WO2011035922A2

WO2011035922A2 - Inhibitors of fgf2 phosphorylation and their use for treating angiogenesis-related diseases

Info

Publication number: WO2011035922A2
Application number: PCT/EP2010/005859
Authority: WO
Inventors: Walter Nickel; Antje D. Ebert; Rainer Pepperkok; Sabine Wegehingel
Original assignee: European Molecular Biology Laboratory (Embl); Landesstiftung Baden-Württemberg GmbH
Priority date: 2009-09-25
Filing date: 2010-09-24
Publication date: 2011-03-31
Also published as: WO2011035922A3

Abstract

The present invention relates to methods for identifying (i) a compound which reduces/inhibits the cellular export of FGF2 by inhibiting FGF2 phosphorylation by Tec kinase, (ii) a compound which reduces/inhibits the interaction between Tec kinase and FGF2, and (iii) a compound which reduces/inhibits the cellular export of FGF2 by binding specifically to phosphorylated FGF2. Furthermore, the present invention provides the compounds identifiable by said methods and pharmaceutical compositions comprising said compounds, in particular, for treating and/or preventing angiogenesis- and/or neovascularization-related diseases such as cancer.

Description

INHIBITORS OF FGF2 PHOSPHORYLATION AND THEIR USE FOR TREATING

ANGIOGENESIS-RELATED DISEASES TECHNICAL FIELD OF INVENTION

The present invention relates to the field of treating and/or preventing angiogenesis- and/or neovascularization-related diseases, in particular, by inhibiting the secretion of fibroblast growth factor 2 (FGF2). In particular, the present invention provides methods for identifying a compound (i) which reduces/inhibits the cellular export of FGF2 by inhibiting FGF2 phosphorylation by Tec kinase, (ii) which reduces/inhibits the cellular export of FGF2 by reducing/inhibiting the interaction between Tec kinase and FGF2, and (iii) which reduces/inhibits the cellular export of FGF2 by binding specifically to phosphorylated FGF2. Furthermore, the present invention provides the compounds identifiable by said methods and pharmaceutical compositions comprising said compounds, in particular, for treating and/or preventing angiogenesis- and/or neovascularization-related diseases such as cancer.

BACKGROUND OF THE INVENTION

Fibroblast growth factor 2 (FGF2) belongs to the FGF family of proteins and is a potent mitogen with pro-angiogenic activity. While the growth and development of new blood vessels may be desired, for example, for wound healing and tissue development, it also contributes to the pathogenesis of several diseases such as cancer or atherosclerosis. It is frequently the local uncontrolled release of angiogenic growth factors such as FGF2 which contributes to neovascularization that takes place during angiogenesis-related diseases. FGF2 has been associated with a number of angiogenesis- and neovascularization-related diseases such as cancer, inflammation, and angio-proliferative diseases. Thus, FGF2 may represent a target for anti-angiogenic therapies.

Several approaches have been suggested for neutralizing the angiogenic activity of FGFs in general, for example, by inhibiting FGF production, by sequestering FGFs in an inactive from in the extracellular environment, by inhibiting the expression of the different FGF receptors in endothelial cells, by masking FGF receptors, thus preventing their engagement by FGFs, by interrupting the signal transduction pathway(s) triggered by FGFs in endothelial cells, or by neutralizing FGF-induced effectors/biological responses whose function is essential in mediating the angiogenic potential of FGFs. An alternative approach for interfering with FGF function is the inhibition of its release from cells. FGF2 differs from other secretory proteins, since its export from cells is mediated by an ER/Golgi-independent mechanism. Initial sorting of FGF2 into its secretory route occurs by recruitment to the inner leaflet of plasma membranes mediated by the phosphoinositide PI(4,5)P₂. Recently, it has been demonstrated that this initial step of FGF2 secretion represents an intrinsic mechanism of quality control as only folded forms of FGF2 can enter this pathway. Albeit the molecular mechanism remains unclear, the ability of FGF2 to translocate across the plasma membrane has been demonstrated both in vitro using affinity-purified plasma membrane inside-out vesicles and in living cells. Membrane translocation of FGF2 does neither depend on ATP hydrolysis nor on a membrane potential. These findings are in line with direct evidence that FGF2 secretion is driven by an extracellular trapping mechanism mediated by membrane- proximal HSPGs. Thus, HSPGs do not only function as extracellular storage sites and components of FGF2 signalling complexes but also directly participate in FGF2 secretion.

In an attempt to identify putative components of the FGF2 secretory pathway, the present inventors combined siRNA arrays with a quantitative analysis of FGF2 secretion. Thereby, the present inventors achieved to provide a comprehensive analysis of more than 9000 human gene products to unveil factors as putative components of the FGF2 secretion machinery. This approach revealed 123 gene products as strong candidates as potential core or regulatory components of FGF2 secretion. Among those, the Tec protein tyrosine kinase, also known as Tec or Tec kinase, has been identified, the first factor shown to be directly involved in unconventional secretion of FGF2. The Tec kinase belongs to the Tec family of non-receptor protein-tyrosine kinases. The Tec family of kinases comprises in addition to Tec, Itk, Txk, Btk and Bmx kinases. Tec family kinases were known to function in various signalling pathways (Mano, 1999; Zemans and Arndt, 2009). For example, they are known as important mediators of antigen receptor signalling in lymphocytes, shown to regulate activation and development of T cells, B cells and mast cells. (Yang et al., 2000; Berg et al., 2005; Felices et al., 2007). Furthermore, Tec kinases have been implicated in various cellular processes such as cell adhesion and migration as well as in the reorganization of the actin cytoskeleton during the formation of the immunological synapse (Finkelstein and Schwartzberg, 2004; Finkelstein et al., 2005; Gomez-Rodriguez et al., 2007). The inventors of the present invention found a new functional relationship as they provide conclusive evidence for a direct role of the Tec kinase in FGF2 secretion, a novel and unexpected function of the Tec kinase.

Almost all Tec kinases share a common domain structure consisting of the Tec homology domain, a PH domain, SH2 and SH3 domains as well as a kinase domain (Mano, 1999; Yang et al., 2000). While they can be recruited to plasma membrane signalling complexes via protein- protein interactions involving their SH2 and SH3 domains (Yang et al., 2001; Garcon et al., 2004), direct membrane association of Tec kinases involves their PH domains that mediate recruitment by phosphoinositides at the inner leaflet of plasma membranes (Ferguson et al., 1995; McLaughlin et al., 2002; Behnia and Munro, 2005; Di Paolo and De Camilli, 2006; Lemmon, 2008). This is particularly interesting since FGF2 binds to phosphoinositides as well, mediated by a basic cluster of amino acids residing in its C-terminal part (Temmerman et al., 2008). Recruitment by phosphoinositides represents a critical initial step in FGF2 secretion (Nickel and Seedorf, 2008; Temmerman et al., 2008) and has recently been identified as an intrinsic quality control mechanism of the FGF2 export pathway (Cespon-Torrado et al., 2009). The established role of phosphoinositides in both FGF2 plasma membrane translocation (Temmerman et al., 2008; Nickel and Rabouille, 2009) and recruitment of Tec kinases to plasma membranes (Chamorro et al., 2001; Finkelstein et al., 2005; Schwartzberg et al., 2005) also imply a local enrichment of both factors since phosphoinositides have been suggested to reside mainly in specialized membrane microdomains.

The inventors of the present invention could show that RNAi-mediated down-regulation and pharmacological inhibition of the Tec kinase causes a substantial drop in FGF2 export efficiency. The present inventors further demonstrate the formation of a heterodimeric complex between FGF2 and Tec kinase that results in FGF2 phosphorylation.

The findings by the present inventors for the first time establish phosphorylation of FGF2 as an essential posttranslational modification required for FGF2 secretion as well as a novel and essential role of Tec kinase as a central component of the FGF2 export machinery. These findings provide a new perspective for the development of drugs such as anti-angiogenic drugs targeting the interaction between FGF2 and Tec kinase as an essential step in the release of FGF2 from cells.

As already mentioned above, beside the Tec kinase also other gene products as candidates as potential core or regulatory components of FGF2 secretion have been identified. These candidates provide a promising tool for the development of drugs which inhibit the secretion of FGF2 export machinery.

SUMMARY OF THE INVENTION

In a first aspect, the invention provides a method for identifying a compound which reduces/inhibits the cellular export of fibroblast growth factor 2 (FGF2) by inhibiting FGF2 phosphorylation, wherein said method comprises the step of contacting a test compound with Tec kinase and FGF2. In a second aspect, the invention provides a method for identifying a compound which reduces/inhibits the cellular export of fibroblast growth factor 2 (FGF2) by reducing/inhibiting the interaction between Tec kinase and FGF2, wherein said method comprises the step of contacting a test compound with Tec kinase and FGF2.

In a third aspect, the invention provides a method for identifying a compound which reduces/inhibits the cellular export of FGF2 by binding specifically to phosphorylated FGF2, wherein said method comprises the step of contacting a test compound with FGF2 which is phosphorylated at the amino acid residue corresponding to amino acid residue Tyr82 in SEQ ID NO: 1 or which comprises a phosphomimetic amino acid residue at this position.

In a fourth aspect, the invention provides a compound identifiable by the method according to the first, second or third aspect.

In a fifth aspect, the invention provides a pharmaceutical composition comprising the compound according to the fourth aspect and one or more pharmaceutically acceptable excipient(s), diluent(s), and/or carrier(s).

In a sixth aspect, the invention provides a compound according to the fourth aspect or a pharmaceutical composition according to the fifth aspect for treating and/or preventing an angiogenesis- and/or neovascularization-dependent disease or condition.

DETAILED DESCRIPTION OF THE INVENTION

Before the present invention is described in detail below, it is to be understood that this invention is not limited to the particular methodology, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

In the following, the elements of the present invention will be described. These elements are listed with specific embodiments, however, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The variously described examples and preferred embodiments should not be construed to limit the present invention to only the explicitly described embodiments. This description should be understood to support and encompass embodiments which combine the explicitly described embodiments with any number of the disclosed and/or preferred elements. Furthermore, any permutations and combinations of all described elements in this application should be considered disclosed by the description of the present application unless the context indicates otherwise.

Preferably, the terms used herein are defined as described in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", H.G.W. Leuenberger, B. Nagel, and H. Kolbl, Eds., Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995). To practice the present invention, unless otherwise indicated, conventional methods of chemistry, biochemistry, and recombinant DNA techniques are employed which are explained in the literature in the field (cf, e.g., Molecular Cloning: A Laboratory Manual, 2^nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989). Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents, unless the content clearly dictates otherwise. For example, the term "a test compound" also includes "test compounds". As used herein, the term "test compound" refers to an agent comprising a compound, molecule, or complex that is being tested for its ability to reduce/inhibit the cellular export of fibroblast growth factor 2 (FGF2) (i) by inhibiting FGF2 phosphorylation, (ii) by inhibiting the interaction between Tec kinase and FGF2, or (iii) by binding specifically to phosphorylated FGF2. Test compounds can be any agents including, but not restricted to, oligopeptides, peptoids, polypeptides, proteins (including antibodies), lipids, metals, nucleotides, nucleotide analogs, nucleosides, polynucleotides, small organic or inorganic molecules, chemical compounds, saccharides, isotopes, carbohydrates, lipoproteins, glycoproteins, enzymes, polyamines, and combinations thereof. In addition, a test compound according to the present invention may optionally comprise a detectable label. Such labels include, but are not limited to, enzymatic labels, radioisotope or radioactive compounds or elements, fluorescent compounds or metals, chemiluminescent compounds and bioluminescent compounds. Well known methods may be used for attaching such a detectable label to a test compound. The test compound of the invention may also comprise complex mixtures of substances, such as extracts containing natural products, or the products of mixed combinatorial syntheses. These can also be tested and the component that reduces/inhibits the cellular export of fibroblast growth factor 2 (FGF2) (i) by inhibiting FGF2 phosphorylation, (ii) by inhibiting the interaction between Tec kinase and FGF2, or (iii) by binding specifically to phosphorylated FGF2 can be purified from the mixture in a subsequent step.

Test compounds can be derived or selected from libraries of synthetic or natural compounds. For instance, synthetic compound libraries are commercially available from Maybridge Chemical Co. (Trevillet, Cornwall, UK), ChemBridge Corporation (San Diego, CA), or Aldrich (Milwaukee, WI). A natural compound library is, for example, available from TimTec LLC (Newark, DE). Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal cell and tissue extracts can be used. Additionally, test compounds can be synthetically produced using combinatorial chemistry either as individual compounds or as mixtures. A collection of compounds made using combinatorial chemistry is referred to herein as a combinatorial library. In a further embodiment of the invention, the test compound applied in any of the above described methods is a small molecule. In a preferred embodiment, said small molecule is derived from a library, e.g., a small molecule inhibitor library. In another embodiment, said test compound is a peptide or protein. In a further preferred embodiment, said peptide or protein is derived from a peptide or protein library.

The term "small molecules" as used in the context of the present invention refers to molecules that have a molecular weight between 50 and about 2,500 Daltons, preferably in the range of 200-800 Daltons. A small molecule can be of inorganic or organic origin. The term "peptides" (from the Greek πεπτίδια, "small digestibles") means short polymers formed from the linking, in a defined order, of a-amino acids. The link between one amino acid residue and the next is called an amide bond or a peptide bond. The term "oligopeptide" means a peptide composed of between two and twelve amino acids. The term "polypeptide" means a peptide composed of between thirteen and 100 amino acids, preferably a peptide composed of thirteen and 50 amino acids. The term "peptoids" (also known as N-substituted glycines) refers to a specific subclass of peptidomimetics. They are closely related to their natural peptide counterparts, but differ chemically in that their side chains are appended to nitrogen atoms along the molecule's backbone, rather than to the a-carbons (as they are in amino acids).

As used herein, the term "nucleotides" refers to structural components, or building blocks, of DNA and RNA. Nucleotides consist of a base (one of four chemicals: adenine, thymine, guanine, and cytosine) plus a molecule of sugar and one of phosphoric acid. The term "nucleosides" refers to glycosylamine consisting of a nucleobase (often referred to simply base) bound to a ribose or deoxyribose sugar. Examples of nucleosides include cytidine, uridine, adenosine, guanosine, thymidine and inosine. Nucleosides can be phosphorylated by specific kinases in the cell on the sugar's primary alcohol group (-CH2-OH), producing nucleotides, which are the molecular building blocks of DNA and RNA. The term "polynucleotides" means a single or double- stranded polymer of deoxyribonucleotide or ribonucleotide bases and includes DNA and RNA molecules, both sense and anti-sense strands. The term comprises cDNA, genomic DNA, and recombinant DNA. A polynucleotide may consist of an entire gene, or a portion thereof.

As used herein, the term "lipoproteins" refers to a biochemical assembly that contains both proteins and lipids. The lipids or their derivatives may be covalently or non-covalently bound to the proteins. Many enzymes, transporters, structural proteins, antigens, adhesins and toxins are lipoproteins. The term "glycoproteins" means proteins that contain oligosaccharide chains (glycans) covalently attached to their polypeptide side-chains. The carbohydrate is attached to the protein in a cotranslational or posttranslational modification. This process is known as glycosylation.

The term "antibody" as used in the context of the present invention refers to both monoclonal and polyclonal antibodies, i.e. any immunoglobulin protein or portion thereof which is capable of recognizing an antigen within FGF2 or Tec kinase. In a preferred embodiment, the antibody is capable of binding the enzymatically active center within FGF2 or Tec kinase. Antigen-binding portions may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. In some embodiments, antigen-binding portions include Fab, Fab', F(ab')₂, Fd, Fv, dAb, and complementarity determining region (CDR) variants, single chain antibodies (scFv), chimeric antibodies such as humanized antibodies, diabodies, and polypeptides that contain at least a portion of an antibody that is sufficient to confer specific antigen binding to the polypeptide.

Residues in two or more polypeptides are said to "correspond" to each other if the residues occupy an analogous position in the polypeptide structures. It is well known in the art that analogous positions in two or more polypeptides can be determined by aligning the polypeptide sequences based on amino acid sequence or structural similarities. Such alignment tools are well known to the person skilled in the art and can be, for example, obtained on the World Wide Web, e.g., ClustalW (www.ebi.ac.uk/clustalw) or Align

(http://www.ebi.ac.uk/emboss/align/index.html) using standard settings, preferably for Align EMBOSS -needle, Matrix: Blosum62, Gap Open 10.0, Gap Extend 0.5.

In a first aspect, the present invention relates to a method for identifying a compound which reduces/inhibits the cellular export of fibroblast growth factor 2 (FGF2) by inhibiting FGF2 phosphorylation, wherein said method comprises the step of contacting a test compound with Tec kinase and FGF2.

Preferably, the Tec kinase and FGF2 used in the method of the first aspect of the present invention is of human origin.

It is preferred that the ability of the test compound to inhibit the phosphorylation of FGF2 is assessed by comparing FGF2 phosphorylation in presence and absence of the test compound. Preferably, the compound inhibits FGF2 phosphorylation by inhibiting Tec kinase activity, by binding to one or more phosphorylation sites within FGF2, and/or by reducing/inhibiting the interaction between Tec kinase and FGF2.

In a preferred embodiment of the method of the first aspect of the present invention, the test compound binds to one or more phosphorylation sites and due to its binding it directly prevents the Tec kinase to phosphorylate FGF2 at these phosphorylation sites. In a further preferred embodiment, the test compound binds to one or more phosphorylation sites and due to its binding it prevents the Tec kinase from recognizing the other one or more phosphorylation sites not occupied by the test compound and, thus, it prevents the Tec kinase to phosphorylate FGF2 at these phosphorylation sites.

In another preferred embodiment of the method of the first aspect of the present invention, the test compound which inhibits FGF2 phosphorylation by reducing or inhibiting the interaction between the Tec kinase and FGF2 can be a molecule that directly binds to the Tec kinase and/or FGF2, preferably that binds to the recognition motifs by which the Tec kinase normally binds to FGF2. The test compound which inhibits FGF2 phosphorylation by inhibiting Tec kinase activity is, in a preferred embodiment, a molecule that binds to the Tec kinase and that due to its binding, e.g. on the active site of the Tec kinase, it hinders the Tec kinase from catalysing the enzymatic reaction generally leading to the phosphorylation of FGF2. The inhibitory binding of the test compound to the Tec kinase can be either reversible or irreversible. Irreversible inhibitors usually react with the enzyme and change it chemically. These inhibitors modify key amino acid residues needed for enzymatic activity. In contrast, reversible inhibitors bind non-covalently and do not modify key amino acid residues need for enzymatic activity. In addition, different types of inhibition can be produced depending on whether the inhibitory test compound binds the Tec kinase, the Tec kinase/FGF2 complex, or both.

The identified test compound which (i) binds to one or more phosphorylation sites within FGF2, (ii) inhibits the Tec kinase activity and/or (iii) inhibits the interaction between Tec kinase and FGF2 is judged by its specificity (its lack of binding to other proteins) and its potency (its dissociation constant, which indicates the concentration needed (i) to bind, (ii) to inhibit the Tec kinase and/or (iii) to inhibit the interaction of Tec kinase and FGF2). A high specificity and potency ensure that the test compound as drug will be highly effective, have few side effects and thus low toxicity.

The term "a test compound is considered to inhibit FGF2 phosphorylation if the phosphorylation is reduced" is used in the context of the present invention to indicate that a test compound is considered to inhibit FGF2 phosphorylation, if the phosphorylation of FGF2 molecules is reduced, preferably, if the phosphorylation of a given number of FGF2 molecules is reduced, most preferably, if the phosphorylation of 1, 10, 100 or 1000 pmol of FGF2 molecules is reduced.

Preferably, a test compound is considered to inhibit FGF2 phosphorylation if the phosphorylation, e.g. of 1, 10, 100 or 1000 pmol of FGF2 molecules, is reduced by at least 60%, preferably by at least 70%, more preferably by at least 80%, more preferably by at least 90%, i.e. by at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99,9%, or even 100%, in presence of the test compound when compared to the phosphorylation, e.g. of 1, 10, 100, or 1000 pmol of FGF2 molecules, in absence of the test compound. For example, if 0.2 pmol of 1 pmol FGF2 molecules are phosphorylated in the presence of the test compound and if 1 pmol of 1 pmol FGF2 molecules are phosphorylated in the absence of the test compound, the test compound is considered to inhibit FGF2 phosphorylation as the phosphorylation of FGF2 is reduced by 80%.

The skilled person can easily assess experimentally if the phosphorylation is reduced, e.g. by (i) incubating Tec kinase and FGF2 with radioactive labeled ATP in the presence and absence of the test compound, (ii) subjecting the samples to SDS PAGE and analyzing the samples by both direct protein staining using Coomassie and autoradiography using a phosphor imager, and (iii) comparing the levels of FGF2 phosphorylation in samples with and without the test compound.

Preferably, the FGF2, used in the method according to the first aspect of the present invention, comprises, essentially consists of, or consists of an amino acid sequence selected from the group consisting of:

(i) the amino acid sequence set forth in SEQ ID NO: 1 or a functionally equivalent part thereof,

(ii) an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 99,9%, preferably at least 80%, identical to any of the amino acid sequences of (i), and

(iii) an amino acid sequence consisting of at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 amino acid residues corresponding to at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 consecutive amino acid residues of any of the amino acid sequence of (i) or (ii) and comprising at least one of the amino acid residues corresponding to the amino acid residues Tyr82, Tyrl 12, and Tyrl24 of the amino acid sequence set forth in SEQ ID O: 1. In the context of the present invention, the term "functional equivalent part of the amino acid sequence set forth in SEQ ID NO: 1" refers to a portion of FGF2 which comprises or consists of an amino acid sequence corresponding to the amino acid sequence set forth in SEQ ID NO: 1 and comprising at least one of the amino acid residues corresponding to the amino acid residues Tyr82, Tyrl 12, and Tyrl 24 of the amino acid sequence set forth in SEQ ID NO: 1. Such a part of FGF2 can be recognized and/or phosphorylated by the Tec kinase, which is essential for the cellular export of FGF2 and is, thus, functional equivalent to (wild-type) FGF2.

Preferably, the functional equivalent part of FGF2 as used in the methods of the present invention comprises, essentially consists of or consists of at least 60, 61, 65, 70, 75, 80, 85, 90, 95, 105, 110, 115, 120, 125, 130, 135, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154 amino acid residues corresponding to at least 60, 61, 65, 70, 75, 80, 85, 90, 95, 105, 110, 115, 120, 125, 130, 135, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154 consecutive amino acid residues of the amino acid sequence of SEQ ID NO: 1 and comprises at least one of the amino acid residues corresponding to the amino acid residues Tyr82, Tyrl 12, and Tyrl 24 of the amino acid sequence set forth in SEQ ID NO: 1. Such a part of FGF2 can be recognized and/or phosphorylated by the Tec kinase, which is essential for the secretion of FGF2 and is, thus, functional equivalent to (wild-type) FGF2. It is possible that the functional equivalent part of FGF2 (i.e. the functional equivalent FGF2 fragment) may comprise C-terminal/N-terminal or internal deletions or additions, e.g., through N- or C-terminal fusions.

In some preferred embodiments of the methods of the present invention, the functional equivalent part of (wild-type) FGF2 comprises or consists of a portion of the amino acid sequence set forth in SEQ ID NO: 1 phosphorylated at position Tyr82. It is preferred that FGF2 (SEQ ID NO: 1) or a functional equivalent part thereof, which is used in the methods of the present invention, comprising, essentially consisting of or consisting of an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 99,9%, preferably at least 80%, identical to the amino acid sequence set forth in SEQ ID NO: 1 (hereinafter designated as a FGF2 variant) or a functionally equivalent part thereof (hereinafter designated as a functionally equivalent part of FGF2 variant), is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 99,9%, preferably at least 80%, identical to the amino acid sequence set forth in SEQ ID NO: 1 or a functionally equivalent part thereof over a continuous stretch of at least 40, 50, 60, 70, 80, 90, 100, 110, 120, 125, 130, 135, 140, 145, 150, or more amino acids, preferably over the whole length of the amino acid sequence of (wild-type) FGF2 set forth in SEQ ID NO: 1 or a functionally equivalent part thereof.

It is particularly preferred that the FGF2 variant or functionally equivalent part of FGF2 variant, which is used in the methods of the present invention, is at least 80% identical over the whole length, is at least 85% identical over the whole length, is at least 90% identical over the whole length, is at least 95% identical over the whole length, is at least 98% identical over the whole length, or is at least 99% identical over the whole length of the amino acid sequence of (wild- type) FGF2 set forth in SEQ ID NO: 1 or a functionally equivalent part thereof.

It is also particularly preferred that the FGF2 variant or functionally equivalent part of FGF2 variant, which is used in the methods of the present invention, is at least 80% identical over a continuous stretch of at least 40, 50, 60, 70, 80, 90, 100, 110 or 120 amino acids, is at least 85% identical over a continuous stretch of at least 40, 50, 60, 70, 80, 90, 100, 110 or 120 amino acids, is at least 90% identical over a continuous stretch of at least 40, 50, 60, 70, 80, 90, 100, 110 or 120 amino acids, is at least 95% identical over a continuous stretch of at least 40, 50, 60, 70, 80, 90, 100, 110 or 120 amino acids, is at least 98%» identical over a continuous stretch of at least 40, 50, 60, 70, 80, 90, 100, 110 or 120 amino acids, or is at least 99% identical over a continuous stretch of at least 40, 50, 60, 70, 80, 90, 100, 110 or 120 amino acids of the amino acid sequence of (wild-type) FGF2 set forth in SEQ ID NO: 1 or a functionally equivalent part thereof.

Additionally, a FGF2 variant or a functionally equivalent part of FGF2 variant is only regarded as a FGF2 variant or a functionally equivalent part of FGF2 variant within the context of the present invention, if it can still be recognized and/or phosphorylated by the Tec kinase. This is the case if the FGF2 variant or the functionally equivalent part of FGF2 variant still comprises or consists of an amino acid sequence comprising at least one of the amino acid residues corresponding to the amino acid residues Tyr82, Tyrl 12, and Tyrl 24 of the amino acid sequence set forth in SEQ ID NO: 1. As already mentioned above, the skilled person can readily assess whether a FGF2 variant or a functionally equivalent part of FGF2 variant is still recognized and/or phosphorylated by the Tec kinase. Suitable assays to determine whether phosphorylation and/or recognition still occurs are well known in the art. However, as an example, a suitable assay to determine whether phosphorylation still occurs comprises the steps of (i) incubating the Tec kinase and the FGF2 variant or a functionally equivalent part of FGF2 variant with radioactive labeled ATP in a first sample and incubating the Tec kinase and (wild-type) FGF2 or a functional equivalent part of (wild-type) FGF2 with radioactive labeled ATP as a positive control in a second sample, (ii) subjecting the samples to SDS PAGE and analyzing the samples by both direct protein staining using Coomassie staining and autoradiography using a phosphor imager, and (iii) comparing the levels of FGF2 phosphorylation in both samples. Co- immunoprecipitation assays can be used to test, whether the FGF2 variant or functionally equivalent part of FGF2 variant can still be recognized by the Tec kinase.

It should be noted that the inventors of the present invention found that replacement of tyrosine 82 by glutamate resulted in a rescue of FGF2 secretion. Thus, a FGF2 variant or functionally equivalent part of FGF2 variant comprising such an amino acid substitution can be regarded as a FGF2 variant or functionally equivalent part of FGF2 variant within the context of the present invention even if the amino acid residues Tyrl 12 and Tyrl 24 are additionally replaced by other amino acids. In some embodiments of the methods of the present invention, the FGF2 variant or a functionally equivalent part of FGF2 variant comprises or consists of an amino acid sequence which is phosphorylated at the amino acid position corresponding to amino acid residue Tyr82 of the amino acid sequence set forth in SEQ ID NO: 1 or comprises a glutamate or aspartate residue at this position, preferably a glutamate residue to mimic phosphorylated tyrosine.

Preferably, the compound binds to at least one of the amino acid residues within FGF2 corresponding to amino acid residues Tyr82, Tyrl 12, and Tyrl24 of the amino acid sequence set forth in SEQ ID NO: 1. In a preferred embodiment of the method of the first aspect of the present invention, the compound binds to the amino acid residue within FGF2 which corresponds to the amino acid residue Tyr82 and, thus, directly inhibits phosphorylation by the Tec kinase at this phosphorylation site. In another preferred embodiment of the method of the first aspect of the present invention, the compound binds to the amino acid residues within FGF2 which correspond to the amino acids Tyrl l2 and Tyrl24 of the amino acid sequence set forth in SEQ ID NO: 1 and, thus, prevents the Tec kinase from binding to the amino acid residue within FGF2 which corresponds to the amino acid Tyr82 which is not occupied by the compound. As a consequence, the compound prevents/inhibits phosphorylation of FGF2 by the Tec kinase at the phosphorylation site Tyr82.

Preferably, the method according to the first aspect of the present invention is performed using an in vitro assay or a cell-based (ex vivo) assay or a combination thereof. It is preferred that the method of the first aspect of the present invention comprises incubating Tec kinase and FGF2 with labeled ATP in presence or absence of the test compound and comparing the levels of FGF2 phosphorylation in reactions with and without test compound. Preferably, the labeled ATP is radioactively labeled ATP, preferably gamma-32P-ATP, or fluorescently labeled ATP, preferably pyrene-labeled ATP.

To determine whether a compound inhibits FGF2 phosphorylation by binding to one or more phosphorylation sites within FGF2, (i) Tec kinase and FGF2 with radioactively labeled ATP, preferably gamma-32P-ATP, or with fluorescently labeled ATP, preferably pyrene-labeled ATP, may be incubated in presence or absence of the test compound, (ii) the specific binding of the test compound to FGF2 may be verified using co-immunoprecipitation experiments, (iii) the samples may be subjected to SDS PAGE and analyzed by direct protein staining using a Coomassie staining, Western blotting and autoradiography using a phosphor imager or spectrofluorometry using a spectrofluorometer for quantitation of FGF2 phosphorylation, and (iv) the levels of FGF2 phosphorylation in samples with and without the test compound may be compared.

Mass spectrometry studies can also be conducted to directly address whether a compound inhibits FGF2 phosphorylation (see examples provided herein for details). To determine whether a compound inhibits FGF2 phosphorylation by reducing or inhibiting the interaction between Tec kinase and FGF2, a pull down assay, preferably a Glutathione S Transferase (GST) pull down assay, in combination with Western blotting and autoradiography or spectrofluorometry may be conducted. For example, (i) the FGF2 may be purified and may be immobilized on beads, preferably glutathione beads, (ii) the FGF2 immobilized on beads, preferably glutathione beads, may be contacted with radioactively labeled ATP, preferably gamma-32P-ATP, or with fluorescently labeled ATP, preferably pyrene-labeled ATP, and with Tec kinase, e.g. purified Tec kinase or with a cell or tissue extract comprising Tec kinase, in the presence or absence of a test compound, (iii) binding of Tec kinase to FGF2 immobilized on beads and the grade of FGF2 phosphorylation in the presence or absence of a test compound may be verified by polyacrylamide gel electrophoresis in combination with Coomassie staining, by Western blotting and by autoradiography using a phosphor imager or by spectrofluorometry using a spectrofluorometer for quantitation, and (iv) the levels of FGF2 phosphorylation in samples with and without the test compound may be compared (see also experiments provided herein).

Alternatively, phosphorylation within FGF2 may be detected using an anti-phospho-tyrosine monoclonal antibody. A Phos-tag can also be used to detect phosphorylated FGF2 (Junya Tomida, Hiroyuki Kitao, Eiji Kinoshita and Minora Takata, "Detection of phosphorylation on large proteins by western blotting using Phos-tag containing gel", Nature protocols). A Phos-tag is a dinuclear metal complex that acts as a phosphate-binding tag. Phos-tag molecules preferentially capture phosphomonoester dianions bound to Tyr residues. Phosphorylated FGF2 can be detected as slower migrating species by electrophoresis and Western blotting using a SDS PAGE gel containing Phos-tag acrylamide.

To determine whether a compound inhibits FGF2 phosphorylation by inhibiting Tec kinase activity, (i) Tec kinase and FGF2 with radioactively labeled ATP, such as gamma-32P-ATP, may be incubated in the presence and absence of a test compound, (ii) in both samples the incorporation of radioactive phosphate residues, e.g. 32P, into FGF2 may be measured over time using a scintillation counter, and (iii) the levels of FGF2 phosphorylation in the samples with and without the test compound may be compared.

Alternatively, the Tec kinase activity in the absence or presence of a test compound may be detected using synthetic peptides as substrates. Peptide substrates typically contain one tyrosine in a phosphorylation site motif. They should have a net positive charge to facilitate binding to phosphocellulose paper at low pH to separate the phosphorylated peptides from other assay components. The assay may be performed in the presence of radioactive labeled ATP, preferably gamma-32P-ATP. The phosphocellulose paper may be placed in a scintillation counter to measure 32P incorporation.

The FGF2 used in the assays described above may comprise the amino acid sequence set forth in SEQ ID NO: 1 (wild-type FGF2), or a functionally equivalent part thereof. The FGF2 used can also be a FGF2 variant or a functionally equivalent part of FGF2 variant. The Tec kinase used in the assays described above may comprise, for example, the amino acid sequence set forth in SEQ ID NO: 2 (wild-type Tec kinase), or a functionally equivalent part thereof. The Tec kinase used can also be a Tec kinase variant, or a functionally equivalent part of Tec kinase variant.

Preferably, the compound exhibits the capability to inhibit FGF2 phosphorylation of at least one of the amino acid residues within FGF2 corresponding to amino acid residues Tyr82, Tyrl l2, and Tyrl24 of the amino acid sequence set forth in SEQ ID NO: 1, preferably of the amino acid residue within FGF2 corresponding to the amino acid residue Tyr82 or Tyrl24 of the amino acid sequence set forth in SEQ ID NO: 1. Most preferably, of the amino acid residue within FGF2 corresponding to the amino acid residue Tyr82 of the amino acid sequence set forth in SEQ ID NO: 1.

In a second aspect, the present invention provides a method for identifying a compound which reduces/inhibits the cellular export of fibroblast growth factor 2 (FGF2) by reducing/inhibiting the interaction between Tec kinase and FGF2, wherein said method comprises the step of contacting a test compound with Tec kinase and FGF2.

It is preferred that the Tec kinase and FGF2 used in the method of the second aspect of the present invention is of human origin.

Preferably, the ability of the test compound to reduce/inhibit the interaction between Tec kinase and FGF2 is assessed by comparing the interaction between Tec kinase and FGF2 in presence and absence of the test compound.

It is preferred that the test compound is added after Tec kinase and FGF2 have been incubated to interact with each other, or wherein Tec kinase, FGF2, and the test compound are added concomitantly to the test reaction, or wherein one of Tec kinase and FGF2 are added first, then the test compound is added, and then the other of Tec kinase and FGF2 is added.

The term "a test compound is considered to inhibit the interaction between Tec kinase and FGF2 if the interaction is reduced" is used in the context of the present invention to indicate that a test compound is considered to inhibits the interaction between Tec kinase and FGF2 if the interaction of Tec kinase molecules and FGF2 molecules is reduced, preferably, if the interaction of a given number of FGF2 molecules and Tec kinase molecules is reduced, most preferably, if the interaction of 1, 10, 100 or 1000 pmol of FGF2 molecules and Tec kinase molecules is reduced.

Preferably, a test compound is considered to inhibit the interaction between Tec kinase and FGF2 if the interaction, e.g. of 1, 10, 100 or 1000 pmol of FGF2 molecules and Tec molecules, is reduced by at least 60%, preferably by at least 70%, more preferably by at least 80%, more preferably by at least 90%, i.e. by at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99,9%, or even 100%, in presence of the test compound when compared to the interaction in absence of the test compound. For example if 0.2 pmol of 1 pmol FGF2 molecules and Tec kinase molecules interact in the presence of the test compound and if 1 pmol of 1 pmol FGF2 molecules and Tec kinase molecules interact in the absence of the test compound, the test compound is considered to inhibit the interaction between Tec kinase and FGF2 as the interaction is reduced by 80%.

The skilled person can easily assess experimentally if the interaction between FGF2 and Tec kinase is reduced, for example, by performing a pull down assay, e.g. a GST pull down assay, or a co-immunoprecipitation assay.

Preferably, the FGF2, used in the method according to the second aspect of the present invention, comprises, essentially consists of, or consists of an amino acid sequence selected from the group consisting of:

(ii) an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 99,9%, preferably at least 80%, identical to any of the amino acid sequences of (i),

(iii) an amino acid sequence corresponding to any of the amino acid sequences of (i) or (ii) which comprises the amino acid residue corresponding to amino acid residue Tyr82 of the amino acid sequence set forth in SEQ ID NO: 1 and is phosphorylated at said amino acid residue,

(iv) an amino acid sequence corresponding to any of the amino acid sequences of (i) or (ii), in which the amino acid residue corresponding to the amino acid residue Tyr82 of the amino acid sequence set forth in SEQ ID NO: 1 is a glutamate or aspartate residue, or a functionally equivalent part thereof comprising said glutamate or aspartate residue, and

(v) an amino acid sequence consisting of at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 amino acid residues corresponding to at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 consecutive amino acid residues of any of the amino acid sequences of (i), (ii), (iii), or (iv) and comprising at least one of the amino acid residues corresponding to amino acid residues Tyr82, Tyrl l2, and Tyrl24 of the amino acid sequence set forth in SEQ ID NO: 1.

It is preferred that the compound binds to FGF2, preferably to at least one of the amino acid residues corresponding to amino acid residues Tyr82, Tyrl l2, and Tyrl24 of the amino acid sequence set forth in SEQ ID NO: 1. It is particularly preferred that the compound binds to the amino acid residues corresponding to amino acid residues Tyrl 12 and Tyrl24 of the amino acid sequence set forth in SEQ ID NO: 1. Preferably, the Tec kinase, used in the methods of the present invention, comprises, essentially consists of, or consists of an amino acid sequence selected from the group consisting of:

(i) the amino acid sequence set forth in SEQ ID NO: 2 or a functionally equivalent part thereof, and

(ii) an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 99,9%, preferably at least 80%, identical to any of the amino acid sequences of (i).

In the context of the present invention, the term "functional equivalent part of the amino acid sequence set forth in SEQ ID NO: 2" refers to a portion of Tec kinase which comprises or consists of an amino acid sequence corresponding to the amino acid sequence of (wild-type) Tec kinase set forth in SEQ ID NO: 2 and still possesses the enzymatic activity to phosphorylate FGF2 and/or the ability to recognize FGF2.

Preferably, the functional equivalent part of Tec kinase as used in the methods of the present invention comprises or consists of at least 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 600, 605, 610, 615, 620, 625, or 628 amino acid residues corresponding to at least 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 600, 605, 610, 615, 620, 625, or 628 consecutive amino acid residues of the amino acid sequence of SEQ ID NO: 2 and still possesses the enzymatic activity to phosphorylate FGF2, preferably on at least one of the tyrosine residues at position 82, 112 and 124, and/or the ability to recognize FGF2. It is possible that the functional equivalent part of Tec kinase (i.e. the functional equivalent Tec kinase fragment) may comprise C-terminal N-terminal or internal deletions or additions, e.g., through N- or C-terminal fusions.

It is preferred that the Tec kinase (SEQ ID NO: 2) or a functionally equivalent thereof, which is used in the methods of the present invention, comprising, essentially consisting of or consisting of an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 99,9% identical to the amino acid sequence set forth in SEQ ID NO: 2 (hereinafter designated as a Tec kinase variant) or a functionally equivalent part thereof (hereinafter designated as a functionally equivalent part of Tec kinase variant),, is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 99,9% identical to the amino acid sequence set forth in SEQ ID NO: 2 or a functionally equivalent part thereof over a continuous stretch of at least 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 600, 605, 610, 615, 620, 625, or 628, or more amino acids, preferably over the whole length of the amino acid sequence of (wild-type) Tec kinase set forth in SEQ ID NO: 2 or a functionally equivalent part thereof. It is particularly preferred that the Tec kinase variant or functionally equivalent part of Tec kinase variant, which is used in the methods of the present invention, is at least 80% identical over the whole length, is at least 85% identical over the whole length, is at least 90% identical over the whole length, is at least 95% identical over the whole length, is at least 98% identical over the whole length, or is at least 99% identical over the whole length of the amino acid sequence of (wild-type) Tec kinase set forth in SEQ ID NO: 2 or a functionally equivalent part thereof.

It is also particularly preferred that the Tec kinase variant or functionally equivalent part of Tec kinase variant, which is used in the methods of the present invention, is at least 80% identical over a continuous stretch of at least 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 520, 540, 560, 580, or 600 amino acids, is at least 85% identical over a continuous stretch of at least 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 520, 540, 560, 580, or 600 amino acids, is at least 90% identical over a continuous stretch of at least 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 520, 540, 560, 580, or 600 amino acids, is at least 95% identical over a continuous stretch of at least 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 520, 540, 560, 580, or 600 amino acids, is at least 98% identical over a continuous stretch of at least 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 520, 540, 560, 580, or 600 amino acids, or is at least 99% identical over a continuous stretch of at least 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 520, 540, 560, 580, or 600 amino acids of the amino acid sequence of (wild-type) Tec kinase set forth in SEQ ID NO: 2 or a functionally equivalent part thereof.

Additionally, a Tec kinase variant or a functionally equivalent part of Tec kinase variant is only regarded as a Tec kinase variant or a functionally equivalent part of Tec kinase variant within the context of the present invention, if it is functionally equivalent to the protein consisting of the amino acid sequence of (wild-type) Tec kinase set forth in SEQ ID NO: 2 or a part thereof. The relevant "biological activity" in the context of the present invention is the "enzyme activity", i.e. the activity of the Tec kinase variant or functionally equivalent part of Tec kinase variant to phosphorylate and/or recognize FGF2. The skilled person can readily assess whether a Tec kinase variant or a functionally equivalent part of Tec kinase variant has still the ability to recognize and/or phosphorylate FGF2, preferably on at least one of the tyrosine residues at positions Tyr82, Tyrl l2 and Tyrl24. Suitable assays to determine whether phosphorylation and/or recognition still occurs are well known in the art. However, as an example, a suitable assay to determine whether phosphorylation still occurs comprises the steps of (i) incubating the Tec kinase variant or a functionally equivalent part of Tec kinase variant and FGF2 with radioactive labeled ATP in a first reaction batch and incubating (wild-type) Tec kinase or a functional equivalent part of (wild-type) Tec kinase and FGF2 with radioactive labeled ATP as a positive control in a second reaction batch, (ii) subjecting the reaction batches to SDS PAGE and analyzing the samples by both direct protein staining using Coomassie and autoradiography using a phosphor imager, and (iii) comparing the levels of FGF2 phosphorylation in both samples. Co-immunoprecipitation assays can be used to test, whether the Tec kinase variant or functionally equivalent part of Tec kinase variant still recognizes FGF2.

Preferably, the method according to the second aspect of the present invention is performed using an in vitro assay or a cell-based (ex vivo) assay or a combination thereof.

In one embodiment, the inhibition or reduction of the interaction between Tec kinase and FGF2 by a test compound may be analyzed in form of a pull down assay, preferably Glutathione S Transferase (GST) pull down assay. For example, (i) the FGF2 may be purified and may be immobilized on beads, preferably glutathione beads, (ii) the FGF2 immobilized on beads may be contacted, for example, with purified Tec kinase or with a cell or tissue extract comprising Tec kinase in the presence or absence of a test compound, (iii) binding of Tec kinase to FGF2 immobilized on beads, preferably glutathione beads, may be verified by polyacrylamide gel electrophoresis in combination with Coomassie staining and by Western blotting, and (iv) the levels of interaction between FGF2 and Tec kinase in samples with and without the test compound may be compared. To quantify the interaction between GST-FGF2 and Tec kinase in samples with and without the test compound, the Tec kinase can be labeled with a radioisotope, e.g. 1-125, or with a fluorescent tag, e.g. GFP or EGFP, and a phosphor imager or a spectrofluorometer can be used for quantitation.

In another embodiment, the inhibition or reduction of the interaction between Tec kinase and FGF2 by a test compound may be analyzed in form of an enzyme-linked immunosorbent assay (ELISA)-based experiment. In one embodiment, FGF2 may be immobilized on the surface of an ELISA plate and contacted (i) with the Tec kinase and the test compound, or (ii) with only the Tec kinase. Binding of Tec kinase may be verified, for example, by antibodies specific for Tec kinase. These antibodies might be directly coupled to an enzyme or detected with a secondary antibody coupled to said enzyme that - in combination with the appropriate substrates - carries out chemiluminescent reactions (e.g., horseradish peroxidase) or colorimetric reactions (e.g., alkaline phosphatase). In another embodiment, binding of Tec kinase in the presence or absence of the test compound might be verified by labels directly coupled to the Tec kinase. Such labels may include enzymatic labels, radioisotope or radioactive compounds or elements, fluorescent compounds or metals, chemiluminescent compounds and bioluminescent compounds. In another embodiment, the Tec kinase might be immobilized on the ELISA plate and contacted (i) with FGF2, labeled FGF2, or tagged FGF2 and the test compound, or (ii) with only FGF2, labeled FGF2, or tagged FGF2. Binding of FGF2 in the presence or absence of the test compound may be verified by a FGF2 specific antibody, a FGF2 tag specific antibody, chemiluminescence or colorimetric reactions as described above. In all embodiments described above, the levels of interaction between FGF2 and Tec kinase in samples with and without the test compound may be compared. A fluorescent compound, for example, emits a signal which may be detected, for example, by an ELISA reader for quantification of the interaction between FGF2 and Tec kinase in the presence or absence of a test compound.

Another assay to assess whether a compound inhibits or reduces the interaction between Tec kinase and FGF2 is the Fluorescence correlation spectroscopy (FCS).

The term "recombinant host cell", as used herein, refers to a host cell that comprises (i) a polynucleotide that codes for FGF2 and a polynucleotide that codes for Tec kinase, (ii) a polynucleotide that codes for FGF2, or (iii) a polynucleotide that encodes for Tec kinase. The recombinant host cell can further comprise a polynucleotide that codes for a test compound. Thus, in preferred embodiments of the invention, the recombinant host cell comprises polynucleotides encoding FGF2, Tec kinase and a test compound. In other preferred embodiments of the invention, the recombinant host cell comprises polynucleotides encoding FGF2 and a test compound. In further preferred embodiments of the invention, the recombinant host cell comprises polynucleotides encoding Tec kinase and a test compound. Said polynucleotides may be found inside the recombinant host cell (i) freely dispersed as such, (ii) incorporated in a recombinant vector or in two or three recombinant vectors, respectively, or (iii) integrated into the host cell genome or mitochondrial DNA. The recombinant host cell can be used for expression of said polynucleotides or recombinant vector(s) (i.e. recombinant expression vectors) comprising said polynucleotides, or for amplification of said polynucleotides or recombinant vector(s) (i.e. recombinant cloning vectors). The term "recombinant host cell" includes the progeny of the original cell which has been transformed, transfected, or infected with said polynucleotides or with the recombinant vector(s) comprising said polynucleotides. A recombinant host cell may a bacterial cell such as an E. coli cell, a yeast cell, a plant cell, an insect cell, or a vertebrate cell, preferably a mammalian cell. Preferred examples of mammalian cells are Chinese hamster ovary (CHO) cells, green African monkey kidney (COS) cells, human embryonic kidney (HEK293) cells, HeLa cells, and the like.

The term "recombinant vector" as used herein includes any vectors known to the skilled person including plasmid vectors, cosmid vectors, phage vectors such as lambda phage, viral vectors such as adenoviral or baculoviral vectors, or artificial chromosome vectors such as bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC), or PI artificial chromosomes (PAC). Said vectors include expression as well as cloning vectors. Expression vectors comprise plasmids as well as viral vectors and generally contain a desired coding sequence and appropriate DNA sequences necessary for the expression of the operably linked coding sequence in a particular host organism (e.g., bacteria, yeast, plant, insect, or mammal) or in in vitro expression systems. Cloning vectors are generally used to engineer and amplify a certain desired DNA fragment and may lack functional sequences needed for expression of the desired DNA fragments. In another embodiment, the inhibitory effect of the compound on the interaction between FGF2 and Tec kinase may be tested in an in vivo setting. For example, the recombinant host cell may be contacted with a test compound. This may be achieved by co-expression of test proteins or polypeptides and verification of reduced/inhibited interaction between FGF2 and Tec kinase in the presence of the test compound, for example, by fluorescence resonance energy transfer (FRET) or co-immunoprecipitation. A recombinant host cell not contacted with a test compound may be used as positive control. In another embodiment, directly labeled test compounds may be added to the medium of the recombinant host cells expressing Tec kinase and FGF2. The potential of the test compound to penetrate membranes and to bind to FGF2, Tec kinase or to both proteins of the FGF2 and Tec kinase complex may be, for example, verified by immunoprecipitation and verification of the presence of the label.

Another cell based assays to assess whether a compound inhibits or reduces the interaction between Tec kinase and FGF2 is the assay of crosslinking of protein complexes. The assay of crosslinking of protein complexes using photo-reactive amino acid analogs is described in detail in Suchanek, M., Radzikowska, A., and Thiele, C. (2005) "Photo-leucine and photo-methionine allow identification of protein-protein interactions in living cells", Nature Methods 2: 261-268. In this method, the recombinant host cell is grown with photoreactive diazirine analogs to leucine and methionine, which are incorporated into proteins. Upon exposure to ultraviolet light, the diazirines are activated and bind to interacting proteins that are within a few angstroms of the photo-reactive amino acid analog.

The FGF2 used in the assays described above may comprise, for example, the amino acid sequence set forth in SEQ ID NO: 1 (wild-type FGF2), or a functionally equivalent part thereof, an amino acid sequence corresponding to the amino acid sequence set forth in SEQ ID NO: 1 which comprises the amino acid residue corresponding to amino acid residue Tyr82 of the amino acid sequence set forth in SEQ ID NO: 1 and is phosphorylated at said amino acid residue (wild- type FGF2 Tyr82P), or an amino acid sequence corresponding to the amino acid sequence set forth in SEQ ID NO: 1 in which the amino acid residue corresponding to amino acid residue Tyr82 of the amino acid sequence set forth in SEQ ID NO: 1 is a glutamate or aspartate residue (wild-type FGF2 Tyr82E). The FGF2 used can also be a FGF2 variant or a functionally equivalent part of FGF2 variant, a FGF2 Tyr82P variant, or a FGF2 Tyr82E variant. The Tec kinase used in the assays described above may comprise, for example, the amino acid sequence set forth in SEQ ID NO: 2 (wild-type Tec kinase), or a functionally equivalent part thereof. The Tec kinase used can also be a Tec kinase variant, or a functionally equivalent part of Tec kinase variant.

In a third aspect, the present invention refers to a method for identifying a compound which reduces/inhibits the cellular export of FGF2 by binding specifically to phosphorylated FGF2, wherein said method comprises the step of contacting a test compound with FGF2 which is phosphorylated at the amino acid residue corresponding to amino acid residue Tyr82 in SEQ ID NO: 1 or which comprises a phosphomimetic amino acid residue, e.g. a glutamate or an aspartate residue, at this position.

Preferably, the FGF2 used in the method of the third aspect of the present invention is of human origin.

It is preferred that the ability of the test compound to specifically bind to phosphorylated FGF2 is assessed by determining the ability of the test compound to bind to FGF2 which is phosphorylated at the amino acid residue corresponding to amino acid residue Tyr82 in SEQ ID NO: 1 or which comprises a phosphomimetic amino acid residue at this position compared to its ability to bind to FGF2 without phosphorylation or a phosphomimetic amino acid residue at this position. A suitable assay to detect whether a test compound specifically binds to phosphorylated FGF2 may be, for example, a co-immunoprecipitation assay. Using co-immunoprecipitation, FGF2 may be isolated with a FGF2 specific antibody, the phosphorylation of the isolated FGF2 may be verified using an anti-phospho-tyrosine monoclonal antibody. The test compound which may be stick to this protein can subsequently be identified by Western blotting. For comparison, a reaction batch comprising non-phosphorylated FGF2 and the compound may be used.

Preferably, the FGF2 which is contacted in the method according to the third aspect of the present invention with the test compound comprises, essentially consists of, or consists of an amino acid sequence selected from the group consisting of:

(i) the amino acid sequence set forth in SEQ ID NO: 1 phosphorylated at position Tyr82 or a functionally equivalent part thereof comprising the phosphorylated residue Tyr82,

(ii) the amino acid sequence set forth in SEQ ID NO: 1, in which the amino acid residue Tyr82 is a glutamate or aspartate residue, or a functionally equivalent part thereof comprising said glutamate or aspartate residue,

(iii) an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 99,9%, preferably at least 80%, identical to any of the amino acid sequences of (i) or (ii) and is phosphorylated at the amino acid position corresponding to amino acid residue Tyr82 of the amino acid sequence set forth in SEQ ED NO: 1 or comprises a glutamate or aspartate residue at this position, and

(iv) an amino acid sequence consisting of at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 amino acid residues corresponding to at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 consecutive amino acid residues of any of the amino acid sequences of (i), (ii), or (iii) and comprising the amino acid residue corresponding to the amino acid residue Tyr82 of the amino acid sequence set forth in SEQ ID NO: 1. It is particularly preferred that the methods according to the first, second and third aspect of the present invention further comprise the step of testing whether the test compound is capable of penetrating cell membranes, preferably mammalian cell membranes.

The potential of the test compound to penetrate mammalian cell membranes may be, for example, tested by (i) directly coupling a test compound to a label, e.g. a radioisotope or radioactive compound, a fluorescent compound, a chemiluminescent compound, or a bioluminescent compound, (ii) adding the directly labeled test compound to the medium of a cell, preferably mammalian cell, such as HeLa cell, and (iii) verifying of the presence of the labeled test compound within the cell, e.g. using fluorescence microscopy.

It is also particularly preferred that the methods according to the first, second and third aspect of the present invention further comprise the step of testing whether the test compound is capable of reducing/inhibiting cellular export of FGF2. The term "the cellular export of FGF2 is considered inhibited if the export is reduced" is used in the context of the present invention to indicate that the cellular export of FGF2 is considered inhibited if the export of FGF2 molecules is reduced, preferably, if the export of a given number of FGF2 molecules is reduced, most preferably, if the export of 1, 10, 100 or 1000 pmol of FGF2 molecules is reduced.

Preferably, the cellular export of FGF2 is considered inhibited if the export, e.g. of 1, 10, 100 or 1000 pmol of FGF2 molecules, is reduced by at least 60%, preferably by at least 70%, more preferably by at least 80%, more preferably by at least 90%, i.e. by at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99,9%, or even 100%, in presence of the test compound when compared to the cellular export in absence of the test compound. For example, if 0.2 pmol of 1 pmol FGF2 molecules are exported in the presence of the test compound and if 1 pmol of 1 pmol FGF2 molecules are exported in the absence of the test compound, the cellular export of FGF2 is considered inhibited as the export is reduced by 80%.

It is preferred that the testing for cellular export is performed using a cell selected from the group consisting of a cell that endogenously expresses Tec kinase and/or FGF2 or a cell that is exogenously provided with nucleic acid sequences that encode Tec kinase and/or FGF2. The term "a cell that endogenously expresses Tec kinase and/or FGF2" means that Tec kinase and/or FGF2 are expressed from the genome of said cell. The term "that the cell is exogenously provided with nucleic acid sequences that encode Tec kinase and/or FGF2" means that the nucleic acid sequences that encode Tec kinase and/or FGF2 are introduced into the respective cell, e.g. via transfection, lipofection or electroporation. Preferably, the compound is capable of reducing/inhibiting cellular export of FGF2 from a cell selected from the group consisting of a tumor cell, an inflammatory cell (leukocyte), a stromal cell, a keratinocyte, a fibroblast, and an endothelial cell. It is preferred that the methods of the first, second and third aspect of the present invention are performed in a high-throughput setting. The term "in a high-throughput setting" refers to high- throughput screening assays and techniques of various types which are used to screen libraries of test compounds for their ability, e.g. to inhibit phosphorylation of FGF2, or to inhibit the interaction between Tec kinase and FGF2. Typically, the high-throughput assays are performed in a multi-well format and include cell-free as well as cell-based assays.

It is further preferred that the methods of the first, second and third aspect of the present invention are performed using a test compound which is selected from the group consisting of a small molecule, a peptide, a protein, and an antibody. In preferred embodiments, the test compound applied is a small molecule. Preferably, said small molecule is derived from a library, e.g., a small molecule inhibitor library. In other preferred embodiments, the text compound applied is a peptide or protein. Preferably, said peptide or protein is derived from a peptide or protein library. Preferably, the test compound is encoded by a nucleic acid. It is preferred that the nucleic acid is comprised in a vector, preferably a viral vector or a plasmid vector. The vector preferably comprises expression control sequences. Such a vector is named expression vector. The term "expression control sequences" refers to nucleotide sequences which affect the expression of coding sequences to which they are operably linked in cells (e.g. mammalian cells). Expression control sequences are sequences which control the expression, e.g. promoters, TATA-box, enhancers; post-transcriptional events, e.g. polyadenylation and translation of nucleic acid sequences.

Exported FGF2 is preferably determined by a FGF2 cell surface assay (Zehe et al., 2006, PNAS, 103, 15479-15484) or by measuring the amount of secreted FGF2 in the cell medium, preferably after treatment with heparin or heparan sulfates.

The potential of a test compound to reduce or inhibit the cellular export of FGF2 may be, for example, tested by (i) providing a recombinant host cell expressing FGF2, preferably as a GFP fusion (i.e. FGF2-GFP), and Tec kinase, (ii) introducing a recombinant vector comprising a polynucleotide encoding the test compound, preferably via transfection, into said cell (iii) cultivating said cell for a certain period of time, e.g. for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 cell divisions, or for 6, 12, 16, 20, 24, 48, or 72 hours in a medium containing no FGF-2, (iv) removing the medium after a certain period of time, e.g. after 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, or 12 cell divisions, or after 12, 16, 20, 24, 48, 72 or 80 hours, preferably after treatment with heparin or heparan sulfates, and quantifying the amount of (phosphorylated) FGF-2, preferably of (phosphorylated) FGF2-GFP, secreted in the medium using standard detection methods, e.g. standard ELISA detection methods, preferably using GFP fluorescence as read-out., and (v) comparing the amount of secreted (phosphorylated) FGF-2, preferably FGF2-GFP, with the amount of secreted (phosphorylated) FGF2, preferably FGF2-GFP, from a recombinant host cell not expressing the test compound.

The potential of a test compound to reduce or inhibit the cellular export of FGF2 may also be, for example, tested by (i) providing a recombinant host cell expressing FGF2 as a GFP fusion (i.e. FGF2-GFP) and Tec kinase, (ii) introducing a recombinant vector comprising a polynucleotide encoding the test compound, preferably via transfection, into said cell (iii) cultivating said cell for a certain period of time, e.g. for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 cell divisions, or for 6, 12, 16, 20, 24, 48, or 72 hours in a medium containing no FGF-2, (iv) verifying (phosphorylated) FGF2- GFP transported to the cell surface using GFP fluorescence and/or cell surface staining using anti-GFP antibodies and determining the amount of (phosphorylated) FGF2-GFP transported to the cell surface by flow cytometry, and (v) comparing the result with the result of a recombinant host cell not expressing the test compound.

In preferred embodiments of the methods of the present invention, it is (i) tested in an in vitro assay, whether the compound is capable of inhibiting FGF2 phosphorylation or the interaction between FGF2 and Tec kinase and (ii) tested in an cellular-based {ex vivo) assay, whether the compound is able to reduce or inhibit the cellular export of FGF2.

Alternatively, is also possible to evaluate, (i) whether a compound is capable of inhibiting FGF2 phosphorylation or the interaction between FGF2 and Tec kinase, and (ii) whether the compound is able to reduce or inhibit the cellular export of FGF2 exclusively in a cellular-based {ex vivo) assay.

The potential of a test compound to inhibit the interaction between FGF2 and Tec kinase and to inhibit the cellular export of FGF2 may be, for example, tested by (i) directly coupling a test compound to a label, e.g. a fluorescent compound, a chemiluminescent compound, or a bioluminescent compound, (ii) adding the directly labeled test compound to the medium of a recombinant host cell expressing FGF2 and Tec kinase, both preferably tagged with an epitope which can be recognized by an antibody and/or labeled with a fluorescent compound, (iii) cultivating said cell for a certain period of time, e.g. for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 cell divisions, or for 6, 12, 16, 20, 24, 48, or 72 in a medium containing no FGF-2, (iv) verifying the presence of the labeled test compound within the cell and verifying the binding of the labeled test compound to FGF2, Tec kinase or to both proteins of the FGF2 and Tec kinase complex, e.g. using fluorescence microscopy, and (v) removing the medium after a certain period of time, e.g. after 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 cell divisions, or after 12, 16, 20, 24, 48, 72 or 80 hours, preferably after treatment with heparin or heparan sulfates, and (vi) quantifying the amount of (phosphorylated) FGF-2 secreted in the medium using standard ELISA detection method. The amount of secreted (phosphorylated) FGF-2 may finally be compared with the amount of secreted (phosphorylated) FGF2 from a recombinant host cell which has not been treated with the test compound.

In all in vitro assays described above, the samples (e.g. a sample comprising wild-type FGF2, wild-type Tec kinase and a test compound) and control samples (e.g. a sample only comprising wild-type FGF2 and wild-type Tec kinase) are comparably treated and the same reaction conditions are chosen. In all cell-based (ex vivo) assays described above, the cells (e.g. cells expressing wild-type FGF2, wild-type Tec kinase and a test compound) and control cells (e.g. cells only expressing wild-type FGF2 and wild-type Tec kinase) are treated and cultivated under identical culture conditions to exclude variations, e.g. in phosphorylation pattern, that may be due to differences in culture conditions.

Preferably, the methods according to the first, second and third aspect of the present invention further comprise the step of formulating said compound or a pharmaceutically acceptable salt thereof with one or more pharmaceutically acceptable excipient(s), diluent(s), and/or carrier(s). The term "pharmaceutically acceptable salt" refers to a salt of a compound identifiable by the methods of the present invention or a compound of the present invention. Suitable pharmaceutically acceptable salts include acid addition salts which may, for example, be formed by mixing a solution of compounds of the present invention with a solution of a pharmaceutically acceptable acid such as hydrochloric acid, sulfuric acid, fumaric acid, maleic acid, succinic acid, acetic acid, benzoic acid, citric acid, tartaric acid, carbonic acid or phosphoric acid. Furthermore, where the compound carries an acidic moiety, suitable pharmaceutically acceptable salts thereof may include alkali metal salts (e.g., sodium or potassium salts); alkaline earth metal salts (e.g., calcium or magnesium salts); and salts formed with suitable organic ligands (e.g., ammonium, quaternary ammonium and amine cations formed using counteranions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, alkyl sulfonate and aryl sulfonate). Illustrative examples of pharmaceutically acceptable salts include, but are not limited to, acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, butyrate, calcium edetate, camphorate, camphorsulfonate, camsylate, carbonate, chloride, citrate, clavulanate, cyclopentanepropionate, digluconate, dihydrochloride, dodecylsulfate, edetate, edisylate, estolate, esylate, ethanesulfonate, formate, fumarate, gluceptate, glucoheptonate, gluconate, glutamate, glycerophosphate, glycolylarsanilate, hemisulfate, heptanoate, hexanoate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, hydroxynaphthoate, iodide, isothionate, lactate, lactobionate, laurate, lauryl sulfate, malate, maleate, malonate, mandelate, mesylate, methanesulfonate, methylsulfate, mucate, 2- naphthalenesulfonate, napsylate, nicotinate, nitrate, N-methylglucamine ammonium salt, oleate, oxalate, pamoate (embonate), palmitate, pantothenate, pectinate, persulfate, 3-phenylpropionate, phosphate/diphosphate, picrate, pivalate, polygalacturonate, propionate, salicylate, stearate, sulfate, subacetate, succinate, tannate, tartrate, teoclate, tosylate, triethiodide, undecanoate, valerate, and the like (see, for example, S. M. Berge et al., "Pharmaceutical Salts", J. Pharm. Sci. 66:1-19 (1977)).

The term "excipient" when used herein is intended to indicate all substances in a pharmaceutical formulation which are not active ingredients such as, e.g., carriers, binders, lubricants, thickeners, surface active agents, preservatives, emulsifiers, buffers, flavoring agents, or colorants.

Acceptable carrier or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R Gennaro edit. 1985). Examples of suitable carriers include, for example, magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, a low melting wax, cocoa butter, and the like. Examples of suitable diluents include ethanol, glycerol and water. The choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice. The pharmaceutical compositions may comprise as, or in addition to, the carrier, excipient or diluent any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilising agent(s). Examples of suitable binders include starch, gelatin, natural sugars such as glucose, anhydrous lactose, freeflow lactose, beta-lactose, corn sweeteners, natural and synthetic gums, such as acacia, tragacanth or sodium alginate, carboxymethyl cellulose and polyethylene glycol. Examples of suitable lubricants include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like. Preservatives, stabilizers, dyes and even flavoring agents may be provided in the pharmaceutical composition. Examples of preservatives include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. Antioxidants and suspending agents may be also used.

It is a fourth aspect of the invention to provide a compound identifiable by the above-described methods. The compound identifiable by the above described methods can be a protein, a peptide, or a small molecule. Preferably, the compound is an antibody. It is preferred that the antibody is capable of binding to Tec kinase or to FGF2, preferably to at least one of the amino acid residues within FGF2 which correspond to the amino acid residues Tyr82, phosphorylated Tyr82, Tyrl 12, and Tyrl 24 of the amino acid sequence set forth in SEQ ID NO: 1.

In a preferred embodiment, the antibody binds to the active centre within the Tec Kinase, and thus, inhibits/reduces the enzyme activity of the Tec kinase. In another embodiment, the antibody binds to the amino acid residue within FGF2 which corresponds to the amino acid residue Tyr82 and, thus, inhibits/reduces the phosphorylation of FGF2 by the Tec Kinase. In a further embodiment, the antibody binds to the amino acid residues within FGF2 which correspond to the amino acid residue sites Tyrl 12 and Tyrl 24 and, thus, inhibits/reduces recognition of FGF2 by the Tec kinase. In a further embodiment, the antibody binds to the amino acid residue within FGF2 which corresponds to the amino acid residue Tyr82, which is phosphorylated, and, thus, inhibits/reduces the cellular export of phosphorylated FGF2.

The antibody may be a monoclonal or polyclonal antibody or portions thereof. Antigen-binding portions may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. In some embodiments, antigen-binding portions include Fab, Fab', F(ab')₂, Fd, Fv, dAb, and complementarity determining region (CDR) fragments, single-chain antibodies (scFv), chimeric antibodies such as humanized antibodies, diabodies, and polypeptides that contain at least a portion of an antibody that is sufficient to confer specific antigen binding to the polypeptide.

Preferably, the compound identifiable by the methods according to the first, second and third aspect of the present invention has the ability of reducing/inhibiting angiogenesis and/or neovascularization. It particularly preferred that the compound that has the ability of reducing inhibiting angiogenesis and/or neovascularization is an antibody, preferably an antibody that is capable of binding to Tec kinase or to FGF2, preferably to at least one of the amino acid residues within FGF2 which correspond to the amino acid residues Tyr82, phosphorylated Tyr82, Tyrl 12, and Tyrl24 of the amino acid sequence set forth in SEQ ID NO: 1.

A compound according to the present invention can be administered alone but, in human therapy, will generally be administered in admixture with a suitable pharmaceutical excipient, diluent, or carrier selected with regard to the intended route of administration and standard pharmaceutical practice (see hereinafter).

In a fifth aspect, the present invention provides a pharmaceutical composition comprising the compound of the fourth aspect and one or more pharmaceutically acceptable excipient(s), diluent(s), and/or carrier(s).

The present invention also provides a compound according to the fourth aspect for use as a medicament. Further, the present invention provides a pharmaceutical composition according to the fifth aspect for use as a medicament.

In a sixth aspect, the invention provides a compound according to the fourth aspect or a pharmaceutical composition according to the fifth aspect for treating and/or preventing an angiogenesis- and/or neovascularization-dependent disease or condition. In a seventh aspect, the invention provides a compound or a pharmaceutical composition according to the invention for treating and/or preventing a disease or condition selected from the group consisting of cancer, atheroscleorsis, psoriasis, warts, pyogenic granuloma, allergic oedema, keloid scars, disease of hormonal growth control (benign prostatic hyperplasia, thyroid hyperplasia), thyroiditis, Crohn's disease, endometriosis, pre-eclampsia, recurrent miscarriage, dysfunctional uterine bleeding, follicular cysts, ovarian hyperstimulation, macular degeneration, diabetic retinopathy, choroidal and other intraocular disorders, hemangioma, hemangioendothelioma, retinopathy of prematurity, obesity, arthritis (e.g., rheumatoid arthritis), synovitis, osteomyelitis, pannus growth, osteophyte formation, nasal polyps. In an eighth aspect, the present invention provides a method for treating and/or preventing an angiogenesis- and/or neovascularization-dependent disease or condition in an individual in need thereof comprising the step of administering the compound according to the fourth aspect or the pharmaceutical composition according to the fifth aspect in a pharmaceutically effective amount to said individual.

Preferably, the individual is a mammal, preferably a human. It is preferred that the angiogenesis- and/or neovascularization-dependent disease or condition is selected from the group consisting of cancer, atheroscleorsis, psoriasis, warts, pyogenic granuloma, allergic oedema, keloid scars, disease of hormonal growth control (benign prostatic hyperplasia, thyroid hyperplasia), thyroiditis, Crohn's disease, endometriosis, pre-eclampsia, recurrent miscarriage, dysfunctional uterine bleeding, follicular cysts, ovarian hyperstimulation, macular degeneration, diabetic retinopathy, choroidal and other intraocular disorders, hemangioma, hemangioendothelioma, retinopathy of prematurity, obesity, arthritis (e.g., rheumatoid arthritis), synovitis, osteomyelitis, pannus growth, osteophyte formation, nasal polyps.

The administration is preferably parenteral, preferably by intravenous, intramuscular, intraperitoneal, or subcutaneous injection.

In preferred embodiment, the individual, preferably a mammal, most preferably a human, is also treated with one or more other therapeutic agents and/or therapies.

Preferably, the angiogenesis- and/or neovascularization-dependent disease is cancer and the individual is also treated with other anti-tumor therapeutics, preferably anti-cancer chemotherapeutics and/or anti-cancer immunotherapeutics.

The pharmaceutical composition contemplated by the present invention may be formulated in various ways well known to one of skill in the art. For example, the pharmaceutical composition of the present invention may be in solid form such as in the form of tablets, pills, capsules (including soft gel capsules), cachets, lozenges, ovules, powder, granules, or suppositories, or in liquid form such as in the form of elixirs, solutions, emulsions, or suspensions. Solid administration forms may contain excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate, glycine, and starch (preferably corn, potato, or tapioca starch), disintegrants such as sodium starch glycolate, croscarmellose sodium, and certain complex silicates, and granulation binders such as polyvinylpyrrolidone, hydroxypropylmethyl cellulose (HPMC), hydroxypropylcellulose (HPC), sucrose, gelatin, and acacia. Additionally, lubricating agents such as magnesium stearate, stearic acid, glyceryl behenate, and talc may be included. Solid compositions of a similar type may also be employed as fillers in gelatin capsules. Preferred excipients in this regard include lactose, starch, a cellulose, milk sugar, or high molecular weight polyethylene glycols.

For aqueous suspensions, solutions, elixirs, and emulsions suitable for oral administration the compound may be combined with various sweetening or flavoring agents, coloring matter or dyes, with emulsifying and/or suspending agents and with diluents such as water, ethanol, propylene glycol, and glycerin, and combinations thereof.

The pharmaceutical composition of the present invention may contain release rate modifiers including, for example, hydroxypropylmethyl cellulose, methyl cellulose, sodium carboxymethylcellulose, ethyl cellulose, cellulose acetate, polyethylene oxide, Xanthan gum, Carbomer, ammonio methacrylate copolymer, hydrogenated castor oil, camauba wax, paraffin wax, cellulose acetate phthalate, hydroxypropylmethyl cellulose phthalate, methacrylic acid copolymer, and mixtures thereof.

The pharmaceutical composition of the present invention may be in the form of fast dispersing or dissolving dosage formulations (FDDFs) and may contain the following ingredients: aspartame, acesulfame potassium, citric acid, croscarmellose sodium, crospovidone, diascorbic acid, ethyl acrylate, ethyl cellulose, gelatin, hydroxypropylmethyl cellulose, magnesium stearate, mannitol, methyl methacrylate, mint flavoring, polyethylene glycol, fumed silica, silicon dioxide, sodium starch glycolate, sodium stearyl fumarate, sorbitol, xylitol.

For preparing suppositories, a low melting wax, such as a mixture of fatty acid glycerides or cocoa butter, is first melted and the active component is dispersed homogeneously therein, as by stirring. The molten homogeneous mixture is then poured into convenient sized molds, allowed to cool, and thereby to solidify. The pharmaceutical composition of the present invention suitable for parenteral administration is best used in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood. The aqueous solutions should be suitably buffered (preferably to a pH of from 3 to 9), if necessary.

The pharmaceutical composition suitable for intranasal administration and administration by inhalation is best delivered in the form of a dry powder inhaler or an aerosol spray from a pressurized container, pump, spray or nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, a hydrofluoroalkane such as 1,1,1,2-tetrafluoroethane (HFA 134A.TM.) or 1,1,1,2,3,3,3-heptafluoropropane (HFA 227EA.TM.), carbon dioxide, or another suitable gas. The pressurized container, pump, spray or nebulizer may contain a solution or suspension of the active compound, e.g., using a mixture of ethanol and the propellant as the solvent, which may additionally contain a lubricant, e.g., sorbitan trioleate.

For treating, ameliorating, or preventing said disease conditions the medicament of the present invention can be administered to an animal patient, preferably a mammalian patient, preferably a human patient, orally, buccally, sublingually, intranasally, via pulmonary routes such as by inhalation, via rectal routes, or parenterally, for example, intracavernosally, intravenously, intra- arterially, intraperitoneally, intrathecally, intraventricularly, intraurethrally intrasternally, intracranially, intramuscularly, or subcutaneously, they may be administered by infusion or needleless injection techniques.

The pharmaceutical compositions of the present invention may be formulated in various ways well known to one of skill in the art and as described above.

The pharmaceutical preparation is preferably in unit dosage form. In such form the preparation is subdivided into unit doses containing appropriate quantities of the active component. The unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, such as packeted tablets, capsules, and powders in vials or ampoules. Also, the unit dosage form can be a capsule, tablet, cachet, or lozenge itself, or it can be the appropriate number of any of these in packaged form.

The quantity of active component in a unit dose preparation administered in the use of the present invention may be varied or adjusted from about 1 mg to about 1000 mg per m², preferably about 5 mg to about 150 mg/m² according to the particular application and the potency of the active component.

The compounds employed in the medical use of the invention are administered at an initial dosage of about 0.05 mg/kg to about 20 mg/kg daily. A daily dose range of about 0.05 mg/kg to about 2 mg/kg is preferred, with a daily dose range of about 0.05 mg/kg to about 1 mg/kg being most preferred. The dosages, however, may be varied depending upon the requirements of the patient, the severity of the condition being treated, and the compound being employed. Determination of the proper dosage for a particular situation is within the skill of the practitioner. Generally, treatment is initiated with smaller dosages, which are less than the optimum dose of the compound. Thereafter, the dosage is increased by small increments until the optimum effect under circumstances is reached. For convenience, the total daily dosage may be divided and administered in portions during the day, if desired. In a further aspect, the invention provides a method for identifying a compound which causes a reduced efficiency of FGF2 secretion by inhibiting or enhancing the activity of a target component of the FGF2 secretory pathway, wherein said method comprises the step of contacting a test compound with a target component selected from the components listed in Table 1 or Table 2.

Preferably, the test compound is considered to inhibit the activity of a target component of the FGF2 secretory pathway, if the activity is reduced by at least 60 %, preferably by at least 70%, more preferably by at least 80%, more preferably by at least 90%, i.e. by at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99,9%, or even 100%, in presence of the test compound compared to the activity in absence of the test compound.

It is also preferred that the test compound is considered to enhance the activity of a target component of the FGF2 secretory pathway, if the activity is enhanced by at least 60 %, preferably by at least 70%, more preferably by at least 80%, more preferably by at least 90%, i.e. by at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99,9%, or even 100%, in presence of the test compound compared to the activity in absence of the test compound. In another aspect, the invention provides a method for identifying a compound which causes a reduced efficiency of FGF2 secretion by inhibiting/reducing or enhancing the interaction between the target component of the FGF2 secretory pathway and FGF2, wherein said method comprises the step of contacting a test compound with a target component selected from the components listed in Table 1 or Table 2 and FGF2.

Preferably, the test compound is considered to inhibit/reduce the interaction between the target component of the FGF2 secretory pathway and FGF2, if the interaction is reduced by at least 60 %, preferably by at least 70%, more preferably by at least 80%, more preferably by at least 90%, i.e. by at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99,9%, or even 100%, in presence of the test compound compared to the interaction in absence of the test compound.

It is also preferred that the test compound is considered to enhance the interaction between the target component of the FGF2 secretory pathway and FGF2, if the interaction is enhanced by at least 60 %, preferably by at least 70%, more preferably by at least 80%, more preferably by at least 90%, i.e. by at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99,9%, or even 100%, in presence of the test compound compared to the interaction in absence of the test compound.

It is particularly preferred that said methods further comprise the step of testing whether the test compound causes a reduced efficiency of FGF2 secretion.

Preferably, the efficiency of FGF2 secretion is considered reduced if the secretion is reduced by at least 60%, preferably by at least 70%, more preferably by at least 80%, more preferably by at least 90%, i.e. by at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99,9%, or even 100%, in presence of the test compound when compared to the secretion in absence of the test compound.

In the context of the method for identifying a compound which causes a reduced efficiency of FGF2 secretion by inhibiting or enhancing the activity of a target component of the FGF2 secretory pathway, or in the context of the method for identifying a compound which causes a reduced efficiency of FGF2 secretion by inhibiting/reducing or enhancing the interaction between the target component of the FGF2 secretory pathway and FGF2, all preferred and particularly preferred embodiments previously described in the context of the methods of the first, second and third aspect of the present invention are similarly preferred if applicable.

The invention further provides a method for identifying a compound which causes an increased efficiency of FGF2 secretion by inhibiting or enhancing the activity of a target component of the FGF2 secretory pathway, wherein said method comprises the step of contacting a test compound with a target component selected from the components listed in Table 1 or Table 2.

In another aspect, the invention provides a method for identifying a compound which causes an increased efficiency of FGF2 secretion by inhibiting/reducing or enhancing the interaction between the target component of the FGF2 secretory pathway and FGF2, wherein said method comprises the step of contacting a test compound with a target component selected from the components listed in Table 1 or Table 2 and FGF2.

In another aspect the present invention relates to methods for the identification of inhibitors of the proteins identified in Table 1 below as well as the use of such inhibitors to treat the diseases exemplified in more detail above with respect to inhibitors identified in the method according to the first to third aspect of the invention. In a further aspect the present invention relates to methods for the identification of activators of the proteins identified in Table 2 below as well as the use of such activators to treat the diseases exemplified in more detail above with respect to inhibitors identified in the method according to the first to third aspect of the invention.

Various modifications and variations of the invention will be apparent to those skilled in the art without departing from the scope of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in the relevant fields are intended to be encompassed by the present invention.

The following figures and examples are merely illustrative of the present invention and should not be construed to limit the scope of the invention as indicated by the appended claims in any way. BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1: Large scale RNAi screening to identify gene products involved in unconventional secretion of FGF2. Scores (normalized signal intensities) and p-values were calculated as described under 'Materials and methods' and the result from each individual siRNA is represented by a grey dot. In case only one out of three siRNAs was functional, a gene product was classified a hit when its mean score was either larger than 4 (negative regulators) or smaller than -3 (core components or positive regulators) combined with p-values smaller than 0.05. In case two or even three siRNAs were functional, a gene product was classified a hit when its mean score was either larger than 1.5 or smaller than -1.5. For details see 'Materials and methods'. (A) Mean scores (based on four replicates) were plotted against p-values for each individual siRNA tested. P-values were computed using a t-test to identify significant differences from zero. As controls, siRNAs against Tec kinase (green dots; 156 individual measurements) and GFP (red dots; 624 individual measurements) were used. The three different siRNAs targeting FGF2 as part of the siRNA library are shown as blue dots. For hit classification, the corresponding thresholds are indicated in pink (score > 4 or < -3), blue (score >1.5 or < -1.5) and orange (p-value < 0.05). (B) Mean scores were plotted for each individual siRNA in the sequence they were tested during the screening procedure. Controls and thresholds used for hit classification are depicted as explained for panel A. The black curve summarizes all mean scores sorted by magnitude.

Fig. 2: Classification of gene products with a putative role in unconventional secretion of FGF2 as core components, positive regulators or negative regulators, respectively. Analysis of membrane association and previously reported functions are based on annotations listed in the Swiss-Prot protein knowledgebase (http://www.expasy.ch/sprot/) and NCBI (http://www.ncbi.nlm.nih.gov/), respectively.

Fig. 3: RNAi-based screening identifies Tec kinase as a novel factor involved in FGF2 secretion. Stable HeLa cell lines expressing FGF2-GFP in a doxycycline-dependent manner were used to quantify FGF2 export to cell surfaces by either the On-cell Western assay (RNAi primary screening) or flow cytometry (RNAi data validation). (A) RNAi primary screening. A high content screening based on an On-cell Western assay in 96-well plates was used to quantify FGF2-GFP localized to the cell surface of HeLa cells. Cells were treated with three independent siRNAs per gene product in independent experiments. Knock-down times were as indicated followed by 24 hours of induction of FGF2-GFP expression. Cells were fixed and stained with anti-GFP- and fluorescently labeled secondary antibodies. Additionally, cells were treated with Syto-60, a red fluorescent nucleic acid stain used to normalize cell surface signals by cell numbers. Signals were quantified using the LI-COR infrared imaging platform. The relative amount of cell-surface-localized FGF2-GFP under Tec kinase knock-down conditions is given as the percentage of the mean of all values across the RNAi array. Standard deviations are given (n=4). (B) RNAi data validation. Flow cytometry was used to simultaneously measure FGF2- GFP expression levels (green bars) and cell-surface-localized FGF2-GFP (red bars). In addition to the functional siRNA Tec #1 identified in the experiments shown in panel A, a second functional siRNA with a different sequence could be identified (Tec #4). Standard deviations are given (n=4). (C) To address potential pleiotropic effects on for example cell viability, transport of FGF4-GFP to cell surfaces was analyzed by flow cytometry following a knock-down of Tec kinase using the siRNAs Tec #1 and #4, respectively. (D) To control siRNA-mediated knockdown efficiencies, following transfection with the siRNAs Tec #1 and Tec #4, mRNA levels were monitored by RT-PCR. GAPDH mRNA levels were analyzed as a control. (E) Quantification of the data shown in panel D. Data were normalized by GAPDH mRNA levels.

Fig. 4: FGF2 secretion is inhibited in the presence of a pharmacological inhibitor of Tec kinases, LFM-A13. HeLa cells stably expressing FGF2-GFP (A) or FGF4-GFP (B) were grown for 24 h in the presence of doxycycline. Cells were either left untreated (mock) or incubated in the presence of LFM-A13 at a final concentration of 275 μΜ. Expression levels and cell-surface- localized material of the GFP fusion proteins indicated were simultaneously measured by flow cytometry. Standard deviations are shown (n=4).

Fig. 5: FGF2 and Tec kinase form a heterodimeric complex in vitro. Recombinant GST-FGF2 and GST, respectively, were coupled to glutathion beads to conduct pull-down experiments. Protein-coupled beads were incubated with recombinant Tec kinase containing a His-tag. Bound proteins were eluted with SDS sample buffer and analyzed by SDS-PAGE/Coomassie staining (A) and Western blotting (B). Both anti-His antibodies to detect recombinant Tec kinase and anti-GST antibodies to detect GST-FGF2 and GST, respectively, were used.

Fig. 6: Tec kinase phosphorylates FGF2 in vitro. Recombinant GST-FGF2 and GST used as negative control, respectively, were incubated with or without recombinant His-Tec in the presence or absence of [γ32Ρ] ATP as indicated. The reaction was terminated by the addition of guanidine hydrochloride (final concentration: 7M). Mixtures were boiled in SDS sample buffer and analyzed by SDS-PAGE. Gels were first stained in Coomassie solution (A), dried and then exposed on a phosphoimaging screen followed by visualization using a phosphoimager (B).

Fig. 7: The surface-exposed tyrosine residues Y82, 122 and 124 in FGF2 are essential for Tec- kinase-dependent phosphorylation of FGF2. Recombinant GST-FGF2 wild-type and the variant forms Y82A, Y112A, and Y124A were analyzed for Tec-kinase-dependent phosphorylation as described in the legend to Fig. 4. Samples were analyzed by Coomassie staining (A) and phosphoimaging (B). The experiment shown in panels A and B represents one example out of a total of four independent experiments. A statistical analysis of all four experiments quantifying the signals obtained by phosphoimaging (normalized for total protein amounts) is shown in panel C. Standard deviations are given (n=4).

Fig. 8A, 8B: Direct demonstration of Tec-kinase-dependent FGF2 phosphorylation at tyrosine residues Y82 and Y124, respectively. GST-FGF2 was incubated with Tec kinase in the presence of ATP followed by protease digestion and peptide analysis using a LTQ Orbitrap Hybrid mass spectrometer as described under 'Materials and Methods'. (A) MS spectrum at retention time 6.1 min. The arrow indicates the putative phosphopeptide containing Yp82 (m/z = 581.752). (B) MS spectrum at retention time 21.59 min. The arrow indicates the putative phosphopeptide containing Ypl24 (m/z = 498.922) (C) MS2 fragmentation of the peptide specified in panel A. Y and b ions of the fragmented peptide and the deduced amino acid sequence are as indicated. (D) MS2 fragmentation of the peptide specified in panel C. Y and b ions of the fragmented peptide and the deduced amino acid sequence are as indicated.

EXAMPLES

The Examples are designed in order to further illustrate the present invention and serve a better understanding. They are not to be construed as limiting the scope of the invention in any way.

Material and methods

Constructs and Generation of Stable Cell Lines

Various FGF-2-GFP fusion constructs (FGF2 wild-type, Y82A/E, Y112A/E and Y124A/E, respectively) were cloned into the retroviral vector pRevTre2. Stable HeLa cell lines expressing FGF-2-GFP variant forms in a doxycyline-dependent manner were generated as described before (Engling et al., 2002). Recombinant forms of FGF-2 were expressed in E. coli using the pGEX- 2T expression system (GE healthcare).

Large-scale RNAi screening to identify gene products involved in FGF2 secretion

For RNAi screening purposes, the LI-COR infrared imaging platform (LI-COR Biosciences) was employed to analyze FGF2-GFP cell surface expression using the On-Cell-Western staining procedure. For siRNA transfection under screening conditions, either 96 well plates (pilot screening; Ambion Silencer® Human Kinase siRNA Library V3) or 384 well plates (large-scale screening; Ambion Silencer® Human Extended Druggable Genome siRNA Library V3) were used. Plates were pre-coated with transfection reagent-siRNA complexes and used for solid- phase transfection (Erfle et al., 2008). Following RNAi-mediated knockdowns (56h and 96h for pilot screening; 48 h for large-scale screening), expression of FGF-2-GFP was induced by addition of 2 μ¾^/ιη1 doxycycline for 16 h. Cells were then fixed on ice for 20 min with PFA (3% in PBS). All following steps were performed at room temperature. Cells were washed (PBS) and treated for 1.5 hours with Odyssey blocking solution (LI-COR Biosciences). Fixed cells were incubated for 2 hours with affinity-purified anti-GFP antibodies diluted in 1% BS A/PBS. Following washing with PBS, cells were treated for 1 hour with both secondary antibodies (IRD800 goat anti-rabbit, LI-COR Biosciences) and Syto-60 (LI-COR Biosciences) to stain nucleic acids as a relative measure for cell numbers. After extensive washing, plates were imaged on a LI-COR Biosciences Odyssey Infrared Imaging System. Antibody-derived signals were taken as a relative measure for cell surface-localized FGF-2-GFP that was normalized by the Syto-60 signal (cell number). HeLa cells expressing GFP in a doxycyline-dependent manner were used to control background signals. RNAi screening data were analyzed using the RNAither (Rieber et al., 2009) and cellHTS (Boutros et al., 2006) packages (R 2.8.0; R Development Core Team; http://www.R-project.org). In brief, wells with low cell counts (<5%) were excluded from further analysis to account for apoptotic effects of siRNA knockdowns. Within-plate normalization was carried out using locally weighted scatterplot smoothing (Cleveland, 1979). This normalization corrects artificial effects on signal intensity based on varying cell counts between wells. B-score normalization was further used to remove spatial effects within plates (Brideau et al., 2003). Variability between plates was addressed by subtracting the plate mean from each measurement divided by the plate standard deviation. Replicate experiments (4) were summarized and expressed as mean scores, a value that describes the deviation of the average of four individual measurements from the corresponding plate medians expressed as the number of standard deviations measured for the corresponding plates. Furthermore, a t-test was carried out to analyze whether mean scores for each siRNA differed significantly from zero. A siRNA was classified to have an effect if its p-value was <0.05 and if its mean score was smaller than a threshold of -1.5 for core components and positive regulators (i.e. a knockdown caused reduced efficiency of FGF2 secretion) or >1.5 for negative regulators (i.e. a knockdown caused an increased efficiency of FGF2 secretion). A gene product was classified significant if either two or three siRNAs targeted against the mRNA were significant according to these criteria, or if a single siRNA had a score smaller than -3 or larger than 4, respectively, combined with a p-value <0.05. siRNAs targeted against Tec kinase and determination of mRNA levels using RT-PCR

The siRNAs Tec #1, Tec #2 and Tec #3 (Fig. 1A) were purchased from Ambion (catalogue ID numbers: siRNA 1, Ambion ID # 383; siRNA 2, Ambion ID # 384; siRNA 3, Ambion ID # 385). A siRNA of different sequence and origin, Tec #4 (Fig. IB and 1C), was purchased Qiagen (Cat. # SI02223165). As negative control, a scrambled siRNA was used (Ambion ID # 4611). To monitor knock-down efficiency at the mRNA level, total RNA was isolated from siRNA-treated HeLa cells (RNeasy Micro Kit, Invitrogen) and mRNA levels were analyzed by RT-PCR (Im- Prom-IITM Reverse Transcription System, Promega). A RT-PCR analysis of GAPDH mRNA was used to normalize data (Fig. ID and IE). Flow cytometry to quantify cell surface expression of FGF2-GFP and FGF4-GFP

In validation experiments, both additional siRNAs and an independent read-out method was used. To quantify both FGF2-GFP expression levels and the corresponding cell surface population, a well-characterized flow cytometry assay was used (Seelenmeyer et al., 2005; Zehe et al., 2006; Temmerman et al., 2008). As a further control, HeLa cells expressing FGF4-GFP were used as FGF4 contains a signal peptide and is secreted via the classical secretory pathway. RNAi-mediated knock-downs were now performed in 24-well plates using liquid-phase transfection and Oligofectamine as a transfection reagent (Invitrogen). SiRNAs were used at a final concentration of 100 nM. Knock-down times were as described in the respective experiment resulting in a final cell confluency of about 80%. Cells were stained with affinity- purified anti-GFP antibodies and anti-rabbit APC-labeled secondary antibodies (Molecular Probes). Samples were analyzed by flow cytometry using a FACSCalibur instrument (BD Biosciences). Where indicated, HeLa cells either expressing FGF2-GFP or FGF4-GFP were grown in the presence of doxycycline and LFM-A13 inhibitor (Calbiochem/Milipore; 275 μΜ final concentration), a specific inhibitor of Tec kinase family members (Fig. 2). Experiments were conducted in 24 well plates. Following for 24 h of incubation, a final confluency of about 80 % was observed. Cells were stained and analyzed by flow cytometry as described above.

Biochemical interaction studies

Recombinant GST-FGF-2 and GST were expressed in E. coli and purified using standard protocols. Recombinant Tec kinase (His-tagged) was purchased from Millipore (# 14-801). For binding experiments, 200 μg recombinant GST-FGF-2 or GST was incubated with 20 □! 4FastFlow Glutation-S-Sepharose beads (GE Healthcare) for 2 h at 4°C in buffer A (50 mM Tris HCl, pH 7.5, 150 mM NaCl, ImM EDTA, 0.1 % β-Mercaptoethanol, and protease inhibitors [Roche]). After thorough washing with buffer A, GST-FGF2- and GST beads, respectively, were incubated with 0.2 μg of recombinant Tec kinase for 12 hours at 4°C. Beads were washed with buffer A and bound material was eluted with SDS sample buffer. Samples were subjected to SDS-PAGE followed by either Western blotting or colloidal Coomassie staining. For Western blotting, both anti-His antibodies and anti-GST-antibodies were used. In case anti-His antibodies were used, 70% of bound and 20% of unbound material analyzed. In case anti-GST antibodies were used, 10% of both bound and unbound material was analyzed. For colloidal Coomassie staining, 70% of bound and 20% of unbound material was analyzed. To detect GST-FGF2 and GST by Coomassie staining, 10% of both bound and unbound material was analyzed. Tec-kinase-catalyzed phosphorylation of FGF2

Recombinant GST-FGF-2 and GST (25 μg each) were incubated with recombinant Tec kinase (0.1 μg) for 30 min at 30 °C in the presence of 20 μΜ ATP containing 0.2 μθ/μΐ [γ32Ρ]ΑΤΡ. The mixture was supplemented with 0.1 mM sodium vanadate and PTK assay buffer from a Sigma TECTTM PTK Assay System kit (Promega). After stopping the reaction with 7.5 M guanidine hydrochloride, samples were boiled in SDS sample buffer and separated on 4 -12 % pre-casted gradient gels (Invitrogen). With regard to the various samples indicated, 54 % of GST-FGF2 wt, 81 % of GST, 54 % of GST-FGF2-Y82A, 54 % of GST-FGF2-Y112A and 54 % of GST-FGF2-Y124A, respectively, were loaded. Gels were stained with Coomassie brilliant blue, dried on a Vacuum Gel Dryer, and exposed for 48 h at room temperature on a phosphor- screen imaging plate (BAS-MS 2040, Fujifilm). The screen was imaged on a phosphor-imager (Fujifilm FLA-7000) and data were analyzed by Aida Image Analyzer software.

Mass spectrometry

20 μg of recombinant GST-FGF2 were incubated with 1 μg of recombinant His-Tec kinase and 0.25 mM ATP, supplemented with 50 μΜ sodium vanadate in 50 μΐ PTK assay buffer (Signa TECTTM PTK Assay System kit [Promega]). 25μ1 of the sample was adjusted to 1 mM DDT and incubated at 37°C for 30min. Subsequently, iodoacetamide was added to (final concentration = 2 mM) and the sample was incubated for 20min at 20°C. After the addition of 120 nmol DDT, the sample was adjusted to 6M guanidine hydrochloride and further incubated at 95°C for 15 min. The sample was then diluted to 1M guanidine hydrochloride and digested with 0.45 μg trypsin at 37°C for 15 hours. Subsequently, 25 μΐ of the sample were subjected to LC-ESI- MS/MS (LTQ Orbitrap, Thermo) as described in (Tegha-Dunghu et al., 2008).

Results

Large scale RNAi screening to identify gene products involved in unconventional secretion of FGF2

Based on stable HeLa cells expressing FGF2-GFP in a doxycyline-dependent manner, we have developed a high content screening assay to quantify FGF2 secretion based on the LI-COR infrared imaging platform. This system is based on a multi-well format and allows for the quantitation of cell-surface associated FGF2 normalized by the number of cells being analyzed for each experimental condition. Primary screening involved a pilot phase in which all kinases known in the human genome were analyzed. To improve chances of success, this relatively small set of siRNAs (2157 siRNAs targeting 719 kinases) was tested at two different time scales of down-regulation, 56 and 96 hours, respectively, followed by 16 hours of induction of FGF2-GFP expression. In a second step, 27306 siRNAs targeting 9102 human gene products as defined by Ambion's 'extended druggable genome' were tested as potential core components, positive regulators or negative regulators of FGF2 secretion. In this case, knockdown times were 48 hours followed by 16 hours of induction of FGF2-GFP expression. Primary screening was conducted in four replicates resulting in almost 120,000 individual measurements of FGF2 secretion. In Fig. 1, the results of the large-scale screening procedure are summarized providing mean scores (normalized signal intensities based on four replicates) and corresponding p-values to address statistical significance. In Fig. 1A, mean scores are plotted against p-values for every single siRNA that was analyzed. Gene products were classified as potential factors required for FGF2 secretion based on two different criteria. In case only one out of three siRNAs caused a phenotype, a gene product was considered a hit when the mean score was either larger than 4 (positive regulators) or smaller than -3 (core components or negative regulators) combined with a p-value smaller than 0.05. In case two or even all three siRNAs caused a phenotype, a gene product was considered a hit when the mean score was either larger than 1.5 or smaller than -1.5. As a positive control, we used a validated siRNA against GFP to target the FGF2-GFP fusion protein itself. This control was expected to cause a strong phenotype in this experimental system and, therefore, was used to evaluate phenotypes caused by siRNA from the library. As evident from Fig. 1, the vast majority of a total of 624 individual control experiments using a siRNA targeting GFP (red dots) showed mean scores smaller than -3 and p-values below 0.05. Similarly, two out of three siRNAs present in the siRNA library that target FGF2 showed mean scores smaller than -3 and -4, respectively, combined with p-values below 0.02. These control experiments established the principal potential of the experimental system described to identify novel gene products required for FGF2 secretion.

Using the criteria defined above and as summarized in Tables 1 and 2, based on one siRNA, we identified 25 gene products as potential core components or positive regulators and 36 gene products as potential negative regulators with mean scores smaller than -3 or larger than 4, respectively. Additionally, based on two or even three siRNAs, we found 23 gene products with mean scores smaller than -1.5 and 39 gene products with mean scores larger than 1.5. Taken together, we report a total of 123 gene products as strong candidates potentially involved in FGF2 secretion. This number corresponds to about 1.4% of the 9102 gene products that were analyzed in this study.

Table 1

Putative core components and positive regulators of FGF2 secretion

Key Gene ID Refseq siRNA ID siRNA sequence Score p-Value

GUCUUGGCCUUAAGCUGACtt

AK 7A2 8574 NM_003689 122533 (SEQ ID NO: 3) -3.39 0.0012

GGUCUUGCUUCUAUCUGGGtt

ALDH3B2 222 NM_000695 106209 (SEQ ID NO: 4) -3.84 0.0399

CCUAUCAGGACCAGAGAGAtt

ASTL 431705 NM_001002036 216185 (SEQ ID NO: 5) -3.07 0.0020

GCCGUUGUAAUCAUAACUGtt

ATP1A1 476 NM_001001586 120183 (SEQ ID NO: 6) -3.12 0.0451

GGCCAGUGAAGCAGAGAUGtt

BCL6 604 NM_001706 2809 (SEQ ID NO: 7) -3.31 0.0009

GCUCUUCCUAUGACUAUGGtt

CXCR3 2833 NM_001504 45982 (SEQ ID NO: 8) -3.15 0.0013

GGUCUUUCUGCCCUGCAUGtt

CXCR6 10663 NM_006564 2040 (SEQ ID NO: 9) -3.24 0.0025

GGAUGAACGUGUGCAAAAGtt

GPR61 83873 NM_031936 6369 (SEQ ID NO: 10) -3.05 0.0018

GCAGAUCAAACGGGUGAAGtt

HRAS 3265 NM_176795 120898 (SEQ ID NO: 11) -3.07 0.0072

CCAGUGAGUGCUGUUUUCAtt

IL12RB1 3594 NM_153701 111582 (SEQ ID NO: 12) -4.10 0.0190

CCGGAUCAAAGUAUGGAAGtt

ITIH5 80760 NM_032817 258842 (SEQ ID NO: 13) -3.37 0.0077

GGUCCAAGCUGAGAGAGAUtt

ASTL 84930 M_032844 1109 (SEQ ID NO: 14) -3.31 0.0041

GGUACCUAAGUUGGAGCUGtt

MGC11134 83707 NM_031472 112249 (SEQ ID NO: 15) -3.22 0.0015

GGCACGGUCAAUGAAGGAAtt

MGRN1 23295 NM_015246 148547 (SEQ ID NO: 16) -3.67 0.0016

GGUUGAUGCGGAGCUGUUUtt

MMP13 4322 NM_002427 104025 (SEQ ID NO: 17) -3.07 0.0013

GUAUGUGGACAUGACAUUGtt

NAT8 9027 NM_003960 46071 (SEQ ID NO: 18) -3.56 0.0057

GGCUGUAGGAAAUCUAGUUtt

NME1 4830 NM_198175 43 (SEQ ID NO: 19) -4.14 0.0287

CCCUCCACUGAGUGAAGAAtt

PHF12 57649 NM_020889 116276 (SEQ ID NO: 20) -3.23 0.0012

GGACAUCUAGCUCAAUACAtt

SCN10A 6336 NM_006514 104572 (SEQ ID NO: 21) -4.07 0.0173

GGUAUCAAGCAGCAGUGAUtt

SEC23IP 11196 N _007190 19205 (SEQ ID NO: 22) -3.71 0.0104

CCCUGG U ACAAG AU AU U U Utt

SLC7A7 9056 NM_003982 117207 (SEQ ID NO: 23) -3.31 0.0074

TAP1 6890 NM 000593 116873 GGUAUGCUGCUGAAAGUGGtt -3.09 0.0061 (SEQ ID NO: 24)

GGAAGAACCGGGACUAUGGtt

TPTE2 93492 NM_ .199254 122520 (SEQ ID NO: 25) -3.45 0.0366

CCUCGUUGGUGAACUACAAtt

UNQ9391 203074 NM. .198464 114014 (SEQ ID NO: 26) -3.17 0.0055

GCAAGAUUGAAGAGAUGGUtt

UXS1 80146 NM. .025076 118827 (SEQ ID NO: 27) -3.07 0.0099

CCUUGGAAAGUACGAUGAUtt

ABCE1 6059 NM. .002940 117102 (SEQ ID NO: 28) -1.68 0.0429

CCUCAAUAUGUAGCCAGAUtt

117103 (SEQ ID NO: 29) -1.93 0.0042

CCACCACAUCUACCUGGAAtt

ALDOA 226 NM. .184041 122362 (SEQ ID NO: 30) -1.97 0.0211

GUAUGUGACCGAGAAGGUGtt

122368 (SEQ ID NO: 31) -2.02 0.0287

GGUCCACAGGGAGAUUGUCtt

ARHGDIG 398 NM. .001176 119597 (SEQ ID NO: 32) -2.31 0.0069

GGAAGAGCCUCU UGGAGAUtt

9458 (SEQ ID NO: 33) -2.82 0.0049

GGAUCAGAUGAACUUACGCtt

ASNS 440 NM. .183356 117863 (SEQ ID NO: 34) -2.39 0.0356

GCCAAUUCGAGUGAAGAAAtt

117862 (SEQ ID NO: 35) -2.99 0.0111

GGAAAUGGAACCCUAACUAtt

CA6 765 NM. .001215 118897 (SEQ ID NO: 36) -1.96 0.0298

GCACUACUACACCUACCAUtt

118896 (SEQ ID NO: 37) -3.50 0.0110

GGCCUUCAAGAAGGAGCUGtt

CNP 1267 NM. .033133 35220 (SEQ ID NO: 38) -1.70 0.0067

GGGUAGAAAAAGGAGCUUAtt

35305 (SEQ ID NO: 39) -1.95 0.0199

CCAAAAGAAGAUGUGAUGAtt

DDX5 1655 NM. .004396 121482 (SEQ ID NO: 40) -1.79 0.0102

GGCGAUGGGCCUAUUUGUUtt

121481 (SEQ ID NO: 41) -2.08 0.0375

GGAUUAAGUUACAUCAUGCtt

PPM1K 152926 NM. .152542 6899 (SEQ ID NO: 42) -1.59 0.0234

GGAGAAGAACUUGGAAACUtt

6723 (SEQ ID NO: 43) -1.63 0.0114

GGCAGGCAGAACCCUCUUAtt

DUSP19 142679 NM. .080876 104969 (SEQ ID NO: 44) -1.58 0.0316

GGCAAAAU U UAGACCAG U Att

104970 (SEQ ID NO: 45) -1.59 0.0242

GGCUUUCAUAUGCUGUCAUtt

104968 (SEQ ID NO: 46) -1.80 0.0147

GGAACUGGGAUGUGUACAAtt

FCER1A 2205 NM. .002001 10815 (SEQ ID NO: 47) -1.61 0.0240

CCUAAGGUCUCCUUGAACCtt

111052 (SEQ ID NO: 48) -1.70 0.0008

GGAGUUGUGUCUAUCAAAGtt

FGF2 2247 NM. .002006 4190 (SEQ ID NO: 49) -3.19 0.0176

GGAGUGUGUGCUAACCGUUtt

4286 (SEQ ID NO: 50) -4.61 0.0005

GGAACCUUCUGGCAUAUUUtt

GLP2R 9340 NM. .004246 5237 (SEQ ID NO: 51) -1.66 0.0057

GGCAUGUCUGAGAGACUUAtt

5146 (SEQ ID NO: 52) -2.26 0.0261

GGAACAAACAUAUGCACACtt

GPR74 10886 NM. .053036 5332 (SEQ ID NO: 53) -1.80 0.0422

GGAUGGCCAUUUGGAAACAtt

5423 (SEQ ID NO: 54) -2.78 0.0066

GAAAGAUCUGAAGGACUACtt

HNRPD 3184 NM. .031370 46390 (SEQ ID NO: 55) -1.74 0.0193

GGACAUGAUUAAAAUUGCAtt

33290 (SEQ ID NO: 56) -1.88 0.0240

GCUGGGAUCUCCCAAUUAUtt

KCNC1 3746 NM. .004976 105455 (SEQ ID NO: 57) -1.57 0.0103

GUGAGCACACGCACUUUAAtt

105454 (SEQ ID NO: 58) -1.74 0.0170

GGUAACUGUGCCAGAAUAAtt

KIAA1950 155185 NM. .133463 219728 (SEQ ID NO: 59) -1.57 0.0146

GCUCAGAUGUAGCAUUAGGtt

219726 (SEQ ID NO: 60) -1.60 0.0106

LIMS2 55679 NM .017980 132221 CGCUUGCACAAUGAAGGCUtt -1.84 0.0345 (SEQ ID NO: 61)

GGGACAGGAGCAAAUUGCAtt

25975 (SEQ ID NO: 62) -2.59 0.0026

GGACAACAAGGUUGCUGUAtt

MCM2 4171 NM. _004526 14854 (SEQ ID NO: 63) -1.61 0.0200

CCAUCUAUCAGAACUACCAtt

143883 (SEQ ID NO: 64) -2.40 0.0127

GGGUCAUAAAGGAUUUCAUtt

PPIH 10465 NM. .006347 111675 (SEQ ID NO: 65) -1.66 0.0174

GGGUGGAGAUUUUGUUAAUtt

7458 (SEQ ID NO: 66) -1.74 0.0091

GGAAGAAAUCCAGGAGAAUtt

TE F2IP 54386 NM. .018975 27569 (SEQ ID NO: 67) -1.70 0.0274

GGAGCAUAAGUACCUGCUGtt

27475 (SEQ ID NO: 68) -1.90 0.0149

GGAUGUCUCCCGGUUUUUUtt

THRAP4 9862 NM. .014815 147536 (SEQ ID NO: 69) -1.98 0.0080

CCUUAUGGCUAAGCGCAAAtt

147534 (SEQ ID NO: 70) -2.33 0.0144

GCAGAAAUCGGGAGAGAAUtt

147535 (SEQ ID NO: 71) -2.86 0.0020

GGAAGAUGAUAAGAUCAGAtt

TM9SF2 9375 NM. .004800 15396 (SEQ ID NO: 72) -1.66 0.0035

GGAGACCUGUAAGCUUGUUtt

15304 (SEQ ID NO: 73) -1.82 0.0099

GGCCAAGGCCUUGAAAGGAtt

UCK1 83549 NM. .031432 103370 (SEQ ID NO: 74) -2.01 0.0339

GGACAGGUUCUACAAGGUCtt

1197 (SEQ ID NO: 75) -2.95 0.0099

CCAGUAUGGACCGAUGGGUtt

ZMIZ1 57178 NM. .020338 133056 (SEQ ID NO: 76) -2.12 0.0046

GGCGUAUAACAGCCAAUUCtt

133057 (SEQ ID NO: 77) -2.16 0.0040

GGACAACAAGGUUGCUGUAtt

Tec kinase 4171 NM. .004526 14854 (SEQ ID NO: 78) -0.44 <0.0001

(48h knockdown)

GGUUGUUCAUGAUGCUAACtt

Tec kinase 4171 NM. .004526 383 (SEQ ID NO: 79) -3.90 0.0020

(96h knockdown)

CAAGCUGACCUGAAGU UCtt

GFP 4626 (SEQ ID NO: 80) -3.70 0.0125

(48h knockdown)

Table 1: Putative core components and positive regulators of FGF2 secretion. A list of gene products whose down-regulation caused reduced efficiency of FGF2 secretion. In case one siRNA was found functional a score smaller than -3 combined with a p-value smaller than 0.05 was used as threshold. In case two or three siRNAs were found functional a score smaller than - 1.5 combined with a p-value smaller than 0.05 was used as threshold. The results for Tec kinase and GFP used as controls in large-scale screening are shown at the bottom.

Table 2

Putative negative regulators of FGF2 secretion

Key Gene ID siRNA ID Sequence Score p-Value

GGCACCUACCUUGCAUCUAtt

HSDL2 84263 NM. .032303 109746 (SEQ ID NO: 81) 4.26 0.0080

GGCGAGAGGUACAAUUUUUtt

CCT7 10574 NM. .006429 135794 (SEQ ID NO: 82) 4.16 0.0354

GGUGAAGAUCACUCUCAUCtt

CDH23 64072 NM. .052836 29845 (SEQ ID NO: 83) 4.29 0.0087

CHRM2 1129 NM 000739 1920 GGAUAGUGAAGCCAAACAAtt 6.52 0.0307 (SEQ ID NO: 84)

GGCAACUGAGGAGCAGUUAtt

CSTF2T 23283 NM. .015235 22650 (SEQ ID NO: 85) 4.27 0.0019

GGAUAUGUACACCUUUGACtt

DLG4 1742 NM. .001365 145816 (SEQ ID NO: 86) 3.95 0.0143

CGGUGUGGACCUCCGAAAUtt

FOXJl 2302 NM. .001454 3275 (SEQ ID NO: 87) 4.68 0.0116

GGUACAACAAAUGCUAAUUtt

GAD2 2572 NM. .000818 106265 (SEQ ID NO: 88) 4.34 0.0437

GGGUUUGUUCCUUUCCUCGtt

GJB1 2705 NM. .000166 7112 (SEQ ID NO: 89) 4.47 0.0432

GGAGGAAUAGUGCUUGCUGtt

GRB10 2887 NM. .001001550 16015 (SEQ ID NO: 90) 4.16 0.0032

GGAGGAAGACCAGCAGUUUtt

HOOK2 29911 NM. .013312 20392 (SEQ ID NO: 91) 4.92 0.0151

GGAGACUCUGAUUUUAUAUtt

HSP90Bf 541611 NM. .001014441 258199 (SEQ ID NO: 92) 5.70 0.0435

CCAUCAAACUCUACGUCCUtt

KCNA10 3744 NM. .005549 105465 (SEQ ID NO: 93) 4.07 0.0023

GCUUCCGGGAGACUAUGCUtt

PPWD1 23398 NM. .015342 118110 (SEQ ID NO: 94) 4.57 0.0056

CGUGAGUUGGAGAAGGUUGtt

LYPLA1 10434 NM. .006330 17864 (SEQ ID NO: 95) 5.36 0.0193

GGUAUAAAUGAGCAGUUAAtt

MAPK11 5600 NM. .002751 103311 (SEQ ID NO: 96) 4.66 0.0171

GGACCUGAGCAGCAUCUUCtt

MLL 4297 NM. .005933 107891 (SEQ ID NO: 97) 4.23 0.0138

GGAGAUUCAGGUACUUCCUtt

MPL 4352 NM. .005373 114521 (SEQ ID NO: 98) 4.17 0.0023

GCUGCCUCAUCUCAGGACUtt

MYBPC3 4607 NM. .000256 118345 (SEQ ID NO: 99) 4.12 0.0079

GGUGCUUUUUUCUCCUCAAtt

P2RY10 27334 NM. .014499 5589 (SEQ ID NO: 100) 5.19 0.0008

GGUGUCUGUCGGCAGUUUUtt

POMT2 29954 NM. .013382 111838 (SEQ ID NO: 101) 4.86 0.0059

GGUUGAGGCAUUUUCCUGCtt

PRIC285 85441 NM. .033405 35445 (SEQ ID NO: 102) 4.25 0.0115

GGCAAAUUGGGCCAUUAAAtt

SDR16C5 195814 NM. .138969 110022 (SEQ ID NO: 103) 5.35 0.0023

CCCUCUCUUUUAGAGCAGGtt

RHO 6010 NM. .000539 2171 (SEQ ID NO: 104) 4.03 0.0125

GGGAAAGAAAACUAGCACAtt

SLC2A4 6517 NM. .001042 118928 (SEQ ID NO: 105) 4.16 0.0052

CCUCCAGUCUUCUCUCGUCtt

THNSL1 79896 " NM. .024838 31900 (SEQ ID NO: 106) - 4.49

GCAGAAGAGCUACCUUUGCtt

ZFP57 346171 NM. .001109809 266043 (SEQ ID NO: 107) 4.21 0.0015

GGCAAGUACCUUCCUAGCCtt

ZSCAN21 7589 NM. .145914 6803 (SEQ ID NO: 108) 4.47 0.0031

GGUGCUGGCUGUCAACCAAtt

ACTN4 81 NM. .004924 15570 (SEQ ID NO: 109) 1.89 0.0335

GGACAUGUUCAUCGUCCAUtt

15662 (SEQ ID NO: 110) 1.57 0.0411

GCAUUUCAGGAGGACUUUUtt

ADAMTS15 170689 NM. .139055 105393 (SEQ ID NO: 111) 2.42 0.0492

GGUGCUGCUUCUUAGAGAUtt

37186 (SEQ ID NO: 112) 2.33 0.0193

GGAGGAAUUGUUGAUAAAGtt

AIP1 9863 NM. .012301 19870 (SEQ ID NO: 113) 2.34 0.0241

GGACACGAAAAUAGUUACAtt

20059 (SEQ ID NO: 114) 2.04 0.0463

GGUAGUUGACCAAGCUCAAtt

ARP3BETA 57180 NM. .020445 28503 (SEQ ID NO: 115) 3.14 0.0357

GGUUCAUGGAGCAAGUGGUtt

28599 (SEQ ID NO: 116) 2.20 0.0041

GGCGAGAGGUACAAUUUUUtt

CCT7 10574 NM. .006429 135794 (SEQ ID NO: 117) 4.16 0.0354

GGUACAAUUUUUUUACUGGtt

18113 (SEQ ID NO: 118) 1.61 0.0033

GGCCCGCUCUACAUCUUCUtt

CDKN1A 1026 NM. _078467 1436 (SEQ ID NO: 119) 2.38 0.0297

GGCGGUUAUGAAAUUCACCtt

1531 (SEQ ID NO: 120) 2.09 0.0035

CENTB5 116983 NM. .030649 32840 GGAGACAAAGAAGCAGUUUtt 2.18 0.0044 (SEQ ID NO: 121)

GGAUGUGCGGAAGUUCAAGtt

32752 (SEQ ID NO: 122) 1.63 0.0454

GAAACUUAAUAGCCAGUAUtt

CITEDl 4435 NMJ304143 45072 (SEQ ID NO: 123) 3.24 0.0018

GCAGUGGCCAUUCUGCACUtt

44980 (SEQ ID NO: 124) 1.76 0.0032

CCAAGCCUUCUUAUCUCAAtt

EBP 10682 N _006579 111697 (SEQ ID NO: 125) 2.09 0.0001

GGGAGACAGCCGAUACAUCtt

111698 (SEQ ID NO: 126) 1.94 0.0078

GGAAGAGUUGGAAGUUGCGtt

ELK1 2002 NM_005229 2829 (SEQ ID NO: 127) 3.51 0.0022

GGGUUUGUGCCAGAAACCAtt

2923 (SEQ ID NO: 128) 2.06 0.0086

GGAUUUGAAGAUAUUACGAtt

E CC4 2072 NM_005236 16181 (SEQ ID NO: 129) 1.74 0.0283

GGAGUAUUUUAUCAAUCAGtt

16095 (SEQ ID NO: 130) 1.68 <0.0001

GAUGAAGACGGUGACCAACtt

GPR8 2832 NMJX55286 41935 (SEQ ID NO: 131) 3.02 0.0046

GUAUGUCCUACGUCAUCACtt

42098 (SEQ ID NO: 132) 2.33 0.0225

GGAGGAAGACCAGCAGUUUtt

GRB10 2887 NM_001001550 16015 (SEQ ID NO: 133) 4.92 0.0151

GGAUGUUAAAGUCUUUAGUtt

16193 (SEQ ID NO: 134) 2.52 0.0019

GAACGGCCAAUGCAACGUGtt

KCNJ4 3761 NM_152868 42003 (SEQ ID NO: 135) 1.89 0.0246

GGUGGACUACUCACGUUUUtt

42079 (SEQ ID NO: 136) 1.55 0.0015

GGUAGUGGAGUUAUUACGUtt

MPP4 58538 NMJD33066 35189 (SEQ ID NO: 137) 2.29 0.0126

GGUGUUAUCCUAUGAGGUAtt

35096 (SEQ ID NO: 138) 1.52 0.0047

GGAUUUAUACCGCUGCGCUtt

MYCBP2 23077 NM_015057 22390 (SEQ ID NO: 139) 1.99 0.0078

GGAGAUGAAGAUAAAAACAtt

22582 (SEQ ID NO: 140) 1.53 0.0004

GGAGGUAAAUUCGUCCCUGtt

NICAL 64780 NM_022765 30280 (SEQ ID NO: 141) 4.89 0.0080

GGACCAGCUCAACUACUGGtt

30185 (SEQ ID NO: 142) 1.87 0.0289

GGAGUUGAACAGUAAAGAGtt

NME4 4833 NM_005009 110871 (SEQ ID NO: 143) 2.70 0.0008

GGGUACAAUGUCGUCCGCGtt

610 (SEQ ID NO: 144) 2.02 0.0207

GGUGCUUUUUUCUCCUCAAtt

P2RY10 27334 NM_014499 5589 (SEQ ID NO: 145) 5.19 0.0008

GGAAACCAUCAUUAGCAGUtt

5777 (SEQ ID NO: 146) 1.52 0.0327

GGAGGAAUUUUCUUCCUUUtt

PABPC5 140886 NM_080832 36357 (SEQ ID NO: 147) 2.64 0.0106

GGGUUAUGCCUAUGUUCACtt

3626 (SEQ ID NO: 148) 2.51 0.0042

GCCUAGCAAUAGCAAGAUAtt

PGDS 27306 NM_014485 45408 (SEQ ID NO: 149) 5.73 0.0107

CCUGUUAGACAACCAUCCAtt

118109 (SEQ ID NO: 150) 3.05 0.0221

GGAGCAGUUGGAGAACCUGtt

PMVK 10654 NM_006556 820 (SEQ ID NO: 151) 3.67 0.0014

GGAACAGUAUGCUCAGGAGtt

818 (SEQ ID NO: 152) 2.90 0.0378

GCAGUUCCAAGCUUUCCAUtt

P0LDIP3 84271 NM_178136 147927 (SEQ ID NO: 153) 1.77 0.0013

GGCACAUGCCGAAGAUACUtt

147929 (SEQ ID NO: 154) 1.64 0.0147

GGCGAUGGUGCAACUCAUAtt

PPARGC1B 133522 NM_133263 109964 (SEQ ID NO: 155) 2.60 0.0208

GGCCCUUCCAAUAUGUUUAtt

109965 (SEQ ID NO: 156) 1.80 0.0199

GGAAUGAAAGGGCCAUGCUtt

PRRX2 51450 NM_016307 5653 (SEQ ID NO: 157) 2.28 0.0192

46254 GAACUUCUCGGUGAGCCACtt 1.54 0.0137 (SEQ ID NO: 158)

GCUGACCCUCAAAUACGGAtt

PSEN2 5664 M_ .000447 105551 (SEQ ID NO: 159) 2.31 0.0254

GGAAAAGCCAGUUCCCUACtt

105012 (SEQ ID NO: 160) 1.69 0.0101

GGAGUGAUUAGUUCGGGUUtt

SCN3B 55800 M_ _018400 104693 (SEQ ID NO: 161) 1.73 0.0084

GGAGGCAACUAAGACUCAUtt

104695 (SEQ ID NO: 162) 1.51 0.0026

GGUGUGGUCAUGGUGUUCAtt

SLC2A8 29988 NM. .014580 21522 (SEQ ID NO: 163) 3.81 0.0289

GCCAAGCUUACCCUUCACAtt

117500 (SEQ ID NO: 164) 1.94 0.0471

GCCUCAAUACAAGGCAGCCtt

SLC30A3 7781 NM. .003459 120108 (SEQ ID NO: 165) 2.37 0.0363

CCAAUAAACUUGUGUCUCAtt

120109 (SEQ ID NO: 166) 1.53 0.0034

GGAGAAAUCAAAAUUGUGCtt

SOCS5 9655 NM. .144949 20575 (SEQ ID NO: 167) 2.36 0.0039

GGUACUGUUAAGUAAACCAtt

20660 (SEQ ID NO: 168) 1.97 0.0244

GUUGAAGAUCCCAUACGAGtt

SPC18 23478 NM. .014300 105714 (SEQ ID NO: 169) 1.75 0.0004

GGUGGUCGUAAGUCUUAACtt

104289 (SEQ ID NO: 170) 1.71 0.0233

GGCUUCAUCAAGUUUCCUGtt

SPRR3 6707 NM. .005416 16242 (SEQ ID NO: 171) 2.38 0.0114

GGAGCCAUGCCACUCAAAGtt

16160 (SEQ ID NO: 172) 2.35 0.0117

GGAAGGCACACCACUACACtt

SYK 6850 NM. .003177 381 (SEQ ID NO: 173) 2.65 0.0151

GGAUAAGAACAUCAUAGAAtt

382 (SEQ ID NO: 174) 2.36 0.0301

GGAGGAUGAACUUACUGCUtt

TRALPUSH 116931 NM. .053002 35660 (SEQ ID NO: 175) 2.05 0.0185

GGCUUCAAUAAUCAGCCAGtt

35756 (SEQ ID NO: 176) 1.68 0.0092

GGAUGCCCUGACAUCUUCUtt

TRPM8 79054 NM. .024080 104798 (SEQ ID NO: 177) 3.05 0.0017

GGUGCUUGGAUUCUCACGGtt

104796 (SEQ ID NO: 178) 1.61 0.0038

GGAAAAGACCAACCGGGUUtt

TSKS 60385 NM. .021733 125327 (SEQ ID NO: 179) 2.30 0.0322

CCAACGUGUCACUGCUCAAtt

125326 (SEQ ID NO: 180) 2.03 0.0032

GCCAUUUACAUUAAUGAGCtt

UBE2D3 7323 NM. .181892 120770 (SEQ ID NO: 181) 1.86 0.0046

GCUAUUUGGAUAGUGUAGCtt

120769 (SEQ ID NO: 182) 1.72 0.0069

GGAAGAACCCAGAUUCACAtt

UMP-CMPK 51727 NM. .016308 1006 (SEQ ID NO: 183) 2.73 0.0020

GGAAAGAUUGUACCAGUUGtt

1007 (SEQ ID NO: 184) 1.91 0.0479

GGACAAGAAGCUCUUCCUGtt

ZAP70 7535 NM. .001079 103736 (SEQ ID NO: 185) 1.58 0.0402

GGGCAUUGCUUACACGGAUtt

110759 (SEQ ID NO: 186) 1.58 0.0041

Table 2: Putative negative regulators of FGF2 secretion. A list of gene products whose down- regulation caused increased efficiency of FGF2 secretion. In case one siRNA was found functional a score larger than 4 combined with a p-value smaller than 0.05 was used as threshold. In case two siRNAs were found functional a score larger than 1.5 combined with a p-value smaller than 0.05 was used as threshold.

Membrane association and previously reported functions of candidate gene products In Fig. 2, based on annotations available through the Swiss-Prot protein knowledgebase as well as NCBI, membrane association as well as putative or previously reported functions of the gene products contained in table 1 and 2 are summarized. Results for core components and positive regulators as well as negative regulators are shown separately. We find a large portion of membrane-associated molecules in the range of about 40%. Within the group of soluble factors, the majority is localized intracellularly, however, a substantial population is comprised by secreted factors. In terms of putative functions, a group of proteins was identified that are likely to affect FGF2 secretion in a rather indirect manner. This group makes up about 25% and includes putative transcription factors and metabolic enzymes. Other groups contain transporters, cell surface receptors and factors associated with cytoskeletal elements. Additional prominent groups contain potential regulatory factors such as kinases and phosphatases as well as lipid- modifying factors. The group of gene products with so far unknown functions represents 17% of the factors listed in Tables 1 and 2. Identification of Tec kinase as a novel factor required for unconventional secretion of FGF2

As described above, to establish the experimental system used for large-scale screening, we first conducted a small-scale screen analyzing a siRNA library targeting all kinases known in the human genome. These experiments revealed Tec kinase as a potential factor involved in FGF2 secretion. As depicted in Fig. 3A, in primary screening, one out of three siRNAs directed against Tec kinase was found to severely block FGF2 secretion when cells were down-regulated for 96 hours followed by the induction of FGF2-GFP expression (Tec #1 siRNA, dark orange bars). Statistical analysis of raw data revealed a mean score of -3.9 with a p-value of 0.002 and, therefore, the corresponding phenotype was considered highly significant. Based on these results, the Tec #1 siRNA was also used as a control in the large-scale screen depicted in Fig. 1A and IB (green dots), however, as expected, at a knockdown time of 48 hours, this siRNA did not cause a phenotype based on the criteria defined above. However, due to the large number of individual control experiments (624) using the Tec #1 siRNA, the calculated mean score of -0.44 combined with a p-value smaller than 0.0001 indicated weak but highly significant inhibition of FGF2 secretion under these conditions.

Primary screening data were validated in a well-characterized FGF2 secretion assay that is based on flow cytometry (Engling et al., 2002; Backhaus et al., 2004; Zehe et al., 2006; Temmerman et al., 2008). As shown in Fig. 3B, when both expression level and FGF2-GFP cell surface staining were measured simultaneously, down-regulation of Tec kinase did not affect expression levels, however, caused a substantial drop in FGF2-GFP cell surface expression. In the course of validation experiments, we identified a second independent siRNA directed against Tec kinase (Tec #4) that caused a very similar phenotype (Fig. 3B) arguing against an off-target effect potentially caused by the siRNA Tec #1. We also analyzed whether the siRNAs Tec #1 and #4 may cause pleiotropic effects generally compromising cellular functions under knockdown conditions. Similar to previous studies (Temmerman et al., 2008), we quantified cell surface expression of FGF4-GFP, a signal-peptide containing FGF family member that is transported along the classical secretory pathway. As shown in Fig. 3C, neither siRNA (Tec #1 and #4) had an impact on FGF4-GFP cell surface expression suggesting that down-regulation of Tec kinase specifically blocks FGF2 secretion rather than causing general effects on for example cell viability. This was confirmed by RT-PCR-mediated analyses of Tec kinase mRNA levels that were found reduced when cells were treated with either Tec #1 or Tec #4 siRNAs (Fig. 3D). Quantitation of these data revealed a reduction of Tec kinase mRNA levels by about 50% for both siRNAs under the conditions used (Fig. 3E). FGF2 secretion is blocked in the presence of a pharmacological inhibitor of Tec kinases

In order to obtain independent evidence for a role of Tec kinase, we tested whether an inhibitor of the Tec family of kinases, LFM-A13, has an impact on FGF2 secretion efficiency. As shown in Fig. 4A, compared to control conditions, LFM-A13 did not affect FGF2-GFP expression levels, however, transport of FGF2-GFP to the cell surface was significantly inhibited. Similar to the RNAi experiments described in Fig. 3, LFM-A13 did not affect ER/Golgi-dependent cell surface expression of a FGF4-GFP fusion protein (Fig. 4B). These findings suggest that LFM- A13-mediated inhibition of FGF2 secretion is not caused by pleiotropic effects on for example cell viability but rather indicate that LFM-A13 specifically targets FGF2 secretion by inhibition of Tec kinase.

A direct interaction between Tec kinase and FGF2

To explore the mechanism by which Tec kinase affects FGF2 secretion we analyzed whether Tec kinase and FGF2 are capable of forming a heterodimeric complex. Using recombinant proteins with FGF2 fused to a GST tag, we conducted binding experiments in the absence of other proteinaceous factors. In Fig. 5, following SDS PAGE, both a Western analysis (panel A) and direct protein staining using Coomassie is shown (panel B). While the majority of Tec Kinase was found to interact with GST-FGF2, Tec kinase failed to bind to GST. Using purified components, these data establish a direct and efficient interaction between FGF2 and Tec kinase. Tec-kinase-mediated phosphorylation of FGF2 Due to the inhibitory effect of LFM-A13 on FGF2 secretion (Fig. 4) and based on the direct interaction between FGF2 and Tec kinase as demonstrated in Fig. 5, we reasoned that FGF2 may represent a direct target for Tec-kinase-mediated phosphorylation. Both GST-FGF2 and GST used as a control were incubated in the presence of Tec kinase and [γ³²Ρ] ATP (Fig. 6). Samples were subjected to SDS PAGE and analyzed by both direct protein staining using Coomassie (Fig. 6 A) and autoradiography using a phosphor imager (Fig. 6B). In these experiments a known autophosphorylation property of Tec kinase served as a positive control. Intriguingly, in addition, phosphorylation of GST-FGF2 could indeed be detected when this fusion protein was incubated in the presence of Tec kinase and [γ³²Ρ]ΑΤΡ (Fig. 6B). By contrast, GST was not phosphorylated under these experimental conditions demonstrating that Tec kinase targeted the FGF2 domain in the GST-FGF2 fusion protein. These findings establish Tec-kinase-mediated phosphorylation of FGF2 in the absence of other proteinaceous factors.

Tec kinase phosphorylates tyrosine 82 in FGF2

To further analyze Tec-mediated phosphorylation of FGF2 and to pave the way for functional studies in the cell-based system used in Figs. 3 and 4, we aimed at the identification of the phosphorylation sites in FGF2. Tec kinase targets tyrosine residues and, based on the known crystal structure of FGF2, three out of a total of seven tyrosine residues are surface-exposed. We, therefore, generated variant forms of the GST-FGF2 fusion protein in which single tyrosine residues in position 82, 112 and 124, respectively, were replaced by alanine residues (Fig. 7). These fusion proteins were analyzed for Tec-mediated phosphorylation as described in the experiments shown in Fig. 6. Again we analyzed all samples by both direct protein staining (Fig. 7A) and autoradiography (Fig. 7B) as read-out. All three variant forms of FGF2 were severely impaired in phosphorylation efficiency with a block of overall phosphorylation by about 90% in every single case (Fig. 7C). These findings suggest that the tyrosine residues 82, 112 and 124 in FGF2 are all targeted by Tec kinase and that they represent interdependent phosphorylation sites. Alternatively, it was also appears possible that not all of these tyrosines are getting phosphorylated by Tec kinase but rather some of them may represent essential elements of the FGF2 motif recognized by Tec kinase.

To obtain further insight into Tec-kinase-mediated phosphorylation of FGF2, we conducted mass spectrometry studies to directly address phosphorylation of the three tyrosine residues discussed above. Following incubation of FGF2 in the presence of ATP and Tec kinase, samples were digested with trypsin and the resulting peptides were analyzed by LC-ESI-MS/MS. This revealed a double charged peptide with a m/z value of 581.752 (Fig. 8A). The calculated mass of this peptide (1161.489 Da) correlated with the tryptic FGF2 peptide YLAMKEDGR (amino acids 82-90) carrying one phosphate residue (error = 0.15 ppm). The fragmentation pattern (Figure 8C) confirmed the identity of this peptide demonstrating phosphorylation of Y82. Similarly, we found a peptide with a m/z value of 498.922 (Fig. 8B). The calculated mass of this peptide (1493.744 Da) correlated with the tryptic FGF2 peptide KYTSWYVALKR (amino acids 119- 129) again carrying one phosphate residue (error = 0.35 ppm). The fragmentation pattern of this peptide (Fig. 8D) confirmed its identity demonstrating phosphorylation of Y124. With regard to Y112, only the non-phosphorylated form of the corresponding FGF2 peptide LESNNYNTYR (amino acids 107-116; m/z value = 637.294; calculated mass = 1272.574 [error = 0.30 ppm]) was detected. In conclusion, employing mass spectrometry as a direct method to identify phosphorylated tyrosine residues, we demonstrate that the FGF2 tyrosine residues 82 and 124 are phosphorylated by Tec kinase.

Tec-kinase-mediated phosphorylation is essential for FGF2 secretion

To analyze the functional relevance of the FGF2 phosphorylation sites identified in the experiments shown in Figs. 6-8, we generated FGF2-GFP fusion proteins containing single amino acid substitutions of all three surface tyrosines (Y82, Yl 12 and Y124, respectively) using either alanine or glutamate residues as replacements. While alanine variants were used to prevent Tec-kinase-mediated phosphorylation, glutamate variants were made to structurally mimic a phosphorylated tyrosine residue. Stable HeLa cells were generated by retroviral transduction expressing FGF2-GFP variant forms in a doxycyline-dependent manner. As shown in Fig. 9, using GFP fluorescence as read-out, the expression levels of all of these fusion proteins were similar (Fig. 9, green bars). By contrast, secretion of all FGF2-GFP variant forms was severely impaired in which the tyrosine residues 82, 112 and 124, respectively, were changed to alanine. A similar effect could be observed when tyrosines 112 and 124 were substituted by glutamate, however, replacement of tyrosine 82 by glutamate resulted in a rescue of FGF2 secretion to wild- type levels. All variant forms analyzed in the experiments shown in Fig. 7 were characterized by normal binding efficiencies at wild-type levels towards heparin and heparan sulfates (data not shown). These controls demonstrate that a block of secretion of the FGF2 variant forms Y82A, Y1 12A/E and Y124A/E was not due to a failure to bind to cell surface heparan sulfate proteoglycans (Zehe et al., 2006). Together with the results shown in Figs. 6-8 providing direct proof of Tec-kinase-mediated phosphorylation of tyrosine 82 in FGF2, our findings establish this posttranslational modification to be essential for FGF2 secretion. REFERENCES

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Claims

1. A method for identifying a compound which reduces/inhibits the cellular export of fibroblast growth factor 2 (FGF2) by inhibiting FGF2 phosphorylation, wherein said method comprises the step of contacting a test compound with Tec kinase and FGF2.

2. The method of claim 1, wherein the ability of the test compound to inhibit the phosphorylation of FGF2 is assessed by comparing FGF2 phosphorylation in presence and absence of the test compound.

3. The method of claim 1 or 2, wherein the compound inhibits FGF2 phosphorylation by inhibiting Tec kinase activity, by binding to one or more phosphorylation sites within FGF2, and/or by reducing/inhibiting the interaction between Tec kinase and FGF2.

4. The method of any one of claims 1 to 3, wherein a test compound is considered to inhibit FGF2 phosphorylation if the phosphorylation is reduced by at least 60%, preferably by at least 70%, more preferably by at least 80%, more preferably by at least 90% in presence of the test compound when compared to the phosphorylation in absence of the test compound.

5. The method of any one of claims 1 to 4, wherein FGF2 comprises, essentially consists of, or consists of an amino acid sequence selected from the group consisting of:

(ii) an amino acid sequence that is at least 80% identical to any of the amino acid sequences of (i), and

(iii) an amino acid sequence consisting of at least 10 amino acid residues corresponding to at least 10 consecutive amino acid residues of any of the amino acid sequence of (i) or (ii) and comprising at least one of the amino acid residues corresponding to the amino acid residues Tyr82, Tyrl l2, and Tyrl24 of the amino acid sequence set forth in SEQ ID NO: 1.

6. The method of any one of claims 1 to 5, wherein the compound binds to at least one of the amino acid residues within FGF2 corresponding to amino acid residues Tyr82, Tyrl l2, and Tyrl24 of the amino acid sequence set forth in SEQ ID NO: 1.

7. The method of any one of claims 1 to 6, which is performed using an in vitro assay or a cell-based assay or a combination thereof.

8. The method of any one of claims 1 to 7, wherein the method comprises incubating Tec kinase and FGF2 with labeled ATP in presence or absence of the test compound and comparing the levels of FGF2 phosphorylation in reactions with and without test compound.

9. The method of claim 8, wherein the labeled ATP is gamma-32P-ATP.

10. The method of any one of claims 1 to 9, wherein the compound exhibits the capability to inhibit FGF2 phosphorylation of at least one of the amino acid residues within FGF2 corresponding to amino acid residues Tyr82, Tyrl 12, and Tyrl24 of the amino acid sequence set forth in SEQ ID NO: 1 , preferably of the amino acid residue within FGF2 corresponding to the amino acid residue Tyr82 of the amino acid sequence set forth in SEQ ID NO: 1.

11. A method for identifying a compound which reduces/inhibits the cellular export of fibroblast growth factor 2 (FGF2) by reducing/inhibiting the interaction between Tec kinase and FGF2, wherein said method comprises the step of contacting a test compound with Tec kinase and FGF2.

12. The method of claim 1 1, wherein the ability of the test compound to reduce/inhibit the interaction between Tec kinase and FGF2 is assessed by comparing the interaction between Tec kinase and FGF2 in presence and absence of the test compound.

13. The method of claim 11 or 12, wherein the test compound is added after Tec kinase and FGF2 have been incubated to interact with each other, or wherein Tec kinase, FGF2, and the test compound are added concomitantly to the test reaction, or wherein one of Tec kinase and FGF2 are added first, then the test compound is added, and then the other of Tec kinase and FGF2 is added.

14. The method of any one of claims 11 to 13, wherein a test compound is considered to inhibit the interaction between Tec kinase and FGF2 if the interaction is reduced by at least 60%, preferably by at least 70%, more preferably by at least 80%, more preferably by at least 90% in presence of the test compound when compared to the interaction in absence of the test compound.

15. The method of any one of claims 11 to 14, wherein FGF2 comprises, essentially consists of, or consists of an amino acid sequence selected from the group consisting of:

(ii) an amino acid sequence that is at least 80% identical to any of the amino acid sequences of (i),

(v) an amino acid sequence consisting of at least 10 amino acid residues corresponding to at least 10 consecutive amino acid residues of any of the amino acid sequences of (i), (ii), (iii), or (iv) and comprising at least one of the amino acid residues corresponding to amino acid residues Tyr82, Tyrl l2, and Tyrl24 of the amino acid sequence set forth in SEQ ID NO: 1.

16. The method of any one of claims 11 to 15, wherein the compound binds to FGF2, preferably to at least one of the amino acid residues corresponding to amino acid residues Tyr82, Tyrl 12, and Tyrl24 of the amino acid sequence set forth in SEQ ID NO: 1.

17. The method of any one of claims 1 to 16, wherein Tec kinase comprises, essentially consists of, or consists of an amino acid sequence selected from the group consisting of:

(ii) an amino acid sequence that is at least 80% identical to any of the amino acid sequences of (i).

18. A method for identifying a compound which reduces/inhibits the cellular export of FGF2 by binding specifically to phosphorylated FGF2, wherein said method comprises the step of contacting a test compound with FGF2 which is phosphorylated at the amino acid residue corresponding to amino acid residue Tyr82 in SEQ ID NO: 1 or which comprises a phosphomimetic amino acid residue at this position.

19. The method of claim 18, wherein the ability of the test compound to specifically bind to phosphorylated FGF2 is assessed by determining the ability of the test compound to bind to FGF2 which is phosphorylated at the amino acid residue corresponding to amino acid residue Tyr82 in SEQ ID NO: 1 or which comprises a phosphomimetic amino acid residue at this position compared to its ability to bind to FGF2 without phosphorylation or a phosphomimetic amino acid residue at this position.

20. The method of claim 18 or 19, wherein the FGF2 which is contacted with the test compound comprises, essentially consists of, or consists of an amino acid sequence selected from the group consisting of:

(ϋ) the amino acid sequence set forth in SEQ ID NO: 1, in which the amino acid residue Tyr82 is a glutamate or aspartate residue, or a functionally equivalent part thereof comprising said glutamate or aspartate residue,

(iii) an amino acid sequence that is at least 80% identical to any of the amino acid sequences of (i) or (ii) and is phosphorylated at the amino acid position corresponding to amino acid residue Tyr82 of the amino acid sequence set forth in SEQ ID NO: 1 or comprises a glutamate or aspartate residue at this position, and

(iv) an amino acid sequence consisting of at least 10 amino acid residues corresponding to at least 10 consecutive amino acid residues of any of the amino acid sequences of (i), (ii), or (iii) and comprising the amino acid residue corresponding to the amino acid residue Tyr82 of the amino acid sequence set forth in SEQ ID NO: 1.

21. The method of any one of claims 1 to 20, wherein said method further comprises the step of testing whether the test compound is capable of penetrating cell membranes, preferably mammalian cell membranes.

22. The method of any one of claims 1 to 21, wherein said method further comprises the step of testing whether the test compound is capable of reducing/inhibiting cellular export of FGF2.

23. The method of any one of claims 1 to 22, wherein the cellular export of FGF2 is considered inhibited if the export is reduced by at least 60%, preferably by at least 70%, more preferably by at least 80%, more preferably by at least 90% in presence of the test compound when compared to the cellular export in absence of the test compound.

24. The method of claim 22 or 23, wherein the testing for cellular export is performed using a cell selected from the group consisting of a cell that endogenously expresses Tec kinase and/or FGF2 or a cell that is exogenously provided with nucleic acid sequences that encode Tec kinase and/or FGF2.

25. The method of any one of claims 1 to 24, wherein the compound is capable of reducing/inhibiting cellular export of FGF2 from a cell selected from the group consisting of a tumor cell, an inflammatory cell (leukocyte), a stromal cell, a keratinocyte, a fibroblast, and an endothelial cell.

26. The method of any one of claims 1 to 25 performed in a high-throughput setting.

27. The method of any one of claims 1 to 26, wherein said test compound is selected from the group consisting of a small molecule, a peptide, a protein, and an antibody.

28. The method of any one of claims 1 to 27, wherein the test compound is encoded by a nucleic acid.

29. The method of claim 28, wherein the nucleic acid is comprised in a vector, preferably a viral vector.

30. The method of any one of claims 1 to 29, wherein said method further comprises the step of formulating said compound or a pharmaceutically acceptable salt thereof with one or more pharmaceutically acceptable excipient(s), diluent(s), and/or carrier(s).

31. A compound identifiable by the method of any one of claims 1 to 30.

32. The compound of claim 31 which is an antibody.

33. The compound of claim 32, wherein the antibody is capable of binding to Tec kinase or to FGF2, preferably to at least one of the amino acid residues within FGF2 which correspond to the amino acid residues Tyr82, phosphorylated Tyr82, Tyrl l2, and Tyrl24 of the amino acid sequence set forth in SEQ ID NO: 1.

34. The compound of any one of claims 31 to 33, which has the ability of reducing/inhibiting angiogenesis and/or neovascularization.

35. A pharmaceutical composition comprising the compound of any one of claims 31 to

34 and one or more pharmaceutically acceptable excipient(s), diluent(s), and/or carrier(s).

36. A compound of any one of claims 31 to 34 or a pharmaceutical composition of claim

35 for treating and/or preventing an angiogenesis- and/or neovascularization-dependent disease or condition.

37. A compound of any one of claims 31 to 36 or a pharmaceutical composition of claims 35 or 36 for treating and/or preventing a disease or condition selected from the group consisting of cancer, atheroscleorsis, psoriasis, warts, pyogenic granuloma, allergic oedema, keloid scars, disease of hormonal growth control (benign prostatic hyperplasia, thyroid hyperplasia), thyroiditis, Crohn's disease, endometriosis, pre-eclampsia, recurrent miscarriage, dysfunctional uterine bleeding, follicular cysts, ovarian hyperstimulation, macular degeneration, diabetic retinopathy, choroidal and other intraocular disorders, hemangioma, hemangioendothelioma, retinopathy of prematurity, obesity, arthritis (e.g., rheumatoid arthritis), synovitis, osteomyelitis, pannus growth, osteophyte formation, nasal polyps.

38. A method for treating and/or preventing an angiogenesis- and/or neovascularization- dependent disease or condition in an individual in need thereof comprising the step of administering the compound of any one of claims 31 to 34 or the pharmaceutical composition of claim 35 in a pharmaceutically effective amount to said individual.

39. The method of claim 38, wherein the individual is a mammal, preferably a human.

40. The method of claim 38 or 39, wherein the angiogenesis- and/or neovascularization- dependent disease or condition is selected from the group consisting of cancer, atheroscleorsis, psoriasis, warts, pyogenic granuloma, allergic oedema, keloid scars, disease of hormonal growth control (benign prostatic hyperplasia, thyroid hyperplasia), thyroiditis, Crohn's disease, endometriosis, pre-eclampsia, recurrent miscarriage, dysfunctional uterine bleeding, follicular cysts, ovarian hyperstimulation, macular degeneration, diabetic retinopathy, choroidal and other intraocular disorders, hemangioma, hemangioendothelioma, retinopathy of prematurity, obesity, arthritis (e.g., rheumatoid arthritis), synovitis, osteomyelitis, pannus growth, osteophyte formation, nasal polyps.

41. The method of any one of claims 38 to 40, wherein administration is parenteral, preferably by intravenous, intramuscular, intraperitoneal, or subcutaneous injection.

42. The method of any one of claims 38 to 41, wherein the individual is also treated with one or more other therapeutic agents and/or therapies.

43. The method of any one of claims 38 to 42, wherein the angiogenesis- and/or neovascularization-dependent disease is cancer and the individual is also treated with other anti-tumor therapeutics, preferably anti-cancer chemotherapeutics and/or anti-cancer immunotherapeutics .