the Cannabinoid Receptors 1 and 2 (CB1R and CB2R) and the endocannabinoids (endogenous ligands such as 2-arachidonoylglycerol and anandamide) as well as metabolic enzymes.

It is involved in several pathophysiological processes such as Parkinson’s disease, depression, addiction, eating disorders and has synergistic effects with anticancer agents.[1–3] These insights highlight the potential in modulating the ECS to fulfill unmet medical needs. New scaffolds modulating the CBRs in both the orthosteric and allosteric sites have been developed, supported by resolved crystal structures of both CBRs.[4–8]

A key challenge in CBR modulator development is separating the therapeutic effects from the side effects, especially the psychotropic effects CB1R causes in the Central Nervous System. This can be appreciated when talking about SCRAs (Synthetic Cannabinoid Agonists), as one of the series developed by Pfizer, including CP5594, gave rise to a new class of abused psychoactive substances, thanks to their higher potency in the CB1R. [9,10]

1.2 Novel fluorescent strategies for CBR binding affinity assays

Thus, the need for novel, robust and cost-effective methodologies for screening compound libraries targeting the CBRs becomes key for identifying drugs with a clinical profile. The most used probe right now is the radioactive CP-55940, [3H-CP-55940].[11]

Figure 1. Chemical structure of [3H-CP-55940].

Selective fluorescent CB1R ligands have been validated for flow cytometry [12], and selective CB2R probes have been validates in TR-FRET[13], as well as receptor visualization in living cells[14], mice[15], and zebrafish[13].

Homogeneous Time-Resolved Fluorescence (HTRF) is a TR-FRET-based assay[16] conducted in homogeneous conditions, using lanthanide (europium, terbium) as donors.[17] Lanthanides provide unique advantages as donors, since they have extended fluorescence duration, enable delayed emission readings, have narrow bands and high stokes shift, which prevent cross-excitation and cross-emission phenomena.[18] Thus, they reduce background noise, improving both the signal-to-noise ratio and the sensitivity of the assays. [18–20]

Several strategies can be used to attach donor fluorophores to the target, among them antibodies bearing the donor. GPCRs are not suitable for antibody use due to steric hindrance and reverse binding, thus, other strategies must be applied. Among them, we have chosen the SNAP-tag, a suicide enzyme technology. The SNAP-tag is an engineered mutant of O5-alkylguanine-DNA alkyltransferase (AGT) that reacts specifically with O6-benzylguanine (BG) derivatives. This modification is small enough to not affect GPCR expression or activity.[21–23]

The Tag-lite® binding assay combines the HTRF detection method with the SNAP-tag. It has been applied to different GPCR binding assays, such as CXCR4, opioid receptors, CCK1 and CCK2. We have previously used this technology using CELT-335, a dual hCB1/hCB2 fluorescent ligand, which was validated in CB1R as a binding assay probe with three natural CBR ligands.

Herein we report the first example of a Tag-lite® binding assay for both CBs using CELT-335, which exhibits a high specific binding (signal-to-noise ratio), and FERT signal, providing a reliable, robust and cost-effective alternative for CB1R and CB2R screening campaigns.[24]

2.     Results

2.1  CELT-335 Binding at CB1 and CB2 Receptors

The first step for the development of the assay is to obtain a fluorescent ligand suitable for the Tag-lite® binding assays. In this case, CELT-335 exhibits exc and em at 650nm and 673nm, which are compatible with the lanthanide Terbium.

Their binding affinity was assessed through radioligand binding, showing a nanomolar affinity for both CBRs, with it being 6-fold higher for CB2R.

Table 1. Comparison of CELT-335 affinity for CB1 and CB2 receptors measured by competition radioligand binding assay (Ki) and saturation Tag-lite® binding assay (Kd). 1 Competition radioligand binding assay. 2 Saturation assay by Tag-lite® binding assay. 3 Displacement of specific [3H]-CP55940 binding in human HEK-CB1 cells expressed as Ki ± SEM in nM (n = 3) or percentage displacement of specific binding at a concentration of 1 μM (n = 2). 4 Displacement of specific [3H]-CP55940 binding in human HEK-CB2 cells expressed as pKi ± SEM in nM (n = 3) or percentage displacement of specific binding at a concentration of 1 μM (n = 2). 5 pKd calculated through saturation of CELT-335 in human HEK-293T cells transiently expressing Tb-labeled SNAP-CB1R. 6 Kd calculated through saturation of CELT-335 in human HEK-293T cells transiently expressing Tb-labeled SNAP-CB2R.

In the saturation experiments, specific binding was also checked, employing appropriate competitors (Figure 2). The high affinity of CELT-335 for CB1R was maintained in the Tag-lite® assay (Ki=44.8nM in radioligand assay and Kd= 42nM in Tag-lite®).

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 triplicates).

2.2 CELT-335 HTRF assay validation in hCB1R Expressing adherent Cells.

To validate the potential of CELT-335 as a probe, seven reference compounds were selected.

Table 2. Comparison of affinity data for CB1 receptor of the set of reference compounds obtained through the radioligand competition binding assay and CB1 competition binding assays with Tag-lite® technology using CELT-335. Values represent the mean ± SEM of triplicate determinations. Reference is not indicated for those compounds whose affinity through the radioligand binding assay was measured experimentally following published protocols.[14]

Figure 3. Chemical structures and functional activity of the reference compounds used for CELT-335 validation in Taglite® binding assays.

Using these results as a starting point, the Tag-lite® binding assay was developed. In Figure 4 coherent and well-defined sigmoidal concentration/response curves can be found for all seven molecules tested, and their pKi values compared to literature data in Table 2.

Figure 4. Competition experiments of binding of fluorescent ligand CELT-335 to living HEK-293 T cells expressing the SNAP-CB1R. Tb labelling was performed as described in Materials and Methods. Tag-Lite® competition binding curves were obtained by using 100 nM of CELT-335 and increasing concentrations of compounds tested (0–10 μM). HTRF Ratio = 665 nm acceptor signal/620 nm donor signal × 10,000; the percentage is calculated by taking the highest value as 100%. Data represent the mean ± SEM (n = 5 in triplicates). (a) concentration/response curves obtained for agonists; (b) concentration/response curves obtained for the antagonists.

2.3  CELT-335 HTRF Assay Validation in hCB2R Expresing adherent cells

The Kd obtained through the Tag-lite® saturation binding assay at the CB2R is 24.2nM, confirming the one observed in radioligand assay, while maintaining the appropriate properties for the HTRF signal. Thus, the same set of compounds used for CB1R competition assays was used in CB2R.

Table 3. Comparison of affinity data for CB1 receptor of the set of reference compounds obtained through the radioligand competition binding assay and CB1 competition binding assays with Tag-lite® technology using CELT-335. Values represent the mean ±SEM of triplicate determinations. Reference is not indicated for those compounds whose affinity through the radioligand binding assay was measured experimentally following published protocols.[14]

Figure 5. Competition experiments of binding of fluorescent ligand CELT-335 to living HEK-293 T cells expressing the SNAP-CB2R. Tb labelling was performed as described in Materials and Methods. Competition binding curves were obtained through Tag-lite® technology using 10 nM of CELT-335 and increasing concentrations of compounds tested (0–10 μM). HTRF Ratio = 665 nm acceptor signal/620 nm donor signal × 10,000; the percentage is calculated by taking the highest value as 100%. Data represent the mean ± SEM (n = 5 in triplicates). (a) concentration/response curves obtained for agonists; (b) concentration/response curves obtained for the antagonists.

3.       Discussion

The saturation experiments revealed a lower 2-fold difference in the Kd for each CBR receptor, which was originally 6-fold in the radioligand binding assays, while keeping an excellent FRET signal. High specific binding was observed by measuring 10uM concentrations of the appropriate competitors (CP55490 and GW405833 for CB1R and CB2R).

To fully optimize the Tag-lite assays, the concentration of probe was based on the Kd obtained for each receptor, 100nM for CB1R and 10nM for CB2R. The curves obtained using this concentration of probe in the Tag-lite® assays are reproducible sigmoidal concentratio/response curves and the affinity data obtained (Ki) has a high correlation to those obtained through tradional radioligand binding assays.

Figure 6. Schematic representation of the high correlation between reference compounds affinity data obtained through radioligand binding assays and the Tag-lite® binding assay developed in this work. (a) CB1R binding affinities; (b) CB2R binding affinities.

The set of reference compounds were chosen to provide the highest diversification in affinity, selectivity, functional acitvity and chemical structure. A graphic highlighting these differences can be found in Figure 7.

Figure 7. Chemical structures, selectivity (radioligand binding assays Ki), and functional activity (blue for antagonists and red for agonists) of the reference compounds used for CELT-335 validation in Tag-lite® competition binding assays.

CELT-335 proved itself as a good competitor for both synthetic and naturally derived cannabinoid ligands, both agonists and antagonists. It highlights the difference in affinity of compounds such as SCRAs and other synthetic cannabinoids. Furthermore, the affinity trend is similar to those obtained in literature and traditional radioligand binding assays. Indeed, the difference is lower than 1pKi value between both experiments. For further validation, a larger range of compounds will be tested, including screening chemical libraries with even more diversity in chemical structures and affinities for other receptors.

If you want to read the materials and methods section check out the link to the full article.

If you have any questions about CELT-335 or the assays themselves, feel free to contact me!

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