pAB ester prodrugs of Rp-isomers are effective inhibitors of PKA. Modifications at position 8 of the adenine nucleobase increases their efficiency. (A–D) HEK293 cells were transfected with isoform-specific BRET sensors composed of mutant Renilla luciferase–conjugated R-subunits hRIα-RLuc8 (A), RIβ-RLuc8 (B), hRIIα-RLuc8 (C), and hRIIβ-RLuc8 (D) as bioluminescent donor proteins and GFP-tagged Cα-subunits as acceptor proteins. Close proximity of luciferase and GFP in the inactive PKA complex results in BRET signal (white bars), which is reduced after activation by Fsk/IBMX (striped white bars, 50/100 µM; 20 min). Pretreatment with 10 µM Rp-8-Br-cAMPS-pAB (green), Rp-8-pCPT-cAMPS-pAB (blue), or Rp-8-Br-cAMPS-pAB (red; 15 min) inhibited the reduction in BRET signals, whereas the control, 4-ABnOH (gray), was ineffective. Values are means ± SD; n = 3; one-way ANOVA with Bonferroni’s test. *, P < 0.05; **, P < 0.01; ***, P < 0.001 indicate significance between control and Fsk-stimulated conditions; ^§, P < 0.05; ^§§, P < 0.01; ^§§§, P < 0.001 between Fsk stimulations in the absence/presence of Rp-isomers; ^#, P < 0.05; ^##, P < 0.01; ^###, P < 0.001 between the unstimulated controls in the absence/presence of Rp-isomers.