Development of a sensitive and HTS-compatible reporter gene assay for functional analysis of human adenosine A2a receptors in CHO-K1 cells.
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.
Tang
Yuting
Y
Department of Drug Discovery, Endocrine Therapeutics and Metabolic Disorders, The Johnson & Johnson Pharmaceutical Research and Development, L.L.C., 1000 Route 202, Raritan, NJ 08869, USA. ytang@prdus.jnj.com
Luo
Jingqing
J
Fleming
Charles R
CR
Kong
Yan
Y
Olini
Gilbert C
GC
Wildey
Mary Jo
MJ
Cavender
Druie E
DE
Demarest
Keith T
KT
D058908:Adenosine A2 Receptor Agonists; D019830:Adenosine-5'-(N-ethylcarboxamide) / Q000494:pharmacology; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D004305:Dose-Response Relationship, Drug; D017930:Genes, Reporter / Q000187:drug effects / Q000235:genetics*; D006801:Humans; D043705:Receptor, Adenosine A2A / Q000235:genetics* / Q000378:metabolism; D014162:Transfection / Q000379:methods*; D001616:beta-Galactosidase / Q000378:metabolism
D058908:Adenosine A2 Receptor Agonists; D043705:Receptor, Adenosine A2A; D019830:Adenosine-5'-(N-ethylcarboxamide); D001616:beta-Galactosidase