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Pharmacol Res Perspect. 2016 Aug 8;4(5):e00250. eCollection 2016 Oct.

Use of a new proximity assay (NanoBRET) to investigate the ligand-binding characteristics of three fluorescent ligands to the human β1-adrenoceptor expressed in HEK-293 cells.

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Cell Signalling and Pharmacology Research Group School of Life Sciences University of Nottingham Nottingham NG7 2UH United Kingdom.
Heptares Therapeutics Ltd. Bio Park Welwyn Garden City AL7 3AX United Kingdom.


Previous research has indicated that allosteric interactions across the dimer interface of β1-adrenoceptors may be responsible for a secondary low affinity binding conformation. Here we have investigated the potential for probe dependence, in the determination of antagonist pKi values at the human β1-adenoceptor, which may result from such allosterism interactions. Three fluorescent β1-adrenoceptor ligands were used to investigate this using bioluminescence energy transfer (BRET) between the receptor-bound fluorescent ligand and the N-terminal NanoLuc tag of a human β1-adrenoceptor expressed in HEK 293 cells (NanoBRET). This proximity assay showed high-affinity-specific binding to the NanoLuc- β1-adrenoceptor with each of the three fluorescent ligands yielding KD values of 87.1 ± 10 nmol/L (n = 8), 38.1 ± 12 nmol/L (n = 7), 13.4 ± 2 nmol/L (n = 14) for propranolol-Peg8-BY630, propranolol- β(Ala-Ala)-BY630 and CGP-12177-TMR, respectively. Parallel radioligand-binding studies with 3H-CGP12177 and TIRF microscopy, to monitor NanoLuc bioluminescence, confirmed a high cell surface expression of the NanoLuc- β1-adrenoceptor in HEK 293 cells (circa 1500 protein-1). Following a 1 h incubation with fluorescent ligands and β1-adrenoceptor competing antagonists, there were significant differences (P < 0.001) in the pKi values obtained for CGP20712a and CGP 12177 with the different fluorescent ligands and 3H-CGP 12177. However, increasing the incubation time to 2 h removed these significant differences. The data obtained show that the NanoBRET assay can be applied successfully to study ligand-receptor interactions at the human β1-adrenoceptor. However, the study also emphasizes the importance of ensuring that both the fluorescent and competing ligands are in true equilibrium before interpretations regarding probe dependence can be made.


Bioluminescence energy transfer; ligand binding; probe dependence; β‐adrenoceptors

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