A new sensitive and subunit-selective molecular tool for investigating protein kinase A in the brain

Despite cellular complexity, a limited number of small molecules act asintracellular second messengers. Protein kinase A (PKA) is the main transducerof the information carried by cyclic adenosine monophosphate (cAMP). Recently,cellular imaging has achieved major technical advancements, although the searchfor more specific and sensitive low‐molecular‐weight probes to exploresubcellular events involving second messengers is still in progress. Theconvergent synthesis of a novel, fluorescent small molecule comprising thecAMP structure and a rhodamine‐based fluorescent residue, connected througha flexible linker, is described here. The interaction motif of this compound withPKA was investigated in silico using a blind docking approach, comparing itstheoretical binding energy with the one calculated for cAMP. Moreover, thepredicted pharmacokinetic properties were also computed and discussed. Thenew probe was tested on three areas of the mouse central nervous system(parietal cerebral cortex, hippocampus, and cerebellar cortex) with differentfixation methods demonstrating remarkable selectivity towards the PKA RIαsubunit. The probe showed overall better performances when compared toother commercially available fluorescent cAMP analogues, acting at lowerconcentrations, and providing stable labeling.