Current Topics in Medicinal Chemistry, 2011, 11, 1393-1405 1393

The cAMP-Dependent Protein Kinase Pathway as Therapeutic Target –

Possibilities and Pitfalls

Rune Kleppe1,*, Camilla Krakstad2, Frode Selheim1,3, Reidun Kopperud1 and Stein Ove Døskeland1

1
Department of Biomedicine, University of Bergen, Jonas Lies vei 91, N-5009 Bergen, Norway, 2Department of Clinical
Medicine, Haukeland University Hospital, 5021 Bergen, Norway, 3Proteomic Unit (PROBE), University of Bergen, Ber-
gen, Norway

Abstract: The prototype second messenger cAMP and its major mediator, the cAMP-dependent protein kinase (PKA), is
able to control simultaneously multiple processes within the same cell. This appears to be achieved through its unique dis-
sociative regulation and the spatiotemporal regulation of both cAMP and PKA. The widespread tissue distribution and
physiological function of this pathway makes it an attractive, but challenging pharmacological target. We will discuss cur-
rent progress in manipulating the fine-tuning of PKA, and outline so far underexploited possibilities for therapy, such as
novel ways to target specific substrates and catalytic cycle intermediates of PKA. An attractive strategy to achieve a more
focused pharmacological treatment is to combine more traditional targeting of extracellular receptors or ligands with that
of intracellular signaling pathway components. The cAMP signaling pathway provides a variety of possibilities for such
an approach.
Keywords: Cell signaling, PKA, cAMP, multi-targeting, disease.

1. INTRODUCTION
Intracellular signaling is instrumental in coordinating
cellular responses to external cues, but the implication of
aberrant signaling in disease is only recently becoming ap-
preciated. The complexity of signaling and the relative recent
status of signaling components as drug targets suggest that
signaling modulation will be a rich source of potential drug
candidates for years. The cyclic nucleotides are prototype
second messengers whose major downstream mediators are
protein kinases. The cAMP-dependent protein kinase (PKA)
is considered a protein kinase paradigm. The compact cata-
lytic subunit is inhibited by a (pseudo)substrate sequence
located on a separate cAMP binding regulatory subunit,
rather than by an inhibitory domain in the same polypeptide
chain [1]. Activation of PKA therefore involves subunit dis-
sociation, which is unique among kinases, and differs
strongly from the activation of its evolutionary offspring the
cGMP-dependent protein kinase (PKG), whose cGMP bind-
ing sites reside in the same polypeptide chain as the catalytic
site [2]. The activity of PKA and its cGMP-activated relative
are already being modulated pharmacologically through ex-
tracellular receptor agonists/antagonists and by inhibitors of
cNMP degrading phosphodiesterases. At the receptor level,
antagonists for the ß1-adrenergic receptors have been used in
treatment of angina and hypertension (e.g. metoprolol),
while the ß2-adrenergic receptor agonists (e.g. salmeterol)
are used for treating asthma or chronic obstructive pulmo-
nary disease (COPD) [3, 4]. Both general and more selective
PDE-inhibitors have reached the clinic. The general PDE
inhibitor theophylline is used for treatment of respira-
*Address correspondence to this author at the Department of Biomedicine,
University of Bergen, Jonas Lies vei 91, N-5009 Bergen, Norway;
Tel: +47-555-86360; Fax: +47-555-86375;
E-mail: rune.kleppe@biomed.uib.no
tory diseases like COPD and asthma [5]. More recently, iso-
type selective inhibitors, such as the PDE5 inhibitor silde-
nafil (Viagra), and the PDE3 inhibitor milrinone have been
developed. Sildenafil has proven successful in treating erec-
tile dysfunction [6], while short-term administration of mil-
rinone increase vasodilation and cardiac contraction after
heart failure [7].
The limited distribution of PKG restricts the number of
diseases that can be targeted, but makes it also easier to
achieve focused therapy with fewer side effects. In contrast,
the widespread tissue-distribution and broad functional rele-
vance of cAMP signaling provides more opportunities, but
also higher risk of side effects. A possible solution to the
dilemma of low specificity may be found in the great variety
of signaling proteins and signaling mechanisms involved in
controlling the cAMP pathway. The rich isotypic variation
combined with extensive spatiotemporal regulation and
cross-signaling provides plentiful of opportunities to fine-
tune the selectivity. Some of these will be discussed here
with emphasis on specific targeting of the PKA isozymes
and their downstream protein substrates.
2. THE cAMP SIGNALING SYSTEM - OVERVIEW
2.1. Regulation of cNMP Turnover
The second messenger cAMP is synthesized from ATP
by adenylyl cyclases (ACs) (see Fig. (1) for overview),
whereas cGMP is synthesized from GTP by guanylyl cy-
clases (GCs). The eukaryotic AC families belong to AC class
III, and can be either soluble (sAC) or of the more com-
monly transmembrane (tmAC) type (reviewed in [8]). Nine
genes (I-IX) code for mammalian tmAC, and each can exist
in several splice variants. TmACs have low basal activity,
are activated by the heterotrimeric G
s, are often subjected
to additional positive and negative modulation by other G-

1568-0266/11 $58.00+.00 © 2011 Bentham Science Publishers Ltd.

1394 Current Topics in Medicinal Chemistry, 2011, Vol. 11, No. 11 Kleppe et al.

tmAC
GPCR GPCR
Ligand
Ligand

Gαs Gα12 Gβγ

PM Gβγ
ATP Epac
AMP
cAMP( ) C GTP- Rap1 Rap1 -GDP
P

RII RII
PDE AKAP-lbc GAP
P P Adhesion
C P
Secretion
C Novel targets
C RI RI PKAII
PDE
C PDE C
PKAI RI
R I
C RI
RI C
C

C C
C sA
C
PKI ATP
P
CREB

RI
cAMP( )

RI
C
ICER

Nucleus Activating
Cytosol Inhibiting
Translocation

Fig. (1). Overview of the cAMP signaling pathway. The Figure shows a cartoon of the cAMP pathway initiated by ligand binding to G-
protein coupled receptors (GPCR) and activation of heterotrimeric G-proteins. The dissociated G
S activated transmembrane adenylyl cy-
clases (tmAC) to synthesize cAMP (grey dots, grey shaded color represents cAMP concentration gradients), which is degraded by phosphodi-
esterases (PDEs). Downstream targets of cAMP are PKA type I and II and Epac. The dimeric regulatory subunits of PKA-I (RI) and (RII) are
shown with empty or filled cAMP-binding sites. Binding of two cAMP molecules/R-subunit enables dissociation of the catalytic subunit,
which may then phosphorylate downstream targets in different compartments within the cell. Anchored PKA-II is shown bound to AKAP-
lbc. Nuclear proteins are cAMP-response element binding protein (CREB), PKA-inhibitor (PKI) and immediate cAMP-induced early re-
pressor (ICER). Soluble AC is shown as sAC.

protein subtypes (see also [9] for review). ACI and ACVIII
are activated by Ca2+/calmodulin [10], and several AC iso-
types are regulated by phosphorylation. One gene is identi-
fied for sAC. This ubiquitously expressed enzyme is stimu-
lated by bicarbonate, calcium and lactate [8, 11]. For this
reason the sACs have been suggested to represent an evolu-
tionary conserved family of proteins involved in sensing the
cellular metabolic state [12].
The level of free cAMP and cGMP (cNMP) in cells is
controlled by the opposing action of AC/GC and PDEs Fig.
(1). The mammalian PDE superfamily so far constitutes 11
families, each of which originates from 1-4 genes, with sev-
eral isoforms (see [13,14,15] for recent reviews). Altogether,
the more than 50 enzyme variants span a range of enzyme
kinetic types with different Km, cNMP specificity, and regu-
lation by other second messengers such as calcium. For the
dual nucleotide PDEs the cNMPs are mutual competitive
inhibitors, depending on their relative affinities and Vmax
values. A special example of this is PDE3A, which has high
affinities for both cAMP and cGMP, and is referred to as a
cGMP-inhibited cAMP-PDE due to a 10 fold higher V max for
cAMP compared to cGMP [16]. Several of the PDE enzyme
types have cGMP binding GAF domains (mammalian
cGMP-binding PDEs, Anabaena Adenylyl cyclases, and
Escherichia coli FhlA), which mediate allosteric activation
of the catalytic domain [17]. In addition to the different ki-
netic characteristics, several splice variants locate to differ-
The cAMP-Dependent Protein Kinase Pathway as Therapeutic Target
ent cell compartments, through membrane association and
binding to proteins, such as to A-Kinase anchoring proteins
(AKAPs) in the case of the PDE4 family [18]. The fact that
so many gene products are devoted to the synthesis and deg-
radation of cyclic nucleotides indicates that the regulation of
the cNMP concentration is of high priority. The multitude of
splice variants, post-translational modifications and protein
interaction partners obviously provides cells with rich oppor-
tunity to control the dynamics of cNMPs and to couple
cAMP and cGMP signaling together as well as to other sig-
naling pathways.
2.2. Receptors for cAMP – PKA
In its inactive states, PKA is a holoenzyme, consisting of
a dimeric regulatory moiety, composed of two regulatory (R)
subunits, and up to two catalytic (C) subunits. The C-
subunit(s) becomes catalytically competent only upon disso-
ciation from the regulatory subunits, which is facilitated by
binding of cAMP to the R-subunit to which it is associated
(see Fig. (1), text below). The N-terminal of the R subunit
contains a dimerization domain, and a localization domain
responsible for binding the R dimer to A-kinase anchor pro-
teins (AKAPs). The N-terminal domains of R are followed
by an autophosphorylation/autoinhibitory motif, and two
homologous cAMP binding sites, referred to as A- and B-
site, at the C-terminal [19,20]. The cAMP binding sites have
different kinetic properties and both sites must be occupied
to achieve activation [21].
Two mammalian PKA isotypes (PKA-I and II) have been
identified, composed by dimeric type I or type II R subunits.
Each isotype of R can be further subdivided into
and 
isoforms, encoded by different genes [22]. The different iso-
forms of R-isotypes show somewhat different tissue expres-
sion. In general, the
isoform of RI and RII are ubiquitously
expressed, whereas the  isoforms are mainly expressed in
nervous system, lung and gonads [23]. Knock-out of RI
in
mouse results in early embryonic death [24], suggesting that
this isoform play a key role in the PKA-signaling system.
Consistent with that notion, this isoform was found to be
regulated in compensation to genetic deletion of R isoforms
and during overexpression of the catalytic subunit
[23,24,25]. Deletion of the neuronal RI isoform, RI, show
reduced short-term memory, despite compensatory expres-
sion by RI
and invariant total PKA activity [25]. Unlike for
RI
, mice lacking RII
appear healthy, whereas the RII
knock-out show protection against diet-induced obesity
[25,26].
Four genes are known for the mammalian C subunit;
C
[27], C [28], C [29,30] and PrKX [31]. C
is further
divided into three different forms caused by post-
translational modification (C
1 and C
2) and alternative
splicing (C
-s) [32,33,34]. The C
1 is ubiquitously ex-
pressed while C
-s is found in sperm, where it is involved in
mobility [35]. The C gene transcribes several splice vari-
ants; C1, C2, C3 and C4 [36] of which C1 is ubiqui-
tously expressed [37], C2 is predominantly expressed in
lymphoid cells [38,39], and C3 and C4 variants are found
solely in neuronal tissues [40]. The difference between these
splice variants is in the N-terminal region of the protein [38].
Genetic null mutations of both C
and C result in death at
Current Topics in Medicinal Chemistry, 2011, Vol. 11, No. 11 1395
early embryonic development [41]. The C
-/- knockout
mouse is viable, but has a severely growth retarded pheno-
type and approximately 73 % of the animals die before
adulthood [35]. In contrast, the C-/- mouse has no apparent
alteration of phenotype [42].
The C seems to be specific to human testis [29]. PrKX
(X chromosome-encoded protein kinase X) differ from the
other C-subunits by selectivity binding to the RI subunits
and by phosphorylation of the RII subunits in a cAMP inde-
pendent manner. PrKX is expressed in several tissues, with
highest mRNA level detected in fetal and adult brain, kidney
and lung [43].
2.3. Receptors for cAMP – Epac and cAMP-gated Ion
Channels
Epac proteins function as guanine nucleotide exchange
factors for both Rap1 and Rap2, which belong to the Ras
family of small GTP-binding protein [44] (Fig. (1)). Two
isoforms have been identified, Epac1 and Epac2, which are
encoded by two different genes [45]. Recently, three splice
variants of Epac2 were identified and named Epac2A, B and
C [46,47]. Epac1 and Epac2 share the same functional do-
mains with a N-terminal regulatory region that contains one
(Epac1, Epac2B, Epac2C) or two (Epac2A) cyclic nucleotide
binding-domains and a Dishewelled/Egl-10/Pleckstrin do-
main (Epac1, Epac2A, Epac2B). The catalytic region is at
the C-terminal and contains a CDC25 homology domain for
GTP-exchange activity in addition to a Ras exchange motif
domain and a Ras association domain [44]. Upon binding of
cAMP, a conformational change take place and the active,
catalytic region is exposed [48,49]. This conformational
change has also been linked to subcellular localization of
Epac1, e.g. to the plasma membrane [50].
The Epac proteins are expressed at different levels in
most tissues. While Epac1 is expressed ubiquitously, recent
studies indicate that the expression of Epac2 splice variants
is tissue specific. Epac2A dominates in pituitary glands and
pancreatic -cells, while Epac2B is the main variant in adre-
nal glands. Epac2B is expressed in adrenocortical cells and is
involved in cAMP-induced actin reorganization and cell mi-
gration together with PKA [51]. The third variant, Epac2C,
is specifically expressed in the liver [46,47].
Ion channels regulated by cAMP are part of the cyclic
nucleotide-gated ion channel family. They are nonselective
cation channels that allow the passage of K+, Na+ and Ca2+
(see [52] for review), and their activation represents an im-
portant step in the signal transduction pathway in both vision
(cGMP) and olfaction (cAMP and cGMP) [53]. Cyclic nu-
cleotide-gated ion channels have also been found in a variety
of other cell types [54,55].
2.4. Activation of PKA – a Unique Mechanism
Dissociative activation makes PKA unique within the
mammalian kinome. This feature has been kept throughout
evolution from yeast even though it has been lost in evolu-
tionary related proteins such as the cGMP-dependent kinase.
Separated regulatory (R) and catalytic (C) protomers enables
different possibilities for regulation than for single chained
kinases. A consequence of separated catalytic and regulatory
1396 Current Topics in Medicinal Chemistry, 2011, Vol. 11, No. 11
chains, is that the degree of dissociation is controlled by
mass action kinetics. Thus, the degree of activation depends
on the concentration of kinase in cellulo and the dissociation
constant between the catalytic subunit and the cAMP-
saturated regulatory subunit. It is generally believed that
PKA-I is half activated between 50-100 nM cAMP and that
full saturation of the holoenzyme leads to complete dissocia-
tion and activation [56, 57]. However, more recent findings
suggest that this is not the case. In fact the dissociation con-
stants for ubiquitous PKA-I
and II
isotypes are lower than
previously thought and in a range that sensitizes the dissocia-
tive activation to the expression levels of the regulatory
subunits [58,59]. The thermodynamic driving force for the
activation is a 1000 fold higher affinity for cAMP to free
regulatory subunit relative to the holoenzyme [60]. Still it
seems that cAMP is not capable of fully activating PKA, at
least in cells that express high levels of kinase. In this re-
spect, the possible existence of super-activators is particular
interesting.
Recent progress in the structural understanding of PKA
activation has come from comparison of cAMP-bound R-
subunit and the resolved structures of PKAI and II holoen-
zymes [1,61,62,63,64,65]. The structures of the holoenzymes
show large surface interactions between the R and C
subunits which are greatly diminished upon binding of
cAMP (reviewed in [66]). Resolving the interface between C
subunit and cAMP-saturated R subunit at the structural level
is however a major challenge that is probably only possible
through high-field NMR due to fast on-/off- interactions of
the subunits. Possibly, the use of single cAMP-site mutants
of the R-subunit can resolve some of the structural enigmas
of PKA activation by cAMP binding, as the affinity between
cAMP-bound R and C-subunit is still in the nanomolar
range. Recent NMR studies have revealed some of the struc-
tural mechanisms underlying communication between the
cAMP-binding sites of the RI subunit [62,65].
Mass action controlled activation enables additional lay-
ers of regulation by controlling the level of R- and C-
subunits. Thus, different R to C stoichiometries as well as
feedback and feedforward regulatory mechanisms can be
envisioned, some of which have been found experimentally
(see [67] for review). Separated catalytic and regulatory
subunits will also influence the use of PKA for compartmen-
talized signaling as diffusion will act to separate concentra-
tion differences within the cell. In that respect, single chain
kinases such as PKG would be expected to be better suited
for kinase activation in signaling domains that are spatially
restrained. Still, spatial regulation of PKA seems to be more
widespread than for many of the evolutionary related
kinases.
2.5. Spatially Confined Signaling Through PKA
Localization of PKA to cellular structures was described
in the early seventies in brain tissue where a majority of
PKA is associated with non-soluble fractions [68]. However,
the ability of proteins to physically bind PKA was first de-
scribed for the microtubule associated protein 2 [69] and
found to be mediated through an A-kinase anchoring do-
main, an amphipathic helix motif first described in Ht31/
AKAP-Lbc/AKAP13 [70]. The anchoring domain specifi-
Kleppe et al.
cally recognizes the far N-terminal region of the regulatory
subunits, which form a docking domain in the immediate
spatial vicinity of the dimerization motifs of the regulatory
subunits [71,72,73]. Presently, about 50 different A-kinase
anchoring proteins (AKAPs) have been described (including
splice variants) (see [74] for recent review), the majority of
which bind PKA-II. However, PKA-I specific AKAPs have
been reported also, such as the
4 integrin receptor subunit
[75], and dual specificity AKAPs that are capable of binding
both PKA isotypes [76]. The battery of different AKAPs
therefore present a wealth of possibility for cells to localize
PKA isotypes to the cytosolic face of almost any organelle or
cellular structure. Many of the AKAPs also bind to other
signaling proteins, such as PDEs (PDE4 isoforms), protein
phosphatases and other kinases. In particular the co-
localization with PDE and recently also AC, is thought to be
a crucial feature of spatially confined cAMP-signaling do-
mains [77, 78].
Several issues are however unclear regarding PKA com-
partments, such as the local concentration of the active com-
ponents in cAMP signaling or the diffusion flux of these
proteins and cAMP itself in and out of these compartments.
Localized PDE has been shown to control cAMP leakage out
of compartments (see for [79] review) in addition to regulat-
ing the local cAMP level and duration of the cAMP signal.
Selective inhibition of PDE is therefore one approach to in-
crease the compartment signal, but might generate signifi-
cant spillover of cAMP and activate cAMP-receptors outside
the borders of compartment if not properly dosed. For PDEs
that are controlled by additional allosteric modulators such
as cGMP and Ca2+/calmodulin, selective pharmacotargeting
offers a possibility to modulate the cross-signaling between
cAMP and other signaling pathways within the compart-
ment.
The responsiveness of a compartment to a signal is how-
ever not only determined by the cAMP dynamics. The PKA
activation dynamics and the protein phosphatase activity are
also important determinants. These issues are much less re-
solved than for cAMP, in particular the well known fact that
the catalytic subunit can diffuse freely when activated. The
repetitive signaling capacity of compartments might there-
fore be limited by their ability to contain or reload with cata-
lytic subunit. The observation that the R-subunit and the dif-
fusing catalytic subunit form complexes, even at high levels
of cAMP, may provide answers to some of these questions.
In particular to address the different characteristics of com-
partments containing type I versus type II kinase.
3. THE PHYSIOLOGY AND PATHOPHYSIOLOGY
OF cAMP SIGNALING
3.1. Metabolism, Energy Expenditure and Deposition
PKA was the first discovered effector of cAMP and was
first implicated in regulation of glycogen metabolism [80].
Thus, regulation of energy metabolism and blood glucose
levels were the first described functions of PKA. Perhaps not
surprisingly, a function in regulation of energy store usage
was found for PKA rather simultaneously in tissues such as
skeletal muscle, heart, liver and adipose tissue. More re-
cently, the concerted action of the cAMP effectors PKA and
Epac was described in differentiation of white adipose tissue
The cAMP-Dependent Protein Kinase Pathway as Therapeutic Target
[81]. Thus, it seems that cAMP signaling is linked to both
fatty acid usage and increase of white adipose tissue. Studies
on knock-out mice of PKA and AC3 supports a role for
cAMP-signaling through PKA in the regulation of energy
usage and appetite [82,83,84,85], and this pathway may
therefore play an important role in the development of meta-
bolic syndrome and obesity.
3.2. Cardiovascular System, Blood Cells
In the cardiovascular system, cAMP and cGMP play im-
portant roles in most cell types. Increased cardiac perform-
ance is a well described function of cAMP and PKA through
adrenergic amplification of cardiomyocyte calcium signal-
ing. Together with Epac, PKA is also implicated in -
adrenergic stimulated hypertrophy [86], but this is yet to be
confirmed in transgenic animal models. For vascular endo-
thelial cells, stabilization of the barrier function of the endo-
thelial cell layer in response to permeabilizing agents (such
as LPS, thrombin, TNF-
and TGF-) is the best described
effect of cAMP [87,88,89]. This is reported to occur through
both Epacs and PKA, where modulation of the activities of
the Rho-GTPases is a major consensus mechanism [89, 90,
91, 92]. Intriguingly, cAMP has been implicated in adhesion
and chemotaxis of leukocytes, as well as in the inhibition of
smooth muscle proliferation and constriction, which together
with its beneficial effect on endothelial barrier function, have
made cAMP-signaling a possible target in atherosclerosis
and pulmonary hypertension. The PDE4 inhibitor roflumilast
has shown promising potential in several preclinical trials for
treatment of COPD [93].
In platelets, cyclic nucleotide signaling through cAMP
and cGMP are important antagonistic pathways that inhibits
activation and aggregation of platelets in response to agents
such as thrombin, collagen, von Willebrand Factor and ADP.
These inhibitory pathways are necessary to control the for-
mation of thrombi (see [94] for review), which may result in
stroke. Anti-platelet therapy is therefore used for secondary
prevention of stroke, typically using acetylsalicylic acid (cy-
clo-oxygenase inhibitor) or clopidogrel (ADP-receptor in-
hibitor) [95]. However, as these agents modify their target
irreversibly, there is a risk of excessive bleeding. The benefit
of cNMPs in platelets is that they reversibly inhibit all the
various platelet activation pathways, in response to vascular
endothelial stress. In platelets, cNMP levels are controlled by
PDE2, 3 and 5, whereas prostaglandins, adenosine and nitric
oxide stimulates the synthesis of cAMP and cGMP, respec-
tively [96,97]. This set of PDEs provides interesting possi-
bilities of cross-talk between the two pathways – positive
through cGMP-inhibited PDE3 and negative through cGMP-
activated PDE2. In particular PKA activation through PDE3
is implicated in NO mediated inhibition of platelet shape
change, which is the initial reversible step in platelet activa-
tion [98]. Arguably, platelet shape change is an interesting
target for long term anti-platelet therapy due to its reversibil-
ity and upstream placement in platelet activation.
3.3. Nervous System, Memory, Neurodegereration, Mood
Disorders
Apart from platelets, the central nervous system (CNS)
contains the highest amount of PKA. Not surprisingly, a
Current Topics in Medicinal Chemistry, 2011, Vol. 11, No. 11 1397
multitude of functions have been ascribed to this kinase in
the CNS. There is now clear evidence that PKA signaling
has a key role in a variety of implicit and explicit memory
processes in both invertebrate and mammals [99,100]. Re-
cently, a transgenic mouse expressing a dominant-negative
form of the PKA regulatory subunit (RI
) in two regions of
hippocampus, was found to have a deficit in several forms of
long-term synaptic potentiation (LTP) and a lowered thresh-
old for the consolidation of long-term synaptic depression
(LTD) [101]. PKA is crucial for the dynamic balance be-
tween phosphatases and protein kinases, which regulate the
consolidation of LTP and LTD. A dominant negative RI

results in decreased kinase activity and thereby less phos-
phorylation of the AMPA glutamate receptor, leading to sus-
tained LTD [102].
Serotonergic and catecholaminergic neurotransmission
are implicated in the modulation of gross neurological func-
tions such as mood, sleep, memory, motivation, pleasure and
motor function. The neurotransmission of these monoamines
is mediated through a range of receptors of which several are
linked to cAMP signaling [103,104]. In addition, PKA is
implicated in the regulation of biosynthesis of catechola-
mines and serotonin [105,106]. Not surprisingly, cAMP-
signaling has been suggested as a potential target in mood
disorders and Parkinson’s disease [107,108]. This is also
supported by examination of PKA protein levels in human
post-mortem brain tissue of normal and depressed persons
[109,110]. In patients with the neurodegenerative Alz-
heimer’s disease, the level of PKA was reduced, resulting in
deficient CREB function. This down regulation was due to
degradation of the PKA subunits and may result from over-
activated calpain [111].
3.4. Immune System, Pathogenic Bacteria
The cAMP-pathway is a well described repressor of
macrophage activation and can therefore suppress inflamma-
tory responses (reviewed in [112]). Much of this effect is
PKA-mediated through inhibition of Toll-like-receptors,
hence inhibiting the synthesis of inflammatory cytokines
(e.g. TNF
, macrophage inflammatory protein-1
), but also
through both PKA and Epac1 by inhibition of phagocytic
receptors and production of reactive oxygen species [113,
114]. Not surprisingly, the repressing action of cAMP is hi-
jacked by several pathogenic bacteria, most notably Myco-
bacterium tuberculosis. They grow within the phagosome of
the macrophages, thereby causing disease. Interestingly, it is
found that an increased level of cAMP by bacterial AC,
stimulated growth of bacteria and virulence through host
PKA. The opposite situation was found at low cAMP levels,
resulting in bacterial destruction [115].
An immunomodulatory function of PKA is also found in
T-cells, where localized PKA inhibits T-cell receptor activa-
tion through C-terminal Src Kinase (see [116] for review).
The cAMP/PKA pathway is also implicated in T-cell dys-
function. Acquired immunodeficiency syndrome results from
the dramatic loss of CD4+T-lymphocytes following human
immunodeficiency virus type 1 (HIV-1) infection [117]. Re-
cently, PKA was found to phosphorylate and activate the
HIV-1 viral protein R, which contributes to the CD4+ cell
death [118]. Blocking PKA activity or the phosphorylation
1398 Current Topics in Medicinal Chemistry, 2011, Vol. 11, No. 11
of HIV-1 viral protein R may thereby represent a strategy for
therapy against HIV-1 infection. In addition, it has been re-
ported that elevated cAMP leads to a PKA-dependent in-
crease in viral replication [119].
4. STRATEGIES TO TARGET PKA-SIGNALING
4.1. Targeting cAMP Levels
Manipulation of second messenger levels by activation of
AC (e.g. forskolin, from the plant Coleus forskohlii) or inhi-
bition of PDEs (3-isobutyl-1-methylxanthine – IBMX) has
been a common strategy to activate PKA experimentally.
This will however nonspecifically activate all downstream
targets of cAMP, but has the potential of gaining specificity
by targeting particular isoforms within the AC and PDE en-
zyme families. For at least two decades, there has been an
interest in finding isoform specific inhibitors of the different
PDEs, and this has led to several compounds with high se-
lectivity. Inhibition of PDE will unequally activate cyclic
nucleotide signaling, however, targeting several of the PDEs
also has the potential of inhibiting cAMP (and cGMP) sig-
naling. Several isoforms of the PDE family have allosteric
activator domains, GAF-domains, which upon ligand occu-
pation can increase the catalytic activity of the enzyme. The
cGMP-activated PDE2 and 5 isoforms are examples of such.
The PDE2 isoforms conduct negative cross-talk between
cGMP and cAMP in several tissues such as the nervous sys-
tem, the heart, platelets and vascular endothelium [120,121].
Thus, in addition to PDE activation, specific antagonists to-
wards the GAF-domain of PDE2 would enable direct inhibi-
tion of this cross communication, without interfering with
the basal activity of the enzyme. Recently, GAF-ligands to-
wards PDE2 and 5 have been described [122]. This extra
cNMP-binding domain provides exciting possibilities for
pharmacological intervention.
More recently, the use of forskolin derivatives have been
used to generate selectivity in the activation of different iso-
forms of AC (see [123] for review). The AC5 selective
forskolin derivative, colforsin daropate hydrochloride, is
now entering clinical use for advanced congestive heart fail-
ure [124]. Generation of isoform specific inhibitors of ACs
has proven challenging, due to the high conservation of the
active site between the isoforms. However, the inhibitor
KH7 shows selectivity towards sAC as compared to tmAC
[125].
4.2. Targeting the cAMP Binding Sites
The cyclic nucleotide activated kinases have been in the
forefront of developing specific kinase activators, through
chemical modifications of the cyclic nucleotide. The discov-
ery of Epac revealed that commonly used cAMP analogs like
8-Br-cAMP and 8-pCPT-cAMP activated both PKA and
Epac. In recent years, cAMP-analogs have been developed
which discriminate well between Epacs and PKAs and even
between different isotypes of the PKAs. In Table 1 we sug-
gest cAMP-analogs that can be used to obtain selective acti-
vation of cAMP receptors, which in addition are quite well
characterized in terms of stability and interference with PDE
activities. The 6'-modified cAMP analogs N6-benzoyl-cAMP
and N6-monobutyryl-cAMP are selective activators of PKA,
Kleppe et al.
and poor activators of Epac [126]. Analogs with hydropho-
bic N6-substituents at the adenine ring of cAMP appear to
bind site selective to the A-sites of RI and RII [127]. Thus,
N6-benzoyl-cAMP, which preferentially bind the A-sites of
both PKA-I and PKA-II can be combined with BII-site bind-
ing analogs, like Sp-5,6-DCl-cBIMPS or 8-piperidino-
cAMP, for selective activation of PKA-II. However, combi-
nation of 8-piperidino-cAMP (AI- and BII-site selective)
with BI-site preferring analogs like 8-AHA-cAMP will lead
to selective synergistic activation of PKA-I (Table 1). By
using analogs such as the strong BI-site selective 8-AHA-
cAMP, it was successfully demonstrated that the PKA-
holoenzyme associated RI subunit bound cAMP with posi-
tive cooperativity through an interchain site A-B interaction
mechanism [60]. As mentioned above, selective synergistic
activation of PKA-I, PKA-II or both can be achieved by us-
ing different pairs of site selective cAMP analogs [67]. Such
synergy between pairs of analogs provides strong evidence
that the biological effect is mediated through PKA-signaling.
Highly Epac-selective cAMP analogs possess a ribose 2'-
O-methyl substituent, which impairs binding to PKA, and an
8-substituent at the adenine ring of cAMP. 8-pCPT-2'-O-Me-
cAMP is a strong Epac activator (Ka = 1.9
M vs Ka = 45

M for cAMP) that has more than 500 fold higher affinity
for Epac1 than for the A-site of PKA-I (Table 1)
[81,126,128]. The Epac-activator synergized strongly with
both the PKA-activator and NGF to promote adipogenesis
and PC-12 differentiation, as determined by neurite out-
growth [126,128]. Later it was shown that Epac activation
per se induced differentiation of human SH-SY5Y neuro-
blastoma cells, an effect that was blocked by Epac1 ShRNA
expression [129]. Importantly, recent work has implicated
that 8-pCPT-2'-O-Me-cAMP can be hydrolyzed to bioactive
metabolites with anti-proliferative effects in certain cell
types [130]. In long term studies such undesirable side ef-
fects can be avoided by using the Sp-modified and hydroly-
sis-resistant form of 8-pCPT-2'-O-Me-cAMP. Unfortunately,
by using isothermal microcalorimetry, it is evident that Sp-8-
pCPT-2'-O-Me-cAMPS can inhibit some cGMP- and cAMP-
specific PDEs [131]. Moreover, the hydrolysis-resistant BII-
selective Sp-5,6-DCl-cBIMPS showed inhibitory effect
against some PDEs, especially PDE1B. In intact cells, ana-
log-mediated inhibition of PDEs would lead to an increase in
the intracellular levels of cyclic nucleotides. Thus, to avoid
possible misinterpretation of data, it is important to perform
cAMP studies with a variety of analogs. Moreover, the use
of inhibitory Rp-modified cyclic nucleotide analogs, like Rp-
cAMPS and Rp-8-Br-cAMPS, should have effects opposite
to the PKA analogs, but should not affect the action of Epac
analogs.
4.3. Targeting the Catalytic Subunit
Kinase inhibitors are often based on the ATP site of the
catalytic domain/subunit, and act as competitive inhibitors
for this substrate, so called type I inhibitors (not to be con-
fused with type I PKA). More recently, allosteric inhibitors
of type II have been described, which stabilizes the inactive
kinase conformation [132]. A successful example of this
inhibitor type is the tyrosine kinase inhibitor imatinib used to
target Bcr-Abl in Chronic myelogenous leukemia (see [133]
for review). However, a valuable lesson has been learned
The cAMP-Dependent Protein Kinase Pathway as Therapeutic Target
Table 1. Relative affinities of selected cAMP-analogs to cAMP-binding sites. The table lists the affinity of analogs relative to cAMP
(Kd' = Kd,cAMP/ Kd, analog). Analog pair selectivity = (Kd' AI* Kd'BI)1/2 : (Kd' AII* Kd'BII)1/2. The data are from [60, 126]
RI
(Kd',
M)
Analog
Site A Site B
N6-Bnz-cAMP 4.0 0.26
Sp-5,6-DCL-cBIMPS 0.022 0.13
8-Pip-cAMP 2.1 0.06
8-AHA-cAMP 0.055 4.1
8-pCPT-2'-O-Me-cAMP 0.043 0.0016
about the price to pay for high specificity, which is higher
risk of resistance through missense mutations [133]. This is a
particular risk with cancer drugs where genetic instability
compared with high selection pressure drive the disease pro-
gression and its response to therapy. Other diseases are gen-
erally less prone to such problems, except through natural
polymorphisms within the population.
For the PKAs, no ATP-site competitive inhibitors are to
our knowledge available commercially, which display satis-
factory selectivity. The ATP-competitive kinase inhibitors
H89 (of the isoquinolinesulfonyl group) and KT5720, which
are often used as PKA-specific inhibitors of the catalytic
subunits, have been shown to inhibit other kinases more
potently than PKA [134]. H89 inhibits S6Kinase-1, mitogen-
and stress-activated protein kinase 1, and Rho dependent
protein kinase-II (ROCK-II) with similar and higher potency
[134]. Thus, when investigating the influence of PKA on
processes that are controlled by Rho kinase, the use of H89
may be particularly misleading as H89 would inhibit PKA,
but still inhibit the ROCK, and RhoA inhibition is one the
established functions of PKA (see Fig. (2)).
Compared to the inhibitory pseudosubstrate region on the
regulatory subunits of PKA, the natural PKA heat stable in-
hibitors, PKI type I and II, inhibits the catalytic subunit by a
similar and highly specific mechanism. The amino acid se-
quence of PKI containing the inhibition motif has been used
to generate kinase inhibitors with high selectivity towards
PKA, e.g. PKI-14-22 (amino acids 14-22, myrostoylated for
increased permeability). These peptide-based inhibitors are
competitive, however, for kinases in the MAPK pathways,
inhibitor have been developed that are non-competitive to
both ATP and protein substrate (reviewed in [135]). A recent
strategy based on staurosporine has provided non-
competitive PKA inhibitors with increased selectivity [136].
However, as with present inhibitors targeting the catalytic
subunit, there is to our knowledge no selectivity towards the
different isoforms of the C-subunit, and isotype selectivity
will be difficult to attain. This would potentiate the risk of
severe side-effects with systemic delivery, however, spatially
controlled or cell-type directed delivery are alternatives to
cope with such problems.
4.4. Targeting Localization
Disruption of localized PKA signaling can be an attrac-
tive strategy to obtain focused negative targeting, by dissoci-
Current Topics in Medicinal Chemistry, 2011, Vol. 11, No. 11 1399
RII
(Kd',
M) Epac1 Analog Pair Selectivity
Site A Site B Kd' (
M) RI
/RII

3.8 0.037 1.3
1:10
0.034 14 0.69
0.047 2.7 0.21
18:1
0.010 0.39 1.3
0.00059 0.024 4.6
ating PKA from its anchoring site. Perhaps the most chal-
lenging point of this strategy is to find compounds with both
disruptive and pharmacological potential, due to a large hy-
drophobic interphase between AKAPs and PKA. In addition,
specificity can be difficult to achieve due to high similarity
at the anchoring domain of most AKAPs, particularly be-
tween the PKAII specific AKAPs. However, a PKA isotype
directed disruption seems feasible in light of recent progress
on RI-anchoring disruptor peptides, and may have promising
potential for treating certain T-cell dysfunctions [137,138].
5. NEW POSSIBILITIES - SUBSTRATE DIRECTED
CONTROL OF SIGNALING INVOLVING PROTEIN
PHOSPHORYLATION
5.1. Modulation of Protein Phosphorylation Downstream
of PKA
The highest specificity of pharmacological intervention
of phosphorylation events is through manipulation at the
level of discrete kinase substrates. This can to some extent
be achieved by modulating the kinase or phosphatase activi-
ties, but this will only provide specificity in the rare cases
when the targeted kinase phosphorylates only very few sub-
strates. This is truly not the case for PKA. However, for
many protein substrates their phosphorylated state(s) is again
affected by association to other proteins, like the 14-3-3 fam-
ily members. 14-3-3 bind the phosphorylated target protein
and thereby may change its activity, localization or interac-
tion with other proteins (see [139] for review). Many binding
partners are described for the 14-3-3 proteins, amongst other
the related Rho-GEF proteins AKAP-Lbc and Lfc, which
both bind 14-3-3 upon phosphorylation by PKA (see Fig.
(2)) [140,141]. Binding of 14-3-3 to PKA-phosphorylated
Rho-GEF inhibits its exchange activity, thereby mediating a
negative signaling connection between cAMP- and Rho-
signaling Fig. (2A and B). Therefore, as long as 14-3-3 is
bound, little Rho activation can occur through this Rho-GEF.
In addition to its primary effect, binding of 14-3-3 inhib-
its dephosphorylation and thereby prolongs the signal. The
strength of the inhibition is expected to depend on the affin-
ity between 14-3-3 and the phospho-protein. This affinity
can be modulated by substances that disrupt or enforce the
interaction between 14-3-3 and its phospho-target. Since the
protein interaction interface is unique to each protein com-
plex, it provides an opportunity to specifically target kinase
1400 Current Topics in Medicinal Chemistry, 2011, Vol. 11, No. 11 Kleppe et al.

substrates. This strategy is illustrated in Fig. (2C) for the
AKAP-Lbc, where a drug that act on the association strength
between 14-3-3 and PKA-phosphorylated AKAP-Lbc (in
Fig. (2C) to enforce the association) will act to modulate the
strength of the negative coupling between the cAMP and
Rho signaling pathways. Interestingly, the engineering of a
compound that stabilize the interaction between 14-3-3 and a
binding partner is used by the fungus Fusicoccum amygdali,
which produces the wilt-inducing phytotoxin fusicoccin. The
molecular mechanism for this toxin seems to be activation of
the plant plasma membrane H+-ATPase by stabilizing its
interaction with 14-3-3 [142].
5.2. New Options to Exploit the Catalytic Mechanism of
Phospho-Transfer for Kinase Activity Modulation
The catalytic mechanism of PKA has been well de-
scribed. The phosphorylation appears to obey an ordered
kinetic mechanism, with Mg/ATP binding occurring first
[143]. Still there does not seem to be any mutual interference
between Mg/ATP and peptide substrate binding, at least for
small peptides [144]. After formation of the ternary kinase-
Mg/ATP-peptide complex, the phospho-transfer occurs rap-
idly, followed by an even faster phospho-substrate release (at
least for the model heptapeptide substrate Kemptide) [145].
Interestingly, the slow dissociation of ADP from the C-
subunit is rate limiting for the steady state kinase activity
[145,146], and the Mg/ADP-bound form of the C-subunit
will be the dominating C subunit species when the kinase is
active. The reaction scheme for PKA mediated phosphoryla-
tion is shown in Fig. (3A).
Similarly to G-proteins where GDP to GTP exchange is
stimulatory, the catalytic efficiency of PKA could probably
be modulated by changing the exchange rate of ADP to
ATP. Binding of Mg2+ to a low affinity site, distinct from the
Mg/ATP binding site, slows down the dissociation of ADP
considerably [146], indicating that this type of control is pos-
sible. Given that dissociation of Mg/ADP is rate limiting for
the catalysis of PKA it is puzzling that even small peptide
substrates are phosphorylated with different Vmax values un-
less the ones with slow turnover stabilize the rate limiting
Mg/ADP-bound state of the kinase. This requires that sub-
strate binds to Mg/ADP-bound C-subunit, an option com-
monly not considered in schemes describing protein kinase
kinetics. We have evidence that the standard peptide sub-
strate kemptide binds to Mg/ADP-bound C-subunit with
similar affinity as to Mg/ATP-bound C-subunit, making it a
realistic option [59] Fig. (3A,B). Expectedly, high affinity
substrates would preferentially associate with the Mg/ADP-
bound form of the C-subunit (right reaction path Fig. (3)),
whereas low affinity substrates are expected to bind the C-
subunit after nucleotide exchange to Mg/ATP (left reaction
path Fig. (3)). Compounds that accelerate ADP to ATP ex-
change would therefore be expected to discriminate in their
activation between these two substrate classes, depending if
they facilitate exchange of the peptide-substrate free or
bound form of the C-subunit (Fig. (3B)). It is however not
currently known to what degree different PKA substrates
associate with the C-subunit in its different ligand states, and
to what degree their association affects nucleotide exchange.
The catalytic mechanism for different substrates becomes
relevant during development of ATP-selective inhibitors,
which may bind poorly to protein substrate associated forms
of the C-subunit. Ligand free C-subunit only exists very
transiently in cells, making it a poor target for inhibitors. On
the other hand, the Mg/ADP-bound form is the most abun-
dant peptide-substrate-free form of the C-subunit and this
provides an opportunity to make compounds that trap the C-
subunit in a catalytically inactive state (I2 in Fig. (3B)). This
type of inhibitors would be expected to inhibit both high
affinity and low affinity substrates equally well. On the other
hand, inhibitors that trap the Mg/ATP bound C-subunit
(without substrate) in an inactive state are expected to pref-
erentially inhibit low affinity substrates (I1 in Fig. (3B)).

A Low cAMP B High cAMP C High cAMP & drug ( )

AC AC AC GPCR
GPCR GPCR

Gα12 Gα12 Gα12

PM PMGαs PMGαs
ATP cAMP ATP cAMP
C C
C
PDE
PDE PDE
RII RII

RII RII
RII RII

AKAP-lbc AKAP-lbc
P GEF AKAP-lbc
P GEF P GEF
14-3-3 14-3-3
PDE C 14-3-3 + PDE C + PDE C +

GDP- Rho Rho -GTP GDP- Rho Rho -GTP GDP- Rho Rho -GTP
PDE PDE PDE

Fig. (2). Targeting specific kinase substrates. Compartmentalized cross signaling between PKA and Rho signaling through the AKAP-lbc is
illustrated at conditions of low cAMP (A) where G
12AKAP-RhoGEFRho signaling is undisturbed (rapid exchange of Rho(GDP) to
Rho(GTP)). (B) at high cAMP-levels (cAMP concentration gradient shown as gray shade, cAMP bound to the RII subunits shown as dark
gray), activated PKA can phosphorylate AKAP-Lbc (P enclosed by a circle), thereby providing a binding site for 14-3-3 proteins. Binding of
14-3-3 blocks access of Rho(GDP) to the RhoGEF domain, and thereby lowers Rho activation (formation of Rho(GTP)). Panel (C) shows
pharmacological intervention by a small molecule drug (black oval) that passively enters the cell (dotted reversible arrow) and binds the in-
terphase between 14-3-3 and PKA-phosphorylated AKAP-lbc. Binding of the drug further strengthen the interaction between 14-3-3 and
phospho-AKAP-lbs, thereby prolonging the PKA-mediated inhibition of Rho.
The cAMP-Dependent Protein Kinase Pathway as Therapeutic Target Current Topics in Medicinal Chemistry, 2011, Vol. 11, No. 11 1401

These are opportunities that are so far unexploited, but have specific drugs, simply because the same targets are used in
the potential of generating compounds that can discriminate different cells of the body. A possible solution to this can
at the substrate level. The screening method to detect prom- come from multi-targeting, e.g. by combining receptor or
ising compounds will however be more challenging than ligand manipulation with intracellular targets of the pathway
traditional kinase assays. of interest, to gain cell-type specificity. A synergistic interac-
tion between the different drugs would then enforce the
A specificity, making adverse side effects less pronounced. The
cAMP-signaling pathway provides many opportunities for
S such a strategy, extending from its coupling to multiple ex-
CATP CATP,S
tracellular receptor types, to the many downstream intracel-
Low affinity
lular targets such as kinases and their substrates. Perhaps
ATP ATP these opportunities of multi-targeting, and the range of dis-
S catalysis
C CS eases for which it bears relevance, can make the cAMP-
ADP Rate- ADP pathway a prototype for selective pharmacotargeting of cell
limiting signaling pathways.
High affinity ACKNOWLEDGEMENTS
CADP CADP,S
S This work was supported by the Norwegian Research
Council, the Norwegian Cancer Society and Western Nor-
pS way Regional Health Authority.

Inhibitor binding Mg/ATP ABBREVIATIONS

bound C-subunit
CATP,l1
B AC = Adenylyl cyclase

l1 tmAC = Transmembrane AC
S sAC = Soluble AC
CATP CATP,S
Low affinity AKAP = A-kinase anchoring protein
ATP ATP cNMP = Cyclic nucleotide monophoshate (either
S catalysis
C CS cAMP or cGMP)
ADP ADP CNS = Central nervous system
Exchange Exchange
stimulation, stimulation COPD = Chronic obstructive pulmonary disease
No substrate High affinity with substrate
CADP CADP,S CREB = cAMP response element binding protein
l2 S Epac = Exchange protein directly activated by cAMP
CADP,l2 pS GC = guanylyl cyclase
Inhibitor binding Mg/ADP Phosphorylated
bound C-subunit Substrate
ICER = Immediate cAMP-induced early repressor
PDE = Phosphodiesterase
PKA = cAMP dependent protein kinase
Fig. (3). PKA catalysis and opportunities for pharmacological
modulation. The kinetic mechanism for peptide phosphorylation is PKAI = PKA isotype I
shown (A) where the different ligand forms of C-subunit (CX) are PKAII = PKA isotype II
shown (X = ADP – Mg/ADP; = ATP – Mg/ATP; = ADP,S –
Mg/ADP+peptide; = ATP,S – Mg/ATP+peptide; = S – peptide). (B) PKG = cGMP-dependent protein kinase
shows targeting of different ligand-bound forms of C-subunit with ROCK = Rho-dependent protein kinase
inhibitors (I1) and (I2), binding to Mg/ATP and Mg/ADP-bound C-
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