Differential Pharmacophore Definition of the cAMP Binding Sites of Neuritogenic cAMP Sensor-Rapgef2, Protein Kinase A, and Exchange Protein Activated by cAMP in Neuroendocrine Cells Using an Adenine-Based Scaffold

Article abstract of DOI:10.1021/acschemneuro.6b00462

We recently reported that the adenylate cyclase (AC) inhibitor SQ22,536 (9-tetrahydrofuranyl-adenine) also has inhibitory activity against the neuroendocrine-specific neuritogenic cAMP sensor-Rapgef2 (NCS-Rapgef2), a guanine nucleotide exchanger and activator for the small effector GTPase Rap1. Cell-based assays that distinguish signaling through the three intracellular cAMP sensors NCS-Rapgef2, exchange protein activated by cAMP (Epac), and protein kinase A (PKA), as well as AC, were used. These, collectively, assess the activities of adenine (6-amino-purine) derivatives modified at several positions to enhance selectivity for NCS-Rapgef2 by decreasing affinity for adenylate cyclase (AC), without increasing affinity for PKA or Epac. Testing of each adenine derivative in whole-cell assays incorporates features of cell permeability, target selectivity, and intrinsic potency into a single EC₅₀ or IC₅₀, making robust extrapolation to compound activity in vivo more likely. N6-MBC-cAMP is a selective PKA activator (EC₅₀ = 265 μM) with low efficacy at NCS-Rapgef2. 8-CPT-2′-O-Me-cAMP and ESI-09 are confirmed as Epac-selective, for stimulation and inhibition, respectively, versus both PKA and NCS-Rapgef2. The compound N6-Phe-cAMP is a full agonist of NCS-Rapgef2 (EC₅₀ = 256 μM). It has little or no activity against Epac or PKA. The compound N6-phenyl-9-tetrahydrofuranyladenine is a novel and potent NCS-Rapgef2 inhibitor without activity at PKA, Epac, or ACs, as assayed in the neuroendocrine NS-1 cell line. This line has been engineered to allow high-content screening for activation and inhibition of AC, PKA, Epac, and NCS-Rapgef2 and the cellular activities initiated by these signaling pathway protein components.

Full text of DOI:10.1021/acschemneuro.6b00462

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Article
Differential Pharmacophore Definition of the cAMP
Binding Sites of NCS-Rapgef2, PKA and Epac in
Neuroendocrine Cells Using an Adenine-based Scaffold
Andrew Clayton Emery, Ryan A. Alvarez, Maribeth V. Eiden, Wenqin Xu, Fabrice G Simeon, and Lee E. Eiden
ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.6b00462 • Publication Date (Web): 14 Mar 2017
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Page 1 of 15
ACS Chemical Neuroscience
Differential Pharmacophore Definition of the cAMP Binding Sites of
NCS-Rapgef2, PKA and Epac in Neuroendocrine Cells Using an Ade-
nine-based Scaffold
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Andrew C. Emery , Ryan A. Alvarez , Maribeth V. Eiden , Wenqin Xu , Fabrice G. Simeon , Lee E.
Eiden *
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Section on Molecular Neuroscience, Section on Directed Gene Transfer, PET Radiopharmaceutical Sciences Section, Naꢀ
tional Institute of Mental Health Intramural Research Program, 9000 Rockville Pike, Bethesda MD 20892
*To whom correspondence should be addressed: 9000 Rockville Pike, Building 49, Room 5Aꢀ38, Bethesda, MD 20892,
Email: eidenl@mail.nih.gov, Tel: 1ꢀ301ꢀ496ꢀ4110
ABSTRACT
We recently reported that the adenylate cyclase (AC) inhibitor SQ22,536 (9ꢀtetrahydrofuranylꢀadenine) also has inhibitory
activity against the neuroendocrineꢀspecific neuritogenic cAMP sensorꢀRapgef2 (NCSꢀRapgef2), a guanine nucleotide exꢀ
changer and activator for the small effector GTPase Rap1. Cellꢀbased assays that distinguish signaling through the three inꢀ
tracellular cAMP sensors NCSꢀRapgef2, exchange protein activated by cAMP (Epac), and protein kinase A (PKA), as well
as AC, were used. These, collectively, assess the activities of adenine (6ꢀaminoꢀpurine) derivatives modified at several posiꢀ
tions to enhance selectivity for NCSꢀRapgef2 by decreasing affinity for adenylate cyclase (AC), without increasing affinity
for PKA or Epac. Testing of each adenine derivative in wholeꢀcell assays incorporates features of cell permeability, target
selectivity, and intrinsic potency into a single EC50 or IC , making robust extrapolation to compound activity in vivo more
50
likely. N6ꢀMBCꢀcAMP is a selective PKA activator (EC50 = 265 ꢀM) with low efficacy at NCSꢀRapgef2. 8ꢀCPTꢀ2’ꢀOꢀMeꢀ
cAMP and ESIꢀ09 are confirmed as Epacꢀselective, for stimulation and inhibition, respectively, versus both PKA and NCSꢀ
Rapgef2. The compound N6ꢀPheꢀcAMP is a full agonist of NCSꢀRapgef2 (EC = 256 ꢀM). It has little or no activity against
50
Epac or PKA. The compound N6ꢀphenylꢀ9ꢀtetrahydrofuranyladenine is a novel and potent NCSꢀRapgef2 inhibitor without
activity at PKA, Epac or ACs, as assayed in the neuroendocrine NSꢀ1 cell line. This line has been engineered to allow highꢀ
content screening for activation and inhibition of AC, PKA, Epac and NCSꢀRapgef2, and the cellular activities initiated by
these signaling pathway protein components.
KEYWORDS: Cyclic AMP, Epac, Rapgef2, ERK, p38, CREB
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INTRODUCTION
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For several decades after the discovery of protein kinase A (PKA), it was assumed that cAMP binding to the regulatory
subunit of PKA (PKAR), with activation of the catalytic subunit of PKA (PKAc) was the sole mode through which cAMP
signal transduction occurs in mammalian cells . PKAc is inactive in association with PKAR. PKAc dissociates from PKAR
when homodimers of PKAR (RIαꢀRIα, RIβꢀRIβ, RIIαꢀRIIα or RIIβꢀRIIβ; products of the RIα, RIβ, RIIα and RIIβ genes),
are each occupied by two molecules of cAMP at sites A and B, found on each R monomeric subunit. The A and B sites of
each of the four monomeric subunits RIα, RIβ, RIIα and RIIβ differ in their affinity for cyclic AMP, with the A site having a
faster, and the B site having a slower dissociation rate for cAMP . In addition, the absolute affinity for cAMP differs
somewhat at both A and B sites, when compared across the four isoforms RIα, RIβ, RIIα and RIIβ. Consequently, a large
number of pharmacological agents directed to sites A and B have been developed, and used in combination, to differentially
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activate the four forms of PKAR (RIαꢀRIα, RIβꢀRIβ, RIIαꢀRIIα and RIIβꢀRIIβ in various tissue and cell types . These
compounds have given significant insight into the cAMP pharmacophore for each of the two sites A and B, but have not
been particularly useful for pharmacological experiments in vivo. For this reason, pharmacological study of PKAꢀdependent
cellular signaling has relied mainly on competitive inhibition of cAMP binding to PKAR (e.g. by the Rp isomer of cyclic
adenosineꢀ3′,5′ monophosphorothioate (RpꢀcAMPS) or of the kinase activity of PKAc, with isoquinolines such as H89 and
5ꢀ6
KT5720. Each of these approaches has its own limitations of specificity and potency
.
In the late 1990s, a second set of sensors for cAMP in mammalian cells was discovered. These were not protein kinases,
but rather guanine nucleotide exchange factors (GEFs): the Exchange Proteins Activated by cAMP (Epac 1 and 2, also called
cAMPꢀGEFꢀI/II, or Rapgef3/4). These proteins were shown to catalyze the activation of the small GTPase effector molecule
Rap, a member of the Ras superfamily, by releasing GDP from, and allowing GTP binding to, Rap . The existence of a
new set of cAMP sensors, besides PKAR, made it necessary to reexamine the specificity of existing pharmacological activaꢀ
tors and inhibitors of PKA, for their actions at Epacs. RpꢀcAMPS and SpꢀcAMPS were found to have similar activities at
both PKAR and Epac. Thus, these compounds could no longer be considered specific for PKA
developed, however with a high degree of specificity for Epacs, compared to PKA. These were 8ꢀchlorophenylthioꢀ2’ꢀOꢀ
methylꢀcAMP, an Epac activator, and ESIꢀ05/09, which are Epac inhibitors
7ꢀ8
9ꢀ11
. Two compounds were
10ꢀ13
.
This operational definition of specificity seemed to be sufficient for the analysis of cAMP signal transduction in most
cell types. A third potential cAMP sensor, CNrasGEF/PDZꢀGEF1 was dismissed as a cAMPꢀregulated GEF, since it seemed
not to function in a cAMPꢀdependent manner
nucleotide binding site (CNBD) was deemed unlikely to mediate cAMPꢀdependent steric alteration/activation of the GEF
functionality of the protein in vivo . Subsequently, however, we have identified a protein product of the Rapgef2 gene,
termed NCS (for neuritogenic cAMP sensor)ꢀRapgef2, which, although apparently not expressed in nonꢀneuronal tissues of
1
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16ꢀ18
, except perhaps in melanoma cells
. In fact, its nonꢀcanonical cyclic
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adult mammals, appears to mediate cAMPꢀdependent activation of ERK, through Rap1, in neuronal and endocrine cells
.
The discovery of this third type of intracellular cyclic AMP sensor necessitates the development of a new cAMPꢀrelated neuꢀ
ropharmacology which takes into account differential affinities and effects of cAMP at its binding site(s) at PKAR, Epac,
and NCSꢀRapgef2. Herein, we describe cellꢀbased signaling assays that probe the specificity of existing PKAꢀ and Epacꢀ
directed cAMP analogs, as extended to interaction with NCSꢀRapgef2. Furthermore, we report the synthesis and pharmacoꢀ
logical properties of some novel adenine derivatives with specific activity at NCSꢀRapgef2 compared to PKA and Epac,
based on a previous report of NCSꢀRapgef2 inhibitory activity of the adenylyl cyclase inhibitor 9ꢀtetrahydrofuranylꢀadenine
21
.
RESULTS AND DISCUSSION
Overview
Our strategy for development of potentially specific NCSꢀRapgef2 inhibitor proceeds from the observation that 9ꢀTHFꢀ
adenine, an adenine derivative modified at the 9ꢀp0sition relative to cyclic AMP, is both an AC inhibitor and an NCSꢀ
Rapgef2 inhibitor, with apparently little activity to block cAMPꢀdependent activation of Rap by Epac or PKAꢀdependent
transcriptional activation of a CRE reporter gene, in NSꢀ1 cells ; and systematic modification at the N6ꢀposition of adenine
to further improve potency and selectivity for NCSꢀRapgef2 inhibition.
21
In order to achieve this, we have engineered a battery of cellꢀbased biochemical assays that quantitatively distinguish
between PKAꢀ, Epacꢀ and NCSꢀRapgef2ꢀdependent signaling in the NSꢀ1 neuroendocrine cell line, based on our previous
demonstration using genetic and pharmacological manipulations, that CREB phosphorylation, p38 activation and growth
arrest, and ERK phosphorylation/activation and neurite extension are uniquely and exclusively stimulated by PKA, Epac and
NCSꢀRapgef2, respectively, in NSꢀ1 cells . To validate the use of phosphospecific antibody cellꢀbased ELISAs (PACE) to
assess the phosphorylation (activation) status of ERK, CREB, and p38 to be used as biochemical surrogates for NCSꢀ
Rapgef2, PKAc, and Epac, NSꢀ1 cells were treated for 30 minutes with the panꢀspecific cAMP analog 8ꢀCPTꢀcAMP (100
22
ꢀM) in the absence or presence of inhibitors of a component of one of the three signaling pathways. Compounds included
H89 (PKAc inhibitor), U0126 (MEK inhibitor), ESIꢀ09 (Epac inhibitor), and SB2239036 (p38 inhibitor). Inhibitors were
applied to cells for 30 minutes prior to the addition of 8ꢀCPTꢀcAMP. As seen in Figure 1AꢀD, 8ꢀCPTꢀcAMP caused a robust
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increase in ERK phosphorylation that was only inhibited by U0126 (Figure 1B). CREB phosphorylation (Figure 1 EꢀH) was
blocked by inhibition of PKAc with H89 (Figure 1E), but unaffected by the other compounds. P38 phosphorylation (Figure 1
IꢀL) was sensitive to Epac inhibitions with ESIꢀ09 (Figure 1K) and SB239036 (Figure 1L).
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Fig. 1
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Figure 1 Measurements of phosphorylated ERK, CREB, and p38 by PACE (phosphospecific antibody cell-based ELISA). NS-1 cells were
grown overnight in 96-well plates at a density of 30,000 cells/well. The next day, inhibitors or DMSO was added as 10x stocks to the fol-
lowing final concentrations: 10
ꢀM H89 or 0.02% DMSO; 10
ꢀM U0126 or 0.02% DMSO; 10
ꢀM ESI-09 or 0.02% DMSO; and 10
ꢀM
SB 239036 or 0.1% DMSO. Cells were pre-treated for 10 min followed by the addition of 100
ꢀM 8-CPT-cAMP (8-CPT) or an equal vol-
ume of media. 30 minutes later, media were aspirated, cells were fixed and immunodetection of phospho-proteins was carried out as de-
scribed in Methods. Bars are means from three experiments are errors correspond to standard errors of the mean. Data were analyzed by
2
#
-way ANVOA and Bonferroni-corrected t-tests, *P < 0.05, **P < 0.01, *** P < 0.001 comparing the effect of 8-CPT-cAMP to media;
#P < 0.01, ###P < 0.001 comparing these effect of each inhibitor within a treatment condition (media or 8-CPT-cAMP).
Identification of sensor-selective agonists
ꢀCPTꢀ2’ꢀOꢀMeꢀcAMP, while inactive at PKAR/CREB (as previously described ) or NCSꢀRapgef2/ERK (Figure 2Aꢀ
13
8
B), was a more potent Epac agonist than the parent compound 8ꢀCPTꢀcAMP (Figure 2C). Together these data show that 8ꢀ
CPTꢀ2’ꢀOꢀMeꢀcAMP is indeed a highly Epacꢀselective agonist. Since 8ꢀCPTꢀcAMP does not discriminate among the three
sensors and 8ꢀCPTꢀ2’ꢀOꢀMeꢀcAMP is Epacꢀspecific, we wished to see whether additional known cAMP analogs could be
used as subtypeꢀpreferring agonists. A number of cyclic nucleotide analogs that preferentially bind to a single site of a reguꢀ
latory subunit of PKA have been identified. The typical motif for these is that N6 modifications confer selectivity for the A
site (also referred to as site I, or the rapid site), while halogenation of C2 and addition of some functional groups to C8 (meꢀ
thylamino or piperidino) confers selectivity for the Bꢀsite (also called site II, or the slow site). Highly selective ligands for
either the A or B site would not be predicted to act as activators of the PKA holoenzyme unless used in combination. Acꢀ
cordingly, we compared the potencies and efficacies of five readilyꢀavailable cAMP analogs on ERK phosphorylation in NSꢀ
1
cells to 8ꢀCPTꢀcAMP (Figure 3A). 8ꢀCPTꢀcAMP caused a maximal increase in ERK activation similar to that observed in
cells treated with N6ꢀBenzoylꢀcAMP (N6ꢀBnꢀcAMP), N6ꢀBenzylꢀcAMP (N6ꢀBzꢀcAMP), N6ꢀPhenylꢀcAMP (N6ꢀPheꢀ
cAMP), and N6ꢀPhenylꢀ8ꢀCPTꢀcAMP (N6ꢀPheꢀ8ꢀCPTꢀcAMP). In contrast, 5,6ꢀDichloroꢀ1ꢀβꢀDꢀ
ribofuranosylbenzimidazoleꢀ 3', 5'ꢀ cyclic monophosphorothioate (cBIMPS) and N6ꢀmonoꢀtert.butylcarbamoylꢀcAMP (N6ꢀ
MBCꢀcAMP) had approximately 60% and 80% less intrinsic activity than other analogs examined, respectively. In rank orꢀ
der of potency for ERK activation: N6ꢀPheꢀ8ꢀCPTꢀcAMP > N6ꢀBnꢀcAMP = 8ꢀCPTꢀcAMP > N6ꢀBzꢀcAMP = N6ꢀPheꢀ
cAMP. As seen in Figure 3B, 8ꢀCPTꢀcAMP, SpꢀDClꢀ5,6ꢀcBIMPS, N6ꢀBnꢀcAMP, N6ꢀBzꢀcAMP, N6ꢀMBCꢀcAMP are full
PKA agonists in these cells. N6ꢀPheꢀcAMP is inactive at up to 1 mM at PKAR, i.e. to activate CREB phosphorylation. N6ꢀ
Pheꢀ8ꢀCPTꢀcAMP, while a full agonist of PKA, was approximately 15 times more potent at NCSꢀRapgef2. N6ꢀMBCꢀcAMP
and Spꢀ5,6ꢀDClꢀcBIMPS were the only agonists of PKA tested with limited intrinsic activity at NCSꢀRapgef2. To see
whether the N6ꢀPheꢀcAMP can be used as a selective agonist of NCSꢀRapgef2 and N6ꢀMBCꢀcAMP is a PKAꢀselective agoꢀ
nist, the ability these two compounds to stimulate Epacꢀdependent signaling (p38 phosphorylation) was evaluated in an assay
with 8ꢀCPTꢀ2’ꢀOꢀMeꢀcAMP as a point of reference. Both N6ꢀPheꢀcAMP and N6ꢀMBCꢀcAMP were inactive at Epac at conꢀ
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centrations as high as 1 mM (Figure 3C). Using 8ꢀCPTꢀcAMP as a point of reference, the relative potencies of all comꢀ
pounds tested are listed in Table 1.
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Fig. 2
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-CPT-2'-O-Me
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Cyclic AMP analog], log (M)
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[Cyclic AMP analog], log (M)
[
[Cyclic AMP analog], log (M)
Figure 2 Activity of 8-(4-Chlorophenylthio)-cAMP (8-CPT) and 8-(4-Chlorophenylthio)-2’-O-Me-cAMP (8-CPT-2’-O-Me) on the neu-
ronal cyclic AMP sensors NCS-Rapgef2, PKAR, and Epac2. NS-1 cells were treated with varying concentrations of either compound for
30 min followed by fixation and measurement of (A) phosphorylated ERK, (B) phosphorylated CREB, and (C) phosphorylated p38 MAP
kinase. Data points are means from 3-4 experiments performed in duplicate or triplicate with error bars corresponding to standard errors
of the mean.
Fig. 3
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cBIMPS
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10-4
10-3
01 001 0
1 01 001 0
[Cyclic AMP analog], log (M)
[Cyclic AMP analog], log (M)
[Cyclic AMP analog], log (M)
Figure 3 Effects of N6-substituted cAMP analogs on (A) ERK and (B) CREB phosphorylation. 8-CPT-cAMP, N6-Benzyl (N6-Bn)-cAMP,
N6-Benzoyl (N6-Bz)-cAMP are each full agonists at both ERK and CREB at approximately equivalent potencies. N6-mono-tert. butylcar-
bamoyl (N6-MBC)-cAMP and Sp-5,6-dichloro-1-β-D-ribofuranosylbenzimidazole-3',5'-monophosphorothioate (cBIMPS) are low efficacy
agonists for ERK and full agonists of PKAR/CREB. By contrast, N6-Phenyl (N6-Phe)-cAMP is active at ERK while inactive at CREB at
concentrations as high as 1 mM. (C) Neither N6-Phe-cAMP nor N6-MBC-cAMP caused a significant increase in p38 phosphorylation
relative to the effect of 8-CPT-2’-O-Me-cAMP.
In order to confirm the NCSꢀRapgef2 efficacy and specificity of the cAMP analogs that were either ERK or CREB seꢀ
lective in phosphorylation assays, we measured neurite elongation (an NCSꢀRapgef2 and ERKꢀdependent phenotypic readꢀ
out) in NSꢀ1 cells treated for 48 hours with vehicle control (Figure 4A), 100 ꢀM 8ꢀCPTꢀcAMP (Figure 4B), which caused
neurite elongation, 200 ꢀM N6ꢀPheꢀcAMP (Figure 4C), which also caused neurite elongation, or 100 ꢀM N6ꢀMBCꢀcAMP
(
Figure 4D), which failed to elicit cause neurite elongation, suggesting that even though this compound had some efficacy to
cause ERK phosphorylation, its intrinsic activity is likely insufficient to cause a sufficient magnitude of sustained ERK actiꢀ
25
vation required for neurite elongation .
N6ꢀPheꢀcAMP has been reported to activate the PKA holoenzyme when applied in combination with a Bꢀsiteꢀpreferring
agonist. As a control, we determined that additive effects on CREB phosphorylation were evident in cells treated with N6ꢀ
PheꢀcAMP (100 ꢀM) and 1 ꢀM of the B site ligand Spꢀ5,6ꢀDClꢀcBIMPS (Figure S1), supporting the notion that N6ꢀPheꢀ
cAMP may be quite specific for PKA’s A sites over B sites, explaining why it is not an agonist of the PKA holoenzyme
when applied alone.
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Fig. 4
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C. N6-Phe-cAMP (200 ꢀM)
D. N6-MBC-cAMP (100 ꢀM)
Figure 4 Cyclic AMP analog-dependent neurite elongation. NS-1 cells were treated for 48 hours with (A) vehicle control, (B) 8-CPT-
cAMP, 100
cAMP, 200
ꢀ
ꢀ
M, slightly greater than the half-maximal concentration for the compounds effect on ERK phosphorylation, (C) N6-Phe-
M, an approximately half-maximal concentration for its effect on ERK phosphorylation, and (D) N6-MBC-cAMP, 100 M, a
supramaximal concentration for its effect on ERK phosphorylation. Photomicrographs were randomly acquired and are representative
images from four determinations obtained in three independent experiments.
ꢀ
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Table 1. Potency of cAMP analogs to activate PKAꢀ, NCSꢀRapgef2ꢀ, and Epac2ꢀdependent signaling processes relative to 8ꢀCPTꢀcAMP.
Results are means and standard errors from 2ꢀ4 independent experiments performed in triplicate. EC50 values were calculated by 4ꢀ
parameter logistic regressions. Values >1000 ꢀM indicates that compound did not reach the halfꢀmaximal effect of 8ꢀCPTꢀcAMP when
applied at up to 1 mM; n.d., not determined.
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Compound
PKA/CREB Rapgef2/ERK Epac2/p38 Structure
EC (ꢀM)
EC (ꢀM)
EC (ꢀM)
5
0
50
50
8
8
ꢀBrꢀcAMP
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158 + 32
163 + 31
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74 + 8
68 + 14
79 + 17
8
ꢀCPTꢀ2’ꢀOꢀMeꢀcAMP >1000
>1000
25 + 6
25 + 3
>1000
N6ꢀPheꢀ8ꢀCPTꢀcAMP
426 + 91
N6ꢀPheꢀcAMP
N6ꢀBnꢀcAMP
>1000
256 + 33
77 + 29
>1000
n.d.
231 + 38
N6ꢀBzꢀcAMP
88 + 8
171 + 46
>1000
n.d.
N6ꢀMBCꢀcAMP
265 + 14
>1000
Spꢀ5,6ꢀDClꢀcBIMPS
54 + 7
>1000
>1000
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Activity of 9-tetrahydrofuranyl adenine (SQ22,536) to inhibit cAMP-dependent activation of PKA, Epac and NCS-
Rapgef2
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Given the specificity of known inhibitors of PKA and Epac shown in Figure 1, we were able to use cAMPꢀdependent
ERK activation as a convenient assay for inhibition of NCSꢀRapgef2 and AC, or combinations thereof, in the same cells.
Previously, we have shown that cAMPꢀdependent ERK activation is completely abrogated by silencing NCSꢀRapgef2 exꢀ
23
pression in NSꢀ1 cells . As seen in Figure 5A, 9ꢀTHFꢀadenine is a relatively potent AC inhibitor (IC50 = 8.9 + 0.9 ꢀM).
Although its potency at NCSꢀRapgef2 is diminished relative to its action at AC (the IC50 for inhibition of 8ꢀBrꢀcAMP stimuꢀ
lation of NCSꢀRapgef2 is 825 + 12 ꢀM), 9ꢀTHFꢀadenine is capable of causing significant inhibition of activation of NCSꢀ
Rapgef2 by cAMP to a much greater extent than its effects of cAMPꢀmediated actions on PKA (Figure 5B) or Epac (Figure
5
C).
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Forskolin
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Forskolin
100
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8-Br-cAMP
Forskolin
1
00 -11 10-7 10-6 10-5 10-4 10-3
1 00 -11 10-7 10-6 10-5 10-4 10-3
[SQ22536], log (M)
1 00 -11 10-7 10-6 10-5 10-4 10-3
[SQ22536], log (M)
[SQ22536], log (M)
Figure 5 SQ22,536 (9-THF-Ade) inhibits adenylate cyclase and NCS-Rapgef2. Measurements of (A) ERK phosphorylation, (B) CREB
phosphorylation, and (C) p38 phosphorylation in NS-1 cells pre-treated for 30 min with varying concentrations of SQ22,536 or vehicle
(0.3% DMSO) followed by treatment with EC80 concentrations of either 8-Br-cAMP (575 ꢀM) or forskolin (3 ꢀM) for 30 min. Data points
correspond to means from three experiments performed in triplicate with error bars equal to the standard errors of the mean. As deter-
mined by ANOVA, 9-THF-Ade had significant inhibitory activity on the effects of both forskolin and 8-Br-cAMP on ERK activation (A), but
only significantly blocked the effects of forskolin on (B) CREB and (C) p38 phosphorylation.
Modification of 9-THF-Adenine to generate an NCS-Rapgef2-selective inhibitor
Previously we reported that the nucleoside 9ꢀtetrahydroꢀ2ꢀfuranylꢀadenine (SQ 22,536, referred to here as 9ꢀTHFꢀAde)
inhibits both adenylate cyclase and NCSꢀRapgef2. Since this compound has two mechanisms of action in the same signaling
pathway, the only way to use it to examine NCSꢀRapgef2 signaling is to examine the sensitivity to inhibition of a cAMPꢀ
mimicking agent that acts downstream of adenylate cyclase (i.e. a cAMP analog). For more experimental flexibility, and as a
potential first step in NCSꢀRapgef2ꢀdirected drug development, we wished to develop compounds with selectivity for NCSꢀ
Rapgef2 at the expense of AC, with a secondary goal of increasing overall potency/cell permeability. Since NCSꢀRapgef2
was the only neuronal cAMP sensor that allowed the addition of an N6ꢀphenyl group, and this group also increases the lipoꢀ
philicity of nucleotides/nucleosides, we synthesized N6ꢀphenylꢀ9ꢀtetrahydroꢀ2ꢀfuranylꢀadenine (N6ꢀPheꢀ9ꢀTHFꢀAde).
As seen in Figure 6A, N6ꢀPheꢀ9ꢀTHFꢀAde inhibited forskolin and 8ꢀBrꢀcAMPꢀinduced ERK phosphorylation at similar
potencies (IC50 values of 9 + 1 ꢀM). In contrast, as seen in Figure 6B, 9ꢀTHFꢀAde was a more potent inhibitor of forskolinꢀ
dependent ERK phosphorylation (IC50 = 9 + 2 ꢀM) than of 8ꢀBrꢀcAMPꢀdependent ERK phosphorylation (IC50 = 110 ꢀM).
Taken together, these data suggest that the observable mechanism of action of N6ꢀPheꢀ9ꢀTHFꢀAde is inhibition of NCSꢀ
Rapgef2 while 9ꢀTHFꢀAde is about an order of magnitude more potent at adenylate cyclase than at NCSꢀRapgef2.
To determine whether N6ꢀPheꢀ9ꢀTHFꢀAde is selective for NCSꢀRapgef2 over PKA and Epac, NSꢀ1 cells were pretreatꢀ
ed with 100 ꢀM of either 9ꢀTHFꢀAde or N6ꢀPheꢀ9ꢀTHFꢀAde and exposed to EC80 doses of forskolin (3 ꢀM) or 8ꢀBrꢀcAMP
(
575 ꢀM) followed by measurements of CREB (Figure 6D) and p38 phosphorylation (Figure 6E). Doseꢀresponse data of 9ꢀ
THFꢀAde and N6ꢀPheꢀ9ꢀTHFꢀAde on 8ꢀBrꢀcAMPꢀ and forskolinꢀinduced CREB and p38 phosphorylation are shown in Figꢀ
ure S2. As seen in Figure 6D, N6ꢀPheꢀ9ꢀTHFꢀAde did not block the effect of forskolin or 8ꢀBrꢀcAMP on CREB phosphoryꢀ
lation. On the other hand, 9ꢀTHFꢀAde blocked the effect of forskolin, but not the effect of 8ꢀBrꢀcAMP, consistent with its
known profile as an AC inhibitor. Similar results were observed for p38 phosphorylation (Figure 6E), suggesting that neither
9ꢀTHFꢀAde nor N6ꢀPheꢀ9ꢀTHFꢀAde block PKA or Epac and that 9ꢀTHFꢀAde blocks adenylate cyclase, while N6ꢀPheꢀ9ꢀ
THFꢀAde does not.
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Fig. 6
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-Br-cAMP
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-Br-cAMP
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[N6-Phe-9-THF-Ade], log (M)
[9-THF-Ade], log (M)
C
D
E
DMSO
-THF-Ade
N6-Phe-9-THF-Ade
DMSO
9-THF-Ade
N6-Phe-9-THF-Ade
9
DMSO
N6-Phe-9-THF-Ade
9-THF-Ade
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Forskolin
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Forskolin
8-Br-cAMP
Forskolin
Figure 2 N6-Phe-9-THF-Ade is a specific inhibitor of cAMP-dependent ERK phosphorylation and acts a single cAMP sensor, NCS-
Rapgef2. (A) Measurements of ERK phosphorylation in NS-1 cells pre-treated for 30 min with varying concentrations of N6-Phe-9-THF-
Ade or vehicle (0.3% DMSO) followed by treatment with either 8-Br-cAMP (575
lation in NS-1 cells after pre-treatment with varying concentrations of 9-THF-Ade followed by either 8-Br-cAMP (575
M). (C) ERK phosphorylation in NS-1 cells pre-treated with either N6-Phe-9-THF-Ade or 9-THF-Ade (100 M each) followed by either
75 M 8-Br-cAMP or 3 M forskolin. Data are replotted from those shown in Panels A and B, ### P < 0.001 by Bonferroni-corrected t-
test following 2-way ANOVAs comparing the effects observed in cells pretreated with N6-Phe-9-THF-Ade and 9-THF-Ade to those pre-
treated with 0.3% DMSO (DMSO). Measurements of (D) CREB phosphorylation and (E) p38 phosphorylation after pretreatment with 100
ꢀM) or forskolin (3
ꢀM) for 30 min. (B) ERK phosphory-
ꢀ
M) or forskolin (3
ꢀ
5
ꢀ
ꢀ
ꢀ
ꢀ
M N6-Phe-9-THF-Ade or 9-THF-Ade followed by 575 ꢀM 8-Br-cAMP or 3 ꢀM forskolin; N = 3-6 separate experiments.
20, 26
The neuropeptide PACAPꢀ38 is a GPCR ligand that stimulates cAMP elevation in NSꢀ1 cells and in vivo
. It seemed
appropriate to show that the results of stimulating the three cAMP pathways pharmacologically, and assessing the activities
and potencies of inhibitors of the pathways, should be recapitulated with a physiological activator (i.e. GPCR ligand/first
messenger) such as PACAP. To this end, we used a phenotypic assay (neurite elongation following 48 h of treatment with
PACAPꢀ38) to determine whether N6ꢀPheꢀ9ꢀTHFꢀAde can be used to effectively inhibit NCSꢀRapgef2 for an extended time.
To this end, NSꢀ1 cells were treated PACAP (100 nM) and either 0.3% DMSO, or N6ꢀPheꢀ9ꢀTHFꢀAde, applied at either 3
M or 10 ꢀM, approximating the IC30 and IC50 of this compound determined by ERK phosphorylation measurements. After
8, cells were imaged, counted, and neurites were traced by a blinded observer. As seen in Figures 7A and 7B, both concenꢀ
trations significantly reduced the effect of PACAP, to a similar magnitude seen when using the shorterꢀterm (1 h) ERK
phosphorylation assay. Inhibition of adenylate cyclase was measured in cells pretreated with either 9ꢀTHFꢀAde or N6ꢀPheꢀ
ꢀTHFꢀAde followed by incubation with 3 nM PACAPꢀ38 (Fig. 7C), which activates the Gsꢀcoupled PAC1 receptor, or 3
ꢀ
4
9
ꢀ
M forskolin (Fig. 7D), which directly activates adenylate cyclase. Cyclic AMP elevation caused by either agent was inhibꢀ
ited by 9ꢀTHFꢀAde, but not significantly blocked by N6ꢀPheꢀ9ꢀTHFꢀAde at concentrations as high as 100 ꢀM, thus demonꢀ
strating that N6ꢀPheꢀ9ꢀTHFꢀAde acts to inhibit PACAPꢀinduced neurite elongation solely via direct inhibition of NCSꢀ
Rapgef2.
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Fig 7
ACS Chemical Neuroscience
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Forskolin + 9-THF-Ade
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0
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10-6
Inhibitor], log (M)
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10-4
0100
10100
[
Figure 3 N6-Phe-9-THF-Ade inhibits PACAP-dependent neurite elongation and cAMP elevation. (A) Neurite outgrowth was measured
after cells were treated for 48 hours. Five images from each well were acquired, N=4. Bars correspond to means and errors represent
standard errors of the mean. Data were analyzed by 2-way ANOVA followed by Bonferroni-corrected t-tests. Neurite extension promoted
by PACAP was significant (***P<0.001) compared to untreated controls. 6-Phe-9-THF-Ade (either 3 or 10
effect of PACAP on neurite outgrowth (###P<0.001). (B) Representative phase-contrast micrographs from each condition plotted in A. D
and E, measurements of intracellular cAMP in NS-1 cells pretreated for 30 min with 500 M IBMX and either 9-THF-Adenine or N6-Phe-
-THF-Adenine or vehicle (0.3% DMSO) for 20 min. Cells were then treated with (C) PACAP-38 (3 nM) or (D) Forskolin (3 M) for 20
ꢀM) significantly blocked the
ꢀ
9
ꢀ
min, N = 3. As determined by one-way ANOVAs, the inhibitory, 9-THF-Ade significantly inhibited the effects of both PACAP (C) and for-
skolin (D), while N6-Phe-9-THF-Ade did not.
Summary and Conclusions
Our survey of the activities of some of the most commonly used cAMP analogs at each of three cAMP sensors exꢀ
pressed in NSꢀ1 cells (relative potencies summarized in Table 1) has led to several conclusions. Modification to the N6 posiꢀ
tion is variably allowed by PKAR and NCSꢀRapgef2 but is not permitted by Epac2. In fact, several analogs, typically used as
PKAꢀspecific ligands have equal apparent potencies at NCSꢀRapgef2.
Where do we stand with specific intracellular cAMP sensor neuropharmacology that is potentially extendable to in vivo
experimentation, and potentially therapeutics? As regards cAMP agonists, we show selectivity of N6ꢀMBCꢀcAMP and Spꢀ
5
,6ꢀDCIꢀcBIMPS for PKA (versus Epac and NCSꢀRapgef2); selectivity of 8ꢀCPTꢀ2’ꢀOꢀmethylꢀcAMP for Epac (versus
NCSꢀRapgef2 and PKA); and selectivity of N6ꢀphenylꢀcAMP and N6ꢀphenylꢀ8ꢀCPTꢀcAMP for NCSꢀRapgef2 (versus PKA
and Epac). As regards cAMP antagonists, we have established selectivity of ESIꢀ09 for Epac (versus PKA and NCSꢀ
Rapgef2) and selectivity of N6ꢀphenylꢀ9ꢀTHFꢀAde for NCSꢀRapgef (versus PKA and Epac). There is currently no candidate
for development of a PKARꢀspecific inhibitor. Future development of agonists for NCSꢀRapgef2 will need to focus on enꢀ
hancing both the potency and the selectivity of the lead compounds N6ꢀPheꢀcAMP and N6ꢀPheꢀ8ꢀCPTꢀcAMP. Regarding
further inhibitor development, the compound space defined by the novel adenineꢀbased NCSꢀRapgef2 inhibitor looks promꢀ
ising not only visꢀaꢀvis other intracellular cAMP sensors (PKA and Epac) but also plasma membrane cAMPꢀgated ion chanꢀ
nels, and the adenosine receptors, both of which have structureꢀactivity relationships for adenineꢀbased compounds considerꢀ
ably different than those defined here for the cAMPꢀbinding domain of NCSꢀRapgef2
27 28
.
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Development of potent and selective NCSꢀRapgef2 inhibitors is likely to be of therapeutic relevance. Recent data indiꢀ
cates that cAMPꢀdependent activation of ERK in primary cortical, hippocampal and cortical rodent neurons in culture has
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pharmacological characteristics of NCSꢀRapgef2 dependence
. NCSꢀRapgef2 mediates ERKꢀdependent aspects of psyꢀ
30
chomotor stimulant drug action in vivo . Exploring the adenineꢀbased structure space in which selectivity and efficacy for
NCSꢀRapgef2 appears greatest suggests that focusing at the eight position (likely to increase potency but without gains in
selectivity) and the six position (likely to alter selectivity as well as potency) may yield compounds deployable in vivo, e.g.
3
1ꢀ32
for inhibition of ERK in the context of psychomotor stimulant dependency, and stressꢀinduced anxiety
, to name two Gsꢀ
GPCR, ERKꢀdependent pathophysiological arenas of CNS neuropharmacology. In the meantime, we would propose as an
urgent basic research priority deployment of N6ꢀphenylꢀ9ꢀTHFꢀAde in ERKꢀdependent GsꢀGPCR signaling in vivo, in aniꢀ
mal models.
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METHODS
Drugs and reagents All reagents and solvents used for chemical synthesis were obtained from SigmaꢀAldrich in the
highest purity available (≥98%) and were used as purchased. Cyclic AMP analogs were made by the Biolog Life Sciences
Institute (Breman, Germany) and purchased through their US distributor (Axxora). Small molecule inhibitors, forskolin, and
IBMX were purchased from Tocris (Ellisville, MO). PACAPꢀ38 was purchased from Anaspec (Fremont, CA). Solutions
used for cell culture were purchased from Life Technologies (Grand Island, NY) unless otherwise specified.
Chemical synthesis. Nꢀphenylꢀ9ꢀ(tetrahydrofuranꢀ2ꢀyl)ꢀ9Hꢀpurinꢀ6ꢀamine 2 was prepared in two steps based on established
33ꢀ34
methods
(Figure 8). 6ꢀChloropurine was reacted with aniline under microwave irradiation to give the Nꢀphenylꢀ9Hꢀ
purinꢀ6ꢀamine 1 in 75%. Reaction of 1 with 2,3ꢀdihydrofuran in the presence of paraꢀtoluenesulfonic acid (pꢀTSA) at reflux
of acetonitrile gave the desired compound 2 in high yield (88%). Compound 2 was soluble to > 100 mM in DMSO. For bioꢀ
logical experiments, this compound was dissolved in DMSO (50 mM), from which stable aqueous working solutions were
prepared. Highꢀresolution mass spectra of 2 were acquired with electrospray ionization (BioꢀSynthesis, Lewisville, TX).
HRMS (ESI+): calculated for C H H O 282.1349, measured 282.1351.
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Figure 4 Synthesis of N-phenyl-9-(tetrahydrofuran-2-yl)-9H-purin-6-amine (2) from 6-Chloropurine.
20
Cell culture Neuroscreenꢀ1 (NSꢀ1) cells were cultured as described previously and were used between passages four and
17 for the experiments reported here. Cells were grown in RPMI 1640 media supplemented with 10% horse serum (Hyꢀ
Clone, Logan, UT), 5% heatꢀinactivated fetal bovine serum (Atlanta Biologicals, Flowery Branch, GA), 2 mM Lꢀglutamine,
100 U/ml penicillin, and 100 ꢀg/ml streptomycin. Cultures were maintained in a humidified environment at 37 °C containing
95% air/5% CO . Cells grown in antibioticꢀfree media were routinely tested for mycoplasma.
2
Cellꢀbased ELISA Phosphorylated ERK, p38, and CREB were measured using cellꢀbased ELISA according to a protocol
35
described previously . As cAMP signaling, in NSꢀ1 cells, is parcellated via activation of NCSꢀRapgef2, Epac2, and PKA, to
ERK, p38, and CREB, respectively, activation (phosphorylation) of the latter represent reliable biochemical surrogates for
22
5
each of the cAMP sensors . NSꢀ1 cells were seeded at density of 3 x 10 per well in 96ꢀwell plates and grown overnight.
Cells were treated for 30 minutes followed by removal of media and fixation in 4% formaldehyde in PBS for 20 min at room
temperature. Fixed cells were permeabilized by three washes in 0.1% Triton Xꢀ100 in PBS (PBST) and endogenous peroxiꢀ
dase activity was quenched by a 20ꢀminute incubation in PBS containing 0.6% H O . Following three washes in PBST, samꢀ
2
2
ples were blocked (10% FBS in PBST) for 1 h and incubated overnight with primary antibodies against phosphoꢀERK1/2
Cell Signaling Technology #9101) at 1:500, phosphoꢀp38 (CST, #4155) at 1:500, or phosphoꢀCREB (CST #9198) at 1:250;
at 4°C. Samples were washed three times in PBST and twice in PBS. An HRPꢀcoupled antiꢀrabbit secondary antibody (CST
7074), diluted 1:500 in PBST containing 5% BSA, was added for one hour at room temperature. Following five washes in
PBST, samples were exposed to the colorimetric substrate OneꢀStep Ultra TMBꢀELISA (Pierce, catalog number 34028).
(
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After development in the dark for 10 minutes, the reaction was stopped by adding 4 M sulfuric acid, and absorbance was
read at 450 nm.
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Cyclic AMP measurements Cyclic AMP was measured using colorimetric cAMP Direct Biotrak EIA kits (GE, Catalog
#RPN2255). Assays were performed following the protocol provided by the manufacturer without acetylating cAMP. NSꢀ1
5
cells were dispensed into 96ꢀwell plates at a density of 3 x 10 cells per well and grown overnight. The next morning, media
were aspirated and culture media containing IBMX (500 ꢀM) and inhibitors or DMSO were added. After 30 minutes, agoꢀ
nists were added as 10x stocks at the indicated concentrations, followed by a 20ꢀminute incubation at 37° C. Media were
then aspirated and cells were lysed using the Novel Lysis Reagent provided by the manufacturer. Intracellular cAMP from
lysates was measured by EIA conducted according the protocol provided by the manufacturer. Raw data were fit to cAMP
standard curves and are expressed as pmol of cAMP per well.
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High content analysis (HCA) An NSꢀ1 cell line stably expressing enhanced GFP was generated for HCA. Cells were disꢀ
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pensed into collagen type Iꢀcoated 96ꢀwell plates in growth medium at a density 1 x 10 cells per well. Two hours after platꢀ
ing, cells were photographed using an inverted fluorescence microscope outfitted with motorized objectives, shutters, filters,
stage, automated focusing and focus correction (TiE Eclipse, Nikon). Five pairs of phaseꢀcontrast and fluorescent images
were captured per well during each acquisition. Following each acquisition, plates were returned to an incubator. Eighteen
hours after plating, cells were treated as indicated. Images were then acquired every 24 hours for five days. Cell number in
each field was determined by Nikon NISꢀElements High Content Software. Cell growth data were normalized to values obꢀ
served in each well two hours postꢀplating and are expressed as a percent of cell growth over time. Neurite elongation was
measured in NISꢀElements by manually tracing neurites in fields captured 48 hours following addition of agonists. Neurite
outgrowth data were normalized as the ratio of neurite length per field to number of cell number per field.
Calculations and statistics SigmaPlot 11.0 (Systat, San Jose CA) was used for all calculations and statistical analysis. For
doseꢀresponse experiments, curves were fit to data using 4ꢀparameter logistic regressions. Simultaneously with curve fitting,
whether or not each test compound had a statistically significant effect was determined by a oneꢀway ANOVA. Unless othꢀ
erwise specified, data from test compounds to which curves were fit had a statistically significant effect as determined by a
oneꢀtailed P value of 0.05 or less. For experiments using two or fewer concentrations of agonists and antagonists, data were
analyzed by oneꢀ or twoꢀway ANOVA followed by Bonferroniꢀcorrected tꢀtests. Statistical tests were nonꢀdirectional (twoꢀ
tailed) and P < 0.05 was the criteria for statistical significance.
Supporting Information
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Supplemental Materials and Methods and Supplemental figures S1ꢀS3
Abbreviations
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Ade, Adenine; AC, Adenylate cyclase; Bn, Benzoyl; Br, Bromo; Bz, Benzyl; cAMP, Cyclic adenosine 3′,5′ꢀmonophosphate;
cAMPS, cyclic adenosineꢀ3′,5′ monophosphorothioate; CREB, Cyclic AMPꢀresponsive elementꢀbinding protein; CPT, 4ꢀ
Chlorophenylthio; ERK, Extracellular signal–regulated kinase; Epac, Exchange protein activated by cyclic AMP; ESIꢀ09, αꢀ
[(2ꢀ(3ꢀChlorophenyl)hydrazinylidene]ꢀ5ꢀ(1,1ꢀdimethylethyl)ꢀβꢀoxoꢀ3ꢀisoxazolepropanenitrile; GEF, Guanine nucleotide exꢀ
change factor; H89, Nꢀ[2ꢀ[[3ꢀ(4ꢀBromophenyl)ꢀ2ꢀpropenyl]amino]ethyl]ꢀ5ꢀisoquinolinesulfonamide dihydrochloride;
MAPK, Mitogenꢀactivated protein kinase; MBC, monoꢀt.butylcarbamoyl; NCSꢀRapgef2, Neuritogenic cyclic AMP sensorꢀ
protein product of the Rapgef2 gene; NSꢀ1, Neuroscreenꢀ1; p38, p38 Mitogenꢀactivated protein kinases α, β, γ, and δ;
PACAP, Pituitary adenylate cyclaseꢀactivating polypeptide; Phe, Phenyl; PKA, Protein kinase A; SB 239063, transꢀ4ꢀ[4ꢀ(4ꢀ
Fluorophenyl)ꢀ5ꢀ(2ꢀmethoxyꢀ4ꢀpyrimidinyl)ꢀ1Hꢀimidazolꢀ1ꢀyl]cyclohexanol; SQ22,536, 9ꢀ(Tetrahydroꢀ2ꢀfuranyl)ꢀadenine;
THF, Tetrahydroꢀ2ꢀfuranyl; U0126, 1,4ꢀDiaminoꢀ2,3ꢀdicyanoꢀ1,4ꢀbis[2ꢀaminophenylthio]butadiene
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9
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9
0
Author Information
Please address correspondence to Lee E. Eiden, 9000 Rockville Pike, Building 49, Room 5Aꢀ27, Bethesda, MD 20892, eiꢀ
denl@mail.nih.gov, 1ꢀ301ꢀ496ꢀ4110.
Author Contributions
A.C.E. and L.E.E. designed experiments and wrote the manuscript. W.X. and M.V.E. designed and created new molecular &
cell biological reagents used in this work. A.C.E., R.A.A., and W.X. performed experiments. F.S. designed and synthesized
novel compounds reported herein. L.E.E. oversaw the project.
Funding Sources
This work was supported by NIMH Intramural Research Program Project Z01 MH002386 (L.E.E.) and by 2014 NARSAD
Young Investigator Grant 21356 from the Brain and Behavior Research Foundation (A.C.E.).
Conflict of Interest
The authors declare no competing financial interests.
Acknowledgements
The authors wish to thank Dr. Zhihong Jiang (NIHM Intramural Research Program) for her insightful comments about this
work and manuscript.
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For Table of Contents Use Only: Graphical abstract
Differential Pharmacophore Definition of the cAMP Binding Sites of NCS-Rapgef2, PKA and Epac in
Neuroendocrine Cells Using an Adenine-based Scaffold
Andrew C. Emery, Ryan A. Alvarez, Maribeth V. Eiden, Wenqin Xu, Fabrice G. Simeon, and Lee E. Eiden
3
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