WO2018073409A1

WO2018073409A1 - Specific ac5 inhibitor

Info

Publication number: WO2018073409A1
Application number: PCT/EP2017/076839
Authority: WO
Inventors: Frank Ulrich MUELLER; Alexander HEINICK; Christina ROLFES; Wilhelm Schmitz; Volker Gerke
Original assignee: Westfälische Wilhelms-Universität Münster
Priority date: 2016-10-21
Filing date: 2017-10-20
Publication date: 2018-04-26
Also published as: EP3528832A1

Abstract

The present invention relates to a polypeptide capable of specifically inhibiting human adenylyl cyclase (5) wherein said polypeptide does essentially not inhibit human adenylyl cyclase (6). The present invention also relates to the polypeptide for use in specifically inhibiting human adenylyl cyclase (5) in a subject. The present invention further relates to an in vitro use of the polypeptide for inhibiting human adenylyl cyclase (5) and an in vitro method for inhibiting human adenylyl cyclase (5).

Description

SPECIFIC A C5 INHIBITOR

Background

[1] G protein-coupled receptor (GPCR)/adenylyl cyclase (AC)/cyclic AMP (cAMP) signaling is crucial for all cellular responses to physiological and pathophysiological stimuli. ACs catalyse the conversion of ATP to cAMP and pyrophosphate. Thus, cAMP is synthesized by adenylyl cyclases following activation of G protein-coupled receptors. There are nine AC isoforms, encoded by different genes and differentially expressed across tissues in mammals. The cAMP signaling pathway is defined by its precise spatial and temporal organization and regulates a multitude of cellular responses and orchestrates a network of intracellular events.

[2] In the heart cAMP is the main second messenger of the β-adrenergic receptor (β-AR) pathway producing positive chronotropic, inotropic, and lusitropic effects during stimulation by catecholamines. These effects involve mainly the activation of cAMP dependent protein kinase (PKA) and the phosphorylation of several key proteins involved in the excitation- contraction coupling. In the heart the main AC isoforms responsible for cAMP synthesis are AC5 and AC6, which regulate heart rate and contractility.

[3] Whereas short-term stimulation of β-AR/AC/cAMP has beneficial effects on cardiac function in times of need, such as in fight of flight, chronic activation of this pathway triggers pathological cardiac remodeling, which may ultimately lead to heart failure. Therefore, it is beneficial to treat patients with heart failure with β-AR antagonist. β-AR antagonists acutely reduce cardiac performance by competing with endogenous catecholamines for binding to their receptor and thereby block β-AR/AC/cAMP signaling. This treatment reduces mortality when administered at slowly escalating doses in patients with chronic heart failure. However, the treatment with β- adrenoceptor antagonists decreases cardiac contractility and is thus encompassed by adverse effects, such as reduced cardiac performance, fatigue, dizziness, heart failure, bradycardia, heart block, and others.

[4] While AC5 and AC6 share 65 % amino acid homology and many regulatory characteristics, the pathophysiological roles of the two major AC-isoforms in the heart are quite different. Overexpression of AC5 in cardiomyocytes of the heart improves baseline cardiac function whereas it impairs the ability of the heart to withstand stress and causes myocardial remodeling with cellular degeneration and fibrosis and compensatory hypertrophy of the remaining cardiomyocytes (Guellich et al., 2014, Eur J Physiol, vol. 466, pp. 1 163- 1 175). Conversely, AC5 knockout mice are resistant to cardiac stress and have an increased median lifespan of approximately 30%. Aged AC5 knockout mice were shown to be protected from aging-induced cardiomyopathy and, in comparison with wild -type mice, had decreased left ventricular (Lv) hypertrophy, increased Lv ejection fraction, decreased Lv apoptosis and Lv fibrosis. In addition, these mice were also protected from reduced bone density and had a decreased susceptibility to age dependent fractures. Accordingly, AC5 rather mediates adverse effects upon chronic β-AR stimulation by constantly high concentrations of catecholamines, as present in patients with chronic heart failure. In contrast, overexpression of AC6 exerted robust and sustained beneficial effects on cardiac function, such as increased cardiac responsiveness and advantageous effects on the failing heart (Pierre et al., 2009, Nature Reviews Drug Discovery, vol. 8, pp. 321 -335; Brand et al., 2013, J Pharmacol Exp Ther, vol. 347, pp. 265-275).

[5] As both AC5 and AC6 are downstream targets of the β- adrenoceptor, it is clear that blocking the β-AR using β-AR antagonists does not only prevent the deleterious effects mediated by AC5 but also the beneficial effects mediated by AC6, shedding light on the cause of the negative side effects of β-AR antagonists. Blocking β-AR/AC/cAMP signaling downstream of the β-AR would allow to specifically hinder deleterious effects without hindering beneficial effects and could thus solve this problem. Targeting specific adenylyl cyclase isoforms is therefore an interesting innovative therapeutic approaches for chronic heart failure. Such a novel therapy would preferably selectively inhibit AC5 without affecting activity of AC6. A specific AC5 inhibitor could exert similar benefits as beta-blockers with less negative inotropic effects on the heart.

[6] There have been reports in the art that adenine-9- β-D-arabinofuranoside (Ara-A) show a selective inhibition of AC5 (Vatner et al., 2013, Am J Physiol Heart Circ Physiol, vol. 305, pp. H1-H8; Iwatsubo et al., 2012, Am J Physiol Heart Circ Physiol, vol. 302, pp. H2622- H2628). However, the specificity of Ara-A was questioned by two independent groups that showed that this inhibitor was unable to discriminate between AC5 and AC6 (Brand et al., 2013, J Pharmacol Exp Ther, vol. 347, pp. 265-275; Braeunig et al., 2013, PLoS One, 8:e68009). Accordingly, there still exists an urgent need in the art for selective AC5 inhibitors. Thus, the technical problem underlying the present invention is to comply with this need.

[7] The present invention provides as a solution to the technical problem new means and methods to specifically inhibit AC5. These means and methods are described herein, illustrated in the Examples, and reflected in the claims. [8] In particular, the present inventors surprisingly uncovered that Annexin A4 specifically inhibits AC5 but not AC6. This was surprising as both ACs share 65 % amino acid homology (Guellich et al., 2014, Eur J Physiol, vol. 466, pp. 1 163-1 175). Therefore, a skilled person would not have expected that Annexin A4 is capable of discriminating between AC5 and AC6 and thus inhibiting AC5 without inhibiting AC6. Even more, although there is an urgent need in the art for AC5 specific inhibitors, several attempts to provide such an inhibitor have failed, as described herein. This further indicates that providing such a specific AC5 inhibitor was a challenging task which has not been solved up to now. The polypeptide of the present invention provides for the first time the possibility of specifically inhibiting AC5 and therefore meets an urgent need in the art (Pierre et al., 2009, Nature Reviews Drug Discovery, vol. 8, pp. 321-335).

[9] Moreover, the present inventors uncovered that an N-terminal fragment of Annexin A4 mediates the inhibition of AC5. This as such was surprising, because also the C-terminal calcium binding repeat domains are known to interact with proteins and to also exert an effect (Gerke and Moss, Physiol Rev, vol. 82, pp. 331 -371 , 2002). This was all the more surprising as truncation variants of Annexin A4 lacking either the N-terminus or certain fragments of the C-terminus are not capable of inhibiting AC5. Thus, a skilled artisan would have assumed that both termini, and thus the full-length Annexin A4 polypeptide, are necessary for the inhibition of AC5. Accordingly, the present inventors provide for the first time an AC5-specific inhibitor. Such a specific AC5 inhibitor may be used in the specific inhibition of AC5 in a patient and has thus the potential to improve treatment of chronic heart failure. Moreover, such an AC5 inhibitor may further be used in the treatment of cardiac stress, pain, diabetes and obesity as described in the art (Vatner et al., 2013, Am J Physiol Heart Circ Physiol, vol. 305, pp. H1 -H8; Brand et al., 2013, J Pharmacol Exp Ther, vol. 347, pp. 265-275).

Description

[11] The present inventors surprisingly uncovered that Annexin A4 specifically inhibits human adenylyl cyclase 5 (AC5) but essentially not human adenylyl cyclase 6 (AC6). The present inventors further uncovered that this specific inhibition is mediated by an N-terminal fragment of Annexin A4. This was surprising as truncation variants of Annexin A4 lacking either the N-terminus or certain fragments of the C-terminus are not capable of inhibiting AC5, as showed herein. Thus, a skilled artisan would have assumed that both termini, and thus the full-length Annexin A4 polypeptide, are necessary for the inhibition of AC5. Deletion of a C-terminal fragment (e.g. deletion of calcium binding repeat domain no. 4 or deletion of calcium binding repeat domain nos. 3 and 4) of Annexin A4 results in a loss of its inhibiting capacity on AC5. As a consequence a skilled artisan would not have assumed that an N- terminal fragment as small as 22 amino acids of Annexin A4 is capable of inhibiting AC5. Such a specific inhibition of AC5 is desirable as AC5 mediates adverse effects upon chronic β-AR stimulation by constantly high concentrations of catecholamines as present in patients with chronic heart failure, whereas AC6 exerts beneficial effects on cardiac function, such as increased cardiac responsiveness and advantageous effects on the failing heart. Therefore, a specific AC5 inhibitor prevents the deleterious effects mediated by AC5 but does not hinder beneficial effects mediated by AC6. This is a reasonable advantage over β-AR antagonists which block the β-AR and thus inhibit both AC5 and AC6, whereas AnnexinA4 blocks the signaling cascade downstream of the β-AR by specifically targeting AC5. This specific inhibition of AC5 is surprising, as AC5 and AC6 share 65 % amino acid homology. Therefore, a skilled person would not have expected that Annexin A4 is capable of discriminating between AC5 and AC6 and thus inhibiting AC5 without inhibiting AC6.

[12] Accordingly, the present provides an AnnexinA4 polypeptide for use in a method for specifically inhibiting human AC5 in a subject. The AnnexinA4 polypeptide can thus be used in a method for treating a disease that can be treated using β-AR antagonists in a subject, wherein the use is characterized in that human adenylyl cyclase 5 is specifically inhibited.

[13] In a preferred embodiment of the invention, the use is characterized in that human adenylyl cyclase 6 is essentially not inhibited, as determined in an in vitro assay. [14] In another preferred embodiment of the invention, the subject suffers from cardiac stress, cardiomyopathy, heart failure, diabetes, obesity, aging, pain ischemic cardiomyopathy, such as chest pain or angina, high blood pressure, arrhythmia, heart attacks, migraine, tremor or tumor growth, and preferably from heart failure. [15] In a further preferred embodiment of the invention the use comprises administering to a subject a pharmaceutically effective amount of AnnexinA4.

[16] In an even further embodiment the AnnexinA4 polypeptide is characterized in that it (a) has the amino acid sequence shown in SEQ ID NO: 2,

(b) is 60 % identical to the amino acid sequence of SEQ ID NO: 2, or

(c) is a fragment of the amino acid sequence of (a) or (b), which is capable of specifically inhibiting human AC5.

[17] Members of the annexin family generally contain 4 calcium-binding domains, forming the annexin core, and a member specific N-terminal domain that mediates interactions with other proteins. All annexins bind negatively charged phospholipids, primarily in a calcium- dependent, but also in a calcium-independent manner. Exemplarily, the structure of Annexin A4 (AnxA4) is shown in Figure 1A. AnxA4 is capable of Ca²⁺-dependent self-association on membrane surfaces, enabling membrane aggregation. Upon Ca²⁺ binding, AnxA4 undergoes conformational changes that lead to oligomerization and the formation of mobile trimers and immobile aggregates. At the same time, Ca²⁺ binding of AnxA4 provokes translocation from the cytosol and nucleoplasm to the plasma and nuclear membrane. The immobile nature of AnxA4 aggregates on membrane surfaces negatively modulates the mobility of transmembrane and membrane-associated proteins. By way of example, a full-length sequence of AnxA4 is set forth in SEQ ID NO: 2. However, SEQ ID NO:2 used herein can be replaced with SEQ ID NO: 27. However, the term "AnxA4" also encompasses AnxA4 polypeptides having an amino acid sequence which shares a certain degree of identity with the amino acid sequence shown in SEQ ID NO: 2 and also encompasses polypeptides having mutations relative to the reference sequence shown in SEQ ID NO: 2 as long as such an Annexin A4 polypeptide is capable of specifically inhibiting AC5 having a sequence as shown in SEQ ID NO: 3. Accordingly, an AnnexinA4 polypeptide as used herein

(a) has the amino acid sequence shown in SEQ ID NO: 2,

(b) is 60 % identical to the amino acid sequence of SEQ ID NO: 2, or

(c) is a fragment of the amino acid sequence of (a) or (b), which is capable of specifically inhibiting human AC5. [18] However, an AnnexinA4 polypeptide of the invention may also have an amino acid sequence which is at least 50%, at least 55%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, identical to the amino acid sequence shown in SEQ ID NO: 2. In case of an AnxA4 polypeptides having a sequence that is less than 100% identical to the amino acid sequence shown in SEQ ID NO: 2 the degree of inhibition on AC5 may be subject to alterations, however the degree of inhibition is preferably not decreased compared to an AnxA4 polypeptide having a sequence set forth in SEQ ID NO: 2.

[19] In a further preferred embodiment, the present invention provides a preferred fragment of AnnexinA4, which is capable of inhibiting human adenylyl cyclase 5, said polypeptide consisting of:

(a) amino acids 1 to 22 of the amino acid sequence shown in SEQ ID NO: 1 ;

(b) a fragment of the amino acid sequence of (a), i.e. a fragment of the preferred fragment;

(c) an amino acid sequence which is at least 60% identical to the amino acid sequence of (a); or

(d) the amino acid sequence of (a), wherein one, two, three, four, five, six, seven or eight amino acids are substituted. However, SEQ ID NO: 1 used herein can be replaced with SEQ ID NO: 26. [20] Accordingly, the present invention relates to the preferred fragment of AnnexinA4 as described herein for use in a method for specifically inhibiting human AC5 in a subject.

[21] The term "polypeptide of the invention" as used herein relates to AnnexinA4 as described herein and to the preferred fragment of AnnexinA4 as described herein. A polypeptide of the invention can be used in the method for specifically inhibiting human AC5 in a subject as described herein.

[22] It is envisioned herein that substituting one, two, three, four, five, six, seven, eight or more amino acids of the polypeptide of the present invention is possible without interfering with its specificity, i.e. such a substituted polypeptide is still capable of inhibiting human AC5. Accordingly, such amino acid substitutions may even increase the inhibiting activity of a polypeptide of the present invention on AC5 without interfering with its specificity.

[23] The term "substitution" as used herein relates to the replacement of an amino acid in a polypeptide sequence by another amino acid of the naturally occurring 20 amino acids or by other amino acids than the naturally occurring 20 amino acids, such as, for example, ornithine, hydroxyproline, citrulline, homoserine, hydroxylysine, norvaline or pyrrolysine. However, such substitutions are preferably conservative, i.e. an amino acid residue is replaced with a chemically similar amino acid residue. Examples of conservative substitutions are the replacements among the members of the following groups: 1 ) alanine, serine, and threonine; 2) aspartic acid and glutamic acid; 3) asparagine and glutamine; 4) arginine and lysine; 5) isoleucine, leucine, methionine, and valine; and 6) phenylalanine, tyrosine, and tryptophan.

[24] A "polypeptide" refers to a molecule comprising a polymer of amino acids linked together by peptide bonds. Said term is not meant herein to refer to a specific length of the molecule and is therefore herein interchangeably used with the term "protein". A polypeptide as used herein may encompass both naturally-occurring and non-naturally-occurring amino acids. Polypeptides may be a polypeptide homologous (native) or heterologous to the host cell.

[25] The term "fragment", as used herein with respect to the polypeptide of the invention, relates to N-terminally and/or C-terminally shortened polypeptides, which retain the capability of inhibiting human adenylyl cyclase 5. A fragment of the preferred fragment may consist of at least 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 amino acids. Accordingly, amino acids 1 -22 of SEQ ID NO: 1 may be shortened from the N- or C-terminus by 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15 or 16 amino acids. [26] "Identity" is a property of sequences that measures their similarity or relationship. The term "sequence identity" or "identity" as used in the present disclosure means the percentage of pair-wise identical residues - following (homologous) alignment of a sequence of a polypeptide of the disclosure with a sequence in question - with respect to the number of residues in the longer of these two sequences. Sequence identity is measured between at two polypeptide sequences aligned using a standardized algorithm. Such an algorithm may insert, in a standardized and reproducible way, gaps in the sequences being compared in order to optimize alignment between two sequences, and therefore achieve a more meaningful comparison of the two sequences. For purposes of the present invention, the sequence identity between two amino acid sequences is determined using the NCBI BLAST program version 2.3.0 (Jan-13-2016) (Altschul et al., Nucleic Acids Res. (1997) 25:3389- 3402). Sequence identity of two amino acid sequences can be determined with blastp set at the following parameters: Matrix: BLOSUM62, Word Size: 3; Expect value: 10; Gap cost: Existence = 1 1 , Extension = 1 ; Compositional adjustments: Conditional compositional score matrix adjustment. [27] A preferred fragment of AnnexinA4 consists of amino acids 1 to 22 of the amino acid sequence shown in SEQ ID NO: 1 or the amino acid sequence shown in SEQ ID NO: 1 . It is also envisioned herein that a preferred fragment of AnnexinA4 consists of amino acids 1 to n of the amino acid sequence shown in SEQ ID NO: 1 , wherein n is any integer between 22 and 83 or of the amino acid sequence shown in SEQ ID NO: 2. However, a polypeptide of the present invention can also consist of an amino acid sequence which is at least 50%, at least 55%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, and preferably at least 60% identical to said amino acid sequences. Accordingly, a preferred fragment of AnnexinA4 can also consist of any one of said amino acid sequences, wherein 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14,15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 amino acids are substituted.

[28] The polypeptide of the present invention is capable of inhibiting human AC5. The term "inhibiting" as used herein means that the catalytic activity of the enzyme AC5 is reduced compared to AC5 which has not been inhibited. By way of example, catalytic activity and thus inhibition of AC5 can be measured by quantifying cAMP levels. Accordingly, inhibition of AC5 can be measured by comparing the cAMP level in cells comprising AC5 and the polypeptide of the present invention to the cAMP level in cells that comprise AC5 but not the polypeptide of the present invention or that comprise less of the polypeptide of the present invention. In such an assay reduced cAMP levels are indicative of AC5 inhibition. Such a measurement and/or comparison of cAMP levels is described in detail elsewhere herein and is exemplarily shown in Figure 4. Therefore, a skilled Artisan can easily determine if AC5 is inhibited by measuring the cAMP level, wherein a reduction of cAMP is indicative of an inhibition of AC5. The term "specifically inhibiting AC5" with respect to the polypeptide of the present invention means that only AC5 is inhibited but not AC6. [29] The terms "adenylyl cyclase 5", "human adenylyl cyclase 5", "AC5" and "human AC5" are used interchangeably herein and relate to the human enzyme expressed in cardiomyocytes, immune cells and neuronal cells. AC5 is one of the two major ACs in the adult mammalian heart where it is responsible for cAMP synthesis. An exemplary sequence of AC5 is shown in SEQ ID NO: 3. However, the term "AC5" also encompasses AC5 polypeptides having an amino acid sequence which shares a certain degree of identity with the amino acid sequence shown in SEQ ID NO: 3 and also encompasses polypeptides having mutations relative to the reference sequence shown in SEQ ID NO: 3 as long as such an AC5 polypeptide is capable of converting ATP to cAMP in a cell and its catalytic activity is inhibited by AnxA4 having a sequence as shown in SEQ ID NO: 2 or an N-terminal fragment thereof consisting of amino acids 1 to 22 of the amino acid sequence shown in SEQ ID NO: 1.

[30] In a further embodiment, the present invention provides a polypeptide which is capable of specifically inhibiting human adenylyl cyclase 5, said polypeptide consisting of: (a) amino acids 1 to n of the amino acid sequence shown in SEQ ID NO: 1 , wherein n is any integer between 22 and 83; (b) a fragment of the amino acid sequence of (a); or

(c) an amino acid sequence which is at least 60% identical to the amino acid sequence of (a); or optionally

(d) the amino acid sequence of (a), wherein up to 30% of the amino acids are substituted.

[31] However, it is also envisioned herein that n is any integer between 10 and 83, 1 1 and 83, 12 and 83, 13 and 83, 14 and 83, 15 and 83, 16 and 83, 17 and 83, 18 and 83, 19 and 83, 20 and 83, 21 and 83, 22 and 23, 22 and 24, 22 and 25, 22 and 26, 22 and 27, 22 and 28, 22 and 29, 22 and 30, 22 and 31 , 22 and 32, 22 and 33, 22 and 34, 22 and 35, 22 and 36, 22 and 37, 22 and 38, 22 and 39, 22 and 40, 22 and 41 , 22 and 42, 22 and 43, 22 and 44, 22 and 45, 22 and 46, 22 and 47, 22 and 48, 22 and 49, 22 and 50, 22 and 51 , 22 and 52, 22 and 53, 22 and 54, 22 and 55, 22 and 56, 22 and 57, 22 and 58, 22 and 59, 22 and 60, 22 and 61 , 22 and 62, 22 and 63, 22 and 64, 22 and 65, 22 and 66, 22 and 67, 22 and 68, 22 and 69, 22 and 70, 22 and 71 , 22 and 72, 22 and 73, 22 and 74, 22 and 75, 22 and 76, 22 and 77, 22 and 78, 22 and 79, 22 and 80, 22 and 81 , 22 and 82 or 22 and 83.

[32] In a preferred embodiment of the invention the preferred fragment of AnnexinA4 consists of:

(a) the amino acid sequence shown in SEQ ID NO: 1 ;

(b) a fragment of the amino acid sequence of (a);

(d) the amino acid sequence of (a), wherein 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14,15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33 or 34 amino acids are substituted. [33] In a further preferred embodiment the polypeptide of the present invention is fused with a linker. The term "linker" as used herein refers to a polypeptide having a length of 1 to 25 amino acids of any amino acid sequence as long as it does not interfere with folding of the polypeptide of the present invention, such that said polypeptide is no longer capable of specifically inhibiting human AC5. Such a linker can be fused to C-terminus of the polypeptide of the present invention. However, it is preferred that such a linker is fused to the N-terminus of the polypeptide of the present invention. Preferred linkers are selected from the group consisting of (GGGGS)n (SEQ ID NO: 6), Gly , (Gly)8 (SEQ ID NO: 8), (Gly)6 (SEQ ID NO: 9), (EAAK)n (SEQ ID NO: 10), A( EAAAK)4 AL E A( E AAAK)4 A (SEQ ID NO: 1 1 ), PAPAP (SEQ ID NO: 12), AEAAAKEAAAKA (SEQ ID NO: 13), (Ala-Pro)n (SEQ ID NO: 14), wherein n is 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or more and wherein it is even more preferred that the linker is a Glycine residue.

[34] In a further preferred embodiment the polypeptide of the present invention is fused with a cell penetrating peptide (CPP). The CPPs as comprised by such a conjugate of the present invention are short peptides that facilitate cellular delivery of the polypeptide of the present invention. The function of the CPPs in this respect is to deliver the polypeptide of the present invention into cells of interest. Accordingly, a CPP of the present invention allows the polypeptide of the present invention to cross cellular membranes and thereby enter a living cell, preferably a human cell. Therefore, a conjugate of a CPP and the polypeptide of the present invention is capable of entering into a cell and to inhibit AC5 is said cell. Preferably, a CPP according to the present invention is a trans-activating transcriptional activator (TAT) peptide delivery domain comprising the amino acid sequence GRKKRRQRRR (SEQ ID NO: 5). However, also other CPPs having the unique ability to gain access to the interior of any type of cell as disclosed for example in Gautam et al. (2012), Database 2012:1 -7, can be equally applied for the conjugates of the present invention.

[35] The CPP can be fused to the C-terminus of the polypeptide of the present invention. However, it is preferred that the CPP is fused to the N-terminus of the polypeptide of the present invention. It is even more preferred that the CPP is fused to the linker, wherein the linker is fused to the N-terminus of the polypeptide of the present invention. A preferred conjugate of the present invention consists of CPP-linker-polypeptide of the present invention, wherein the CPP and the linker are preferably fused to the N-terminus of the polypeptide of the present invention.

[36] Accordingly, the present invention provides a polypeptide which is capable of inhibiting human adenylyl cyclase 5, said polypeptide comprising:

(a) amino acids 1 to 22 of the amino acid sequence shown in SEQ ID NO: 1 ;

(b) a fragment of the amino acid sequence of (a);

(d) the amino acid sequence of (a), wherein one, two, three, four, five, six or seven amino acids are substituted; and

a cell penetrating peptide, wherein the polypeptide consisting of the amino acid sequence of any one of (a) to (d) and the cell penetrating peptide are optionally fused via a linker, wherein the linker is fused to the N-terminus of said polypeptide.

[37] Moreover, the present invention provides a polypeptide which is capable of inhibiting human adenylyl cyclase 5, said polypeptide comprising: (a) the amino acid sequence shown in SEQ ID NO: 1 ;

(b) a fragment of the amino acid sequence of (a);

(d) the amino acid sequence of (a), wherein 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14,15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33 or 34 amino acids are substituted; and

[38] In some embodiments the polypeptide of the present invention and/or its potential interaction partners (e.g. AC5 or AC6) may be fused at its N-terminus or its C-terminus to a protein, a protein domain or a peptide, for instance, a signal sequence and/or an affinity tag.

[39] Affinity tags such as the Strep-tag® or Strep-tag® II (Schmidt, T.G.M. et al. (1996) J. Mol. Biol. 255, 753-766), the myc-tag, the FLAG-tag, the Hiss-tag or the HA-tag or proteins such as glutathione-S-transferase also allow easy detection and/or purification of recombinant proteins are further examples of suitable fusion partners. Finally, proteins with chromogenic or fluorescent properties such as the green fluorescent protein (GFP) or the yellow fluorescent protein (YFP) are suitable fusion partners and allow detecting and/or isolation of the polypeptide of the present invention or its possible interaction partners (e.g. AC5 or AC6). By way of example, for isolation of a polypeptide a GFP-Trap can be used, which is a monovalent matrix of magnetic particles coupled with GFP-binding proteins. This allows the one-step isolation of fluorescent fusion proteins (e.g. YFP-AC5, YFP-AC6, GFP- AC5, or GFP-AC6) and optionally also co-immunoprecipitation of their interacting partners (e.g. the polypeptide of the present invention), as described in detail elsewhere herein. However, such an isolation of the polypeptide of the present invention with an optional co- immunoprecipitation of an interacting partner can also be performed using a myc-Trap, allowing the one-step isolation of proteins fused to a myc-tag, as described in detail elsewhere herein. [40] In general, it is possible to label the polypeptide of the present invention with any appropriate chemical substance or enzyme, which directly or indirectly generates a detectable compound or signal in a chemical, physical, optical, or enzymatic reaction. An example for a physical reaction and at the same time optical reaction/marker is the emission of fluorescence upon irradiation or the emission of X-rays when using a radioactive label. Alkaline phosphatase, horseradish peroxidase and β-galactosidase are examples of enzyme labels (and at the same time optical labels) which catalyze the formation of chromogenic reaction products. The polypeptide of the present invention may also be conjugated with any suitable therapeutically active agent, e.g., for the targeted delivery of such agents to a given cell, tissue or organ or for the selective targeting of cells, e.g., of tumor cells without affecting the surrounding normal cells.

[41] Furthermore, the polypeptide of the present invention may in some embodiments be conjugated to a moiety that extends the serum half-life of the polypeptide of the present invention. The moiety that extends the serum half-life may be a polyalkylene glycol molecule, hydroxyethyl starch, fatty acid molecules, such as palmitic acid (Vajo & Duckworth 2000, Pharmacol. Rev. 52, 1-9), an Fc part of an immunoglobulin, a CH3 domain of an immunoglobulin, a CH4 domain of an immunoglobulin, albumin, an albumin binding peptide, or an albumin binding protein, transferrin to name only a few.

[42] In another preferred embodiment the polypeptide of the present invention does essentially not inhibit human adenylyl cyclase 6, as determined in an in vitro assay. AC5 and AC6 are closely related and share a significant homology of 65% in amino acid sequence. Therefore, the specific inhibition of AC5 but not of AC6 mediated by the polypeptide of the present invention was surprisingly uncovered by the present inventors. As both ACs share such a high sequence homology, a skilled person would not have expected that the polypeptide of the present invention inhibits AC5 without inhibiting AC6. Even more, although there is an urgent need in the art for AC5 specific inhibitors, several attempts to provide such an inhibitor have failed, as described herein. This further indicates that providing such a specific AC5 inhibitor was a challenging task. Thus, the polypeptide of the present invention provides for the first time the possibility of specifically inhibiting AC5 and therefore meets an urgent need in the art. [43] The terms "adenylyl cyclase 6", "human adenylyl cyclase 6", "AC6" and "human AC6" are used interchangeably herein and relate to the second major human AC, beside AC5, in the adult mammalian heart where it is responsible for cAMP synthesis. An exemplarily sequence of AC6 is shown in SEQ ID NO: 4. However, the term "AC6" also encompasses AC6 polypeptides having an amino acid sequence which shares a certain degree of identity with the amino acid sequence shown in SEQ ID NO: 4 and also encompasses polypeptides having mutations relative to the reference sequence shown in SEQ ID NO: 4 as long as such an AC6 polypeptide is capable of converting ATP to cAMP in a cell and its catalytic activity is essentially not inhibited by AnxA4 having a sequence as shown in SEQ ID NO: 2 or an N- terminal fragment thereof consisting of amino acids 1 to 22 of the amino acid sequence shown in SEQ ID NO: 1. [44] A suitable in vitro assay that allows to determine the absence of AC6 inhibition by the polypeptide of the present invention is a co-immunoprecipitation (co-IP) followed by immunoblot analysis. Such an assay allows determining the absence of direct interaction of the polypeptide of the invention and AC6, wherein the absence of direct interaction is indicative for the absence of AC6 inhibition by the polypeptide of the invention (i.e. in case no interaction between the polypeptide of the invention and AC6 can be detected, AC6 is not inhibited). By way of example, such a co-IP can be performed using lysates of HEK293 cells transiently co-transfected with pcDNA3-YFP-hAC6 as bait-protein and myc-AnxA4-wt as prey protein or with GFP and myc-AnxA4-wt as a control. After binding of YFP-AC6 to magnetic beads (GFP-trap) co-immunoprecipitation of myc-AnxA4-wt from the lysate can be determined in a subsequent immunoblot analysis, wherein no co-immunoprecipitation occurs in case of YFP-AC6 indicative of a lack of inhibition of AC6 by AnxA4 or the polypeptide of the present invention. A detailed description of such an in vitro assay can be found elsewhere herein. [45] The term "essentially not" as used herein with respect to the inhibition of AC6 means that less than 25%, 20%, 15%, 10%, 5%, 2%, 1 %, and ideally 0% interaction between the polypeptide of the invention and AC6 can be detected in the in vitro assay described elsewhere herein.

[46] In a further preferred embodiment the present invention provides a pharmaceutical composition, comprising the preferred fragment of AnnexinA4 and optionally a pharmaceutical acceptable carrier. The present invention also relates to a pharmaceutical composition, comprising the polypeptide of the invention and optionally a pharmaceutical acceptable carrier.

[47] The pharmaceutical composition preferably comprises a pharmaceutically effective amount of polypeptide of the invention.

[48] The term "pharmaceutical acceptable carriers" as used herein includes, but is not limited to diluents (fillers, bulking agents, e.g. lactose, microcrystalline cellulose), disintegrants (e.g. sodium starch glycolate, croscarmellose sodium), binders (e.g. PVP, HPMC), lubricants (e.g. magnesium stearate), glidants (e.g. colloidal Si02), solvents/co- solvents (e.g. aqueous vehicle, Propylene glycol, glycerol), buffering agents (e.g. citrate, gluconates, lactates), preservatives (e.g. Na benzoate, parabens (Me, Pr and Bu), BKC), anti-oxidants (e.g. BHT, BHA, Ascorbic acid), wetting agents (e.g. polysorbates, sorbitan esters), anti-foaming agents (e.g. Simethicone), thickening agents (e.g. methylcellulose or hydroxyethylcellulose), sweetening agents (e.g. sorbitol, saccharin, aspartame, acesulfame), flavouring agents (e.g. peppermint, lemon oils, butterscotch, etc), humectants (e.g. propylene, glycol, glycerol, sorbitol). Further pharmaceutically acceptable carriers are (biodegradable) liposomes; microspheres made of the biodegradable polymer poly(D,L)- lactic-coglycolic acid (PLGA), albumin microspheres; synthetic polymers (soluble); nanofibers, protein-DNA complexes; protein conjugates; erythrocytes; or virosomes. Various carrier based dosage forms comprise solid lipid nanoparticles (SLNs), polymeric nanoparticles, ceramic nanoparticles, hydrogel nanoparticles, copolymerized peptide nanoparticles, nanocrystals and nanosuspensions, nanocrystals, nanotubes and nanowires, functionalized nanocarriers, nanospheres, nanocapsules, liposomes, lipid emulsions, lipid microtubules/microcylinders, lipid microbubbles, lipospheres, lipopolyplexes, inverse lipid micelles, dendrimers, ethosomes, multicomposite ultrathin capsules, aquasomes, pharmacosomes, colloidosomes, niosomes, discomes, proniosomes, microspheres, microemulsions and polymeric micelles. Other suitable pharmaceutically acceptable excipients are inter alia described in Remington's Pharmaceutical Sciences, 15th Ed., Mack Publishing Co., New Jersey (1991 ) and Bauer et al., Pharmazeutische Technologie, 5th Ed., Govi-Verlag Frankfurt (1997). The person skilled in the art will readily be able to choose suitable pharmaceutically acceptable carriers, depending, e.g., on the formulation and administration route of the pharmaceutical composition. The term "pharmaceutically acceptable" preferably means a non-toxic material that does not interfere with the effectiveness of the biological activity of the polypeptide of the present invention. [49] In a further preferred embodiment the present invention relates to a polypeptide of the present invention or the pharmaceutical composition of the invention for use in inhibiting human adenylyl cyclase 5 in a subject, wherein the use is preferably characterized in that human adenylyl cyclase 6 is essentially not inhibited, as determined in an in vitro assay.

[50] The term "subject" as used herein relates to an animal, preferably a mammal, which can be, for instance, a mouse, rat, guinea pig, hamster, rabbit, dog, cat, or primate. Preferably, the subject is a human.

[51] In a preferred embodiment of the present invention the subject suffers from cardiac stress, cardiomyopathy, heart failure, diabetes, obesity, aging, pain ischemic cardiomyopathy, such as chest pain or angina, high blood pressure, arrhythmia, heart attacks, migraine, tremor or tumor growth, and preferably from heart failure. Accordingly, the present invention relates to a polypeptide of the present invention or the pharmaceutical composition of the invention for use in inhibiting human adenylyl cyclase 5 in a subject, wherein the use is preferably characterized in that human adenylyl cyclase 6 is essentially not inhibited, as determined in an in vitro assay, wherein the subject suffers from cardiac stress, cardiomyopathy, heart failure, diabetes, obesity, aging, pain ischemic cardiomyopathy, such as chest pain or angina, high blood pressure, arrhythmia, heart attacks, migraine, tremor or tumor growth, and preferably from heart failure.

[52] In particular De Lorenzo et al. (2014, Aging Cell, vol. 13, pp. 102-1 10) have shown that an AC5 knockout results in decreased tumor growth. Therefore, the polypeptide of the present invention, inhibiting AC5, may be a novel therapeutic option in the treatment of a subject suffering from a tumor.

[53] AC5 is a downstream target of the β-adrenoceptor, which can be blocked using β-AR antagonists (β-blocker). Therefore, the polypeptide of the present, which specifically inhibits AC5, has a comparable mode of action to β-AR antagonists. Thus, the polypeptide of the present invention can be used in the treatment of any medical disorder that is known in the art to be treated using β-AR antagonists.

[54] In a further preferred embodiment of the present invention the use of the polypeptide of the present invention or the pharmaceutical composition of the invention in inhibiting human adenylyl cyclase 5 in a subject further comprises administering to a subject a pharmaceutically effective amount of the polypeptide or the pharmaceutical composition.

[55] A "pharmaceutically effective amount" is an amount sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations. The exact amount of the polypeptide or the pharmaceutical composition of the invention which is administered to a subject may depend on the purpose of the treatment (e.g. treatment of acute disease vs. prophylactic treatment), route of administration, age, body weight, general health, sex, diet, time of administration, drug interaction and the severity of the condition, and will be ascertainable with routine experimentation by those skilled in the art.

[56] The polypeptide of the present invention or the pharmaceutical composition of the invention can be administered via any parenteral or non-parenteral (e.g. enteral) route that is therapeutically effective. A therapeutically effective route provides for delivery of an agent to a desired compartment, system, or location. For example, a therapeutically effective route is one through which an agent can be administered to provide at the desired site of action an amount of the agent sufficient to effectuate a beneficial or desired clinical result.

[57] In a further preferred embodiment the present invention relates to the in vitro use of the polypeptide of the present invention or the pharmaceutical composition of the present invention for inhibiting human adenylyl cyclase 5, wherein the in vitro use is preferably characterized in that human adenylyl cyclase 6 is essentially not inhibited. [58] In another preferred embodiment the present invention relates to an in vitro method for inhibiting human adenylyl cyclase 5, the method comprising bringing into contact

(i) human adenylyl cyclase 5; and

(ii) the polypeptide of the present invention or the pharmaceutical composition of the present invention; and

thereby inhibiting human adenylyl cyclase 5, wherein the method is preferably characterized in that human adenylyl cyclase 6 is essentially not inhibited.

[59] In a preferred embodiment the in vitro method of the present invention is characterized in that the human adenylyl cyclase 5 and the human adenylyl cyclase 6 are comprised by a cell, preferably a human cell.

[60] In a further preferred embodiment the present invention relates to a polynucleotide encoding the preferred fragment of AnnexinA4.

[61] In a further preferred embodiment the polynucleotide encoding the preferred fragment of AnnexinA4 is comprised by a vector allowing expression of the polynucleotide. [62] The terms "polynucleotide", "nucleotide sequence" or "nucleic acid molecule" are used interchangeably herein and refer to a polymeric form of nucleotides which are usually linked from one deoxyribose or ribose to another. The term "polynucleotide" preferably includes single and double stranded forms of DNA or RNA. A nucleic acid molecule of this invention may include both sense and antisense strands of RNA (containing ribonucleotides), cDNA, genomic DNA, and synthetic forms and mixed polymers of the above. They may be modified chemically or biochemically or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those of skill in the art. Such modifications include, for example, labels, methylation, substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), pendent moieties (e.g., polypeptides), intercalators (e.g., acridine, psoralen, etc.), chelators, alkylators, and modified linkages (e.g., alpha anomeric nucleic acids, etc.) Also included are synthetic molecules that mimic polynucleotides in their ability to bind to a designated sequence via hydrogen bonding and other chemical interactions. Such molecules are known in the art and include, for example, those in which peptide linkages substitute for phosphate linkages in the backbone of the molecule. [63] In this regard, a nucleic acid being an expression product is preferably a RNA, whereas a nucleic acid to be introduced into a cell is preferably DNA or RNA, e.g. synthetic DNA, genomic DNA or cDNA.

[64] The term "vector" as used herein refers to a nucleic acid sequence into which an expression cassette comprising the polynucleotide encoding the polypeptide of the present invention may be inserted or cloned. Furthermore, the vector may encode an antibiotic resistance gene conferring selection of the host cell. Preferably, the vector is an expression vector. The vector can contain elements for propagation in bacteria (e.g. E. coli), yeast (e.g. S. cerevisiae), insect cells and/or mammalian cells. The vector may have a linear, circular, or supercoiled configuration and may be complexed with other vectors or other material for certain purposes.

[65] In yet another preferred embodiment the present invention relates to a kit comprising the polypeptide of the present invention, the pharmaceutical composition of the present invention or the polynucleotide encoding the preferred fragment of AnnexinA4 and/or a vector as described herein.

[66] It must be noted that as used herein, the singular forms "a", "an", and "the", include plural references unless the context clearly indicates otherwise. Thus, for example, reference to "a reagent" includes one or more of such different reagents and reference to "the method" includes reference to equivalent steps and methods known to those of ordinary skill in the art that could be modified or substituted for the methods described herein.

[67] Unless otherwise indicated, the term "at least" preceding a series of elements is to be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.

[68] The term "and/or" wherever used herein includes the meaning of "and", "or" and "all or any other combination of the elements connected by said term".

[69] The term "about" or "approximately" as used herein means within 20%, preferably within 10%, and more preferably within 5% of a given value or range. It includes, however, also the concrete number, e.g., about 20 includes 20. [70] The term "less than" or "greater than" includes the concrete number. For example, less than 20 means less than or equal to. Similarly, more than or greater than means more than or equal to, or greater than or equal to, respectively.

[71] Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integer or step. When used herein the term "comprising" can be substituted with the term "containing" or "including" or sometimes when used herein with the term "having". [72] When used herein "consisting of" excludes any element, step, or ingredient not specified in the claim element. When used herein, "consisting essentially of" does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim.

[73] In each instance herein any of the terms "comprising", "consisting essentially of and "consisting of" may be replaced with either of the other two terms.

[74] It should be understood that this invention is not limited to the particular methodology, protocols, material, reagents, and substances, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims.

[75] All publications and patents cited throughout the text of this specification (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. To the extent the material incorporated by reference contradicts or is inconsistent with this specification, the specification will supersede any such material.

[76] A better understanding of the present invention and of its advantages will be obtained from the examples, offered for illustrative purposes only. The examples are not intended to limit the scope of the present invention in any way. An AnnexinA4 polypeptide for use in a method for specifically inhibiting human AC5 in a subject.

The AnnexinA4 polypeptide of item 1 , wherein the use is characterized in that human adenylyl cyclase 6 is essentially not inhibited, as determined in an in vitro assay.

The AnnexinA4 polypeptide of item 1 or 2, wherein the subject suffers from cardiac stress, cardiomyopathy, heart failure, diabetes, obesity, aging, pain ischemic cardiomyopathy, such as chest pain or angina, high blood pressure, arrhythmia, heart attacks, migraine, tremor or tumor growth, and preferably from heart failure.

The AnnexinA4 polypeptide of anyone of items 1 to 3, wherein the use comprises administering to a subject a pharmaceutically effective amount of AnnexinA4.

The AnnexinA4 polypeptide for the use of anyone of items 1 -4, wherein said AnnexinA4 polypeptide

(a) has the amino acid sequence shown in SEQ ID NO: 2,

(b) is 60 % identical to the amino acid sequence of SEQ ID NO: 2, or

(c) is a fragment of the amino acid sequence of (a) or (b).

The Annexin A4 polypeptide for the use of item 5, wherein said fragment consists of

(a) amino acids 1 to 22 of the amino acid sequence shown in SEQ ID NO: 1 ;

(b) a fragment of the amino acid sequence of (a);

(d) the amino acid sequence of (a), wherein one, two, three, four, five, six, seven or eight amino acids are substituted.

The Annexin A4 polypeptide for the use of item 6, wherein the amino acid sequence of (a) is the amino acid sequence shown in SEQ ID NO: 1.

The Annexin A4 polypeptide for the use of anyone of items 1 to 7, wherein the polypeptide is fused with a linker.

The Annexin A4 polypeptide for the use of item 8, wherein the linker is selected from the group consisting of (GGGGS)n, Gly, (Gly)8, (Gly)6, (EAAK)n, A(EAAAK)4ALEA(EAAAK)4A, PAPAP, AEAAAKEAAAKA, (Ala-Pro)n, wherein the linker is preferably a Glycine residue.

The Annexin A4 polypeptide for the use of anyone of items 1 to 9, wherein said polypeptide is fused with a cell penetrating peptide.

The Annexin A4 polypeptide for the use of anyone of items 1 to 10, wherein the cell- penetrating polypeptide allows the polypeptide to cross cellular membranes and thereby enter a living cell, preferably a human cell.

The Annexin A4 polypeptide for the use of anyone of items 1 to 1 1 , wherein said polypeptide does essentially not inhibit human adenylyl cyclase 6, as determined in an in vitro assay.

A polypeptide which is capable of inhibiting human adenylyl cyclase 5, said polypeptide consisting of:

(a) amino acids 1 to 22 of the amino acid sequence shown in SEQ ID NO: 1 ;

(b) a fragment of the amino acid sequence of (a);

The polypeptide of item 13, wherein the amino acid sequence of (a) is the amino acid sequence shown in SEQ ID NO: 1.

The polypeptide of item 13 or 14, wherein the polypeptide is fused with a linker.

The polypeptide of item 15, wherein the linker is selected from the group consisting of (GGGGS)n, Gly, (Gly)8, (Gly)6, (EAAK)n, A(EAAAK)4ALEA(EAAAK)4A, PAPAP, AEAAAKEAAAKA, (Ala-Pro)n, wherein the linker is preferably a Glycine residue.

The polypeptide of any one of items 13 to 16, wherein said polypeptide is fused with a cell penetrating peptide. The polypeptide of item 17, wherein the cell-penetrating polypeptide allows th polypeptide to cross cellular membranes and thereby enter a living cell, preferably human cell. 19. The polypeptide of any one of items 13 to 18, wherein said polypeptide does essentially not inhibit human adenylyl cyclase 6, as determined in an in vitro assay.

20. A pharmaceutical composition, comprising the polypeptide of any one of items 13 to 19 and optionally a pharmaceutical acceptable carrier.

21 . A polynucleotide encoding the polypeptide of any one of items 13 to 19.

22. A vector comprising the polynucleotide of item 21 . 23. A kit comprising the polypeptide of any one of items 13 to 19, the pharmaceutical composition according to item 20, the polynucleotide according to item 21 , and/or the vector of item 22.

In vitro use of the polypeptide as defined in of any one of items 5 to 19 or the pharmaceutical composition of item 20 for inhibiting human adenylyl cyclase 5, wherein the in vitro use is characterized in that human adenylyl cyclase 6 is essentially not inhibited.

An in vitro method for inhibiting human adenylyl cyclase 5, the method comprising bringing into contact

(i) human adenylyl cyclase 5; and

(ii) the polypeptide as defined in any one of claims 5 to 19 or the pharmaceutical composition of claim 20; and

thereby inhibiting human adenylyl cyclase 5, wherein the method is characterized in that human adenylyl cyclase 6 is essentially not inhibited.

The in vitro method according to item 25, wherein the human adenylyl cyclase 5 and the human adenylyl cyclase 6 are comprised by a cell, preferably a human cell. Description of the Figures

[77] Figure 1 : (A) Schematic overview of AnxA4 domain structure. Schematic overview of wildtype AnxA4 and AnxA4 lacking the N-Terminus both containing a myc-tag and four (1 -4) calcium binding repeat domains. (B) Expression of wildtype AnxA4 and AnxA4 lacking the N- terminus. HEK293 expression control of myc-AnxA4-wt and myc-AnxA4-dN by immunoblot using a myc antibody.

[78] Figure 2: (A) Schematic overview of GFP-Trap assay (Figure from Chromotek).The GFP-Trap is a monovalent matrix of magnetic particles coupled with GFP-binding proteins. It was used for the one-step isolation of fluorescent fusion proteins (here YFP-AC5 or YFP- AC6) and their interacting partners (here AnxA4). The GFP-Trap is based on antibodies from the Camelidae family. Camelidae antibodies bind to their antigens via the VHH domains. The VHH domains are small, chemically stable and have specificity and high affinities to their antigens. (B) Schematic overview of myc-AnxA4-wt domain structure. Schematic overview of wildtype AnxA4 containing a myc-tag and four (1 -4) calcium binding repeat domains. (C) Direct interaction of myc-AnxA4-wt and YFP-AC5 using GFP-Trap. "GFP-Trap" based co- immunoprecipitation assays using lysates of HEK293 cells transiently co-transfected with pcDNA3-YFP-hAC5 as bait-protein and myc-AnxA4-wt as prey protein or with GFP and myc- AnxA4-wt as a control. After binding of YFP-AC5 to magnetic beads myc-AnxA4-wt was co- precipitated from the lysate whereas no co-immunoprecipitation of myc-AnxA4-wt was detected in control experiments employing GFP alone.

[79] Figure 3: (A) Schematic overview of AnxA4 lacking the N-Terminus and containing a myc-tag and four (1 -4) calcium binding repeat domains. (B) No direct interaction of myc- AnxA4dN and YFP-AC5 using GFP-Trap. "GFP-Trap" based co-immunoprecipitation assays using lysates of HEK293 cells transiently co-transfected with pcDNA3-YFP-hAC5 as bait- protein and myc-AnxA4-dN as prey protein or with GFP and myc-AnxA4-dN as a control. After binding of YFP-AC5 to magnetic beads myc-AnxA4-dN was not co-precipitated from the lysate. No co-immunoprecipitation of myc-AnxA4-dN was detected in control experiments employing GFP alone.

[80] Figure 4: No decrease of cAMP level in stimulated HEK293 cells with overexpression of AnxA4 lacking the N-terminus. Real time analysis of cAMP levels of HEK293 cells, co- transfected with myc-anxA4-wt or myc-anxA4-dN and a cAMP-sensitive FRET sensor (CFP- EPAC-Ven us-Venus) containing a cyan fluorescent protein (CFP) and an improved version of the yellow fluorescent protein (YFP). Cells were stimulated with NKH477 a direct activator of adenylyl cyclases, and treated with 3 isobutyl-1 -methylxanthin (IBMX), an inhibitor of phosphodiesterases. Ratiometric quantification of CFP/YFP fluorescences of the FRET sensor served as a direct measure of the cAMP produced in the cells. In contrast to cells transfected with a control plasmid or myc-anxA4-dN, myc-anxA4-wt-transfected HEK293 cells, treated with NHK/IBMX, showed a reduction of the CFP/YFP ratio rise indicative of reduced cAMP levels in the first minutes following treatment but not at late time points. [81] Figure 5: Direct interaction of myc-AnxA4-wt and YFP-AC5 using myc-Trap. "myc- Trap" based co-immunoprecipitation assays using lysates of HEK293 cells transiently co- transfected with myc-AnxA4-wt as bait-protein and pcDNA3-YFP-hAC5 as prey protein. After binding of myc-AnxA4-wt to beads YFP-hAC5 protein was co-precipitated from the lysate.

[82] Figure 6: No direct interaction of myc-AnxA4-wt and YFP-AC6 using GFP-Trap. "GFP-Trap" based co-immunoprecipitation assays using lysates of HEK293 cells transiently co-transfected with pcDNA3-YFP-hAC6 as bait-protein and myc-AnxA4-wt as prey protein or with GFP and myc-AnxA4-wt as a control. After binding of YFP-AC6 to magnetic beads myc- AnxA4-wt was not co-precipitated from the lysate. No co-immunoprecipitation of myc-AnxA4- wt was detected in control experiments employing GFP alone. [83] Figure 7: Decrease of cAMP level in stimulated HEK293 treated with AnxA4 N- terminus peptide. Analysis of real time cAMP levels of HEK293 cells, treated with TAT control-peptide or TAT-peptide linked to the 22 amino acid long AnxA4-N-terminus and a cAMP-sensitive FRET sensor (CFP-EPAC-Venus-Venus) containing a cyan fluorescent protein (CFP) and an improved version of the yellow fluorescent protein (YFP). Cells were stimulated with NKH477 a direct activator of adenylyl cyclases, and treated with 3 isobutyl-1 - methylxanthin (IBMX), an inhibitor of phosphodiesterases. Ratiometric quantification of CFP/YFP fluorescences of the FRET sensor served as a direct measure of the cAMP produced in the cells. In contrast to cells treated with the TAT control peptide anxA4-N- terminus-treated HEK293 cells, treated with NHK/IBMX, showed a reduction of the CFP/YFP ratio rise indicative of reduced cAMP levels in the first minutes following treatment but not at late time points.

[84] Figure 8: Functional activity of pcDNA3-AC5 and YFP-AC5 proteins in Hek293 cells. Analysis of cAMP levels by a biochemical assay showed a great increase of cAMP levels in HEK293 cells overexpressing pcDNA3-AC5 and YFP-AC5 treated with NKH477/IBMX for 5 minutes, confirming high activity of adenylyl cyclase protein. Treatment of HEK293 cells with suitable control plasmids pcDNA3 and GFP showed no increase in cAMP levels.

[85] Figure 9: Strong increase of cAMP level in stimulated HEK293 cells with overexpression of pcDNA3-AC5 confirming activity. Analysis of real time cAMP levels of HEK293 cells, co-transfected with pcDNA3, pcDNA3-AC5 and a cAMP-sensitive FRET sensor (CFP-EPAC-Venus-Venus) containing a cyan fluorescent protein (CFP) and an improved version of the yellow fluorescent protein (YFP). Cells were stimulated with forskolin (FSK) a direct activator of adenylyl cyclases, and treated with 3 isobutyl-1 -methylxanthin (IBMX), an inhibitor of phosphodiesterases. Ratiometric quantification of CFP/YFP fluorescences of the FRET sensor served as a direct measure of the cAMP produced in the cells. In contrast to cells transfected with the pcDNA3 control plasmid, pcDNA3-AC5- transfected HEK293 cells treated with FSK/IBMX, showed a much faster and higher increase of the CFP/YFP ratio rise indicative of much faster increased cAMP levels in the first minutes following treatment but not at late time points. HEK293 cells transfected with pcDNA3 and pcDNA3-AC5 without FSK/IBMX treatment showed no rise in CFP/YFP ratio indicative of low cAMP levels over time elapsed.

[86] Figure 10: Schematic overview of generated AnxA4 domain structure mutants. Schematic overview of wildtype AnxA4, AnxA4 lacking the N-Terminus, AnxA4 lacking calcium binding repeat domain no. 4, AnxA4 lacking calcium binding repeat domain no. 3 and 4 and AnxA4 lacking calcium binding repeat domain no. 2, 3 and 4. All constructs contain an N-terminal myc-tag.

[87] Figure 11 : (A) Direct interaction of myc-AnxA4-wt and YFP-AC5 using GFP-Trap. "GFP-Trap" based co-immunoprecipitation assays using lysates of HEK293 cells transiently co-transfected with pcDNA3-YFP-hAC5 as bait-protein and myc-AnxA4-wt as prey protein or with GFP and myc-AnxA4-wt as a control. After binding of YFP-AC5 to magnetic beads myc- AnxA4-wt was co-precipitated from the lysate whereas no co-immunoprecipitation of myc- AnxA4-wt was detected in control experiments employing GFP alone. (B) No direct interaction of myc-AnxA4-wt and YFP-AC6 using GFP-Trap. "GFP-Trap" based co- immunoprecipitation assays using lysates of HEK293 cells transiently co-transfected with pcDNA3-YFP-hAC6 as bait-protein and myc-AnxA4-wt as prey protein or with GFP and myc- AnxA4-wt as a control. After binding of YFP-AC6 to magnetic beads myc-AnxA4-wt was not co-precipitated from the lysate. No co-immunoprecipitation of myc-AnxA4-wt was detected in control experiments employing GFP alone.

[88] Figure 12: (A) No direct interaction of myc-AnxA4-dN and YFP-AC5 using GFP-Trap. "GFP-Trap" based co-immunoprecipitation assays using lysates of HEK293 cells transiently co-transfected with pcDNA3-YFP-hAC5 as bait-protein and myc-AnxA4-dN as prey protein or with GFP and myc-AnxA4-dN as a control. After binding of YFP-AC5 to magnetic beads myc- AnxA4-dN was not co-precipitated from the lysate. No co-immunoprecipitation of myc- AnxA4-dN was detected in control experiments employing GFP alone. (B) No direct interaction of myc-AnxA4-d4 and YFP-AC5 using GFP-Trap. "GFP-Trap" based co- immunoprecipitation assays using lysates of HEK293 cells transiently co-transfected with pcDNA3-YFP-hAC5 as bait-protein and myc-AnxA4-d4 as prey protein or with GFP and myc- AnxA4-d4 as a control. After binding of YFP-AC5 to magnetic beads myc-AnxA4-d4 was not co-precipitated from the lysate. No co-immunoprecipitation of myc-AnxA4-d4 was detected in control experiments employing GFP alone. (C) No direct interaction of myc-AnxA4-d34 and YFP-AC5 using GFP-Trap. "GFP-Trap" based co-immunoprecipitation assays using lysates of HEK293 cells transiently co-transfected with pcDNA3-YFP-hAC5 as bait-protein and myc- AnxA4-d34 as prey protein or with GFP and myc-AnxA4-d34 as a control. After binding of YFP-AC5 to magnetic beads myc-AnxA4-d34 was not co-precipitated from the lysate. No co- immunoprecipitation of myc-AnxA4-d34 was detected in control experiments employing GFP alone.

[89] Figure 13: cAMP measurement via cAMP-Glo Assay. Dose-response-curve of NKH477 (water-soluble derivative of Forskolin) in myc-AnxA4-wt or pcDNA3-myc-transfected cells (control), respectively. In AnxA4-transfected cells compared to control cells, higher dRLU values, indicative to lower cAMP levels, were observed.

[90] Figure 14: Amino acid sequences of polypeptides used herein.

[91] Figure 15: Functional activity of pcDNA3-hAC5, YFP-hAC5, YFP-cAC6, YFP- hAC6 and pcDNA3-hAC6 protein in HEK293 cells. Analysis of cAMP levels by a biochemical assay showed a great increase of cAMP levels in HEK293 cells overexpressing pcDNA3-hAC5, YFP-hAC5, YFP-cAC6, YFP-hAC6 and pcDNA3-hAC6 treated with NKH477/IBMX for 5 minutes, confirming high activity of adenylyl cyclase protein. Treatment of HEK293 cells with suitable control plasmids pcDNA3 and GFP showed no increase in cAMP levels.

[92] Figure 16: Decrease of cAMP-level in stimulated HEK293 cells treated with 10 μΜ AnxA4 N-terminus peptide. Analysis of cAMP levels by a biochemical assay showed a significant decrease of cAMP levels in HEK293 cells treated with 10 μΜ AnxA4 N-terminus peptide after stimulation with NKH477/IBMX for 5 minutes. Treatment of HEK293 cells with 50 μΜ AnxA4 N-terminus peptide showed no decrease in cAMP levels compared to TAT- treated control. [93] Figure 17: Comparison of cAMP level in non-, sc22- and TAT-treated pcDNA3- hAC5-overexpressing HEK293 cells. Analysis of real time cAMP levels of HEK293 cells, co-transfected with pcDNA3-AC5 and a cAMP-sensitive FRET sensor (CFP-EPAC-Venus- Venus) containing a cyan fluorescent protein (CFP) and an improved version of the yellow fluorescent protein (YFP). 24 h after transfection these cells were either not treated or treated with TAT-peptide and sc22 peptide, containing TAT sequence and the 22 aa of AnxA4 N- terminus in a scrambled order. Cells were stimulated with NKH477 a direct activator of adenylyl cyclases, and treated with 3 isobutyl-1-methylxanthin (IBMX), an inhibitor of phosphodiesterases. Ratiometric quantification of CFP/YFP fluorescences of the FRET sensor served as a direct measure of the cAMP produced in the cells. The normalized emission ratios were not significant different between non-treated, sc22- or TAT-treated HEK293 cells after stimulation with NKH477/IBMX indicating no difference between these controls used in further experiments.

[94] Figure 18: Decrease of cAMP level in stimulated pcDNA3-hAC5 overexpressing HEK293 cells treated with AnxA4 N-terminus peptide. Analysis of real time cAMP levels of HEK293 cells, co-transfected with pcDNA3-AC5 and a cAMP-sensitive FRET sensor (CFP-EPAC-Venus-Venus) containing a cyan fluorescent protein (CFP) and an improved version of the yellow fluorescent protein (YFP). 24 h after transfection these cells were treated with TAT control-peptide or AnxA4 N-terminus peptide (TAT-A4N_1-22) and stimulated with 0,5 μΜ NKH477 a direct activator of adenylyl cyclases, and treated with 100 μΜ 3 isobutyl-1-methylxanthin (IBMX), an inhibitor of phosphodiesterases. Ratiometric quantification of CFP/YFP fluorescences of the FRET sensor served as a direct measure of the cAMP produced in the cells. TAT-A4N_{1 -2}2-treated HEK293 cells showed a significant decrease in cAMP level in the first minutes following stimulation compared to TAT-treated control. At late time points no difference could be observed.

[95] Figure 19: No decrease of cAMP level in stimulated pcDNA3-hAC6 overexpressing HEK293 cells treated with AnxA4 N-terminus peptide. Analysis of real time cAMP levels of HEK293 cells, co-transfected with pcDNA3-AC6 and a cAMP-sensitive FRET sensor (CFP-EPAC-Venus-Venus) containing a cyan fluorescent protein (CFP) and an improved version of the yellow fluorescent protein (YFP). 24 h after transfection these cells were treated with TAT control-peptide or AnxA4 N-terminus peptide (TAT-A4N_{1 -2}2) and stimulated with 0,5 μΜ NKH477 a direct activator of adenylyl cyclases, and treated with 100 μΜ 3 isobutyl-1 -methylxanthin (IBMX), an inhibitor of phosphodiesterases. Ratiometric quantification of CFP/YFP fluorescences of the FRET sensor served as a direct measure of the cAMP produced in the cells. TAT-A4N_1-22-treated HEK293 cells showed no difference in cAMP level compared to TAT-treated control cells.

[96] Figure 20: Decrease of cAMP level in stimulated pcDNA3-hAC5 overexpressing HEK293 cells and coexpressing myc-AnxA4 WT. Analysis of real time cAMP levels of HEK293 cells, co-transfected with pcDNA3-hAC5, myc-AnxA4 WT or pcDNA3-myc (as control) and a cAMP-sensitive FRET sensor (CFP-EPAC-Venus-Venus) containing a cyan fluorescent protein (CFP) and an improved version of the yellow fluorescent protein (YFP). Cells were stimulated with NKH477 as a direct activator of adenylyl cyclases, and treated with 3 isobutyl-1-methylxanthin (IBMX), an inhibitor of phosphodiesterases. Ratiometric quantification of CFP/YFP fluorescences of the FRET sensor served as a direct measure of the cAMP produced in the cells. In contrast to cells transfected with the pcDNA3-myc control plasmid, myc-AnxA4 WT-transfected HEK293 cells treated with NKH477/IBMX, showed a smaller increase of the CFP/YFP ratio rise indicative of slower increased cAMP levels in the first minutes following treatment but not at late time points. This is in line with an inhibition of adenylyl cyclase activity under inhibition of cAMP degradation by the use of the phosphodiesterase inhibitor IBMX.

[97] Figure 21 : No decrease of cAMP level in stimulated pcDNA3-hAC6 overexpressing HEK293 cells and coexpressing myc-AnxA4 WT. Analysis of real time cAMP levels of HEK293 cells, co-transfected with pcDNA3-hAC6, myc-AnxA4 WT or pcDNA3-myc (as control) and a cAMP-sensitive FRET sensor (CFP-EPAC-Venus-Venus) containing a cyan fluorescent protein (CFP) and an improved version of the yellow fluorescent protein (YFP). Cells were stimulated with NKH477 as a direct activator of adenylyl cyclases, and treated with 3 isobutyl-1-methylxanthin (IBMX), an inhibitor of phosphodiesterases. Ratiometric quantification of CFP/YFP fluorescences of the FRET sensor served as a direct measure of the cAMP produced in the cells. In cells transfected with the pcDNA3-myc control plasmid and myc-AnxA4 WT-transfected HEK293 cells treated with NKH477/IBMX, no difference in cAMP levels could be detected.

[98] Figure 22: Decrease of cAMP level in stimulated pcDNA3-hAC5 overexpressing HEK293 cells and coexpressing myc-AnxA4 WT but not in stimulated pcDNA3-hAC6 overexpressing cells coexpressing myc-AnxA4 WT. Analysis of real time cAMP levels of HEK293 cells, co-transfected with pcDNA3-hAC6 + myc-AnxA4 WT, pcDNA3-hAC6 + pcDNA3-myc (as control), pcDNA3-hAC5 + myc-AnxA4 WT or pcDNA3-hAC5 + pcDNA3- myc (as control) and a cAMP-sensitive FRET sensor (CFP-EPAC-Venus-Venus) containing a cyan fluorescent protein (CFP) and an improved version of the yellow fluorescent protein (YFP). Cells were stimulated with NKH477 as a direct activator of adenylyl cyclases, and treated with 3 isobutyl-1 -methylxanthin (IBMX), an inhibitor of phosphodiesterases. Ratiometric quantification of CFP/YFP fluorescences of the FRET sensor served as a direct measure of the cAMP produced in the cells. In cells transfected pcDNA3-hAC6 + myc-AnxA4 WT and pcDNA3-hAC6 + pcDNA3-myc-transfected HEK293 cells treated with NKH477/IBMX, no difference in cAMP levels could be detected. In contrast to cells transfected with the pcDNA3-hAC6 plasmid, pcDNA3-hAC5 + myc-AnxA4 WT -transfected HEK293 cells treated with NKH477/IBMX, showed a smaller increase of the CFP/YFP ratio rise indicative of slower increased cAMP levels in the first minutes following treatment but not at late time points (compared to pcDNA3-hAC5 + pcDNA3-myc control). This is in line with an inhibition of adenylyl cyclase activity under inhibition of cAMP degradation by the use of the phosphodiesterase inhibitor IBMX. [99] Figure 23: Decrease of cAMP-Level in stimulated AC5-overexpressing HEK293 cells treated with AnxA4 N-terminus peptide. Analysis of cAMP levels by a biochemical assay showed a significant decrease of cAMP levels in HEK293 cells transfected with pcDNA3-hAC5 and treated with 10 μΜ AnxA4 N-terminus peptide after stimulation with NKH477/IBMX for 5 minutes compared to control cells, treated with sc22 containing TAT sequence and the 22 aa of AnxA4 N-terminus in a scrambled order.

[100] Figure 24: No decrease of cAMP -Level in stimulated AC6-overexpressing HEK293 cells treated with AnxA4 N-terminus peptide. Analysis of cAMP levels by a biochemical assay showed no difference in cAMP levels in HEK293 cells transfected with pcDNA3-hAC5 and treated with 10 μΜ AnxA4 N-terminus peptide after stimulation with NKH477/IBMX for 5 minutes compared to control cells, treated with sc22, containing TAT sequence and the 22 aa of AnxA4 N-terminus in a scrambled order.

[101] Figure 25: (A) Direct interaction of myc-AnxA4-wt and YFP-AC5 using GFP-Trap. "GFP-Trap" based co-immunoprecipitation assays using lysates of HEK293 cells transiently co-transfected with myc-AnxA4-wt as prey-protein and pcDNA3-YFP-hAC5 as bait-protein. After binding of YFP-hAC5 to beads myc-AnxA4-wt protein was co-precipitated from the lysate; (B) and (C) No direct interaction of myc-AnxA4-wt and YFP-AC6 using GFP-Trap. "GFP-Trap" based co-immunoprecipitation assays using lysates of HEK293 cells transiently co-transfected with pcDNA3-YFP-hAC6 as bait-protein and myc-AnxA4-wt as prey protein or with GFP and myc-AnxA4-wt as a control. After binding of YFP-AC6 to magnetic beads myc- AnxA4-wt was not co-precipitated from the lysate. No co-immunoprecipitation of myc-AnxA4- wt was detected in control experiments employing GFP alone.

Examples

Example 1:

Antibody cDNA plasmid constructs and peptides. [103] To obtain a construct for overexpression of the full length AnxA4 cDNA with an N- terminal myc-Tag in HEK293 cells (myc-AnxA4-wt), a pcDNA3-myc vector was cut with BamHI and EcoRI (vector kindly provided by Prof. Dr. Heumann, Ruhr-Universitat Bochum). Then AnxA4 cDNA was generated by PCR using the primers 5 -AAAGGATCCATGGAA- GCCAAAGGAGGAACC-3^' (BamHI-AnxA4wt-fwd, SEQ ID NO: 15) and 5 - CCCGAATTCTTAATCATCTCCTCCACAGAG-3^' (EcoRI-AnxA4wt-rev, SEQ ID NO: 16) and wildtype AnxA4 as template. The AnxA4 PCR product was digested with BamHI and EcoRI and ligated into the linearized pcDNA-myc vector.

[104] AnxA4 consists of a specific N-terminal domain and four calcium binding repeats. The constructs for overexpression of different AnxA4 mutants lacking only the N-terminus (SEQ ID NO: 28), lacking only repeat 4 (SEQ ID NO: 29), lacking only repeat 3+4 (SEQ ID NO: 30) or lacking only repeat 2+3+4 (SEQ ID NO: SEQ ID NO: 1 ), respectively, were cloned by amplification of the appropriate AnxA4 sequence with the corresponding primer pairs.

AnxA4-dN:

BamHI-ANXAdN-fwd (SEQ ID NO: 17): 5 -AAAGGATCCATGCTGAGGAAGGCCATGAAAG- 3^'

EcoRI-AnxA4wt-rev (SEQ ID NO: 18): 5^'-CCCGAATTCTTAATCATCTCCTCCACAGAG-3^' AnxA4-d4:

BamHI-ANXA4wt-fwd (SEQ ID NO: 19): 5 -AAAGGATCCATGGAAGCCAAAGGAGGAACC- 3^'

EcoRI-ANXA4-d4-rev (SEQ ID NO: 20): 5^'-

CCCGAATTCTTACTTCACTATAGCCAACAGGG-3^'

AnxA4-d34:

BamHI-ANXA4wt-fwd (SEQ ID NO: 21 ): 5 -AAAGGATCCATGGAAGCCAAAGGAGGAACC- 3^'

EcoRI-ANXA4-d34-rev (SEQ ID NO: 22): 5^'-GATGAATTCTTACGACAGGGACACCAGTACT C-3^' AnxA4-d234

BamHI-ANXA4wt-fwd (SEQ ID NO: 23): 5 -AAAGGATCCATGGAAGCCAAAGGAGGAACC- 3^'

EcoRI-ANXA4-d234-rev (SEQ ID NO: 24): 5^'- G ATG AATTCTTACAG CCCCAG G ATCACCTG-3 ^'

The PCR products were cut with BamHI and EcoRI and inserted into the pcDNA3-myc vector, which was also digested with BamHI and EcoRI.

[105] The CFP-EPAC-Venus-Venus construct (SEQ ID NO: 34) herein referred to as EPAC-FRET) was a kind gift from Kees Jalink from the "Netherlands Cancer Institute" in Amsterdam, NL.

[106] The pcDNA3-AC5 (SEQ ID NO: 31 ), the pcDNA3 YFP-hAC5 (SEQ ID NO: 32) and the pcDNA3 YFP-hAC6 (SEQ ID NO: 33) constructs were kindly provided by Dr. Carmen W. Dessauer (Department of Integrative Biology and Pharmacology, The University of Texas, Health Science Center, Houston, USA). [107] The AnxA4 N-terminus peptide consists of the first 22 amino acids of the murine AnxA4-wt protein (AnxA41 -22), a glycine linker and a TAT-sequence. The TAT-sequence (GRKKRRQRRR) corresponds to the amino acid sequence 48-57 of the HIV-TAT-protein and enables fused peptides or molecules to penetrate the cell membrane. The full TAT- AnxA41-22 peptide (GRKKRRQRRRGMEAKGGTVKAASGFNATEDAQT; SEQ ID NO: 25) was synthesized by EMC microcollections (Tubingen, Germany), purified by HPLC (purity > 95 %) and tested by mass spectroscopy. The control peptide TAT48-57 was purchased from anaspec (Gottingen, Germany).

Example 2

Cell culture [108] Human embryonic kidney cells (HEK293) were cultured in Dulbecco^'s modified Eagle^'s medium (PAA, Pashing, Austria) supplemented with 10% (vol/vol) fetal bovine serum, 100 U penicillin, and 0.1 mg streptomycin per ml at 37°C in an atmosphere of 5% C0₂. Cells were transfected using X-tremeGENE HP Transfection Reagenz (Roche, Basel, Switzerland) according to manufacturer's protocol. Example 3

myc-AnxA4-wt and myc-AnxA4-dN immunoblotting in transiently transfected HEK293.

[109] HEK293 cells were transfected with 5 pg of myc-AnxA4-wt or myc-AnxA4-dN for 48 h. The cells were harvested and homogenized in mammalian lysis buffer [50 mM Tris-CI (pH 7.5), 150 mM NaCI, 1 % SDS, and 1 % Triton-X100] supplemented with protease inhibitor cocktail (Promega). The cells were homogenized through a 27-gauge needle attached to a syringe and incubated on ice for another 20 min, with extensive pipetting every 10 min. After centrifugation (8 min, 20,000 g, 4°C) the supernatant was used as the cell lysate and the protein content of the supernatant was determined according to Lowry with BSA as standard. After SDS-PAGE on 10% polyacrylamide gels, the transfer onto nitrocellulose membranes (BA 85 Protran Nitrocellulose, Whatman™, GE Healthcare Life Sciences, Littele Chalfont, UK) was performed by tank blotting in sodium phosphate buffer for 180 min at 1.5 A and 4°C. For detection of myc-tagged AnxA4 mutants a primary myc-antibody from Cell Signaling Technology® was used (anti-myc (9B1 1 ), dilution 1 :2000 in 5% dry fat milk in TBS-T). Membranes were blocked with 5% dry fat milk in TBS-T for 1 h at room temperature before over night incubation with the primary antibody at 4°C. Membranes were washed with TBS/TBS-T/TBS each for 10 min before and after incubation with secondary anti-mouse antibody for 2 h at room temperature (ECL™ Anti-rabbit IgG, HRP linked, GE Healthcare, Little Chalfont, UK; 1 : 10000 in 5% dry fat milk in TBS-T). Example 4

Co-lmmunoprecipitation and immunoblotting via "GFP-Trap"

[110] For co-immunoprecipitation (co-IP), HEK293 cells were transfected with myc-AnxA4- wt (SEQ ID NO: 35) and pcDNA3 YFP-hAC5 DNA, myc-AnxA4-wt and pcDNA3 YFP-hAC6 DNA or myc-AnxA4-wt and eGFP as control (pEGFP-N1 , Clontech, La Yolla, USA). All AnxA4-mutants (AnxA4 dN (SEQ ID NO: 36), AnxA4d4 (SEQ ID NO: 37), AnxA4d34 (SEQ ID NO: 38), AnxA4d234(SEQ ID NO: 39)) were also used for co-immunopreciptiation with pcDNA3 YFP-hAC5 DNA or eGFP as control. 24-48 h after transfection cells were harvested and homogenized in lysis buffer (10 mM Tris/CI (pH 7.5), 150 mM NaCI, 0.5 mM EDTA (Ethylendiamintetraacetat), 0.5% IGEPAL CA-630) supplemented with Protease Inhibitor Cocktail (Promega, Madison, USA). Cells were homogenized through a G-27 needle attached to a syringe and additionally incubated on ice for 20 minutes with extensively pipetting every 10 minutes. Following centrifugation (8-10 min, 20000 g, 4°C) the supernatant was used as "cell lysate" in the "GFP-Trap" based co-IP (GFP-Trap^®, ChromoTek, Planegg- Martinsried, Germany). "GFP-Trap" was used according to manufacturer's protocol. Protein concentration has been determined using the Lowry protein Assay. The corresponding volume of 750 pg cell lysate protein was adjusted to 500 μΙ with cold dilution buffer (10 mM Tris/CI (pH 7.5), 50 mM NaCI, 0.5 mM EDTA) (conditions for experiment shown in figure 25). In case of the other GFP-Trap experiments 170 μΙ cell lysate was diluted with 500 μΙ cold dilution buffer (10 mM Tris/CI (pH 7.5), 150 mM NaCI, 0.5 mM EDTA) and incubated with equilibrated magnetic beads under constant mixing for 1 h at 4°C. The "GFP-Trap" magnetic beads are also able to bind YFP-linked proteins. After 3-4 washing steps with cold dilution buffer, bound proteins were eluted by boiling the beads for 10 min at 95°C in 80 μΙ SDS Elution Buffer (10 mM Tris/CI (pH 7.5), 1 % SDS). Five samples were loaded on 10% SDS- polyacrylamide gels: 3 μΙ of cell lysate + 12 μΙ SDS Elution Buffer and 15 μΙ of the not bound fraction with 5 μΙ 4xl_ammli (supplemented with DTT (6mg/ml) and 10% SDS), were used. 37.5 μΙ of the wash step supernatants 1 , 3 and the eluate were mixed with 12.5 μΙ 4xLammli and also loaded on the gel (conditions for experiment shown in figure 25). Five samples were loaded on Mini-Protean TGX 4-15% gels (Bio-Rad). 1.1 μΙ of cell lysate and of the not bound fraction + 13.65 μΙ SDS Elution Buffer, 13.2 μΙ of the wash step supernatant and of the eluate + 1.65 μΙ SDS Elution Buffer, to every sample 4.95 μΙ 4xLammli was added (supplemented with DTT (6mg/ml) and 10% SDS). After SDS-PAGE the proteins were electroblotted onto PVDF membrane (Whatman™ Westran™ CS PVDF Membrane, Schleicher & Schullj in ethanol buffer (25 mM Tris, 192 mM Glycin, 20% ethanol ad. 1 1 H₂0) for 1 h at 100 V and 4°C using the tank blotting method. Membranes were blocked with 5% dry fat milk in Tris-Buffered Saline-T (TBS-T, 9 g NaCI, 1.58 g Tris, ad 1 I H₂0, pH 7.4) with 1 % Tween 20) for 1 h at room temperature before the myc-tagged AnxA4 was detected using the myc 9B1 1 antibody as described above; dilution 1 :2000 in 5% dry fat milk in TBS- T) overnight at 4°C. Immunoblot with anti-myc antibody (9B1 1 ) was additionally probed with an anti-GFP antibody (GFP [3H9] Rat monoclonal, ChromoTek; dilution 1 :1000 in 2-5% BSA in PBS or TBS-T) and immunological detection was performed overnight at 4°C after blocking with 2-5% BSA in PBS or TBS-T for 1 h at room temperature. Previously to and after the application of secondary antibody the membranes were washed tree times for 10 min with TBS-T. The secondary antibody was applied for 2 h at room temperature for myc anti-mouse (Anti-mouse IgG, HRP linked, Sigma-Aldrich, Steinheim, Germany) 1 : 10000 in 5% dry fat milk in TBS-T; for GFP anti-rat (ECL™ Anti-rat IgG, HRP linked, GE Healthcare, Little Chalfont, UK) 1:4000 in 2-5% BSA in PBS or TBS-T. Signals were visualized and quantified using the ECL plus detection system (Amersham ECL Plus; GE Healthcare, Little Chalfont, UK) and ChemiDoc™ MP Imaging System (Bio-Rad Laboratories, Mijnchen, Germany).

Example 5

Co-immunoprecipitation and immunoblotting via "myc-Trap" [111] For co-immunoprecipitation (co-IP) HEK293 cells were transfected with myc-AnxA4- wt and pcDNA3 YFP-hAC5 DNA, myc-AnxA4-wt and pcDNA3 YFP-hAC6 DNA or pcDNA3 DNA and pcDNA3 YFP-hAC5 DNA or pcDNA3 YFP-hAC6 (as control), respectively. 24 h after transfection cells were harvested and homogenized in lysis buffer (10 mM Tris/CI (pH 7.5), 150 mM NaCI, 0.5 mM EDTA (Ethylendiamintetraacetat), 0.5% IGEPAL CA-630) supplemented with Protease Inhibitor Cocktail (Promega, Madison, USA). Cells were homogenized through a G-27 needle attached to a syringe and additionally incubated on ice for 20 minutes with extensively pipetting every 10 minutes. Following centrifugation (8 min, 20000 g, 4°C) the supernatant was used as "cell lysate" in the "Myc-Trap" based co-IP (Myc- Trap^®, ChromoTek, Planegg-Martinsried, Germany). "Myc-Trap" was used according to manufacturer's protocol. The whole cell lysate was diluted with 300 μΙ cold dilution buffer (10 mM Tris/CI (pH 7.5), 150 mM NaCI, 0.5 mM EDTA) and incubated with equilibrated Myc- Trap_A beads under constant mixing for 1 h at 4°C. The "Myc-Trap" agarose beads are also able to bind myc-tagged proteins. After 4 washing steps with cold dilution buffer, bound proteins were eluted by adding a myc-peptide in a final concentration of 25 μg diluted in 50 μΙ water for 15 min at room temperature. To increase the elution efficiency this step was repeated. Five samples were loaded on Mini-Protean TGX 4-15% gels (Bio-Rad). 1.1 μΙ of cell lysate and of the not bound fraction + 13.65 μΙ SDS Elution Buffer, 13.2 μΙ of the wash step supernatant and of the eluate + 1.65 μΙ SDS Elution Buffer, to every sample 4.95 μΙ 4xLammli was added (supplemented with DTT (6mg/ml) and 10% SDS). After SDS-PAGE the proteins were electroblotted onto PVDF membrane (Whatman™ Westran™ CS PVDF Membrane, Schleicher & SchullJ in ethanol buffer (25 mM Tris, 192 mM Glycin, 20% ethanol ad. 1 1 H₂0) for 1 h at 100 V and 4"C using the tank blotting method. Membranes were blocked with 5% dry fat milk in Tris-Buffered Saline-T (TBS-T, 9 g NaCI, 1 .58 g Tris, ad 1 I H₂0, pH 7.4) with 1 % Tween 20) for 1 h at room temperature before the myc-tagged AnxA4 was detected using the myc 9B1 1 antibody as described above; dilution 1 :2000 in 5% dry fat milk in TBS-T) overnight at 4°C. Immunoblot with anti-myc antibody (9B1 1 ) was additionally probed with an anti-GFP antibody (GFP [3H9] Rat monoclonal, ChromoTek; dilution 1 :1000 in 2% BSA in PBS) and immunological detection was performed overnight at 4°C after blocking with 2% BSA in PBS for 1 h at room temperature. Previously to and after the application of secondary antibody the membranes were washed tree times for 10 min with TBS-T. The secondary antibody was applied for 2 h at room temperature for myc anti-mouse (Anti-mouse IgG, HRP linked, Sigma-Aldrich, Steinheim, Germany) 1 :10000 in 5% dry fat milk in TBS-T; for GFP anti-rat (ECL™ Anti-rat IgG, HRP linked, GE Healthcare, Little Chalfont, UK) 1:4000 in 2% BSA in PBS. Signals were visualized and quantified using the ECL plus detection system (Amersham ECL Plus; GE Healthcare, Little Chalfont, UK) and ChemiDoc™ MP Imaging System (Bio-Rad Laboratories, Munchen, Germany).

BLANK PAGE

Example 6

Dynamic FRET monitoring.

[112] For real time cAMP measurements HEK293 were co-transfected with 1000 ng EPAC- FRET DNA and 1000 ng of myc-AnxA4-wt and myc-AnxA4-dN DNA or equal pcDNA3-myc amounts as control, respectively. The aim of another experiment was to determine the activity of pcDNA3-AC5 in HEK293 cells. In this experiment HEK293 were co-transfected with 1000 ng EPAC-FRET DNA and 500 ng pcDNA3-AC5 or equal pcDNA3 amounts as control. The measurement was performed 48 h (AnxA4) or 24 h (AC5) after transfection. For testing the peptide cells were transfected with 1000 ng EPAC-FRET DNA and incubated for 30 min with 10 μΜ TAT-AnxA4_{1 -2}2 or TAT_48-57 (as control) in serum free medium (37 °C, 5 % C0₂) before the start of the measurement. The basal FRET signals were detected for 3 min before the bath was changed to phosphate buffered saline (PBS) solution (137 mM NaCI, 2.86 mM KCI, 10.14 mM Na₂HP0₄, 1.76 mM KH₂P0₄, pH 7.4) containing 50 μΜ of the potent adenylyl cyclase activator NKH (NKH477, SigmaAldrich, Steinheim, Germany) + 100 μΜ of the PDE inhibitor 3-isobutyl-1 -methylxanthine (IBMX; Sigma-Aldrich, Steinheim, Germany).

[113] A. AnxA4 deletion mutants

For real time cAMP measurements HEK293 were co-transfected with 1000 ng EPAC-FRET DNA and 1000 ng of myc-AnxA4-wt and myc-AnxA4-dN DNA or equal pcDNA3-myc amounts as control, respectively. The measurement was performed 48 h (AnxA4) after transfection. The basal FRET signals were detected for 3 min before the bath was changed to phosphate buffered saline (PBS) solution (137 mM NaCI, 2.86 mM KCI, 10.14 mM Na₂HP0 , 1 .76 mM KH₂P0 , pH 7.4) containing 50 μΜ of the potent adenylyl cyclase activator NKH (NKH477, SigmaAldrich, Steinheim, Germany) + 100 μΜ of the PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX; Sigma-Aldrich, Steinheim, Germany). [114] B. AC5-activity

For real time cAMP measurements HEK293 were co-transfected with 1000 ng EPAC-FRET DNA and 500 ng pcDNA3-AC5 or equal pcDNA3 amounts as control. The measurement was performed 24 h after transfection. The basal FRET signals were detected for 3 min before the bath was changed to phosphate buffered saline (PBS) solution containing 50 μΜ of the potent adenylyl cyclase activator NKH (NKH477, SigmaAldrich, Steinheim, Germany) + 100 μΜ of the PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX; Sigma-Aldrich, Steinheim, Germany).

[115] C. Effect of AnxA4 WT on AC5 and AC6 activity For real time cAMP measurements HEK293 were co-transfected with 1000 ng EPAC-FRET DNA, 500 ng pcDNA3-AC5, pcDNA3-hAC6 and myc-AnxA4 WT or equal pcDNA3-myc amounts as control. The measurement was performed 24 h after transfection. The basal FRET signals were detected for 3 min before the bath was changed to phosphate buffered saline (PBS) solution containing 0.5 μΜ of the potent adenylyl cyclase activator NKH (NKH477, SigmaAldrich, Steinheim, Germany) + 100 μΜ of the PDE inhibitor 3-isobutyl-1- methylxanthine (IBMX; Sigma-Aldrich, Steinheim, Germany).

[116] D. Effect of TAT-AnxA4_{1 -2}2 without AC-overexpression

For real time cAMP measurements HEK293 were co-transfected with 1000 ng EPAC-FRET DNA and incubated for 30 min with 10 μΜ TAT-AnxA4_1-22 or TAT_48-57 (as control) in serum free medium (37 °C, 5 % C0₂) before the start of the measurement. The basal FRET signals were detected for 3 min before the bath was changed to phosphate buffered saline (PBS) solution containing 50 μΜ of the potent adenylyl cyclase activator NKH (NKH477, SigmaAldrich, Steinheim, Germany) + 100 μΜ of the PDE inhibitor 3-isobutyl-1- methylxanthine (IBMX; Sigma-Aldrich, Steinheim, Germany).

[117] E. Effect of TAT-AnxA4i_-22 with AC5 and AC6 overexpression

For real time cAMP measurements HEK293 were co-transfected with 1000 ng EPAC-FRET DNA, 500 ng pcDNA3-AC5, pcDNA3-hAC6 and incubated for 5 min with 10 μΜ TAT-AnxA4i. ₂₂ or TAT-sc22 (as control) in serum free medium (37 °C, 5 % C0₂) before the start of the measurement. The basal FRET signals were detected for 3 min before the bath was changed to phosphate buffered saline (PBS) solution containing 0.5 μΜ of the potent adenylyl cyclase activator NKH (NKH477, SigmaAldrich, Steinheim, Germany) + 100 μΜ of the PDE inhibitor 3-isobutyl-1 -methylxanthine (IBMX; Sigma-Aldrich, Steinheim, Germany).

[118] Fluorescence was recorded using a Zeiss LSM 710 confocal fluorescence microscope (Zeiss, Oberkochen, Germany) with excitation at 458 nm. Emission of CFP and YFP was detected simultaneously through 463-509 nm and 519-621 nm band-pass filters. FRET was expressed as ratio of CFP to YFP signals, the value of which was set to 100% at the onset of the experiments. Ratio changes correlate with the changes of cAMP levels within the cells and were expressed as percent deviation from the initial value of 100% or values of AnxA4 overexpressing cells as percent deviation from the control value at one time point. For analysis of cells treated with NKH + IBMX, only cells with a predefined range of initial CFP signal were used, in order to ensure a comparable EPAC-FRET protein expression level in the cells. Example 7

In vitro measurement of intracellular cAMP-levels. [119] A. AC-activity

HEK293 cells, grown in cell culture flasks were transiently transfected with 4.2 μg pcDNA3 YFP-hAC5 DNA, 4.2 μg eGFP, 4.2 μg pcDNA3-AC5, 4.2 μg pcDNA3, 4.2 μg YFP-cAC6, 4.2 μg YFP-hAC6, 4.2 μg pcDNA3-hAC6 and treated 24 h after transfection with 50 μΜ NKH477 + 100 μΜ IBMX in PBS for 10 min, while maintained at 37°C and 5% C0₂.

[120] B. Coexpression of myc-AnxA4 WT and pcDNA3-hAC5/pcDNA3-hAC6

HEK293 cells, grown in cell culture flasks were transiently transfected with 4.2 μg pcDNA3- hAC5+ myc-AnxA4 WT, 4.2 μg pcDNA3-hAC5 + pcDNA3-myc (as control), 4.2 μg pcDNA3- hAC6+ myc-AnxA4 WT, 4.2 μg pcDNA3-hAC6 + pcDNA3-myc (as control) and treated 24 h after transfection with 0.5 μΜ NKH477 + 100 μΜ IBMX in PBS for 5 min, while maintained at 37°C and 5% C0₂.

[121] C. Treatment with Annexin A4 N-Terminus peptide HEK293 cells, grown in cell culture flasks were transiently transfected with 4.2 μg pcDNA3- hAC5, 4.2 μg pcDNA3-hAC6 and incubated 24 h after transfection for 5 min with 10 μΜ TAT- AnxA4-|.22 or TAT-sc22 (as control) in serum free medium (37 °C, 5 % C0₂) before the start of stimulation with 0.5 μΜ NKH477 + 100 μΜ IBMX in serum free medium for 5 min, while maintained at 37°C and 5% C0₂. [122] D. Test of different peptide concentrations

HEK293 cells, grown in cell culture flasks were incubated for 30 min with 10 μΜ ΤΑΤ-ΑΠΧΑ4_!. ₂₂ or TAT₄₈-57 (as control) in serum free medium (37 °C, 5 % C0₂) before the start of stimulation with 50 μΜ NKH477 + 100 μΜ IBMX in serum free medium for 5 min, while maintained at 37°C and 5% C0₂. [123] After quick-freezing in N₂ and resuspension in 400 μΙ ice cold PBS, cell disruption was achieved by addition of 0.05 M HCI and incubation at 95°C for 10 min. The cAMP-carrying cytosolic fraction of HEK293 cells was obtained in the supernatant after centrifugation (15 minutes, 14000 g, 4°C). The pellet was dissolved in 500 μΙ NaOH (0.1 M) and the protein concentration was determined according to Bradford with BSA as standard (29). The cAMP concentration was measured with the Amersham cAMP Biotrak Enzymeimmunoassay (EIA) System (GE Healthcare, Little Chalfont, UK). The enzyme immunoassay was performed according to manufacturer's protocol. For the measurement of the NKH/IBMX-treated samples 0.3 μg protein was used in the non-acetylation protocol to measure the cAMP content. The enzymatic reaction of peroxidase-labeled cAMP conjugate was stopped by addition of 1.0 M sulfuric acid. The optical density was detected at 450 nm within 30 min employing a Mithras LB 940 Microplate Analyser (Berthold Technologies, Bad Wildbad, Germany).

Example 8

cAMP measurement via cAMP-Glo Assay [124] HEK293 were grown in cell culture flasks and transfected with 3 myc-AnxA4-wt DNA or 3 μg pcDNA3-myc DNA, respectively (as control). 24 h after transfection, transfected cells were seeded in a density of 10.000 cells per well on a 96-well plate (37 °C, 5 % C0₂). After 48 h cells were treated with different NKH477 (water-soluble derivative of Forskolin) concentrations (0; 5;10;20;30;40;50;60;75;100 μΜ) and 100 μΜ IBMX in PBS for 5 min, while maintained at 37 °C and 5% C0₂. The cAMP-Glo Assay (Promega, Madison, USA) was performed according to manufacturer's protocol and luminescence was measured 10 min after addition of Kinase-Glo Reagent. Luminescence units were normalized to non-treated samples. The assay is based on the principle that protein kinase A (PKA) activity is stimulated by cAMP, which increases after stimulation with NKH477 and IBMX. In turn this decreases the available ATP concentration leading to a decreased light production in a coupled luciferase reaction. A decreased cAMP level leads to a higher RLU value.

[125]

Claims

1. An AnnexinA4 polypeptide for use in a method for treating a disease that can be treated using β-AR antagonists in a subject, wherein the use is characterized in that human adenylyl cyclase 5 is specifically inhibited.

2. The AnnexinA4 polypeptide of claim 1 , wherein the use is characterized in that human adenylyl cyclase 6 is essentially not inhibited, as determined in an in vitro assay.

3. The AnnexinA4 polypeptide of claim 1 or 2, wherein the subject suffers from cardiac stress, cardiomyopathy, heart failure, diabetes, obesity, aging, pain ischemic cardiomyopathy, such as chest pain or angina, high blood pressure, arrhythmia, heart attacks, migraine, tremor or tumor growth, and preferably from heart failure.

4. The AnnexinA4 polypeptide of anyone of claims 1 to 3, wherein the use comprises administering to a subject a pharmaceutically effective amount of AnnexinA4.

5. The AnnexinA4 polypeptide for the use of anyone of claims 1 -4, wherein said AnnexinA4 polypeptide

(a) has the amino acid sequence shown in SEQ ID NO: 2,

(b) is 60 % identical to the amino acid sequence of SEQ ID NO: 2, or

(c) is a fragment of the amino acid sequence of (a) or (b).

6. The Annexin A4 polypeptide for the use of claim 5, wherein said fragment consists of

(a) amino acids 1 to 22 of the amino acid sequence shown in SEQ ID NO: 1 ;

(b) a fragment of the amino acid sequence of (a);

7. The Annexin A4 polypeptide for the use of claim 6, wherein the amino acid sequence of (a) is the amino acid sequence shown in SEQ ID NO: 1.

8. The Annexin A4 polypeptide for the use of anyone of claims 1 to 7, wherein the polypeptide is fused with a linker.

9. The Annexin A4 polypeptide for the use of claim 8, wherein the linker is selected from the group consisting of (GGGGS)n, Gly, (Gly)8, (Gly)6, (EAAK)n, A(EAAAK)4ALEA(EAAAK)4A, PAPAP, AEAAAKEAAAKA, (Ala-Pro)n, wherein the linker is preferably a Glycine residue.

10. The Annexin A4 polypeptide for the use of anyone of claims 1 to 9, wherein said polypeptide is fused with a cell penetrating peptide.

1 1. The Annexin A4 polypeptide for the use of anyone of claims 1 to 10, wherein the cell- penetrating polypeptide allows the polypeptide to cross cellular membranes and thereby enter a living cell, preferably a human cell.

12. The Annexin A4 polypeptide for the use of anyone of claims 1 to 1 1 , wherein said polypeptide does essentially not inhibit human adenylyl cyclase 6, as determined in an in vitro assay.

13. A polypeptide which is capable of inhibiting human adenylyl cyclase 5, said polypeptide consisting of:

(a) amino acids 1 to 22 of the amino acid sequence shown in SEQ ID NO: 1 ;

(b) a fragment of the amino acid sequence of (a);

14. The polypeptide of claim 13, wherein the amino acid sequence of (a) is the amino acid sequence shown in SEQ ID NO: 1.

15. The polypeptide of claim 13 or 14, wherein the polypeptide is fused with a linker.

16. The polypeptide of claim 15, wherein the linker is selected from the group consisting of (GGGGS)n, Gly, (Gly)8, (Gly)6, (EAAK)n, A( E AAAK)4 AL E A( E AAAK)4 A, PAPAP, AEAAAKEAAAKA, (Ala-Pro)n, wherein the linker is preferably a Glycine residue.

17. The polypeptide of any one of claims 13 to 16, wherein said polypeptide is fused with a cell penetrating peptide.

18. The polypeptide of claim 17, wherein the cell-penetrating polypeptide allows the polypeptide to cross cellular membranes and thereby enter a living cell, preferably a human cell.

19. The polypeptide of any one of claims 13 to 18, wherein said polypeptide does essentially not inhibit human adenylyl cyclase 6, as determined in an in vitro assay.

20. A pharmaceutical composition, comprising the polypeptide of any one of claims 13 to 19 and optionally a pharmaceutical acceptable carrier.

21 . A polynucleotide encoding the polypeptide of any one of claims 13 to 19.

22. A vector comprising the polynucleotide of claim 21 .

23. A kit comprising the polypeptide of any one of claims 13 to 19, the pharmaceutical composition according to claim 20, the polynucleotide according to claim 21 , and/or the vector of claim 22.

24. In vitro use of the polypeptide as defined in of any one of claims 5 to 19 or the pharmaceutical composition of claim 20 for inhibiting human adenylyl cyclase 5, wherein the in vitro use is characterized in that human adenylyl cyclase 6 is essentially not inhibited.

25. An in vitro method for inhibiting human adenylyl cyclase 5, the method comprising bringing into contact

(i) human adenylyl cyclase 5; and

The in vitro method according to claim 25, wherein the human adenylyl cyclase 5 and the human adenylyl cyclase 6 are comprised by a cell, preferably a human cell.