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

Abstract

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-A3 receptor (A3AR; 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-A1 receptor, and β1 and β2 adrenoceptors (β1AR and β2AR; BODIPY-TMR-CGP-12177) were also carried out. Affinity values determined for the binding of the fluorescent ligands CA200645 and AV039 to A3AR for a range of classical adenosine receptor antagonists were consistent with A3AR pharmacology and correlated well (R2 = 0.94) with equivalent data obtained using a confocal imaging plate reader (ImageXpress Ultra). The binding of BODIPY-TMR-CGP-12177 to the β1AR was potently inhibited by low concentrations of the β1-selective antagonist CGP 20712A (pKi 9.68) but not by the β2-selective antagonist ICI 118551(pKi 7.40). Furthermore, in experiments conducted in CHO K1 cells expressing the β2AR this affinity order was reversed with ICI 118551 showing the highest affinity (pKi 8.73) and CGP20712A (pKi 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 A3AR. 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 A3AR (pKi ≥6) were successfully identified. We found three novel compounds in the library that displayed unexpected sub-micromolar affinity for the A3AR. These were K114 (pKi 6.43), retinoic acid p-hydroxyanilide (pKi 6.13) and SU 6556 (pKi 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 A3AR 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.

Pharmacological Characterisation of Novel Adenosine Receptor A3R Antagonists

“…In addition, the dichloro-phenyl ring can be oriented to the unique lipophilic area of A3R including V169 5.30 , M177 5.40 , I249 6.54 and L264 7.34 stabilized in that cleft through attractive van der Waals interactions; K18 is further stabilized through π-π aromatic stacking interactions between isoxazole ring and the phenyl group of F168 5.29 and the thiazole group is oriented deeper into the receptor favoring interactions with L246 6.51 and L90 3.32 and possibly with I268 7.39 . In combination with our mutagenesis data, the final binding pose of K18 appears to be within the orthosteric binding site, involving residues previously described to be involved in binding of A3R compounds (Arruda et al, 2017). We reported no detectable Gi/o response following co-stimulation with forskolin and NECA or IB-MECA for A3R mutants F168A 5.29 , L246A 6.51 , N250A 6.55 and I268A 7.39 (Stamatis et al, 2019, in preparation).…”

Fluorescent ligands: Bringing light to emerging GPCR paradigms

“…This has been shown at the early endosome following receptor internalisation. (e) Interestingly, there is also evidence of active GPCRs signalling from the Golgi apparatus prior to trafficking to cell membrane receptor (Young et al, 2005), apelin receptor (Iturrioz et al, 2010), and adenosine A 3 receptor (Arruda et al, 2017). These studies demonstrate the potential for fluorescent ligands to be used in screening large compound libraries at a GPCR, without the need for radioligands.…”

“…These high signal‐to‐noise ratios mean fluorescent ligands can be used at much higher concentrations than radioligands, and consequently, ligands with relatively low affinity ( K D ~500 nM) have been successfully used in a BRET‐based binding assay for the P2Y 2 receptor (Conroy et al ). Fluorescent ligands have been used in a number of small‐scale screens (~1,000 compounds) to successfully identify novel compounds that bind to the formylpeptide receptor (Young et al, ), apelin receptor (Iturrioz et al, ), and adenosine A 3 receptor (Arruda et al, ). These studies demonstrate the potential for fluorescent ligands to be used in screening large compound libraries at a GPCR, without the need for radioligands.…”

Adenosine-Related Mechanisms in Non-Adenosine Receptor Drugs

“…Sulmazole 65 (11 µM) and DMCM (1.6 µM) also inhibit rat A 1 AR binding [119,120]. A 3 AR binding is inhibited by other drug substances, identified using high-throughput fluorescent screening of a chemical library: K114 (67, K i 372 nM), Src inhibitor SU6656 (68, K i 676 nM), and retinoic acid p-hydroxyanilide (69, K i 741 nM, fenretinide) [121].…”