AUTHOR=Arruda Maria Augusta , Stoddart Leigh A. , Gherbi Karolina , Briddon Stephen J. , Kellam Barrie , Hill Stephen J. 

TITLE=A Non-imaging High Throughput Approach to Chemical Library Screening at the Unmodified Adenosine-A3 Receptor in Living Cells

JOURNAL=Frontiers in Pharmacology

VOLUME=8

YEAR=2017

URL=https://www.frontiersin.org/journals/pharmacology/articles/10.3389/fphar.2017.00908

DOI=10.3389/fphar.2017.00908

ISSN=1663-9812

ABSTRACT=<p>Recent advances in fluorescent ligand technology have enabled the study of G protein-coupled receptors in their native environment without the need for genetic modification such as addition of N-terminal fluorescent or bioluminescent tags. Here, we have used a non-imaging plate reader (PHERAstar FS) to monitor the binding of fluorescent ligands to the human adenosine-A<sub>3</sub> receptor (A<sub>3</sub>AR; CA200645 and AV039), stably expressed in CHO-K1 cells. To verify that this method was suitable for the study of other GPCRs, assays at the human adenosine-A<sub>1</sub> receptor, and β<sub>1</sub> and β<sub>2</sub> adrenoceptors (β<sub>1</sub>AR and β<sub>2</sub>AR; BODIPY-TMR-CGP-12177) were also carried out. Affinity values determined for the binding of the fluorescent ligands CA200645 and AV039 to A<sub>3</sub>AR for a range of classical adenosine receptor antagonists were consistent with A<sub>3</sub>AR pharmacology and correlated well (<italic>R</italic><sup>2</sup> = 0.94) with equivalent data obtained using a confocal imaging plate reader (ImageXpress Ultra). The binding of BODIPY-TMR-CGP-12177 to the β<sub>1</sub>AR was potently inhibited by low concentrations of the β<sub>1</sub>-selective antagonist CGP 20712A (pK<sub>i</sub> 9.68) but not by the β<sub>2</sub>-selective antagonist ICI 118551(pK<sub>i</sub> 7.40). Furthermore, in experiments conducted in CHO K1 cells expressing the β<sub>2</sub>AR this affinity order was reversed with ICI 118551 showing the highest affinity (pK<sub>i</sub> 8.73) and CGP20712A (pK<sub>i</sub> 5.68) the lowest affinity. To determine whether the faster data acquisition of the non-imaging plate reader (~3 min per 96-well plate) was suitable for high throughput screening (HTS), we screened the LOPAC library for inhibitors of the binding of CA200645 to the A<sub>3</sub>AR. From the initial 1,263 compounds evaluated, 67 hits (defined as those that inhibited the total binding of 25 nM CA200645 by ≥40%) were identified. All compounds within the library that had medium to high affinity for the A<sub>3</sub>AR (pK<sub>i</sub> ≥6) were successfully identified. We found three novel compounds in the library that displayed unexpected sub-micromolar affinity for the A<sub>3</sub>AR. These were K114 (pK<sub>i</sub> 6.43), retinoic acid <italic>p</italic>-hydroxyanilide (pK<sub>i</sub> 6.13) and SU 6556 (pK<sub>i</sub> 6.17). Molecular docking of these latter three LOPAC library members provided a plausible set of binding poses within the vicinity of the established orthosteric A<sub>3</sub>AR binding pocket. A plate reader based library screening using an untagged receptor is therefore possible using fluorescent ligand opening the possibility of its use in compound screening at natively expressed receptors.</p>