EP1891028A1

EP1891028A1 - Non-peptidic inhibitors of akap/pka interaction

Info

Publication number: EP1891028A1
Application number: EP06742376A
Authority: EP
Inventors: Enno Klussmann; Walter Rosenthal; Jörg Rademann; Frank Christian; Sina Meyer
Original assignee: Forschungsverbund Berlin FVB eV
Current assignee: Forschungsverbund Berlin FVB eV
Priority date: 2005-05-18
Filing date: 2006-05-18
Publication date: 2008-02-27
Also published as: US20090176773A1; WO2006122546A1; DE112006002052A5; JP2008540586A; CA2611896A1

Abstract

The invention relates to non-peptidic molecules which modulate, especially inhibit, the interaction of protein kinase A (PKA) and A kinase anchor proteins (AKAP) and to a host or target organism that comprises said non-peptidic compounds or recognition molecules directed to said compounds, such as e.g. antibodies or chelating agents. The invention also relates to a pharmaceutical agent, especially for use in the treatment of diseases that are associated with a disturbance of the cAMP signal path, especially insipid diabetes, hypertonia, pancreatic diabetes, duodenal ulcer, asthma, heart failure, obesity, AIDS, edema, hepatic cirrhosis, schizophrenia and others. The invention also relates to the use of the inventive molecules.

Description

Nonpeptidic inhibitors of the AKAP-PKA interaction

Besehreibung

The invention relates to non-peptidic molecules which modulate, in particular inhibit, the interaction of protein kinase A (PKA) and protein kinase A anchor proteins (AKAP), and a host or target organism which encodes these non-peptidic compounds or recognition molecules are directed, such. Antibodies or chelators; The invention furthermore relates to a pharmaceutical agent, in particular for the treatment of diseases associated with a disorder of the cAMP signaling pathway, in particular diabetes insipidus, hypertension, diabetes mellitus, duodenal ulcer, asthma, cardiac insufficiency, obesity, AIDS, edema, Cirrhosis, schizophrenia and others. The invention also relates to the use of the molecules according to the invention.

The ability of cells to receive and respond to external signals is fundamental to their survival and function. The signals are detected by receptors and converted into a cellular response, which always involves a chemical process.

Since there are a large number of possible signals and an even greater number of possible reactions, many signal paths are required. A possible signaling pathway that is the cAMP-dependent signaling pathway. It begins by activating a G protein-coupled, heptahelical transmembrane receptor by an extracellular signal, e.g. By a neurotransmitter or a hormone. The most well-studied example of such a signaling pathway is the beta-adrenergic receptor system in which adrenaline or norepinephrine activate G-protein-coupled, beta-adrenergic receptors (GPCR v. G protein-coupled receptor). In the case of the beta-adrenergic receptors, a distinction is made between the subtypes beta _x , beta2 and beta ₃ , which differ in terms of their tissue distribution and ligand affinity.

The receptor thus activated transmits the signal to a heterotrimeric, guanosine triphosphate-binding protein (GTP-binding or G protein); this then exchanges bound GDP for GTP and is also activated.

After GTP binding, the trimer dissociates into an alpha subunit and a betagamma subunit. Both subunits are membrane-bound via acylations and can each activate or inhibit their own effectors.

G proteins have an intrinsic GTPase activity that can cleave the bound GTP and thus deactivate the G protein, and alpha and beta gamma subunits then form a trimer again. So the G-proteins take over the function of a molecular switch. Different classes of G proteins are distinguished (G ₃ , Gi, G _q , G _olf ) whose subunits can activate or deactivate different effector molecules. The effectors include Ca ²⁺ and K ⁺ channels, phospholipase C-beta and adenylate cyclase (AZ). The adenylate cyclase - also a membrane-bound, occurring in several variants - enzyme is stimulated by G _alpha . It converts ATP into the second messenger cAMP. The cAMP can freely diffuse into the cytosol and activate different effectors. These include cyclic nucleotide-gated (CNG) cation channels that open or close upon cAMP binding, and a family of guanine nucleotide exchange factors (GEF), the so-called Epac (ex- change proteins actϊvated by cAMP). The latter regulate the small monomeric GTPases Rap-1 and Rap-2, which act as suppressors of Ras. CAMP binding to Epac leads to a conformational change that exposes and activates the GEF domain. Thus, the suppressive effect is abolished, and Ras can be activated by the GEF function. Ras is involved in MAP kinase (mitogen-activated protein kinase) signaling pathways, which are affected by a variety of proliferation or differentiation-inducing signals, e. As of cytokines activated. Via cAMP-responsive element binding proteins (CREB), cAMP can also influence gene expression; CREB bind to the cAMP-responsive elements (CRE) of DNA and thus act as transcription factors. However, the most extensively described and best characterized effector of cAMP is the cAMP-dependent protein kinase (PKA or A kinase). When inactive, the protein is a heterotetramer consisting of two regulatory (R) and two catalytic (C) subunits. In this bound state, the catalytic subunits are inactive. If the intracellular cAMP concentration increases after stimulation, two cAMP molecules bind to each R subunit. By the resulting conformational change releases the catalytic subunits. They then phosphorylate nearby target proteins at Ser and Thr residues, which in turn undergo these conformational and thus functional changes. The recognition of the target proteins takes place via consensus sequences. Since many different extracellular stimuli converge in the cAMP-PKA pathway, and since PKA is an enzyme with broad substrate specificity, the question arises as to how the numerous different stimuli can trigger different specific cell responses.

A first contribution to the specificity in the cAMP-PKA signaling pathway is provided by the R- and C-subunits (RIalpha, RIbeta, RIIalpha, RKbeta or Calpha, Cbeta, Cgamma, PrKX), which are present in different isoforms depending on the cell type PKA isoforms can assemble together. The RI generally have a higher affinity for cAMP than the RII subunits. R subunits can form both homo- and heterodimers, allowing many combinations with fine affinity gradients.

In addition, local aspects play an important role in the specificity of the signaling pathway: cAMP is generated at a certain location near the G protein-coupled receptor through the AZ, and free diffusion in the cytosol is limited by phosphodiesterases (PDEs) - these hydroly - cAMP to adenosine monophosphate. After an extracellular stimulus, only PKA tetramers are activated, which are located in the local PDE-limited cAMP activity range. Not only does an A-kinase anchor protein (AKAP) play an important role in anchoring PKA in such an area, but also in the vicinity of substrates to be phosphorylated.

AKAP proteins are a family of currently about 50 proteins without significant sequence homology but with similar function. AKAP proteins are defined by the binding domain feature for the R subunits of PKA. This conserved motif is an amphipathic alpha helix of 14 to 18 amino acids, whose hydrophobic side interacts with a hydrophobic pocket of PKA. The hydrophobic pocket is formed by the N-termini of dimerized R subunits. The N-termini assemble in antiparallel fashion, with two helix-turn-helix yiotics forming a four-helix bundle. The alpha helices of each subunit closest to the N-terminus form the hydrophobic pocket required for AKAP binding, while the C-terminal alpha helices of the bundle allow dimerization. In addition to the amino acids involved in the alpha helices, other N-terminal amino acid side chains are involved in interactions with AKAP proteins.

The N-terminal part of the RI and RII subunits differs in sequence, resulting in different AKAP binding specificities. Most AKAP proteins bind to RII subunits (K _d in the nanomolar range), distinguishing low- and high-affinity AKAP proteins. While AKAP Lbc / Ht31, AKAP79 and AKAP95 _d with ~ K values between 1 and 50 nM for the high-affinity include AKA proteins, include Gravin and the

Ezrin / radixin / moesin protein family AKAP

Proteins to the low affinity (K _d values in the micromolar

Area) . In addition, there are also several dual-specific AKAP proteins (D-AKAP) that bind both types of R. To date, only AKAPCE has been known as RI-specific AKAP proteins

(from Caenorhabditis elegans), AKAP82, PAP7 [peripheral-type benzodiazepine receptor (PBR) associated protein 7) and hAKAP220. Another characteristic domain of AKAP proteins is the so-called targeting domain, which anchors each AKAP protein in a specific subcellular compartment in the cell. In addition to PKA and subcellular structures, A-KAP proteins can bind additional signaling molecules and enzymes important in the cAMP-PKA system, such as PDE and phosphatases. The latter reverse the phosphorylation reactions by hydrolysis and can thus terminate the action of a stimulus. In this way, AKAP proteins close to the substrate provide a scaffold that can recruit all the proteins needed to initiate and terminate the signal. The basically diffuse cAMP signal emanating from the AZ is thus spatially and spatially focused with substrate phosphorylation by the AKAP-fixed PKA.

AKAP proteins are involved in central sites of many cellular processes. One possibility of subdivision is the localization of the AKAP proteins in the cell; there are ion channel, cytoskeletal and mitochondrial-associated AKAP proteins. Another possibility is given by the tissue-specific expression of the AKAP proteins.

In order to demonstrate the importance of AKAP proteins for signal transduction pathways in cells, the functions of three AKAP proteins are described in more detail below: Gravin, AKAP79 and AKAP18.

Gravin is an actin-associated A-KAP; polymerized actin is an important component of the cytoskeleton. Gravin serves as a multivalent scaffold protein, which in addition to actin also binds PKA and the Ca ²⁺ -dependent protein kinase (PKC). This complex is important for the desensitization of the beta ₂ adrenergic receptor (see above) after stimulation by agonists. At rest, the gravin complex is bound to the receptor. If the receptor is activated, for. B. by adrenaline binding, this leads to the formation of cAMP (see above). Graken-anchored PKA releases its catalytic subunits, which themselves phosphorylate gravin. Phosphorylation initially strengthens the bond. If the stimulation by the agonist persists, then Gravin is also phosphorylated by the likewise anchored PKC. This phosphorylation causes the dissociation of the gravin complex from the receptor. Instead, the adapter protein linked to the ubiquitin ligase mouse double minute-2 (MDM2) binds beta-arrestin to the receptor. MDM2-catalyzed ubiquitination characterizes the receptor for proteasomal degradation and endocytosis, thus desensitizing the beta ₂ adrenergic receptor system.

AKAP79 is an ion channel-associated AKAP which is involved in the regulation of synaptic plasticity in the hippocampus. pus is involved. There, PKA-mediated Na ⁺ currents or Ca ²⁺ currents are enhanced, which were induced by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. AMPA receptors have an intrinsic Ca ²⁺ / Na ⁺ channel function. The enhancement is achieved by modulating the activity of the glutamate receptor by phosphorylation. The task of the AKAP79 is to anchor the PKA in the vicinity of the AMPA receptors. For this it is endowed with N-terminal, basic regions which allow binding to the membrane lipid phosphatidylinositol-4,5-bisphosphate. The binding of AKAP79 to the receptor is not direct, but it binds to membrane-associated guanylate kinase (MAGÜK) proteins, which in turn bind to the receptor.

AKAP18 was first described as a membrane-associated, 15 or 18 kDa protein anchoring PKA to the basolateral plasma membrane of epithelial cells and to skeletal muscle cells near the L-type Ca ²⁺ channels on the plasma membrane, with AKAP18 attached directly to the C Terminus of the Ca ²⁺ channels interacts.

The protein consists of 81 amino acids containing the PKA-binding amphipathic helix and N-terminal myristoyl and palmitoyl lipid anchors that anchor the PKA-AKAP complex to the plasma membrane. The interaction between AKAP18 and the Ca ²⁺ channel occurs via the C-terminal domain of the alpha subunits of the channel; this is a leucine zipper-like motif involved. In this way, the PKA is positioned near an important phosphorylation site in the alpha subunit, allowing for specific phosphorylation. Phosphorylation increases the likelihood of the channel. Further studies have shown that several splice variants emerge from the AKAP18 gene; therefore, the AKAP18 protein described above was designated AKAP18alpha (also AKAP15). Other splice variants are AKAP18b, gamma and delta.

AKAPlδbeta consists of 104 amino acids and contains the same membrane binding domain as the alpha variant. An additional 23 amino acid domain directs the protein to the apical plasma membrane in polarized epithelial cells. The function of AKAPlδbeta is so far unclear.

Unlike most AKAP proteins, the 326 amino acid AKAP18gamma is also localized in soluble cell fractions. In mouse oocytes, it anchors PKA-RI subunits in the nucleus, suggesting an involvement in the regulation of transcription. AKAP18delta is a 353 amino acid protein whose RII binding domain is almost identical to those of the alpha-gamma variants. It is for the most part homologous to AKAPlδgamma. AKAPlδdelta was discovered during the search for AKAP proteins involved in the translocation of the aquaporin-2 (AQP2) water channel from intracellular vesicles into the apical plasma membrane of kidney seed tube cells (see below). It could be shown that AKAPlδdelta is localized to the AQP2-containing vesicles.

In addition, because its distribution is very similar to that of AQP2, and as it is translocated to the plasma membrane along with AQP2 following appropriate stimulation of the kidney cells, involvement in AQP2 translocation is suspected. AQP2 translocation is induced by the antidiuretic hormone arginine vasopressin (AVP, also known as ADH). The formation of the hormone and its secretion into the bloodstream are controlled by osmoreceptors in the hypothalamus, which constantly monitor the osmolarity of the blood. If the osmolarity increases, for example as a result of reduced fluid absorption of the organism, AVP is synthesized and secreted into the bloodstream. The start signal for AQP2 redistribution delivers the binding of AVP to the vasopressin receptor (V2R) on the basolateral membrane side of the main renal collecting tube. The V2 receptor, which is a GPCR, initiates the signaling cascade described above, so that more cAMP is formed. The PKA anchored to the AQP2-containing secretory vesicles by AKAPlδdelta releases its catalytic subunits due to the cAMP-induced conformational change, which phosphorylates the nearby AQP2 water channels. This phosphorylation ensures the transport of the vesicles and their fusion with the apical plasma membrane. In this way, the water permeability of this membrane is increased, and it can be increasingly water from the - located on the other side of the apical membrane in the manifold - reabsorbed primary urine. This counteracts the initial stimulus of the signal chain.

In addition to AKAPlδdelta, other proteins are associated with the AQP2 vesicles, but unlike A-KAPlδdelta they do not reach the plasma membrane. This could mean that the PKA anchored to the vesicle membranes by AKAPlδdelta is not limited to the AQP2 Phosphorylation, but also, for example, for the transport process to the plasma membrane regulating phosphorylations is required. Such a function has also been demonstrated for the L-type Ca ²⁺ channel-associated AKAP79 (see above).

It is possible, as described above, to inhibit the binding of PKA to AKAP proteins by using synthetic peptides that mimic the amphipathic helix of the PKA binding domain. Although these inhibitory peptides play an important role in elucidating the physiological importance of PKA compartmentalization by AKAP proteins, they have disadvantages. As peptides they are z. B. not very stable and expensive in the synthesis. Because AKAP proteins provide specificity and tissue-specific expression in many cellular processes, they are an attractive target for new drugs. However, before developing new drugs, the AKAP proteins must be validated as a target. As a result, the interaction between AKAP proteins and PKA in the animal model and later also in the human model must be reversed. However, peptides can not be administered to experimental animals because they are degraded in the gastrointestinal tract. Another important model for the study of AKAP functions are cell culture models. For use in cell cultures, however, peptides must be acylated to ensure membrane permeability. Prior art peptides dock more tightly to the hydrophobic pocket of the regulatory PKA subunits due to lack of steric hindrance or better interacting (hydrophobic) amino acid side chains complete AKAP proteins. In this way, it can be experimentally verified how the abolition of PKA compartmentalization by AKAP proteins has a physiological effect. The first introduced and hitherto most widely used anchor inhibitor peptide is the 22 to 24 amino acid Ht31 peptide derived from the PKA binding domain of the AKAP protein Ht31, also known as AKAP-Lbc. Using bioinformatic methods and peptide-aerray screenings, in which amino acids were systematically exchanged from a consensus binding site for RII subunits calculated from various AKAP proteins and the peptides were coupled to cellulose membranes for binding experiments, a more inhibitory Peptide generated AKAPi ₃ (in silico). This is a 17-mer and can undergo additional hydrophobic interactions with the RII subunit through altered positions of two amino acid side chains, and it can form an additional salt bridge that stabilizes its conformation.

Higher affinity inhibitory peptides could be generated from the RII binding domain of the AKAPlδdelta, which belongs to the high affinity AKAP proteins. These have u.a. the advantage that they can be dosed lower, which makes nonspecific effects of the peptide less likely.

First, all amino acids of a 25-mer containing the A-KAP18delta-RII binding domain were individually replaced with all sorts of other biogenic amino acids. Starting from the higher affinity peptides, further amino acid substitutions were made based on a Structural model of the binding of the peptide in the hydrophobic pocket, made. However, a further affinity enhancement was not possible. The resulting peptides were tested for their specificity and biological function as global inhibitors of AKAP-RII binding.

Surprisingly, it has been shown that the non-peptidic molecules according to Table A can be used as inhibitors or decouplers of PKA and AKAP or as substances for blocking the PKA anchoring. The non-peptidic inhibitors are also lead compounds for new drugs. Such drugs are a new tool for blocking PKA anchoring for in vitro experiments and they are the basis for a new class of drugs. which, unlike classical drugs, attack protein-protein interactions and do not modulate enzyme or receptor activities.

All these molecules have in common that they are non-peptidic blockers or decouplers of the PKA-AKAP interaction, which have not yet been disclosed in the prior art. All these substances can also be used to block the PKA anchorage. Targeted inhibition of an AKAP-RII complex is possible with the claimed compounds. Surprisingly, the chemical and physical properties of the substances according to the invention make it possible to use them directly in cell cultures, animal models and in the primate and human regions, thus making it possible for the first time to study the function of AKAP proteins in vivo. So far, in the prior art, the investigation of the function of AKAP proteins not described in vivo. This in vivo capability is a common feature of all compounds of the invention.

Not all of the compounds according to the invention have a new structural element, but this does not mean that the uniformity of the teaching according to the application no longer exists. The claimed alternative chemical compounds have a common property or effect.

In a further aspect of the invention, surprisingly, preferred claimed compounds have a multiplicity of common physicochemical properties which result in the compounds being very useful as pharmaceutical agents since they comply to the so-called rules of five of Lipinski. correspond. These common physicochemical or structural features of the components according to the invention relate to their molecular weight, which is in the range from 150 to 600 g / mol, preferably from 190 to 300 g / mol, a distribution coefficient of logP ≦ 10, preferably ≦ 8, especially preferably ≥ 1 to ≤ 5 with a maximum of 10 hydrogen bond donors and a maximum of 10 hydrogen bridge acceptors, a solubility value of logSw of -400 to 0 and a BrotN value of 0 to 7, wherein preferably the AKAP18delta-RII interaction is at least 40% is inhibited. The sum of the structural similarities leads to the functional connection of the inhibition of the action of PKA and AKAP or to the blocking of PKA anchoring. The common physicochemical features therefore do not represent an arbitrary sum of but are, so to speak, the common fingerprint of the claimed compounds, which advantageously allows and characterizes the good in vivo suitability of the compounds. The molecules according to the invention particularly preferably bind to regulatory subunits of the PKA, in particular to RIalpha or RII-alpha or RII-alpha or R-beta. The molecules according to the invention make it possible to modify, inhibit or decouple AKAP, preferably AKAP18, more preferably AKAP18delta and PKA, depending on the species used. It will be readily apparent to those skilled in the art, by means of simple routine experimentation, to provide recognition molecules against the molecules of the invention. These may be, for example, antibodies, chelators, complexing agents or other structures known in the art. Such recognition molecules are easy to generate when their carrying is revealed - as in the present case the decouplers. For example, the decouplers can be administered together with an adjuvant into organisms, thereby producing antibodies that can be obtained by known methods. With the aid of these recognition molecules or with the aid of the molecules according to the invention, it is possible to provide organisms in which the AKAP-PKA interaction is tissue- and / or cell-specifically modified by bringing the organisms mentioned into contact with the molecules according to the invention or the recognition molecules.

Preferred decouplers have a logP value of <9, 8, 7, 6, 5, preferably 4, more preferably 3 and very particularly preferably <2 and at most 10, 9, 8, 7, 6, preferably 5, particularly preferably 4, 3 and / or 2 water Substance bridge donors and / or acceptors and / or a BrotN value of in particular 1, 2, 3, 4, 5, or 6 and / or a logSw value of -350, -300, -250, -200, - 150, -100 or -50 and a molecular weight of 150 to 550, from 150 to 500, from 150 to 450, from 150 to 400 or from 150 to 350.

The decouplers preferably have a maximum of 6 hydrogen-bond donors and / or a maximum of 4 hydrogen-bond acceptors and / or a distribution coefficient of logP of ≥ 1 to ≦ 5.

In a particularly preferred embodiment of the invention, the decouplers according to the invention inhibit the interaction of AKAP and PKA subunits by at least 80%. The person skilled in sufficient methods are known with which he can determine the inhibition of the interaction. There are, for example, numerous disclosures which describe how the inhibition of AKAP-PKA binding can be realized with the aid of Ht31 peptide. For the purposes of the invention, however, inhibition means any form of modification of the interaction between AKAP and PKA against unaffected interaction with these two molecules. Although it is preferred to inhibit the binding between AKAP and PKA, it may be desired according to the invention that the interaction of AKAP and PKA be increased.

Particularly preferred decouplers are those shown in Table A. These decouplers exhibit physicochemical

Properties that not only inhibit the interaction of AKAP and PKA, but that Furthermore, they can be very well absorbed in a target organism, so that it is possible to treat diseases resulting from an imbalance or disruption of cAMP-dependent signal transduction. The skilled person is aware of such diseases by the current state of the art; So the expert knows wel ^¬ che diseases are recorded and meant. Known diseases include diabetes insipidus, diabetes mellitus, obesity, edema, chronic obstructive pulmonary disease, AIDS, schizophrenia, cirrhosis, heart failure, coronary heart disease, hypertension and / or asthma. However, the diseases caused by the disruption of cAMP-dependent signal transduction are not limited to these. The other diseases mentioned below are also among the preferred diseases which can be treated very well with the agents according to the invention.

Further preferred molecules of the invention are disclosed in Table B. These preferred compounds share common physicochemical properties that result in the compounds being optionally used in conjunction with a pharmaceutically acceptable carrier for the surgical and / or therapeutic treatment of the human or animal body and diagnostic procedures performed on the human or animal body can. Advantageously, the compounds have substance properties that comply with the rules of five by Lipinski et al. (Lipinski et al., Adv. Drug Deliv. Rev. 46, 3-26, 2001). A further preferred embodiment of the invention relates to decouplers selected from Table C. These preferred compounds have such a good membrane permeability that they can very well be used in vivo, ie, in particular as a pharmaceutical agent, for PKA anchorage or to modulate the AKAP-PKA interaction, in particular to inhibit.

Advantageous molecules according to the invention are disclosed in Table A, B and / or C. Because of their physicochemical properties, such as their low molecular weight, advantageous partition coefficient and solubility and bread N value, as well as the fact that they contain no more than 10 H-bond acceptors, and in particular substantially no more than 7, preferably 6, all particularly preferably have 5 H-bridge donors, these agents are very good in prophylaxis, therapy, post-treatment and / or diagnosis in in vivo systems such. B. target organisms, preferably in humans or in experimental animals such. Primates, rats or mice. Further preferred decouplers are disclosed in Table D.

In a further preferred embodiment of the invention decoupler of the invention have the general ^¬ my formula I which can be guided into one another by resonance ^¬ (R ² and R ³ are treated as interchangeable) LV

wherein X is a non-hydrogen atom, preferably a sulfur atom, wherein R ^{1 is} an alkyl or aryl radical, preferably a 1-Naphthylmethylrest wherein R ² and R ^{3 is} a hydrogen atom or an alkyl or aryl radical, preferably R ² and R are ^{3 is} two hydrogen atoms, two methyl radicals, one benzyl radical and one methyl radical or one shortcoming radical and one tert-butyl radical, more preferably R ² and R ^{3 are} a 2-thiazolidinyl radical and a methyl or tert-butyl radical, and a 1-naphthyl radical and Isoproyl, cyclohexyl, benzyl or methyl radical. The invention accordingly also relates to compounds according to the general formula I for use as medicaments or as pharmaceutical active ingredients. In a further preferred embodiment, the invention also relates to the use of the compounds according to the general formula I for the treatment of the diseases disclosed in the application.

In a further preferred embodiment of the invention düng, the decouplers of the invention have the general formula II, wherein Rl to R3 have the meaning as above:

Rotation around single bond

The ratio 1/5 depends on the basicity of the nitrogen next to R ³ . Advantageously, from the structure 2, the arrangement of the radicals R ² and R ¹ exchangeable. Particularly preferred are the protonated compounds, such as. Hydrochloride, for use as a decoupler of the AKAE-PKA interaction. For compounds with several basic see centers such. For example, the compound JG5 (see table) is particularly preferred the dihydrochloride. Preferably, the lead structure is 990 (see table).

The IC values decrease as the aliphatic residues become larger and, as with JG31 (see Table), the anthracene has additional effects. This means a higher electron thrust to amidine and a higher basicity. For certain applications, the electron density on the amidine is important, so that the radicals R ¹ and R ^{2 are} electron donating units (see compounds 6 and 7; ortho- or para-methoxyphenyl, ortho- or para-diaminophenyl).

Also advantageous may be certain degradation products of the decouplers according to the invention. The thiocarbamimic acid esters (such as # 990) are labile to nucleophiles such as e.g. B. Amines. This produces thiols and guanidines, which can also be used for example for the treatment of diseases. Accordingly, in a preferred embodiment, the invention relates to the cyclic imidazoles (6) and the dihydroimidazoles (7) for use in medicine, in particular as decouplers of AECAP and PKA, and as tools in basic research in vivo and in vitro. The radicals R ¹ and R ² are preferably aryl radicals or cyclic aliphatic radicals (SM 61, SM63 and SM65). The 6 ring on the amidine is also preferred (compound 8 is based on SM71).

8th

It is also advantageous to separate the amidine from the sulfur and attach it to the 2-position of naphthalene (compound 9). In an advantageous embodiment, the distance N / S remains almost the same. Preferably, R ¹ contains SR so that the electron density on the amidine is comparable to the active compounds. Preferably, R is an alkyl radical. Accordingly, the isomer 10 is also preferred.

10

Accordingly, decouplers according to the general formula 11 and 12 are preferred.

Claimed are the compounds themselves, as well as their HCl salts. In preferred embodiments, R ¹ and R ^{2 may be} independent of each other:

acyclic aliphatic with a chain length of Cl to Cβ; cyclic aliphatic having a ring size of C3 to C9, including one or more of a plurality of heteroatoms of the type O or N; aromatic as a monocyclic, heteroaryl and mono- to trisubstituted monocycle radical. Reference has been made above to the electron-donating substituents (eg OMe or NMe2) The use of heteroaromatics is particularly advantageous with regard to bioavailability It is furthermore advantageous to use the structure 12 with X as a linker group of the acyclic aliphatic type a chain length of Cl to C6 and R ³ with the same groups as for R ¹ and R ² independently formulated.

Concerning the chemistry of said compounds, it is stated that the sulfur and adjacent position 10 may be sensitive to oxidation in certain embodiments of the invention. In the event that only the sulfur is oxidized, it is advantageous that the active form does not have the framework of the lead structure (see above), but is either the sulfoxide 2 or the sulfone 3. In addition, the position 10 may be acidic after oxidation of the sulfur and may be after deprotonation z. B. undergo a Claisen aldol addition. The latter reaction is exemplified by an acetic acid building block as compound 16 (see below). If R ^{2 is} a hydrogen atom, an intramolecular ring closure also occurs.

Advantageously, the two positions (sulfur and No. 10) are very reactive.

Furthermore, it is preferred that, for the effectiveness of structure 11, position 10 is not modified. As already stated, these azides can be sensitive to oxidation, as explained below. This position can advantageously without significant change of Raumerfüllung be provided with fluorine atoms, which prevent diversification under physiological conditions and receive the activity advantageously. In particular, the structure 16 is thus protected against premature loss.

As a preferred embodiment for secondary products from the oxidation of the 10-position (oxidation gives compound 18), the addition product of an acetyl unit is depicted by way of example as structure 8 after elimination of water in 20 (see below). Advantageously, the position 10 can also be substituted. This relates in particular to the radicals R ³ and R ⁴ as shown in connection 21, which may preferably also be F as an extension to R ¹ and R ² . The above to the spacer between N and R ¹ / R ² and the oxidation of the sulfur also applies to the compounds listed below.

18 19 20

21

R ¹ , R ² , R ³ and R ⁴ may independently, for example, be alkyl and / or aryl radicals.

Preference is thus given to the use of the 1-substituted naphthalene. In the lipophilic pockets of a target molecule, however, their affinity for a lipophilic side group, such as an aromatic bicyclic, can make an important contribution to the acceptance of the substrate. Accordingly, preferred are compounds having the general formula according to compounds 22, 23 and 24, which advantageously have an equivalent affinity.

22 23 24, R = H or F

Such bicyclic compounds according to the compounds 22 to 24 have in advantageous embodiments X = oxygen, but also N, NH or S. Triple-anilated ring systems may also be preferred (see also compounds SM39 and SM44, Table).

In a further preferred embodiment, guanidines are preferred when R ¹ or R ^{2 is} a cyanide (example compound 25).

25

Preference is furthermore given to a sulfur amidine which may have different stabilities in vitro and in vivo (lead structure according to compound 11). In the case of oral administration, the compounds according to the invention have a solubility in water of 1 mg / ml, preferably at a pH of 2 to 7. In the case of an injection with a daily At a dose of 200 to 400 mg, the water solubility is even higher, to avoid injecting 400 ml solution with a syringe.

Preference is therefore also given to a compound in which the naphthalene is replaced by quinoline or isoquinoline, which can be administered, for example, as HCl salt, in particular in order to increase the water solubility.

Quinoline isoquinoline

Accordingly, a decoupler with the general formula III, d. H. according to the structure 1 'and the formula 2' in equilibrium with it

When 2 ', 2 "is rotated around the single bond SC and compared with 1', the double bond and the hydrogen come to coincide, while R2 comes to R3 (and vice versa). Further preferred decouplers have a general structure according to FIG. 19 and / or FIG. 20. The preferred decouplers have many surprising advantages over the peptidic inhibitors or decouplers known in the art. First, the non-peptidic decouplers according to the invention represent ei ^¬ ne shift away from conventional technologies, which follows a new task. With the structures according to the invention a long-unresolved, urgent need is solved, which the experts have hitherto attempted in vain. In particular, the simplicity of the solution speaks in favor of an inventive step as it replaces the more complicated teachings of the prior art. The development of the scientific technique with respect to the treatment of the above and below diseases has taken a different direction, so that the teaching according to the invention represents a development-firming achievement, which eliminates misconceptions of the experts over the solution of the corresponding problem. The technical advancement, which is realized by the teaching according to the invention, manifests itself in particular through an improvement, increase in performance, cheapening, savings in time, material, work stages, costs or difficult-to-obtain raw materials, increased reliability, elimination of errors, quality enhancement, Freedom from maintenance, greater effectiveness, higher yield, increase of technical possibilities, provision of further means, opening of a further way to the treatment of the diseases, opening of a new area, first solution of a task, provision of reserve resources, alternatives, the possibility of rationalization, automation resp Miniaturization and enrichment of the drug. Accordingly, the teachings of the present invention represent a fortunate handle in which, of the multitude of possibilities, a particular one has been selected whose result could not be predicted. It is in the teaching of the invention is a very young field of technology, which is praised by the art and leads to economic success or licensing. In particular, the preferred molecules according to the invention can be used for the treatment of diseases which are caused by a disorder or by a defect of cAMP-dependent signal transduction. This characterization of the disease is not intended to functionally define the ailments to be treated, but the presentation as diseases associated with a modification or defect of compartmentalized cAMP-dependent signal transduction serves as a generic term for a well-defined group of diseases in the sense of the invention. That is, the person skilled in the art can judge which diseases are covered by the definition according to the preamble and thus fall under the claims of the teaching according to the invention of the claim. By naming the individual concrete diseases, which fall under the generic term, no difficultly manageable number of therapeutic options for which no tests are presented should be claimed, but merely clarification and the concrete definition of the claimed diseases a disturbance of the above-mentioned signal transduction caused. By means of the molecules according to the invention, the abovementioned AKAP proteins can be influenced in their interaction with PKA molecules. In particular, the o. G. AKAP18 proteins and their interaction with PKA molecules are modified, and in particular A-KAPl8delta proteins and their interaction with PKA molecules, in particular with subunits and particularly preferably with Rllalpha and / or RIIbeta molecules. That is, it can, for example, the interaction of AKAP79, Gravin, AKAP 82 or other AKAPs mentioned above, but especially of AKAP18 as AKAP18alpha, -beta, -gamma, -delta, but more preferably modulated by AKAP18delta in their interaction with PKA , in particular be inhibited. It is preferred that the decouplers inhibit substantially 100%, it is also preferred to inhibit 90%, 80%, 70%, 60%, 50%, 40%, preferably 30%, particularly preferably 20% and in particular 10%. Any of these percentage inhibitions may be preferred.

The decouplers of the invention are on the one hand claimed as new molecules; on the other hand, preferred molecules are claimed as compounds for which a use in medicine is first disclosed.

In a further preferred embodiment of the invention, the novel molecules, in particular the new molecules in the field of medicine, are claimed for the treatment of diseases which according to the definition of the invention are included under the term of the defects associated with the compartmentalized cAMP-dependent signal transduction Illnesses fall.

In a particularly preferred embodiment, these are diseases selected from the group consisting of diabetes insipidus, diabetes mellitus, obesity, edema, chro- obstructive pulmonary disease, AIDS, schizophrenia, cirrhosis, heart failure, coronary heart disease, hypertension, duodenal ulcer and / or asthma.

The compounds according to Table B, preferably of Table C or D, are new molecules which have not been described in the prior art. For the further disclosed compounds (preferably according to Table A) it has not yet been disclosed that they can be used in the field of therapy or diagnostic methods. However, these connections can be regarded as completely new connections even if their novelty is given by the fact that they were admitted in an archive or library, but the public did not learn about their existence for lack of cataloging (in particular, Tables A and B) ).

The invention also relates to recognition molecules which are directed against the non-peptidic molecules according to the invention. Because of the disclosure of the non-peptide molecules of the present invention, one of ordinary skill in the art can readily, without undue burden, provide recognition molecules against the non-peptidic molecules of the present invention by routine experimentation so that the recognition molecules are clearly and completely disclosed. The recognition molecules according to the invention are either antibodies, complexing agents or chelators or peptides which interact with the non-peptidic decouplers such that they are impaired in their biological activity, the modulation of the AKAP-PKA interaction , By means of the recognition molecules, it is also possible to use the decouplers in an in vitro or in vivo system. For this purpose, it may be advantageous if the recognition molecules are provided with a detectable probe. The skilled person is aware of such probes.

The invention also cells, cell aggregates, tissue cultures or tissue pieces, but also organisms ^¬ men, such as mice, rats, cattle, horses, donkeys, sheep, camels, goats, pigs, rabbits, guinea pigs, hamsters, cats, monkeys , Dogs or humans comprising the decouplers of the invention and / or the recognition molecules of the invention. By means of these cells, tissue cultures or organisms, various diseases can be investigated, whereby the examination of the diseases relates, for example, to their causes or to possible methods of diagnosis and treatment. Preferred diseases are asthma, hypertension, hypertrophy of the heart, coronary heart disease, duodenal ulcer, heart failure, liver cirrhosis, schizophrenia, AIDS, diabetes mellitus, diabetes insipidus, obesity, chronic obstructive pulmonary disease, learning difficulties, edema (pathological water retention), infectious diseases and / or cancer , Of course, it may also be preferred to use an organism for the study of these diseases, which does not have the recognition molecules according to the invention, but the decouplers according to the invention or both structures at the same time. On the basis of the disclosure of the teaching according to the invention, the person skilled in the art knows which decouplers or which recognition molecules he has to use in which concentration, since these are directly and unambiguously determined by routine experiments from the disclosed inventive technology. see doctrine as well as from the prior art, as set forth, for example, in reference works. The organisms can be used to develop drugs that modify, preferably decouple, the PKA-AKAP interaction. Of course, the decouplers according to the invention may of course be used as test molecules for pharmaceuticals, but also as lead structures from which the pharmaceuticals are developed. The organisms can also be used in vivo to study metabolic processes in which the AKAP-PKA interaction plays a role or in which it should be clarified whether, for a particular event, such as a specific pathogenic change such as, for example, Degeneration of cells involved in AKAP-PKA interaction. The decouplers or recognition molecules used according to the invention may also be modified decouplers or recognition molecules which have been obtained by combinatorial methods from the compounds according to the invention. The structures thus obtained, in which the molecules according to the invention serve as a guide structure, are essentially functionally analogous to the abovementioned decouplers and recognition molecules according to the invention. Functional analogy means that the homoge- neous structures obtained also allow conclusions to be drawn regarding the interaction of AKAP and PKA or their significance for certain diseases. Accordingly, functionally analogous molecules in the sense of the invention are molecules which the person skilled in the art can identify as having essentially the same effect. The invention accordingly also relates to modified molecules which have substantially the same function in substantially the same way and produce substantially the same result as the invention Decouplers or recognition molecules, or equivalent compounds, in which it will be apparent to one of ordinary skill in the art that the same can be achieved with them, which is achieved with the molecules disclosed in the claims. These functionally analogous structures are thus obtained by using the decouplers or recognition molecules according to the invention as lead structures. The functional analogues can be obtained by structure-based, combinatorial or another drug design. The term drug design is clearly defined by those skilled in the art; he refers z. B. on the reference drug design. The path to the drug, or the textbook of clinical pharmacy or standard works.

Accordingly, the invention also relates to a pharmaceutical composition which comprises a decoupler according to the invention or a recognition molecule directed against it in the form of a chelator, complexing agent or antibody, if appropriate together with a pharmaceutically acceptable carrier and / or excipients. The adjuvants may be, for example, adjuvants, vehicles or others. The carriers may be, for example, fillers, extenders, binders, humectants, disintegrants, dissolution inhibitors, absorption accelerators, wetting agents, adsorbents and / or lubricants. In this case, ie if carriers, adjuvants and / or vehicles - such. B. Liposomes - present together with the decouplers according to the invention or their recognition molecules, these are referred to as drugs or as a pharmaceutical agent. In a further preferred embodiment of the invention, the agent according to the invention is used as gel, powder, powder, tablet, sustained-release tablet, premix, emulsion, pour-on formulation, drops, concentrate, granules, syrup, pellet, fluid, capsule, aerosol, Spray and / or inhalant prepared and / or used in this form. The tablets, dragees, capsules, pills and granules may be provided with the usual coatings and sheaths which optionally contain opacifying agents and may also be composed in such a way that they release the active substance only or preferably delayed in a specific part of the intestinal tract , wherein as embedding masses, for example, polymer substances and waxes can be used.

For example, the pharmaceutical compositions of this invention may be used for oral administration in any orally acceptable dosage form including, but not limited to, capsules, tablets, and aqueous suspensions and solutions. In the case of tablets for oral use, carriers that are commonly used include lactose and corn starch. Lubricants, such as magnesium stearate, can typically be added. For oral administration in capsule form, useful diluents such as lactose and dried corn starch are used. When aqueous suspensions are administered orally, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening and / or flavoring and / or coloring agents may be added. The active substance (s) may optionally also be present in microencapsulated form with one or more of the excipients specified above.

Suppositories can fe of these agents in addition to the active substance contain the customary water-soluble or water-insoluble excipients, for example polyethylene glycols, fats, for example cocoa fat and higher esters (for example _Ci-C4 alcohol with C ₆ fatty acid) 'or mixtures thereof.

Ointments, pastes, creams and gels may contain the customary excipients in addition to the active substance (s), for example animal and vegetable fats, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide or mixtures of these substances.

Powders and sprays may contain the customary carriers in addition to the active substance (s), for example lactose, talc, silicic acid, aluminum hydroxide, calcium silicate and polyamide powder or mixtures of these substances. Sprays may additionally contain the usual propellants, for example chlorofluorohydrocarbons.

Solutions and emulsions may, in addition to the active compounds, ie the compounds according to the invention, the customary carriers such as solvents, solubilizers and emulsifiers, for example water, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, Propylene glycol, 1,3-butylene glycol, dimethylformamide, oils, in particular cottonseed oil, soil nut oil, maize germ oil, olive oil, castor oil and sesame oil, glycerol, glycerol formal, tetrahydofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan or mixtures of these substances. For parenteral administration, the solutions and emulsions may also be in sterile and blood isotonic form.

Suspensions may contain ^{, in} addition to the active ingredients, the customary carriers such as liquid diluents, for example water, ethyl alcohol, propylene glycol, suspending agents, for example ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar Agar and tragacanth or mixtures of these substances.

The pharmaceutical compositions may be in the form of a lyophilized sterile injectable preparation, for example as a sterile injectable aqueous or oily suspension. This suspension may also be formulated by methods known in the art using suitable dispersing or wetting agents (such as Tween 80) and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example, a solution in 1,3-butanediol. Compatible vehicles and solvents that may be used include mannitol, water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile non-volatile oils are commonly used as the solvent or suspending medium. For this purpose, any mild non-volatile oil, including synthetic mono- the diglycerides are used. Fatty acids, such as oleic acid and its glyceride derivatives, are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated forms. These oil solutions or suspensions may also contain a long-chain alcohol or similar alcohol as a diluent or dispersant.

The formulation forms mentioned may also contain colorants, preservatives and odour- and taste-improved additives, for example peppermint oil and eucalyptus oil, and sweeteners, for example saccharin. The compounds of the invention should preferably be present in the listed pharmaceutical preparations in a concentration of about 0.01 to 99.9, preferably from about 0.05 to 99 wt .-% of the total mixture.

Apart from the compounds, the listed pharmaceutical preparations may contain further pharmaceutically active substances, but in addition to other pharmaceutical active substances also salts, buffers, vitamins, sugar derivatives, in particular saccharides, enzymes, plant extracts and others. The buffers and sugar derivatives advantageously reduce pain on subcutaneous administration, and enzymes such as hyaluronidase increase efficacy. The preparation of the abovementioned pharmaceutical preparations is carried out in the customary manner by known methods, for example by mixing the active substance (s) with the excipient (s). The preparations mentioned can be used in humans and animals either orally, rectally, parenterally (intravenously, intramuscularly, subcutaneously), intracisternally, intravaginally, intraperitoneally, locally (powder, ointment, drops) and for the therapy of the diseases mentioned below. Suitable preparations are injection solutions, solutions and suspensions for oral therapy, gels, infusion formulations, emulsions, ointments or drops. For local therapy, ophthalmic and dermatological formulations, silver and other salts, ear drops, eye ointments, powders or solutions may be used. In animals, uptake can also take place via the feed or drinking water in suitable formulations. Furthermore, the drugs can be incorporated into other carrier materials such as plastics - plastic chains for local therapy -, collagen or bone cement.

In a further preferred embodiment of the invention, the compounds are introduced in a concentration of from 0.1 to 99.5, preferably from 0.5 to 95, particularly preferably from 20 to 80,% by weight in a pharmaceutical preparation. That is, the compounds are present in the pharmaceutical compositions listed above, for example, tablets, pills, granules and others, preferably in a concentration of 0.1 to 99.5% by weight of the total mixture. The amount of active ingredient, that is, the amount of compounds of the present invention combined with the carrier materials to produce a single dosage form will vary depending on the subject to be treated and the particular mode of administration. After In view of the condition of the patient, the proportion of the active compound in the preparation may be changed so as to provide a maintenance dose that arrests the disease. Thereafter, the dose or frequency of administration, or both as a function of the symptoms, may be reduced to a level at which the improved condition is maintained. When the symptoms have been alleviated to the desired level, treatment should cease. However, patients may require long-term intermittent treatment after any recurrence of disease symptoms. Accordingly, the proportion of the compounds, that is to say their concentration, in the overall mixture of the pharmaceutical preparation, as well as their composition or combination, is variable and can be modified and adapted by a person skilled in the art on the basis of his specialist knowledge.

It is known to the person skilled in the art that the compounds according to the invention can be brought into contact with an organism, preferably a human or an animal, in various ways. Furthermore, it is known to the person skilled in the art that, in particular, the pharmaceutical agents can be administered in various dosages. The application should be carried out in such a way that the disease is combated as effectively as possible or the onset of a disease in a prophylactic administration is prevented. The concentration and the type of application can be determined by the skilled person through routine experimentation. Preferred applications of the compounds according to the invention are oral administration in the form of powders, tablets, juice, drops, capsules or the like, rectal administration in the form of suppositories, solutions and the like, parenterally in the form of injections, infusions and solutions, and locally in the form of ointments, patches, envelopes, rinses and the like. The contacting of the compounds according to the invention preferably takes place prophylactically or therapeutically.

The suitability of the chosen forms of application as well as the dose, the application regimen, the adjuvant choice and the like can be determined, for example, by taking serum aliquots from the patient, ie human or animal, and testing for the presence of disease indicators during the course of the treatment protocol become. Alternatively and concomitantly, the state of the kidneys, the liver or the like, but also the amount of T cells or other cells of the immune system or other markers that characterize or document a course of disease or recovery, can be determined concomitantly in a conventional manner Overall overview of the immunological constitution of the patient and in particular the constitution of metabolically important organs to obtain. In addition, the patient's clinical condition can be monitored for the desired effect. If inadequate therapeutic efficacy is achieved, the patient may be further treated with agents of the invention modified with other known drugs of which an improvement in overall constitution can be expected. Of course, it is also possible to modify the carriers or vehicles of the pharmaceutical agent or to vary the route of administration. In addition to the oral intake, it may then be provided, for example, that injections, for example intramuscularly or subcutaneously or into the blood vessels, are a further preferred route for the therapeutic administration of the compounds according to the invention. At the same time, the delivery via catheters or surgical tubes can be used; For example, via catheters that lead directly to specific organs such as the kidneys, liver, spleen, intestine, lungs, etc.

The compounds according to the invention can be used in a preferred embodiment in a total amount of preferably 0.05 to 500 mg / kg of body weight per 24 hours, preferably from 5 to 100 mg / kg of body weight. This is advantageously a therapeutic amount used to prevent or ameliorate the symptoms of a disorder or responsive pathological physiological condition.

Of course, the dose will depend on the age, the health and weight of the recipient, the degree of the disease, the nature of a necessary concomitant treatment, the frequency of treatment and the nature of the effects desired and the side effects. The daily dose of from 0.005 to 500 mg / kg, preferably from 0.05 to 500 mg / kg of body weight may be applied once or several times to obtain the desired results. Typically, in particular, pharmaceutical agents are used for about 1 to 10 times daily administration or alternatively or additionally as a continuous infusion. Such administrations can be used as a chronic or acute therapy. The Amounts of active ingredient combined with the carrier materials to produce a single dosage form may, of course, vary depending on the host to be treated and the particular mode of administration. It is preferred to divide the daily dose into 2 to 5 applications, with each Application, for example, 1 to 2 tablets are administered with an active ingredient content of 0.05 to 500mg / kg body weight. Of course it is possible to choose the active ingredient content also higher, for example up to a concentration of up to 5000 mg / kg. The tablets may also be retarded, reducing the number of applications per day to 1 to 3. The active ingredient content of the delayed-release tablets can be 3 to 3000 mg. As stated, if the active ingredient is administered by injection, it is preferred to contact the host with the compounds of the invention 1 to 10 times a day or by continuous infusion, with amounts of 1 to 4000 mg per day being preferred. The preferred total amounts per day have proven to be advantageous in human medicine and in veterinary medicine. It may be necessary to deviate from the stated dosages, depending on the type and body weight of the host to be treated, the nature and severity of the disease, the method of preparation of the drug and the period or interval within which the administration takes place. Thus, in some cases it may be preferable to bring the organism into contact with less than the stated amounts, while in other cases the stated amount of active substance must be exceeded. The definition of the respectively required optimal dosages and the The type of application of the active ingredients can be carried out by a person skilled in the art on the basis of his specialist knowledge.

In a further particularly preferred embodiment of the invention, the pharmaceutical agent is used in a single dose of 1 to 100, in particular from 2 to 50 mg / kg of body weight. As well as the total amount per day (see above), the amount of the single dose per application can also be varied by the person skilled in the art on the basis of his specialist knowledge. The compounds used according to the invention can also be used in veterinary medicine in the individual concentrations and preparations mentioned together with the feed or with feed preparations or with the drinking water. A single dose preferably contains the amount of active ingredient administered in one application, which usually corresponds to a whole, a half daily dose or a third or a quarter of a daily dose. The dosage units may accordingly preferably contain 1, 2, 3 or 4 or more single doses or 0.5, 0.3 or 0.25 of a single dose. The daily dose of the compounds according to the invention is preferably distributed over 2 to 10 applications, preferably 2 to 7, more preferably 3 to 5 applications. Of course, a continuous infusion of the inventive means is possible.

In a particularly preferred embodiment of the invention, 1 to 2 tablets are given for each oral application of the compounds according to the invention. The tablets according to the invention may be provided with coatings and casings known to the person skilled in the art and may also be composed in such a way that they contain only the active substance (s) in preferred, release in a particular part of the host.

In a further embodiment of the invention, it is preferred that the individual constituents of the compounds are optionally associated with one another or bound to a carrier in liposomes, wherein the inclusion in liposomes in the sense of the invention need not necessarily mean that the compounds are inside the liposomes available. An inclusion in the sense of the invention may also mean that the compounds are associated with the membrane of the liposomes, for example, so that they are anchored on the outer membrane. Such a representation of the compounds according to the invention in or on the liposomes is advantageous if the person skilled in the art selects the liposomes in such a way that they have an immunostimulating action. The expert is known from DE 198 51 2 82 different ways to modify the immune stimulating effect of liposomes. The lipids may be simple lipids such as esters and amides or complex lipids such as glycolipids such as cerebrosides or ganglionides, sphingolipids or phospholipids.

Preferred diseases that can be treated with the agent according to the invention are selected from the group comprising AIDS, acne, albuminuria (proteinuria), alcohol withdrawal syndrome, allergies, alopecia (hair loss), ALS (amyotrophic lateral sclerosis), Alzheimer's disease, AMD (age-related macular degeneration) , Anemia, anxiety disorders, anthrax (anthrax), aortic sclerosis, arterial occlusive disease, arteriosclerosis, arterial Conclusion, Temporal Arteritis, Atherosclerosis, Arteriovenous Fistulas, Arthritis, Osteoarthritis, Asthma, Respiratory Insufficiency, Autoimmune Disease, AV Block, Acidosis, Herniated Disc, Peritonitis, Pancreatic Cancer, Becker Muscular Dystrophy, Benign Prostate Hyperplasia (BPH), Bladder Carcinoma, Hemorrhage (Haemophilia) , Bronchial carcinoma, breast cancer, BSE, Budd-Chiari syndrome, bulimia nervosa, bursitis (bursitis), Byler syndrome, bypass, chlamydia infection, chronic pain, cirrhosis, cerebral commu- ni (concussion), Creutzfeld-Jakob disease, Colon carcinoma, Colon cancer, Intestinal tuberculosis, Depression, Diabetes insipidus, Diabetes mellitus, Juvenile diabetes mellitus, Diabetic retinopathy, Duchenne muscular dystrophy, Duodenal carcinoma, Dystrophia musculorum progressiva, Dystrophy, Ebola, Eczema, Erectile dysfunction, Obesity, Fibrosis, Cervical cancer, Cervical cancer, cerebral hemorrhage, encephalitis, hair loss, hemisphere paralysis, hemolytic anemia, haemophilia, pet allergy (animal hair allergy), skin cancer, herpes zoster, myocardial infarction, heart failure, heart valve inflammation, brain metastasis, stroke, brain tumor, testicular cancer, ischaemia, Kahler's disease (plasmocytoma), polio (poliomyelitis), bone loss, contact dermatitis , Paralysis, liver cirrhosis, leukemia, pulmonary fibrosis, lung cancer, pulmonary edema, lymph node cancer (Hodgkin's disease), lymphogranulomatosis, lymphoma, lyssa, gastric carcinoma, breast carcinoma, meningitis, anthrax, cystic fibrosis, multiple sclerosis (MS), myocardial infarction, atopic dermatitis, neurofibrosis - matose, neural tumors, kidney cancer (renal cell carcinoma), osteoporosis, pancreatic carcinoma, pneumonia, polyneuropathies, potency disorders, progressive systemic skiing rose (PSS), prostate cancer, urticaria, cross-sectional syndrome, traumatic, rectal cancer, pleurisy, traumatic brain injury, vaginal carcinoma, sinusitis, esophageal cancer, tremor, tuberculosis, tumor pain, vaginal carcinoma, burn, scald, poisoning , Viral meningitis, menopause, soft tissue sarcoma, soft tissue tumor, cerebral circulatory disorders and / or CNS tumors.

In a further preferred embodiment, the pharmaceutical compositions according to the invention can also be used for the treatment of cancers selected from the group of cancers or neoplasms of the ear, nose and throat, the lung, the mediastinum, the gastrointestinal tract, the genitourinary system , the gynecological system, the breast, the endocrine system, the skin, bone and soft tissue sarcomas, mesotheliomas, melanomas, neoplasms of the central nervous system, cancers or childhood tumors, lymphomas, leukemias, paraneoplastic syndromes, metastases without a known primary tumor (CUP syndrome ), peritoneal carcinomatosis, immunosuppression-related malignancies and / or tumor metastases. In particular, the tumors may be the following cancers: adenocarcinoma of the breast, prostate and colon; all forms of lung cancer emanating from the bronchi; Bone marrow cancer; the melanoma; the hepatoma; neuroblastoma; the papilloma; the Apudo, the Choristom; the branchioma; the malignant carcinoid syndrome; carcinoid heart disease; carcinoma (for example, Walker's carcinoma, basal cell carcinoma, basosquamous carcinoma, Brown-Pearce's carcinoma, ductal carcinoma, Ehrlich- Tumor, in situ carcinoma, cancer 2 carcinoma, Merkel cell carcinoma, mucous carcinoma, non-small cell lung carcinoma, oat cell carcinoma, papillary carcinoma, cirrhotic carcinoma, bronchiolo-alveolar carcinoma, bronchial carcinoma, squamous cell carcinoma and transiting nacellar carcinoma); histiocytic dysfunction; Leukemia (for example in connection with B-cell leukemia, mixed cell leukemia, null cell leukemia, T-cell leukemia, chronic T-cell leukemia, HTLV-II-associated leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, mast cell leukemia and myeloid leukemia); malignant histiocytosis, Hodgkin's disease, non-Hodgkin's lymphoma, solitary plasma cell tumor; Reticuloendotheliosis, chondroblastoma; Chondroma, chondrosarcoma; fibroma; fibrosarcoma; Giant cell tumors; histiocytoma; lipoma; liposarcoma; Leukosarkom; mesothelioma; myxoma; myxosarcoma; osteoma; osteosarcoma; Ewing's sarcoma; Synovioma; Adenofribrom; Adenolymphom; carcinosarcoma; chordoma; craniopharyngioma; dysgerminoma; hamartoma; Mesenchymal; Mesonephrom; myosarcoma; ameloblastoma; Cementom; O-dontom; teratoma; thymoma; Chorio-blastoma; adenocarcinoma; adenoma; cholangiomas; cholesteatoma; cylindroma; Cystadenocarcinoma; Cystadenom; Granulosa cell tumor; Gyne- droblblastoma; Hidradenoma; Islet cell tumor; Leydig cell tumor; papilloma; Sertoli cell tumor; Theca-tumor; leiomyoma; leiomyosarcoma; myoblastoma; fibroid; myosarcoma; rhabdomyoma; rhabdomyosarcoma; Ependynom; ganglioneuroma; glioma; medulloblastoma; meningioma; neurilemmoma; neuroblastoma; neuroepithelioma; neurofibroma; neuroma; Parangaloma; non-chromaffin paraganglioma; Angiokeratoma; angiolymphoid hyperplasia with eosinophilia; sclerosing angioma; angiomatosis; Glomus Tumor; Hemangioendothe- liom; hemangioma; Hemangiopericytoma, hemangiosarcoma; Lymphangioma, lymphangio-myoma, lymphangiosarcoma; pinealoma; Cystosarcoma phyllodes; Hemangiosarkom; lymphangiosarcoma; myxosarcoma; ovarian cancer; Sarcoma (for example Ewing sarcoma, experimental, Kaposi's sarcoma and mast cell sarcoma); Neoplasms (for example, bone neoplasms, breast neoplasms, neoplasms of the digestive system, colo-rectal neoplasms, liver neoplasms, pancreatic neoplasms, pituitary neoplasms, testicular neoplasms, orbital neoplasms, neoplasms of the head and neck, Central nervous system, neoplasms of the auditory organ, pelvis, respiratory tract and the orrogenital tract); Neurofibromatosis and cervical squamous epithelial dysplasia.

In a further preferred embodiment, the cancer or tumor being treated or prevented is selected from the group: tumors of the ear, nose and throat, including tumors of the internal nose, sinuses, nasopharynx, lips, and the like Oral cavity, oropharynx, larynx, hypopharynx, ear, salivary glands and paragangliomas, tumors of the lung comprising non-small cell lung carcinomas, small cell lung carcinomas, tumors of the mediastinum, tumors of the gastrointestinal tract, tumors of the esophagus, stomach, of the pancreas, liver, gall bladder and biliary tract, small intestine, colon and rectal carcinomas and anal carcinomas, genitourinary tumors including kidney, ureter, bladder, prostate, urethra, penile and thoracic tumors, gynecological tumors including tumors of the cervix, vagina, vulva, corpus carcinoma, malignant trophoblast disease, ovarian carcinoma, Tumors of the Fallopian tubes, tumors of the abdominal cavity, breast cancers, endocrine tumors including tumors of the thyroid, parathyroid, adrenal, endocrine pancreatic tumors, carcinoid tumors and carcinoid syndrome, multiple endocrine neoplasms, bone and soft tissue sarcomas, mesotheliomas, skin tumors, melanomas comprising cutaneous and intraocular melanomas, central nervous system tumors, childhood tumors comprising retinoblastoma, Wilms tumor, neurofibromatosis, neuroblastoma, Ewing's sarcoma tumor family, rhabdomyosarcoma, lymphomas comprising non-Hodgkin's lymphomas, cutaneous T-cell lymphomas, primary lymphomas of the central nervous system , Hodgkin's disease, leukemias including acute leukemias, chronic myeloid and lymphatic leukemias, plasma cell neoplasms, myelodysplastic syndromes, paraneoplastic syndromes, metastases without known primary tumor (CUP syndrome), peritoneal carcinomastosis, immunosuppression-related malignancy, including AIDS-b related malignancies such as Kaposi's sarcoma, AIDS-associated lymphomas, AIDS-associated central nervous system lymphomas, AIDS-associated Hodgkin's disease and AIDS-associated anogenital tumors, transplant-related malignancies, metastatic tumors including brain metastases, lung metastases, Liver metastases, bone metastases, pleural and pericardial metastases and malignant ascites.

In a further preferred embodiment, the cancer or tumor being treated or prevented is selected from the group consisting of cancers or tumors of mammal carcinomas, gastrointestinal tumors including colon carcinomas, gastric carcinomas, pancreatic carcinomas, colonic carcinomas. cancer, small intestine cancer, ovarian cancer, cervical carcinoma, lung cancer, prostate cancer, renal cell carcinoma and / or liver metastases.

In a further preferred embodiment of the invention, the disease is selected from the group comprising diseases which are referred to in the context of the invention as infectious diseases associated with a modulation of compartmentalized cAMP-dependent signal transduction, namely: monkey pox, AIDS, anthrax (Bacillus anthracis , Anthrax), avian influenza (avian influenza, bird flu), Lyme disease, Borrelia recurrentis (louse fever), botulism (Clostridium botulinum), brucellosis, Campylobacter infections, chlamydiosis, cholera (Vibrio cholerae), Creutzfeldt-Jakob disease, Coxiella burnetii (Q Fever), Cryptosporidium parvuum (cryptosporidiosis), dengue fever, diphtheria, Ebola virus infections, echinococcosis (fox tapeworm, dog tapeworm), EHEC infections (STEC infections, VTEC infections), enteroviruses, typhus fever (Rickettsia prowazeckii), Francisella tularensis (tularemia), early summer meningoencephalitis (TBE), yellow fi eczema, giardiasis, gonorrhea, flu (influenza), haemophilis influenzae, hantavirus, helicobacter pylori, hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E, herpes, HUS (hemolytic uremic syndrome), keratoconjunctivitis epidemica, whooping cough ( pertussis), polio (poliomyelitis), lice infestation, scabies mites, Crimean-Congo hemorrhagic fever, Lassa fever, food-related diseases, legionellosis, leishmaniasis, leprosy, Leptospi ^¬ rose, listeriosis, Lyme disease, lymphogranuloma Vene- reum, malaria (Plasmodien- Infections), Marburg virus Infections, measles, meliodosis, meningococcal disease, MRSA (staphylococci), mumps, mycoses (fungal infections), new and increased infectious diseases, noroviruses, ornithosis (parrots), papillomaviruses, paratyphoid fever, plague (Yersinia pestis), pneumococcal infections (Streptococcus pneumoniae), smallpox, rice-associated infectious diseases, human cattle tapeworm infection, rotaviruses, rubella, RSV infections, salmonellosis, scarlet fever, severe acute respiratory syndrome (SARS), sexually transmitted infections, shigellosis, syphilis, tetanus, rabies, toxoplasmosis, Thrichinellosis, tuberculosis, typhus, varicella (chicken pox), variant Creutzfeldt-Jakob disease, viral haemorrhagic fever, West Nile fever, yersiniosis and / or tick-borne diseases. It is not mandatory that agents according to the invention inhibit the enzymes of these pathogens, in particular the phosphatase. The agents may also be membrane destabilizing or otherwise acting. For the purposes of the invention, it is preferably essential that the pathogenicity of the pathogens is reduced.

In a further preferred embodiment of the invention, the disease to be treated is essentially triggered by bacteria or co-triggered by bacteria; the bacteria may be Legionella, Streptococci, Staphylococci, Klebsiella, Haemophilis influenzae, Rickettsia (spotted fever), Mycobacteria, Mycoplasmas, Ureaplasmas, Neisseria (Meningitis, Waterfridge-Friedrichsen syndrome, Gonorrhea), Pseudomonas, Bordetella (pertussis) , Corynobacteria (diphtheria), chlamydia, Campylobacter (diarrhea), Escherichia coli, pro- teus, Salmonella, Shigella, Yersinia, Vibrio, Dethococci, Clostridia, Listeria, Borrelia, Treponema pallidum, Brucella, Francisellen and / or Leptospira.

The invention also relates to the use of the decoupler for specific binding to AKAP, preferably AKAP18, particularly preferably AKAP18delta and / or specific binding to PKA, preferably subunits thereof, very particularly preferably to RII subunits.

The invention also relates to the inhibition of the interaction of RIalpha, RIIalpha, RIbeta and / or RIIbeta subunits of PKA with AKAP, wherein inhibition in the sense of the invention is any form of modification.

In a particularly preferred embodiment of the invention, the decouplers can be used as aquaretics, contraceptives, as an anti-infective agent, as an anxiolytic agent and / or as an anti-tumor agent.

In a further advantageous embodiment, the diseases are selected from the group comprising asthma of any kind, etiology or pathogenesis, or asthma from the group of atopic asthma, non-atopic asthma, allergic asthma, IgE-induced atopic asthma, bronchial asthma, essential asthma, primary asthma, by pathophysiological Disorders induced endogenous asthma, environmental factors, exogenous asthma, essential asthma of unknown or inapparent cause, non-atopic asthma, bronchitis, emphysematous asthma, stress-induced asthma ma, occupational asthma, infectious allergic asthma caused by bacterial, fungal, protozoan or viral infections, non-allergic asthma, inhaled asthma, wheezy infant syndrome; chronic or acute bronchoconstriction, chronic bronchitis, small airway obstruction, and emphysema; obstructive or inflammatory respiratory disease of any kind, etiology or pathogenesis, or an obstructive or inflammatory respiratory disease from the group of asthma; Pneumoconiosis, chronic eosinophilic pneumonia; chronic obstructive pulmonary disease (COPD); COPD, including chronic bronchitis, pulmonary emphysema or associated respiratory distress, COPD characterized by irreversible progressive airway obstruction, adult respiratory distress syndrome (ARDS) and exacerbation of respiratory hypersensitivity due to treatment with other drugs; Pneumoconiosis of any kind, etiology or pathogenesis, or the avian pneumonia from the group aluminosis or aluminum pneumonia, anthracosis (asstosis), asbestosis or asbestos pneumonia, chalikosis or calcified pneumonia, ptilosis caused by inhalation of ostrich feather dust, siderosis caused by inhalation of iron particles, silicosis or stone dust, byssinosis or cotton dust neumoconiosis, and talc pneumoconiosis;

Bronchitis of any kind, etiology or pathogenesis, or bronchitis of the group acute bronchitis, acute jaynegotracheal bronchitis, peanut-induced bronchitis, bronchial catarrh, croupous bronchitis, bronchitis without expectoration, infectious asthma bronchitis, bronchitis chiti with sputum, staphylococcal or streptococcal bronchitis; as well as vesicular bronchitis;

Bronchiectasis of any kind, etiology or pathogenesis, or bronchiectasis from the group of cylindrical bronchiectasis, saccular bronchiectasis, spindle-shaped bronchiectasis, bronchiolar dilatation, cystic bronchiectasis, bronchiectasis without sputum, and follicular bronchiectasis; Seasonal allergic rhinitis, perennial allergic rhinitis, or sinusitis of any kind, etiology or pathogenesis, or sinusitis from the group of purulent or non-conductive sinusitis, acute or chronic sinusitis, ethmoiditis, frontal sinusitis, maxillary sinusitis, or sphenoiditis; rheumatoid arthritis of any kind, etiology or pathogenesis, or rheumatoid arthritis from the group of acute arthritis, acute gouty arthritis, primary chronic polyarthritis, osteoarthritis, infectious arthritis, Lyme arthritis, progressive arthritis, psoriatic arthritis, and spondylarthritis;

Gout, inflammation-associated fever, or inflammation-associated pain; an eosinophil-related pathological disorder of any kind, etiology or pathogenesis, or an eosinophil-related pathological disorder from the group of eosinophilia, eosinophilic lung infiltrate, Löffler's syndrome, chronic eosinophilic pneumonia, tropical pulmonary eosinophilia, bronchopneumonic aspergillosis, aspergilloma, eosinophilic granuloma, allergic granulomatous anguish or Churg-Strauss syndrome, Polyarterütis nodosa (PAN), as well as systemic vasculitis necroticans; atopic dermatitis, allergic dermatitis, or allergic or atopic dermatitis;

Hives of all types, aetiology or pathogenesis, or hives from the group of immune-related nettle hives, complement-dependent hives, hives-inducing hives, hives induced by physical stimuli, stress-induced hives, idiopathic hives, acute hives, chronic hives, angioneurotic edema Urticaria cholinergica, cold urticaria in its autosomal dominant form or in its acquired form, contact urticaria, urticaria gigantean and papular urticaria; Conjunctivitis of any kind, etiology or pathogenesis, or conjunctivitis from the group of actinic conjunctivitis, acute catarrhal conjunctivitis, acute contagious conjunctivitis, allergic conjunctivitis, atopic conjunctivitis, chronic catarrhal conjunctivitis, purulent conjunctivitis, and spring conjunctivitis; Uveitis of any type, etiology or pathogenesis or uveitis from the group inflammation of the entire uvea or a part thereof, anterior uveitis, iritis, cyclitis, iridocyclitis, granulomatous uveitis, non-granulocytic uveitis, phakoantigen uveitis, posterior uveitis, choriditis and choriorethinitis; Psoriasis; multiple sclerosis of any kind, etiology or pathogenesis, or multiple sclerosis from the group of primarily progressive multiple sclerosis as well as multiple sclerosis with relapses and a tendency to remissions; Autoimmune / inflammatory diseases of any kind, etiology or pathogenesis, or an autoimmune / inflammatory disease from the group autoimmune hematology disorders, haemolytic anemia, aplastic anemia, aregenerative anemia, idiopathic thrombocytopenic purpura, systemic lupus erythematosus, polychondritis, scleroderma, Wegener's granulomatosis, light disease, chronic active hepatitis, myasthenia gravis, Stevens-Johnson syndrome, idiopathic sprue, autoimmune - irritable bowel disease, ulcerative colitis, Crohn's disease, endocrine opthamopathy, Graves' disease, sarcoidosis, alveolitis, chronic hypersensitivity pneumonitis, primary biliary cirrhosis, insulin deficiency diabetes or type 1 diabetes mellitus, anterior uveitis, granulomatous uveitis or uveitis posterior, keratoconjunctivitis sicca, keratoconjunctivitis epidemica, ( diffuse) interstitial pulmonary fibrosis, pulmonary cirrhosis, cystic fibrosis, psoriatic arthritis, glomerulonephritis with and without nephrosis, acute glomerulonephritis, idiopathic nephrosis, minimal-change nephropathy, inflammatory / hyperproliferative skin disorders, psoriasis, atopic dermatitis itis, contact dermatitis, allergic contact dermatitis, familial benign pemphigus, pemphigus erythematosus, pemphigus foliaceus and pemphigus vulgaris; Prevention of xenograft rejection after organ transplantation, inflammatory bowel disease (IBD) of any kind, etiology or pathogenesis, or irritable bowel from the group of ulcerative colitis (UC), collagenous colitis, colitis polyposa, transmural colitis and Crohn's disease (CD); septic shock of any kind, aetiology or pathogenesis, or septic shock from the group of kidney failure, acute renal failure, cachexia, malaria cachexia, pituitary cachexia, uremic cachexia, cardiac cachexia, cachexia suprarenalis and Addison's disease, carcinoma nasty cachexia and cachexia due to infection by Human Immunodeficiency Virus (HIV); Liver damage; pulmonary hypertension and pulmonary hypertension due to lack of oxygen;

Bone wasting, primary osteoporosis and secondary osteoporosis; disorders of the central nervous system of any type, etiology or pathogenesis, or a disorder of the central nervous system from the group depression, Parkinson's disease, learning and memory disorders, dodecious dyskinesia, drug dependence, arteriosclerotic dementia, and dementia as a concomitant of Huntington's disease, Wilson's disease Paralysis agitans and thalamatic atrophy;

Infections, particularly viral infections, which viruses increase the production of TNF-α in their host, or which are susceptible to up-regulation of TNF-α in their host, so as to hamper their replication or other important activities, including viruses from the Group HIV-1, HIV-2 and HIV-3, cytomegalovirus, CMV; Influenza, adenoviruses and herpesviruses, including herpes zoster and herpes simplex; Yeast and fungal infections, wherein these yeasts and fungi are susceptible to upregulation by TNF-α or induce TNF-α production in their host, preferably fungal meningitis, especially when co-administered with other drugs of choice for the treatment of systemic yeast and fungal infections, including the polymycins, preferably polymycin B, imidazoles, preferably clotrimazole, econazole, miconazole and / or ketoconazole, the triazoles, preferably fluconazole and / or itrananazole, and the amphotericins, preferably amphotericin B and / or liposomal amphotericin B.

The invention also relates to a method for the modification, in particular an inhibition of an AKAP-PKA interaction comprising the steps:

(a) providing the decoupler according to the invention or a recognition molecule which is directed against it and

(b) contacting at least one product according to (a) with a cell, a cell culture, a tissue and / or a target organism.

In a preferred embodiment of the invention, this method is characterized in that the modification takes place on a regulatory RII subunit of the PKA, wherein particularly preferably the RII subunits are RIIalpha and / or Rllbeta subunits.

The invention also relates to a kit comprising the products according to the invention - particularly preferably the decouplers and the recognition molecules directed against them - and / or a pharmaceutical composition according to the invention, optionally together with information - eg. As a leaflet or an Internet address that refers to homepages with more information, etc. - about the handling or about the combination of the contents of the kit includes. The information for handling the kit, for example, a therapy scheme for the above diseases, especially for the preferred diseases include. However, the information may also include information on how to use the products of the invention within a diagnosis of diseases associated with, or their decoupling, AKAP-PKA interactions. The kit according to the invention can also be used in basic research. Within basic research, the kit is preferably used to detect whether a metabolic phenomenon is associated with the ACL or PKA interaction and the non-existent interaction. In particular, it is possible with the aid of the kit according to the invention to determine which subunits of AKAP and / or PKA are responsible for the interaction of these two molecules or for the non-existence of the interaction between them.

In an advantageous embodiment, the products according to the invention may comprise peptides, vectors, nucleic acids, amino acids, carbohydrates or lipids. For example, it may be preferred that the products be coupled to a fat remnant so that their membrane permeability is changed. By comparison with substances that bind PKA with a different affinity, it will still be possible to make quantitative statements that define to what degree a PKA-AKAP interaction is necessary to ensure the progression of a physiological process. In particular, the kits of the invention may be used to study this course of the physiological process. It is advantageous here that the molecules according to the invention can be selected such that they bind the RII subunits of the PKA more strongly than the typical PKA binding domains from AKAP, preferably AKAP18, in particular from AKAP18delta. Since selected molecules according to the invention are advantageously RIIalpha or RIIbeta-specific or, for certain RI-subunits, z. For example, the kit provides particularly detailed insights into the interaction of these molecules. The decoupling with one or the other regulatory subunit of the PKA from AKAP proteins can in particular give information on which PKA, type IIalpha or type IIbeta or the type I, are involved in the particular process under investigation.

The invention also relates to a process for the preparation of pharmaceutical agents, which comprises the following steps:

(a) provision of a decoupler according to the invention, preferably as a lead structure,

(B) chemical modification of the lead structure, preferably by means of a combinatorial and / or structure-based drug design, wherein substances are recovered and optionally

(c) testing the substances for their ability to affect the AKAP-PKA interaction, and optionally

(d) Selection of suitable substances as pharmaceutical agents. In a preferred embodiment of the invention, this method also encompasses the formulation of the tested substances in a pharmaceutically acceptable form.

The invention also relates to the process product obtained directly by this process.

In the following, the invention will be explained in more detail by means of examples, without being limited to these examples. The inhibitors or decouplers according to the invention modulate the interaction of AKAP and PKA. By way of example, the invention will be described in more detail below with reference to a few selected ones.

1. Material and methods

The reagents and chemicals used, unless otherwise stated, come from Merck (Darmstadt), Sigma (Deisenhofen) or Carl Roth (Karlsruhe).

1.1 Media and buffers

Luria-Bertani (LB) medium 10 g / l tryptone 10 g / l NaCl 5 g / l yeast extract pH 7.0

The solution was autoclaved after intensive stirring.

LB agar

To LB medium, 15 g / L agar-agar was added and then autoclaved.

For the preparation of agar plates, the LB agar was heated in a microwave oven until all solid particles were dissolved. After cooling to 40 ^° C., the required antibiotic was added and the solution poured into plates.

ampicillin

Ampicillin is a penicillin derivative and prevents cell wall synthesis in proliferating bacteria. It was (mg / ml 100) stored as a stock solution at -20 ⁰ C, just before use to a concentration of 100 ug / ml in LB agar or LB medium diluted.

2Ox tris-acetic acid-ethylenediaminetetraacetate (EDTA) - buffer (TAE)

242 g of tris (hydroxymethyl) aminomethane (Tris) in 0.5 l

H ₂ O

40 ml of 0.5 M EDTA pH 8 22.8 ml of acetic acid ad 1 1 H ₂ O.

Ethidium bromide solution

1 g ethidium bromide 100 ml H ₂ O

βx deoxyribonucleic acid (DNA) sample buffer 250 mg bromophenol blue 250 mg xylene cyanol 33 ml 150 mM Tris-HCl pH 7.6 60 ml glycerol 7 ml H ₂ O Isopropyl-beta-D-thiogalactoside (IPTG) solution 1.79 g IPTG 50 ml H ₂ O

Phosphate-buffered saline (PBS) 28.5 g Na ₂ HPO ₄ ^• 2 H ₂ O 5.5 g NaH ₂ PO ₄ ^• H ₂ O 164 g NaCl ad 1 H ₂ O

Protease inhibitors mix

1.4 μg / μl trasylol (aprotinin) 0.5 mM benzamidine 3.2 μg / μl soybean trypsin inhibitor (STI)

Phenylmethylsulfonyl fluoride (PMSF) solution (40 mM)

14 mg PMSF

2 ml of ethanol

Dithiothreitol (DTT) stock solution (0.5 M)

771 mg DTT

10 ml H ₂ O

lysis buffer

PBS

Protease Inhibitor Mix (1: 125)

PMSF solution (1:80)

DTT stock solution (1: 100)

Lysozyme solution

10 mg lysozyme 10 ml H ₂ O

Glutathione elution buffer

40 mM reduced glutathione 200 mM NaCl

0.2% Tween 20

100 mM Tris-HCl pH 8.5

Sodium Dodecyl Sulfate (SDS) Sample Buffer 100 mM Tris-HCl pH 6.8

4% SDS

20% glycerol 10% beta-mercaptoethanol 0.02% bromophenol blue 30 mM DTT

Separating gel (10%); the indicated amounts are sufficient for two gels:

3.75 ml of acrylamide 30% with bisacrylamide 0.8% 5.625 ml of Tris-HCl 0.75 M, pH 8.8

56.5 μl SDS 20%

2.5 ml H ₂ O

79 μl ammonium peroxodisulfate (APS) 10%

5.65 ul N, N, N ^1, N 'tetramethylethylenediamine (TEMED)

stacking; the indicated amounts are sufficient for two gels:

835 μl acrylamide 30% with bisacrylamide 0.8%

625 μl Tris-HCl 0.625 M, pH 6.8

25 μl SDS 20% 3.5 ml H ₂ O

25 μl of APS 10%

5 μl TEMED SDS polyacrylamide gel electrophoresis (PAGE) running buffer 250 mM glycine 25 mM Tris 0.1% SDS

Coomassie staining solution

2 g Coomassie Brilliant Blue G 250 75 ml acetic acid 500 ml methanol ad 1 1 H ₂ O

destaining

75 ml of acetic acid 100 ml of ethanol ad 1 1 H ₂ O.

Semi-dry transfer buffer

25 mM Tris 190 mM glycine

20% methanol

Tris-buffered saline (TBS) 10mM Tris-HCl pH 8.0 150mM NaCl

TBS-Tween 20 (TBST) as TBS, with 0.01% Tween 20

Ponceau S staining solution

2 g Ponceau S 30 g trichloroacetic acid 30 g sulfosalicylic acid 100 ml H ₂ O

Blotto

50 g / l skimmed milk powder in TBST

Bradford Reagent 100 mg Coomassie Brilliant Blue G 250

50 ml ethanol, undegraded

800 ml H ₂ O

Let solution stand overnight, then add 100 ml of phosphoric acid ad 1 1 H ₂ O.

The solution was shaken and filtered,

Binding buffer 80 μl Protease inhibitor mix 125 μl PMSF solution 20 μl DTT stock solution 0.5 M ad 10 ml PBS

blocking solution

150 mg skimmed milk powder

100 μl of 0.5 M DTT stock solution

0.05% Tween-20

625 μl PMSF solution 400 μl protease inhibitor mix ad 50 ml PBS PBST

0.05% Tween 20 in PBS

1.2 Transformation of competent bacteria with plasmid DNA

For the transformation, competent E. coli cells of the strain BL21 (DE3) were prepared by the method of Cohen and Wang.

The bacteria stored at -80 ° C were thawed on ice and 2 ng of the plasmid DNA to be transformed was added to 50 μl of bacterial suspension. After incubation on ice for 30 minutes, the cells were exposed to a heat shock of 42 ° C for 45 seconds to collect the plasmid DNA. Immediately afterwards 250 ul of pre-warmed to 37 ° C was LB medium was added and the cells shaken for 1 h at 37 ⁰ C so that it could express the encoded by the plasmid antibiotic resistance. From the cell suspension then 30 .mu.l were removed and plated on antibiotic-containing LB agar; the residue was centrifuged at 8000 xg for 1 min, resuspended in 100 μl of medium and also plated out. The agar plates were incubated at 37 ° C overnight. The next day, the colonies were counted to quantify the transformation efficiency.

1.3 Plasmid DNA preparation

For the expression of GST fusion proteins, the pGEX-4T3 plasmid (Figure 1) with the cloned, pro- teinkoding sequence used. By means of such a vector, foreign DNA can be introduced into bacterial cells, and the plasmid DNA can, for. B. for control purposes, also be isolated from the cells again. To isolate 5-10 μg of plasmid DNA from E. coli, 2-3 ml of LB medium were inoculated with the appropriate antibiotic with single colonies of agar plates. The cultures were incubated at 37 ⁰ C overnight with shaking. The cells were centrifuged at 13,000 xg and the supernatant was discarded as completely as possible.

The plasmid isolation from the bacterial sediments was carried out according to the plasmid minipreparation protocol of the QIAprep Spin Miniprep Kit (Qiagen GmbH, Hilden). The cells were then lysed in alkaline lysis buffer to remove proteins and protein-associated chromosomal DNA. The soluble plasmid DNA was bound to silica gel (in a column) in the presence of a highly concentrated saline solution, washed, and finally eluted using a low concentration saline solution. The isolated plasmid DNA was analyzed by restriction endonuclease digestion followed by agarose gel electrophoresis.

1.4 DNA restriction

Restriction endonucleases such as EcoRI (isolated from E. coli) cut DNA at specific sites defined by the sequence. The enzymes recognize these sequences, bind and cut the DNA hydrolytically. Microorganisms such as E. coli have restriction endonucleases to degrade foreign DNA. The distinction between own and foreign DNA is made possible by different methylation patterns of the DNA.

The endonucleases obtained from various manufacturers (New England BioLabs (NEB), Beverly, USA, Fermentas GmbH, St. Leon-Rot, Vitrogener GmbH, Karlsruhe) were used according to the manufacturer's instructions. DNA restriction (treatment with restriction endonucleases) was normally carried out at 37 ^° C. for 1 h. A typical reaction mixture contained 5 μg of DNA, 1 μl of 10x enzyme buffer and 0.5 μl of enzyme solution and was made up to a final volume of 10 μl with sterile water. After incubation, the entire reaction was analyzed by agarose gel electrophoresis.

1.5 Agarose gel electrophoresis

In electrophoresis, molecules are separated according to their size and electrical charge in a gel by applying an electrical voltage. 1% agarose gels were used. For preparation, 1% solid agarose was added to TAE buffer and solubilized by boiling in a microwave oven. The solution was clear cooled to 45 ⁰ C, and per 100 ml agarose solution 5 ul of ethidium bromide (EtBr) was added.

EtBr intercalates into the bases of the nucleic acids and allows their detection under UV light. EtBr fluorescence is enhanced by the delocalized pi-electron systems of the purine and pyrimidine bases of the DNA, between which EtBr inserts.

The liquid gel was then poured into a plastic mold where it could cool and polymerize. The DNA samples to be tested were spiked with sample buffer containing TAE, glycerol and bromophenol blue. Glycerol increased the density of the solution, making it easier to run into and stay in the gel pockets. Bromphenol blue allowed the visualization of the sample. In order to determine the size of the DNA fragments in the sample, 5 μl of a DNA molecular weight standard were analyzed with DNA fragments of defined size in one lane per gel (HyperLadder I, Bioline GmbH, Luckenwalde, Fig. 2).

The electrophoresis was carried out at a voltage of 120 V for 30 minutes. The gels were then photographed with Boehringer Mannheim's Lillimager Fl and analyzed with the associated Lumi Analyst 3.0 software.

1.6 Expression and Purification of GST Fusion Proteins

150 ml of ampicillin-containing LB medium were inoculated with E. coli cells of a transformed clone from an agar plate. The culture solution was incubated at 30 ⁰ C overnight (overnight). (At 37 ^° C, the cells proliferate too fast, so the next day the culture has already passed the logarithmic phase of growth, and the higher incubation temperature leads to stronger basal expression of the fusion protein, which can be due to the formation of insoluble inclusion bodies (ϊnclusion bo-this) make the purification more difficult.)

The next day, 20 ml of the overnight culture were transferred to 500 ml of fresh, ampicillin-containing LB medium. During the incubation at 37 ^° C., the multiplication of the bacteria was monitored by means of measurements of the optical density (OD) at 600 nm. After reaching an OD _60O of approx. 0.6 - then the bacteria are in the logarithmic growth phase, in which the protein expression is maximal - the expression of the glutathione S-transferase (GST) fusion protein was induced by addition of IPTG in a final concentration of 1.5 mM. IPTG is a synthetic inducer of the lac operon. It binds to the repressor and thus inhibits its binding to the DNA. After incubation at 37 ⁰ C for 30 min, the bacteria were collected by centrifugation xg for 10 min at 5000 and 4 ⁰ C se- dimentiert.

From here all further purification steps were carried out on ice to keep the activity of proteases low. The sediment was resuspended in 30 ml of cold lysis buffer and added to DTT at a final concentration of 5 mM. The cell suspension was mechanically lysed three times with the French Pressure Cell Press ("French Press") During this treatment, the bacterial cell walls were destroyed by the high pressure, facilitating subsequent lysis of the plasma membranes by a detergent.

The detergent used was Triton X-100 at a final concentration of 1%. In order to solve the plasma membranes and release the cytosolic proteins in the solution, the suspension was for 30 min at 4 ⁰ C slightly shakes overall.

The lysate thus obtained was centrifuged at 17,000 xg and 4 ° C for 10 minutes to remove cell debris. The ^supernatant contained the GST fusion proteins, and to purify them, it was incubated with 600 μl glutathione-Sepharose 4B (50% suspension in lysis buffer, Amersham GmbH & Co. KG, Braunschweig) for 30 min at room temperature with gentle Shaking incubated. In vivo, the enzyme GST plays an important role in detoxification reactions, for example in the cytosol of hepatocytes. It catalyzes the reaction of glutathione with various electrophilic, hydrophobic substrates. In protein purification, the GST fused to the desired protein binds with high affinity and high specificity to glutathione, which is coupled to Sepharose beads. Sepharose is a pearl-shaped, agarose-derived base material that is linked to a three-dimensional network. Due to their large volume, the beads and anything that has bound to them can be sedimented by centrifugation at 500 xg for 5 min. Alternatively, GST fusion proteins can also be purified by affinity chromatography over a column filled with glutathione-Sepharose. This application usually results in greater purity, but also in lower yield of proteins. The Sepharose sediment with the GST fusion proteins was then washed three times by resuspending each in 3 ml of lysis buffer and recentrifuging. To elute the purified proteins from the glutathione-Sepharose, the pellet was mixed with 300 μl elution buffer and shaken for 10 min at room temperature. The elution buffer contains excess glutathione in excess and thus competitively displaces the glutathione sepharose from the GST fusion protein. After centrifugation at 500 xg for 5 min of the fusion protein containing supernatant was carefully pipetted off, combined with the same volume chen glycerol, aliquoted and stored at -20 ⁰ C. Glycerin prevents the formation of ice crystals, which lead to the degradation of proteins (especially after several freezing and thawing cycles). To control the purification process, samples were taken at various points in the procedure and analyzed by SDS-PAGE (see below): before and after the induction of protein synthesis with IPTG, before and after centrifugation of the cell lysate, before and after addition of glutathione -Sepharose and after washing the Sepharose sediment.

1.7 SDS PAGE

The method for analyzing the purity and the molecular weight of proteins is the Laemmli discontinuous sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

In order to detect the molecular weight, the influence of the charge and tertiary structure on the migration speed of the proteins in the gel must be eliminated. Then the (logarithmic) mobility of the proteins depends solely on their molecular weight.

This situation can be almost achieved by denaturing the native proteins with the detergent SDS. It leads to the dissociation of oligomeric proteins into their subunits. The negative charges of the sulfonic acid groups of SDS overlay the intrinsic charges of the individual amino acid side chains and thus ensure a uniformly negative charge of the proteins.

Disulfide bridges are reduced and cleaved by DTT and beta-mercaptoethanol, which are next to SDS in the sample buffer. The denatured proteins bind SDS according to their size, that is the length of the amino acid chain. As in the agarose gel electrophoresis of nucleic acid acids, a molecular weight standard consisting of proteins of known molecular weight is loaded onto the gel. The BenchMark protein ladder molecular weight standard was used (Figure 3, Invitrogen GmbH, Karlsruhe).

The SDS-PAGE is referred to as discontinuous because the gel consists of two parts, a collection and a separation gel. They differ with regard to their pore size and their pH. Such a discontinuous system provides sharper bands than a continuous one, thus allowing a more precise determination of protein size and molecular weight.

Bound protein-linked sepharose jbeads can be used directly for SDS-PAGE because the SDS sample buffer has a high reducing power that results in the elution of the proteins from the beads.

The separating gel was prepared using the reagents mentioned in Section 2.1. After pipetting TEMED, polymerization began and 3.75 ml of the solution was pipetted between glass plates clamped in a special plastic holder. Subsequently, the gel was covered with 2-propanol to eliminate air bubbles and to keep air from the gel (oxygen prevents the polymerization reaction). After complete polymerization, the 2-propanol was removed and the stacking gel prepared (see Section 1.1). After overlaying the release gel with the collection gel, the comb forming the sample pockets was placed in the gel solution. The protein samples were denatured by boiling in sample buffer for 5 min at 95 ° C. The samples were briefly centrifuged and injected into the. With a Hamilton syringe Transferred gel cakes.

After about 15 min of electrophoresis at 90 V, the protein samples reached the separation gel and the voltage was increased to 120 V for 1.5 h. After completion of the electrophoresis, the separated proteins in the gel were either stained with Coomassie brilliant blue G 250 solution or further analyzed by Western blot analysis (see below). For Coomassie staining, the gel was shaken for 30-60 minutes in Coomassie staining solution and then decolorizing solution until the excess stain was removed and the contrast between unstained gel and stained proteins was sufficiently strong. Finally, a photo was taken with the Bio-Rad ChemiDoc EQ and the belonging Software Quantity One. 1.8 Western Blot

In a Western blot, after separation by SDS-PAGE, the proteins are transferred by electrical voltage to a nitrocellulose or polyvinylidene fluoride (PVDF) membrane and detected with specific antibodies (Ab). After binding of the Ab, excess Ab are washed away and the membrane is incubated with a secondary Ab which specifically recognizes and is labeled the primary Ab. The label may consist of radioactive isotopes or an enzyme that catalyzes a dye-forming reaction.

First, an Immobilon P-PVDF membrane was cut to the size of the gel, wetted with ethanol for 15 seconds and then moistened with Semidry transfer buffer for at least 5 minutes. Whatman filter papers were also tailored to the correct size and in Moisten transfer buffer.

Protein transfer from the gel to the membrane was carried out with a BioRad TransBlot SD Semi Dry Transfer Cell onto which 2 filters, the membrane, the gel, and again 2 filters were layered. Air bubbles have been removed as they impede protein transfer. After pipetting 4 ml of transfer buffer onto the stack, the transfer cell was closed and the proteins were transferred to the membrane at 10 V for 1 h. Subsequently, the proteins on the membrane were stained with Ponceau S solution. For this purpose, the membrane was washed off with water and shaken for 20 minutes at room temperature in Ponceau S solution. The bands of the molecular weight standard were traced with a pencil, and the membrane was wrapped in cling film to take a photograph with the Lumilmager Fl (7 s exposure under top illumination).

Before the Ak incubation, the free binding sites on the membrane must be blocked, as otherwise large amounts of Ab can adhere unspecifically to disturb the specific signals. The saturation of the membrane was achieved with shaking for 1 h in Blotto. The primary A18delta3-Ab was already present in the workgroup. It was generated by immunizing rabbits against a peptide whose sequence was identical to amino acids 60-76 of the AKAP18delta sequence. The antisera thus obtained had been purified by affinity chromatography via the peptides used for the immunization, coupled to thiopropyl-Sepharose 6B (Amersham GmbH & Co. KG, Braunschweig). The A18delta3-Ab binds both AKAPlδdelta and -gamma. The Ak solution was diluted 1: 500 in Blotto. In order to To reduce the required Ak, the membranes were shaken in incubation bags with 3 ml of air-free Ak solution for 2 h at room temperature or overnight at 4 ⁰ C. After washing three times with TBST for 5 min, the membranes were shaken for 1 h in a 1: 100 O dilution of the secondary Ab (anti-rabbit F (ab) ₂ fragments) in Blotto. The secondary Ak was coupled to horseradish peroxidase (POD) (Dianova, Hamburg). The membranes were washed three times with TBST for 10 min and for detection, 4 ml of solutions 1 and 2 of the LumiLight Western Blotting Substrate (Roche Diagnostics GmbH, Mannheim) were added to the membrane and shaken for 5 min at room temperature. The detection solution contains luminol, which is oxidized by the coupled POD to produce light (chemiluminescence). A photograph of the membrane wrapped in cling film was taken under the setting of chemiluminescence (exposure time 1 min) on the lumi-lager Fl.

1.9 Bradford Protein Concentration Determination

For the determination of the protein concentration in the GIu- tathion-Sepharose eluates is the Bradford method best suited as the chromogen used herein (Coomassie brilliant blue G 250) does not bind to the likewise located in the eluate excess glutathione, son ^¬ countries va to cationic and hydrophobic amino acid side chains.

For the measurement, 50 μl of the protein-containing sample (diluted eluate) were added to 50 μl of 2 M NaOH and incubated for 10 min incubated at 60 ° C. Then 1 ml Bradford reagent was added, the sample transferred to a cuvette and the absorbance at 595 nm determined.

The protein concentration was determined by comparison with a calibration set of different ovalbumin dilutions.

1.10 Enzyme-Linked Immunosorbent Assay (ELISA)

ELISA is a method of detecting an antigen with a specific antibody. The detection takes place via an enzyme covalently linked to one of the binding partners, which catalyzes a chromogen conversion. In this case, for example, a dye or chemiluminescence is produced quantitatively

(as already described for the Western Blot).

Since one of the two binding partners is immobilized, the removal of unbound antibodies or

Antigens very easily. Detection takes place by recording the intensity (I) of the chemiluminescence with the help of a photomultiplier in a special reader (ELISA reader). The measured values are output in relative light units (RLU). In the present case, it should be investigated whether the binding of one protein (PKA) to another (AKAP18delta-GST) is inhibited by the presence of small organic molecules. Therefore, a sandwich ELISA was developed in which the PKA subunit RIIalpha, to which AKAP proteins bind, was bound to 384-well microtiter plates (MTP).

White MTPs of high protein binding capacity polystyrene were used (384 well white fiat bottom po- lystyrene High Bind Microplate, product no. 3703, Corning GmbH, Wiesbaden). The white color improves the detection of the resulting chemiluminescence by reflection and at the same time prevents the irradiation of neighboring wells. After binding, free binding sites were blocked (see below), and the second protein GST-AKAP18delta was added together with the potential low-molecular-weight inhibitors or-as control-inhibitory peptides (see below). Subsequently, the bound GST-AKAP18delta was quantitated by antibody and chemiluminescence. The principle of the ELISA is shown in FIG.

The buffer system used was PBS (pH: 4, phosphate-buffered saline, pH 7.4) in order to maintain a physiological pH for the proteins used. A mix of protease inhibitors and PMSF as a non-specific protease inhibitor was added to the buffer to delay the degradation of the proteins. In addition, DTT was used to protect the proteins from oxidation. The blocking solution additionally contained 0.3% skimmed milk powder in order to saturate the unspecific binding sites on the MTP with the milk proteins contained. For the establishment of the ELISA, which is described in detail in the results section, in addition to the aforementioned and described in Section 1.8 (Western Blot) still further antibodies for the detection of PKA RIIalpha were needed: The commercially available mouse PKA _R n _a ipha antibody (BD Biosciences, San Jose, USA) and an anti-mouse POD Ab (Dianova, Hamburg).

1 . 11 Calculation of Binding Curves and ICso Values The data obtained from the experiments was partially evaluated using GraphPad Prism (GraphPad Software, San Diego, USA).

The adaptation of binding curves to the measured values (see results) was based on the one-site binding model, which applies to the binding of GST-AKAP18delta to RIIalpha. The corresponding formula was

The ICso calculation readings were fitted to the one-site competition model. It was determined that the lower asymptote (bottom) should be greater than 0.0 to exclude erroneous adjustments with negative binding values. The formula used was

1.12 Screening of the substance library

The FMP20000 substance library was used for screening - the systematic, in part automated search for potential inhibitors of AKAP18delta-RIIalpha binding. It contained 574 MTP, each with 384 wells 20064 different, commercially available substances (ChemDiv, San Diego, USA) in 10 mM stock solutions in DMSO. The substances of the library were selected according to certain criteria, which also apply to previously known pharmacological agents. These include, for example, the Lipinski rules (see discussion).

To prepare an MTP for the screening, 20 μl of RIIalpha solution with a concentration of 0.75 ng / μl (in binding buffer) were filled in 380 wells using electronically controlled 24-channel pipettes (Eppendorf). 10 μl of AKAP18delta-GST (8 ng / μl) were pipetted into the remaining 4 wells in order to obtain a positive control for detection (see also MTP assignment in Table 1.1 and pipetting scheme in Table 1.2). After centrifugation of the MTP (1 min at 2500 x g), the MTPs were added for at least 1 h

Tab. 1.1: Assignment of an MTP with control reactions (columns 1 and 24) and low-molecular substances (2-23). See also pipetting scheme in Tab. 1.2.

Room temperature incubated. After removal of the protein solution by knocking off on a paper stack, the non-specific binding sites were blocked with 100 μl of blocking solution / well for at least 1 h at room temperature. The blocking solution was removed using the automatic Power Washer 384 (Tecan) and all wells were rinsed with TBST. After completion of the washing process, any remaining solvents on a paper stack were knocked off. In the next step, the MTPs were prepared for the automated pipetting of low-molecular substances. 10 μl of blocking solution were initially introduced into each well or the controls were pipetted according to the pipetting scheme (Table 1.2). The controls used were reactions without inhibitors, but with increasing amounts of AKAP18delta GST (0, 80, 160 ng / well), as well as with the inhibitory peptide L314E and the control peptide 18d-PP (25 nM and 1 μM final concentration). The plates were centrifuged briefly. At the same time, the library MTPs were prepared for the pipetting procedure as follows: After thawing (37 ^° C. for 30 minutes), centrifugation was carried out briefly to collect the solutions at the bottom of the plate. In order to dissolve any solid particles, the plates were immersed in an ultrasonic bath for 1 min. Water adhering to the plates was evaporated by treatment in the Eppendorf Concentrator 5301 at 30 ^° C. for 5 minutes, and the protective film was carefully removed.

Using the Sciclone ALH 3000 Workstation (Caliper Liberties, Hopkinton, USA), 0.5 μl of each potential low molecular weight inhibitor (10 mM stocks in dimethylsulfoxide (DMSO), final concentration 244 μM) was transferred from the library MTP to the screening Transfer MTP. In Control Columns 1 and 24 were each transferred to 0.5 μl of DMSO, so that the controls were also spiked with the same DMSO content as the remaining samples. After centrifuging the screening MTP, according to the pipetting scheme (Table 2.2) AKAP18delta-GST (8 ng / μl in blocking solution) was added, centrifuged and incubated for at least 30 minutes at room temperature. After removal of excess protein by washing with PBST the Power Washer the amount of the bound in the presence of the potential inhibitor AKAPl8delta-GST was determined using 20 ul Al8delta3- (1: 1000 in blocking solution for at least 1 h at room temperature or overnight at 4 ⁰ C ) and 20 μl of anti-rabbit POD-Ab (1: 3000 in blocking solution, at least 15 min at room temperature) per well. Between the individual pipetting steps each time with the help of the Power Washer with PBST was washed. The quantification was carried out by pipetting 20 μl of LumiLight Western blotting substrate (Roche) per well and incubating for 5 min at room temperature. The generated chemiluminescence was measured using a Tecan Genios Pro MTP reader and Magellan 5.02 software {Chemiluminescence endpoint measurement, integration time 10 ms / 'well; Tecan Germany GmbH, Crailsheim).

Tab. 1.2: Pipetting scheme for an MTP of the screening. In the columns 1 and 24 are the control reactions, in 2-23 the reactions with the low-molecular substances.

* In 1E / 1F / 24K / 24L: After pipetting the small molecules, the 10 μl blocking buffer was replaced with 20 μl GST-AKAP18delta.

2 results

2.1 Purification of Recombinant AKAP18delta

2.1.1 The plasmid encoding AKAP18delta-GST fusion protein

The pGEX-4T3 vector suitable for the expression of GST fusion proteins (FIG. 1) with the A-KAP18delta-coding sequence cloned in was already present in the work group. The 1059 base pair A-KAP18delta cDNA fragment was located at the 3 'end of the GST coding sequence, so that in the expressed protein the GST was N-terminal to AKAP18delta. The correctness of the sequence was confirmed shortly before the work started by sequencing.

For expression, the GST-AKAP18delta construct was transformed into competent BL21 cells. In order to verify the transformation, the plasmid DNA was isolated from four of the clones obtained and a DNA restriction was carried out using the restriction endonucleases EcoRI and Xhol. Since these enzymes were also used for cloning, it was expected that fragments of the size of the AKAP18delta (1.0 kb) insert and the linearized pGEX vector (4.9 kb) could be detected by agarose gel electrophoresis (FIG 5).

2.1.2 Optimization of lysis of GST-AKAP18delta-expressing bacteria

Bacteria that had been transformed with the pGEX-AKAP18delta vector could be induced to express the recombinant protein by incubation in IPTG-containing medium. After the subsequent lysis of the Bak cells, the GST fusion protein released into the solution was bound to glutathione-Sepharose, washed and finally eluted with excess glutathione. In order to achieve the highest possible yield of protein, were based on a standard method different Pa ^¬ varied parameters of the GST-AKAPl8delta purification. The purification procedure thus determined is described in Section 1.6. For the purified, recombinant GST-AKAP18delta, a size of 75 kDa was expected, for the control purposes purified GST alone of 25 kDa.

After transformation of competent E. coli BL21 cells with the pGEX-AKAP18delta plasmid, cells were expanded and protein synthesis was induced with IPTG. It was first investigated whether and how the lysis of the bacteria under different conditions on the protein yield. Besides variations of the standard method, lysis with the French Press, cells from the same induction culture were also lysozyme lysed and after DTT addition. The following variations were examined:

^■ once with the French Press

^■ three times with the French Press ^■ Addition of 5 mM DTT before a single French Press lysis

^■ 30 min at RT with lysozyme (1 mg / ml)

■ 30 min at 37 ° C with lysozyme (1 mg / ml)

After the further purification procedure and the binding to the glutathione-Sepharose (see Section 1.6), 5 μl of beads were taken from each batch and analyzed by SDS-PAGE with subsequent Coomassie staining (Figure 7). When Control were carried with cells transformed with an empty pGEX vector; for these, only the GST was expected as a purification product.

As can be seen from Figures 6A and 6B, proteins of the expected sizes could be detected.

The addition of 5 mM DTT to the cell suspension prior to lysis resulted in a higher protein yield (Figure 6A). A visual comparison of the Coomassie stained gels showed that lysozyme was less effective than the French Press to lyse the cells (Figure 6A).

The highest protein yield could be achieved by incubation at room temperature for 30 minutes (FIG. 6B).

2.1.3 The binding efficiency of the GST fusion protein to glutathione-Sepharose has been improved

By varying the incubation time and the incubation temperature of the cell lysate with the Sepharose jbeads, it was attempted to optimize the efficiency of the binding of GST-AKAP18delta to the glutathione-Sepharose (FIG. 6B).

It could be shown that an incubation of the cell lysate at room temperature for 30 min yielded the highest protein yield.

2.1.4 The elution conditions have been optimized

Since elution of GST-AKAP18delta from the Sepharose beads proved to be less than standard under standard conditions, elution conditions were adjusted to maximize protein yield.

Since variations in the elution time and temperature compared to the manufacturer's instructions (10 min at Raumtempe- temperature) did not lead to improvements, the composition of the elution buffer was changed. Its composition was recommended by the manufacturer with 10 mM glutathione (GSH) in 50 mM Tris-HCl pH 8.0. After the addition of 100 mM NaCl or 0.1% Triton X-100 had not been found to enhance efficiency (Figures 7, A and B), the GSH concentration gradually increased to 40 mM, the Tris concentration to 100 mM , the pH increased to 9.0, and the NaCl concentration increased to 200 mM. In addition, 0.2% Tween-20 was added to the elution buffer. Finally, it was checked whether more protein could be obtained by repeated elution (FIG. 7, CI). Western blotting (with A18delta3 and peroxidase (POD) -coupled anti-rabbit Ab) identified the purified protein as GST-AKAP18delta. (Fig. 7J).

The protein concentration of pre-optimization purified and used for ELISA establishment was determined by Bradford assay; it was 0.4 μg / μl.

Screening required further purification of the recombinant GST-AKAP18delta protein, which was performed under the optimized conditions. The concentration of eluate from this purification was determined by the established ELISA by comparison to the first purification; it was 8 μg / μl.

2.2 Establishment of an ELISA for the quantification of protein-protein interactions For the quantitative detection of protein-protein binding between the RIIalpha subunit of PKA and GST-AKAP18delta, ELISA-based detection was developed; which can be used optimally for all other AKAP proteins and PKA subunits by routine experimentation.

The advantages of this method are, as already described in Section 1.10, above all in the high sensitivity and ease of use. In addition, the required substances were already established and were available in sufficient quantities. While the anti-rabbit POD-Ab and the chemiluminescent solution were commercially available, the A18delta3-Ab and GST-AKAP18delta were self-produced (see above). First of all, the basic question was whether GST-AKAP18b should first be bound to the microtiter plates (MTP) and then the binding of RIIalpha should be detected, or vice versa. Since sensitivity was lower and protein requirement was higher in the former, and the required PKa _RIIalpha antibody could not be self-produced, only the second option, ie binding of RIIalpha to the MTP and then detection of binding, became available after the first steps of establishment from GST-AKAP18delta. The following important aspects were to be investigated during the development of the assay:

• Composition of suitable binding or blocking buffer

Amount of RIIalpha protein to be bound to the MTP; amount of GST-AKAP18delta binding to the plate-bound RIIalpha • Appropriate dilution of antibodies for detection

• Influence of the solvent dimethyl sulfoxide (DMSO) on the ELISA (peptides and organic substances were dissolved in DMSO)

• Concentration of inhibitory peptides for control reactions

• Incubation times for RIIalpha, GST-AKAP18delta, antibody and chemiluminescence detection solution • Storage conditions for bound protein MTP

• Detection of chemiluminescence

The following describes the establishment of the ELISA, taking these aspects into account. Unless otherwise stated, duplicate determinations were made in all the experiments of establishment, that is, two wells each having the same amounts of protein and under the same conditions were examined.

Although only two measurements were made for each experiment, the standard error was calculated for the respective mean to obtain a measure of the precision of the mean. The information should not be used to determine significance. The error indicators in the figures indicate the standard error of the mean; in some cases (eg Fig. 9A) the standard errors of the means were so small that the indicators are not recognizable.

2.2.1 Skim milk is best suited to block free binding sites on MTP

Skimmed milk powder, bovine serum albumin (BSA, bovine serum albumin) and BSA, which was specially formulated for ELISA applications pretreated (ELISA-BSA), as blocking reagents examined (Figure 8A, these are individual determinations).

BSA often contains substances which generate nonspecific luminescence signals, as could also be shown here. However, ELISA-BSA still showed considerable nonspecific signals, while skimmed milk powder produced virtually no chemiluminescence. Thus, skimmed milk powder ^¬ was used at a final concentration of 0.3% in binding buffer for blocking free binding sites on MTP.

2.2.2 An MTP-well can be saturated with 50 ng of PKA-RIIalpha

Binding buffer solutions with increasing RIIalpha concentration were pipetted into the wells of a MTP. After blocking free binding sites with skim milk _solution , the protein _amounts bound to the plates were _detected with the PKA _RIIalpha- Ab and the anti-mouse POD-Ab. Since the optimal antibody concentrations were still unknown, dilutions of 1: 5000 (PKA _RIIa ipha) and 1: 10000 (anti-mouse POD) were used. The measured chemiluminescence intensities were plotted against protein concentration to determine if and when saturation of the wells with protein was achieved (Figure 8B).

From 45 ng RIIalpha per well, the binding capacity was saturated. In the following experiments were carried out with RIIalpha amounts of 40 ng / well and 25 ng / well.

2.2.3 The chemiluminescence intensity reaches a maximum at 200 ng GST-AKAP18 delta per well 25 ng or 40 ng of RIIalpha were bound to the wells of each two rows of an MTP. After blocking, blocking buffer solutions were pipetted into the wells with increasing GST-AKAP18delta concentration (between 0 and 200 ng / well). This test arrangement is referred to below as the bond series. The detection of the bound protein was carried out with A18delta3 and anti-rabbit POD Ab (1: 5000 and 1: 10000, respectively). The measured signal intensities were plotted against the mass of GST-AKAP18delta per well (Figure 8C). It turned out that from 200 ng GST-AKAP18 delta per well, the binding curve almost reached saturation. An initial evaluation of the readings indicated a value of 80 ng GST-AKAP18delta per well to achieve half-maximal saturation. At half maximum saturation of a bond, the highest possible sensitivity is achieved. Therefore, the following experiments were performed with a GST-AKAPδδ concentration of 80 ng / well. A later evaluation with the adaptation of the one-binding-site model (see Section 1.11) to the measured values is shown in FIG. from this, a lower value of 50-60 ng GST-AKAP18delta for the half-maximal saturation could be deduced. Since there was no significant difference between the luminescence intensities of the 25 ng Rllalpha and 40 ng Rllalpha coated wells, the following experiments were performed with 25 ng Rllalpha / well to save protein. It was later investigated whether the screening also required an RIIalpha amount of 15 ng / well to save protein (FIG. 9A). Again, there was no impairment Determination of the luminescence intensity was determined so that the screening with this amount of protein could be performed.

2.2.4 The A18delta3 antibody was diluted 1: 1000

As in the previous experiment, a RIIalpha-GST AKAPlδdelta binding series was prepared, which is then excreted with ver ^¬ dilutions of A18delta3-Pc detected (Fig. 9B). The dilution of the anti-rabbit POD Ab was initially left at 1: 10,000.

The binding curves showed that at a dilution of 1: 5000 not all GST-AKAP18delta-RIIalpha complexes could be detected. At a concentration five times higher, significantly stronger signals were obtained. Since the background signal, ie the luminescence intensity at 0 ng GST-AKAP18delta, remained low at the same time, a greater sensitivity was thus also obtained.

2.2.5 For the secondary antibody, a 1: 3000 dilution is useful

The same experiment was done as in the previous section, with the dilution of the primary Al8delta3 antibody being 1: 1000 (Figure 9C). Also, for the anti-rabbit POD Ab, a signal intensity and sensitivity increase could be detected when the dilution was reduced from 1: 10,000 to 1: 3,000. Therefore, the following experiments and substance screening with the determined dilutions were made 1: 1000 for the A18delta3-Ab and 1: 3000 for the anti-rabbit POD-Ab. 2.2.6 DMSO does not affect the ELISA

The influence of the organic solvent dimethylsulphoxide (DMSO) on the protein-protein binding or its detection had to be investigated, since the inhibitory peptides L314E and Ht31 used as controls as well as the substance library; even in DMSO were solved. In a first experiment, the effect of 1% DMSO relative to the total reaction volume (of 20 μl) was tested, again using different secondary Ak dilutions and GST-AKAP18delta concentrations simultaneously (Figure 10A). This showed that DMSO had no significant influence on the results; The previously obtained results for the different GST-AKAP18delta concentrations and Ak dilutions could be confirmed.

In a further experiment in which increasing DMSO concentrations of up to 2.5% were added to constant RII-alpha and GST-AKAP18delta levels (Figure 10B), the signal obtained was only slightly reduced. In the screening, the maximum DMSO concentration was also 2.5%. In a control experiment, the RIIalpha bound to the MTP was detected by means of PKA _RIIa _pha and anti-mouse POD-Ab (FIG. 10A, penultimate value).

In another control (also Fig. 10A, last value) no RIIalpha was bound to the plate and the remaining assay was performed as known. Surprisingly, a high chemiluminescence intensity was measured here, so that the question arose whether the measured signals at all the amount of bound GST- AKAP18delta reflected. For verification, another experiment (see next section) was performed.

2.2.7 The detection of RIIalpha-bound GST-AKAP18delta is specific

In order to control the signal specificity, 25 ng of RIIalpha / well were bound in one row of an MTP, whereas in a second row no RIIalpha was bound. After blocking unspecific binding sites, solutions with increasing concentrations of GST-AKAP18delta were pipetted into both rows and detected using the antibody specifically bound protein (Figure 10C). While the signals in the RIIalpha series increased and finally reached saturation, in the series without RIIalpha only a slight increase due to nonspecific binding of GST-AKAP18delta was observed. The result showed that the detection was specific. The control in Fig. 10A, last value, was therefore probably a pipetting or other error.

2.2.8 Inhibitory peptides prevent binding of GST-AKAP18delta to RIIalpha

It is known that the binding between AKAPs and the R subunits of PKA can be specifically and competitively inhibited by AKAP-derived peptides. Such peptides were therefore used for control reactions in which the interaction of RIIalpha and GST-AKAP16delta with peptides should be prevented. In order to obtain the most sensitive detection possible, First, the IC ₅₀ values for the established anchor inhibitor peptide Ht31 and for AKAP18-L314E were determined. The IC ₅₀ value is the peptide concentration at which only 50% of the maximum possible protein-protein complexes are formed.

To determine these values, 25 ng of RIIalpha / well were coupled to MTP and in each case 80 ng of GST-AKAP18b added. Subsequently, the peptides were pipetted in increasing concentrations. Because the L314E peptide inhibited binding to a greater extent, it was used for the control reactions at the IC ₅₀ near-25 nM concentration (Figure 11). The peptide inhibition was specific because the proline-containing control peptides 18d-PP and Ht31-P did not or only weakly inhibited the binding.

2.2.9 An incubation period of 30-60 min is sufficient for protein-protein binding

The time factor plays a not inconsiderable role in the planning and implementation of a substance library screening. For this reason, a few experiments were carried out, which gave an idea of the incubation times required. It was first tested in which time sufficient protein complexes are formed to obtain sufficient chemiluminescence intensity. Binding sequences were again pipetted and the formation of the protein-protein bonds was interrupted by washing of the MTP after various times - 15, 30 and 60 min. Subsequently, the bound GST-AKAP18delta amount was detected with the Ak (FIG. 12). Already after 30-60 minutes, sufficiently high chemiluminescence signals could be detected.

2.2.10 30-60 min are sufficient for inhibition by inhibitory peptides

The binding of GST-AKAP18delta to RIIalpha was also assayed in the presence of the competitive inhibiting peptide L314E and the control peptide 18d-PP. The peptides were added to the reactions at concentrations of 0.01 or 10 μM for 15, 30 or 60 min, and then the amount of bound GST-AKAP18delta was detected (Figure 13A). Again, the previously determined incubation time of 30-60 min proved sufficient.

2.2.11 Optimum antibody incubation times are 60 or 15 min

The binding of antibodies to their antigens is time-dependent, whereby the specificity initially increases with a longer incubation period. Later, z. B. due to the degradation of proteins decrease the specificity again. In order to obtain the incubation time required for sensitive measurements, the A18delta3 and anti-rabbit POD antibodies were added to RIIalpha-GST-AKAP18delta complexes and the binding reactions were interrupted by washing at 15, 30 and 60 min. All possible combinations of incubation times for the primary and secondary antibodies were considered (FIG. 13B).

It was found here that the highest signal intensity was achieved with 60 min incubation with both antibodies. de. However, it seemed reasonable to shorten the incubation time of the secondary antibody if the primary could bind for 60 min.

2.2.12 Chemiluminescence can be detected over a period of 8 min

The generation of chemiluminescence by the oxidation of luminol is a time-dependent reaction. Substrate consumption and denaturation of the peroxidase in the course of the reaction, the intensity decreases after a few minutes.

However, addition of auxiliary reagents that slows the decrease in intensity does not pose a major problem with commercially available products such as the LumiLight solution used here. However, the time frame after which the substrate solution was added to the reaction mixtures was checked Measurement should be performed. For this purpose, a bond series was pipetted (see above) and the measurement of the chemiluminescence was repeated 2, 4 and 8 minutes after addition of the Lumi-Light solution (FIG. 13C).

Within 8 minutes of adding LumiLight, the measurement could be performed without dramatic signal intensity loss. Within this timeframe, the entire measurement process could be concluded without any problems.

2.2.13 Protein-coated MTPs can be stored for extended periods of time To speed up the screening, it was useful to coat several MTPs with RIIalpha and then store them until further use. To find out if the protein was not degraded by storage, it was bound to two rows of an MTP RIIalpha as described, blocking non-specific binding sites. Then, in a series was the after 1 h of storage at room temperature, is completed in the other h after 66 storage at 4 ⁰ C, the ELISA and the amount of bound GST-AKAPl8delta detected (Fig. 14).

In the time frame examined, the storage period had no discernible influence on the result.

With the concentrations and incubation times thus determined, the substance library was screened.

2.3 Screening of a substance library for finding low molecular weight inhibitors of the RIIalpha ~ GST-AKAP18delta ~ interaction

After pipetting and measuring the MTPs as described in Section 1.12, it was first checked whether the measured chemiluminescence intensities were high enough. If this was the case, the chemiluminescence of each well was converted to the corresponding proportion of RIIalpha-GST-AKAP18delta binding relative to the control in percent using the Microsoft Excel spreadsheet program. For this purpose, the mean of the measured values of the controls with 80 ng GST-AKAP18delta [wells Cl, Dl, M24, N24; s. Tab. 1.1 and 1.2) as 100%. The results were then compared with an automated color assignment to simplify the optical evaluation (by using a Microsoft Visual

al Basic written program), whereby a different color was used at 10% intervals. As an example, the raw data of an MTP (chemiluminescence in RLU) and the associated conversion as a percentage of the binding with staining are shown in Table 2.1. The complete data of the screening can be found in the appendix. After staining, in some MTP abnormalities were evident, for example, many inhibitor candidates in a particular plate area. If the control values showed that the 100% values in column 1 and 24 were very different (more than 20% difference), this MTP was re-evaluated. In this case, for each half of the plate only the control values of this half were used (eg for columns 1-12 the control values from column 1).

If the conspicuous plate areas still persisted, the entire plate was pipetted again, measured and evaluated. This was required for plates 4, 6, 7, 13, 21, 38 and 50. Each substance of the library was assigned a so-called compound ID, which facilitated the EDP-supported evaluation of the data. After completion of the screening, all measurement results in a table were assigned to the corresponding compound IDs. By sorting the results according to increasing percentages of binding, the substances shown in Table 2.2 were determined, with which an inhibition of protein-protein binding between RIIalpha and GST-AKAP18delta could be achieved to 20% of the control value or less. Tab. 2.2: By screening the library comprising 20064 substances, 19 candidates could be found which, at a substance concentration of 244 μM, inhibited the binding between RIIα and GST-AKAP18δ to 20% or less compared to the controls. PlateJD = Library MTP No., PosJD = position of the well with the candidate substance. 5

Tab. 2.3: Pipetting scheme and measurement results of the validation. For each potential inhibitor (A7-H15, I1-P10, the compound IDs are given) and for the peptides (A3-H6, I11-P12), a dilution series was prepared to examine the concentration dependence of the inhibitory effect. In addition, binding sequences without inhibitors were prepared (A1-H2, I13-P14). The luminescence intensities were also converted into percentages and stained as described in Table 3.1 (see Table 3.4). Comp.-ID = compound ID, I = intensity

2.4 Validation of Potential Inhibitors

For the validation of the potential inhibitors found in the screening of the substance library, these were picked out from the library MTP and, according to the pipetting scheme shown in Tab. 2.3, dilution series were prepared together with control reactions. The serial dilutions should show the concentration dependence of the potential inhibitors found, which could only be expected in the case of specific inhibition of GST-AKAPlδdelta-RIIalpha binding. For these compounds (Tab. 2.4) were the IC ₅ o values calculated as described in Section 1.11 (Fig. 15) using the determined data. These values indicate at which concentration of the substance half of the maximum possible inhibition is reached. Individual determinations were made because of the limited amounts of substances, so no error indicators are shown in FIG. 15 except for the controls. The identity and purity of the identified substances were confirmed by liquid chromatography mass spectrometry (LC-MS). The substances were reordered for further investigation in larger quantities at ChemDiv (San Diego, USA).

Tab. 2.4: Percentage binding between GST-AKAP18S and RIIα in the presence of inhibitory molecules (A7-H15, I1-P10), peptides (A3-H6, I11-P12), or without inhibitors (binding sequences in A1-H2, I13- P14), calculated from the results in Tab. 2.3. The color coding corresponds to that in Tab. 2.1.

Tab. 2.5: Properties of the nine substances that showed in the validation concentration-dependent inhibition of RIIalpha-GST-AKAP18delta binding. MW = molecular weight in g / mol, logP = partition coefficient, H-accept. = Number of atoms that can act as acceptors in hydrogen bonds, H donor. = Number of atoms that can act as donors in hydrogen bonds

3rd discussion

Protein kinase A anchor proteins (AKAP proteins) have important functions in cAMP-dependent signaling pathways. They are expressed tissue-specific, and various AKAP proteins anchor protein kinase A (PKA) to various subcellular compartments. At the same time, they can be scaffold proteins for key components of other signaling pathways. It was u. a. developed an ELISA for the screening of a library of small molecule, drug-like substances. This method was used to find n concentration-dependent inhibitors of AKAP18delta-RIIalpha binding. Specific inhibition of PKA anchoring in combination with the properties of small molecules could not only serve as potential targets for novel drugs for the validation of AKAP proteins, but could also serve as a guide to their development.

3.1 The inhibitors of RIIalpha-AKAP18delta binding have pharmacologically interesting properties

An important requirement for active ingredients is the ability to cross biological membranes. Above all, their availability depends on cells, both in the organism and in cell culture models. The prerequisite for membrane permeability is the lipophilicity of a substance. On the other hand, the driving force for a membrane transfer is a possible: high concentration on a membrane side, which only through good solubility in an aqueous medium can be achieved, which is inversely proportional to lipophilicity. One of several models for predicting membrane permeability is the distribution of the substance in a 1-octanol-water mixture. From the distribution the distribution coefficient P (v. English, partition coefficient) can be calculated:

P = [substance] i_octanoi / [substance] _H 2o

The logarithm of this value is usually given, log P. For the effective inhibitors, the log P values are given in Tab. 2.5. If the value is greater than 1, the largest part of the substance accumulates in the organic phase, if it is less than 1, then the largest part accumulates in the aqueous phase. Since the log P values for the substances found are greater than 1, they are all expected to have good membrane permeability. One factor that adversely affects the membrane permeability of organic molecules is the ability to form hydrogen bonds. This type of noncovalent binding ensures that the molecules are surrounded by a hydrate shell, which must be stripped off again before the membrane is passed through with energy expenditure. However, in addition to the hydrophobic interactions, the hydrogen bonds are also the most important forces that lead to the noncovalent binding of an active substance to its target structure - in this case to the RIIalpha subunit of PKA or AKAPlδdelta. One way to assess hydrogen bonding ability is to simply count hydrogen bond donors and acceptors. The number The hydrogen bond donors and acceptors are also given in Tab. 2.5.

Another possibility is the computer-aided creation of a model; For example, for an HIV-1 protease inhibitor, the contribution of, inter alia, hydrogen bonds to the total bond was determined by calculating possible trajectories of the binding partners taking into account their molecular dynamics. Then, the free-energy fractions were analyzed to obtain information about the forces that drive the formation of the protein-inhibitor complex. Another property that affects the availability of small molecules in cells is their spatial extent. The spatial extent of a molecule can be approximated by its molecular weight. Both absorption of a substance and its elimination via bile have been shown to depend on molecular weight: lower molecular weights result in better absorption and less gelling elimination. An essential feature of all substances contained in the substance library is the relatively low molecular weight of on average 250 g / mol (see Table 2.5). Since none of the above methods alone makes reliable predictions possible, especially since the hydrogen bond binding potential must be considered in the context of lipophilicity, Lipinski et al. set up the so-called rules of five. These rules define which substance properties cause poor membrane permeability: ^■ More than five H-bond donors (= sum of OH and NH groups) ^■ more than ten ^{hydrogen bond} acceptors (sum of N and O atoms)

■ molecular weight above 500 g / mol

^■ Distribution coefficient log P> 5 The active substances identified by the screening fulfill all these conditions (Table 2.5), so that they are permeable to membranes.

Nevertheless, as a goal for new active substances and for the systematic modification of the structures found to improve their effectiveness, these values should be lowered as far as possible; Preferred structures according to the invention have these lowered values.

3.2 The applications of the inhibitors are determined by their specificities

After the screening, validation (Table 2.4, Fig. 2.11 and 2.12) for certain inhibitors was shown to inhibit the binding of the PKA subunit RII-alpha to GST-AKAP18delta in a concentration-dependent manner. Since all of the identified substances have hydrophobic aromatic ring systems, binding in the hydrophobic pocket formed by the RII subunits is possible. On the other hand, hydrogen bonding also allows binding directly to the RII binding domain of the AKAP protein.

Since selected substances specifically bind to AKAP18delta, the consequences of abolishing the compartmentalization of this signaling pathway can also be investigated in vivo (see below). Other substances bind specifically to other A-KAP proteins, so that they can be used to study other signaling pathways. Advantageous substances specifically bind AKAP18 proteins, ie all splice variants alpha, beta, gamma and delta, thus excluding them from interaction with RII subunits, since all isoforms have the same RII binding domain (Figure 1.5).

However, when one of the substances binds to the RII subunit, one has a tool that can generally inhibit interactions between AKAP proteins and RII subunits. The elucidation of the binding sites is possible with computer-assisted modeling, in which the structures of the determined substances can be checked for possible interacting regions with the structures of RIIalpha or the AKAP-RII binding domain.

3.3 The AKAP-PKA inhibitors are lead compounds for new drugs

The inhibitors of RIIalpha-GST-AKAP18delta binding are advantageously further developed into a new class of drugs.

One possibility of using inhibitors of PKA-AKAP18delta binding (but also of binding with other AKAP molecules) as novel drugs results from the involvement of AKAPlδdelta in the AVP-induced translocation of AQP2 into the apical membrane of collection tube cells of the Kidney (AQP2 shuttle).

AKAPlδdelta anchors the PKA to the AQP2-containing vesicles, allowing the catalytic subunits to specifically phosphorylate the water channel proteins following AVP stimulation, thus transporting and fusing the vesicles to the apical plasma membrane entails. This increases the water permeability of the membrane.

In the rat model, it was shown that water retention can occur in certain diseases, such as heart failure, cirrhosis of the liver, hypertension, as well as during pregnancy. This can u. a. in edema.

So far, the retention of water is treated with so-called diuretics. Diuretics indirectly increase the excretion of water through the kidneys by inhibiting Na ⁺ , K ⁺ , and Ca ²⁺ ion transporters, which normally carry ions from the primary urine back to the collecting duct epithelial cells. As a result, the osmolarity in the collecting tube increases and water follows osmotically through AQP2 molecules that are constitutively located in the apical cell membrane of the collecting tube epithelial cells. In addition to water retention, diuretics are also used in heart failure and hypertension, although adverse drug reactions (ADRs) can occur as a result of the severe loss of electrolytes.

One class of drugs without the electrolyte lost UAW are the aquaretics. They cause an increased water excretion and represent a potential replacement for conventional diuretics. As aquaretically acting drugs so far only the vasopressin receptor antagonists are known.

Due to the involvement of AKAP18delta in the AQP2 translocation, the inhibitors of RIIalpha-GST-AKAP18delta binding can be further developed into novel aquarium chemicals. Decoupling of PKA from the AQP2-bearing vesicles by specific inhibition of A-KAP18delta-PKA binding (see Section 1.7) interrupts the AVP-mediated signaling cascade, so that in spite of the AVP stimulus, the incorporation of AQP2 molecules is inhibited in the membrane. Thus, the water reabsorption can not be increased and there is an increased water extraction with the urine. In this mode of action - in contrast to that of diuretics (see above) - the ion transport back into the collecting duct epithelial cells is not impaired. An active substance with this property would thus be an aquarium, with which the diuresis could be specifically enforced.

A prerequisite for the development to a drug is an optimization of the inhibitory molecules. These are effective in lower concentrations so that they can also be used in animal models to experimentally verify the expected effect as an aquarium. For example, if rats excreted more water after the administration of optimized inhibitors, AKAP proteins would also be validated as a possible target for pharmaceuticals. AKAPlδdelta is not only expressed in the kidney, but also in other tissues. Particularly strong is the expression in the heart, where other AKAP proteins take over important functions. It could be shown in the cell culture model of cardiomyocytes that beta-adrenergic regulation of Ca ²⁺ currents via L-type Ca ²⁺ channels (Ca _v l .2 channels) leads to AKAP-mediated PKA anchoring Requirement is. AKAP18alpha (= AKAP15) binds via a leucine zipper motif to the C terminus of the pore-forming alpha subunit of the Ca ²⁺ channel. The TIALLY the AKAP-PKA protein ^¬ dene sets after activation of beta-adrenergic re zeptor- / cAMP pathway their catalytic subunits which can then phosphorylate a serine residue in the alpha subunit of the channel. This increases the probability of opening the channel and increases the Ca ²⁺ influx. This has a positive i-notrop and a positive chronotropic effect.

In the same study, it was shown that the use of the anchor inhibitor peptide Ht31 abrogates the beta adrenergic receptor-activated PKA-dependent increase in Ca _v l .2 channel activity. The low-molecular-weight inhibitors according to the invention have the same effect as beta-blockers in heart muscle cells. Beta-blockers are drugs that competitively inhibit the action of the neurotransmitters epinephrine and norepinephrine on the beta-adrenergic receptors of the respective target cells. A distinction is betai-, beta2 ~ and beta3 ~ receptors, which are expressed tissue-specific. Norepinephrine has stronger effects on the former and weaker effects on the latter. In heart muscle cells, predominantly beta-receptors are expressed, and there are beta-selective (= cardioselective) and nonselective beta-blockers. Beta-blockers have negative inotropic and negative chronotropic effects on the heart. Since this leads to a reduced oxygen demand of the heart muscle, beta-blockers are used for the treatment of coronary heart disease. In addition, beta-blockers act via an unknown mechanism relaxing on the vascular musculature, which is why they are also used to treat high blood pressure. Inhibitors of binding of AKAP proteins to RII subunits are a potential alternative to beta-blockers. Targeted blocking of the AKAP18alpha-dependent signaling pathway may make them more specific than beta-blockers, as the latter block all signaling pathways. to block the receptor. The AKAP18 inhibitors could also be used in contraindication of beta-blockers, e.g. In asthma.

However, the new drugs could lead to ADRs in skeletal muscle, as they also express L-type Ca ²⁺ channels and AKAP18alpha.

Apart from heart muscle cells, Ca _v l.2 channels and A-KAP18alpha ^' also occur in dendrites and cell bodies of neurons in the brain. Therefore, it is possible that AKAPlδalpha is also involved in the regulation of Ca _v l .2 channels. This opens up another potential application for the inhibitory molecules.

3.4 Substances are new tools to block PKA anchorage

As described in Section 1.8, the protein-protein interaction between AKAP proteins and RII subunits of PKA has so far been inhibited with peptides (Ht31, AKA-Pis). In this way, however, no targeted inhibition of a specific AKAP-RII complex is possible because the peptides of the RII binding domain of Ht31 correspond or were generated from a calculated from different AKAP proteins consensus binding site and so all in the investigated Block existing regulatory subunits.

Since all RII-binding domains of AKAP proteins are very similar, it is questionable whether with small molecules a specific inhibition of certain AKAP-PKA complexes is possible. On the other hand, the slight differences in the RII binding domains may allow a specific inhibition of the AKAP-PKA interaction after optimization (see below) of the substances. Another disadvantage of the peptides is that they are not membrane permeable. For experiments in which membrane permeability is needed, for example on cell culture models, the peptides therefore had to be acylated. With the help of the low molecular weight inhibitors described here, this disadvantage can be overcome. The chemical-physical properties of the substances according to the invention allow the direct use in cell cultures and also in animal models, thus for the first time the investigation of the function of AKAP proteins in vivo is possible.

In addition, the almost unlimited structural diversity of small organic molecules can be exploited in order to optimize the identification of starting substances with the aim of stronger binding to the protein surface, better stability, less nonspecific side effects and increased in vivo availability.

The low molecular weight inhibitors of the preferred RIIalpha GST-AKAP18delta interaction allow additional experiments that provide further information on their properties, their mode of action and their specificity. First of all, the established ELISA can be used to check whether the substances do not exert non-specific effects - such as denaturation - on proteins. For this purpose, RIIalpha or GST-AKAP18delta can be bound to MTP and admixed with increasing concentrations of the substances. Subsequently, the proteins can use the PCA _R ii _a ipha- Al8delta3 or antibodies to be detected. An initial review of the specificity of the low molecular weight inhibitors is also possible with the ELISA by binding RIIalpha to MTP and adding various recombinant AKAP proteins in the presence of increasing inhibitor concentrations. If all AKAP-Rallla interactions are inhibited, this indicates binding of the inhibitor to RIIalpha. If the inhibition of AKAP protein to AKAP protein is different, the inhibitors probably bind to the slightly different RII binding domains of the AKAP proteins.

In addition to these investigations, three further experiments are mentioned here, which can be carried out immediately with already proven reagents and methods. They can provide information about which of the substances determined are also effective in cells, in particular if the cells are part of an organism, and therefore can be considered for optimization.

First, collecting tube cells from the inner medulla of rat kidneys (IMCD cells, v. Inner medullary collecting duct) in the presence or absence of the substances can be stimulated with AVP to stimulate AQP2 translocation. Subsequently, immunofluorescent stains can be performed. The cells are fixed and permeabilized after incubation. Using fluorescence-dye-coupled antibodies, the intracellular localization of AQP2 can then be determined under the fluorescence microscope. In this way, the influence of the substances on the presumably AKAPlδdelta-dependent AQP2 translocation can be investigated. Another experiment is the measurement of Ca ²⁺ currents by L-type Ca ²⁺ channels in primary cultured neonatal rat heart muscle cells using patch clamps. Technology. Membrane sections of a single cell are fixed with a buffer-filled glass capillary and a constant voltage is applied across the membrane. By means of a current pulse, a measurable increase in the Ca ²⁺ current can be triggered by the voltage-dependent L-type Ca ²⁺ channels located in the fixed membrane region.

After stimulation of the cells with the nonspecific beta-adrenoceptor agonist isoproterenol, an increase in the Ca ²⁺ channel current is to be expected, since the activation of the receptor triggers the A KAP18alpha-dependent signaling cascade described in Section 3.3. As a result, the L-type Ca ²⁺ channel is phosphorylated, which increases its probability of opening. Such measurements can be carried out in the presence or absence of the substances so that the effect on the signal path can be quantified indirectly via the measured currents. Finally, another experiment with the said heart muscle cells is possible, in which the cells are preincubated with isoproterenol and the substances. Thereafter, a membrane-permeable, fluorescent Ca ²⁺ indicator is added to the cells. In this way, the Ca ²⁺ distribution in the cell can be visualized, and with the aid of a laser scarming microscope (LSM), the Ca ²⁺ concentration changes can also be analyzed in a time-resolved manner. In the presence of the substances, weaker changes in the Ca ²⁺ concentration are expected if they have an inhibitory effect on the AKAP-PKA interaction and thus on the phosphorylation of the Ca ²⁺ channel. Should it be shown in these experiments that the substances in the cell culture model also allow an AKAP18delta-specific or a non-specific inhibition of the interaction with the PCA, an optimization can be carried out with the aim of improving the concentration-dependent efficacy.

The first step in an optimization is computational modeling to get an idea of which functional groups of the molecules could interact with which amino acids in the proteins.

On this basis, functional groups of the molecules can be systematically exchanged in accordance with the Lipinski rules (see Section 3.1) in order to increase the affinity for the binding partner.

In addition, it can be tried whether an additive or synergistic effect can be achieved by a combined use of the inhibitors. If improvements can be made in this way, an attempt can be made to purposely synthesize a molecule that links the most effectively interacting functional groups.

Of the substances thus obtained, lower concentrations would be required to achieve the same effect. This advantageously allows use in vivo, reducing the likelihood of nonspecific or toxic effects.

The figures represent the following facts: Fig. 1:

Schematic representation of the expression of GST

Fusion proteins used vector pGEX-4T3.

Fig. 2:

The molecular weight standard HyperLadder I DNA Marker.

3:

The molecular weight standard Invitrogen BenchMark prote-in ladder for SDS-PAGE.

4:

Schematic representation of the ELISA for the quantitative detection of protein-protein binding between PKA-RIIalpha and GST-AKAP18delta. In each well of a 384-well MTP

(left) one of the following reactions takes place: A - without inhibitor binds GST-AKAP18delta to the plate-bound RIIalpha and is therefore detectable with the antibodies by chemiluminescence. B - If small molecules are added which bind to one of the two binding partners (here GST-AKAP18delta), both GST-AKAPlδdelta and the antibodies are removed in the subsequent washing steps and no chemiluminescence can be generated. C - The same applies to the inhibition of binding by inhibitory peptides. However, these always bind to Rllalpha.

Fig. 5:

Competent E. coli cells were transformed with pGEX-AKAPlδdelta, the plasmid DNA was prepared from 4 clones, subjected to restriction digestion and analyzed by agarose gel electrophoresis. Clone 1 ung = untreated plasmid from clone 1, clones 1-4 = EcoRI and Xhol-treated plasmids of clones 1-4. The digested plasmids show the expected fragments of 4.9 kb and 1 kb.

Fig. 6 A and B:

Analysis of SDS-PAGE sample buffer eluates of glutathione-Sepharose beads to which the recombinant GST-AKAP18b (75 kDa) or the GST (25 kDa) had been linked after purification with different lysis and binding conditions by SDS-PAGE Coomassie staining. In each case 5 .mu.l of the sample buffer eluates were applied.

A: Variation of lysis conditions. GST = product of the empty pGEX vector; GST-I8delta = product of pGEX-

AKAP18delta; FP = French Press; DTT = addition of DTT before one-time French Press lysis; Lyso = lysis with lysozyme; RT = room temperature; M standard = molecular weight standard.

B: variation of binding conditions. The bacterial suspension was in each case Iysed three times by French Press.

Fig. 7: By stepwise alteration of the elution buffer composition, elution of GST-AKAP18delta from the glutathione (GSH) -sepharose beads could be optimized. In the first and last column, excerpts from Coomassie-stained SDS-PAGE (AI) and Western Blots (J) can be seen. The arrows in the last column indicate which elution conditions were applied to the beads before being analyzed. It will be respectively the section between 70 and 80 kDa shown. In the second and third column, the applied sample volumes are shown, in the penultimate the elution fraction and in the remaining columns the composition of the elution buffer. V = volume, c = concentration, TX = Triton X-100, T-20 = Tween 20, E = eluate no.

Fig. 8:

A - BSA, BSA ELISA and skimmed milk powder were placed in binding buffer. After addition of chemiluminescent substrate, the intensity of the nonspecific signals was determined (individual determinations).

B binding buffer with increasing RIIalpha concentrations (expressed as total mass m (RIIalpha)) was pipetted into MTP, blocking nonspecific binding sites with blocking buffer, and the amount of bound protein with PKA _RIIa ip _ha - and anti-mouse POD-Ab detected. C - Bindepuf ^¬ fer with constant amounts Rllalpha (25 and 40 ng / well) was pipetted onto MTP, nonspecific binding sites with blocking buffer, blocked, blocking buffer with increasing concentrations of GST-AKAP18delta added and the amount of bound GST-AKAP18delta with A18delta3- and anti Rabbit POD Ab detected. I = intensity, RLU = relative light units, m = mass.

Fig. 9:

A-C - Binding series were each made (as in Fig. 8C).

A - For binding sequences with 15 and 25 ng of constant RII-alpha, the chemiluminescence intensities were determined. B - binding sequences were detected with different Al8delta3 and constant anti-rabbit POD-Ak dilutions.

C - binding sequences were detected with constant A18delta3 and various anti-rabbit POD-Ak dilutions.

Fig. 10:

A - Constant RIIalpha levels were given 0, 80 or 160 ng GST-AKAP18delta. The detection was carried out with constant A18delta3 dilutions and with 1: 3000 (3k) or 1: 10000 (10k) dilutions of the secondary (2) anti-rabbit POD Ab. Each experiment was performed in the presence (+) or absence (-) of 1% DMSO. B - Binding of constant GST-AKAP18delta levels to constant RIIalpha levels was monitored in the presence of increasing DMSO concentrations (0-2.5 %) detected. C - Bonding sequences were prepared with 0 and 25 ng Rllalpha and detected to detect the specificity of the signals.

Fig. 11:

To calculate IC _ö o values, the binding between constant RIIalpha and GST-AKAP18delta levels was detected in the presence of increasing peptide concentrations. A single binding site model was adapted to the measured values (see Section 1.11). While L314E and Ht31 competitively inhibit binding, 18d-PP and Ht31-P are used as negative controls. Fig. 12:

Binding series were prepared in which the RIIalpha-GST-AKAP18delta interaction was disrupted by washing after 15, 30 or 60 minutes. Then the amount of bound GST-AKAP18delta was detected.

Fig. 13:

A - For constant RIIalpha and GST-AKAP18delta levels, the peptides L314E and 18d-PP were added at concentrations of 0.01 or 10 μM for 15, 30 or 60 min. Subsequently, the amount of bound GST-AKAP18b was detected

B - RIIalpha-GST-AKAP18delta complexes at constant concentrations were detected with the A18delta3 and the anti-rabbit POD Ab for all possible combinations of incubation times 15, 30 and 60 min. The display of error indicators was not possible in this figure. C - To the same RIIalpha-GST-AKAP18delta binding series were added the Ab and the chemiluminescent substrate. The chemiluminescence intensity was measured after 2, 4 and 8 minutes.

Fig. 14: MTP were coated with equal amounts of RIIalpha for 1 h. Then blocking buffer was added and the MTP incubated for 1 h at room temperature or for 66 h at 4 ⁰ C. Subsequently, GST-AKAP18delta was added in increasing concentrations. The amount of bound GST-AKAPlδdelta was detected with A18delta3 and anti-rabbit POD-Ab. Fig. 15:

A - The RIIalpha-GST-AKAP18delta binding curve shows that the remaining experiments, all performed with 50 nM GST-AKAP18delta, were in the sensitive range.

B - Binding was also inhibited in a concentration-dependent manner using the inhibitory peptide L314E. C - Summary of validation experiments with increasing inhibitor concentrations. Shown are the 9 hits and, for comparison, the non-active compound 2348. D- L - Concentration-dependent effect of each inhibitor, structural formula and adaptation of a one-binding-site competition model (see Section 1.11) to the measured values (individual determinations, therefore no error indicators) calculated IC ₅₀ values (DL: individual determinations). Continued (HL) on the next page.

FIG. 16: Part 2 of FIG. 15. Part 1 and legend see FIG. 15.

Fig. 17:

Schematic representation of the ELISA for the quantitative detection of the protein-protein binding between PKA-RIIalpha or PKA-RIIbeta and GST-AKAP18delta (glutathione-S)

Transferase fusion with AKAP18delta). The interaction occurs in the absence or presence of low molecular weight substances in 384-well ELISA plates. Proof of protein interaction is provided by primary anti-AKAP18delta and secondary peroxidase-coupled antibodies and chemiluminescence. Fig. 18: Inhibition of vasopressin (AVP) -dependent redistribution of the aquaporin-2 (AQP2) water channel in renal main cells by the indicated substances (see Table B, concentrations of inhibitors in μM). Primarily cultured main cells from the inner medulla of rat kidneys (IMCD

Cells) represent a model for the AVP-induced translocation of AQP2. This redistribution forms the molecular basis of AVP-induced water reabsorption in the kidney. AQP2 was detected by a specific antiserum and Cy3-labeled secondary antibody in untreated (control) and AVP- or substance-treated cells with the laser scanning microscope {Tamma et al. , 2003; Henn et al. , 2004). FIG. 19: Clusters of substances that influence the interaction of A-

KAPlδdelta with regulatory Rllalpha subunits of PKA inhibit. All substances inhibiting the interaction (see Tables A and B) were examined for structural similarities. The numbers under the names Structures 1-18 correspond to the Com_ID (see Tables A and B). There was a structural similarity between substances 1-4, 5 and 6, 7 - 9, 11 and 12, 13 and 14, 15 and 16 and between 17 and 18. Substance 10 had no resemblance to other interaction-inhibiting substances. Generalizations of the structures are shown in FIG. Fig. 20

Generalization of Structures 1-17 Illustrated in FIG. 19 FIG. 21: FIG.

Effect of the low molecular weight substances 18882, 990 and the 990 derivatives SM61 and SM65 on the AVP-induced conversion Distribution of AQP2 in IMCD cells (see Fig. 18). The structures and chemical properties of the substances are shown in Tab. A, B and C (concentrations of inhibitors in μM). AQP2 was detected by means of a specific antiserum and Cy3-labeled secondary antibodies in untreated (control) and AVP- or substance-treated cells with the laser scanning microscope {Tamma et al. _r 2003; Henn et al., 2004). The low-molecular weight substance 990 inhibits the interaction of AKAP18δelta with regulatory RIIalpha subunits of PKA in IMCD cells (see Fig. 22). Similarly, 990 prevents AVP-induced AQP2 redistribution. Substance SM61, a 990 derivative, also inhibits AVP-induced AQP2 redistribution. In contrast, the substances 18882 and 990 derivative SM65 have no influence on the AVP-induced AQP2 redistribution at the concentrations used.

FIG. 22: The low molecular weight substance 990 inhibits the interaction of AKAP18delta with regulatory RIIalpha subunits of PKA in IMCD cells. The substance (see TABLES A, B and D) was identified by screening a substance library comprising 20064 compounds by means of the ELISA-based experimental set-up shown in FIG. Primarily cultured IMCD cells remained untreated (control), were incubated with substance 990 (100 μM) or as a negative control with cAMP for 30 min. Subsequently, the cells were lysed and subjected to cAMP agarose precipitation (Henn et al., JBC 279, 26654, 2004). A. AKAPlδdelta was used in the lysates and the cAMP agarose precipitates (cAMP agarose) of the anti-AKAP18delta specific antibody A18delta4 detected in Western blot (Henn et al., 2004). As a negative control, cAMP was added to the batch. This competitively inhibits the binding of regulatory subunits of PKA to cAMP agarose. Therefore, no AKAPs are precipitated and thus AKAP18delta can not be detected in this sample. The positive control used was recombinantly produced AKAP18delta. B. To detect AKAPs in the lysates and cAMP agarose precipitates, a RII overlay was performed (Henn et al., JBC 279, 26654, 2004). As a positive control, recombinantly produced AKAP18delta was detected. AKAP18delta is indicated by arrows in A. and B.

Fig. 23:

The small molecule 18882 inhibits the interaction of AKAP18δelta with regulatory RIIalpha subunits of PKA in cardiomyocytes. In contrast, substance 990 has no influence on this interaction. The substances (see Tables A, B and D) were identified by screening a substance library comprising 20064 compounds by means of the ELISA-based experimental set-up shown in FIG. Primarily cultured neonatal cardiomyocytes remained untreated (control), were incubated with the substances 990, 18882 (100 μM each) or as a negative control with cAMP for 30 min. Subsequently, the cells were lysed and subjected to cAMP agarose precipitation (Henn et al., JBC 279, 26654, 2004). A. AKAP18delta was detected in the lysates and cAMP agarose precipitates (cAMP agarose) using the anti-AKAP18delta specific antibody A18delta4 detected in Western blot (Henn et al., JBC 279, 26654, 2004). As a negative control, cAMP was added to the batch. This competitively inhibits the binding of regulatory subunits of PKA to cAMP agarose. As a result, no AKAPs are precipitated and A-KAPlδdelta can not be detected in this sample. The positive control used was recombinantly produced AKAP18 delta. B. Regulatory Rllbeta subunits of PKA were detected by Western blot in the same samples as in A. As expected, no Rllbeta was detected in the sample containing cAMP (see A.). As expected, the antibody does not recognize AKAP18delta. C. For non-selective detection of AKAPs in the lysates and cAMP agarose precipitates, a RII overlay was performed (Henn et al., JBC 279).

26654, 2004). As a positive control, recombinantly produced AKAP18delta was detected.

Fig. 24: Inhibition of L-type Ca2 + channel currents by the substance

18882. L-type Ca2 + channel currents were measured by patch-clamp technique in rat neonatal cardiomyocytes. The cells were maintained at -70 mV and repeatedly depolarized to a test potential of 0 mV (after 400 ms increase to -35 mV). Upper Fig.: The substances 18882 (see Tables A and B) and the solvent DMSO as a control were added to the cells in the concentrations indicated. Lower figure: Substances 990 and the 990 derivative SM61 (see Table C) were added to the cells at the concentrations indicated. Currents were measured 400 seconds after the establishment of the whole-cell configuration. The cells were incubated with isoproterenol (1 μM, ISO; ta receptor agonist) at the indicated times. Isoproterenol was washed out through the ES medium. The figure shows time courses of the normalized currents (normalized current, n corresponds to the number of measured cells, the error bars are means + SEM).

Tables A, B, C and D represent the following facts:

Table A Low molecular weight substances that inhibit the interaction of A-KAP18delta with regulatory RIIalpha subunits of PKA. The table shows 142 substances that inhibit interaction by at least 40%. Binding (in%), this value indicates the relative binding of GST-AKAP18delta to PKA-RIIalpha in the presence of the respective substance in percent relative to the control (binding of GST-AKAP18delta to PKA-RIIalpha in the absence of substance). The substances were identified by screening a substance library comprising 20064 compounds by means of the ELISA-based experimental set-up shown in FIG. Structures of the substances are shown. The substances are sorted according to their inhibitory effect. The list begins with the substances that most potently inhibit the interaction of AKAPlδdelta with PKA regulatory RIIa subunits.

MW (in g / mol), this value indicates the molecular mass of the substance. The molecular mass also represents an approximation of the spatial extent of the substance that is important for bioavailability (small molecules more easily pass through cellular membranes). Comp_ID, these numbers represent the passing substance Coding of the Leibniz Institute for Molecular Pharmacology (20064). Plate ID number of the sub-library ^¬ punched plate in which the substance was stored; Pos ID, position in the substance library plate where the substance was stored. Formula, the sum formula mentioned here, indicates the composition of the substances from the elements. logP, this value is the calculated logarithm of the distribution coefficient of the substance. It makes it possible to estimate the solubility of the substance in polar (aqueous) or non-polar ones

(membranous (lipid)) phases and therefore provides a first statement about the bioavailability in cellular systems. logSw, this value is the calculated logarithm of the solubility coefficient, which gives an indication of the solubility of the substance in water. H_acceptor, indicates the number of hydrogen bond acceptors (N and O atoms) in the substance. H_donor, indicates the number of hydrogen-bond donors (NH and OH groups) in the substance. Hydrogen bonds are the most important type of noncovalent bond between small molecules and proteins; therefore, hydrogen-bond donors and acceptors favor the potential of the substances to interact with proteins. B_rotN, this value corresponds to the number of atomic bonds in the substance, around which there is free rotation. A higher number of such bonds increases the conformational flexibility of the substance and thus the probability of accurately binding to a protein surface. Taken together, these data provide predictions about the bioavailability of chemical substances, as reported by Lipinski et al. (Lipinski, CA, Lom bardo, F., Dominy, BW & Feeney, PJ Experimental and Computational approaches to estimate solubility and per- meability in drug discovery and development settings. Adv. Drug Deliv. Rev. 46, 3-26 (2001).); For this, substances should fulfill the so-called "rule of five":

^■ a maximum of five H-bond donors (= sum of the OH and NH groups)

■ a maximum of ten H-bond acceptors (sum of N and O atoms) ^■ molecular weight max. 500 g / mol

^■ Distribution coefficient log P <= 5

Table B

Selected low-molecular-weight substances that inhibit the interaction of AKAP18delta with regulatory RIIalpha subunits of PKA. The table shows 9 substances from Table A that inhibit interaction by at least 80%. Binding (in%), this value indicates the relative binding of GST-AKAP18delta to PKA-RIIalpha in the presence of the respective substance in percent relative to the control (binding of GST-AKAP18delta to PKA-RIIalpha in the absence of substance). The IC50 value was determined by titration of the respective substance. The IC50 for inhibition of interaction was determined in the ELISA experiment described in FIG. MW (in g / mol), this value indicates the molecular mass of the substance. The molecular mass also represents an approximation of the spatial extent of the substance, which is important for bioavailability (small molecules easily pass through cellular membranes). Comp_ID, these numbers represent the continuous substance coding of the Leibniz Institute of Molecular Pharmacology (in particular overall 20064). Plate ID, number of the substance library plate in which the substance was stored; Pos ID, position in the substance library plate where the substance was stored. Formula, the formulas mentioned here, indicate the composition of the substances in the elements. logP, this value is the calculated logarithm of the distribution coefficient of the substance. It allows an estimation of the solubility of the substance in polar (aqueous) or non-polar (membrane (lipid)) phases and therefore provides a first statement about the bioavailability in cellular systems. logSw, this value is the calculated logarithm of the solubility coefficient, which gives an indication of the solubility of the substance in water. H_acceptor, indicates the number of hydrogen bond acceptors (N and O atoms) in the substance. H_donor, indicates the number of hydrogen-bond donors (NH and OH groups) in the substance. Hydrogen bonds are the most important type of noncovalent bond between small molecules and proteins; therefore, hydrogen-bond donors and acceptors favor the potential of the substances to interact with proteins. B_rotN, this value corresponds to the number of atomic bonds in the substance, around the free rotation prevails. A higher number of such bonds increases the conformational flexibility of the substance and thus the probability of accurately binding to a protein surface.

Table C Low molecular weight substances that modulate the interaction of A-KAP17δ with regulatory RIIalpha subunits of PKA. The substances represent derivatives the substance 990 shown in Tables A and B, which were developed by chemical modification of the substance 990. The structures, their chemical formulas (formula), their molecular weight (MW) and the logP value, the number of H-acceptors and H donors of each substance are shown. (Explanations of these parameters in the legends to Tab. A and B.) The inhibitory effect of each substance on the interaction of A-KAP18delta with regulatory Rllalpha subunits of PKA was up to three times (Screening no. 1-3) in the in Fig. 17 is tested ELISA experiment and determines the IC50 (mean in μM). The ratio of the IC50 of the derivative and the substance 990 was calculated (ratio IC50 (comp / 990). Values> 1 reflect a greater inhibition of the interaction with the original substance and values <1 mean that IC50 gives the results of the experiments carried out average IC 50 normalized and normalized to the mean IC50 of 990. For substances that did not show inhibition, a "-" was entered.

Table D

Low-molecular-weight substances SM-FP and 990, which inhibit the interaction of AKAP18delta with regulatory RIIalpha subunits of PKA. The substance SM-FP represents a derivative of the substance 990 shown in Tables A and B. SM-FP is characterized by the presence of a fluorescein group. The structures, their chemical formulas (formula), their molecular weight (MW) and the logP value, the number of H-acceptor and H-donors of each substance are shown. (Explanations of these parameters in the legends to Tab. A and B.) The inhibitory effect of each substance on the interaction of AKAP18delta with regulatory RIIalpha subunits of PKA was checked up to three times (screening no. 1-3) in the ELISA experiment depicted in FIG. 17, and from this the IC50 (mean in μM ) certainly. The ratio of the IC50 of the derivative and the substance 990 was calculated (ratio IC50 (comp / 990). Values> 1 reflect a greater inhibition of the interaction with the parent substance and values <1 mean IC50 gives the results of the experiments carried out average IC 50 normalized and normalized to the mean IC50 of 990. For substances that did not show inhibition, a "-" was entered.

Claims

claims

1. Non-peptidic protein kinase A / protein kinase A anchor protein decoupler according to Table A.

2. Non-peptidic protein kinase A / protein kinase A anchor protein decouplers having a molecular weight in the range of 150 to 600 g / mol, preferably from 190 to 300 g / mol, a distribution coefficient of logP ≤ 10, preferably ≤ 8, with a maximum of 10 Hydrogen bridge donors and a maximum of 10 hydrogen-bond acceptors, a solubility value of logSw -400 to 0 and a BrotN value of 0 to 7.

3. decoupler according to claim 2, characterized in that they have a maximum of 7, preferably 6 H-bridge donors, a maximum of 6, preferably 5, more preferably 4 hydrogen bridge acceptors and / or a logP value of ≥ 1 to ≦ 8, preferably ≥ 1 to ≦ 5.

4. Decoupler according to one of the preceding claims, characterized in that it detects the interaction of AKAP and PKA

Subunits by at least 40%, preferably by at least 80% inhibit.

5. Decoupler according to one of the preceding claims, selected from Table B.

6. Decoupler according to one of the preceding claims, selected from Table C.

7. Decoupler according to one of the preceding claims, selected from Table D.

8. decoupler according to one of the preceding claims according to the general formula I, which are interconvertible by mesomerism (R ² and R ³ are treated as exchangeable)

wherein X is a non-hydrogen atom, preferably a sulfur atom,

where R ^{1 is} an alkyl or aryl radical, preferably a 1-naphthylmethyl radical,

where R ² and R ^{3 are} a hydrogen atom or an alkyl or aryl radical, preferably R ² and R ^{3 are} two hydrogen atoms, two methyl radicals, one benzyl radical and one methyl radical or one benzyl radical and one tert-butyl radical,

R ² and R ³ particularly preferably represent a 2-thiazolidinyl radical and a methyl or tert-butyl radical

Butyl radical, as well as a 1-naphthyl radical and an isoproyl radical pyl, cyclohexyl, benzyl or methyl radical; or above according to the general formula II

(Rotation about single bond ^

1 2 3 4 5

wherein X, R ¹ , R ² and R ^{3 have} the same meaning as in formula .1.

9. Decoupler according to one of the preceding claims, characterized in that they comprise a structure according to FIG. 19.

10. Decoupler according to one of the preceding claims, characterized in that they comprise a structure according to FIG. 20.

11. Decoupler according to one of the preceding claims, characterized in that the bonds of AKAP18 proteins, preferably A-KAPlδdelta proteins and / or RI alpha and / or RII-alpha and / or RIbeta and / or Rllbeta be inhibited.

12. decoupler according to one of the preceding claims for the surgical or therapeutic treatment of the human or animal body and / or for the use of diagnostic procedures that are performed on the human or animal body.

13. Pharmaceutical agent comprising at least one decoupler according to any one of claims 1 to 12, additionally comprising at least one pharmaceutical carrier and / or excipients.

14. Pharmaceutical agent according to the preceding claim, characterized in that the carrier is selected from the group comprising fillers, disintegrants, binders, humectants, extenders, dissolution inhibitors, resorpti- onsbeschleuniger, wetting agents, adsorbents and / or lubricants.

15. Pharmaceutical agent according to one of the preceding claims, characterized in that it is a capsule, a tablet, a dragee, a suppository, an ointment, a cream, a patch, an injection solution and / or an infusion solution.

16. A recognition molecule directed against the decoupler according to one of claims 1 to 12, wherein the recognition molecule is an antibody, a complexing agent and / or a chelator.

17. A kit comprising a decoupler according to one of claims 1 to 12, a pharmaceutical agent according to any one of claims 13 to 15 and / or a recombinant The detection molecule according to claim 16, optionally together with information about the combination and / or handling of the components of the kit.

18. Use of the decoupler and / or the pharmaceutical agent according to any one of the preceding claims and / or the recognition molecule according to claim 16 for the modification, in particular an inhibition of an AKAP-PKA interaction.

19. Use according to the preceding claim, characterized in that the modification of the interaction in a cell, a cell culture, a tissue and / or a target organism are performed.

20. Use according to the preceding claim, characterized in that the modification of the interaction, the vasopressin-induced redistribution of AQP2 is modified, in particular is prevented.

21. Use according to one of the preceding claims, characterized in that the interaction of RIalpha, Rllalpha, Rlbeta and / or Rllbeta Üntereinheiten the PKA modified with AKAP, in particular is inhibited.

22. Use according to one of the preceding claims, characterized in that the agents according to claim 1 to 17 specifically to A-KAP, preferably AKAP18, particularly preferably A- KAPlδdelta and / or specific and PKA, preferably subunits of these, most preferably bind to RII subunits.

23. Use according to the preceding claim, characterized in that the subunits are of human and / or murine origin and / or are obtained from rats.

24. Use of the decoupler or of the pharmaceutical agent according to one of the preceding claims in vitro or in vivo for influencing a preferably compartmentalized cAMP-dependent signal transduction.

25. Use of the decoupler or pharmaceutical

Composition according to one of the preceding claims for the manufacture of a medicament for the prophylaxis or treatment of diseases associated with defects of a compartmentalized cAMP-dependent

Signal transduction, selected from the group comprising asthma of any kind, etiology or pathogenesis, or asthma from the group of atopic asthma, non-atopic asthma, allergic asthma, IgE-induced atopic asthma, bronchial asthma, essential asthma, primary asthma, endogenous asthma caused by pathophysiological disorders , exogenous asthma caused by environmental factors, essential asthma of unknown or inapparent cause, non-atopic asthma, bronchial asthma, emphysematous asthma, exercise-induced asthma, occupational asthma Bacterial, fungal, protozoal or viral infections induced infectious allergic asthma, non-allergic asthma, inhaled asthma, wheezy in- fant syndrome; chronic or acute bronchoconstriction, chronic bronchitis, small airway obstruction, and emphysema; obstructive or inflammatory respiratory disease of any kind, etiology or pathogenesis, or an obstructive or inflammatory respiratory disease from the group of asthma; Pneumoconiosis, chronic eosinophilic pneumonia; chronic obstructive pulmonary disease (COPD); COPD, including chronic bronchitis, pulmonary emphysema or associated respiratory distress, COPD characterized by irreversible progressive airway obstruction, adult respiratory distress syndrome (ARDS) and exacerbation of respiratory hypersensitivity due to treatment with other drugs; Airborne pneumonia of any kind, etiology or pathogenesis, or pneumoconiosis from the group aluminosis or aluminum pneumoconiosis, anthracosis (asstosis), asbestosis or asbestos sting, chalicosis or calcified pneumonia, ptilosis caused by inhalation of ostrich feather dust, siderosis caused by inhalation of iron particles, silicosis or stinging stone pneumonia .

Byssinosis or cotton dust pneumoconiosis as well

Talk pneumoconiosis;

Bronchitis of any kind, etiology or pathogenesis, or bronchitis from the group of acute bronchitis, acute laryngotracheal bronchitis, peanut-induced bronchitis, bronchial catarrh, croupous Bronchitis, bronchitis without expectoration, infectious asthma bronchitis, bronchitis with sputum, staphylococcal or streptococcal bronchitis; as well as vesicular bronchitis; Bronchiectasis of any kind, etiology or pathogenesis, or bronchiectasis from the group of cylindrical bronchiectasis, saccular bronchiectasis, spindle-shaped bronchiectasis, bronchiolar dilatation, cystic bronchiectasis, bronchiectasis without sputum, and follicular bronchiectasis; Seasonal allergic rhinitis, perennial allergic rhinitis, or sinusitis of any kind, etiology or pathogenesis, or sinusitis from the group of purulent or non-conductive sinusitis, acute or chronic sinusitis, ethmoiditis, frontal sinusitis, maxillary sinusitis or sphenoiditis; rheumatoid arthritis of any kind, etiology or pathogenesis, or rheumatoid arthritis from the group of acute arthritis, acute gouty arthritis, primary chronic polyarthritis, osteoarthritis, infectious arthritis, Lyme arthritis, progressive arthritis, psoriatic arthritis, and spondylarthritis; Gout, inflammation-associated fever, or inflammation-associated pain; a morbid disorder of any kind, etiology or pathogenesis associated with eosinophils, or a morbid disorder associated with eosinophils from the group of eosinophilia, eosinophilic lung infiltrate, spoonbill

Syndrome, chronic eosinophilic pneumonia, tropical pulmonary eosinophilia, bronchopneumonic aspergillus Aspergillus, eosinophilic granuloma, allergic granulomatous angiitis or Churg-Strauss syndrome, polyarteritis nodosa (PAN), as well as systemic vasculitis necroticans; atopic dermatitis, allergic dermatitis, or allergic or atopic dermatitis;

Hives of all types, aetiology or pathogenesis, or hives from the group of immune hives, complement hives, hives-inducing hives, hives induced by physical stimuli, stress-induced hives, idiopathic hives, acute hives, chronic hives, angioneurotic edema Urticaria cholinergica, cold urticaria in its autosomal dominant form or in its acquired form, contact urticaria, urticaria gigantean and papular urticaria; Conjunctivitis of any kind, etiology or pathogenesis, or conjunctivitis from the group of conjunctivitis actinica, acute catarrhal conjunctivitis, acute contagious conjunctivitis, allergic conjunctivitis, atopic conjunctivitis, chronic catarrhal conjunctivitis, purulent conjunctivitis and spring conjunctivitis;

Uveitis of any kind, etiology or pathogenesis or uveitis from the group inflammation of the entire uvea or a part thereof, anterior uveitis, iritis, cyclitis, iridocyclitis, granulomatous uveitis, non-granulomatous uveitis, phakoantigen uveitis, posterior uveitis, choroiditis and choriorethinitis; Psoriasis; multiple sclerosis of any kind, etiology or pathogenesis, or multiple sclerosis from the group of primarily progressive multiple sclerosis as well as multiple sclerosis with relapses and a tendency to remissions;

Autoimmune / inflammatory diseases of any kind, etiology or pathogenesis, or an autoimmune / inflammatory disease from the group autoimmune hematological disorders, hemolytic anemia, aplastic anemia, aregenerative anemia, idiopathic thrombocytopenic purpura, systemic lupus erythematosus, polychondritis, scleroderm, Wegener's disease Granulomatosis, light disease, chronic active hepatitis, myasthenia gravis, Stevens-Johnson syndrome, idiopathic sprue, autoimmune

Irritable bowel diseases, ulcerative colitis, Crohn's disease, endocrine opthamopathy, Graves' disease, sarcoidosis, alveolitis, chronic hypersensitivity pneumonitis, primary biliary cirrhosis, insulin deficiency diabetes or type 1 diabetes mellitus, anterior uveitis, granulomatous uveitis or uveitis posterior, keratoconjunctivitis sicca, keratoconjunctivitis epidemica, (diffuse) interstitial pulmonary fibrosis, pulmonary cirrhosis, cystic fibrosis, psoriatic arthritis, glomerulonephritis with and without nephrosis, acute glomerulonephritis, idiopathic nephrosis, minimal-change nephropathy, inflammatory / hyperproliferative skin diseases, psoriasis, atopic dermatitis, contact dermatitis , allergic contact dermatitis, familial benign Pemphigus, pemphigus erythematosus, pemphigus foliaceus, and pemphigus vulgaris;

Prevention of allograft rejection after organ transplantation, inflammatory bowel disease (IBD) of any kind, etiology or pathogenesis, or irritable bowel from the group of ulcerative colitis (UC), collagenous colitis, politis polyposa, transmural colitis and Crohn's disease (CD); septic shock of any kind, aetiology or pathogenesis, or septic shock from the group of renal failure, acute renal failure, cachexia, macularachexia, pituitary cachexia, uremic cachexia, cardiac cachexia, cachexia suprarenalis or adadon's disease, carcinomatous cachexia, and cachexia Infection by Human Immunodeficiency Virus (HIV); Liver damage; pulmonary hypertension and pulmonary hypertension caused by hypoxia;

Bone wasting, primary osteoporosis and secondary osteoporosis; disorders of the central nervous system of any kind, etiology or pathogenesis, or a disorder of the central nervous system from the group depression, Parkinson's disease, learning and memory disorders, tardive dyskinesia, drug dependence, atherosclerotic dementia, and dementia as a concomitant of Huntington's chorea, Wilson's disease, paralysis agitans and thalamic atrophies; Infections, particularly viral infections, these viruses being the production of TNF-α in their host or which viruses are susceptible to upregulation of TNF-α in their host, so as to hinder their replication or other important activities, including viruses from the group consisting of HIV-1, HIV-2 and HIV-3, cytomegalovirus, CMV; Influenza, adenoviruses and herpesviruses, including herpes zoster and herpes simplex; Yeast and fungal infections, where these yeasts and fungi are sensitive to upregulation by TNF-α or TNF-α production in their

Host, preferably fungal meningitis, especially when coadministered with other drugs of choice for the treatment of systemic yeast and fungal infections, including the polymycins, preferably polymycin B, imidazoles, preferably clotrimazole, econazole, miconazole and / or ketoconazole, the triazoles Fluconazole and / or ilanazole, as well as the amphotericins, preferably amphotericin B and / or liposomal amphotericin B.

26. Use according to one of the preceding claims, characterized in that the pharmaceutical agent according to any one of claims 13 - 15 as gel, powder, powder, tablet, sustained-release tablet, premix, emulsion, infusion formulation,

Drop, concentrate, granules, syrup, pellet, boiled egg, capsule, aerosol, spray and / or inhalant is prepared and applied.

27. Use according to the preceding claim, characterized in that a pharmaceutical agent according to any one of claims 13 - 15 in a concentration of 0.1 to 99.5, preferably from 0.5 to 95.0, particularly preferably from 20.0 to 80.0 wt .-% in a preparation ,

28. Use according to the preceding claim, characterized in that the preparation is set orally, subcutaneously, intravenously, intramuscularly, intraperitoneally and / or topically.

29. Use according to one of the preceding claims, characterized in that a pharmaceutical agent according to any one of claims 13 - 15 in total amounts of 0.05 to 500 mg per kg, preferably from 5 to 100 mg per kg body weight, per 24 hours is used.

30. Use according to one of the preceding claims, characterized in that at least one pharmaceutical agent according to any one of claims 13 to 15 are brought into contact with an organism, preferably a human or an animal.

31. Use according to one of the preceding claims, characterized in that the contacting takes place orally, via injection, topically, vaginally, rectally and / or nasally.

32. Use of the decoupler or of the pharmaceutical agent according to one of the preceding claims Manufacture of a medicament for the prophylaxis or treatment of asthma, hypertension, coronary heart disease, hypertrophy of the heart, duodenal ulcer, cardiac insufficiency, liver cirrhosis, schizophrenia, AIDS, diabetes mellitus, diabetes insipidus,

Obesity, chronic obstructive pulmonary disease and / or edema.

33. Use according to one of the preceding claims as aquaretics, as contraceptives, as an anti-infective agent, as an anxiolytic agent and / or as an antitumor agent.

34. An organism comprising the decoupler according to one of claims 1 to 12 and / or the recognition molecule according to claim 16.

35. An organism according to the preceding claim, characterized in that the organism, preferably by the presence of the recognition molecule, a disease selected from the group comprising asthma, hypertension, hypertrophy of the heart, coronary heart disease, duodenal ulcer, heart failure, liver cirrhosis, schizophrenia , AIDS, diabetes mellitus, diabetes insipidus, obesity, cancer, chronic obstructive pulmonary disease, learning difficulties and / or edema.

36. Use according to one of the preceding claims, characterized in that The organism according to claim 28 is used as a model for the tissue and / or cell-specific AKAP-PKA interaction, in particular as a model for diabetes insipidus, diabetes mellitus, adipositas, edema, chronic obstructive pulmonary diseases, AIDS, schizophrenia, liver cirrhosis, cardiac insufficiency , coronary heart disease, hypertrophy of the heart, improvement of learning, hypertension, duodenal ulcer and / or asthma.

37. A method of modification, preferably inhibition of an AKAP-PKA interaction comprising the steps

(A) providing a decoupler according to one of claims 1 to 13 and / or a recognition molecule according to claim 16 and

38. Method according to the preceding claim, characterized in that the modification takes place at a regulatory RII subunit of the PKA.

39. Method according to the preceding claim, characterized in that the RII subunits are RIIalpha and / or RIIbeta üntereinheiten.

40. Use of the decoupler according to one of claims 1 to 12 as lead structures for the development of pharmaceutical agents, in particular by means of combinatorial and / or structure-based drug design.

41. Process for the preparation of pharmaceutical agents, which comprises the following steps:

(a) providing a decoupler according to one of claims 1 to 12 as a lead structure,

(b) chemical modification of the lead structure, preferably by means of a combinatorial and / or structure-based active substance design, wherein substances are obtained and, if appropriate,

(c) testing the substances for their ability to influence the AKAP-PKA interaction and

Selection of suitable substances as pharmaceutical agents.

42. A method according to the preceding claim comprising the formulation of the substance in a pharmaceutically acceptable form.

281, 137 3502 10 N22 22.33 1.7106 -4.86333

260.34 19588 56 D16 24.43 4.185 -5.6794

181,238 18803 54 C11 24,88 2,84 -4,00302

ul

298.273 14617 42 113 43.50 2,7939 -4,91081

185,226 14542 42 N08 45,52 2,6414 -3,17019

166,224 4712 14 H10 45,55 1,6398 -1,406