Identification of a Novel, Small Molecule Partial Agonist for the Cyclic AMP Sensor, EPAC1

Abstract

Screening of a carefully selected library of 5,195 small molecules identified 34 hit compounds that interact with the regulatory cyclic nucleotide-binding domain (CNB) of the cAMP sensor, EPAC1. Two of these hits (I942 and I178) were selected for their robust and reproducible inhibitory effects within the primary screening assay. Follow-up characterisation by ligand observed nuclear magnetic resonance (NMR) revealed direct interaction of I942 and I178 with EPAC1 and EPAC2-CNBs in vitro. Moreover, in vitro guanine nucleotide exchange factor (GEF) assays revealed that I942 and, to a lesser extent, I178 had partial agonist properties towards EPAC1, leading to activation of EPAC1, in the absence of cAMP, and inhibition of GEF activity in the presence of cAMP. In contrast, there was very little agonist action of I942 towards EPAC2 or protein kinase A (PKA). To our knowledge, this is the first observation of non-cyclic-nucleotide small molecules with agonist properties towards EPAC1. Furthermore, the isoform selective agonist nature of these compounds highlights the potential for the development of small molecule tools that selectively up-regulate EPAC1 activity.

Figure 1

8-NBD-cAMP Competition Assay Using Purified EPAC1-CNB and EPAC2-CNB Proteins. (a)The 8-NBD-cAMP competition assay was carried out as previously described. Briefly, purified EPAC1-CNB (0.8 µM), EPAC2-CNB (0.8 µM), GST (0.8 µM) or assay buffer (no protein) was incubated in the presence of cAMP (50 µM) or DMSO. 8-NBD-cAMP (0.1 µM) was then added to every well and the fluorescence intensity was measured after four hours. Significant inhibition of fluorescence in the presence of cAMP is indicated, ^#P < 0.0001 (n = 3). (b) Inhibition of 8-NBD-cAMP (62.5 nM) fluorescence, resulting from interaction with EPAC1-CNB (0.8 µM), is plotted in the presence of varying concentrations of known competitor compounds; cAMP (red squares), 007 (blue circles) or 8-CPT (green triangles). IC50 values were calculated for each competition assay and are shown in comparison with published Kd values for each competitor.

Figure 2

Single Point Testing for Compounds Capable of Disrupting. Interaction between EPAC1-CNB and 8-NBD-cAMP in HTS Compounds from the BioAscent library of diverse lead like structures were assessed in 384 well plates at 10 µM with 0.25% DMSO final, n = 1 over two testing days. Responses (% effect) from each compound tested are shown. For the screen a selection cut-off of 15% was calculated using the median plus three times the robust standard deviation (scaled median absolute deviation) of the percentage inhibition across all compound wells, generating 33 hits (indicated with red dots) and a 0.67% hit rate. The screen performed extremely well with consistent levels of maximum (Max) and minimum (Min) control responses, Z’ values and S/B ratios. %CV values were calculated for matching control wells as described (materials and methods) and were below 10%, indicating excellent inter-plate and inter-day variability.

Figure 3

Dose Response Testing of Hits against EPAC1-CNB. The three hit compounds isolated from the Bioascent libraries (structures and molecular weights are shown on the left) were tested in a 7-point dose response format (75 µM top concentration, 1 in 2 serial dilution, n = 6 on one plate) alongside a non-binding compound (I516) in the EPAC1-CNB, 8-NBD-cAMP binding assay with final DMSO concentration of 0.75%. IC₅₀ values are shown in the individual graphs.

Figure 4

GEF activity of EPAC1. (a) Time trace of the exchange reaction with 500 μM cAMP either 500 μM I178, I288, I516 or I942 where indicated. The data are fitted as single exponential decay with off-set (red lines) to obtain the rate constants k_obs, as shown in (b). Rate constants from exchange reactions with either 500 μM or 50 μM cAMP, the presence or absence of 500 μM I178, I288, I516 or I942, as indicated, are presented as a bar graph (n = 4). (c) Exchange activity induced by cAMP, I942 or I178. The dependency of k_obs on the concentration of cAMP (open triangles), I942 (closed circles) or I178 (open circles) is plotted on the left. The lower panel shows a magnification of the I942 and I178 induced activity. Inhibition of cAMP induced exchange activity by I942 and I178 is shown on the right. k_obs were determined in the presence of 500 μM cAMP and various concentrations of I942 or I178.

Figure 5

I942 and I178 have reduced Agonist Potential against EPAC2 Compared with EPAC1. (a) Time trace of the exchange reaction with recombinant EPAC2^280–993 with cAMP (50 μM), I942 (500 μM) or a combination of cAMP (50 μM) and I942 (500 μM). (b) Rate constants from exchange reactions (n = 4)in the presence of I942 (500 μM), I178 (500 μM) and/or cAMP (50 μM). (c) Inhibition of cAMP induces exchange activity of Epac2^280–993. k_obs were determined in the presence of 50 μM cAMP and various concentrations of I942 and I178.

Figure 6

Ligand Observed NMR reveals Hit compounds Interact Directly with EPAC1-CNB and EPAC2-CNB, but not GST alone. (A) One dimensional proton (1D) and waterLOGSY (WL) spectra were performed on samples containing hit compound I942 (50 µM) in the absence and presence of EPAC1-CNB, EPAC2-CNB or GST alone, as indicated (2.5 µM). A representative region of the I942 spectrum is shown (7.1–7.75 ppm). Interaction between compound and protein was assessed through comparison of the compound alone and protein-compound sample spectra. The NMR data are representative of data collected at several protein and ligand concentrations. (B) 1D and WL spectra were performed on samples containing I178 (50 µM) in the absence and presence of EPAC1-CNB, EPAC2-CNB or GST alone, as indicated (2.5 µM). A representative region of the I178 spectrum is shown (7.1–7.51 ppm). (C) 1D and WL spectra were performed on samples containing the commercially available EPAC selective inhibitor ESI-09 (50 µM) in the absence and presence of EPAC1-CNB, EPAC2-CNB or GST alone, as indicated (2.5 µM). A representative region of the ESI-09 spectrum is shown (0.6–1.2 ppm).