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Chronic activation of β-adrenergic (β-AR) pathway causes excessive accumulation of cAMP in cardiomyocytes and participates in pathological cardiac remodeling leading to heart failure (HF). Cyclic nucleotide phosphodiesterases (PDEs) are responsible for the degradation of cAMP and cGMP in subcellular compartments. Among the different PDE families, PDE2 is unique in being activated ˜;5-fold by cGMP, thus mediating a negative cross talk between cGMP and cAMP signaling. Recently, we found that PDE2A activity is increased in human HF and this may represent a protective mechanism limiting â-AR cardiotoxicity in chronic HF. Our therapeutic strategy is to target PDE2 with specific activators to treat HF at the early stages. A PDE2 activity assay has been developed, first using a purified PDE2 from bovine adrenal glands, then using a commercial recombinant protein (full-length human PDE2A). We miniaturized a bioluminescent activity assay in 384 well plates, which was validated for High-Throughput Screening (HTS) with an excellent Z-factor (>0.7) on the Ciblot HTS platform. Moreover, an in silico screening based on the binding of molecules on the GAF-B domain (regulatory domain) of PDE2 has been performed on a large library of molecules. As a counter screen, a PDE4 assay has been employed to eliminate nonspecific modulators. Ranking of hits is based on activation efficacy in comparison to 5 μM cGMP, M EC50, Emax and cytotoxicity following chemical resynthesis in a SAR (Structure Activity Relationship) strategy. Several complementary assays are currently under development to confirm the effect of the activators in a Hit to Lead process (cellular assays, cAMP ELISA, co-cristallisation, Biacore). This challenging project is supported by LabEx LERMIT within its new cardiovascular target program