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. 2014 Mar 21;289(12):8217-30.
doi: 10.1074/jbc.M114.548636. Epub 2014 Feb 4.

Identification and validation of modulators of exchange protein activated by cAMP (Epac) activity: structure-function implications for Epac activation and inhibition

Loren M Brown  1 , Kathleen E Rogers, J Andrew McCammon, Paul A Insel
Affiliations

Identification and validation of modulators of exchange protein activated by cAMP (Epac) activity: structure-function implications for Epac activation and inhibition

Loren M Brown et al. J Biol Chem. .

Abstract

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.

Keywords: Bioluminescence Resonance Energy Transfer (BRET); Cyclic AMP (cAMP); Enzyme Inhibitors; Epac1; Guanine Nucleotide Exchange Factor (GEF); Molecular Docking; Molecular Pharmacology.

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Figures

FIGURE 1.
FIGURE 1.
CAMYEL assay. A, schematic diagram of the activation of the CAMYEL construct: the catalytic region (CR) and regulatory region (RR) (Epac1 amino acids 149–880) with Renilla luciferase attached to the catalytic region and citrine attached to the regulatory region. B, cAMP produces a decrease in the BRET signal upon binding to CAMYEL, as measured by the decrease of 465/535 nm. A.U., arbitrary units.
FIGURE 2.
FIGURE 2.
CAMYEL activation is induced by Epac agonists in a concentration-dependent manner. cAMP and cAMP analogs were added to a 96-well white plate with 100 μl of CAMYEL in HEK293 lysate and incubated for 5 min before measuring activation. A, cAMP analogs activate the CAMYEL construct in a concentration-dependent manner. B, structures of cAMP analogs used in the assay of CAMYEL activation. n = 3.
FIGURE 3.
FIGURE 3.
CAMYEL can assess if cAMP analogs are agonists, super agonists, or partial agonists. cAMP and cAMP analogs were added and assayed as in Fig. 2. A, activation of CAMYEL by cAMP analogs. B, activation of CAMYEL is decreased by cAMP analogs modified with 8-R substitutions. C, maximum decrease in BRET (measured by 465/535 nm) in response to 300 μm cAMP, 8-Me, or CPT. D, activation assays of the cAMP analogs and their EC50 values (E) and maximum activation (F) compared with that by cAMP. n = 3, **, p < 0.01; ***, p < 0.001.
FIGURE 4.
FIGURE 4.
(Rp)-substituted cAMP analogs are competitive inhibitors, and addition of 8-CPT enhances binding affinity. cAMP and cAMP analogs were added and assayed as in Fig. 2. A, (Rp)-cAMPS and (Rp)-CPT inhibit CAMYEL preactivated by 100 μm cAMP in a concentration-dependent manner. B, (Rp)-CPT was preincubated with CAMYEL for 5 min before adding cAMP. The concentration-dependent activation of CAMYEL by cAMP was determined 5 min after activation by cAMP. C, Schild plot obtained from B. n = 3–6.
FIGURE 5.
FIGURE 5.
CE3F4, an Epac-specific inhibitor, inhibits CAMYEL activation. CAMYEL was preincubated with CE3F4 (3, 10, and 20 μm) for 5 min before assessing the ability of cAMP (A), 8-Me (B), or CPT (C) to activate CAMYEL. CE3F4 inhibited the ability of the Epac agonists to activate CAMYEL in a concentration-dependent manner. n = 3.
FIGURE 6.
FIGURE 6.
N6-Modified cAMP analogs are competitive inhibitors of Epac1, as assessed in the CAMYEL assay. A, structures of the adenine ring of cAMP showing various N6 substitutions. N6-Modified cAMP analogs were tested for their ability to activate CAMYEL (B) or inhibit 100 μm cAMP-activated CAMYEL (C). D, CAMYEL activated by 100 μm cAMP is inhibited by N6-Phe or CPT-N6 in a concentration-dependent manner. E, CPT-N6 inhibits CAMYEL activation by cAMP in a concentration-dependent manner. F, Schild plot obtained from data shown in E. n = 3–6. AE, aminoethyl; Bn, benzene; Bnz, benzoyl; Br, bromine; MB, monobutyryl.
FIGURE 7.
FIGURE 7.
cGMP analogs as inhibitors of Epac1, as assessed in the CAMYEL assay. A, concentration-dependent activation of CAMYEL by cGMP and cGMP analogs. B, concentration-dependent inhibition by cGMP and cGMP analogs of CAMYEL preactivated with 100 μm cAMP. n = 3.
FIGURE 8.
FIGURE 8.
Comparison of inhibitors of Epac, as assessed using Epac2-cAMPS or the CAMYEL assay. A, cAMP, (Rp)-cAMPS, or N6-cAMP-Phe were added to 100 μl of Epac2-cAMPS lysate in a black 96-well plate. After 5 min, their ability to activate Epac2-CNBD was measured (results normalized to cAMP). B, activation of CAMYEL by cAMP preincubated for 5 min with 10 μm CPT-N6 or 10 μm (Rp)-CPT. C, CAMYEL was activated with 100 μm cAMP before assessment of the ability of CE3F4, CPT-N6, and (Rp)-CPT to inhibit CAMYEL. n = 3.
FIGURE 9.
FIGURE 9.
Epac1 activates Rap1 and PKA activation mediates VASP phosphorylation in Swiss 3T3 cells. A, QT-PCR analysis of Epac1 and Epac2 mRNA expression in Swiss 3T3 cells and mouse reference cDNA. B, Rap1 activation and VASP phosphorylation were assessed by Western blotting of Swiss 3T3 cells that were untreated (Unt) or incubated with 8-Me, N6, or forskolin (Fsk) for 5 min. C, pooled results (n = 3) for Rap1 activation. D, VASP phosphorylation by 8-Me, N6, or forskolin. E, inhibition of forskolin-promoted Rap1 activation but not VASP phosphorylation by 20 μm CE3F4. F and G, pooled results for effect of 20 μm CE3F4 on forskolin-promoted Rap1 activation and VASP phosphorylation, respectively n = 4, **, p < 0.01.
FIGURE 10.
FIGURE 10.
CPT-N6 and (Rp)-CPT inhibit forskolin-promoted Epac activation in Swiss 3T3 cells. Swiss 3T3 cells were incubated with 50 μm CPT-N6 (A and B) or 100 μm (Rp)-CPT (C and D) for 30 min and were then untreated (Unt) or incubated with forskolin (Fsk) (10 μm). Cells were harvested 5 min later, and the levels of activated Rap1 were quantified. B and D, show pooled results for n = 5, *, p < 0.01.
FIGURE 11.
FIGURE 11.
Molecular model of CPT-N6 bound to Epac1. A, solved structure of Epac2 CNBD (green) with (Sp)-cAMPS (PDB code 3CF6). B, representative structures from the halo-MD of CPT-N6 docked to the Epac1 model (trajectory 1 in purple and trajectory 2 in orange) are shown for comparison. C, closer look at the CPT-N6-bound Epac1 models from trajectory 1. D, closer look at the CPT-N6-bound Epac1 models from trajectory 2, showing potential interactions. Ligands and water molecules are shown as sticks, protein residues as lines, and bonds as dotted lines. Protein secondary structure is shown as a schematic in A and B. Residues are labeled for Epac1 and Epac 2 (in parentheses) for comparison with C and D. Figures were created using PyMOL.
FIGURE 12.
FIGURE 12.
Chembridge compound 5225554 is an allosteric inhibitor of Epac. A, structure of 5225554. B, preincubation with increasing concentrations of 5225554 decreases cAMP-promoted activation of CAMYEL. C, Swiss 3T3 cells were treated with 100 μm compound 5225554 for 30 min prior to incubation with forskolin (Fsk) (10 μm) for 5 min. Activated Rap1 and VASP phosphorylation were then assessed by Western blotting. D and E, pooled results (n = 3) for effect of 5225554 on forskolin-promoted Rap1 activation and VASP phosphorylation, respectively. F and G, virtual docking of compound 5225554 near the hinge region in the inactive Epac1 model. Protein secondary structure is shown as a schematic. 5225554 is yellow, surrounding residues are shown as lines. Leu-315 and Phe-342 are shown as sticks to highlight proximity; black dotted lines depict potential hydrogen bonding. PyMOL was used to generate F and G. *, p < 0.05; **, p < 0.01; Unt, untreated.
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