Development of a Sensitive and HTS-Compatible Reporter Gene Assay for Functional Analysis of Human Adenosine A2a Receptors in CHO-K1 Cells

Abstract

Adenosine A2a receptor, a member of the G protein-coupled receptor superfamily, has been demonstrated to be an important pharmacological target. It couples to stimulatory G protein and activates adenylate cyclase upon agonist stimulation. Here we attempted to stably transfect Chinese hamster ovary (CHO-K1) cells, which lack any known subtypes of adenosine receptors, with recombinant human adenosine A2a receptors (hA2aR). Rapid down-regulation of hA2aR in a clonal cell line, CHOA2a-2, was observed over a short period of time in culture. This is consistent with other groups' findings of low expression and poor G protein coupling of this receptor in several cell systems. To facilitate pharmacological profiling for hA2aR ligand, we introduced a cyclic AMP response element (CRE)-linked β-galactosidase reporter gene into CHOA2a-2 cells to generate a stable cell line, CHOA2a-2CREβgal#26. Robust cyclic AMP signal amplification was obtained using a colorimetric assay measuring β-galactosidase activity. The EC₅₀ of 5′-N-ethylcarboxamidoadenosine (NECA), a potent A2a agonist, for inducing β-galactosidase activity was 23.3 ± 3.5 nM, similar to 22.7 ± 3.9 nM, which was the NECA EC₅₀ in the direct measurement of cyclic AMP of CHOA2a-2 cells in early culture. Subsequently we validated this assay for high throughput screening for hA2aR agonists. The Z′ factor for robotic assay performance was 0.79 ± 0.03, the ratio of signal/noise was 157 ± 36, and the ratio of signal/background was 10.6 ± 1.2, demonstrating that this assay is well suitable for quality high throughput screening. High throughput screening of Johnson & Johnson libraries uncovered a couple of distinct series of nonadenosine small molecules, in addition to adenosine analogues, as potential hA2aR agonists with EC₅₀ values of 2–6 μM. Preliminary characterization of those compounds was presented.