Quantification of cAMP antagonist action in vitro and in living cells.

cAMP-dependent protein kinase (PKA) plays a key role in intracellular signalling. cAMP antagonists, acting as suppressors of PKA activity by preventing PKA-holoenzyme dissociation, have received increasing attention because of their potential use in diagnostics as well as for therapeutic purposes. A large number of cAMP analogs have been described over the last three decades and methodology has been established to monitor cAMP agonists action by either following enzymatic activity or holoenzyme dissociation. This is not the case for cAMP antagonists, where only a few substances have been demonstrated to exhibit effects in the low micromolar range, for example, Rp-8-Br-cAMPS. A main drawback in the development of new compounds is the lack of technologies to assess antagonist action in an in vitro situation as well as in living cells. Here we quantify the effect of several cAMP analogs applying three different biochemical/biophysical assay setups and one in-cell assay. This includes two methods monitoring subunit dissociation in a test tube, namely AlphaScreen, a bead-based proximity assay, and surface plasmon resonance, determining the association and dissociation patterns of the two PKA subunits in real time in response to antagonists. BRET(2), performed in living cells in a 96-well format, allows testing for the efficacy of membrane-permeable cAMP analogs based on a genetically engineered cAMP sensor. Using novel and established experimental strategies side by side, the action of cAMP and cAMP analogs was tested on type Ialpha PKA holoenzyme, thus generating methodology to screen drug libraries for potential cAMP antagonists with high accuracy, reproducibility as well as potential for automation.