Identification and validation of modulators of exchange protein activated by cAMP (Epac) activity: structure-function implications for Epac activation and inhibition.
The signaling molecule cAMP primarily mediates its effects by activating PKA and/or exchange protein activated by cAMP (Epac). Epac has been implicated in many responses in cells, but its precise roles have been difficult to define in the absence of Epac inhibitors. Epac, a guanine nucleotide exchange factor for the low molecular weight G protein Rap, is directly activated by cAMP. Using a bioluminescence resonance energy transfer-based assay (CAMYEL) to examine modulators of Epac activity, we took advantage of its intramolecular movement that occurs upon cAMP binding to assess Epac activation. We found that the use of CAMYEL can detect the binding of cAMP analogs to Epac and their modulation of its activity and can distinguish between agonists (cAMP), partial agonists (8-chlorophenylthio-cAMP), and super agonists (8-chlorophenylthio-2'-O-Me-cAMP). The CAMYEL assay can also identify competitive and uncompetitive Epac inhibitors, e.g. (Rp)-cAMPS and CE3F4, respectively. To confirm the results with the CAMYEL assay, we used Swiss 3T3 cells and assessed the ability of cyclic nucleotide analogs to modulate the activity of Epac or PKA, determined by Rap1 activity or VASP phosphorylation, respectively. We used computational molecular modeling to analyze the interaction of analogs with Epac1. The results reveal a rapid means to identify modulators (potentially including allosteric inhibitors) of Epac activity that also provides insight into the mechanisms of Epac activation and inhibition.
From the Departments of Pharmacology and.
Loren M
LM
Brown
Kathleen E
KE
Rogers
J Andrew
JA
McCammon
Paul A
PA
Insel
D000818:Animals; D002460:Cell Line; D000242:Cyclic AMP / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D055808:Drug Discovery; D020662:Guanine Nucleotide Exchange Factors / Q000819:agonists* / Q000037:antagonists & inhibitors* / Q000378:metabolism; D006801:Humans; D008163:Luminescent Measurements; D051379:Mice; D008958:Models, Molecular; D015398:Signal Transduction / Q000187:drug effects; D013873:Thionucleotides / Q000737:chemistry / Q000378:metabolism*
C482601:Epac protein, mouse; D020662:Guanine Nucleotide Exchange Factors; C116122:RAPGEF3 protein, human; D013873:Thionucleotides; C016957:adenosine-3',5'-cyclic phosphorothioate; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases
Bioluminescence Resonance Energy Transfer (BRET); Cyclic AMP (cAMP); Enzyme Inhibitors; Epac1; Guanine Nucleotide Exchange Factor (GEF); Molecular Docking; Molecular Pharmacology