The use of NanoBRET to determine the cooperative effects of allosteric modulators on the binding of a1 adenosine receptor agonists

S. L. Cooper^1,2, M. Soave^1,2, S. J. Hill^1,2, J. Woolard^1,2. ¹Division of Physiology, Pharmacology and Neuroscience, School of Life Sciences, University of Nottingham, Nottingham, United Kingdom, ²Centre of Membrane Proteins and Receptors, University of Nottingham, Nottingham, United Kingdom.

Introduction: The A₁ adenosine receptor (A1AR) is a vital regulator of many biological functions and is implicated as a therapeutic target for several diseases (1). Here, we used a previously characterised bioluminescence energy transfer (NanoBRET) proximity assay (2) to determine: (a) The binding affinities of agonists and antagonists to the human A1AR and (b) the cooperative effects of allosteric modulators on A1AR agonist-binding.

Methods: Specific binding of ligands to the human A1AR was measured using a NanoBRET proximity assay. The N-terminus of the human A1AR was tagged with NanoLuc and stably-expressed in HEK293 cells. Xanthine amine congener (XAC)-based fluorescent ligand; CA200645, was used to monitor binding of unlabelled ligands to A1AR in living cells, using NanoBRET (3). Total and nonspecific binding of CA200645 was measured using increasing concentrations of CA200645 in the presence and absence of 1μM DPCPX. Competing ligands (adenosine, NECA, capadenoson, PD81723, VCP171 and DPCPX) were added at increasing concentrations, simultaneously with CA200645 (25nM). The Cheng-Prussoff equation was used to determine pK_i values. Binding cooperativities between allosteric modulators (PD81723 and VCP171) and A1AR agonists (adenosine, NECA and capadenoson) were measured using increasing concentrations of agonist, (in the presence or absence of allosteric modulator) using 25nM CA200645. The allosteric ternary complex model (4) was used to estimate the binding cooperativity between the allosteric modulator and the agonist (α_I).

Results: CA200645 exhibited specific binding to the A1AR (K_D=33.84±10.15nM; mean±S.E.M, n=5). Competing ligands inhibited the specific binding of 25nM CA200645 yielding pKi values of: Adenosine pK_i=4.24±0.08, (n=6); capadenoson pK_i=6.60±0.12, (n=6); NECA pK_i=5.17±0.20, (n=6); PD81723, pK_i=4.02±0.24, (n=5); VCP171, pK_i=4.63±0.15, (n=5) and DPCPX pK_i=7.96±0.08 (n=5). Allosteric modulation of A1AR by PD81723 and VCP171 enhanced the binding of all A1AR agonists yielding α_I cooperativity values (for PD81723 and VCP171 respectively) of: adenosine, logα_I=1.1±0.15 and 0.68±0.13; NECA, logα_I=1.02±0.13 and 0.74±0.10 and capadenoson, logα_I=0.22±0.14 and 0.19±0.28. The pK_B-values obtained for PD81723 (4.34±0.05, n=6) and VCP171 (4.69±0.07, n=6) from this allosteric analysis were very similar levels to the pK_i values.

Conclusions: These data highlight the use of the NanoBRET proximity assay to study receptor-ligand interactions. Importantly, these data also demonstrate the ability to measure agonist binding affinities and cooperative effects of allosteric modulators.

References:

1. Borea PA et al. (2016) Trends Pharmacol Sci. 37, 419-434

2. Stoddart LA et al. (2015) Nat. Methods 12, 661-663

3. Soave M et al. (2016) Pharma Res Per 4, e00250

4. Nguyen AT et al. (2016) Mol Pharmacol, 90, 715-725.