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 beta-galactosidase reporter gene into CHOA2a-2 cells to generate a stable cell line, CHOA2a-2CREbetagal#26. Robust cyclic AMP signal amplification was obtained using a colorimetric assay measuring beta-galactosidase activity. The EC(50) of 5'-N-ethylcarboxamidoadenosine (NECA), a potent A2a agonist, for inducing beta-galactosidase activity was 23.3 +/- 3.5 nM, similar to 22.7 +/- 3.9 nM, which was the NECA EC(50) 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 EC(50) values of 2-6 microM. Preliminary characterization of those compounds was presented.