Differential binding of Δ9-tetrahydrocannabinol derivatives to type 1 cannabinoid receptors (CB1)

If you’ve read our previous post, you probably know what CELT-335 is and its high affinity for the cannabinoid 1 receptor (CB1R). During that project we tested its validity as a fluorescent probe for Tag-lite® assays, where we used a set of 7 cannabinoid ligands (both natural and synthetic) to validate and optimize the assay.

Because Tag-lite® is based on FRET, we have collaborated with BMG Labtech to develop an application note where CELT-335 is used to differentiate binding of THC derivatives to CB1R.

Introduction

Cannabinoid receptors are GPCRs, and two main types exist, CB1R and CB2R. CB1R are mainly found in the central nervous system (CNS) and the brain. They regulate mood, memory, appetite and motor function. CB2R are located primarily in the immune system and peripheral tissues, where they modulate inflammation and immune response.

Cannabinoids have the potential to treat a variety of diseases, including neurological and metabolic conditions. This interest has been driving efforts to understand their pharmacology since the 1980s. It has been proven that when different ligands bind to the receptors the effects are different depending on which signaling pathway is activated, which is called ‘bias signaling’.

The bias depends on the agonist’s structure as well as the state of the cannabinoid receptor (whether it’s monomeric or heteomeric).

Here, the PHERAstart FSX microplate reader was used to differentiate binding of natural cannabinoids to CB1R based on TR-FRET assays. In this case, Terbium is used as donor and CELT-335 fluorescent ligand is used as acceptor.

Figure 1.Combinational optical spectra of terbium (Tb) and CELT-335: The terbium (donor) emission spectrum overlaps with the CELT-335 (acceptor) excitation spectrum.

CELT-335 was successfully used in assays to determine the pharmacological properties of Δ9-tetrahydrocannabinol (Δ9-THC), Δ9-tetrahydrocannabinolic acid (Δ9-THCA) and Δ9-tetrahydrocannabivarin (Δ9-THCV) when binding to CB1R.

Assay Principle

CELT-335 is a fluorescently labelled full agonist, which binds to the orthosteric site of the human CBRs. It bears a hydrophilic fluorophore compatible with the terbium donor in TR-FRET assays. It binds to the receptor, which is labelled with Tb. When this happens the proximity between donor and acceptor is sufficient for a transmission of energy to occur between the Tb and fluorophore, which will emit at 665nm.

Figure 2.Assay Principle: TR-FRET assay using CELT-335, a dual (CB1/CB2) fluorescent ligand that serves as a TR-FERT acceptor.

Results and Discussion

The TR-FRET assay performed in living HEK-293 T cells expressing CB1R using CELT-335 as acceptor provides a sensitive and robust measurement for competition binding experiments. The curves were obtained using 100nM of probe and increasing concentrations of Δ9-THC, Δ9-THCA and Δ9-THCV (0 – 10 μM). Competition was similar for Δ9-THC and Δ9-THCV, but considerably lower for Δ9-THCA. The data obtained is comparable to those reported in radioligand binding assays (Table 1).

Figure 3.TR-FRET competition assay between CELT-335 (TR-FRET acceptor) and Δ9-THC, Δ9-THCA and                     Δ9-THCV.Data represent the mean ± SEM (n = 5 in triplicates)

Table 1. Comparison of binding affi nities using radioligand binding or TR-FRET assays. Radioligand binding data are from [2,3].

Conclusion

CELT-335 is a functional TR-FRET acceptor which can be coupled with Tb as donor. It has shown high affinity for the CB1R, and the binding affinity values obtained for natural cannabinoids and their derivatives are comparable values to those reported in literature [2,3]. These assays probide a reliable way to measure binding affinities for CB1R in live cells and the PHERAstar FSX is the ideal microplate reader for characterizing the GPCR-ligand interactions.

If you want to check out the Materials and Methods section as well as the application note, here is the link to our website!

If you are interested in our ligands or technology, feel free to contact me anytime.

References

1. Raïch I et al., Similarities and differences upon binding of naturally occurring Δ9-tetrahydrocannabinolderivatives to cannabinoid CB1 and CB2 receptors, Pharmacological Research (2021) 174: 105970, doi: 10.1016/j.phrs.2021.105970

2. Zagzoog A et al., In vitro and in vivo pharmacological activity of minor cannabinoids isolated from Cannabis sativa, Scientifi c Reports (2020) 10(1):20405. doi: 10.1038/s41598-020-77175-y

3. Palomares B et al., Δ9-Tetrahydrocannabinolic acid alleviates collagen-induced arthritis: Role of PPARγ and CB1 receptors, British Journal of Pharmacology (2020) 177(17): 4034-4054. doi: 10.1111/bph.15155.