Project no. 037189
thera-cAMP
Identification of therapeutic molecules to target
compartmentalised cAMP signalling networks
in human disease
Specific Targeted Research Project
Priority 1 "Life sciences, genomics and biotechnology for health"
Publishable final executive summary
Period covered:
from 01/10/2006 to 30/09/2009
Start date of project: 01/10/2006
Date of preparation: 15 November 2009
Duration:
Dr. Enno Klussmann
Forschungsverbund Berlin e.V.
Version 1
36 months
Publishable executive summary
thera-cAMP (contract no. 037189)
The thera-cAMP project
The project “Identification of therapeutic molecules to target compartmentalised cAMP signalling
networks in human disease (thera-cAMP)” is a Specific Targeted Research Project (STREP) under
the EU’s Sixths Framework Programme. It is funded for three years within the thematic priority 1
“Life sciences, genomics and biotechnology for health“ (contract no. 037189). The following
partners participate:
Table 1: List of contractors
Contractor
Principal investigator
1 FMP
Leibniz-Institut für Molekulare Pharmakologie im
Forschungsverbund Berlin e.V.
DE
Enno Klußmann
2 UNIK
Universität Kassel, Institut für Biochemie
DE
Friedrich W. Herberg
3 UiO
University of Oslo, The Biotechnology Centre of Oslo NO
Kjetil Taskén
4 FTELE
Fondazione Telethon
Manuela Zaccolo
IT
 5B UGLA  University of Glasgow
UK
5 UGLA
University of Glasgow
UK
Miles D. Houslay
6 UGOT
Göteborg University, Institute of Biomedicine
SE
Sven Enerbäck
7 Biaffin
Biaffin GmbH & Co KG
DE
Bastian Zimmermann
8 Lauras
Lauras AS
NO
Vidar Hansson
9 Biolog
Biolog Life Science Institute, Forschungslabor und
Biochemica Vertrieb GmbH
DE
Hans-Gottfried Genieser
 5A UGLA
Co-ordinators contact details:
Dr. Enno Klußmann
Group Leader “Anchored Signalling”
Leibniz-Institut für Molekulare Pharmakologie
Robert-Rössle-Str. 10
D-13125 Berlin
Tel.: +49-(0)30-94793 260
Fax: +49-(0)30-94793 109
E-mail: klussmann@fmp-berlin.de
FMP website: http://www.fmp-berlin.de
Project website: http://www.thera-camp.eu
Major diseases including cardiovascular and renal diseases, diabetes mellitus, obesity, diseases of
the immune system, cancer, and neurological disorders are associated with or caused by
dysregulation of compartmentalised cAMP (cyclic adenosine monophosphate) signalling pathways.
Cyclic AMP is generated by adenylyl cyclases (ACs) in response to a plethora of extracellular
signals. It is degraded by phosphodiesterases (PDEs) hydrolysing cAMP to adenosine
monophosphate (AMP). The main effector of cAMP is protein kinase A (PKA). It binds four
molecules of cAMP, thereby becomes activated and phosphorylates a variety of target proteins.
Often, PKA and further signalling molecules of a signalling cascade are encompassed in multiprotein complexes in which they directly interact. The complexes are confined to defined cellular
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Publishable executive summary
thera-cAMP (contract no. 037189)
compartments by direct protein-protein interactions with anchoring or scaffolding proteins, which
thereby timely and spatially coordinate cellular signalling processes. A kinase anchoring proteins
(AKAPs) are a family of scaffolding proteins participating in the coordination of cAMP signalling by
tethering PKA and other signalling molecules to cellular compartments including the plasma
membrane, the outer mitochondrial membrane, the endoplasmatic reticulum, or exocytic vesicles.
Both compartmentalization and protein-protein interactions permit highly selective pharmacological
interference with a defined cellular process. Due to the specificity and vast diversity of proteinprotein interactions represent a large class of potential drug targets that offer great opportunities
for therapeutic intervention1, 2.
Objectives of thera-cAMP
Thera-cAMP aims at the identification of small “druggable” therapeutic molecules derived from
small molecule libraries which affect the interactions of signalling proteins with anchoring proteins
or the binding of anchoring proteins to cellular compartments. In particular, it is attempted to
identify molecules which
i) disrupt protein-protein interactions of ACs, PDEs, AKAPs, and PKA and/or
ii) displace AKAPs, PKA and PDEs from their cognate intracellular location.
The multidisciplinary approach is based on postgenomic research, established and novel cell lines
representing different diseases. The disease models used for the experiments represent
cardiovascular diseases, nephrogenic diabetes insipidus (NDI), asthma, chronic obstructive
pulmonary disease (COPD), AIDS, obesity, and schizophrenia. Screening of compound libraries
will be performed in living cells and in vitro with purified components of the cAMP signalling
system. The targets of small molecules are to be identified using established and to be developed
tools and bioassays. Cell signature responses to challenges with those compounds will be
elucidated in order to gain mechanistic insight into the effects on the disease phenotypes and to
anticipate side-effects of the identified substances. The small molecules will be valuable tools to
investigate compartmentalised cAMP signalling. Moreover, due to the potentially high specificity of
the lead compounds the consortium hopes to pave the way to alternative treatments with less sideeffects. Conventional therapies typically target receptors, inhibit enzymes, or alter the permeability
of ion channels, and thus inhibit protein functions throughout cells. In addition, many of the affected
targets are present in all cells of the body. This most likely accounts for many undesirable sideeffects of conventional medicine.
Implementation of the work
The work is divided between eight work packages (WPs). WP1 is responsible for the management
of the project. In WPs 2-5 cell models for diseases are utilized for the identification of small
molecules that disrupt protein-protein interactions of ACs, PDEs, AKAPs, and PKA and/or displace
PDEs, AKAPs, and PKA from their cognate intracellular location. WPs 6 and 8 contribute to the
discovery of small molecules by molecular modelling studies and small molecule synthesis. Novel
biosensors based on FRET (fluorescence resonance energy transfer) and BRET (bioluminescence
1
Arkin, MR and Wells, JA (2004) Small-molecule inhibitors of protein-protein interactions: progressing towards the
dream. Nat Rev Drug Discov, 3: 301-317
2
Wells, JA and McClendon, CL (2007) Reaching for high-hanging fruit in drug discovery at protein-protein interfaces.
Nature, 450: 1001-1009
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thera-cAMP (contract no. 037189)
resonance energy transfer) techniques are developed. The sensors will progressively enter the
screening process. Once small molecules have been identified, their interactions with the assumed
target protein and the interference with protein-protein interactions are quantitatively analysed in
WPs 7 and 8. In WP8 the small molecules are chemically modified in order to enhance their
binding to the targets. Modified small molecules are returned into WPs 2-5 for testing in cell
models. The lead structures are transformed into selective high-affinity small therapeutic
molecules. In WP7 the signature responses of cells to challenges with the small molecules are
characterised in order to monitor the specificity of the compounds and to anticipate side-effects.
Thus all work packages are strongly interdependent, which leads to a quick transfer of project
results within the consortium and thereby guarantees an appropriate advancement of the project.
Results of the first reporting period
In the first reporting period several protein-protein interactions in compartmentalized cAMPdependent signalling processes were characterized in detail e.g. 3, 4, 5, 6, 7, 8, 9, 10, 11. These included the
AKAP18-phospholamban (PLN) interaction, which plays a role in the regulation of Ca2+ reuptake
into the sarcoplasmic reticulum (SR) of cardiac myocytes. This interaction may be a target for
treatment of cardiovascular disease6. Other interactions characterized were those within the
PKA/Ezrin/EBP50/Cbp/Csk signalling complex. Interference with the ezrin-PKA interaction resulted
in improved immune responses in a mouse model for AIDS, indicating that targeting the complex
would be a viable strategy for immunomodulation in viral disease7. Targeting the PKA type I – Csk
pathway with the cAMP antagonist Rp-8-Br-cAMPS revealed an improved anti-tumor immune
3
Terrin, A, Di Benedetto, G, Pertegato, V, Cheung, YF, Baillie, G, Lynch, MJ, Elvassore, N, Prinz, A, Herberg, FW,
Houslay, MD, and Zaccolo, M (2006) PGE(1) stimulation of HEK293 cells generates multiple contiguous domains with
different [cAMP]: role of compartmentalized phosphodiesterases. J Cell Biol, 175: 441-451
4
Stefan, E, Wiesner, B, Baillie, GS, Mollajew, R, Henn, V, Lorenz, D, Furkert, J, Santamaria, K, Nedvetsky, P,
Hundsrucker, C, Beyermann, M, Krause, E, Pohl, P, Gall, I, MacIntyre, AN, Bachmann, S, Houslay, MD, Rosenthal, W,
and Klussmann, E (2007) Compartmentalization of cAMP-dependent signaling by phosphodiesterase-4D is involved in
the regulation of vasopressin-mediated water reabsorption in renal principal cells. J Am Soc Nephrol, 18: 199-212
5
Sachs, BD, Baillie, GS, McCall, JR, Passino, MA, Schachtrup, C, Wallace, DA, Dunlop, AJ, MacKenzie, KF,
Klussmann, E, Lynch, MJ, Sikorski, SL, Nuriel, T, Tsigelny, I, Zhang, J, Houslay, MD, Chao, MV, and Akassoglou, K
(2007) p75 neurotrophin receptor regulates tissue fibrosis through inhibition of plasminogen activation via a
PDE4/cAMP/PKA pathway. J Cell Biol, 177: 1119-1132
6
Lygren, B, Carlson, CR, Santamaria, K, Lissandron, V, McSorley, T, Litzenberg, J, Lorenz, D, Wiesner, B, Rosenthal,
W, Zaccolo, M, Tasken, K, and Klussmann, E (2007) AKAP complex regulates Ca2+ re-uptake into heart sarcoplasmic
reticulum. EMBO Rep, 8: 1061-1067
7
Ruppelt, A, Mosenden, R, Gronholm, M, Aandahl, EM, Tobin, D, Carlson, CR, Abrahamsen, H, Herberg, FW, Carpen,
O, and Tasken, K (2007) Inhibition of T cell activation by cyclic adenosine 5'-monophosphate requires lipid raft targeting
of protein kinase A type I by the A-kinase anchoring protein ezrin. J Immunol, 179: 5159-5168
8
Smith, KJ, Baillie, GS, Hyde, EI, Li, X, Houslay, TM, McCahill, A, Dunlop, AJ, Bolger, GB, Klussmann, E, Adams, DR,
and Houslay, MD (2007) 1H NMR structural and functional characterisation of a cAMP-specific phosphodiesterase-4D5
(PDE4D5) N-terminal region peptide that disrupts PDE4D5 interaction with the signalling scaffold proteins, beta-arrestin
and RACK1. Cell Signal, 19: 2612-2624
9
Willoughby, D, Baillie, GS, Lynch, MJ, Ciruela, A, Houslay, MD, and Cooper, DM (2007) Dynamic regulation,
desensitization, and cross-talk in discrete subcellular microdomains during beta2-adrenoceptor and prostanoid receptor
cAMP signaling. J Biol Chem, 282: 34235-34249
10
Baillie, GS, Adams, DR, Bhari, N, Houslay, TM, Vadrevu, S, Meng, D, Li, X, Dunlop, A, Milligan, G, Bolger, GB,
Klussmann, E, and Houslay, MD (2007) Mapping binding sites for the PDE4D5 cAMP-specific phosphodiesterase to the
N- and C-domains of beta-arrestin using spot-immobilized peptide arrays. Biochem J, 404: 71-80
11
Yaqub, S, Henjum, K, Mahic, M, Jahnsen, FL, Aandahl, EM, Bjornbeth, BA, and Tasken, K (2008) Regulatory T cells
in colorectal cancer patients suppress anti-tumor immune activity in a COX-2 dependent manner. Cancer Immunol
Immunother, 57: 813-821
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thera-cAMP (contract no. 037189)
response in patient samples ex vivo based on inhibition of regulatory T cells through a COX-2 /
PGE2 / cAMP signalling pathway11. The interaction of PDE4A with the neurotrophin receptor,
p75NTR, is responsible for local regulation of the cAMP level in the vicinity of the receptor, and
regulates fibrin deposition. Clearance of fibrin is a critical step of matrix remodelling that
contributes to tissue repair in a variety of pathological conditions, such as stroke, atherosclerosis,
and pulmonary disease. p75NTR regulates local degradation of cAMP and thereby perpetuates
scar formation after injury5.
Peptidic and non peptidic small molecules as disruptors of AKAP-dependent signalling complexes
were developed11, 12, 13. A peptide derived from the AKAP18 binding site of PLN displaces the
AKAP from PLN in cultured adult cardiac myocytes, and decreases the velocity of Ca2+ reuptake
into the SR of neonatal cardiac myocytes. Such an agent may protect the heart against the
influence of adrenergic stimuli in cardiovascular diseases6. Screening of a small molecule library
led to the identification of a compound, FMP-API-1, disrupting AKAP-PKA interactions. The
molecule prevents -adrenoceptor-mediated increases of L-type Ca2+ channel currents in neonatal
cardiac myocytes, and may thus also be cardio-protective in cardiovascular diseases. A focussed
library of derivatives of the molecule was synthesized and contains compounds preventing the
interaction with higher efficiency than the parent molecule.
Based on the defined and characterized protein-protein interactions, various vectors for expression
of interacting proteins in cells have been constructed and were transiently or stably expressed. For
FRET assays the interacting partners are fused with cyan fluorescent- and yellow fluorescent
proteins (CFP and YFP, respectively), for BRET experiments as fusions with green fluorescence
protein (GFP) and luciferasee.g. 14, 15, 16, 17. In cell-based (or in in vitro) small molecule library screens
the fluorescence tags may either be used for monitoring FRET- or BRET signal changes or the
displacement of one of the partners from a cellular compartment. For example, fusions of PKA
regulatory RII subunits and a fragment of AKAP-Lbc (Ht31) as fusions with CFP and YFP,
respectively, have been generated, and HEK293 cells stably expressing the partners are currently
being established. Screens for small molecules disrupting such interactions will commence at the
beginning of the second reporting period.
Methods for the identification of the targets of small molecules have been established. For
example, a combination of Biacore measurements and mass spectrometric analysis has revealed
the binding site of FMP-API-1 and derivates in RII subunits of PKA. Precipitation experiments
with agarose beads coupled to a new cAMP analogue have pulled down a variety of cAMPdependent signalling molecules as revealed by mass spectrometry. This method will allow
12
Hundsrucker, C, Krause, G, Beyermann, M, Prinz, A, Zimmermann, B, Diekmann, O, Lorenz, D, Stefan, E, Nedvetsky,
P, Dathe, M, Christian, F, McSorley, T, Krause, E, McConnachie, G, Herberg, FW, Scott, JD, Rosenthal, W, and
Klussmann, E (2006) High-affinity AKAP7delta-protein kinase A interaction yields novel protein kinase A-anchoring
disruptor peptides. Biochem J, 396: 297-306
13
Stokka, AJ, Gesellchen, F, Carlson, CR, Scott, JD, Herberg, FW, and Tasken, K (2006) Characterization of A-kinaseanchoring disruptors using a solution-based assay. Biochem J, 400: 493-499
14
Prinz, A, Diskar, M, and Herberg, FW (2006) Application of bioluminescence resonance energy transfer (BRET) for
biomolecular interaction studies. Chembiochem, 7: 1007-1012
15
Zaccolo, M and Movsesian, MA (2007) cAMP and cGMP signaling cross-talk: role of phosphodiesterases and
implications for cardiac pathophysiology. Circ Res, 100: 1569-1578
16
Diskar, M, Zenn, HM, Kaupisch, A, Prinz, A, and Herberg, FW (2007) Molecular basis for isoform-specific
autoregulation of protein kinase A. Cell Signal, 19: 2024-2034
17
Willoughby, D and Cooper, DM (2008) Live-cell imaging of cAMP dynamics. Nat Methods, 5: 29-36
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thera-cAMP (contract no. 037189)
comparison of the effects of identified small molecules on various cell models representing
different diseases.
In summary, we proposed a collaborative interdisciplinary approach to reach the goals of the
project and planned to set up a project that strongly relies on interdependencies between work
packages. During the first reporting period we have implemented this and delivered milestones and
deliverables as proposed. Only minor changes in the deliverables were necessary. Moreover, the
consortium has published nearly 50 scientific paper and reviews. In the second reporting period we
anticipate to identify small molecules interfering with protein-protein interactions within
compartmentalized cAMP signalling networks. Cell signature responses to challenges with small
molecules will be defined in order to gain mechanistic insight into the effects on the disease
phenotypes and to anticipate side effects of identified substances. The small molecules will be
valuable tools to investigate compartmentalised cAMP signalling. Moreover, this approach may
lead to alternative strategies for the treatment of diseases associated with altered cAMP signalling
that are not addressed effectively by conventional pharmacotherapy.
Results of the second reporting period
In the second reporting period relevant protein-protein interactions were further characterized
biochemically and by molecular modelling studies. Screening systems were established and
utilized to identify small molecule disruptors of defined protein-protein interactions within
compartmentalized cAMP signalling networks. For example, the PKA/Ezrin/EBP50/Cbp/Csk
signalling complex was further characterized. The interaction surfaces of the components were
mapped. The molecular interactions were characterized biochemically using in solution methods
and it was tested whether the interactions can be disrupted. Using peptidomimetics approaches a
peptide competing AKAP binding to PKA type I was stabilized, and the Ezrin-PKA interaction could
be targeted in vivo, in an animal model for immunodeficiency, murine AIDS. Next, test systems
were designed and used in high throughput screenings for the identification of small molecules
disrupting anchoring and assembly of the PKA/Ezrin/EBP50/Cbp/Csk and, in addition, of the
PDE/-arrestin/PKB complex. For this, interacting pairs were labelled with suitable fluorochromes
or tags for fluorochrome labelling and signals arising from these pairs in readout assays for proteinprotein interactions were optimized with regard to signal-to-noise. Interacting pairs were used for
BRET-, fluorescence polarization- and amplified luminescence homogeneous ligand proximity
assay (Alpha-screen; Torheim et al, 2009; Jarnæss, et al., 2009; Stokka et al., 2009; Ruppelt et al., 2009;
Yaqub et al., 2008; Rogne et al., 2009; Solheim et al., 2008).
During the second reporting period various screening approaches have been established. One
main emphasis was the establishment of cell-based assays. In particular, BRET assays have been
implemented. More than 100 BRET/FRET vectors were generated in a previously established
microplate-based BRET2 assay in several co-transfected adherent cell lines (Diskar et al., 2007;
Prinz und Herberg, 2009). The utility of fluorescence and bioluminescence-based reporters in live
cells was evaluated (Prinz et al., 2008). Novel cAMP sensors based on EPAC1 and improved
Renilla luciferase (Rluc8) were evaluated. GFP-EPAC-L-Rluc8 (hEpac1(157-881, T781A/F782A)
yielded highest s/n of about 45% in resting vs. cAMP stimulated eukaryote model cell lines,
including F11 cells. A cellular BRET assays was established that identified a novel, dynamic
interaction between PDE4D and Lis, a protein involved in neuronal development and whose
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thera-cAMP (contract no. 037189)
mutation leads to encephalies and with Ndel1, a protein that interacts with the scaffold DISC1,
which is mutated in schizophrenia. This allows novel insights into neurological disease.
Utilizing cell-based assays, different classes of small molecules were identified by screening a
library comprising 20,000 small molecules, which either induce or prevent PDE44-dependent foci
formation in CHO cells, stably expressing PDE4A. The small molecules have therapeutic potential
as they either trigger or inhibit the intracellular re-localisation of PDE4A4, a cAMP specific
phosphodiesterase linked to COPD (Chronic Obstructive Pulmonary Disease), fibrosis and sleep
aberrations. This allows novel insights into COPD and other fibrotic disease mechanisms with
potential for therapeutic development. In addition, small molecule inhibitors of the AVP-dependent
redistribution of AQP2 from intracellular vesicles into the plasma membrane of renal principal cells
were identified by screening a library of 20,000 compounds and utilizing primary cultured renal
inner medullary collecting duct (IMCD) cells. The identified molecules have potential to be
developed into therapeutics for the treatment of diseases associated with water retention such as
chronic heart failure or liver cirrhosis. In addition to the cell-based assays ELISA-and Alpha screen
assays were established that have already been used to identify small molecules inhibitors of
defined protein-protein interactions or will in the future be used for this purpose.
The technology for characterizing the interaction of small molecules with their protein target was
established. For example, surface plasmon resonance (SPR)-based competition assays for testing
potencies of small molecules disrupting AKAP-PKA interactions were established. More than 120
compounds and a number of natural compounds (>20) displaying structural homologies compared
to the identified lead compounds were tested in such assays. Two lead compounds and several
derivatives thereof were identified as promising candidates. The binding site of the compound on
PKA RII subunit was mapped for both lead compounds using SPR approaches. Besides testing
effects of anchoring disruptors on PKA RII isoforms alpha and beta binding to AKAP18, effects on
the PKA holo enzyme complex and on binding of PKA RII to AKAP450 were investigated as well.
Medicinal chemistry allowed the optimization of small molecules and, in addition, provided the
basis for analysing structural similarities between the first set of lead compounds inhibiting AKAPPKA interactions and natural compounds. Out of the identified natural compounds more than 20
flavonoids and polyphenols were tested and some of them were identified as efficient AKAP
disruptors competing for PKA R-subunit binding to AKAP18 in vitro.
Rational approaches were chosen to design and synthesize a large number of cyclic nucleotide
analogues that were screened for inhibitory or activating potential on type I and type II R-subunits
of PKA. Agarose gels were functionalised with cyclic nucleotides. Such immobilized small
molecules can be utilized for affinity precipitations in order to identify targets of the small molecules
and provide a basis for analysis of potential side effects.
Taken together, the second reporting period has yielded detailed analyses of disease-relevant
protein-protein interactions in compartmentalized cAMP signalling networks, screening systems for
the identification of small molecules interfering with such defined interactions, and small molecules
that disrupt such interactions. In addition, identified small molecules were optimized and the
technology was developed to specifically analyse the effects of such molecules on cAMP signalling
networks in a cellular context.
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