WO2013113755A1

WO2013113755A1 - Reagents and methods for the treatment of diseases based on the inhibition of calcineurin - nfat signalling pathway

Info

Publication number: WO2013113755A1
Application number: PCT/EP2013/051798
Authority: WO
Inventors: Mercè PÉREZ-RIBA; Sergio MARTÍNEZ HØYER; Ramon Messeguer Peypoch; Emilio ITARTE FRESQUET
Original assignee: Fundació Institut D'investigació Biomèdica De Bellvitge (Idibell); Lykera Biomed S.A.; Universitat Autònoma De Barcelona
Priority date: 2012-01-30
Filing date: 2013-01-30
Publication date: 2013-08-08

Abstract

The present invention relates to peptides derived from the RCAN proteins for their use in medicine and in the treatment of diseases in a subject occurring with uncontrolled cell proliferation, immune disorders, cardiovascular disorders, neurodegenerative diseases, alopecia, undesired angiogenesis and undesired PMN infiltration.

Description

REAGENTS AND METHODS FOR THE TREATMENT OF DISEASES BASED ON THE INHIBITION OF CALCINEURIN - NFAT SIGNALLING PATHWAY

TECHNICAL FIELD

The present invention relates to peptides derived from the RCAN proteins for their use in medicine and in the treatment of diseases in a subject occurring with uncontrolled cell proliferation, immune disorders, cardiovascular disorders, neurodegenerative diseases, or other conditions in which an exacerbated activation of the calcineurin-NFAT pathway has been associated to its pathogenesis.

BACKGROUND ART

The calcium and calmodulin-dependent serine-threonine protein phosphatase calcineurin (Cn, formally called PP3, formerly PP2B) is a cellular sensor enzyme that plays a pivotal role in transducing Ca²⁺ signals to cellular responses. Calcineurin is involved in many crucial processes such as T cell activation, embryonic and adult angiogenesis, skeletal and cardiac muscle growth and function and in modulating learning, memory and neural plasticity. The activation of calcineurin, which is ubiquitously expressed, results in the dephosphorylation of a set of physiologically relevant substrates, the best characterized of which are the NFAT (nuclear factor of activated T-cells) family of transcription factors. Upon an intracellular increase in the concentration of Ca²⁺, calcineurin becomes activated resulting in NFAT dephosphorylation and thereby triggering their translocation from the cytosol to the nucleus, where in cooperation with other transcription factors, such as MEF2 (myocyte enhancer factor-2), GATA, FOXP3 and API (activator protein 1), they bind DNA and regulate transcriptional activation of a large number of genes. In T cells, NFATcl to c3 regulate the transcription of many inducible genes involved in the activation of these cells such as cytokines, chemokines and cell surface receptors.

The NFAT family of transcription factors has been shown to be important in the development and function of cardiac, skeletal muscle, immune and nervous systems. Consequently, deregulation of calcineurin/NFAT signaling and/or abnormal expression of its components has been associated with cell proliferation diseases such as cancer, autoimmune diseases, cardiovascular diseases, diabetes, and bone diseases to name a few. Recently, it has been described that NFAT transcription factors are involved in cell proliferation, migration and cell invasion in breast carcinoma (Jauliac S et al. 2002 Nat Cell Biol 4:540-544). Furthermore, NFAT antagonists such as cyclosporine A have been shown to be highly effective in the treatment of T-ALL lymphoma murine models where persistent activation of the calcineurin-NFAT signaling pathway is pro-oncogenic (Medyouf H and Ghysdael 2008 J. Cell Cycle 7:297-303). Moreover, Gregory and colleagues showed very recently that the combination of CsA with imatinib, increases the potency of the current treatment in a mouse model of Chronic Mieloid Leukemia (Gregory M et al. 2010 Cancer Cell) putting forward the idea that NFAT inhibition could be benefitial in some types of immune cell cancer.

The mechanism by which the most effective immunosuppressants currently available act is by inhibiting the Ca²⁺-calcineurin- NFAT signalling pathway. Immunosuppression can be achieved by the administration of cyclosporine A (CsA) or FK506 together with other less specific immunosuppressants. Both drugs, when they interact with their intracellular receptors or immunophilins, cyclophilin A and FKBP12, respectively, strongly inhibit calcineurin phosphatase activity. Unfortunately, prolonged treatment with these anti-calcineurinic drugs, which require binding to cytosolic immunophilins and promote a sustained and ubiquitous calcineurin phosphatase inhibition, produce major side effects such as nephrotoxicity, diabetes and cancer among others.

RCAN is a recently described protein family of endogenous inhibitors of calcineurin conserved from yeast to mammals. In vertebrates, the family is comprised by three members: RCANl, RCAN2 and RCAN3. RCAN proteins are evolutionarily conserved on their central and c-terminal amino acid sequence, but each of them presents a divergent N-terminal sequence. Due to the conserved central and c-terminal sequence that include Cn-binding sites, RCANs can specifically bind to calcineurin and regulate the calcineurin- dependent activity of NFATs. RCAN proteins bind to calcineurin in a similar way as the NFAT transcription factors, through two different conserved sequences: the LXXP site and the PxIxIT site. Both sites are conserved among the RCANs. The PxIxIT sequence is comprised within a highly conserved motif termed CIC. This motif has been shown to be sufficient and necessary to achieve Cn-NFATc inhibtion, due to its ability to competitively disrupt the interaction between the PxIxIT site on NFAT and Cn in vitro. In T-cells, the overexpression of a CIC-derived peptide has been shown to inhibit NFAT activation and NFAT-dependent gene transcription in a dose dependent manner (Mulero et al, J Biol Chem. 284:9394-401, 2009). Although the PXIXIT site is considered the primary site of RCAN binding to calcineurin, the LXXP site seems to be important for both stimulatory and inhibitory activities on the calcineurin-NFAT signalling axis. It has been suggested that the LXXP site could be important when low RCAN expression levels are present. However, when there is a high affinity PXIXIT site present the LXXP motif does not seems to affect RCAN- calcineurin interaction.

Importantly enough, while the RCAN PXIXIT site of RCAN interacts at the catalytic region of calcineurin but not in the active site, the LXXP site does. As a consequence, the displacement of the Cn-NFATc interaction mediated by the CIC motif does not alter the general phosphatase activity of Cn over other substrates, implying a high specificity of the CIC motif towards the blockade of NFAT dephosphorylation and subsequent activation.

The signature of the family is the highly conserved serine-proline (SP) motif (known also as a FLISPP motif), which is a calcineurin substrate. However, the SP motif is neither sufficient nor required for the inhibition of calcineurin, although it can act as a competitive inhibitor. The state of phosphorylation of the RCANl SP motif correlates with the protein half-life. RCAN binding to calcineurin does not interfere with binding of either the calcium/calmodulin complex or the regulatory B subunit to the catalytic A subunit of calcineurin.

RCAN proteins have been shown in eukarya to stimulate or to inhibit calcineurin signalling in vivo through direct interactions with the catalytic subunit of the phosphatase (Mehta et al 2009 Mol Cellular Biol 29: 2777-2793). The level of RCAN protein expression and the different calcineurin binding characteristics of the RCAN PXIXIT and LXXP would determine the level of activity of the calcineurin-NFAT signalling cascade in vivo. The patent application WO2007/074198 describes peptides that inhibit the biological activity of calcineurin and whose sequence is derived from the NFAT region involved in the interaction with calcineurin. Mulero MC (J. Biol. Chem., 2009, 284: 9394-9401) describes peptides the sequences of which derive from the RCAN proteins and which inhibit the calcineurin/NFAT interaction.

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Therefore, it is necessary to develop alternative strategies for disrupting Ca^' calcineurin- NFAT signalling pathway that overcome the problems associated to the methods based on the use of currently available immunosupressants to be more specific for the calcineurin/NFAT pathway and that overcome the associated side effects.

BRIEF SUMMARY OF THE INVENTION

The invention relates to peptides derived from human RCAN proteins, compositions thereof, their use in medicine and in the treatment of a disease occurring with uncontrolled cell proliferation.

In a first aspect, the invention relates to a peptide comprising

(i) a first region comprising the amino acid sequence X₁X₂X₃VX₄X₅ (SEQ ID NO: l), wherein Xi is Pro or Gly, X₂ is any amino acid, X3 is He, Val or Leu, X₄ is He, Val or Leu, and X₅ is Glu, Asp, Asn, His or Thr,

(ϋ) a second region selected from the group consisting of a phosphorylated amino acid, a phosphorylatable region and a region comprising a phosphomimetic amino acid,

wherein the C-terminal end of the amino acid sequence in (i) is linked to the N-terminal end of the region in (ii) by a first linker, and

(iii) a third region comprising the amino acid sequence XnX₁₂EL (SEQ ID NO:7), wherein X_n is Lys or Gin and X_n is Ala, Phe or Tyr,

wherein the C- terminal end of said third region is connected to the N-terminal end of the first region by a second linker or a functionally equivalent variant thereof that retains the phosphorylated amino acid, the phosphorylatable region or the region comprising a phosphomimetic amino acid,

wherein the peptide or the functionally equivalent variant thereof comprises fewer than 60 aminoacids,

wherein the phosphorylatable region comprises a sequence for phosphorylation by a serine/threonine kinase that comprises the consensus sequence X₆X₇XsX₉ (SEQ ID NO:3), wherein X₆ is Ser or Thr, X₇ and Xs are any aminoacid and X₉ is Asp or Glu, and

wherein said peptide or functionally equivalent variant thereof is capable of disrupting the interaction between calcineurin and RCAN and/or inhibiting the calcineurin-NFAT signalling pathway.

In a second aspect, the invention relates to a fusion protein comprising the previously mentioned peptide and at least an heterologous polypeptide.

In a third aspect, the invention relates to an antibody that specifically recognizes the previously mentioned peptide.

In a further aspect, the invention relates to a nucleic acid encoding the previously mentioned peptide.

In a further aspect, the invention relates to a gene construct comprising the previously mentioned nucleic acid.

In a further aspect, the invention relates to a vector comprising the previously mentioned nucleic acid or the the previously mentioned gene construct.

In a further aspect, the invention relates to a cell comprising the previously mentioned peptide, fusion protein, nucleic acid, gene construct or vector.

In a further aspect, the invention relates to a pharmaceutical composition comprising a therapeutically effective amount of the peptide, fusion protein, nucleic acid, gene construct, vector or cell of the invention, together with at least one pharmaceutically acceptable excipient.

In a further aspect, the invention relates to the previously mentioned peptide, fusion protein, nucleic acid, gene construct, vector or cell for use in medicine.

In a further aspect, the invention relates to the previously mentioned peptide, fusion protein, nucleic acid, gene construct, vector or cell for its use in the treatment of a disease selected from the group consisting of an inflammatory disease, an autoimmune disorder, a cardiovascular disease, a neurodegenerative disease, a disease occurring with uncontrolled cell proliferation, alopecia, a disease occurring with unwanted angiogenesis and a disease occurring with unwanted polimorphonuclear (PMN) infiltration.

In a further aspect, the invention relates to a method for the identification of a compound capable of disrupting the interaction between calcineurin and RCAN and/or the calcineurin- inducing NFAT signalling that comprises

(i) contacting a candidate compound with a polypeptide comprising the RCAN-binding region of calcineurin and a peptide according to the invention or a fusion protein according to the invention and

(ii) determining if the candidate compound disrupts the interaction between calcineurin and said peptide or said fusion protein

wherein if the compound disrupts the interaction between the polypeptide comprising the RCAN-binding region of calcineurin and the peptide, then said compound is identified as being able to disrupt the interaction between calcineurin and RCAN and/or the calcineurin-inducing NFAT signalling.

In a last aspect, the invention relates to a method for the obtention of a peptide according to the invention comprising

(i) cultivating a host cell of the present invention under conditions suitable for the expression of the peptide and

(ii) recovering the variant.

BRIEF DESCRIPTION OF THE FIGURES

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Figure 1. The overexpression of the RCAN3 ^" peptide inhibits NFAT activation and NFAT-dependent COX-2 gene expression in HEK 293T and MDA-MB-231 cultured cells. (A) A phosphorylated peptide corresponding to the novel extended CIC motif of RCAN3 (amino acids 183-208: Ac- KYELHAGTESTPSVVVHVCE(pS)ETEEE-NH₂) (SEQ ID NO:54) is a better disruptor of the Cn-RCAN3 interaction than a non- phosphorylated peptide spanning the same region of RCAN3 (Ac-KYELHAGTESTPSVVVHVCESETEEE-NH₂) (SEQ ID NO:50). GST-Pulldown assays using GST-RCAN3 bound to Sepharose beads as bait and a soluble protein extract from HEK 293T cells was used as source of CnA. The protein soluble extracts were supplemented with increasing concentrations of both phosphorylated or not phosphorylated CIC-derived peptides prior to incubation with GST-RCAN3. After the pull down, a sample was taken from the flow- through (including unbound CnA) and beads were extensively washed. The retained CnA was eluted from the beads by boiling in Laemmli buffer for 10 minutes. Samples (bound CnA) were analyzed by western blot with anti-CnA. Ponceau staining of the membrane shows equal GST-RCAN3 loading of each lane. p203 refers to RCAN3 phosphorylated Ser 203 residue. (B) A variant of the previously reported RCAN3^178-203 CIC peptide (Mulero et al 2007 Biochim Biophys Acta, 1773:330-341) incorporating a CK2 phosphorylation site in the CIC motif (the RCAN3^178-210 peptide, SEQ ID NO:21) results in an increased capability to inhibit NFAT activation by calcineurin. The S203 A

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mutation in the RCAN3 - , which cannot be phosphorylated, impairs the increased inhibition on the NFAT -promoter. Data is presented as mean percentage with the standard deviation of NFAT activation were the 100% is the activation achieved with ionomycin (Io)/PMA/CaCl₂ alone. Results are representative of at least three independent experiments performed in triplicates. The expression of each construct in each condition was assessed by western blot with antibody against EGFP. (-) no stimulation; (+) stimulated with Io/PMA/CaCl₂; (CsA) stimulated with Io/PMA//CaCl₂/Cyclosporine A. The RCAN3 peptides were expressed as a EGFPc fusion protein: EGFP-RCAN3^178-203 , EGFP-RCAN3^178-210 and EGFP- RCAN3^178-210 S203A. (C) Gene expression ofIL-2, RCANl-4 and IFN-γ analyzed by real-time PCR in transduced Jurkat cells upon Io/PMA/Ca2+ stimulation. Human Jurkat T cells were

178 210 previously transduced with a lentivirus encoding either EGFP alone, EGFP-R3 - or

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EGFP-R3 - S203A fusion peptides. The three groups were sorted under the same criteria depending on the level of EGFP expression. The control group (EGFP) was analyzed only at the maximum level of expression. Data is represented as mean (±SEM) percentage of mRNA levels where 100% is that obtained in the EGFP expressing group in stimulating conditions. ANOVA test was used for statistical analyses (N=3). *p<0.05; **p<0.01; ns= non significant. (D) The overexpression of the EGFP-

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RCAN3 - peptide inhibited Cn-NFAT activity. NFATc-luciferase reporter gene assays performed in MDA-MB-231 breast cancer cells transduced with lentiviruses encoding EGFPR3^178-210 and EGFPR3¹⁷⁸²¹⁰ AAQ (wherein VVH is mutated to AAQ) fusion proteins at different MOI. Luciferase units were normalized to Renilla lucif erase values. Data is presented as mean percentage (±SEM) of NFAT activation were the 100% is the activation achieved with Ionomycin/PMA/CaCl₂ alone. Results are representative of at least three independent experiments performed in triplicates. (E) NFAT- dependent COX-2 gene expression in human breast MDA-MB-231 cell line. The induction of COX-2 protein in these cells was analyzed by western-blot using specific anti-COX-2 antibodies. The expression of each construct in each condition was assessed by western blot using anti-EGFP antibodies. Tubulin is shown as loading control. (-) no stimulation; (+) stimulated with Io/PMA/Ca²⁺; (CsA) stimulated with Io/PMA/Cyclosporine A. These results show that EGFPR3^178-210 inhibits NFAT activation and NFAT-dependent gene expression in human HEK 293 T, JURKAT and MDA-MB-231 cells.

Figure 2. The overexpression of the RCAN3^178-210 peptide in MDA-MB-231 cells used on an orthothopic breast cancer model inhibits tumour growth and NFAT-dependent COX-2 and MMP-9 gene expression. (A) The overexpression of the CIC motif of the RCAN proteins is sufficient to produce inhibition on Cn-NFAT signalling and consequently to reduce tumour growth in an orthotopic mouse model of breast cancer. Graph shows the tumor growth (mm³) in an orthotopic breast cancer model in immunodeficient mice after the injection of human MDA-MB-231 cells infected with

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control empty pWPT lentivirus (pWPI) and with RCAN3 - encoding lentivirus (RCAN3) as a fusion protein to EGFP. N= 7._(B) The overexpression of the EGFP- RCAN3^178-210 peptide inhibits NFAT- dependent COX-2 and MMP9 gene expression in the tumour tissue.

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Figure 3. Overexpression of the RCAN3 - peptide in an orthothopic breast cancer model inhibits tumour angiogenesis (A) Representative CD31 staining images of the tumor sections. Scale bars correspond to 200μιη. (B) Quantification of mean vessel area in mm² per HPF. Data is presented as mean ±SEM. Statistical differences were assessed by ANOVA or T-test (EGFP n= 6; RCAN3^178-210 n= 7). (C) Tumor xenograft niRNA levels of hVEGF were assessed by Real Time PCR. It shows that overexpression of the

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RCAN3 ^" peptide decreases vessels area and human VEGF mRNA levels in tumour mass. HPRT1 gene was used as internal control. (EGFP n= 6; RCAN3^178-210 n= 8). *p<0.05, **p<0.01; ***p<0.001). As the qPCR probe only recognizes the human

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VEGF mRNA we can conclude that overexpression of the RCAN3 ^" peptide inhibits NFAT activation and by these means decreases human VEGF synthesis and secretion by human tumor cells and angiogenesis in the tumor xenograft.

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Figure 4. Overexpression of RCAN3 ^" peptide fused to EGFP inhibits immune cell infiltration to the tumor microenvironment in vivo. (A) Representative images of tumor polymorphonuclear (PMN) cells infiltration on tissue sections stained with hematoxilin- eosin. White arrows spot polymorphonucleated tumor infiltrating cells; scale bars represent 100 μιη (20X) and ΙΟμιη (ΙΟΟχ). (B) PMN cells number determination on 4 μιη haematoxilin-eosin deparaffinized-tumor slices was achieved by quantification of five High Power Field (HPF) (600 x) images for each EGFP and EGFP-R3^178-210 tumor sample (n=8 per group). Neither hemorrhagic nor necrotic tissue was chosen for PMN quantification. Mann- Whitney Test was used for stadistical analysis; (C and E) mRNA levels of hIL-8 (C, EGFP n= 5; RCAN3 n= 7; RCAN3 AAQ n= 7) and hCSF2 (E,

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EGFP n= 6; RCAN3 n= 8) in breast tumor xenografts were assessed by Real-Time PCR. hHPRTl gene expression was used as internal control. Data is presented as mean ±SEM. Statistical differences were assessed by ANOVA or T-test. (D) Human IL-8 protein expression in tumor xenografts is dependent on NFATc activity in the human tumor cell. Actin is shown as loading control. *p<0.05, **p<0.01. (D and E) These

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results allow us to confirm that overexpression of the RCAN3 ^" peptide in human tumor cells inhibit NFAT activation and by these means inhibit human IL-8 mRNA and protein amount synthesis and, therefore, IL-8 secretion and by these means affect tumor microenvironment. DETAILED DESCRIPTION OF THE INVENTION

Peptides of the invention The authors of the present invention have found peptides derived from RCAN proteins which are capable of disrupting the interaction between calcineurin and the transcription factor NFAT, thereby resulting in an inhibtion of the calcineurin-induced NFAT activation. Theses peptides do not substantially affect the phosphatase activity of calcineurin. As can be seen for instance in example 1 of the present invention, a peptide corresponding to amino acids 183 to 208 of RCAN3 (hereinafter RCAN3^183-208 , SEQ ID NO: 50) is more potent in disrupting the interaction of calcineurin and RCAN3 in an in vitro pull-down model as compared to a previously described peptide spanning aminoacids 183-203 of RCAN3 (RCAN3^183-203) (Mulero et al, J Biol Chem 2009). Moreover, a peptide spanning amino acids 178- 210 of RCAN3 (herein after RCAN3^178- ²¹⁰, SEQ ID NO:21) also blocks more effectively the calcineurin-induced transcriptional

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activation of NFAT as compared to RCAN3 ^" . The increased effect of the RCAN3^178-210 peptide compared to the RCAN3^183-203 on the calcineurin-mediated signaling seems to depend on the phosphorylation of the serine at position 203 of

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RCAN3 since a mutant of the RCAN3 - peptide wherein the serine at position 203 has been replaced by alanine (the S203A mutant) shows a substantially reduced ability of disrupting the interaction between RCAN3 and calcineurin and of reducing the calcineurin-mediated activation of the activation the NFAT transcription factor.

Thus, in a first aspect, the invention relates to a peptide comprising

(i) a first region comprising the amino acid sequence X₁X₂X₃VX₄X₅ (SEQ ID NO: l), wherein Xi is Pro or Gly, X₂ is any amino acid,

X₃ is He, Val or Leu, X₄ is He, Val or Leu, and X₅ is Glu, Asp, Asn, His or Thr, and

(ii) a second region selected from the group consisting of a phosphorylated amino acid, a phosphorylatable region and a region comprising a phosphomimetic amino acid,

(iii) a third region comprising the amino acid sequence X_{1 1}X₁₂EL (SEQ ID NO:7), wherein X_n is Lys or Gin and X_n is Ala, Phe or Tyr,

wherein the C- terminal end of said third region is connected to the N-terminal end of the first region by a second linker or

a functionally equivalent variant thereof that retains the phosphorylated amino acid, the phosphorylatable region or the region comprising a phosphomimetic amino acid,

wherein the peptide or functionally equivalent variant thereof comprises fewer than 60 aminoacids,

wherein the phosphorylatable region comprises a sequence for phosphorylation by a serine/threonine kinase that comprises the consensus sequence X₆X₇XsX₉ (SEQ ID NO:3), wherein X₆ is Ser or Thr, X₇ and Xs are any amino acid and X₉ is Asp or Glu, and

The term "peptide", as used herein, refers to a sequence of amino acids, analogues or mimetics having substantially similar or identical functionality. The term "peptide" also includes analogues having synthetic and natural amino acids joined together by peptide bonds. Peptides of this invention preferably comprise fewer than 60 amino acids, and preferably fewer than 30 amino acids, and most preferably ranging from about 4 to 30 amino acids. Peptide, as used here and in the claims, is also intended to include analogs, derivatives, salts, retro-inverso isomers, mimics, mimetics, or peptidomimetics thereof. The peptides of the invention further include other peptide modifications, including analogs, derivatives and mimetics, that retain the function of disrupting the interaction between calcineurin and NFAT and/or inhibiting the calcineurin-NFAT signalling pathway as described herein. For example, a peptidic structure of a modulator of the invention may be further modified to increase its stability, bioavailability, solubility, etc. "Analog", "derivative" and "mimetic" include molecules which mimic the chemical structure of a peptidic structure and retain the functional properties of the peptidic structure.

Approaches to designing peptide analogs, derivatives and mimetics are known in the art. For example, see Farmer, P. S. in Dmg Design (E. J. Ariens, ed.) Academic Press, New York, 1980, vol. 10, pp. 119-143; Ball, J. B. and Alewood, P. F. (1990) J. Mol. Recognition 3:55. Morgan, B. A. and Gainor, J. A. (1989) Ann. Rep. Med. Chem. 24:243; and Freidinger, R. M. (1989) Trends Pharmacol. Sci. 10:270. See also Sawyer, T. K. (1995) Peptidomimetic Design and Chemical Approaches to Peptide Metabolism in Taylor, M. D. and Amidon, G. L. (eds.) Peptide-Based Drug Design: Controlling Transport and Metabolism, Chapter 17; Smith, A. B. 3rd, et al. (1995)J. Am. Chem. Soc. 117: 11113-11123; Smith, A. B. 3rd, et al. (1994) J. Am. Chem. Soc. 116:9947-9962; and Hirschman, R., et al. (1993) J. Am. Chem. Soc. 115: 12550-12568.

A "derivative" (e.g., a peptide or amino acid) includes forms in which one or more reaction groups on the compound have been derivatized with a substituent group. Examples of peptide derivatives include peptides in which an amino acid side chain, the peptide backbone, or the amino- or carboxy-terminus has been derivatized (e.g., peptidic compounds with methylated amide linkages). An "analog" of a compound X includes compounds which retain chemical structures necessary for functional activity, yet which also contains certain chemical structures which differ. An example of an analog of a naturally-occurring peptide is a peptide which includes one or more non- naturally-occurring amino acids.

A "mimetic" of a compound includes compounds in which chemical structures of the compound necessary for functional activity have been replaced with other chemical structures which mimic the conformation of the compound. Examples of peptidomimetics include peptidic compounds in which the peptide backbone is substituted with one or more benzodiazepine molecules (see e.g., James, G. L. et al. (1993) Science 260: 1937-1942).

The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes. The term amino acid includes naturally occurring amino acids (Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, He, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val), uncommon natural amino acids, non natural (synthetic) amino acids. The amino acids are preferably in the L configuration, but also D configuration, or mixtures of amino acids in the D and L configurations.

The term "natural amino acids" comprises aliphatic amino acids (glycine, alanine, valine, leucine and isoleucine), hydroxylated amino acids (serine and threonine), sulfured amino acids (cysteine and methionine), dicarboxylic amino acids and their amides (aspartic acid, asparagine, glutamic acid and glutamine), amino acids having two basic groups (lysine, arginine and histidine), aromatic amino acids (phenylalanine, tyrosine and tryptophan) and cyclic amino acids (proline).

As used herein the term "non natural amino acid" refers to a carboxylic acid, or a derivative thereof, substituted at position a with an amine group and being structurally related to a natural aminoacid. Illustrative non- limiting examples of modified or uncommon amino acids include 2-aminoadipic acid, 3-aminoadipic acid, beta-alanine, 2-aminobutyric acid, 4-aminobutyric acid, 6-aminocaproic acid, 2-aminoheptanoic acid, 2-aminoisobutyric acid, 3-aminoisobutyric acid, 2-aminopimelic acid, 2,4- diaminobutyric acid, desmosine, 2,2'-diaminopimelic acid, 2,3-diaminopropionic acid, N-ethylglycine, N-ethylasparagine, hydroxy lysine, alio hydroxy lysine, 3- hydroxyproline, 4-hydroxyproline, isodesmosine, alloisoleucine, N-methylglycine, N- methyliso leucine, 6-N-methyl-lysine, N-methylvaline, norvaline, norleucine, ornithine, etc.

When more than one amino acid is acceptable at a particular position of the peptide, the possible amino acids for that particular position are shown between square brackets.

The first region comprises the sequence X₁X₂X₃VX₄X₅ wherein the aminoacid at position Xi is proline (Pro) or glycine (Gly), the amino acid at position X₂ is any aminoacid, the aminoacid at position X3 is isoleucine (He), valine (Val) or leucine (Leu), the amino acid at position X₄ aminoacid is isoleucine (He), valine (Val) or leucine (Leu) and the amino acid at position X5 is glutamic acid (Glu), aspartic acid (Asp), asparagine (Asn), histidine (His) or threonine (Thr). In particular embodiments of the invention, the Xi residue is Pro, the X₂ residue is Ser, the X₃ residue is Val, the X₄ residue is Val and/or the amino acid at the X₅ position is His. In another embodiment, the sequence XiX₂X₃VX₄X₅ (SEQ ID NO: 1) is PX₂VVVH (SEQ ID NO:2). In a still more preferred embodiment of the invention, the sequence PX₂VVVH (SEQ ID NO:2) is PSVVVH (SEQ ID NO: 17).

The second region is selected from the group consisting of a phosphorylated amino acid, a phosphorylatable region and a region comprising a phosphomimetic amino acid, wherein the phosphorylatable region comprises a sequence for phosphorylation by a serine/threonine kinase that comprises the consensus sequence X₆X₇X₈X (SEQ ID NO:3), wherein X₆ is Ser or Thr, X₇ and Xs are any amino acid and X₉ is Asp or Glu.

The term "phosphorylated amino acid", as used in the present invention, refers to a covalently modified naturally occurring amino acid with a phosphate group (P0₄ ^3~) on a free hydroxyl group (-OH) within the side chain of said amino acid. Suitable phosphorylated aminoacids include, without limitation, phosphoserine, phosphothreonine or phosphotyrosine.

The term "phosphorylatable region", as used in the present invention, refers to a sequence that comprises one or more amino acids than can be phosphorylated by a protein kinase.

The term "kinase" or "protein kinase" refers to the enzyme that modifies a protein by chemically adding phosphate groups to said protein in a reaction known as phosphorylation. The chemical activity of a protein kinase involves transferring a phosphate group from a nucleoside triphosphate, usually ATP, and covalently attaching it to one of three amino acids that have a free hydroxyl group. The phosphorylatable region, in order to be phosphorylated by an enzyme, must be accessible to said enzyme and contain structural and chemical elements for the formation and reaction of the enzyme-substrate complex. The term "phosphorylation" refers to the addition of a phosphate (P0₄ ^3~) group to a protein. Protein phosphorylation is known to play a significant role in a number of cellular processes, such as enzyme activation/inhibition, protein-protein interactions, intracellular traffic and protein protein degradation.

Techniques to detect whether a protein or peptide is phosphorylated at a particular site are well known in the art. Such techniques comprise, but are not limited to, detection using phosphospecific antibodies (i.e, antibodies able to bind phosphorylation-induced conformational changes in a protein), kinase activity assays, western blot, enzyme-linked immunosorbent assays (ELISA), intracellular flow citometry, immunocytochemistry, immunohistochemistry, detection of posttranslational modification (PTM) isoforms by bidimensional gels, electrophoretic mobility shift assays (EMSA), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS- PAGE), large-scale mass spectrophotometry, fluorescence immunoassays, microscale thermophoresis, fluorescence resonance energy transfer (FRET), fluorescence polarization, fluorescence-quenching, mobility shift, bead-based detection, and cell- based formats.

It is well known to those skilled in the art that detection of kinase-dependent substrate phosphorylation can be effected by a number of means other than measurement of radiolabeled phosphate incorporation into the substrate. For example, incorporation of phosphate groups can affect physicochemical properties of the substrate, such as electrophoretic mobility, light absorbance, fluorescence and/or phosphorescence, chromatographic properties and the like. Such alterations of substrate physicochemical properties can be readily measured by one skilled in the art and used as an indicator of kinase activity.

Suitable phosphorylatable regions that can form part of the peptide according to the present invention include, without limitation, a region comprising a S residue, a region comprising a T residue, or a region comprising a consensus sequence for phosphorylation by kinase.

Suitable phosphorylatable regions that can be used in the present invention include, without limitation, regions phosphorylatable by serine and threonine (serine/threonine protein kinases), regions phosphorylatable by tyrosine (tyrosine protein kinases), regions phosphorylatable by histidine kinases and regions phosphorylatable by dual-specificity kinases, which act on serine, threonine and tyrosine residues.

In a preferred embodiment, the second region is a region phosphorylatable by a serine threonine kinase that comprises the consensus sequence X₆X₇XsX₉ (SEQ ID NO:3), wherein X₆ is Ser or Thr, X₇ and Xs are any amino acid and X₉ is Asp or Glu. Serine/threonine protein kinases (EC 2.7.11.1) phosphorylate the OH group of serine or threonine (which have similar side-chains). Activity of these protein kinases can be regulated by specific events (e.g., DNA damage), as well as numerous chemical signals, including cAMP/cGMP, diacylglycerol, and Ca2+/calmodulin. Serine/threonine protein kinases comprise, but are not limited to, the MAP kinases (mitogen-activated protein kinases), kinases of the ER subfamily, typically activated by mitogenic signals, the stress-activated protein kinases INK and p38, and protein kinase CK2 (also known as casein kinase 2, casein kinase II, or CKII).

Suitable regions phosphorylatable by serine-threonine kinases include, without limitation, the R-R-X-S/T-Y region (phosphorylatable by the cAMP-dependent Protein Kinase (PKA)), the pS-X-X-S/T region (phosphorylatable by casein kinase I (CKI)), the S/T-X-X-E/D region (phosphorylatable by protein knase CK2,the S/T-X-X-X-pS-pT region (phosphorylatable by the Glycogen Synthase Kinase 3 (GSK-3)), the S/T-X-X- X-pS-pT region (phosphorylatable by the CDKl-cyclin B), the R-X-X-S/T region (phosphorylatable by the Calmodulin-dependent Protein Kinase II (CaMK II)), the S/T- P region (phosphorylatable by the p42 MAP Kinase (MAPK)), the S/T-Q region (phosphorylatable by PI3-kinase like kinases (PIKKS), such as ATM, ATR, and DNA- PK), the S/T-P-X-R/K region (phosphorylatable by the CDC2 kinase), the S/T-X-E/pS (phosphorylatable by the GCK-like kinase), the R-X-R-X-X-S/T region (phosphorylatable by AKT kinase), wherein the pS and pT are, respectively, phosphoserine or phosphothreonine and wherein the underlined serine and threnonine residues correspond to the residues that are actually phosphorylated.

In preferred embodiment of the invention, the second region is a region phosphorylatable by protein kinase CK2.

The protein kinase CK2 (CK2, EC 2.7.11.1) is a constitutively active, ubiquitously expressed serine/threonine protein kinase which phosphorylates serine or threonine residues which are N-terminal to acidic residues. The consensus site for phosphorylation by CK2 is X₆X₇X₈X₉, wherein X₆ is Ser or Thr, X₇ is any aminoacid, X₈ is any aminoacid and X₉ is Asp or Glu. In a preferred embodiment of the invention, the phosphorylatable region comprises a sequence for phosphorylation by a serine/threonine kinase that comprises the consensus sequence X₆X₇XsX (SEQ ID NO:3). In a more preferred embodiment of the invention, the phosphorylatable region comprises a sequence selected from the group consisting of SETE (SEQ ID NO:4), SDQE (SEQ ID NO:5) and SDIE (SEQ ID NO:6).

In another embodiment, the second region is a region that comprises a phosphomimetic amino acid. The term "region comprising a phosphomimetic amino acid", as used in the present invention, refers to a phosphopeptide mimetic that closely approximates the functionality of natural phosphorylated residues. For example, the phoshorylatable amino acids are replaced with amino acids that mimic the negative charge of the phosphorus atom, such as glutamic acid or aspartic acid. Preferably, the phosphopeptide mimetic is chemically stable (e.g. resistant to dephosphorylation by phosphatases, For example, the phosphopeptide mimetics contains a nonhydrolyzable linkage between the carbon backbone and the phosphorous atom. This is achieved, with a synthetic molecule that comprises the amino acid atomic structure with a nonhydrolyzable linkage to a phosphate moiety, in lieu of the naturally occurring oxygen bridge. For example, a CF2 group links the amino acid to the phosphate. Mimetics of several amino acids which are phosphorylated in nature can be generated by this approach. Mimetics of phosphoserine, phosphothreonine, and phosphotyrosine are generated by placing a CF2 linkage from the appropriate carbon to the phosphate moiety. Optionally, the mimetic molecule L-2-amino-4-(diethylphosphono-)-4,-4- difluorobutanoic acid substitutes for phosphoserine (Otaka et al. Tetrahedron Letters 36: 927-930 (1995)), L-2-amino-4-phosphono-4,-4-difluoro-3-methylbutanoic acid substitutes for phosphothreonine, and L-2-amino-4- phosphono(difluoromethyl)phenylalanine substitutes for phosphotyrosine. Other methods of making a non- hydro lyzable linkage or otherwise stable phospho mimetics are known to those skilled in the art.

Phosphomimetics of serine, threonine, and tyrosine employ species that mimic the electronic properties of the phosphate group as well as prevent hydrolysis of the phosphate group by endogenous cellular phosphatases, thus maintaining the biological properties and activity of the phosphopeptide. There are two approaches which possess the above properties currently in mainstream usage by the biomedical research community. Aspartic acid and glutamic acid both approximate the side chain and net negative charge of phosphoserine, phosphothreonine, and phosphotyrosine, and have found utility for their ease of incorporation and. More exact mimics of non- hydro lyzable phosphoserine/ phosphothreonine/phosphotyrosine mimetics include phosphonomethylene alanine (Pma) (Shapiro, Buechler et al. 1993), phosphonodifluoromethylene alanine (Pfa) (Berkowitz, Eggen et al. 1996), phosphonomethylene phenylalanine (Pmp), phosphonodifluoromethylene phenylalanine (F2Pmp) (Burke, Smyth et al. 1994) with both substituting the labile oxygen of the phosphate group with a non-labile carbon. These more precise phosphomimetics are more useful because they maintain more of the native hydrogen bonding potential than would be possible with aspartic acid and glutamic acid phosphomimetics.

Phosphotyrosine mimetics have been described by Kulis et al., (Kulis C. et al. 2007 Proceedings of the 4^th International Peptide Symposium), by Erlanson et al., (Erlanson D.A. et al. 2003 J. Am. Chem. Soc. 125(19): 5602-5603). A synthesis of phosphoserine and phosphothreonine mimetics has been described by Ruiz et al. (Ruiz M et al. 2002 Chem Commun 7(15): 1600-1601).

In a particular embodiment, the region comprising a phosphoamino acid mimetic comprises one or more phosphoamino acid mimicking residues. In a preferred embodiment of the invention, the phosphoamino acid mimicking residue is D or E.

The third region comprises the amino acid sequence comprising XnXi₂EL (SEQ ID NO: 7), wherein the C-terminal end of said third region is connected to the N- terminal end of the first region of the peptide of the invention by a second linker. Xn is Lys or Gin. Xi₂ is Ala, Phe or Tyr. In a preferred embodiment of the invention, the sequence XnXi₂EL (SEQ ID NO:7) is KYEL (SEQ ID NO: 18).

In another preferred embodiment of the invention, the amino acid sequence comprising XnXi₂EL (SEQ ID NO:7) is PGXi₀XnXi₂ELXi₃ (SEQ ID NO:8). X₁₀ is Asp or Glu. Xi₃ is His or Gin. In yet another preferred embodiment of the invention, the amino acid sequence PGXi₀XnXi₂ELXi₃ (SEQ ID NO:8) is PGXioKYELXn (SEQ ID NO:9). In a more preferred embodiment of the invention, the amino acid sequence PGXioXi iXi₂ELXi₃ (SEQ ID NO:8) is PGEKYELH (SEQ ID NO: 10). According to the present invention, the first region of the peptides of the invention is linked to the second by a first linker of 1 to 10 amino acids. The C-terminal end of the first region is connected to the N-terminal end of the second region and the C-terminal end of the third region is connected to the N-terminal end of the first region. The first and second regions can be directly connected or can be linked by a first linker region. The third and first region can be directly connected or can be linked by a second linker region.

As used herein, the term "linker" refers to a peptide that connects two peptide sequences. Linker sequences display specific characteristics in composition, conformation, hydrogen-bonding and flexibility that are known in the art (Argos P 1990 J Mol Biol 21 1 : 943-958; Robinson CR and Sauer RT., 1998, Proc. Natl. Acad. Sci. USA 95 : 5929-5934; Crasto CJ & Feng J 2000 Protein Eng 13 : 309-312). Exemplary linkers that can be used in the present invention include glycine polymers, glycine- serine polymers and glycine-alanine polymers, optionally comprising Glu and Lys residues are interspersed within the Gly, Ser and Ala residues linkers for better solubility. In a still more preferred embodiment, the first linker of the peptide of the invention comprises the sequence XiCX₂, wherein Xi is an amino acid having anhydrophobic side chain and X₂ is an amino acid having an acidic side chain. In another preferred embodiment, Xi is Val. In yet another embodiment, the amino acid having an acidic side chain is Glu. In a preferred embodiment, the first linker comprises a sequence selected from the group consisting of VCD and VCE.

The first linker of the peptide of the invention comprises between 1 and 10 amino acid residues, preferably at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 or 10 amino acids. In a particular embodiment, the first linker of the peptide of the invention comprises between 1 and 10 amino acid residues. In a particular embodiment, the first linker of the peptide of the invention comprises between 1 and 6 amino acid residues. In a particular embodiment, the first linker of the peptide of the invention comprises 3 amino acids. In a particular embodiment, if the first linker of the peptide of the invention consists of 3 amino acids, then the linker does not have the GPH sequence.

In another embodiment, the second linker of the peptide of invention comprises between 1 and 10 amino acids. In a particular embodiment, the second linker of the peptide of the invention comprises between 1 and 8 amino acids. In a particular embodiment, the second linker of the peptide of the invention has 6 amino acids. In a preferred embodiment, the second linker of the peptide of the invention has a sequence selected from the group consisting of AGTEST (SEQ ID NO: 1 1), AATDTT (SEQ ID NO: 12) and AGTEST (SEQ ID NO: 13).

The peptide according to the invention is capable of disrupting the interaction between calcineurin and RCAN and/or capable of inhibiting the calcineurin-NFAT signalling pathway. Peptides capable of disrupting the interaction between calcineurin and RCAN can be identified by a method as described in the examples of the present invention based on the ability of peptide to prevent the formation of a complex between calcineurin and RCAN when a mixture of the peptide and calcineurin is contacted with RCAN or a fusion protein comprising RCAN.

The term "RCAN" used in the present invention is referred to any member of a eukaryotic protein family of regulators or calcineurin (previously known as Down syndrome candidate region, DSCR1, calcipressins, CALPs or MCIPs) which are able to bind and regulate calcineurin. Suitable members of the RCAN family for use in the present invention include, without limitation, the RCAN 1-1 and RCANl-4, the RCAN2-3 and RCAN2-4 isoforms which are the protein products of RCAN2 gene, the RCAN3-2, RCAN3-2,3,4b,5 and RCAN3-2,5 isoforms derived from the RCAN3 gene (Davies KJA et al 2007 The FASEB J 21 :3023-3028).

This function can be determined using any method known in the art which allows determining the binding of two molecules (e.g., by means of an affinity assay). Such methods comprise, but are not limited to, coimmunoprecipitation, bimolecular fluorescence complementation (BIFc), affinity electrophoresis, pull-down assays, label transfer, phage display, tandem affinity purification (TAP), chemical cross-linking, proximity ligation assay (PLA), electrophoretic mobility shift assay (EMSA), bio-layer interferometry, dual polarization interferometry (DPI), static light scattering (SLS), dynamic light scattering (DLS), surface plasmon resonance, fluorescence polarization/anisotropy, fluorescence correlation spectroscopy, fluorescence resonance energy transfer (FRET) and microscale thermopheresis. Fluorescent polarization based- assays for the identification of disruptors of the interaction between RCAN1 CIC amino acid sequence and calcineurin A have been described by Mulero CM et al. 2010, Anal. Biochem. 398: 99-103). The peptides according to the present invention are capable of preventing the formation at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% of the complex between calcineurin and RCAN with respect to the amount of complex formed in the absence of peptide.

Alternatively or additionally, the peptides according to the invention are capable of inhibiting the calcineurin-NFAT signalling pathway. Peptides capable of inhibiting the calcineurin-NFAT signalling pathway can be identified by any method known in the art for measuring calcineurin- induced activation of NFAT using a stimulant, e.g., calcium ionophore, a neurotransmitter, or a biologically active peptide, known to trigger activation of NFAT via the calcium/calcineurin pathway (for examples, see Table 1 in Rao et al, Annu. Rev. Immunol. 15:707-747 (1997)). Such methods include, without limitation, the method described in example 1 of the present invention based on the determination of the expression levels of a reporter gene operatively linked to NFAT operator sequences in cells after induction of calcineurin activity by stimulation with ionomycin, PMA and CaCl₂, the method based in the determination of the ability of the peptide to prevent translocation of NFAT to the nucleus in cells expressing the peptide after induction of calcineurin activity by stimulation with ionomycin, PMA and CaCl₂ as described by Aubareda et al. (Cellular Signaling, 2006, 18: 1430-1438), the method based on the determination of NFAT-dependent cytokines in T cells, including one or more of GM-CSF, IFNy, TNFa, IL-2, IL-3 and IL-13 after stimulation of the cells using ionomycin and PMA as described by Aubareda et al. (Cellular Signaling, 2006, 18: 1430-1438) and the methods based on the determination of the ability of the peptide to prevent calcineurin-induced NFAT dephosphorylation, as described by Martinez et al. (Proc.Natl.Acad.Sci.USA, 2009, 106: 6117-6122). The peptides according to the present invention are capable of achieving at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% of the calcineurin-NFAT signalling pathway.

The peptides of the present invention, while being capable of disrupting the interaction between calcineurin and RCAN and/or of inhibiting the calcineurin-NFAT signalling pathway, do not affect the phosphatase activity of calcineurin. Calcineurin activity can be measured using standard assays based on the determination of the phosphatase activity on phosphopeptides which are known substrates of calcineurin such as the RII phosphopeptide.

The present invention also relates to a functionally equivalent variant of the peptide previously described wherein said functionally equivalent variant is capable of disrupting the interaction between calcineurin and NFAT and/or the calcineurin-NFAT signalling pathway and retains the phosphorylated amino acid, the phosphorylatable region or the region comprising a phosphomimetic amino acid.

In the context of the present invention, "functionally equivalent variant" of the peptide of the invention is understood as (i) any peptide resulting from the peptides of the invention by substitution in one or more of the amino acid residues by a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue), wherein such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) any peptide resulting from the peptides of the invention due to an insertion or a deletion of one or more amino acids and having the same function as the peptide of the invention, i.e., disrupting the interaction between calcineurin and RCAN and/or inhibiting the calcineurin-NFAT signalling pathway.

As used herein, the term "conservative aminoacid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. For example, families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Other conserved amino acid substitutions can also occur across amino acid side chain families, such as when substituting an asparagine for aspartic acid in order to modify the charge of a peptide.

The variants according to the invention preferably have sequences similarity with the amino acid sequence of the peptide of the invention of at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%. The degree of similarity between the variants and the peptide of the invention sequences defined previously is determined using algorithms and computer processes which are widely known by the persons skilled in the art. The similarity between two amino acid sequences is preferably determined using the BLASTP algorithm [BLAST Manual, Altschul, S., et al, NCBI NLM NIH Bethesda, Md. 20894, Altschul, S., et al, J. Mol. Biol. 215: 403-410 (1990)].

Optimal alignment of sequences for comparison can be conducted, for instance, by the Smith- Waterman local homology algorithm, by the Needleman-Wunsch homology alignment algorithm, by the Pearson-Lipman similarity search method, by computerized implementations of these algorithms or by manual alignment and visual inspection. See Smith T, Waterman M, Adv. Appl. Math. 1981; 2:482-489; Needleman S, Wunsch C, J. Mol. Biol. 1970; 48:443-453; Pearson W, Lipman D, Proc. Natl. Acad. Sci. USA 1988; 85:2444-2448; the GAP, BESTFIT, FASTA and TFASTA programs, Wisconsin Genetics Software Package, Genetics Computer Group, Madison, WI, USA; Ausubel F, et al, Eds., "Short Protocols in Molecular Biology", 5^th Ed. (John Wiley and Sons, Inc., New York, NY, USA, 2002).

One example of a useful algorithm is PILEUP. This program creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments to show relationship and percent sequence identity. It also plots a tree or dendogram showing the clustering relationships used to create the alignment. See Feng D, Doolittle R, J. Mol. Evol. 1987; 35:351-360. The method is similar to the CLUSTAL algorithm. See Higgins D, Sharp P, Gene 1998; 73:237-244 and CABIOS 1989; 5: 151- 153. The program can align up to 300 sequences, each of a maximum length of 5,000 nucleotides or amino acids. The multiple alignment procedure begins with the pairwise alignment of the two most similar sequences, producing a cluster of two aligned sequences. This cluster is then aligned to the next most related sequence or cluster of aligned sequences. Two clusters of sequences are aligned by a simple extension of the pairwise alignment of two individual sequences. The final alignment is achieved by a series of progressive, pairwise alignments. The program is run by designating specific sequences and their amino acid or nucleotide coordinates for regions of sequence comparison and by designating the program parameters. For example, a reference sequence can be compared to other test sequences to determine the percent sequence identity relationship using the following parameters: default gap weight (3.00), default gap length weight (0.10), and weighted end gaps.

Other examples of algorithms suitable for determining percent sequence identity and sequence similarity are BLAST and BLAST 2.0 algorithms. See Altschul S, et al., Nuc. Acids Res. 1977; 25:3389-3402 and Altschul S, et al, J. Mol. Biol. 1990; 215:403- 410. The BLAST and BLAST 2.0 programs are used, with the parameters described herein, to determine percent sequence identity for the nucleic acids and proteins of the invention. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. See http://blast.ncbi.nlm.nih.gov/blast.cgi, September 2011. This algorithm involves first identifying high scoring sequence pairs (HSPs) through the recognition of short words of length W in the query sequence, which either match or satisfy some positive- valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold. See Altschul, 1997, 1990, supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation (E) of 10, M=5, N=4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word length of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix alignments (B) of 50, expectation (E) of 10, M=5, N4, and a comparison of both strands. See Henikoff S, Henikoff J, Proc. Natl. Acad. Sci. USA 1989; 89: 10915-10919. The BLAST algorithm also performs a statistical analysis of the similarity between two sequences. See Karlin S, Altschul S, Proc. Natl. Acad. Sci. USA 1993; 90:5873-5787. One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01.

In a preferred embodiment, the region formed by the first and second regions comprise a sequence selected from the group consisting of PSVVVHVCESDQE, (SEQ ID NO:36), PSVVVHVCDSDIE (SEQ ID NO:37) and PSVVVHVCESETE (SEQ ID NO:38).

The peptides according to the present invention may further comprise an acidic tail of one or more amino acids having acidic side chains. Said acidic tail is C-terminal position with respect to the phosphorylation site and comprises one or more acidic amino acids (Asp or Glu) which may be connected to the peptide by an acidic amino acid (Lys or Arg). In preferred embodiments, the acidic tail comprises a sequence selected from the group consisting of KEEEEE (SEQ ID NO: 39), EEED (SEQ ID NO:40) and EEEET (SEQ ID NO:41).

In a preferred embodiment, the peptide of the invention comprises a sequence selected from the group consisting of PSVVVHVCESDQEKEEEEE, (SEQ ID NO:42), PSVVVHVCDSDIEEEED (SEQ ID NO:43) and PSVVVHVCESETEEEEET (SEQ ID NO:44).

In a preferred embodiment, the peptide of the invention comprises a sequence selected from the group consisting of KYELHAATDTTPSVVVHVCESDQE, (SEQ ID NO:45), KYELHAGTESTPSVVVHVCDSDIE (SEQ ID NO: 46) and KYELHAGTESTPSVVVHVCESETE (SEQ ID NO:47).

In a preferred embodiment, the peptide of the invention comprises a sequence selected from the group consisting of: Peptide SEQ ID Sequence

designation NO:

RCAN1^{198- 7} 14 KYELHAATDTTPSVVVHVCESDQEKEEEEE

RCAN2^{193- 0} 15 KYELHAGTESTPSVVVHVCDSDIEEEED

RCAN3^183-211 16 KYELHAGTESTPSVVVHVCESETEEEEET

RCAN1^193-227 19 LGPGEKYELHAATDTTPSVVVHVCESDQEKEEEEE

RCAN2^188-220 20 LGPGEKYELHAGTESTPSVVVHVCDSDIEEEED

RCAN3^178-210 21 LGPGEKYELHAGTESTPSVVVHVCESETEEEEE

RCAN1^198-224 48 KYELHAATDTTPSVVVHVCESDQEKEE

RCAN2^193-217 49 KYELHAGTESTPSVVVHVCDSDIEE

RCAN3^183-208 50 KYELHAGTESTPSVVVHVCESETEEE

RCAN1^198-226 51 KYELHAATDTTPSVVVHVCESDQEKEEEE

RCAN2^193-219 52 KYELHAGTESTPSVVVHVCDSDIEEEE

RCAN3^183-210 53 KYELHAGTESTPSVVVHVCESETEEEEE

According to the invention, any of the peptides described above comprising a phosphorylatable region can be phosphorylated. Thus, the invention also comprises peptides comprising a phosphorylatable region as mentioned above which are phosphorylated at said phosphorylatable region. It is understood that the peptide is phosphorylated at least within the amino acids which appear within the phosphorylatable region. However, the invention also relates to peptides which contain additional phosphorylation in other serine, threonine or tyrosine residues. Thus, in preferred embodiments, the invention relates to:

198 227

- the RCANl ^iyo-zz'peptide wherein the serine residue at position 21 is phosphorylated,

- the RCAN2 1ⁱ9^y3^j- 2^z2^z0^u peptide wherein the serine residue at position 21 is phosphorylated,

- the RCAN3^183-211 peptide wherein the serine residue at position 21 is phosphorylated,

193 227

- to the RCAN1 - peptide wherein the serine residue at position 26 is phosphorylated,

188 220

- the RCAN2^100-ZZU peptide wherein the serine residue at position 26 is phosphorylated,

178 210

- the RCAN3^{1 ,0-Z1U} peptide wherein the serine residue at position 26 is phosphorylated, 198 224

- the RCAN1 peptide wherein the serine residue at position 21 is phosphorylated,

193 217

the RCAN2 ^" peptide wherein the serine residue at position 21 is phosphorylated,

183 208

- the RCAN3^10J~ZU0 peptide wherein the serine residue at position 21 is phosphorylated,

198 226

193 219

the RCAN2 ^" peptide wherein the serine residue at position 21 is phosphorylated and

183 210

- the RCAN3^10J_Z1U peptide wherein the serine residue at position 21 is phosphorylated

The term "phosphorylation", as used herein, refers to the post-translational covalent addition of a phosphate group (PO₄ ³ ) to the side chain in an amino acid within the phosphorylatable region of the peptide of the invention. Typically, protein phosphorylation can be carried out by contacting a peptide according to the invention with a protein kinase protein which is capable of specifically phosphorylating the phosphorylatable region found in the peptide of the invention. Alternatively, the peptides of the invention can also be obtained by chemical synthesis using also a phosphoamino acid.

In another embodiment, the invention relates to a fusion protein comprising one or more peptides according to the present invention and at least a heterologous protein. In the context of the present invention, a "heterologous protein" is understood as a protein that is usually not attached to any of the claimed peptides in any naturally occurring RCAN protein, including RCAN1 , RCAN2 or RCAN3 of any organism.

Heterologous protein sequences which can be incorporated in the polynucleotides of the present invention include sequences encoding heterologous proteins which are easily detectable, which allows using polynucleotides of the invention for studying infection processes by means of viewing cells expressing the detectable protein. Detectable heterologous polypeptides the encoding sequences of which can be incorporated in the present invention include but are not limited to luciferase, (green/red) fluorescent protein and variants thereof, such as EGFP (enhanced green fluorescent protein), RFP (red fluorescent protein, such as DsRed or DsRed2), CFP (cyan fluorescent protein), BFP (blue fluorescent protein), YFP (yellow fluorescent protein), β-galactosidase or chloramphenicol acetyltransferase, and the like.

Alternatively or additionally, the heterologous protein can be a polypeptide of therapeutic interest such that the peptides according to the methods of the present invention can be used for the in vitro expression of said polypeptide or for the treatment of diseases requiring the expression of said polypeptide. Examples of sequences of therapeutic interest which can be incorporated in the polynucleotides of the invention include but are not limited to genes or cDNAs encoding erythropoietin (EPO), leptins, corticotropin-releasing hormone (CRH), growth hormone-releasing hormone (GHRH), gonadotropin-releasing hormone (GnRH), thyrotropin-releasing hormone (TRH), prolactin-releasing hormone (PRH), melatonin-releasing hormone (MRH), prolactin- inhibiting hormone (PIH), somatostatin, adrenocorticotropic hormone (ACTH), somatotropin or growth hormone (GH), luteinizing hormone (LH), follicle- stimulating hormone (FSH), thyrotropin (TSH or thyroid-stimulating hormone), prolactin, oxytocin, antidiuretic hormone (ADH or vasopressin), melatonin, Mullerian inhibiting factor, calcitonin, parathyroid hormone, gastrin, cholecystokinin (CCK), secretin, insulin-like growth factor type I (IGF-I), insulin-like growth factor type II (IGF-II), atrial natriuretic peptide (ANP), human chorionic gonadotropin (HCG), insulin, glucagon, somatostatin, pancreatic polypeptide (PP), leptin, neuropeptide Y, renin, angiotensin I, angiotensin II, factor VIII, factor IX, tissue factor, factor VII, factor X, thrombin, factor V, factor XI, factor XIII, interleukin 1 (IL-1), interleukin 2 (IL-2), tumor necrosis factor alpha (TNF- a), interleukin 6 (IL-6), interleukin 8 (IL-8 and chemokines), interleukin 12 (IL-12), interleukin 16 (IL-16), interleukin 15 (IL-15), interleukin 24 (IL-24), interferons alpha, beta, gamma, CD3, ICAM-1, LFA-1, LFA-3, chemokines including RANTES l , MIP- l , ΜΙΡ-Ιβ, nerve growth factor (NGF), platelet-derived growth factor (PDGF), transforming growth factor beta (TGF-beta), bone morphogenetic proteins (BMPs), fibroblast growth factors (FGF and KGF), epidermal growth factor (EGF and related factors), vascular endothelial growth factor (VEGF), granulocyte colony-stimulating factor (G-CSF), glial growth factor, keratinocyte growth factor, endothelial growth factor, alpha 1 -antitrypsin, tumor necrosis factor, granulocyte-macrophage colony- stimulating factor (GM-CSF), cardiotrophin-1 (CT-1), oncostatin M (OSM), amphiregulin (AR), cyclosporine, fibrinogen, lactoferrin, tissue-type plasminogen activator (tPA), chymotrypsin, immunoglobins, hirudin, superoxide dismutase, imiglucerase, β-Glucocerebrosidase, alglucosidase-a, a-L-iduronidase, iduronate-2- sulfatase, galsulfase, human a-galactosidase A, a-1 proteinase inhibitor, lactase, pancreatic enzymes (lipase, amylase, protease), adenosine deaminase, immunoglobulins, albumin, Botulinum toxins type A and B, collagenase, human deoxyribonuclease I, hyaluronidase, papain, L-asparaginase, lepirudin, streptokinase, cell transforming factor beta (TGF-β) inhibitor peptides such as those described in WO0331155, WO200519244 and WO0393293, the content of which is incorporated in the present invention by reference, expression cassettes suitable for the transcription of interference R A molecules (shRNA, siRNA, miRNA, RNA of modified Ul ribonucleoproteins) .

In a preferred embodiment of the invention, the heterologous protein is selected from the group consisting of EGFP (enhanced green fluorescent protein) and HA (haemagglutinin) .

In another embodiment, the peptides according to the present invention may further comprise an additional heterologous protein that allows the peptide to be cell permeable. A variety of naturally-occurring or artificially synthesized polypeptides having cell-membrane permeability (Joliot A. & Prochiantz A., Nat. Cell Biol. 2004; 6: 189-96) have been described and can be used for modifying polypeptides in the present invention. Said peptides include, without limitation, a peptide selected from the group consisting of:

- poly-arginine; Matsushita et al, (2003) J. Neurosci.; 21, 6000-7 constituted by any number of arginine residues. Specifically, for example, it is constituted by consecutive 5-20 arginine residues. The preferable number of arginine residues is 11.

- Tat/RKKRRQRRR/ (SEQ ID NO: 22) Frankel et al, (1988) Cell 55,1189-93 and Green & Loewenstein (1988) Cell 55, 1179-88.

- Penetratin/RQIKIWFQNRRMKWKK (SEQ ID NO: 23) Derossi et al, (1994) J.

Biol. Chem. 269, 10444-50.

- Buforin II / TRS SRAGLQFP VGRVHRLLRK (SEQ ID NO: 24) Park et al, (2000) Proc. Natl Acad. Sci. USA 97, 8245-50. - Transportan / GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO: 25) Pooga et al, (1998) FASEB J. 12, 67-77.

- MAP (model amphipathic peptide) / KLALKLALKALKAALKLA] (SEQ ID NO: 26), Oehlke et al, (1998) Biochim. Biophys. Acta. 1414, 127-39.

- K-FGF/AA V ALLP A VLLALLAP] (SEQ ID NO: 27) Lin et al, (1995) J.

Bioi. Chem. 270, 14255-8.

- Ku70 / VPMLK (SEQ ID NO: 28) Sawada et al, (2003) Nature Cell Biol. 5, 352-7.

- Ku70/ PMLKE] (SEQ ID NO: 29) Sawada et al, (2003) Nature Cell Biol. 5, 352-7.

- Prion / MANLGYWLLALFVTMWTDVGLCKKRPKP (SEQ ID NO: 30) Lundberg et al, (2002) Biochem. Biophys. Res. Commun. 299, 85-90.

- pVEC / LLIILPvRRIRKQAHAHSK] (SEQ ID NO: 31) Elmquist et al, (2001) Exp. Cell Res. 269, 237-44.

- Pep-1 / KETWWETWWTEWSQPKKKRKV (SEQ ID NO: 32), Morris et al, (2001) Nature Biotechnol. 19, 1 173-6.

- SynBl / RGGRLSYSRRRFSTSTGR (SEQ ID NO: 33) Rousselle et al, (2000) Mol. Pharmacal. 57, 679-86.

- Pep-7 / SDL WEMMMVSLACQY (SEQ ID NO: 34) Gao et al, (2002) Bioorg.

Med. Chem. 10, 4057-65.

- HN-1/ TSPLNIHNGQKL] (SEQ ID NO: 35) Hong & dayman (2000) Cancer Res. 60, 6551-6.

In another embodiment, the invention relates to the peptides as described here above, said peptides being modified. The peptides provided herein can be modified by means well-known in the art.

"Protecting groups" are those groups that prevent undesirable reactions (such as proteolysis) involving unprotected functional groups. Specific examples of amino protecting groups include formyl; trifluoroacetyl; benzyloxycarbonyl; substituted benzyloxycarbonyl such as (ortho- or para-) chlorobenzyloxycarbonyl and (ortho- or para-) bromobenzyloxycarbonyl; and aliphatic oxycarbonyl such as t-butoxycarbonyl and t-amiloxycarbonyl. The carboxyl groups of amino acids can be protected through conversion into ester groups. The ester groups include benzyl esters, substituted benzyl esters such as methoxybenzyl ester; alkyl esters such as cyclohexyl ester, cycloheptyl ester or t-butyl ester. The guanidino moiety may be protected by a arylsulfonyl such as tosyl, methoxybenzensulfonyl or mesitylenesulfonyl, even though it does not need a protecting group. The protecting groups of imidazole include tosyl, benzyl and dinitrophenyl. The indole group of tryptophan may be protected by formyl or may not be protected. The modification of the peptides aims in particular to improve their life time in vivo. One type of modification is the addition to the N or C termini of the peptides of polyethylene glycol (PEG). PEG is known by the person skilled in the art to have many properties that make it an ideal carrier for peptides such as high water solubility, high mobility in solution and low immunogenicity. This modification also protects the peptides from exopeptidases and therefore increases their overall stability in vivo. The other modifications used to prevent degradation of the peptides by endopeptidases or exopeptidases include N-terminal modifications such as acetylation or glycosylation, C-terminal modifications such as amidation and use of unnatural amindo acids (beta -amino and a-trifluoromethyl amino acids) at particularly sites within the peptides. Another alternative to increase peptide molecular size is the genetic fusion of the peptides to the Fc domain of human gamma immunoglobulin or the fusion of the peptides to albumin. Another object of the invention is a pharmaceutical composition comprising at least one of the peptide as described here above in combination with pharmaceutically acceptable excipients. Another object of the invention is a method for wound healing comprising the administration to a subject in need thereof of a therapeutically effective amount of at least one of the peptides of the invention. Methods of production of the peptides of the invention

The present invention also relates to methods of producing a peptide according to the invention.

In one aspect, the peptides are produced by a method which comprises

(ii) recovering the variant. The host cells are cultivated in a nutrient medium suitable for production of the variant using methods known in the art. For example, the cell may be cultivated by shake flask cultivation, or small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid state fermentations) in laboratory or industrial fermentors performed in a suitable medium and under conditions allowing the peptide to be expressed and/or isolated. The cultivation takes place in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts, using procedures known in the art. Suitable media are available from commercial suppliers or may be prepared according to published compositions (e.g., in catalogues of the American Type Culture Collection). If the peptide is secreted into the nutrient medium, the peptide can be recovered directly from the medium. If the peptide is not secreted, it can be recovered from cell lysates.

The peptide may be detected using methods known in the art that are specific for the variants. These detection methods may include use of specific antibodies, formation of an enzyme product, or by any of the biological assays mentioned above.

The peptide may be recovered by methods known in the art. For example, the variant may be recovered from the nutrient medium by conventional procedures including, but not limited to, collection, centrifugation, filtration, extraction, spray- drying, evaporation, or precipitation.

The peptide may be purified by a variety of procedures known in the art including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing), differential solubility (e.g., ammonium sulfate precipitation), SDS- PAGE, or extraction (see, e.g., Protein Purification, J.-C. Janson and Lars Ryden, editors, VCH Publishers, New York, 1989) to obtain substantially pure variants.

In another aspect, the peptides of the invention may also be prepared by in vitro synthesis, using conventional methods as known in the art. Various commercial synthetic apparatuses are available, for example automated synthesizers by Applied Biosystems Inc., Beckman, etc. By using synthesizers, naturally occurring amino acids may be substituted with unnatural amino acids, particularly D-isomers (or D-forms) e.g. D-alanine and D-iso leucine, diastereoisomers, side chains having different lengths or functionalities, and the like. The particular sequence and the manner of preparation will be determined by convenience, economics, purity required, and the like.

Chemical linking may be provided to various peptides or proteins comprising convenient functionalities for bonding, such as amino groups for amide or substituted amine formation, e.g. reductive amination, thiol groups for thioether or disulfide formation, carboxyl groups for amide formation, and the like.

If desired, various groups may be introduced into the peptide during synthesis or during expression, which allow for linking to other molecules or to a surface. Thus cysteines can be used to make thioethers, histidines for linking to a metal ion complex, carboxyl groups for forming amides or esters, amino groups for forming amides, and the like.

The polypeptides may also be isolated and purified in accordance with conventional methods of recombinant synthesis. A lysate may be prepared of the expression host and the lysate purified using HPLC, exclusion chromatography, gel electrophoresis, affinity chromatography, or other purification technique. For the most part, the compositions which are used will comprise at least 20 percent by weight of the desired product, more usually at least about 75 percent by weight, preferably at least about 95 percent by weight, and for therapeutic purposes, usually at least about 99.5 percent by weight, in relation to contaminants related to the method of preparation of the product and its purification. Usually, the percentages will be based upon total protein.

Wherein it is intended to obtain a phosphorylated peptide, the peptide may be obtained by in vitro synthesis using the phosphoamino acid precursor instead of the amino acid at the position wherein the phosphorylation is desired. Alternatively, the purified peptide obtained in vitro or in vivo by expression in a suitable host may be phosphorylated by contacting with the suitable kinase. In the particular case that the peptide contains a consensus sequence for phosphorylation by a serine/threonine kinase, the peptide is contacted with the corresponding serine/threonine kinase and phosphate molecules. In a preferred embodiment, the serine/threonine kinase is protein kinase CK2. Conditions adequate for phosphorylation in vitro with serine/threonine kinase are widely known in the art. The phosphopeptides according to the invention can be obtained, e.g., by in vitro phosphorylation of the synthetic peptides with kinase in instances where the synthetic peptide includes flanking residues that form a consensus site for the kinase (Czernik et al, Methods Enzymol 201 :264-283 (1991)), or, e.g., by chemical synthesis of peptides phosphorylated on serine or threonine residues (Perich J W, Methods Enzymol 201 :225- 233 (1991)).

Nucleic acids, gene constructs, vectors and host cells of the invention The present invention is also related to nucleic acids encoding any of the peptides previously described or any fusion protein as previously described, as well as to gene constructs, vectors or host cells comprising said nucleic acid.

The expressions "nucleic acid", "nucleotide sequence" and "polynucleotide" are used interchangeably in this invention to refer to the polymer form of phosphate esters of ribonucleosides (adenosine, guanosine, uridine or cytidine; "R A molecules") or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine or deoxycytidine; "DNA molecules"), or any analogous phosphoester thereof, such as phosphorothioates and thioesters, in single-strand or double-strand form. Thus, helices formed by DNA-DNA, DNA-RNA and RNA-RNA are possible. The term "nucleic acid sequence" and, in particular, DNA or RNA molecule, refers solely to the primary or secondary structure of the molecule and does not limit any particular type of tertiary structure. Thus, this term includes double-chain DNA as it appears in linear or circular DNA molecules, supercoiled DNA plasmids and chromosomes.

In another aspect, the nucleic acid encoding the peptide of the invention can be contained in a gene construct. Said gene construct can incorporate, operatively bound thereto, a regulatory sequence for the expression of said nucleic acid encoding the peptide of the invention, thus forming an expression cassette. As it is used in this description, the expression "operatively bound" means that the peptide of the invention, encoded by a nucleic acid, is expressed in the correct reading frame under the control of the control sequences or the expression regulators. The control sequences are sequences that control and regulate the transcription and, where appropriate, the translation of the protein of the invention and include promoter sequences, transcription regulator encoding sequences, ribosome binding sequences (RBS) and/or transcription termination sequences. Advantageously, the construct further comprises a marker or gene encoding a motif or a phenotype which allows the selection of the organism transformed with said construct. Said gene construct can be obtained by means of using widely known techniques in the state of the art (Sambrook et al., 2001 "Molecular cloning: to Laboratory Manual", 3rd ed., Cold Spring Harbor Laboratory Press, N.Y.).

In another aspect, the invention relates to a vector comprising a nucleic acid or a gene construct according to the invention.

As used in this invention, the term "vector" refers to a vehicle whereby a polynucleotide or a DNA molecule may be manipulated or introduced into a cell. The vector may be a linear or circular polynucleotide, or it may be a larger-size polynucleotide or any other type of construct, such as DNA or RNA from a viral genome, a virion or any other biological construct that allows for the manipulation of DNA or the introduction thereof into the cell. It is understood that the expressions "recombinant vector" and "recombinant system" may be used interchangeably with the term "vector". Those skilled in the art will note that there is no limitation in terms of the type of vector that may be used, since said vector may be a cloning vector suitable for propagation and to obtain the adequate polynucleotides or gene constructs or expression vectors in different heterologous organisms suitable for the purification of the peptides. Thus, suitable vectors in accordance with this invention include expression vectors in prokaryotes, such as pUC18, pUC19, Bluescript and the derivatives thereof, mpl8, mpl9, pBR322, pMB9, CoIEl, pCRl, RP4, phages and "shuttle" vectors, such as pSA3 and pAT28, expression vectors in yeasts, such as vectors of the 2-micron plasmid type, integration plasmids, YEP vectors, centromere plasmids and similar ones, expression vectors in insect cells, such as the vectors in the pAC series and the pVL series, expression vectors in plants, such as vectors from the pIBI, pEarleyGate, pAVA, pCAMBIA, pGSA, pGWB, pMDC, pMY, pORE series and similar ones, and expression vectors in higher eukaryotic cells based on viral vectors (adenoviruses, viruses associated with adenoviruses, as well as retroviruses and lentiviruses) and non- viral vectors, such as pSilencer 4.1-CMV (Ambion), pcDNA3, pcDNA3.1/hyg, pHCMV/Zeo, pCR3.1, pEFl/His, pIND/GS, pRc/HCMV2, pSV40/Zeo2, pTRACER- HCMV, pUB6/V5-His, pVAXl, pZeoSV2, pCI, pSVL and pKSV-10, pBPV-1, pML2d and pTDTl.

The vectors may also comprise a reporter or marker gene which allows identifying those cells that have been incorporated the vector after having been put in contact with it. Useful reporter genes in the context of the present invention include lacZ, luciferase, thymidine kinase, GFP and on the like. Useful marker genes in the context of this invention include, for example, the neomycin resistance gene, conferring resistance to the aminoglycoside G418; the hygromycinphosphotransferase gene, conferring resistance to hygromycin; the ODC gene, conferring resistance to the inhibitor of the ornithine decarboxylase (2-(difluoromethyl)-DL-ornithine (DFMO); the dihydrofolatereductase gene, conferring resistance to methotrexate; the puromycin-N- acetyl transferase gene, conferring resistance to puromycin; the ble gene, conferring resistance to zeocin; the adenosine deaminase gene, conferring resistance to 9-beta-D- xylofuranose adenine; the cytosine deaminase gene, allowing the cells to grow in the presence of N-(phosphonacetyl)-L-aspartate; thymidine kinase, allowing the cells to grow in the presence of aminopterin; the xanthine-guanine phosphoribosyltransferase gene, allowing the cells to grow in the presence of xanthine and the absence of guanine; the trpB gene of E. coli, allowing the cells to grow in the presence of indol instead of tryptophan; the hisD gene of E. coli, allowing the cells to use histidinol instead of histidine. The selection gene is incorporated into a plasmid that can additionally include a promoter suitable for the expression of said gene in eukaryotic cells (for example, the CMV or SV40 promoters), an optimized translation initiation site (for example, a site following the so-called Kozak's rules or an IRES), a polyadenylation site such as, for example, the SV40 polyadenylation or phosphoglycerate kinase site, introns such as, for example, the beta-globulin gene intron. Alternatively, it is possible to use a combination of both the reporter gene and the marker gene simultaneously in the same vector.

The vector of the invention may be used to transform, transfect or infect cells susceptible to being tranformed, transfected or infected by said vector.

Therefore, another aspect of the invention relates to a cell that comprises a peptide, a fusion protein, a nucleic acid, a gene construct or a vector of the invention; to this end, said cell has been transformed, transfected or infected with a construct or vector provided by this invention. Transformed, transfected or infected cells may be obtained by conventional methods known to those skilled in the art (Sambrook et al., 2001 "Molecular cloning: to Laboratory Manual", 3rd ed., Cold Spring Harbor Laboratory Press, N.Y.). In a particular embodiment, said host cell is an animal cell transfected or infected with an appropriate vector.

Host cells suitable for the expression of the conjugates of the invention include, without being limited thereto, cells from mammals, plants, insects, fungi and bacteria. Bacterial cells include, without being limited thereto, cells from Gram-positive bacteria, such as species from the genera Bacillus, Streptomyces and Staphylococcus, and cells from Gram-negative bacteria, such as cells from the genera Escherichia and Pseudomonas. Fungi cells preferably include cells from yeasts such as Saccharomyces, Pichia pastoris and Hansenula polymorpha. Insect cells include, without limitation, Drosophila cells and Sf9 cells. Plant cells include, amongst others, cells from cultivated plants, such as cereals, medicinal plants, ornamental plants or bulbs. Mammalian cells suitable for this invention include epithelial cell lines (porcine, etc.), osteosarcoma cell lines (human, etc.), neuroblastoma cell lines (human, etc.), epithelial carcinomas (human, etc.), glial cells (murine, etc.), hepatic cell lines (from monkeys, etc.), CHO (Chinese Hamster Ovary) cells, COS cells, BHK cells, HeLa, 911, AT 1080, A549, 293 or PER.C6 cells, human NTERA-2 ECC cells, D3 cells from the mESC line, human embryonary stem cells, such as HS293 and BGV01, SHEF1, SHEF2 and HS181, NIH3T3, 293T, REH and MCF-7 cells, and hMSC cells.

Antibodies

In another aspect, the invention relates to an antibody which specifically binds a peptide as in the invention.

An antibody "specifically binds" a given antigen when it binds this antigen with higher affinity and in a specific, as opposed to non-specific fashion, relative to a second non-identical antigen. Stated differently, the "specific binding" of an antibody molecule or preparation can be used to distinguish between two different polypeptides. The invention also provides antibodies which are capable of specific binding to the phosphopeptide without substantially binding to non-phosphopylated peptide. The antibodies can be tested to determine whether they show the ability to discriminate between phosphorylated and unphosphorylated peptides, e.g., by dot immunoblotting or by ELISA (Czernik et al, Methods Enzymol 201 :264-283 (1991)).

An antibody may refer to an immunoglobulin molecule capable of specific binding to formsbinding partner, including a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one binding recognition site (e.g., antigen binding site), including a site located in the variable region of the immunoglobulin molecule. As used herein, the term encompasses not only full length antibodies (e.g., IgG), but also fragments thereof (such as Fab, Fab', F(ab')₂, Fv), single chain (ScFv), heavy chain or fragment thereof, light chain or fragment thereof, V_H or dimers thereof, V_L or dimers thereof, V_HV_L), mutants thereof, fusion proteins comprising an antibody, or any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site of a desired specificity. An antibody fragment may refer to an antigen binding fragment. An antibody includes an antibody of any class, such as IgG, IgA, or IgM (or sub-class thereof), and the antibody need not be of any particular class. Depending on the antibody amino acid sequence of the constant domain of its heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., lgGl, lgG2, lgG3, lgG4, IgAl and lgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three- dimensional configurations of different classes of immunoglobulins are well known.

Techniques for the preparation and use of the various antibodies are well known in the art (Ausubel, et al, ed., Current Protocols in Molecular Biology, John Wiley and Sons, Inc., NY 1987-2001 ; Sambrook, et al, Molecular Cloning: A Laboratory Manual, 2^nd Edition, Cold Spring Harbor, NY, 1989; Harlow and Lane, Antibodies, a Laboratory Manual, Cold Spring Harbor, NY, 1989; Colligan, et al, ed., Current Protocols in Immunology, John Wiley and Sons, Inc., NY 1994-2001; Colligan et al, Current Protocols in Protein Science, John Wiley and Sons, NY, NY, 1997-2001; Kohler et al, Nature 256:495-497, 1975; US 4, 816, 567, Queen et al, Proc. Natl. Acad. Sci. 86: 10029- 10033, 1989). For example, fully human monoclonal antibodies lacking any non-human sequences can be prepared from human immunoglobulin transgenic mice or from phage display libraries (Lonberg et al, Nature 368:856-859, 1994; Fishwild et al, Nature Biotech, 1 :845-851, 1 96; Menriez et al, Nature Genetics 15: 146-156, 1997; Knappik et al, J. Mol. Biol. 296:57-86, 2000; Krebs et al, J. Immunol. Meth. Pharmaceutical compositions of the invention

In another aspect, the invention is related to a pharmaceutical composition comprising a therapeutically effective amount of the peptide of the invention or the fusion protein of the invention or the nucleic acid coding said peptide or said fusion protein of the invention, or the gene construct of the invention of the vector of the invention or the cell of the invention together with at least one pharmaceutically acceptable excipient.

The pharmaceutical composition of the invention can be administered by any suitable route of administration, for example, oral, by inhalation, parenteral (for example, subcutaneous, intraperitoneal, intravenous, intramuscular route, etc.), rectal route, etc.

Illustrative examples of dosage forms for administration by the oral route include tablets, capsules, granulate, solutions, suspensions, etc., and can contain the conventional excipients, such as binders, diluents, disintegrants, lubricants, wetting agents, etc., and can be prepared by conventional methods. The pharmaceutical compositions can also be adapted for their parenteral administration, in the form of, for example, sterile solutions, suspensions or lyophilized products, in the suitable dosage form; in this case, said pharmaceutical compositions will include the suitable excipients, such as buffers, surfactants, etc. In any case, the excipients will be chosen according to the selected pharmaceutical dosage form.

A review of the different dosage forms for the administration of drugs and their preparation can be found in the book "Tratado de Farmacia Galenica", by C. Fauli i Trillo, 10 Edition, 1993, Luzan 5, S.A. de Ediciones.

The composition of the invention is administered in a manner compatible with the dosage formulation and in a therapeutically effective amount. The quantity to be administered and timing depends on the subject to be treated, capacity of the subject's system to utilize the active ingredient, and degree of therapeutic effect desired. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner and are peculiar to each individual.

As used herein, the terms "pharmaceutically acceptable", "physiologically tolerable" and grammatical variations thereof, as they refer to compositions, carriers, diluents and reagents, are used interchangeably and represent that the materials are capable of administration to or upon a mammal without the production of undesirable physiological effects.

A "pharmaceutically acceptable excipient," "pharmaceutically acceptable diluent," or "pharmaceutically acceptable carrier", or "pharmaceutically acceptable vehicle," used interchangeably herein, refer to a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any conventional type. A pharmaceutically acceptable excipient is essentially non-toxic to recipients at the employed dosages and concentrations and is compatible with other ingredients of the formulation. For example, the carrier for a formulation containing polypeptides would not normally include oxidizing agents and other compounds that are known to be deleterious to polypeptides. Suitable carriers include, but are not limited to water, dextrose, glycerol, saline, ethanol, and combinations thereof. The carrier can contain additional agents such as wetting or emulsifying agents, pH buffering agents, or adjuvants that enhance the effectiveness of the formulation.

The term "pharmaceutically acceptable salt" refers to any pharmaceutically acceptable salt, (isomer, solvate, prodrug or any other compound) which, upon administration to the recipient is capable of providing (directly or indirectly) a compound as described herein. However, it will be appreciated that non- pharmaceutically acceptable salts also fall within the scope of the invention since those may be useful in the preparation of pharmaceutically acceptable salts. The preparation of salts (isomers, solvates, prodrugs and derivatives) can be carried out by methods known in the art.

For instance, pharmaceutically acceptable salts of compounds provided herein are synthesized from the parent compound which contains a basic moiety by conventional chemical methods. Generally, such salts are, for example, prepared by reacting the free base forms of these compounds with a stoichiometric amount of the appropriate acid in water or in an organic solvent or in a mixture of the two. Generally, nonaqueous media like ether, ethyl acetate, ethanol, isopropanol or acetonitrile are preferred. Examples of the acid addition salts include mineral acid addition salts such as, for example, hydrochloride, hydrobromide, hydroiodide, sulphate, nitrate, phosphate, and organic acid addition salts such as, for example, acetate, maleate, fumarate, citrate, oxalate, succinate, tartrate, malate, mandelate, methanesulphonate and p-to luenesulphonate .

Therapeutic methods of the invention It has been reported that the inhibition of NFAT signaling results in an immunosuppressive action, suppression of (heart) muscle hypertrophy (Wilkins BJ and Molkentin JD 2004 Biochem Biophys Res Commun 322: 1178-1191), suppression of osteoclastic differentiation and decrease in cancer cell migration, invasion and proliferation (Miiller MR and Rao A 2010 Nat Rev Immunol 10:645-656; Gachet S and Ghysdael 2009 J. Gen. Physiol. Biophys. Spec No F47-54). Thus, inhibitors of the NFAT transcription factor are useful in the treatment of a number of all diseases wherein it is desired to decrease the activity of NFAT. In view of the ability of the peptides of the invention to inhibit calcineurin-mediated NFAT signalling, the peptides according to the invention as well as the previously mentioned fusion proteins, nucleic acids encoding said peptides or fusion proteins, gene constructs, vector or cells as previously described are useful for treating diseases which require inhibition of NFAT. Moreover, the authors of the present invention have found that a RCAN3-derived peptide according to the present invention reduces tumor size, tumor angiogenesis and immune cell infiltration to the tumor microenvironment in an orthotopic breast cancer model (see Figures 2-4 and Examples 2-4).

Thus, in another aspect, the invention is related to a peptide or fusion protein as previously described or to a nucleic acid coding for said peptide or fusion protein or to a gene construct comprising said nucleic acid or to to a vector comprising said nucleic acid or gene construct or to the cell as previously described for its use in medicine.

In another aspect, the invention is then related to peptides as those previously described for their use in the treatment of diseases involving NFAT activation. The term "disease involving NFAT activation", as used herein, refers to diseases which occur with increased NFAT activation, as determined using any of the methods mentioned above based on the determination of the transcriptional activity of NFAT, the phosphorylation status of NFAT or the cellular localization of NFAT. Diseases involving NFAT activation which can be treated with the peptides, fusion proteins, nucleic acids, gene constructs, vectors and cells according to the present invention include, without limitation, an inflammatory disease, an autoimmune disorder, a cardiovascular disease, a neurodegenerative disease, a disease occurring with uncontrolled cell proliferation, alopecia, a disease occurring with unwanted angiogenesis and a disease occurring with unwanted polimorphonuclear (PMN) infiltration.

Thus, in another aspect, the invention relates to a peptide or to a fusion protein as previously described or to a nucleic acid as previously described coding for said peptide or to a gene construct, vector or cell as previously described for use in the treatment of a disease in a subject, wherein said disease is selected from the group consisting of an inflammatory disease, an autoimmune disorder, a cardiovascular disease, a neurodegenerative disease, a disease occurring with uncontrolled cell proliferation, alopecia, a disease occurring with unwanted angiogenesis and a disease occurring with unwanted polimorphonuclear (PMN) infiltration.

Alternatively, the invention relates to a peptide or to a fusion protein as previously described or to a nucleic acid as previously described coding for said peptide or to a gene construct, vector or cell as previously described for the manufacture of a medicament for the treatment of a disease in a subject, wherein said disease is selected from the group consisting of an inflammatory disease, an autoimmune disorder, a cardiovascular disease, a neurodegenerative disease, a disease occurring with uncontrolled cell proliferation, alopecia, a disease occurring with unwanted angiogenesis and a disease occurring with unwanted polimorphonuclear (PMN) infiltration.

Alternatively, the invention relates to a method for the treatment of a disease selected from the group consisting of an inflammatory disease, an autoimmune disorder, a cardiovascular disease, a neurodegenerative disease, a disease occurring with uncontrolled cell proliferation, alopecia, a disease occurring with unwanted angiogenesis and a disease occurring with unwanted polimorphonuclear (PMN) infiltration in a subject in need thereof which comprises the administration to said subject of a peptide or of a nucleic acid according to the present invention.

As used herein, the term "treatment" refers to both therapeutic measures and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological change or disorder, such as the acute rejection after a renal transplant. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented.

By "subject" or "individual" or "animal" or "patient" or "mammal," is meant any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or therapy is desired. Mammalian subjects include humans, domestic animals, farm animals, and zoo, sports, or pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, and so on. In a preferred embodiment of the invention, the subject is a mammal. In a more preferred embodiment of the invention, the subject is a human.

The term "immune disorder" or "immune disease" refers to a condition in a subject characterized by cellular, tissue and/or organ injury caused by an immunological reaction of the subject. The term "autoimmune disorder" or "autoimmune disease" refers to a condition in a subject characterized by cellular, tissue and/or organ injury caused by an immunological reaction of the subject to its own cells, tissues and/or organs. Illustrative, non-limiting examples of autoimmune diseases which can be treated with the peptide or with the nucleic acid of the invention include alopecia areata, ankylosing spondylitis, antiphospho lipid syndrome, autoimmune Addison's disease, autoimmune diseases of the adrenal gland, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune oophoritis and orchitis, autoimmune thrombocytopenia, Behcet's disease, bullous pemphigoid, cardiomyopathy, celiac sprue-dermatitis, chronic fatigue immune dysfunction syndrome (CF1DS), chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, cicatrical pemphigoid, CREST syndrome, cold agglutinin disease, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia- fibro myositis, glomerulonephritis, Graves' disease, Guillain-Barre, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenia purpura (ITP), IgA neuropathy, juvenile arthritis, lichen planus, Meniere's disease, mixed connective tissue disease, multiple sclerosis, type 1 or immune-mediated diabetes mellitus, myasthenia gravis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndromes, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynauld's phenomenon, Reiter's syndrome, sarcoidosis, scleroderma, progressive systemic sclerosis, Sjogren's syndrome, Good pasture's syndrome, stiff-man syndrome, systemic lupus erythematosus, lupus erythematosus, takayasu arteritis, temporal arteristis/giant cell arteritis, ulcerative colitis, uveitis, vasculitides such as dermatitis herpetiformis vasculitis, vitiligo, Wegener's granulomatosis, Anti-Glomerular Basement Membrane Disease, Antiphospho lipid Syndrome, Autoimmune Diseases of the Nervous System , Familial Mediterranean Fever, Lambert-Eaton Myasthenic Syndrome, Sympathetic Ophthalmia, polyendocrinopathies, psoriasis, etc.

The term "immune mediated inflammatory disease" shall be taken to mean any disease mediated by the immune system and characterized by chronic or acute inflammation, resulting from, associated with or triggered by, a dysregulation of the normal immune response e.g. Crohn's disease, type 1 diabetes mellitus, rheumatoid arthritis, inflammatory bowel disease, psoriasis, psoriatic arthritis, ankylosing spondylitis, systemic lupus erythematosus, Hashimoto's disease, graft-versus-host disease, Sjogren's syndrome, pernicious anemia, Addison disease, scleroderma, Goodpasture's syndrome, ulcerative colitis, autoimmune hemolytic anemia, sterility, myasthenia gravis, multiple sclerosis, Basedow's disease, thrombopenia purpura, Guillain-Barre syndrome, allergy, asthma, atopic disease, arteriosclerosis, myocarditis, cardiomyopathy, glomerular nephritis, hypoplastic anemia, and rejection after organ transplantation.

For the purposes of the invention described herein, "immune disorders" include autoimmune diseases and immunologically mediated diseases. The term "inflammatory disease" refers to a condition in a subject characterized by inflammation, e.g., chronic inflammation. Illustrative, non-limiting examples of inflammatory disorders include, but are not limited to, Celiac Disease, rheumatoid arthritis (RA), Inflammatory Bowel Disease (IBD), asthma, encephalitis, chronic obstructive pulmonary disease (COPD), inflammatory osteolysis, allergic disorders, septic shock, pulmonary fibrosis (e.g. , idiopathic pulmonary fibrosis), inflammatory vacultides (e.g. , polyarteritis nodosa, Wegner's granulomatosis, Takayasu's arteritis, temporal arteritis, and lymphomatoid granulomatosus), post-traumatic vascular angioplasty (e.g. , restenosis after angioplasty), undifferentiated spondyloarthropathy, undifferentiated arthropathy, arthritis, inflammatory osteolysis, chronic hepatitis, and chronic inflammation resulting from chronic viral or bacteria infections.

The term "cardiovascular disease or disorder", as used herein, relates to diseases affecting the heart or blood vessels or both or associated with the cardiopulmonary and circulatory systems including but not limited to ischemia, angina, edematous conditions, artherosclerosis, Coronary Heart Disease, LDL oxidation, adhesion of monocytes to endothelial cells, foam-cell formation, fatty-streak development, platelet adherence, and aggregation, smooth muscle cell proliferation, reperfusion injury, high blood pressure, thrombotic disease, arrhythmia (atrial or ventricular or both); cardiac rhythm disturbances; myocardial ischemia; myocardial infarction; cardiac or vascular aneurysm; vasculitis, stroke; peripheral obstructive arteriopathy of a limb, an organ, or a tissue; reperfusion injury following ischemia of the brain, heart or other organ or tissue, endotoxic, surgical, or traumatic shock; hypertension, valvular heart disease, heart failure, abnormal blood pressure; shock; vasoconstriction (including that associated with migraines); vascular abnormality, inflammation, insufficiency limited to a single organ or tissue.

In a preferred embodiment of the invention, the cardiovascular disease is related to cardiac hypertrophy.

The term "neurodegenerative disease", as it is used herein, is related to diseases which result from the degeneration or deterioration of nervous tissue, particularly of neurons, leading over time to a dysfunction or to a disability; the term degeneration includes loss of cell viability, loss of cell function and/or loss of the number of cells (neurons or others). Illustrative, non-limiting, examples of neurodegenerative diseases include Alzheimer's disease, Huntington's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), multiple sclerosis, etc. In a particular embodiment, said neurodegenerative disease is a disease related to neuronal death caused by a substance which, for example, causes oxidative stress or endoplasmic reticulum stress or apoptosis or excitotoxicity or neuronal death in general.

The term "disease occurring with uncontrolled cell proliferation" is related to diseases characterized by disregulation of the mechanisms controlling cell division. An ilustrative, non-limiting, example of a disease occurring with uncontrolled cell proliferation includes cancer.

The term "cancer" is referred to a disease characterized by uncontrolled cell division (or by an increase of survival or apoptosis resistance) and by the ability of said cells to invade other neighbouring tissues (invasion) and spread to other areas of the body where the cells are not normally located (metastasis) through the lymphatic and blood vessels, circulate through the bloodstream, and then invade normal tissues elsewhere in the body. Depending on whether or not they can spread by invasion and metastasis, tumours are classified as being either benign or malignant: benign tumours are tumours that cannot spread by invasion or metastasis, i.e., they only grow locally; whereas malignant tumours are tumours that are capable of spreading by invasion and metastasis. Biological processes known to be related to cancer include angiogenesis, immune cell infiltration, cell migration and metastasis. As used herein, the term cancer includes, but is not limited to, the following types of cancer: breast cancer; biliary tract cancer; bladder cancer; brain cancer including glioblastomas and medulloblastomas; cervical cancer; choriocarcinoma; colon cancer; endometrial cancer; esophageal cancer; gastric cancer; hematological neoplasms including acute lymphocytic and myelogenous leukemia; T-cell acute lymphoblastic leukemia/lymphoma; hairy cell leukemia; chronic myelogenous leukemia, multiple myeloma; AIDS-associated leukemias and adult T-cell leukemia/lymphoma; intraepithelial neoplasms including Bowen's disease and Paget's disease; liver cancer; lung cancer; lymphomas including Hodglun's disease and lymphocytic lymphomas; neuroblastomas; oral cancer including squamous cell carcinoma; ovarian cancer including those arising from epithelial cells, stromal cells, germ cells and mesenchymal cells; pancreatic cancer; prostate cancer; rectal cancer; sarcomas including leiomyosarcoma, rhabdomyosarcoma, liposarcoma, fibrosarcoma, and osteosarcoma; slun cancer including melanoma, Merkel cell carcinoma, Kaposi's sarcoma, basal cell carcinoma, and squamous cell cancer; testicular cancer including germinal tumors such as seminoma, non-seminoma (teratomas, choriocarcinomas), stromal tumors, and germ cell tumors; thyroid cancer including thyroid adenocarcinoma and medullar carcinoma; and renal cancer including adenocarcinoma and Wilms tumor. Other cancers will-be known to one of ordinary skill in the art.

In a preferred embodiment of the invention, the disease occurring with uncontrolled cell proliferation is cancer. The term "cancer" as used in the present invention is related to the multiple phases of tumorigenesis, including tumor growth, invasion, angiogenesis and metastasis. In a more preferred embodiment of the invention, the cancer is selected from the group consisting of breast cancer, colon cancer, prostate cancer and immunological cancer.

The term "breast cancer" or "breast carcinoma" refers to any malignant proliferative mammary cell disorder, usually occurring in the ducts (the tubes that carry milk to the nipple) and lobules (milk producing glands).

The term "colon cancer" or "colon carcinoma" refers to any malignant proliferative colon cell disorder.

The term "prostate cancer" or "prostate carcinoma" refers to any malignant proliferative prostate cell disorder.

The term "immunological cancer" or "immunological carcinoma" refers to any malignant proliferative immune system cell disorder.

The term "angiogenesis", also known as vascularisation, refers to the process of formation of new blood vessels from other pre-existing ones, and includes the processes of vasculogenesis and arteriogenesis. This process is strictly controlled by a balance of activators and inhibitors. When angiogenic growth factors are produced in excess of angiogenic inhibitors, the balance is tipped in favour of blood vessel growth, connecting the so called "angiogenic switch". Angiogenesis is reduced in the adult to some processes related to reproductive cycles (corpus luteum formation, endometrial vascularization, placental development), wound healing and bone repair. In all these cases, angiogenesis takes places as a transient and highly regulated process. On the contrary, a persistent and deregulated angiogenesis is an essential step in the transition of tumors from a dormant state to a malignant state. Nowadays, angiogenesis is considered to be one of the hallmarks of cancer, playing a relevant role in tumor growth, invasion, and metastasis. The role of the angiogenesis switch is not limited to the neoplasic diseases pathogenesis, but it has also been related to other non-neoplasic diseases including wet macular degeneration, diabetic retinopathies, diabetes, psoriasis and rheumatoid arthritis, among others.

The expression "diseases associated to an unwanted angiogenesis" relates to all those diseases where pathogenic angiogenesis occur i.e. when said process is harmful or undesirable, whether cancerous or not. The scope of the present invention thus excludes the treatment of angiogenesis in situations where it is necessary, such as wound healing. Diseases associated to an undesired angiogenesis which may be treated with the compounds in accordance with the present invention, without limitation, are inflammatory diseases, especially chronic inflammatory diseases such as rheumatoid arthritis, psoriasis, sarcoidosis and such like; autoimmune diseases; viral diseases; genetic diseases; allergic diseases; bacterial diseases; ophthalmo logical diseases such as diabetic retinopathy, premature retinopathy, proliferative atrial retinopathy, retinal vein oclusion, macular degeneration, senile discoid macular degeneration, neovascular ocular glaucoma, choroidal neovascularization diseases, retinal neovascularization diseases, rubeosis (angle neovascularization), corneal graft rejection, retrolental fibroplasia, epidermal keratoconjunctivitis, vitamin A deficiency, contact lens exhaustion, atopical keratitis, superior limbic keratitis, pterygium dry eye, Sjogrens syndrome, acne rosacea, phlyctenulosis, syphilis, micobacterial infections, lipid degeneration, burns with corrosive substances, bacterial ulcers, mycotic ulcers, protozoan infections, Kaposi sarcoma, Mooren's ulcer, Terrien marginal degeneration, marginal keratolysis, scleritis, chronic retinal detachment and such like; atherosclerosis; endometriosis; obesity; cardiac insufficiency; advanced renal insufficiency; endotoxemia; toxic shock syndrome; meningitis; silicon-induced fibrosis; asbestos-induced fibrosis; apoplexia; periodontitis; gingivitis; macrocytic anaemia; refractory anaemia; 5q deletion syndrome; conditions where the vascularization is altered as infection by HIV, hepatitis, hemorrhagic telangiectasia or Rendu-Osler- Weber's disease.

In a particular embodiment the diseases associated to an undesired angiogenesis are inflammatory diseases and diseases occurring with uncontrolled cell proliferation. In a preferred embodiment, the disease associated to an undesired angiogenesis is a disease selected from cancer, rheumatoid arthritis, psoriasis, sarcoidosis, diabetic retinopathy, premature retinopathy, retinal vein oclusion, senile discoid macular degeneration, atherosclerosis, endometriosis and obesity, preferably cancer.

The term "metastasis" is understood as the distance propagation, fundamentally by the lymphatic or blood stream, of the cancer causing cells, and the growth of new tumors in the destination sites of said metastasis.

The term "alopecia" includes the involuntary complete or partial hair loss from the head or body of an individual and includes alopecia areata (AA), alopecia totalis (AT), alopecia universalis (AU), androgenetic alopecia (alopecia androgenetica, or male baldness) or post- chemotherapy alopecia (PCA) or chemotherapy- induced alopecia (CIA). Alopecia areata may include diffuse alopecia areata, alopecia areata monolocularis, alopecia areata multilocularis, and alopecia areata barbae.

The term "polimorfonuclear (PMN) infiltration" relates to the process of infiltration of polymorphonuclear neutrophils into tissues during inflammation. Upon pathogen infection or irritant infliction, local macrophages and other cells sense the insult and produce a panel of inflammatory mediators such as cytokines and chemokines that stimulate the nearby microvasculature and attract large numbers of PMN to migrate across the vascular wall and infiltrate into tissues. After arrival at the inflammatory site, PMN perform phagocytosis and also release powerful anti-pathogen and tissue-damaging reagents to kill pathogens and aberrant cells. Thus, the activity of PMN cells is extremely important for host defense. However, PMN activity can also induce adverse effects.

The term "diseases occurring with unwanted polimorphonuclear (PMN) infiltration" relates to diseases due to the adverse effect of PMN infiltration including, without being limited to, inflammatory bowel diseases (IBD), arthritis, some cardiovascular conditions, inflammatory pulmonary and renal diseases, viral/bacterial infection-associated damage, graft versus host disease, transplantation therapy and diseases occurring with unwanted inflammatory diseases. Screening method of the invention

The authors of the present invention have identified peptides derived from the sequence of the RCAN proteins which are capable of competing with RCAN in the binding to calcineurin, thereby inhibiting the phosphatase activity of calcineurin and the activation of the NFAT. Thus, the identification of the interaction between the peptides and calcineurin can be used for the identification of compounds which are capable of displacing the peptides from their binding site in calcineurin and thus inhibit calcineurin and the activation of the NFAT. The candidate compounds identified by the screening method of the invention as disruptors of the interaction between calcineurin and RCAN or inhibitors of the calcineurin- induced NFAT signaling can be used as inhibitors of the calcineurin/NFAT pathway and, therefore, are contemplated to be useful in diseases involving NFAT activation. Thus, the screening method of the invention is related to the identification of candidate compounds for their use in the treatment of a disease in a subject, wherein said disease is selected from the group comprising inflammatory diseases, autoimmune disorders, cardiovascular diseases, neurodegenerative diseases and diseases occurring with uncontrolled cell proliferation.

Thus, in a further aspect, the invention is related to a method for the identification of a compound capable of disrupting the interaction between calcineurin and RCAN and/or of inhibiting the calcineurin-inducing NFAT signalling that comprises

(i) contacting a candidate compound with a polypeptide comprising the RCAN- binding region of calcineurin and a peptide according to the invention or a fusion protein according to the invention and

(ii) determining if the candidate compound disrupts the interaction between the polypeptide comprising the RCAN-binding region of calcineurin and the peptide of the invention or the the fusion protein of the invention wherein if the compound disrupts the interaction between the polypeptide comprising the RCAN-binding region of calcineurin and the peptide, then said compound is identified as being able to disrupt the interaction between calcineurin and RCAN and/or of inhibiting the calcineurin-inducing NFAT signalling.

In a first step, the screening method of the invention comprises identifying the candidate compound with the polypeptide comprising the RCAN-binding region of calcineurin and with a peptide according to the invention. The term "candidate compound" in the present invention is related to any compound wherein the characterization of the ability of the compound to disrupt the interaction between calcineurin and NFAT and/or the interaction between calcineurin and RCAN is desirable.

The test compounds of the present invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; peptoid libraries (libraries of molecules having the functionalities of peptides, but with a novel, non-peptide backbone, which are resistant to enzymatic degradation but that nevertheless remain bioactive); spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the One-bead one-compound' library method; and synthetic library methods using affinity chromatography selection. The biological library and peptoid library approaches are limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam (1997) Anticancer Drug Des. 12: 145). Libraries of compounds may be presented in solution, on beads, chips, bacteria, spores, plasmids or on phage.

The compounds that can be screened by the methods described herein include, but are not limited to, any small molecule compound libraries derived from natural and/or synthetic sources, small non-nucleic acid organic molecules, small inorganic molecules, peptides, peptoids, peptidomimetics, oligonucleotides (e.g., siRNA, antisense RNA, aptamers such as those identified using SELEX), and oligonucleotides containing synthetic components.

As used in the present invention, the "polypeptide comprising the RCAN- binding region of calcineurin" refers to a region of calcineurin that contains the binding site for the RCANs which is located in the hydrophobic pocket defined by β-sheets 11 and 14 in the catalytic subunit of calcineurin (CnA). In a preferred embodiment, the polypeptide comprising the RCAN-binding region of calcineurin comprises aminoacids 269 to 336 of CnA and lacks the CaM-binding, CnB-binding, and linker regions of calcineurin. In a preferred embodiment, the polypeptide comprising the RCAN-binding region of calcineurin is calcineurin. As used in the present invention, "calcineurin", also known as protein phosphatase 3 (PPP3, formerly PP2B), is a calcium-calmodulin-dependent serine- threonine protein phosphatase which is ubiquitously expressed and that plays pivotal roles in many physiological processes, including cell proliferation, development, and apoptosis. Calcineurin activates the nuclear factor of activated T-cells ("NFAT") by dephosphorylating it. Activated NFAT is translocated into the nucleus, where it upregulates the expression of cytokine genes such as IL-2. Calcineurin is the target of calcineurin inhibitors such as cyclosporine, pimecrolimus and tacrolimus. Although the active form of calcineurin is a heterodimeric complex comprising a 61 kD catalytic subunit A (calcineurin A, CnA) and a 19 kD regulatory subunit B (calcineurin B, CnB), the first step in the screening method of the invention involves the use of the catalytic subunit A, since this is the subunit that contains the RCAN binding site and the binding site for the peptides of the invention. Suitable calcineurin catalytic subunits that can be used in the method according to the invention include the gene product of the any of the calcineurin-encoding genes CnAa/PPP3CA, CnAp/PPP3CB and CnAy/PPP3CC or eukaryotic related sequences. In addition to its catalytic domain (aa 70-328), CnA contains a regulatory domain including a CnB-binding domain (aa 333-390), a calmodulin-binding domain (aa 390-414) and a carboxy terminal auto inhibitory domain.

The contacting step can be carried out using the polypeptide comprising the RCAN-binding region of calcineurin can be provided in any degree of purity. Thus, said polypeptide can be provided in substantially pure form or forming part of a more complex mixture. In a preferred embodiment, a lysate of a cell expressing calcineurin can be provided as a source of polypeptide comprising the RCAN-binding region of calcineurin. In another embodiment, the polypeptide comprising the RCAN-binding region of calcineurin is provided as a fusion protein with a carrier polypeptide which specifically binds to a binding partner. In this case, the polypeptide comprising the RCAN-binding region of calcineurin may be affinity purified prior to the contacting step by binding to the binding partner specific for the the carrier protein which forms part of the fusion protein.

As it can be appreciated, the contacting step of the polypeptide comprising the

RCAN-binding region of calcineurin, the peptide of the invention and the candidate compound can be carried out in one step by contacting the three components at the same time, or can be carried out in two steps wherein a complex between the polypeptide comprising the RCAN-binding region of calcineurin and the peptide of the invention is preformed and this complex is then contacted with the candidate compound. Alternatively, the polypeptide comprising the RCAN-binding region of calcineurin and the candidate compound can be premixed and the mixture then contacted with the peptide according to the invention.

In a second step, the screening method of the invention comprises determining whether the candidate compound disrupts the interaction between the polypeptide comprising the RCAN-binding region of calcineurin and the peptide according to the invention. Assays to determine the disruption of the interaction between calcineurin and NFAT and/or the interaction between calcineurin and RCAN have been previously described.

The interaction between two molecules can be detected, e.g., using fluorescence resonance energy transfer (FRET) (see, for example, Lakowicz et al, U.S. Pat. No. 5,631,169; Stavrianopoulos et al, U.S. Pat. No. 4,868,103 ). A fluorophore label is selected such that a first 'donor' label's emission spectrum overlaps with the absorption spectrum of a second, 'acceptor' molecule, which then fluoresces on excitation of the donor, if the labels are in close proximity, due to transfer of energy. Alternately, the 'donor' protein molecule may simply utilize the natural fluorescent energy of tryptophan residues. Labels are chosen that emit different wavelengths of light, such that the 'acceptor' molecule label may be differentiated from that of the 'donor'. Since the efficiency of energy transfer between the labels is related to the distance separating the molecules, the spatial relationship between the molecules can be assessed. In a situation in which binding occurs between the molecules, the fluorescent emission of the 'acceptor' molecule label in the assay is increased over the emission when binding does not occur, or when, e.g., binding is prevented by the excess of unlabelled competitor protein. A FRET binding event can be conveniently measured, in comparison to controls, through standard fluorometric detection means well known in the art (e.g., using a fluorimeter).

In another embodiment, determining the ability of the candidate compound to prevent the formation of a complex between the peptide of the invention and the polypeptide comprising the RCAN-binding region of calcineurin can be accomplished using real-time Biomolecular Interaction Analysis (BIA) (see, e.g., Sjolander and Urbaniczky, 1991, Anal. Chem. 63:2338-2345 and Szabo et al, 1995, Curr. Opin. Struct. Biol. 5:699-705 ). "Surface plasmon resonance" or "BIA" detects biospecific interactions in real time, without labeling any of the interactants (e.g., BIAcore). Changes in the mass at the binding surface (indicative of a binding event) result in alterations of the refractive index of light near the surface (the optical phenomenon of surface plasmon resonance (SPR)), resulting in a detectable signal that can be used as an indication of real-time reactions between biological molecules.

In one embodiment, either the peptide of the invention or the polypeptide comprising the RCAN-binding region of calcineurin is anchored onto a solid phase. The target gene product/test compound complexes anchored on the solid phase can be detected at the end of the reaction. In general, the target gene product can be anchored onto a solid surface, and the test compound, (which is not anchored), can be labeled, either directly or indirectly, with detectable labels discussed herein.

It may be desirable to immobilize the peptide of the invention or the polypeptide comprising the RCAN-binding region of calcineurin to facilitate separation of complexed from non-complexed forms of one or both of the molecules, as well as to accommodate automation of the assay. Interaction of the peptide of the invention with the polypeptide comprising the RCAN-binding region of calcineurin in the presence and absence of a candidate compound, can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes, and micro-centrifuge tubes. In one embodiment, a fusion protein can be provided which adds a domain that allows one or both of the proteins to be bound to a matrix. For example, fusion proteins of the polypeptide comprising the RCAN-binding region of calcineurin or the peptide of the invention and glutathione- S -transferase (GST) can be adsorbed onto glutathione Sepharose™ beads (Sigma Chemical, St. Louis, Mo.) or glutathione-derivatized microtiter plates, which are then combined with the test compound and non-adsorbed peptide of the invention or the polypeptide comprising the RCAN-binding region of calcineurin, and the mixture incubated under conditions resulting in complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtiter plate wells are washed to remove any unbound components, the matrix immobilized in the case of beads, complex determined either directly or indirectly, for example, as described above. Alternatively, the complexes can be dissociated from the matrix, and the level of binding determined using standard techniques.

Other techniques for immobilizing either the peptide of the invention or the polypeptide comprising the RCAN-binding region of calcineurin on matrices include using conjugation of biotin and streptavidin. Biotinylated peptide of the invention or the polypeptide comprising the RCAN-binding region of calcineurin can be prepared from biotin-NHS(N-hydroxysuccinimide) using techniques known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, III.), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemicals).

To conduct the assay, the non-immobilized component is added to the coated surface containing the anchored component. After the reaction is complete, unreacted components are removed (e.g., by washing) under conditions such that any complexes formed will remain immobilized on the solid surface. The detection of complexes anchored on the solid surface can be accomplished in a number of ways. Where the previously non-immobilized component is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed. Where the previously non-immobilized component is not pre-labeled, an indirect label can be used to detect complexes anchored on the surface; e.g., using a labeled antibody specific for the immobilized component (the antibody, in turn, can be directly labeled or indirectly labeled with, e.g., a labeled anti-Ig antibody).

The assay may be performed utilizing antibodies reactive with the peptide of the invention or the polypeptide comprising the RCAN-binding region of calcineurin but which do not interfere with binding of calcineurin to either the polypeptide comprising the RCAN-binding region of calcineurin or the peptide of the invention. Such antibodies can be derivatized to the wells of the plate, and unbound target or candidate compound trapped in the wells by antibody conjugation. Methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies reactive with the candidate compound or target molecule, as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the NFAT regulator protein or target molecule. Alternatively, cell free assays can be conducted in a liquid phase. In such an assay, the reaction products are separated from unreacted components, by any of a number of standard techniques, including, but not limited to: filtration; differential centrifugation (see, for example, Rivas and Minton, 1993, Trends Biochem. Sci. 18:284-7 ); chromatography (gel filtration chromatography, ion-exchange chromatography); electrophoresis (see, e.g., Ausubel et al, eds. Current Protocols in Molecular Biology 1999, J. Wiley: New York.); and immunoprecipitation (see, for example, Ausubel et al, eds. Current Protocols in Molecular Biology 1999, J. Wiley: New York ). Such resins and chromatographic techniques are known to one skilled in the art (see, e.g., Heegaard, 1998, J. Mol. Recognit. 11 : 141-8; Hage and Tweed, 1997, J. Chromatogr. B. Biomed. Sci. Appl. 699:499-525). Further, fluorescence resonance energy transfer may also be conveniently utilized, as described herein, to detect binding without further purification of the complex from solution.

The assay can include contacting the peptide of the invention with the polypeptide comprising the RCAN-binding region of calcineurin to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with the polypeptide comprising the RCAN-binding region of calcineurin, wherein determining the ability of the test compound to interact with the polypeptide comprising the RCAN-binding region of calcineurin includes determining the ability of the test compound to preferentially bind to said polypeptide or of inducing the release of the peptide of the invention from the the polypeptide comprising the RCAN-binding region of calcineurin, as compared to the known compound.

Once a candidate compound is identified as being able to disrupt the interaction between the polypeptide comprising the RCAN-binding region of calcineurin and the peptide according to the invention, its ability to modulate the activity of a NFAT can be confirmed in vivo, e.g., in an animal such as an animal model for a disease (e.g., an animal with leukemia or autoimmune disease or an animal harbouring a xenograft from an animal (e.g., human) or cells from a cancer resulting from a leukemia or other lymphocytic disorder, or cells from a leukemia or other lymphocytic disorder cell line.

The screening method according to the present invention can be easily adapted as a high-throughput screening which allows screening a large number of candidate agents easily and quickly. In preferred embodiments, a plurality of candidate agents are contacted with the the polypeptide comprising the RCAN-binding region and calcineurin. The different candidate agents can be contacted with the other compounds in groups or separately. Preferably, each of the candidate agents is contacted with both the first compound and the second compound in separate wells. For example, the method can screen libraries of potential agents. Libraries are meant to include, e.g., natural product libraries, organic chemical libraries, combinatorial chemical libraries, peptide libraries, and modified peptide libraries, including, e.g., D-amino acids, unconventional amino acids, or N-substituted amino acids. Preferably, the libraries are in a form compatible with screening in multiwell plates, e.g., 96-well plates. The assay is particularly useful for automated execution in a multiwell format in which many of the steps are controlled by computer and carried out by robotic equipment. The libraries can also be used in other formats, e.g., synthetic chemical libraries affixed to a solid support and available for release into microdroplets.

The invention is described in detail below by means of the following examples which are to be construed as merely illustrative and not limitative of the scope of the invention.

EXAMPLE 1. Phosphorylation of the CIC motif of the RCAN3^178'210 peptide on Ser203 enhances the inhibitory potential of the peptide towards NFAT activation and NFAT- dependent gene expression in cultured cells.

GST-pulldown assays using GST-RCAN3 bound Sepharose beads as bait and a soluble protein extract from HEK293T cells as source of calcineurin A (CnA). RCAN3 full length protein was cloned into BamHI sites of a pGEX6P expression plasmid. The resulting plasmid construct was transformed into E. coli BL21 codon plus strain. A single colony was grown in suspension, and at OD (600nm)= 0.6, 0,5mM IPTG was added to the culture to induce protein expression for 3h at 37° C. Then, cells were pelleted and lysed in bacteria lysis buffer (2mM MgCl₂, ImM EGTA, ImM DTT, lmM PMSF, 2μg/ml aprotinin, 2μg/ml leupeptin, l(^g/ml DNAase I and lmg/ml lysozime in phosphate buffered saline, pH 7.2) by sonication, then implemented with 1% Triton X- 100 and then subjected to three freeze/thaw cycles. The resulting soluble extract was clarified by centrifugation and GST-RCAN3 protein was purified from the supernatant using Glutathione Sepharose beads (GE Healthcare). To perform the pull down assay, HEK293T cells were lysed in binding buffer (50mM Tris-HCl pH= 7,5, NaCl lOOmM, 1% NP40, 2mM CaCl₂, 5mM MgCl₂, ImM DTT, 2mM PMSF) supplemented with protease and phosphatase inhibitor cocktails (Roche). The clarified soluble extract was preincubated for 20 minutes with increasing concentrations of both peptides, , non phosphorylated (R3^{183 208} :Ac-KYELHAGTESTPSVVVHVCESETEEE-NH₂) and phosphorylated (R3^{183 208} _pS203: Ac-KYELHAGTESTPSVVVHVCE(pS)ETEEE-NH₂) (SEQ ID NO: 50) prior to incubation with the beads bearing GST-RCAN3 for 60 minutes. After the pull down, a sample was taken from the flow- through (unbound CnA) and beads were extensively washed. The retained CnA was eluted from the beads by boiling in Laemmli buffer for 10 minutes. Samples (bound CnA) were analyzed by western blot against CnA. Ponceau staining of the membrane shows equal GST-RCAN3 loading of the gel.

As shown in Fig. 1 A, phosphorylation of the CIC motif of the peptide modulates

183 208

its binding properties to CnA. The phosphorylated peptide (R3 ^" pS203) is a better competitor than the non-phosphorylated peptide of the binding of RCAN to CnA.

Luciferase reporter assays were performed in HEK 293T cells transfected with 3xNFAT-luc plasmid and with increasing concentrations of the indicated RCAN3 full- length wild type and mutant constructs. HEK293T cells were seeded at 40% confluency on 24 well plates. The following day, cells were transfected with 500ng DNA/well with linear Polyethyleneimine 25KDa using a DNA:PEI ratio of 1 :2,5 (w/w). Empty EGFPc vector was used to equilibrate the amount of plasmid DNA transfected. A Renilla expression vector (pRLNull) was used as an internal transfection control. 24h post- transfection cells were stimulated with ionomycin (Io; ΙμΜ), PMA (lOng/ml) and CaCl₂ (lOmM) for 6 h. After stimulation, cells were washed once with cold PBS and lysed with passive lysis buffer (Promega). Soluble protein extracts were analyzed using the Dual Luciferase Kit (Promega) following manufacturer's instructions in a microplate luminometer (FluoSTAR Optima, BMG). Data is presented as mean percentage of NFAT activation were the 100% is the activation achieved with Ionomycin/PMA/CaCl₂ alone. Results are representative of at least three independent experiments performed in triplicates. Error bars correspond to standard deviation. The expression of each construct in each condition was assessed by western blot using anti-EGFP antibody. (-) no stimulation; (+) stimulated with Io/PMA/Ca²⁺; (CsA) stimulated with Io/PMA/Cyclosporine A. The RCAN3 peptides were expressed as a EGFPc fusion protein: EGFP-RC AN3^{178 203} _s EGFP-RC AN3^{178 210} and EGFP- RCAN3^178-210 S203A.

As shown in Fig. IB, the S203A mutation impairs the increased NF AT -promoter inhibition achieved by the RCAN3^178"210. In particular, pR3^178-210 S203E and pR3^178"210, which is phosphorylated in cultured cells, inhibit NFAT activation stronger than R3^178~ ²¹⁰S203A.

Furthermore, it was analyzed how the phosphorylated CIC motif could influence the expression of NFATc dependent genes by real-time PCR in Jurkat cells after stimulation with ionomycin and PMA for 4h (Fig. 1C). To do so, the cells were

R3178 210 previously transduced with a lentivirus encoding either the EGFP- ^" or

178 210

EGFPR3 ^" S203A peptides and sorted in three different groups depending on their level of EGFP peptide expression. As shown in Fig. IB, the expression of the NFATc- dependent genes IL-2, IFN-y and RCANl-4 was significantly inhibited when the cells

178 210 178 210 expressed EGFP-R3 ^" at non-saturating conditions. Importantly, the EGFP-R3

178 210

S203A peptide expressed in the same conditions as the wildtype EGFP-R3 ^" peptide did not proof to be as effective in inhibiting NFATc-dependent gene transcription.

NFATc- lucif erase reporter gene assay were performed in MDA-MB-231 breast

178 210 cancer cells transduced with lentiviruses encoding either or EGFPR3 ^" and

EGFPR3 178210 AAQ fusion proteins at different multiplicity of infection or MO I (the ratio of infectious agents,i.e,. virus to infection targets, i.e cell) (Fig. 1 D). The induction of COX-2 protein in these cells was analyzed by western-blot using specific anti-COX- 2 antibodies (Fig. IE). The expression of each construct in each condition was assessed by western blot using anti-EGFP antibodies.

As shown in Fig. ID and IE, overexpression of the EGFPR3 178 ^" 210 peptide inhibits NFAT activation (Fig. ID) and NFAT- dependent COX-2 gene expression (Fig. IE) in human breast MDA-MB-231 cancer cell line. EXAMPLE 2. Overexpression of RCAN3 ^~ in an orthotopic breast model reduces tumor size and impairs NFAT-dependent gene expression.

Transduced MDA-MB-231 cells grown in exponential growth were harvested with trypsin-EDTA (0.05%/0.02%) (Invitrogen), washed and examined for viability by trypan blue dye exclusion. Viability was greater than 95%. For primary tumor growth, cells (lxl0⁶/0.1 mL DMEM high glucose) were ortothopically injected into the right fat-pad of nude mice. Tumor growth was followed by measuring tumor diameters with calipers and the tumor volume was calculated using an approximated formula for a prolate ellipsoid: volume= (Dxd²)/2 where D is the longest axis of the tumor and d the shortest. At the end of the experiment animals were killed, tumors were surgically removed, weighed and half tumor were embedded in OCT compound for subsequent CD31 immunostaining analyses and the other half tumor were frozen for RNA and protein analysis or fixed in formaldehyde for subsequent analyses, including parafin embebbed for histochemical analysis. Blood samples from animals bearing MDA-MB-231 transduced cells tumors were collected at the end of the experiment, using EDTA-coated material. Immediately after, samples were centrifuged for 10 min at 5000 rpm, room temperature and stored at -20°C until analysis.

A fusion protein comprising the peptide RCAN3^178-210 and EGFP (EGFP-CIC) was overexpressed in MDA-MB-231 cells, and these cells were injected into mice breast as previously described.

178 210

As shown in Figure 2 A, the overexpression of RCAN3 ^" peptide fused to EGFP (CIC) reduced tumor growth compared to cells expressing EGFP alone (EGFP). In addition, COX-2 and MMP-9 protein expression in the tumor mass is dependent on a fully activated NFATc signalling in the tumor cell (Fig. 2B). Tubulin is shown as loading control.

178 210

It can be concluded from these results that overexpression of the RCAN3 peptide in MDA-MB-231 cells injected on orthothopic breast cancer experiments inhibits tumour growth and NFAT-dependent COX-2 and MMP-9 gene expression. EXAMPLE 3. Overexpression of RCAN3 ^' peptide fused to EGFP inhibits human VEGF gene expression in human tumor cells and tumor angiogenesis.

Tumor samples were frozen in OCT compound and cut in 4μιη of tissue thickness. Sections were fixed 10 min in 10% formaldehyde and permeabilized with Triton X-100 0.1 % in Tris-buffered saline (TBS). Endogenous peroxidase was inhibited in a 3%H₂0₂ in Phosfate-buffered saline (PBS) solution and blocked for 1 h at room temperature by incubation with 20% Goat Serum/PBS. Primary antibody Rat anti- mouse CD31 (BD Pharmigen, 550274) was diluted 1 :20 in PBS lx. Sections were incubated with primary antibody overnight at 4°C. After several washes in Triton X-100 in TBS, the sections were incubated for 30 min at room temperature with secondary antibody: Polyclonal Rabbit anti-rat immunoglobulins biotinylated (Dako, E0468). After washing, sections were incubated with avidin-biotin solution (Vectastain ABC Kit, ATOM PK-4000) following manufacturer's instructions. Finally sections were incubated with DAB chromogen (Dako, K3468), counterstained with hematoxylin, dehydrated and mounted with DPX (Sigma, 06522).

Figure 3 A shows representative CD31 staining images of the tumor sections at day 35. Figure 3B shows the quantification of mean vessel area in mm² per HPF. Figure 3C shows tumor xenograft mRNA levels of VEGF assessed by Real Time PCR. HPRTl gene was used as internal control.

178 210 From this results it is confirmed that overexpression of the RCAN3 ^" peptide in human MDA-MB-231 cells inhibits human VEGF in tumor cells and angiogenesis in the breast tumor xenograft.

EXAMPLE 4. Overexpression of RCAN3^178'210 peptide fused to EGFP inhibits immune cell infiltration to the tumor micro environment in vivo.

Representative images of tumor tissue sections stained with hematoxilin-eosin are shown in Figure 4A. White arrows spot polymorphonucleated tumor infiltrating cells. Figure 4B shows the polimorfonuclears (PMN) cells number determination on 4 μιη haematoxilin-eosin deparaffmized-tumor slices was achived by PMN quantification of five High Power Field (HPF) (600 x) images for each EGFP and EGFP-R3^178-210 tumor sample (n=8 per group). Neither hemorrhagic nor necrotic tissue was chosen for PMN quantification. Mann- Whitney Test was used for stadistical analysis.

mRNA levels of hIL-8 (C, EGFP n= 5; RCAN3 n= 7; RCAN3 AAQ n= 7) and CSF2 (E, EGFP n= 6; CIC n= 8) in breast tumor xenografts were assessed by Real- Time PCR (Figure 4C and 4E). hHPRTl gene expression was used as internal control. Data is presented as mean ±SEM. Statistical differences were assessed by ANOVA or T-test. Figure 4D shows that IL-8 protein expression in tumor xenograftsis is dependent on NFATc activity in the tumor cell. Actin is shown as loading control. *p<0.05, **p<0.01.

178 210

In summary, overexpression of the EGFPR3 ^" peptide inhibits NFAT activation and NFAT-dependent COX-2, MMP9, IL-8 and CSF2 gene expression in human MDA-MB-231 breast cancer cells and by these means inhibits angiogenesis, neutrophil infiltration and, as a consequence, tumour progression.

From these results we can confirm that overexpression of the RCAN3 178 210 peptide in human MDA-MB-231 cells inhibits human IL-8 mRNA and protein amount, and its secretion in human tumor cells and PMN cell infiltration to the tumor microenvironment in vivo

Claims

A peptide comprising

(ii) a second region selected from the group consisting of a phosphorylated amino acid, a phosphorylatable region and a region comprising a phosphomimetic amino acid, wherein the C-terminal end of the amino acid sequence in (i) is linked to the N-terminal end of the region in (ii) by a first linker, and

wherein the phosphorylatable region comprises a sequence for phosphorylation by a serine/threonine kinase that comprises the consensus sequence X₆X₇XsX9 (SEQ ID NO:3), wherein X₆ is Ser or Thr, X₇ and Xs are any amino acid and X9 is Asp or Glu, and

A peptide according to claim 1 wherein Xi is Pro, X₃ and X₄ are Val and/or X5 His.

3. A peptide according to claims 1 or 2 wherein Xi is Pro, X₂ is Ser, X₃ and X₄ are Val and X₅ is His (SEQ ID NO: 17).

A peptide according to any of claims 1 to 3 wherein the phosphorylatable region comprises a sequence selected from the group consisting of SETE (SEQ ID NO:4), SDQE (SEQ ID NO:5) and SDIE (SEQ ID NO:6).

A peptide according to any of claims 1 to 3 wherein the region comprising a phosphomimetic amino acid comprises one or more phosphoamino acid mimicking residues.

A peptide according to claim 5 wherein the phosphoamino acid mimicking residue is Asp or Glu.

A peptide according to any of claims 1 to 6 wherein the first linker comprises 1 to 10 amino acids.

A peptide according to claim 7 wherein the first linker comprises 3 amino acid residues.

9. A peptide according to claim 8 wherein the first inker comprises a sequence selected from the group consisting of VCD and VCE.

10. A peptide according to any of claims 1 to 9 wherein region formed by the first and second regions comprises a sequence selected from the group consisting of PSVVVHVCESDQE (SEQ ID NO:36), PSVVVHVCDSDIE (SEQ ID NO:37) and PSVVVHVCESETE (SEQ ID NO:38).

1 1. The peptide according to any of claims 1 to 10 further comprising an acidic tail which is C-terminal with respect to the second region, said acidic tail comprising at least one amino acid having an acidic side chain.

12. The peptide according to claim 1 1 wherein the acidic tail is connected to the second region by an amino acid having an acidic side chain.

13. The peptide according to claims 1 1 or 12 wherein the acidic tail comprises a sequence selected from the group consisting of KEEEEE (SEQ ID NO:39), EEED (SEQ ID NO:40) and EEEET (SEQ ID NO:41).

14. The peptide according to claim 13 wherein the region formed by the first region, the second region and the acidic tail comprises a sequence selected from the group consisting of PSVVVHVCESDQEKEEEEE, (SEQ ID NO:42), PSVVVHVCDSDIEEEED (SEQ ID NO:43) and PSVVVHVCESETEEEEET

(SEQ ID NO:44).

15. A peptide according to claim any of claims 1 to 14 wherein the region comprising the amino acid sequence X_{1 1}X₁₂EL (SEQ ID NO:7) comprises the sequence KYEL (SEQ ID NO: 18)

16. A peptide according to any of claims 1 to 15 wherein the region comprising the amino acid sequence X_{1 1}X₁₂EL (SEQ ID NO:7) comprises the sequence PGX10X11X12ELX13 (SEQ ID NO:8), wherein X_i0 is Asp or Glu and X_!3 is His or Gin.

17. A peptide according to claim 16 wherein the sequence PGX₁₀X_{1 1}X₁₂ELX₁₃ (SEQ ID NO:8) is PGX₁₀KYELX₁₃ (SEQ ID NO:9).

18. A peptide according to claim 17 wherein the sequence PGX₁₀KYELX₁₃ (SEQ ID NO:9) is PGEKYELH (SEQ ID NO: 10).

19. A peptide according to any of claim 1 to 18 wherein the second linker comprises 6 amino acids.

20. A peptide according to any of claims 1 to 19 wherein the second linker comprises a sequence selected from the group consisting of AGTEST (SEQ ID NO: 11) and

AATDTT (SEQ ID NO: 12).

21. A peptide according to any of claims 1 to 4 and 7 to 20 wherein the peptide comprises a sequence selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21,

SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52 and SEQ ID NO:53.

22. A peptide according to any of claims 1 to 4 and 7 to 21 wherein the phosphorylatable region is phosphorylated.

23. A fusion protein comprising a peptide according to any of claims 1 to 22 and at least an heterologous polypeptide.

24. A fusion protein according to claim 23 wherein the heterologous polypeptide is selected from the group consisting of EGFP, a peptide tag and a peptide for cell permeability across cell membrane.

25. An antibody that specifically recognizes a peptide according to any of claims 1 to 22 or a fusion protein according to any of claims 23 to 24.

26. A nucleic acid encoding a peptide according to any of claims 1 to 22 or a fusion protein according to any of claims 23 to 24.

27. A gene construct comprising a nucleic acid according to claim 26.

28. A vector comprising a nucleic acid according to claim 26 or a gene construct according to claim 27.

29. A cell comprising a peptide according to any of claims 1 to 22 or a fusion protein according to any of claims 23 to 24 or a nucleic acid according to claim 26 or a gene construct according to claim 27 or a vector according to claim 28.

30. A pharmaceutical composition comprising a therapeutically effective amount of a peptide according to any of claims 1 to 22 or a fusion protein according to any of claims 23 to 24 or a nucleic acid according to claim 26 or a gene construct according to claim 27 or a vector according to claim 28 or a cell according to claim 29, together with at least one pharmaceutically acceptable excipient.

31. A peptide according to any of claims 1 to 22 or a fusion protein according to any of claims 23 to 24 or a nucleic acid according to claim 26 or a gene construct according to claim 27 or a vector according to claim 28 or a cell according to claim 29 for use in medicine.

32. A peptide according to any of claims 1 to 22 or a fusion protein according to any of claims 23 to 24 or a nucleic acid according to claim 26 or a gene construct according to claim 27 or a vector according to claim 28 or a cell according to claim 29 for use in the treatment of a disease in a subject, wherein said disease is selected from the group consisting of an inflammatory disease, an autoimmune disorder, a cardiovascular disease, a neurodegenerative disease, a disease occurring with uncontrolled cell proliferation, alopecia, a disease occurring with unwanted angiogenesis and a disease occurring with unwanted polimorphonuclear (PMN) infiltration.

33. A peptide or fusion protein or nucleic acid or gene construct or vector or cell for use according to claim 32 wherein the disease occurring with uncontrolled cell proliferation is cancer.

34. A peptide or fusion protein or nucleic acid or gene construct or vector or cell for use according to claim 33 wherein the cancer is breast cancer.

A method for the identification of a compound capable of disrupting the interaction between calcineurin and RCAN and/or of inhibiting the calcineurin- inducing NFAT signalling that comprises

(i) contacting a candidate compound with a polypeptide comprising the RCAN-binding region of calcineurin and a peptide according to any of claims 1 to 22 or a fusion protein according to any of claims 23 to 24 and

(ii) determining if the candidate compound disrupts the interaction between calcineurin and the peptide according to any of claims 1 to 22 or with a fusion protein according to any of claims 23 to 24

wherein if the compound disrupts the interaction between the polypeptide comprising the RCAN-binding region of calcineurin and the peptide, then said compound is identified as being able to disrupt the interaction between calcineurin and RCAN and/or inhibiting the calcineurin-inducing NFAT signalling.

A method according to claim 35 wherein the polypeptide comprising the RCAN- binding region of calcineurin is calcineurin.

A method for the obtention of a peptide according to any of claims 1 to 22 comprising

(ii) recovering the variant.