CORDIS - EU research results

Identification of therapeutic molecules to target compartmentalised cAMP signalling networks in human disease

Final Report Summary - THERA-CAMP (Identification of therapeutic molecules to target compartmentalised cAMP signalling networks in human disease)

The project THERA-CAMP was a Specific Targeted Research Project (STREP) under the EU's Sixth Framework Programme (FP6). It is funded for three years within the thematic priority 1 'Life sciences, genomics and biotechnology for health'.

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 deregulation of compartmentalised cyclic adenosine monophosphate (cAMP) 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 multi-protein complexes in which they directly interact. The complexes are confined to defined cellular 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 compartmentalisation and protein-protein interactions permit highly selective pharmacological interference with a defined cellular process. Due to the specificity and vast diversity of protein-protein interactions represent a large class of potential drug targets that offer great opportunities for therapeutic intervention.

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.

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 utilised 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 Fluorescence resonance energy transfer (FRET) and Bioluminescence resonance energy transfer (BRET) 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 WPs 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 compartmentalised cAMP-dependent signalling processes were characterised in detail. These included the AKAP18 delta-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 disease. Other interactions characterised 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 disease. Targeting the PKA type I - Csk pathway with the cAMP antagonist Rp-8-Br-cAMPS revealed an improved anti-tumour immune response in patient samples ex vivo based on inhibition of regulatory T cells through a COX-2 / PGE2 / cAMP signalling pathway.

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 luciferase. 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 cAMP-dependent signalling molecules as revealed by mass spectrometry. This method will allow 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 WPs. 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 compartmentalised 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 characterised biochemically and by molecular modelling studies. Screening systems were established and utilised to identify small molecule disruptors of defined protein-protein interactions within compartmentalised cAMP signalling networks. For example, the PKA / Ezrin / EBP50 / Cbp / Csk signalling complex was further characterised. The interaction surfaces of the components were mapped. The molecular interactions were characterised 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 stabilised, 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 / beta-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 protein-protein interactions were optimised with regard to signal-to-noise. Interacting pairs were used for BRET-, fluorescence polarisation- and amplified luminescence homogeneous ligand proximity assay.

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. The utility of fluorescence and bioluminescence-based reporters in live cells was evaluated. 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 versus 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 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.

Utilising 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 Chronic obstructive pulmonary disease (COPD), 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 utilising 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 characterising 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 (over 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 optimisation of small molecules and, in addition, provided the basis for analysing structural similarities between the first set of lead compounds inhibiting AKAP-PKA 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 synthesise 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 immobilised small molecules can be utilised 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 compartmentalised 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 optimised and the technology was developed to specifically analyse the effects of such molecules on cAMP signalling networks in a cellular context.