Functional cAMP assay optimization at CHO cells expressing A2A adenosine receptors and stably transfected with firefly luciferase biosensor

A2A adenosine receptor (A2AAR) ligands have been proposed as an attractive pharmacological tool for the treatment of several diseases, including neurodegenerative disorders. In this work, we perform and validate a new functional cAMP assay suitable for the evaluation of A2AAR ligand activity. This functional assay uses a biosensor expressing a mutant form of Photinus pyralis luciferase into which a cAMP-binding protein moiety has been inserted. When cAMP binds to the biosensor, there is an increased light output proportional to the A2AAR ligand activities. Hence, a new CHO cell line expressing A2AARs and stably transfected with firefly luciferase biosensor was generated. A2AAR agonists and antagonists were tested using cAMP assay in the new cell line, which allowed speeding up and facilitating the screening of the ligands. The results achieved are in agreement with those obtained with binding studies.