WO2009090049A1

WO2009090049A1 - Peptides for the specific binding and regulation of protein targets

Info

Publication number: WO2009090049A1
Application number: PCT/EP2009/000179
Authority: WO
Inventors: Victor Ilia Neduva; Robert Bruce Russell; Gordana Apic; Oliver Wichmann; Carsten Schultz; Alen Piljic
Original assignee: European Molecular Biology Laboratory
Priority date: 2008-01-14
Filing date: 2009-01-14
Publication date: 2009-07-23

Abstract

The present invention relates to peptides comprising an amino acid sequence motif selected from an amino acid sequence motif of the general formula (E/D) YxFxx(E/D) (including the more specific formula EYxFxxD); LQxLDxVL; N/K/RxxYxxHP; NxxEYxxK; YAxxI; TxPxY; FxDL; and (R/K)(V/I/L)xF (including the more specific formula RVxF). Moreover, the present invention relates to a polynucleotide encoding said peptide, a vector comprising said polynucleotide, a host cell comprising the peptide, the vector or the polynucleotide, an antibody specifically recognizing the peptide or an array comprising the peptide, the polynucleotide, the host cell or the antibody. Moreover, encompassed by the invention are pharmaceutical compositions, which comprise the peptide, the polynucleotide, the vector, the host cell or the antibody and the use of such compositions for the treatment of various diseases. Finally, the present invention relates to methods for identifying compounds, which specifically block interactions of the tumor protein p53 (TP53); SMAD-SMAD interactions; interactions of histone deacetylases (HDACs), protein tyrosine phosphatase, non-receptor type 11 (PTPNI11), RNA binding protein with multiple splicing (RBPMS), inhibitor of growth family, member 5 (ING5), cyclin A1 (CCNA1) and/or protein phosphatase 1 (PPP1CA (B,C)).

Description

Peptides for the specific binding and regulation of protein targets

The present invention relates to peptides comprising an amino acid sequence motif selected from an amino acid sequence motif of the general formula (E/D)YxFxx(E/D) (including the more specific formula EYxFxxD); LQxLDxVL; N/K/RxxYxxHP; NxxEYxxK; YAxxI; TxPxY; FxDL; and (R/K)(V/I/L)xF (including the more specific formula RVxF). Moreover, the present invention relates to a polynucleotide encoding said peptide, a vector comprising said polynucleotide, a host cell comprising the peptide, the vector or the polynucleotide, an antibody specifically recognizing the peptide or an array comprising the peptide, the polynucleotide, the host cell or the antibody. Moreover, encompassed by the invention are pharmaceutical compositions, which comprise the peptide, the polynucleotide, the vector, the host cell or the antibody and the use of such compositions for the treatment of various diseases. Finally, the present invention relates to methods for identifying compounds, which specifically block interactions of the tumor protein p53 (TP53); SMAD-SMAD interactions; interactions of histone deacetylases (HDACs), protein tyrosine phosphatase, non-receptor type 11 (PTPNI l), RNA binding protein with multiple splicing (RBPMS), inhibitor of growth family, member 5 (ING5), cyclin Al (CCNAl) and/or protein phosphatase 1 (PPPlCA (B₃C)).

BACKGROUND OF THE INVENTION

There are many proteins, in particular in signalling pathways, which play key roles in the regulation and development of diseases.

TP53 is one of the most important molecules with indications in cell apoptosis and numerous cancers, for example, leukemia, esophageal squamous cell carcinoma, breast carcinomas, soft- tissue sarcomas, brain tumors, osteosarcoma, adreno-cortical carcinoma and many more.

The transforming growth factor (TGF) signalling pathway is involved in many cellular processes including cell growth, cell differentiation, apoptosis, cellular homeostasis and other cellular functions. The transforming growth factor β (TGF-β) regulates a wide variety of physiological processes. The TGF-β signal is mediated by SMAD proteins. SMADs are a class of proteins that modulate the activity of TGF-β ligands. The SMADs form complexes, often with other SMADs, enter the nucleus and serve as transcription factors. There are three classes of SMAD (1) receptor regulated SMAD (R-SMAD) which include SMADl, SMAD2, SMAD3, SMAD5 and SMAD9; (2) the common-mediator SMAD (coSMAD) SMAD4 and (3) inhibitory SMAD (I-SMAD) which include SMAD6 and SMAD7. The SMAD proteins are homologs of both the drosophila protein, mothers against decapentaplegic (MAD) and the C. elegans protein SMA. The name is a combination of the two.

Histone deacetylases (HDACs) are enzymes that catalyze removal of acetyl groups from amino-terminal lysine residues in histones, which leads to chromatin condensation and transcriptional repression. The overall result of histone deacetylation is a global (non specific) reduction in gene expression.

Tyrosyl phosphorylation, which is controlled by protein-tyrosine kinases (PTKs) and protein- tyrosine phosphatases (PTPs), regulates numerous cellular processes. PTPs are known to be signalling molecules that regulate a variety of cellular processes including cell growth, differentiation, mitotic cycle, and oncogenic transformation. PTPNI l is a gene encoding the protein tyrosine phosphatase, non-receptor type 11, also called Shp2. PTPNI l contains two tandem Src homology-2 domains, which function as phospho-tyrosine binding domains and mediate the interaction of PTPNIl with its substrates. PTPNI l is widely expressed in most tissues and plays a regulatory role in various cell signalling events that are important for a diversity of cell functions. Missense mutations in the PTPNl 1 locus are associated with both Noonan syndrome and Leopard syndrome. Patients with a subset of Noonan syndrome PTPNI l mutations also have a higher prevalence of juvenile myelomonocytic leukemias (JMML).

RNA-binding proteins are typically cytoplasmic and nuclear proteins that associate with (bind) and facilitate the translation of RNAs. The RNA binding protein with multiple splicing (RBPMS) also interacts with Ewing Sarcoma breakpoint region 1 (EWSRl), which is known to be involved in cancer and children cancer. ING5 is a member of the ING family of proteins that inhibit growth and is in particular a close homolog to INGl. ING5 binds TP53 and EP300/p300, a component of the histone acetyl transferase complex, and is involved in the TP53-dependent regulatory pathway.

The protein encoded by CCNAl belongs to the highly conserved cyclin family, whose members are characterized by a dramatic periodicity in protein abundance through the cell cycle. Cyclins function as regulators of CDK kinases. The cyclin encoded by CCNAl, cyclin Al, was shown to be expressed in testis and brain, as well as in several leukemic cell lines, and is thought to primarily function in the control of the germline meiotic cell cycle. Cyclin Al binds both CDK2 and CDC2 kinases, which give two distinct kinase activities, one appearing in S phase, the other in G2, and thus regulate separate functions in cell cycle. Cyclin Al was found to bind to important cell cycle regulators, such as Rb family proteins, transcription factor E2F-1, and the p21 family proteins (see 12 and 13).

The protein encoded by PPPlCA is one of the three catalytic subunits of protein phosphatase 1 (PPl). PPl is a serine/threonine specific protein phosphatase known to be involved in the regulation of a variety of cellular processes, such as cell division, glycogen metabolism, muscle contractility, protein synthesis, and HIV-I viral transcription. Increased PPl activity has been observed in the end stage of heart failure. Studies in both human and mice suggest that PPl is an important regulator of cardiac function (see 14-16).

Although it has been well established that the above target proteins TP53, SMADs, HDACs, PTPNI l, RBPMS, ING5, CCNAl and PPPlCA (B,C) are important molecules involved in various disease conditions, means and methods for specifically modulating their activity in the aforementioned disease conditions have not yet been made available.

Accordingly, the technical problem underlying the present invention may be seen as the provision of means and methods for treating or preventing diseases which are caused by an impaired activity of the target proteins TP53, SMADs, HDACs, PTPNI l, RBPMS, ING5, CCNAl and/or PPPlCA (B,C). The technical problem is solved by the embodiments characterized in the claims and herein below. Accordingly, the present invention provides a peptide comprising an amino acid sequence motif of the general formula (E/D)YxFxx(E/D), including the more specific general formula EYxFxxD.

Accordingly, the present invention further provides a peptide comprising an amino acid sequence motif of the general formula LQxLDxVL.

Accordingly, the present invention further provides a peptide comprising an amino acid sequence motif of the general formula N/K/RxxYxxHP.

Accordingly, the present invention further provides a peptide comprising an amino acid sequence motif of the general formula NxxEYxxK.

Accordingly, the present invention further provides a peptide comprising an amino acid sequence motif of the general formula YAxxI.

Accordingly, the present invention further provides a peptide comprising an amino acid sequence motif of the general formula TxPxY.

Accordingly, the present invention further provides a peptide comprising an amino acid sequence motif of the general formula FxDL.

Accordingly, the present invention further provides a peptide comprising an amino acid sequence motif of the general formula (R/K)(V/I/L)xF, including the more specific general formula RVxF.

The amino acid sequence motif of the general formula (R/K)(V/I/L)xF / RVxF comprises the peptide motifs or peptides, respectively, named "RRLIF", "RLF", "RLF6x", "KIQF" (as shown in Table 2).

Wherein "x" refers to any amino acid residue, including modified amino acids, unnatural amino acids, peptidomimetics. Thus, the present invention provides a peptide comprising an amino acid sequence motif selected from the group of

(a) an amino acid sequence motif of the general formula (E/D)YxFxx(E/D);

(b) an amino acid sequence motif of the general formula LQxLDxVL;

(c) an amino acid sequence motif of the general formula N/K/RxxYxxHP;

(d) an amino acid sequence motif of the general formula NxxE YxxK;

(e) an amino acid sequence motif of the general formula YAxxI;

(f) an amino acid sequence motif of the general formula TxPxY;

(g) an amino acid sequence motif of the general formula FxDL; and

(h) an amino acid sequence motif of the general formula (R/K)(V/I/L)xF.

The term "peptide" as used herein refers to peptides, which essentially consists of 200 amino acids in length, preferably, 4 to 150 amino acids in length. More preferably, the peptides of the present invention are at least about 4, 5, 6, 7, 8, 10, 11, 12, 13, 14, 15, 16, 18, 20, 25, 30 or 35 amino acids in length. The peptides shall comprise at least one motif of the general formula selected from (E/D)YxFxx(E/D); LQxLDxVL; N/K/RxxYxxHP; NxxEYxxK; YAxxI; TxPxY; FxDL; and (R/K)(V/I/L)xF. It is to be understood that a peptide of the present invention may comprise multiple copies of one such motif or copies of different motifs.

In a preferred embodiment, the peptide of the invention comprises one or more repeats of the amino acid sequence motif of the general formula selected from (E/D)YxFxx(E/D); LQxLDxVL; N/K/RxxYxxHP; NxxEYxxK; YAxxI; TxPxY; FxDL; and (R/K)(V/I/L)xF. Preferably, the peptide of the invention comprises 2, 3, 4, 5, 6, 7, 8, 9 or 10 repeats of said amino acid sequence motif, more preferably 6 repeats of said amino acid sequence motif.

In this embodiment, the peptide is about 30 to 80 amino acids in length , preferably about 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 or 80 amino acids in length, more preferably 46, 52 or 55 amino acids.

In a preferred embodiment, the peptide comprises 6 repeats of the amino acid sequence motif of the general formula selected from (E/D)YxFxx(E/D) and (R/K)(V/I/L)xF, preferably from EYxFxxD and RVxF. Preferably, the peptides of the invention are capable of specifically binding to a target protein which is preferably selected from tumor suppressor protein p53 (TP53), SMADs, histone deacetylases (HDACs), protein tyrosine phosphatase, non-receptor type 11 (PTPNI l), RNA binding protein with multiple splicing (RBPMS), inhibitor of growth family, member 5 (ING5), cyclin Al (CCNAl) and protein phosphatase 1, catalytic subunit (PPPlCA (B₅C)).

Preferably, the peptides of the invention comprise an amino acid sequence motif of the general formula (E/D)YxFxx(E/D) and are capable of specifically binding to the target protein tumor suppressor protein p53 (TP53) (Genbank refseq accession no. NP 000537).

Preferably, the peptides of the invention comprise an amino acid sequence motif of the general formula LQxLDxVL and are capable of inhibiting SMAD-SMAD interactions, in particular are capable of specifically binding to the target proteins SMADs, in particular SMAD4 (Genbank refseq accession no. NP_005350).

Preferably, the peptides of the invention comprise an amino acid sequence motif of the general formula N/K/RxxYxxHP and are capable of specifically binding to the target proteins histone deacetylases (HDACs), in particular HDACl (Genbank refseq accession no. NP_004955).

Preferably, the peptides of the invention comprise an amino acid sequence motif of the general formula NxxEYxxK and are capable of specifically binding to the target proteins histone deacetylases (HDACs), in particular HDACl (Genbank refseq accession no. NP_004955).

Preferably, the peptides of the invention comprise an amino acid sequence motif of the general formula YAxxI and are capable of specifically binding to the target protein protein tyrosine phosphatase, non-receptor type 11 (PTPNI l) (Genbank refseq accession no. NP_002825).

Preferably, the peptides of the invention comprise an amino acid sequence motif of the general formula TxPxY and are capable of specifically binding to the target protein RNA binding protein with multiple splicing (RBPMS) (Genbank refseq accession no. NP_001008712). Preferably, the peptides of the invention comprise an amino acid sequence motif of the general formula FxDL and are capable of specifically binding to the target protein inhibitor of growth family, member 5 (ING5) (Genbank refseq accession no. NP_115705).

Preferably, the peptides of the invention comprise an amino acid sequence motif of the general formula (R/K)(V/I/L)xF and is capable of specifically binding to cyclin Al (CCNAl) (Genbank refseq accession no. NP_003905).

Preferably, the peptides of the invention comprise an amino acid sequence motif of the general formula (R/K)(V/I/L)xF and is capable of specifically binding to protein phosphatase 1, catalytic subunit, alpha isoform (PPPlCA (B,C)) (Genbank refseq accession no. NP_002699).

Thus, a peptide of the invention comprises

(a) an amino acid sequence motif of the general formula (E/D)YxFxx(E/D) and is capable of specifically binding to the tumor suppressor protein p53; or

(b) an amino acid sequence motif of the general formula LQxLDxVL and is capable of specifically binding to SMADS, in particular SMAD4; or

(c) an amino acid sequence motif of the general formula N/K/RxxYxxHP and is capable of specifically binding to histone deacetylases (HDACs), in particular HDACl; or

(d) an amino acid sequence motif of the general formula NxxEYxxK and is capable of specifically binding to histone deacetylases (HDACs), in particular HDACl; or

(e) an amino acid sequence motif of the general formula YAxxI and is capable of specifically binding to protein tyrosine phosphatase, non-receptor type 11 (PTPNI l); or

(f) an amino acid sequence motif of the general formula TxPxY and is capable of specifically binding to RNA binding protein with multiple splicing (RBPMS); or

(g) an amino acid sequence motif of the general formula FxDL and is capable of specifically binding to inhibitor of growth family, member 5 (ING5); or

(h) an amino acid sequence motif of the general formula (R/K)(V/I/L)xF and is capable of specifically binding to CCNAl and/or PPPlCA (B₅C). "Specifically binding" as referred to herein, means that the peptide shall be capable of physically binding to the target protein (TP53, SMADs, HDACs, PTPNI l, RBPMS, ING5, CCNAl and/or PPPlCA (B₅C)) with a significantly higher affinity than it will be capable of binding to other proteins. How to determine whether a protein or peptide is capable of specifically binding to another protein or peptide can be determined by techniques well known in the art. Preferably, the peptides of the present invention achieve an affinity of at least 150 micromolar (typically peptides of this broad class bind their partners with an affinity of between 0.1 and 150 micromolar [I]).

The affinity of the peptides of the present invention to the target protein (TP53, SMADs, HDACs, PTPNI l, RBPMS, ING5, CCNAl and/or PPPlCA (B,C)) may be influenced by amino acids in adjacent positions to the aforementioned amino acid sequence motif having the sequence depicted in the general formula. Thus, the sequence composition and length of the adjacent sequence stretches of the peptide may be modulated in order to increase or decrease the affinity of the peptide or to increase bioavailability.

The peptides of the present invention comprising the motif(s) described herein, which are motifs derived from interaction partners of target proteins, are versatile means, that can advantageously be used for a "fine tuning" of the activity /inhibition etc of said target proteins.

In a preferred embodiment of the peptide of the present invention said peptide comprises an amino acid sequence as shown in any one of SEQ ID NOs. 1 to 35.

In a preferred embodiment the peptide of the present invention has an amino acid sequence as shown in any one of SEQ ID NOs. 1 to 39.

The peptides shown in said SEQ ID NOs. 1 to 35 are derived from respective protein interaction partners of the target protein TP53, SMADs, HDACs, PTPNI l, RBPMS, ING5, CCNAl and/or PPPlCA (B,C).

The peptides shown in said SEQ ID NOs. 36 to 39 comprise more than one (namely 3 or 6) repeats of the amino acid sequence motif of the general formula selected from (E/D)YxFxx(E/D) (namely EYxFxxD); LQxLDxVL; NxxEYxxK; and (R/K)(V/I/L)xF (namely RLxF, e.g. RLLF).

For further details, see also Tables 1 to 3.

SEQ ID NO. 1 corresponds to amino acid positions 517 to 523 of the protein hypoxia- inducible factor 1 A (HIFlA) (Genbank refseq accession no. NPJ)01521). and SEO ID NO. 2 corresponds to amino acid positions 514 to 526 of said HIFlA.

SEO ID NO. 3 corresponds to amino acid positions 293 to 299 of the protein excision repair cross-complementing rodent repair deficiency, complementation group 3 (ERCC3) (Genbank refseq accession no. NP OOOl 13) and SEQ ID NO. 4 corresponds to amino acid positions 290 to 303 ofsaid ERCC3.

SEQ ID NO. 5 corresponds to amino acid positions 141 to 147 of the protein transcriptional adaptor 3-like A (TADA3L) (Genbank refseq accession no. NP_006345) and SEO ID NO. 6 corresponds to amino acid positions 138 to 151 of said TADA3L.

SEQ ID NO. 7 corresponds to amino acid positions 281 to 287 of the protein transcription factor Dp-I (TFDPl) (Genbank refseq accession no. NP 009042) and SEO ID NO. 8 corresponds to amino acid positions 278 to 290 of said TFDPl .

SEQ ID NO. 9 corresponds to amino acid positions 532 to 539 of the protein SMAD4 (Genbank refseq accession no. NP 005350) and SEQ ID NO. 8 corresponds to amino acid positions 529 to 542 of said SMAD4.

SEQ ID NO. 11 corresponds to amino acid positions 543 to 550 of the protein SMADl (Genbank refseq accession no. NP_005891) and SEQ ID NO. 12 corresponds to amino acid positions 540 to 553 of said SMADl.

SEQ ID NO. 11 also corresponds to amino acid positions 543 to 550 of the protein SMAD5 (Genbank refseq accession no. NP_005894) and SEO ID NO. 12 also corresponds to amino acid positions 540 to 553 of said SMAD5. SEO ID NO. 11 further corresponds to amino acid positions 408 to 415 of the protein SMAD9 (Genbank refseq accession no. NP_005896) and SEO ID NO. 12 also corresponds to amino acid positions 405 to 418 of said SMAD9.

SEQ ID NO. 13 corresponds to amino acid positions 343 to 350 of the protein SATBl (special AT-rich sequence binding protein 1) (Genbank refseq accession no. NP_002962) and SEQ ID NO. 14 corresponds to amino acid positions 341 to 352 of said SATBl.

SEQ ID NO. 15 corresponds to amino acid positions 354 to 361 of the protein HDACl (histone deacylase 1) (Genbank refseq accession no. NP_004955) and SEQ ID NO. 16 corresponds to amino acid positions 351 to 364 of said HDACl.

SEQ ID NO. 17 corresponds to amino acid positions 1128 to 1131 of the protein TIE-I (tyrosine kinase with immunoglobulin-like and EGF-like domains 1) (Genbank refseq accession no. NP 005415) and SEQ ID NO. 18 corresponds to amino acid positions 1123 to 1135 of said TIE-I.

SEQ ID NO. 19 corresponds to amino acid positions 262 to 265 of the protein MPZLl (myelin protein zero-like 1) (Genbank refseq accession no. NP_003944) and SEQ ID NO. 20 corresponds to amino acid positions 259 to 268 of said MPZLl.

SEQ ID NO. 21 corresponds to amino acid positions 470 to 473 of the protein FRS2 (fibroblast growth factor receptor substrate 2) (Genbank refseq accession no. NP_006645) and SEQ ID NO. 22 corresponds to amino acid positions 466 to 477 of said FRS2.

SEO ID NO. 23 corresponds to amino acid positions 119 to 123 of the protein EWSRl (E wing Sarcoma breakpoint region 1) (Genbank refseq accession no. NP_005234) and SEQ ID NO. 24 corresponds to amino acid positions 116 to 126 of said EWSRl .

SEQ ID NO. 25 corresponds to amino acid positions 388 to 392 of the protein DVL2 (dishevelled, dsh homolog 2) (Genbank refseq accession no. NP 004413) and SEO ID NO. 26 corresponds to amino acid positions 385 to 395 of said DVL2. SEO ID NO. 27 corresponds to amino acid positions 18 to 21 of the protein TP53 (Genbank refseq accession no. NP_000537) and SEQ ID NO. 28 corresponds to amino acid positions 15 to 24 of said TP53.

SEQ ID NO. 29 corresponds to amino acid positions 152 to 162 of the protein CDKNlA (cyclin-dependent kinase inhibitor IA) (Genbank refseq accession no. NP 510867) and SEQ ID NO. 30 corresponds to amino acid positions 156 to 159 of said CDKNlA.

SEQ ID NO. 31 corresponds to amino acid positions 11 to 14 of the protein CDC25A (cell division cycle 25 homolog A) (Genbank refseq accession no. NP_963861) and SEQ ID NO. 32 corresponds to amino acid positions 8 to 17 of said CDC25A.

SEO ID NO. 33 corresponds to amino acid positions 9 to 12 of the protein PPRPlA (protein phosphatase 1, regulatory (inhibitor) subunit IA) (Genbank refseq accession no. NP_006732) and SEQ ID NO. 34 corresponds to amino acid positions 6 to 15 of said PPRPlA.

SEQ ID NO. 35 corresponds to a minimalistic peptide (or consensus peptide) binding to protein phosphatase 1 corresponding to the simplest peptide conforming to the well established RVxF motif (i.e. replacing x by A or Alanine, the simplest standard amino acid).

SEQ ID NO. 36 shows a peptide (named "EYFDόx") containing six times the motif EYCFYVD (of SEQ ID NO. 1).

SEQ ID NO. 37 shows a peptide (named "LQLVL3x") containing three times the motif LQWLDKVL (of SEQ ID NO. 11).

SEQ ID NO. 38 shows a peptide (named ''NEYKJx") containing three times the motif NTNEYLEK (of SEQ ID NO. 15)

SEQ ID NO. 39 shows a peptide (named "RLF6x ") containing six times the motif RLLF (of SEQ ID NO. 31). It is to be understood that the amino acids in adjacent positions to the amino acid sequence motifs of the aforementioned general formulas may be modified by substitutions, deletions and/or additions and the resulting peptides shall also be encompassed by the present invention as long as the peptides are still capable of specifically binding to the target protein, namely TP53, SMADs, HDACs, PTPNl 1, RBPMS, ING5, CCNAl and/or PPPlCA (B,C).

Preferably, the peptides comprise or are selected from amino acid sequence of SEQ ID NO. 30 (peptide named "RRLIF", see Tables) amino acid sequence of SEQ ID NO. 36 (peptide named "EYFDόx", see Tables) amino acid sequence of SEQ ID NO. 39 (peptide named "RLF6x", see Tables)

In a preferred embodiment, the peptide comprises or consists of an amino acid sequence of SEQ ID NO. 30.

In a preferred embodiment, the peptide comprises or consists of an amino acid sequence of SEQ ID NO. 36.

In a preferred embodiment, the peptide comprises or consists of an amino acid sequence of SEQ ID NO. 39.

In further preferred embodiments, the peptide comprises or has an amino acid sequence of each one of SEQ ID NOs. 1-39.

The peptides of the present invention also encompass modified peptides, i.e. peptides, which may contain amino acids modified by addition of any chemical residue, such as phosphorylated or myristylated amino acids.

In the studies underlying this invention, short sequence motifs responsible for mediating protein-protein interactions could be revealed which are responsible for the binding of interaction partners to the target protein, namely TP53, SMADs, HDACs, PTPNl 1, RBPMS, ING5, CCNAl and/or PPPlCA (B,C). This was achieved by analyzing proteins previously identified to interaction with the target protein, namely TP53, SMADs, HDACs, PTPNI l, RBPMS, ING5, CCNAl and/or PPPlCA (B,C) using various techniques. Advantageously, these peptides itself may be applied for various therapeutic approaches for diseases caused by impaired TP53, SMADs, HDACs, PTPNI l, RBPMS, ING5, CCNAl and/or PPPlCA (B,C) function and allow to efficiently screen for chemical entities capable of mimicking or blocking disease causing interactions of TP53, SMADs, HDACs, PTPNI l, RBPMS, ING5, CCNAl and/or PPPlCA (B,C).

The present invention further relates to a polynucleotide encoding the peptides of the present invention. Moreover, the present invention relates to a vector comprising the aforementioned polynucleotide or at least one of the polynucleotides. The term "vector" as used herein, preferably, refers to plasmids. Although, the term also encompasses other types of vectors such as BAC or YACs or any other polynucleotide comprising the aforementioned polynucleotide(s) of the present invention as long as the said polynucleotide is either capable of expressing the polynucleotide of the present invention or propagating it. The plasmids referred to in accordance with the present invention are plasmids for propagation of the polynucleotide and/or expression thereof. Preferably, plasmids are suitable for expression of the polynucleotide of the present invention in host cells. Such plasmids usually comprise an expression control sequence, which may consist of promoter elements and/or further enhancer elements. Suitable expression control sequences are well known in the art.

The present invention also relates to a host cell comprising at least one polynucleotide or at least one vector of the present invention.

The host cell can be any prokaryotic or eukaryotic cell, such as a bacterial, insect, fungal, plant, animal, mammalian or, preferably, human cell. Preferred fungal cells are, for example, those of the genus Saccharomyces, in particular those of the species S. cerevisiae. The term "prokaryotic" is meant to include all bacteria, which can be transformed or transfected with a polynucleotide for the expression of a variant polypeptide of the invention. Prokaryotic hosts may include gram negative as well as gram positive bacteria such as, for example, E. coli, S. typhimurium, Serratia marcescens and Bacillus subtilis. Moreover, the present invention relates to an antibody, which specifically recognizes at least one peptide of the present invention.

Antibodies against the peptides of the invention can be prepared by well-known methods using a purified protein according to the invention or a (synthetic) fragment derived therefore as an antigen. Monoclonal antibodies can be prepared, for example, by the techniques as originally described in Kδhler and Milstein, Nature 256 (1975), 495, and Galfre, Meth. Enzymol. 73 (1981), 3, which comprise the fusion of mouse myeloma cells to spleen cells derived from immunized mammals. In a preferred embodiment of the invention, said antibody is a monoclonal antibody, a polyclonal antibody, a single chain antibody, human or humanized antibody, primatized, chimerized or fragment thereof that specifically binds said peptide or polypeptide also including bispecific antibody, synthetic antibody, antibody fragment, such as Fab, Fv or scFv fragments etc., or a chemically modified derivative of any of these. Furthermore, antibodies or fragments thereof to the aforementioned polypeptides can be obtained by using methods which are described, e.g., in Harlow and Lane "Antibodies, A Laboratory Manual", CSH Press, Cold Spring Harbor, 1988.

The present invention also relates to an array comprising at least one peptide, at least one polynucleotide, at least one host cell or at least one antibody of the present invention.

The present invention also relates to a pharmaceutical composition comprising at least one peptide, at least one polynucleotide, at least one vector, at least one host cell or at least one antibody of the present invention.

The term "pharmaceutical composition" as used herein comprises the substances of the present invention and optionally one or more pharmaceutically acceptable carrier. The substances of the present invention may be formulated as pharmaceutically acceptable salts. Acceptable salts comprise acetate, methylester, HCl, sulfate, chloride and the like. The pharmaceutical compositions can be conveniently administered by any of the routes conventionally used for drug administration, for instance, orally, topically, parenterally or by inhalation. The substances may be administered in conventional dosage forms prepared by combining the drugs with standard pharmaceutical carriers according to conventional procedures. These procedures may involve mixing, granulating and compressing or dissolving the ingredients as appropriate to the desired preparation. It will be appreciated that the form and character of the pharmaceutically acceptable character or diluent is dictated by the amount of active ingredient with which it is to be combined, the route of administration and other well-known variables. The carrier(s) must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. The pharmaceutical carrier employed may be, for example, either a solid or liquid. Exemplary of solid carriers are lactose, terra alba, sucrose, talc, gelatine, agar, pectin, acacia, magnesium stearate, stearic acid and the like. Exemplary of liquid carriers are phosphate buffered saline solution, syrup, oil such as peanut oil and olive oil, water, emulsions, various types of wetting agents, sterile solutions and the like. Similarly, the carrier or diluent may include time delay material well known to the art, such as glyceryl mono-stearate or glyceryl distearate alone or with a wax. The substance according to the present invention can be administered in various manners to achieve the desired effect. Said substance can be administered either alone or in the formulated as pharmaceutical preparations to the subject being treated either orally, topically, parenterally or by inhalation. Moreover, the substance can be administered in combination with other substances either in a common pharmaceutical composition or as separated pharmaceutical compositions. The diluent is selected so as not to affect the biological activity of the combination. Examples of such diluents are distilled water, physiological saline, Ringer's solutions, dextrose solution, and Hank's solution. In addition, the pharmaceutical composition or formulation may also include other carriers, adjuvants, or nontoxic, nontherapeutic, nonimmunogenic stabilizers and the like. A therapeutically effective dose refers to that amount of the substance according to the invention which ameliorate the symptoms or condition. Therapeutic efficacy and toxicity of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50 (the dose therapeutically effective in 50% of the population) and LD50 (the dose lethal to 50% of the population). The dose ratio between therapeutic and toxic effects is the therapeutic index, and it can be expressed as the ratio, LD50/ED50. The dosage regimen will be determined by the attending physician and other clinical factors; preferably in accordance with any one of the methods described above. As is well known in the medical arts, dosages for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. Progress can be monitored by periodic assessment. A typical dose can be, for example, in the range of 1 to 1000 μg; however, doses below or above this exemplary range are envisioned, especially considering the aforementioned factors. Generally, the regimen as a regular administration of the pharmaceutical composition should be in the range of 1 μg to 10 mg units per day. If the regimen is a continuous infusion, it should also be in the range of 1 μg to 10 mg units per kilogram of body weight per minute, respectively. Progress can be monitored by periodic assessment. However, depending on the subject and the mode of administration, the quantity of substance administration may vary over a wide range to provide from about 0.01 mg per kg body mass to about 10 mg per kg body mass. The pharmaceutical compositions and formulations referred to herein are administered at least once in accordance with the use of the present invention. However, the said pharmaceutical compositions and formulations may be administered more than one time, for example from one to four times daily up to a non- limited number of days. Specific formulations of the substance according to the invention are prepared in a manner well known in the pharmaceutical art and usually comprise at least one active substance referred to herein above in admixture or otherwise associated with a pharmaceutically acceptable carrier or diluent thereof. For making those formulations the active substance(s) will usually be mixed with a carrier or diluted by a diluent, or enclosed or encapsulated in a capsule, sachet, cachet, paper or other suitable containers or vehicles. A carrier may be solid, semisolid, gel-based or liquid material, which serves as a vehicle, excipient or medium for the active ingredients. Said suitable carriers comprise those mentioned above and others well known in the art, see, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania. The formulations can be adapted to the mode of administration comprising the forms of tablets, capsules, suppositories, solutions, suspensions or the like. The dosing recommendations will be indicated in product labeling by allowing the prescriber to anticipate dose adjustments depending on the considered patient group, with information that avoids prescribing the wrong drug to the wrong patients at the wrong dose.

The present invention furthermore provides the peptides, the polynucleotides, the vectors, the host cells and/or the antibodies of the present invention, as described herein, for treating and/or preventing cancer, in particular for treating and/or preventing cancer related to the target protein, wherein the target protein is selected from TP53, SMADs, HDACs, PTPNI l, RBPMS, ING5, CCNAl and PPPlCA (B,C).

Cancer is referred as "cancer related to target protein" when the target protein (i.e. TP53, SMADs, HDACs, PTPNI l, RBPMS, ING5, CCNAl or PPPlCA (B₅C)) or their variants or mutant form, participate in development, progression and/or regulation of the cancer/tumor.

Providing the peptides, the polynucleotides, the vectors, the host cells and/or the antibodies of the present invention, as described herein, for treating and/or preventing a certain disease refers to the second medical use of these compounds. Accordingly, the present invention furthermore provides the use of the peptides, the polynucleotides, the vectors, the host cells and/or the antibodies of the present invention, as described herein, for the manufacture of a medicament/pharmaceutical preparation for treating or preventing diseases, preferably cancer, in particular for treating and/or preventing cancer related to the target protein, wherein the target protein is selected from TP53, SMADs, HDACs, PTPNl 1, RBPMS, ING5, CCNAl and/or PPPlCA (B,C).

In an embodiment the peptides, the polynucleotides, the vectors, the host cells and/or the antibodies of the present invention, as described herein, are provided for treating and/or preventing defects of the heart, and TP53 related cancers.

Specifically related to the heart failure, HIFlA induces angiogenesis, which prevents cardiac hypertrophy and restores normal heart function. If however, HIFlA is inhibited through interaction with TP53, angiogenesis is prevented and cardiac hypertrophy proceeds to heart failure [2, 3].

Therefore, in an embodiment the peptide, polynucleotide, vector, host cell or antibody of the present invention are provided for treating and/or preventing defects of the heart, wherein said defects of the heart are selected from heart failure and cardiac hypertrophy, wherein the peptide preferably comprises an amino acid sequence motif of the general formula (E/D)YxFxx(E/D) and is capable of specifically binding to TP53.

More preferably, the peptide comprises or consists of SEQ ID NO. 36, see Table 3, Figure 7 and the Examples.

Related to cancer treatment, TFDPl is a transcription factor that heterodimerizes with E2F transcription factor and regulates expression of genes involved in the cell cycle. Inhibition of TFDPl interactions result in inhibition of cell-cycle and apoptosis of mammalian tumor cells. Therefore, inhibition of TFDP1-TP53 interaction would provide strategy for cancer treatment [4, 5]. In an embodiment the peptides, the polynucleotides, the vectors, the host cells and/or the antibodies of the present invention, as described herein, are provided for treating and/or preventing TP53 related cancers, preferably selected from leukemias, esophageal squamous cell carcinoma, breast carcinomas, soft-tissue sarcomas, brain tumors, osteosarcoma, adreno-cortical carcinoma, wherein the peptide preferably comprises an amino acid sequence motif of the general formula (E/D)YxFxx(E/D) and is capable of specifically binding to TP53.

The transforming growth factor (TGF) signalling pathway is involved in many cellular processes including cell growth, cell differentiation, apoptosis, cellular homeostasis and other cellular functions. The transforming growth factor β (TGF-β) regulates a wide variety of physiological processes. The TGF-β signal is mediated by SMAD proteins. SMADs can be receptor-associated (SMADl, SMAD2, SMAD3, SMAD5, SMAD8), inhibitory (SMAD6, SMAD7) and SMAD4 which is called common-mediator SMAD (co-SMAD). The TGF-β interacts with receptor-associated SMADs forming complexes that translocate to the nucleus and activate or repress transcription. The end effect of this TGF signalling cascade is inhibition of cell proliferation, which results in tumor suppression. However, at later stages of tumor development, TGF-β signalling starts to promote tumor growth and metastasis. Therefore, inhibitors of the TGF-β signalling pathway, such as SMAD-SMAD interaction inhibitors, are targets for anticancer therapy.

In an embodiment the peptides, the polynucleotides, the vectors, the host cells and/or the antibodies of the present invention, as described herein, are provided for treating and/or preventing cancers, which are related to TGF-β signalling pathway, in particular SMAD-

SMAD interaction, more particularly related to SMAD4. wherein the peptide preferably comprises an amino acid sequence motif of the general formula LQxLDxVL and is capable of specifically binding to SMADs, in particular to

SMAD4.

Histone deacetylases (HDACs) are enzymes that catalyze removal of acetyl groups from amino-terminal lysine residues in histones, which leads to chromatin condensation and transcriptional repression. The overall result of histone deacetylation is a global (non specific) reduction in gene expression. HDACs are promising targets for anticancer therapy and several classes of HDAC inhibitors (HDIs) are showing promising results [6, 7]. HDACl activity is regulated through association with SATBl (special AT-rich sequence binding protein 1). SATBl acts as a docking site for HDACl and some other chromatin remodelling enzymes and thereby acts as a global regulator of gene expression.

In an embodiment the peptides, the polynucleotides, the vectors, the host cells and/or the antibodies of the present invention, as described herein, are provided for treating and/or preventing cancers, which are related to HDACs, in particular HDACl, wherein the peptide preferably comprises an amino acid sequence motif of the general formula N/K/RxxYxxHP and is capable of specifically binding to HDACs, in particular

HDACl.

Protein tyrosine phosphatases (PTPs) are known to be signalling molecules that regulate a variety of cellular processes including cell growth, differentiation, mitotic cycle, and oncogenic transformation. PTPNl 1 is a gene encoding the protein tyrosine phosphatase, nonreceptor type 11, also called Shp2. PTPNI l contains two tandem Src homology-2 domains, which function as phospho-tyrosine binding domains and mediate the interaction of PTPNl 1 with its substrates. PTPNl 1 is widely expressed in most tissues and plays a regulatory role in various cell signalling events that are important for a diversity of cell functions. Missense mutations in the PTPNI l locus are associated with both Noonan syndrome and Leopard syndrome. Patients with a subset of Noonan syndrome PTPNl 1 mutations also have a higher prevalence of juvenile myelomonocytic leukemias (JMML). Activating Shp2 mutations have also been detected in neuroblastoma, melanoma, acute myeloid leukemia, breast cancer, lung cancer, colon cancer. The PTPNI l mutations lead to an increased activity of phosphatase. Therefore a specific disruption of PTPNI l interactions will reduce its phosphates function in key pathways. Additional advantage in developing inhibitors of PTNI l interactions is prevention of H. pylori infection via virulence factor CagA. This infection causes gastric ulcer and, ultimately, gastric carcinoma.

Tyrosyl phosphorylation, which is controlled by protein-tyrosine kinases (PTKs) and protein- tyrosine phosphatases (PTPs), regulates numerous cellular processes. Altered expression and/or mutations in PTKs are linked to many forms of cancer, yet until recently little was known about the roles of PTPs in normal cells or in cancer. Earlier work established that a member of the PTP superfamily, PTEN, is an important tumor suppressor gene. At least one other PTP, the SH2 domain-containing phosphatase Shp2 (also called PTPNl 19, is a bona fide oncogene that is mutated in several types of leukemia and hyperactivated by other mechanisms in some solid tumors. [8]

In an embodiment the peptides, the polynucleotides, the vectors, the host cells and/or the antibodies of the present invention, as described herein, are provided for treating and/or preventing PTPNl 1 related cancers, preferably selected from leukemia, neuroblastoma, melanoma, acute myeloid leukemia, breast cancer, lung cancer, colon cancer, juvenile myelomonocytic leukemias (JMML), wherein the peptide preferably comprises an amino acid sequence motif of the general formula YAxxI and is capable of specifically binding to PTPNl 1.

In an embodiment the peptides, the polynucleotides, the vectors, the host cells and/or the antibodies of the present invention, as described herein, are provided for treating and/or preventing Noonan syndrome, Leopard syndrome and/or H. pylori infection, wherein the peptide preferably comprises an amino acid sequence motif of the general formula YAxxI and is capable of specifically binding to PTPNl 1.

RNA-binding proteins are typically cytoplasmic and nuclear proteins that associate with (bind) and facilitate the translation of RNAs. RNA binding protein with multiple splicing (RBPMS) has a single RRM domain, which is known to interact with DNA/RNA and peptides using distinct binding surfaces. The interaction of RBPMS with Ewing Sarcoma breakpoint region 1 (EWSRl), which is known to be involved in cancer and children cancer, is of special medical interest. EWSRl is known to interact with other transcription factors via its disordered and proline-rich N-terminal portion, linking the RPBMS/EWSRl complex to many essential pathways. Thus, an interaction inhibition would decrease EWS expression activity, consequently diminishing cell viability and tumor growth.

A recent paper shows that chemicals which affects EWS have a strong essential phenotype, however, the mechanism is unknown [9].

In an embodiment the peptides, the polynucleotides, the vectors, the host cells and/or the antibodies of the present invention, as described herein, are provided for treating and/or preventing Ewing Sarcoma, wherein the peptide preferably comprises an amino acid sequence motif of the general formula TxPxY and is capable of specifically binding to RBPMS.

ING5 is a member of the ING family of proteins that inhibit growth and is in particular a close homolog to INGl, a tumor suppressor protein that can interact with TP53, inhibit cell growth, and induce apoptosis. ING5 contains a PHD-finger, which is a common motif in proteins involved in chromatin remodeling. The PHD domain of ING5 interacts with the DNA binding domain of TP53. Thus, ING5 binds TP53 and EP300/p300, a component of the histone acetyl transferase complex, and is involved in TP53-dependent regulatory pathway. Targeted inhibition of ING5 would reduce apoptosis.

Accordingly, the present invention furthermore provides the peptide, the polynucleotide, the vector, the host cell or the antibody for the regulation of apoptosis, wherein the peptide preferably comprises an amino acid sequence motif of the general formula FxDL and is capable of specifically binding to ING5.

The protein encoded by CCNAl belongs to the highly conserved cyclin family, whose members are characterized by a dramatic periodicity in protein abundance through the cell cycle. Cyclins function as regulators of CDK kinases. The cyclin encoded by CCNAl, cyclin Al, was shown to be expressed in testis and brain, as well as in several leukemic cell lines, and is thought to primarily function in the control of the germline meiotic cell cycle. Cyclin Al binds both CDK2 and CDC2 kinases, which give two distinct kinase activities, one appearing in S phase, the other in G2, and thus regulate separate functions in cell cycle. Cyclin Al was found to bind to important cell cycle regulators, such as Rb family proteins, transcription factor E2F-1, and the p21 family proteins (see 12-13).

Accordingly, the present invention furthermore provides the peptide, the polynucleotide, the vector, the host cell or the antibody for treating and/or preventing cyclin Al related cancers, preferably by modulating cyclin Al (CCNAl) function or those of its binding proteins, such as CDK2 or CDC2 kinases, wherein the peptide preferably comprises an amino acid sequence motif of the general formula (R/K)(V/I/L)xF and is capable of specifically binding to CCNAl. More preferably, the peptide comprises or consists of SEQ ID NO. 30 or the peptide comprises or consists of SEQ ID NO. 39, see Table 3, Figure 8 and the Examples.

Accordingly, the present invention furthermore provides the peptide, the polynucleotide, the vector, the host cell or the antibody for treating and/or preventing defects of the heart, wherein said defects of the heart are preferably selected from heart failure and other cardiac conditions, wherein the peptide preferably comprises an amino acid sequence motif of the general formula (R/K)(V/I/L)xF and is capable of specifically binding to PPPlCA (B,C).

More preferably, the peptide comprises or consists of SEQ ID NO. 30, see Table 3, Figure 9 and the Examples.

The present invention also relates to a method for identifying a compound, which specifically binds to the peptide of the present invention comprising

(a) contacting a compound suspected to specifically bind to the peptide with the said peptide; and

(b) determining whether the compound specifically binds to the said peptide.

Preferably, such a compound also specifically blocks interaction of the target protein with its interaction partners.

target protein interactions partners

TP53 HIFlA, TFDPl, ERCC3, TADA3L

SMAD4 SMAD4, SMADl, SMAD5, SMAD9 HDACs SATBl

PTPNI l TIEl, MPZLl, FRS2

RBPMS EWSRl, DVL2

ING5 TP53

CCNAl CDKNlA, CDC25A

PPPlCA (B₅C) PPPlRlA (2), CDKNlA, CDC25A

See also Tables 1 to 3

As meant herein, the term "specifically blocks interaction" does not necessarily mean that a compound must physically bind to the target protein, namely TP53, SMADS₅ HDACS, PTPNI l, RBPMS, ING5, CCNAl and/or PPPlCA (B,C). Rather, it is sufficient that the activity of the target protein (namely TP53, SMADs, HDACs, PTPNI l, RBPMS, ING5, CCNAl and/or PPPlCA (B₅C)) is blocked due to administration of the said compound. This may be achieved by a specific physical interaction or binding to the target protein. However, the same effect shall be obtained by physical interaction of the compound with the interaction domain of the interaction partners of the target protein (namely TP53, SMADs, HDACs, PTPNI l, RBPMS, ING5, CCNAl and/or PPPlCA (B₅C)), such as the aforementioned proteins.

Moreover, the present invention relates to a method for identifying a compound which specifically blocks interaction of the target protein comprising the steps of

(a) contacting a compound suspected to specifically bind to the target protein with the peptide of the present invention and the target protein; and

(b) determining whether the compound is capable of blocking binding of the said peptide to the target protein, wherein said target protein is selected from TP53, SMADs, HDACs, PTPNI l₅ RBPMS, ING5, CCNAl and/or PPPlCA (B,C).

In such a method, the compound will compete with the peptide of the present invention for specific binding to the target protein. A compound which does not specifically bind to the target protein will allow the formation of a complex between the peptide of the present invention and the target protein while a compound which specifically interacts with the target protein will compete with the peptide for complex formation. Accordingly, determining whether the compound is capable of blocking binding of the peptide to the target protein may be achieved by either measuring the formation of the complex or by measuring the level of the free peptide of the present invention or the free compound suspected to specifically binding to the target protein. Preferably, this is achieved by providing either labeled compounds or labeled peptides. Suitable labels and techniques for labeling are well known in the art.

In a preferred embodiment, the method comprises the use of a host cell according to the invention, which expresses peptide(s) of the invention and which preferably expresses the target protein.

Preferably, the method comprises the use of labeled target protein(s) and/or labeled peptide(s) of the invention, wherein the peptide(s) can be fused to further moieties / tags, such as proteins.

Labels, suitable for in vivo and in vitro applications, are known in the art. Preferred labels are fluorescent tags / fluorophors, chromophors, radioisotopes, protein tags, antibodies and antibody fragments.

Fluorescent tags / fluorophors which can be attached to the peptide(s) of the present invention and/or to target protein(s) are known in the art, including (E)GFP and derivatives thereof, fruit fluorescent proteins (such as mCherry) and derivatives thereof.

Preferred are fluorescent tags / fluorophors suitable for FRET assays (as donor-acceptor FRET pairs), such as

(E)CFP and (E)YFP; (E)GFP and mCherry, (E)GFP and DsRed,

The labels, such as flurorescent tags / fluorophors, are preferably suitable for use in cellular assays, preferably in vivo.

In one embodiment, the method comprises the use of labeled (fluorescent) target protein(s) and/or labeled (fluorescent) annexin A4-protein fusion(s) with the peptide(s).

More preferably, binding or interaction of the peptide(s) of the present invention with their respective target protein(s) is analyzed by an annexin translocation assay utilizing a translocating FRET probe based on annexin A4, as described in reference 10 and 11 and in the Examples.

Preferably, the compound to be identified in the aforementioned methods is an agonist or antagonist of the target protein, namely TP53, SMADs, HDACs, PTPNI l, RBPMS, ING5, CCNAl and/or PPPlCA (B₅C).

An "agonist" of the target protein as used herein is a compound, which will activate the target function or pathway, wherein the target protein is involved. Conversely, an antagonist is a compound which will inhibit the target function or pathway, wherein the target protein is involved.

As set forth above, this may either be achieved by a direct interaction between the compound and the target protein or by an interaction of the compound with an interaction partner of the target protein. In case of an agonist, the compound shall interact with an interaction partner of the target protein which physiologically down regulates the activity of the target protein while an antagonist might be a compound which interacts with an activator of the activity of the target protein.

Suitable compounds which may be used in the methods of the present invention include, but are not limited to small molecule compounds, peptides, proteins, nucleic acids and derivatives of the aforementioned compound classes.

Finally, the present invention relates to a method of manufacturing a pharmaceutical composition comprising the steps of the aforementioned methods and the further step of formulating the compound identified in a pharmaceutically accepted form. It is to be understood that once the compound, e.g. an agonist or antagonist of the target protein, has been identified by any one of the methods of the present invention, the person skilled in the art will be in a position to formulate said compound, i.e. to synthesize the compound in a pharmaceutically acceptable form, without further ado. Table 1 Regions containing the proposed general motifs in the interaction partners of the target proteins TP53, SMAD4, HDACl, PTPNl 1,

RBPMS, ING5, CCNAl and PPPlCA (B,C). The respective specific sequence motifs are underlined and conform to the general motifs. κ>

Table 2 Preferred peptides derived from the interaction partners of the target proteins. κ> -J

Table 3 Preferred peptides derived from the interaction partners of the target proteins that interacted with the target proteins.

KJ

The following examples and drawings illustrate the present invention without, however, limiting the same thereto.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows regions containing the amino acid sequence motif of the general formula (E/D)YxFxx(E/D) in various interactions partners of TP53.

Figure 2 shows regions containing the amino acid sequence motif of the general formula LQxLDxVL in various interactions partners of SMAD4.

Figure 3 shows regions containing the amino acid sequence motif of the general formula N/K/RxxYxxHP in an interactions partner of HDACl.

Figure 4 shows regions containing the amino acid sequence motif of the general formula YAxxI in various interactions partners of PTPNl 1.

Figure 5 shows regions containing the amino acid sequence motif of the general formula TxPxY in various interactions partners of RBPMS.

Figure 6 shows regions containing the amino acid sequence motif of the general formula FxDL in an interactions partner of ING5.

Figure 7 Interaction o/EYxFxxD motif and TP 53. a HeLa cells expressing the EYxFxxD motif from HIFlA repeated six times (EYFDόx,

SEQ ID NO. 36) as bait and TP53 as target. Translocation in both emission channels upon addition of ionomycin indicates protein-protein interaction.

White lines indicate the position of the measured fluorescence profile which is shown in the graphs below the corresponding images (c). b HeLa cells expressing a different (control) sequence from HIFlA as bait and TP53 as target. No interaction was observed. White lines indicate the position of the measured fluorescence profile which is shown in the graphs below the corresponding images (d). Scale bars, 10 μm.

Figure 8 Interaction of RVxF motif and cyclin Al (CCNAl). a HeLa cells expressing a single repeat of the RVxF motif from CDKN 1 A (RRLIF, SEQ

ID NO. 30) (bait) and CCNAl (target). Translocation in both emission channels upon addition of ionomycin indicates protein-protein interaction.

White lines indicate the position of the measured fluorescence profile which is shown in the graphs below the corresponding images (d). b No interaction was observed between a different (control) sequence from CDC25A

(bait) and CCNAl (target) in HeLa cells.

White lines indicate the position of the measured fluorescence profile which is shown in the graphs below the corresponding images (e). c Interaction was observed between the RVxF motif from CDC25A repeated 6 times

(RLFόx, SEQ ID NO. 39) (bait) and CCNAl (target) in HeLa cells.

White lines indicate the position of the measured fluorescence profile which is shown in the graphs below the corresponding images (f).

Scale bars, 10 μm.

Figure 9 Interaction of RVxF motif and protein phosphatase 1. a) HeLa cells expressing a single repeat of the RVxF motif from CDKNlA (RRLIF, SEQ ID NO. 30) (bait) and PPPlCC (target). Translocation in both emission channels upon addition of ionomycin indicates protein-protein interaction.

White lines indicate the position of the measured fluorescence profile which is shown in the graphs below the corresponding images (c). b) No interaction was observed between a different (control) sequence from CDC25A (bait) and PPPlCC (target) in HeLa cells.

White lines indicate the position of the measured fluorescence profile which is shown in the graphs below the corresponding images (d). Scale bars, 10 μm. EXAMPLES

Example 1

The analysis of the interaction of peptides derived from interaction partners of respective target proteins was carried as a annexin translocation assay utilizing a translocating FRET probe based on annexin A4, as described in 10 and 11.

Experimental procedures

Cloning

All constructs were cloned using standard molecular biology methods. Briefly, cDNA's encoding desired proteins were obtained from a commercial provider (Origene) and inserted into fluorescent protein and annexin A4-fluorescent protein containing vectors using restriction enzymes. To generate constructs encoding fluorescent protein and/or annexin A4- fluorescent protein fusions with peptide sequences, primers were designed that encode the desired sequence flanked with restriction sites. The primers were 5'-phosphorylated with the T4 polynucleotide kinase (NEB) according to the manufacturer's instructions, heated to 95°C for 5 min and cooled down to anneal. Such double-stranded primers were ligated into desired vectors digested with matching restriction enzymes. In cases where the inserted sequence was too large for a single set of primers, two or three primer sets were used with 6 nucleotides overlapping regions. Those primers sets were treated as described above and ligated with each other and with the desired vector in a single ligation reaction.

Cell culture and transfection

Experiments were performed in HeLa cells. Cells were maintained using standard tissue culture procedures and transfected with FuGENE 6 reagent (Roche) according to the manufacturers' instructions. For multiple transfections, equal amounts of DNA were used for each construct. Cells were washed 12-24 h after transfection and incubated in imaging medium (20 mM Hepes, pH 7.4, 115 mM NaCl, 1.2 mM CaCl₂, 1.2 mM MgCl₂, 1.2 mM K₂HPO₄, 2 g/L D-glucose) at 37°C with 5% CO₂ for about 30 min before imaging. A DMSO stock of ionomycin (Calbiochem) was prepared and ionomycin prediluted in imaging medium before it was carefully added to the dish to induce translocation.

Equipment and settings AIl experiments were performed on a Leica TCS SP2 AOBS microscope (Leica Microsystems). An HCX PL APO lbd.BL 63.Ox 1.40 oil objective was used. Pinhole was half-opened in all experiments (2.62 airy). Laser power and PMT gain were adjusted from experiment to experiment. Images were taken in 8 bit mode, with 2-4 line averaging. General excitation and emission settings are as follows:

ECFP excitation 405 nm emission 465-495 nm

EGFP excitation 488 nm emission 495-510 nm

EYFP excitation 532 nm emission 545-565 nm mCherry excitation 594 nm emission 605-650 nm

Alternatively, emission settings can be modified. For instance, also 458 nm laser can be used for ECFP excitation, 515 nm for EYFP excitation and 561 nm laser for mCherry excitation. Emmision setting can be modified depending on the excitation settings and can be 450-480 nm for ECFP, or 530-550 nm for EYFP. The optimal excitation and emission settings depend on the fluorescent protein combination used in a particular experiment.

Image processing

All image processing and calculations were performed using ImageJ (http://rsb.info.nih.gov/ij/). Background level was measured outside cells and subtracted globally. Median filter (1 pixel) was used for image smoothening. Profile graphs were obtained by measuring fluorescence signal across cells where it is indicated by the white line.

Example 2

Interaction of EYxFxxD motif and TP53 were tested as described in Example 1.

Results are shown in Figure 7 and Table 3.

HeLa cells expressing the EYxFxxD motif from HIFlA repeated six times as bait (EYFDδx,

SEQ ID NO. 36) and TP53 as target were tested. Translocation in both emission channels upon addition of ionomycin indicates protein-protein interaction (see Figures 7a and c).

HeLa cells expressing a different (control) sequence from HIFlA as bait and TP53 as target were also tested. No interaction was observed (see Figures 7b and d). The result suggests that having 6 repeats of a motif, one after the other, increases the sensitivity of the assay due to increased effective concentration of the motif-bait and/or potential secondary structure elements appearing that might have a higher affinity for the target.

Example 3

Interaction of RVxF motif and cyclin Al (CCNAl) as described in Example 1.

Results are shown in Figure 8 and Table 3.

HeLa cells expressing a single repeat of the RVxF motif from CDKNlA (RRLIF, SEQ ID

NO. 30) (bait) and CCNAl (target) were tested. Translocation in both emission channels upon addition of ionomycin indicates protein-protein interaction (see Figures 8a and d).

No interaction was observed between a different (control) sequence from CDC25A (bait) and

CCNAl (target) in HeLa cells (see Figures 8b and e).

Interaction was observed between the RVxF motif from CDC25A repeated 6 times (RLFόx,

SEQ ID NO. 39) (bait) and CCNAl (target) in HeLa cells (see Figures 8c and f).

RRLIF HSKRRLiFSKR interacted (SEQ ID NO. 30)

RLFόx RRRLLFACRRRLLFACKGRRRLLFACRRRLLFACGKRRRLLFACRRRLLFAC interacted (SEQ ID NO. 39)

Example 4

Interaction of RVxF motif and protein phosphatase 1 were tested as described in Example 1. Results are shown in Figure 9 and Table 3.

HeLa cells expressing a single repeat of the RVxF motif from CDKNlA (RRLIF, SEQ ID NO. 30) (bait) and PPPlCC (target) were tested. Translocation in both emission channels upon addition of ionomycin indicates protein-protein interaction (see Figures 9a and c). No interaction was observed between a different (control) sequence from CDC25A (bait) and PPPlCC (target) in HeLa cells (see Figures 9b and d).

RRLIF HSKRRLIFSKR interacted (SEQ ID NO. 30) The features disclosed in the foregoing description, in the claims and/or in the accompanying drawings may, both separately and in any combination thereof, be material for realizing the invention in diverse forms thereof.

REFERENCES

1. Neduva, V., et al., Systematic discovery of new recognition peptides mediating protein interaction networks. PLoS Biol, 2005. 3(12): p. e405.

2. Sano, M., et al., p53-induced inhibition of Hif-1 causes cardiac dysfunction during pressure overload. Nature, 2007. 446(7134): p. 444-8.

3. Schmid, T., J. Zhou, and B. Brune, HIF-I andp53: communication of transcription factors under hypoxia. J Cell MoI Med, 2004. 8(4): p. 423-31.

4. Sorensen, T.S., et al., Functional interaction between DP-I andp53. MoI Cell Biol, 1996. 16(10): p. 5888-95.

5. O'Connor, D.J., et al., Physical and functional interactions between p53 and cell cycle cooperating transcription factors, E2F1 and DPI. Embo J, 1995. 14(24): p. 6184-92.

6. Beutler, A.S., et al., Carbamazepine is an inhibitor ofhistone deacetylases. Life Sci, 2005. 76(26): p. 3107-15.

7. Gurvich, N., et al., Histone deacetylase is a target of valproic acid-mediated cellular differentiation. Cancer Res, 2004. 64(3): p. 1079-86.

8. Mohi MG, Neel BG, The role ofShp2 (PTPNIl) in cancer. Curr Opin Genet Dev. 2007. 17(1): p.23-30. 9. Stegmaier K, Wong JS₅ Ross KN, Chow KT, Peck D, Wright RD, Lessnick SL, Kung AL, Golub TR, Signature-based small molecule screening identifies cytosine arabinoside as an EWS/FLI modulator in Ewing sarcoma. PLoS Med., 2007. 4(4):el22.

10. Piljic A, Schultz C Simultaneous recording of multiple cellular events by FRET. ACS Chem Biol. 2008. 3(3): 156-60.

11. Piljic A, Schultz C Analysis of protein complex hierarchy in living cells. ACS Chem Biol. 2008. 3(12):749-55.

12. Nevins JR. E2F: a link between the Rb tumor suppressor protein and viral oncoproteins. Science. 1992 Oct 16;258(5081):424-9.

13. Halaban R. Melanoma cell autonomous growth: the Rb/E2F pathway. Cancer Metastasis Rev. 1999 18(3):333-43.

14. Gupta RC, Mishra S, Rastogi S, Imai M, Habib O, Sabbah HN. Cardiac SR-coupled PPl activity and expression are increased and inhibitor 1 protein expression is decreased in failing hearts. Am J Physiol Heart Circ Physiol. 2003 285(6):H2373-81.

15. Grote-Wessels S, Baba HA, Boknik P, El-Armouche A, Fabritz L, Gillmann HJ, Kucerova D, Matus M, Mϋller FU, Neumann J, Schmitz M, Stumpel F, Theilmeier G, Wohlschlaeger J, Schmitz W, Kirchhefer U. Inhibition of protein phosphatase 1 by inhibitor-2 exacerbates progression of cardiac failure in a model with pressure overload. Cardiovasc Res. 2008 79(3):464-71.

16. Chen G, Zhou X, Nicolaou P, Rodriguez P, Song G, Mitton B, Pathak A, Zachariah A, Fan GC, Dorn GW 2nd, Kranias EG. A human polymorphism of protein phosphatase- 1 inhibitor- 1 is associated with attenuated contractile response of cardiomyocytes to beta- adrenergic stimulation. FASEB J. 2008 22(6): 1790-6.

Claims

European Molecular Biology Laboratory"Peptides for the specific binding and regulation of protein targets"E30997PCTClaims

1. A peptide comprising an amino acid sequence motif selected from the group of

(a) an amino acid sequence motif of the general formula (E/D)YxFxx(E/D);

(b) an amino acid sequence motif of the general formula LQxLDxVL;

(c) an amino acid sequence motif of the general formula N/K/RxxYxxHP;

(d) an amino acid sequence motif of the general formula NxxEYxxK;

(e) an amino acid sequence motif of the general formula YAxxI;

(f) an amino acid sequence motif of the general formula TxPxY;

(g) an amino acid sequence motif of the general formula FxDL; and

(h) an amino acid sequence motif of the general formula (R/K)(V/I/L)xF.

wherein "x" refers to any amino acid residue.

2. The peptide of claim 1, wherein said peptide is capable of specifically binding to a target protein selected from tumor suppressor protein p53 (TP53), SMADs, histone deacetylases (HDACs), protein tyrosine phosphatase, non-receptor type 11 (PTPNI l), RNA binding protein with multiple splicing (RBPMS), inhibitor of growth family, member 5 (ING5), cyclin Al (CCNAl) and protein phosphatase 1, catalytic subunit (PPPlCA (B₅C)).

3. The peptide of claim 1 or 2, wherein the peptide comprises

(a) an amino acid sequence motif of the general formula (E/D)YxFxx(E/D) and is capable of specifically binding to TP53; or

(b) an amino acid sequence motif of the general formula LQxLDxVL and is capable of specifically binding to SMADs; or

(c) an amino acid sequence motif of the general formula N/K/RxxYxxHP and is capable of specifically binding to HDACs; or

(d) an amino acid sequence motif of the general formula NxxEYxxK and is capable of specifically binding to HDACs; or

(e) an amino acid sequence motif of the general formula YAxxI and is capable of specifically binding to PTPNl 1 ; or (f) an amino acid sequence motif of the general formula TxPxY and is capable of specifically binding to RBPMS; or

(g) an amino acid sequence motif of the general formula FxDL and is capable of specifically binding to ING5; or

(h) an amino acid sequence motif of the general formula (R/K)(V/I/L)xF and is capable of specifically binding to CCNAl and/or PPPlCA (B,C).

4. The peptide of any of claims 1 to 3, wherein said peptide is 4 to 150 amino acids in length.

5. The peptide of claim 4, wherein said peptide is about 4, 5, 6, 7, 8, 10, 11, 12, 13, 14, 15, 16, 18, 20, 25, 30 or 35 amino acids in length.

6. The peptide of any of claims 1 to 3, wherein said peptide comprises one or more repeats of the amino acid sequence motif, preferably 2, 3, 4, 5, 6, 7, 8, 9 or 10 repeats of said amino acid sequence motif, more preferably 6 repeats of said amino acid sequence motif.

7. The peptide of claim 6, wherein said peptide is about 30 to 80 amino acids in length , preferably about 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 or 80 amino acids in length, more preferably 46, 52 or 55 amino acids.

8. The peptide of any of claims 1 to 7, wherein said peptide comprises an amino acid sequence as shown in any one of SEQ ID NOs. 1 to 39.

9. The peptide of any of claims 1 to 7 selected from a peptide with an amino acid sequence comprising or having SEQ ID NO. 30, 36 or 39.

10. A polynucleotide encoding the peptide of any one of claims 1 to 9.

11. A vector comprising the polynucleotide of claim 10.

12. A host cell comprising at least one peptide of any one of claims 1 to 9, at least one polynucleotide of claim 10 or the vector of claim 11.

13. An antibody specifically recognizing at least one peptide of any one of claims 1 to 9.

14. An array comprising at least one peptide of any one of claims 1 to 9, at least one polynucleotide of claim 10, at least one host cell of claim 12 or at least one antibody of claim 13.

15. A pharmaceutical composition comprising at least one peptide of any one of claims 1 to 9, at least one polynucleotide of claim 10, at least one vector of claim 11, at least one host cell of claim 12 or at least one antibody of claim 13.

16. The peptide of any one of claims 1 to 9, the polynucleotide of claim 10, the vector of claim 11, the host cell of claim 12 or the antibody of claim 13 for treating and/or preventing cancer, in particular cancers related to the target protein, wherein the target protein is selected from TP53, SMADs, HDACs, PTPNl 1, RBPMS, ING5, CCNAl and PPPlCA (B₅C).

17. The peptide, the polynucleotide , the vector, the host cell or the antibody according to claim 16 for treating and/or preventing TP53 related cancers, preferably selected from leukemias, esophageal squamous cell carcinoma, breast carcinomas, soft-tissue sarcomas, brain tumors, osteosarcoma, adreno-cortical carcinoma, the peptide preferably comprising or consisting of SEQ ID NO. 36.

18. The peptide, the polynucleotide , the vector, the host cell or the antibody according to claim 16 for treating and/or preventing PTPNl 1 related cancers, preferably selected from leukemia, neuroblastoma, melanoma, acute myeloid leukemia, breast cancer, lung cancer, colon cancer, juvenile myelomonocytic leukemias (JMML).

19. The peptide of any one of claims 1 to 9, the polynucleotide of claim 10, the vector of claim 11, the host cell of claim 12 or the antibody of claim 13 for treating and/or preventing defects of the heart, preferably selected from heart failure and cardiac hypertrophy, wherein the peptide preferably comprises an amino acid sequence motif of the general formula (E/D)YxFxx(E/D) and is capable of specifically binding to TP53, the peptide preferably comprising or consisting of SEQ ID NO. 36.

20. The peptide of any one of claims 1 to 9, the polynucleotide of claim 10, the vector of claim 11, the host cell of claim 12 or the antibody of claim 13 for treating and/or preventing Noonan syndrome, Leopard syndrome and/or H. pylori infection, wherein the peptide preferably comprises an amino acid sequence motif of the general formula YAxxI and is capable of specifically binding to PTPNl 1.

21. The peptide, the polynucleotide, the vector, the host cell or the antibody according to claim 16 for treating and/or preventing Ewing Sarcoma, wherein the peptide preferably comprises an amino acid sequence motif of the general formula TxPxY and is capable of specifically binding to RBPMS.

22. The peptide of any one of claims 1 to 9, the polynucleotide of claim 10, the vector of claim 11, the host cell of claim 12 or the antibody of claim 13 for the regulation of apoptosis, wherein the peptide preferably comprises an amino acid sequence motif of the general formula FxDL and is capable of specifically binding to ING5.

23. The peptide of any one of claims 1 to 9, the polynucleotide of claim 10, the vector of claim 11, the host cell of claim 12 or the antibody of claim 13 for treating and/or preventing cyclin Al related cancers, wherein the peptide preferably comprises an amino acid sequence motif of the general

(R/K)(V/I/L)xF and is capable of specifically binding to CCNAl, the peptide preferably comprising or consisting of SEQ ID NO. 30 or SEQ ID NO. 39.

24. The peptide of any one of claims 1 to 9, the polynucleotide of claim 10, the vector of claim 11, the host cell of claim 12 or the antibody of claim 13 for treating and/or preventing defects of the heart, preferably selected from heart failure and other cardiac conditions, wherein the peptide preferably comprises an amino acid sequence motif of the general (R/K)(V/I/L)xF and is capable of specifically binding to PPPlCA (B₅C), the peptide preferably comprising or consisting of SEQ ID NO. 30.

25. A method for identifying a compound which specifically binds to the peptide of claim 1 to 9 comprising contacting a compound suspected to specifically binding to the peptide of any of one of claims 1 to 9 with the said peptide and determining whether the compound specifically binds to the said peptide.

26. The method of claim 25, wherein said compound specifically blocks interaction of a target protein selected from TP53, SMADs, HDACs, PTPNI l, RBPMS, ING5, CCNAl and PPPlCA (B,C).

27. A method for identifying a compound which specifically blocks interactions of a target protein, comprising the steps of: contacting a compound suspected to specifically bind to said target protein with the peptide of any one of claims 1 to 9 and said target protein; and determining whether the compound is capable of blocking binding of the said peptide to said target protein, wherein the target protein is selected from TP53, SMADs, HDACs, PTPNl 1, RBPMS, ING5, CCNAl and PPPlCA (B,C).

28. The method of claim 27 comprising the use of a host cell according to claim 12, which expresses peptide(s) of any one of claims 1 to 9 and which preferably expresses the target protein, preferably comprising the use of labeled peptide(s) and/or labeled target protein, wherein the label is preferably a fluorophor, more preferably suitable for FRET, such as comprising the use of fluorescent annexin A4-protein fusions with the peptide(s) and/or fluorescent target protein.

29. The method of claim 25 to 28, wherein said compound is an agonist or antagonist of TP53, SMADs, HDACs, PTPNl 1, RBPMS, ING5, CCNAl and/or PPPlCA (B₅C).

30. A method of manufacturing a pharmaceutical composition comprising the steps of the method of any one of claims 25 to 29 and the step of formulating the compound identified by the said method in a pharmaceutically acceptable form.