HIGH-THROUGHPUT SCREENING ASSAY FOR CAMP TRANSPORT INHIBITORS: SEARCHING FOR A NEW DRUG FOR PDAC TREATMENT

CERVIÑO, RAMIRO HECTOR; DAVIO CARLOS; GANCEDO SAMANTA NEREA; GOMEZ NATALIA; YANEFF, AGUSTÍN

Congreso; LXIV Reunión Anual de la Sociedad Argentina de Investigación Clínica (SAIC); 2019

In a previous work, we validated the inhibition of MRP4-dependant cAMP extrusion process as a promising therapeutic strategy for pancreatic ductal adenocarcinoma. MRP4 inhibition abrogated proliferation in vitro by both, augmenting intracellular cAMP and diminishing extracellular cAMP. In this work we sought to study the activity of a large series of compounds as cAMP transport inhibitors. In view of this objective, we first analyzed the available information about the inhibitory effect of different molecules on MRPs and classified them according to their chemical structure and their transporter-substrate specificity. Since the data set of cAMP transport inhibitors is scarce, we developed a microplate high-throughput föster resonance energy transfer (FRET) assay that monitors the real-time kinetics of intracellular cAMP levels in cell monolayers, using the EPAC-SH187 biosensor. We established a HEK293T- EPAC-SH187 clone that stably expresses this sensor, which exhibited a wide dynamic range and high sensitivity (2-fold delta FRET in a concentration range of 0.01?500 μM cAMP). Using this technique, we analyzed 60 compounds, with diverse chemical structures and pharmacological uses, which were previously identified in literature as substrates or inhibitors of MRP4. We identified 25 compounds that functioned as inhibitors of cAMP transport and characterized them by performing concentration-response experiments. These set of compounds include traditional MRP4 inhibitors as probenecid and MK571, channel blockers, protease inhibitors, nucleotide analogs and non-steroidal anti-inflammatory drugs. The emerging results will serve as a basis for the further development of specific inhibitors of MRP4-mediated cAMP transport potentially applicable in the treatment of PDAC.