EP1940875A2

EP1940875A2 - Fibroblast growth factor receptor-derived peptides

Info

Publication number: EP1940875A2
Application number: EP06791473A
Authority: EP
Inventors: Vladimir Berezin; Vladislav V. Kiselyov; Elisabeth Bock
Original assignee: Enkam Pharmaceuticals AS
Current assignee: Enkam Pharmaceuticals AS
Priority date: 2005-10-19
Filing date: 2006-10-17
Publication date: 2008-07-09
Also published as: WO2007045247A2; WO2007045247A3; JP2009511614A

Abstract

The present invention relates to novel peptides capable of binding to a receptor of the fibroblast growth factor receptor family (FGFRs) and modulating activity of said receptor. The peptides of the invention are fragments of FGFRs, wherein said fragments comprise amino acid residues involved in the reciprocal interaction of the Immunoglobulin-like modules 2 (Ig2) and Immunoglobulin-like module 1 (Ig1) of FGFR. The invention discloses amino acid sequences derived from the reciprocal Ig 1 -to-lg2 binding site of FGFR and relates to use of the peptides comprising said amino acid sequences for the treatment of different pathological conditions, wherein FGFRs play a prominent role. Accordingly, pharmaceutical compositions comprising the compounds of the invention are also concerned.

Description

Fibroblast growth factor receptor-derived peptides

Field of invention

The present invention relates to novel peptides that are capable of binding to a receptor of the fibroblast growth factor receptor family (FGFRs) and modulating activity of said receptor. The peptides of the invention are peptide fragments of FGFRs and derived from a binding site for the reciprocal binding of the immunoglobulin-like modules 1 and 2 of FGFR. The invention discloses amino acid sequences derived from the reciprocal Ig1-to-lg2 binding site of FGFR and relates to use of peptides comprising said amino acid sequences and pharmaceutical compositions comprising thereof for the treatment of different pathological conditions, wherein FGFRs play a prominent role.

Background of invention

Fibroblast growth factor receptors (FGFRs) are a family of closely related transmembrane tyrosine kinases (FGFR1 - FGFR4). FGFR activation and signalling are dependent on dimerization of the receptor which is induced by binding of FGFR natural ligand of the fibroblast growth factor family (FGFs), and it also requires participation of cell surface heparin or heparan sulphate proteoglycans (McKeehan et al., 1998; ltoh and Ornitz, 2004). FGFs (FGF1-FGF23) and FGFRs constitute an elaborate signaling system that participates in many developmental and repair processes of virtually all mammalian tissues (Bottcher and Niehrs, 2005), in particular, they play a prominent role in functioning of the peripheral and central neural system in the adult (of 23 members of the FGF family, ten have been identified in the brain (Jungnickel et al, (2004) MoI Cell Neurosci. 25:21-9; Reuss and von Bohlen und Halbach (2003) Cell Tissue Res. 313:139-57)).

The prototypical FGFR consists of three immunoglobulin-like modules (Ig1 - Ig3), a trans-membrane domain and a cytoplasmic tyrosine kinase domain. The linker region between the Ig1 and Ig2 modules is very long, consisting of 20-30 amino acid residues, including a stretch of acidic amino acids termed the acid box. FGFRs also bind heparin/heparan sulphate, which is required for the high-affinity FGF-FGFR interaction (Yayon et al., 1991 ; Ornitz et al., 1992). Binding studies of several FGFs to various FGFR fragments and crystal structures of several FGFs in complex with fragments of FGFRs consisting of the Ig2 and Ig3 modules indicate that these modules and the Ig2-lg3 linker region are sufficient for the specific FGF-FGFR interaction (Wang et al., 1995a; Plotnikov et a/., 1999; Pellegrini et al., 2000). FGF-FGFR binding results in dimerisation of FGFR leading to auto-phosphorylation of the re- ceptor tyrosine kinase domains. Based on crystal structures of the ternary FGF- FGFR-heparin complex, two models, a symmetric and an asymmetric, of FGFR dimerisation have been proposed. In the symmetric model (Plotnikov et al., 1999; Plotnikov et al., 2000; Schlessinger et al., 2000), dimerisation of the two FGF-FGFR complexes is stabilized by 1) a bivalent FGF-FGFR interaction through a primary and a secondary interactions site, 2) a direct FGFR-FGFR interaction, and 3) hepa- rin-FGF and heparin-FGFR interactions. In the asymmetric model (Pellegrini et al., 2000), there are no protein-protein contacts between the two FGF-FGFR complexes, and the ternary complex is stabilized only by heparin-FGF interactions. Recently, it has been shown that mutations abolishing interactions at the secondary FGF-FGFR interaction site (observed only in the symmetrical model) lead to diminished FGFR activation without affecting the FGF-FGFR binding, supporting the symmetric model (Ibrahimi et al., 2005). Regulation of the binding specificity of FGFs is primarily achieved by alternative splicing of FGFRs. Alternative splicing of exons encoding the C-terminal part of the Ig3 module in FGFR1-3 results in two isoforms (3b and 3c) possessing different FGF binding specificity (Miki. et al., 1992; Yayon et al., 1992). There are also FGFR isoforms lacking the Ig1 module (FGFR1 and 2), the Ig1 module combined with the Ig1-lg2 linker sequence (FGFR2), or the Ig1-lg2 linker alone (in FGFR3) (McKeehan et al., 1998; Shimizu et al., 2001).

The structure of the Ig 1 module is unknown and the physiological significance of the module is not well elucidated. Although the Ig1 module is dispensable for FGF- FGFR binding, the triple Ig-module form of FGFR1 (FGFRIα) has an 8- and 3-fold lower affinity for FGF1 and heparin, respectively, compared to the double Ig-module form (FGFRIβ) (Wang et al., 1995b). Since deletion of the Ig1-lg2 linker in FGFRIα abolishes the inhibitory effect of the Ig 1 module, it has been suggested that the Ig 1- Ig2 linker forms a flexible hinge allowing the Ig 1 module to interact with the Ig2/lg3 modules (Wang et al., 1995b), and this assumption has recently been supported by Olsen et al. (2004) who showed that the Ig 1 module of FGFR3 binds to FGFR3β with a dissociation constant (Kd) of 20 μM. A large body of evidence accumulated by now indicates that FGFR is involved in intracellular signal transduction associated with major neural cell adhesion molecules NCAM, L1 and N-cadherin involved in neural cell differentiation, survival and plasticity (Williams et al. (1994) Neuron 13:83-94; Doherty and Walsh, 1996; Kise-

NCAM has recently been regarded as a member of a new class of putative alternative ligands of FGFR, low affinity ligands (Kiselyov et al., 2003; Kiselyov et al. (2005) J Neurochem 94:1169-1179). There has been obtained evidence for a direct interac- tion between NCAM and the receptor and stimulation of FGFR by NCAM (Kiselyov et al. (2003) Structure (Camb) 11 :691-701 ). The identified NCAM fragment having the sequence EVYWAENQQGKSKA (FGL peptide) involved in the interaction between NCAM and FGFR has been suggested as a new candidate drug for the treatment of a variety of pathologic conditions where the activity of FGFR may play a role (WO 03/016351). WO 03/016351 describes some biological effects of the FGL peptide due to binding and activating FGFR.

The mulfunction of FGFRs has been associated with a number of diseases, e. g. cancer, which signifies the receptors as important targets for therapeutic interven- tion. However, the multitude of biological processes in regulation of which FGFRs play a prominent role and plethora of FGFR ligands make such search to be a very challenging task.

The present invention provides new compounds which may be advantageously used for modulating FGFR activity and FGFR-dependent biological processes, and thus the invention provides new candidate drugs for the treatment of FGFR-related diseases.

Summary of invention

The present invention describes a novel FGFR binding site for the reciprocal binding of FGFR Ig1 and Ig2 modules of same FGFR molecule and discloses peptide sequences that are capable of binding to said binding site and thereby modulate the receptor activation. Thus, in a first aspect the invention relates to novel peptide sequences capable of binding to FGFR and modulating FGFR activity.

According to the invention, a peptide sequence which is capable of binding to the reciprocal Ig1-to-lg2 binding site comprises the amino acid motif Q/E-(x)₃-x^p, wherein x^p is a hydrophobic amino acid residue and

(x)_c is an amino acid sequence of three amino acid resides, wherein x is any amino acid residue, or amino acid motif K/R-x^p-(x)_o-i-K/R, wherein x^p is a hydrophobic amino acid residue, and (x) is a charged amino acid residue.

Preferably, a peptide of the invention comprises of about 25 amino acid residues and comprises a fragment of FGFR derived from the reciprocal Ig1-to-lg2 binding site. The invention discloses amino acid sequences derived from the reciprocal binding site of the invention and relates to pharmaceutical compositions comprising thereof. Invention also relates to uses of the peptides and pharmaceutical compositions comprising thereof for the treatment of different pathological conditions, wherein FGFRs play a role in pathology and/or recovery from disease, for example for a) treatment of conditions of the central and peripheral nervous system associated with postoperative nerve damage, traumatic nerve damage, impaired myelina- tion of nerve fibers, postischaemic damage, e.g. resulting from a stroke, Parkinson's disease, Alzheimer's disease, Huntington's disease, dementias such as multiinfarct dementia, sclerosis, nerve degeneration associated with diabetes mellitus, disorders affecting the circadian clock or neuro-muscular transmission, and schizophrenia, mood disorders, such as manic depression; b) treatment of diseases or conditions of the muscles including conditions with impaired function of neuro-muscular connections, such as after organ transplanta- tion, or such as genetic or traumatic atrophic muscle disorders; or for treatment of diseases or conditions of various organs, such as degenerative conditions of the gonads, of the pancreas such as diabetes mellitus type I and II, of the kidney such as nephrosis and of the heart, liver and bowel; c) promotion of .wound-healing; d) prevention of death of heart muscle cells, such as after acute myocardial infarction; e) promotion of revascularsation; f) stimulation of the ability to learn and/or the short and/or long-term memory; g) prevention of cell death due to ischemia; h) prevention of body damages due to alcohol consumption; i) treatment of prion diseases; j) treatment of cancer.

Description of Drawings

Figure 1. Structure of the FGFR1 Ig1 module. A) Ribbon representation of the structure of the Ig 1 module. B) An overlay of the backbone atoms of 20 superimposed structures of the Ig1 module. C) Comparison of the structures of the A/A' loop region of the Ig1 and Ig3 modules.

Figure 2. Binding of the FGFR1 Ig1 module to the combined FGFR Ig2-lg3 modules. A) A plot of the equilibrium binding level of the Ig1 module to the lg2-3 modules versus the concentration of the Ig 1 module is shown. The binding was studied by means of SPR analysis. The Ig1 module was injected into the sensor chip at the specified concentrations. The binding is given as a response difference between the binding to the sensor chip with the immobilized lg2-3 modules and a blank sensor chip (unspecific binding). The binding is given as an average of six replicates, with the error bar showing standard deviations. The data were fitted with the theoretical curve in order to calculate the dissociation constant (Kd). B) Changes in chemical shifts of 0.5 mM ¹⁵N labeled Ig1 module after addition of 2 mM unlabeled Ig2 module. C) Changes in chemical shifts of 0.5 mM ¹⁵N labeled Ig2 module after addition of unlabeled Ig1 module. The chemical shifts were determined from the ¹⁵N- HSQC spectra. The data are given as averages from two independent experiments. The change of the chemical shift was calculated using the following expression: ((5*ΔH)^Λ2+ (ΔN)^Λ2)^Λ0.5, where ΔH is the change of the ¹H chemical shift and ΔN is the change of the ¹⁵N chemical shift.

Figure 3. Mapping of the various FGFRI-ligand binding sites onto the structures of the FGFR1 Ig1 and Ig2 modules. A) Mapping of the residues of the Ig2 module perturbed by the interaction with the Ig1 module onto the structure of the Ig2 module; and mapping of the residues of the Ig 1 module perturbed by the interaction with the Ig2 module onto the structure of the Ig 1 module. B) Mapping of the residues of the Ig2 module involved in binding to the Ig2 module, heparin, FGF through the primary interaction site (according to both the symmetrical and asymmetrical model) and secondary interaction site (according to the symmetrical model).

Figure 4. Quantitative analysis of the auto-inhibitory effect of the FGFR1 Ig1 module. A) The structure of FGFRIα (triple Ig form) is depicted schematically with various random conformations of the Ig1-lg2 linker (assuming that there is no interaction between the Ig 1 and Ig2 modules). The circle corresponds to the average distance between the N-termini of the FGFR1 Ig1 and Ig2 modules. B) Simulation of the FGF1-FGFR1β (two-lg form) binding, (blue), FGF1- FGFRIα (triple Ig form) binding (green) and the intra-molecular Ig1-lg2 binding (red) for the various Ig1-lg2 linker lengths and FGF1 concentrations. C) The data points for the FGF1- FGFRIα binding (from panel B) were fitted with a curve describing the single site binding in order to calculate the apparent dissociation constant values. D) A plot of the affinity decrease due to the auto-inhibitory effect of the Ig 1 module versus the dissociation constant (Kd) of the Iigand-FGFR1 β (two-lg form) binding.

Figure 5. Effect of the FGFR1 Ig1 and Ig2 modules and the derived peptides on phosphorylation of FGFR1. TREX-293 cells, stably transfected with FGFR1 containing a C-terminal Strepll-tag, were stimulated for 20 min with either 100 ng/ml FGF1, or other compounds at the specified concentrations. FRD1 stands for the Ig1 module, FRD2 - the Ig2 module, FRDIa - peptide corresponding to the Ig1-lg2 binding site in the Ig 1 module, FRD2a and FRD2b - peptides corresponding to the Ig1-lg2 binding site in the Ig2 module. After stimulation, FGFR was immunopurified using anti-phosphotyrosine antibodies and then analyzed by immunoblotting using antibodies against the Strepll-tag (Panel A). Quantification of FGFR1 phosphorylation (Panel B) was performed by densitometric analysis of the band intensity. Phosphorylation was estimated relative to the control (untreated cells), which was normalized to 100%. Error bar represents one standard error of the mean. P<0.05 (marked by ^*) and P<0.01 (marked by ^**) by paired t-test when comparing treated cells with controls (the t-test was performed on six independent sets of non-normalized data).

Figure 6. Estimation of the average distance between the N termini of the Ig1 and Ig2 modules of FGFR1. r is the distance between the N termini of the Ig 1 and

Ig2 modules, / is the linker length, p is the length of the Ig 1 module, and φ is the angle between a long axis of the Ig1 module and the straight line connecting the N terminal of the Ig2 module and the C terminal of the Ig1 module.

Figure 7. Neurite outgrowth of cerebellar granular neurons in response to treatment with the peptides FRD2a (SEQ ID NO:8 )(A) and FRD2b (SEQ ID NO:13) (B) derived from the Ig1-to-lg2 reciprocal binding site of FGFR The length of neurites in cultures presented as a percentage of neurite length in the treated cultures compared to control (untreated cultures).

Detailed description of the invention

1. Receptor

"Fibroblast Growth Factor Receptor" or "FGFR" refers to a polypeptide that specifi- cally binds one or more fibroblast growth factors. A FGFR typically is a single chain transmembrane polypeptide that comprises an extracellular domain consisting of three imunoglogulin-like modules (Ig modules) Ig1 , Ig2 and Ig3 interconnected through the linker regions, a transmembrane domain and cytoplasmic tyrosine kinase domain.

The invention relates to a functional cell-surface FGFR. By "functional cell-surface receptor" is meant a receptor that is located in the outer plasma membrane of the cell and has an identifiable group of extracellular ligands. A "ligand" is any molecule that binds to a specific site in the receptor molecule. Binding of ligands to the recep- tor at specific sites usually occurs extracellularly and typically causes a change in the receptor molecule which is transferred further through the membrane and induces intracellular signal transduction which results in a physiological response of the cell. The physiological response of a cell, such as for example a change in cell metabolism, induction of cell differentiation, termination or induction of cell prolifera- tion, survival or death of the cell, change in motile behavior of the cell, depends on which of the specific binding sites of a receptor is/are occupied by a ligand, on intracellular and extracellular environments of the receptor and/or on particularity of the ligand-receptor interaction, e.g. affinity and/or duration of interaction.

Ligand binding to a receptor often leads to a change in the activation status of the receptor, e.g. the receptor becomes capable of initiating a cascade of biochemical reactions inside the cell resulting in one or more of the above mentioned cellular responses. Binding of the ligand may also results in the inhibition of receptor activity which means that the receptor becomes unable of initiating a cascade of biochemi- cal reactions which is normally initiated due to ligand binding.

A group of FGFR ligands so far described in the art includes fibroblast growth factors (FGFs), heparin and neural cell adhesion molecules NCAM, L1 and N-cadherin. Binding sites for the latter ligands are different and located in the extracellular part of FGFR molecule. One individual FGFR molecule can also extracellularly bind another individual FGFR molecule at a specific binding site located in the Ig2 module making thus a- FGFR dimer. The FGFR-to-FGFR binding in essential for FGFR activity and occurs in the course of the ligand binding, being thus a ligand assisted - receptor binding/dimerization. Spontaneous dimerisation of two individual FGFR molecules takes place seldom and usually associated with pathological conditions.

According to the present invention, an individual FGFR molecule comprising Ig1 and Ig2 modules separated by a linker region comprises a binding site for the recoprocal interaction if the Ig1 and Ig2 module interaction of which prevents interaction of said individual FGFR molecule wit another individual FGFR molecule and thus prevent dimerisation of FGFR. The reciprocal interaction of Ig 1 and Ig2 modules involves specific amino acid residues of the modules. The interacting amino acid residues of the reciprocal binding site include residues of both the Ig1 and Ig2 modules of the same FGFR molecule.

Thus, according to the invention, the reciprocal interaction of the Ig1 and Ig2 modules of the same FGFR polypeptide prevents interaction said FGFR polypeptide with another FGFR polypeptide and thus prevents FGFR self-activation at the absence of FGFs. The reciprocal binding of the Ig 1 and Ig2 modules of the same FGFR mole- cule according to the invention also attenuates activating of FGFR by a ligand, e. g. a FGF, heparin, cell adhesion molecule, by decreasing the affinity of the FGFR - ligand interaction and/or decreasing the stability of the FGFR-ligand receptor complex, inhibiting thus FGFR activity.

Accordingly, in a first aspect, the invention relates to a reciprocal Ig1-to-lg2 binding site in FGFR and to compounds that are capable of modulating the interaction of the Ig1 and Ig2 modules at this binding site.

In one embodiment the compounds of the invention are isolated peptides which comprise a fragment of FGFR comprising amino acid residues involved in the reciprocal interaction of the Ig1 and Ig2 modules. It is a preferred embodiment of the invention that the peptides comprise a fragment of FGFR which comprises a contiguous amino acid sequence derived from the reciprocal Ig1-to-lg2 binding site. In one embodiment, the peptide may comprise a contagious amino acid sequence which is derived from a part of the reciprocal Ig1-to-lg2 binding site which comprises amino acid residues of the Ig 1 module. In another embodiment, the peptide according to the invention may comprise a contiguous amino acid sequence which is derived from a part of the reciprocal Ig1-to-lg2 binding site which comprises amino acid residues of the Ig2 module.

Accordingly, in one embodiment, the peptide according to invention may be capable of interacting with the Ig1 module of a FGFR of at the reciprocal Ig1-to-lg2 binding site. In another embodiment, the peptide according to the invention may be capable of interacting with the Ig2 module of a FGFR at the reciprocal Ig1-to-lg2 binding site.

A peptide of the invention is capable of binding to any FGFR, such as FGFR1 , FGFR2, FGFR3, FGFR4 and FGFR5 or it may bind to a variant of any of FGFR1-5, such as a natural or recombinant FGFR variant, for example a FGFR variant produced due alternative splicing, e.g. FGFRIb or FGFR2b, or genetic polymorphism, or any type of recombinant FGFR. It is to be understood that a FGFR of the invention and a variant thereof comprise the reciprocal Ig1-to-lg2 module binding site described herein, or comprise at least a part of said reciprocal binding site. Examples of FGFRs of the invention which comprise the reciprocal binding site of the invention may be the FGFR polypeptides identified in the GenBank database as Ass. Nos: P11362 (corresponding to human FGFR1), P21802 (corresponding to human FGFR2), P22607 (corresponding to human FGFR3), P22455 (corresponding to human FGFR4) or AAK26742 (corresponding to human FGFR5).

The peptide according to the invention is a peptide which is capable of modulating activity of FGFR. In one embodiment the peptide may be capable of activating FGFR. In another embodiment, the peptide may be capable of inhibiting FGFR.

In a preferred embodiment FGFR of the invention is FGFR1 or a variant thereof. Thus, in one embodiment FGFR1 may be activated by the peptide of the invention. In another embodiment, FGFR1 may be inhibited by the peptide of the invention.

Amino acid sequence

Peptides according to the invention comprise a fragment of FGFR which comprises a contigous amino acid sequence derived from the reciprocal Ig1-to-lg2 binding site. Such amino acid sequence according to the invention may be selected from the following amino acid sequences:

TLPEQAQPWGA (SEQ ID NO: I )

TKYQISQPEV (SEQ ID NO:2) EPGQQEQLV (SEQ ID NO:3)

SLEQQEQKL (SEQ ID NO:4)

SLVEDTTLEPEEP (SEQ ID NO:5)

GTEQRWGRAAEV (SEQ ID NO:6)

EASEEVELEPCLA (SEQ ID NO:7) EKMEKKLHAV (SEQ ID NO:8)

EKMEKRLHAV (SEQ ID NO:9)

ERMDKKLLAV (SEQ ID NO:10)

QRMEKKLHAV (SEQ ID NO:11 )

SKMRRRVIAR (SEQ ID NO:12) AAKTVKFKC (SEQ ID NO:13)

AANTVKFRC (SEQ ID NO:14)

AANTVRFRC (SEQ I D NO: 15)

AGNTVKFRC (SEQ ID NO:16) or

VGSSVRLKC (SEQ ID NO:17). In some preferred embodiments, the peptide according to the invention may comprise a contigious amino acid sequence which is derived from a part of the reciprocal Ig1-to-lg2 binding site which comprises amino acid residues of the Ig1 module of FGFR. Accordingly, in this embodiment the invention concerns the following se- quences:

TLPEQAQPWGA (SEQ ID NO:1)

TKYQISQPEV (SEQ ID NO:2)

EPGQQEQLV (SEQ ID NO:3)

SLEQQEQKL (SEQ ID NO:4) SLVEDTTLEPEEP (SEQ ID NO:5)

GTEQRWGRAAEV (SEQ ID NO:6)

EASEEVELEPCLA (SEQ ID NO:7).

According to the invention the amino acid sequences identified as SEQ ID NOs: 1-7 all comprise a binding motif which is essential for interaction of said sequences with amino acid residues of the Ig1-to-lg2 reciprocal binding site of FGFR located in the Ig2 module, said binding motif has the following formula Q/E-(x)₃-x^p, wherein x^p is a hydrophobic amino acid residue and (x)₃ is an amino acid sequence of three amino acid resides, wherein x is any amino acid residue. According to the invention the residues x^p may be any hydrophobic amino acid residue, however residues V, L or P are preferred. It is also preferred that amino acid sequence (x)₃ comprises at least one residue Q or at least one residue E, even more preferred that the (x)₃ sequence further comprises either a charged amino acid residue or a phydrophobic amino acid residue. A preferred hydrophobic residue may be selected from P, I, L or V. Most prefered the motif which correspond to any the following amino acid sequences: QAQPW (SEQ ID NO: 18) QISQP (SEQ ID NO: 19)

QQEQL (SEQ ID NO: 20) QEQKL (SEQ ID NO: 21) QEQLV (SEQ ID NO: 22) EPEEP (SEQ ID NO: 23) EQRW (SEQ ID NO: 24) EEVEL (SEQ ID NO: 25).

In other preferred embodiments, the peptide according to the invention may com- prise a contigious amino acid sequence which is derived from a part of the reciprocal Ig1-to-lg2 binding site which comprises amino acid residues of the Ig2 module. Accordingly, in this embodiment the invention relates to the following sequences: EKMEKKLHAV (SEQ ID NO:8) EKMEKRLHAV (SEQ ID NO:9)

ERMDKKLLAV (SEQ ID NO:10)

QRMEKKLHAV (SEQ ID NO:11)

SKMRRRVIAR (SEQ ID NO: 12)

AAKTVKFKC (SEQ I D NO: 13) AANTVKFRC (SEQ ID NO:14)

AANTVRFRC (SEQ ID NO:15)

AGNTVKFRC (SEQ ID NO:16)

VGSSVRLKC (SEQ ID NO:17).

According to the invention the sequences SEQ ID NOs:8-17 comprise a binding motif which is essential for interaction of said sequences with amino acid residues of the Ig1-to-lg2 reciprocal binding site of FGFR located in the Ig1 module. The binding motif is defined by the formula K/R-x^p-(x)_Q-rK/R, wherein x^p is a hydrophobic amino acid residue and (x) is a charged amino acid residue. According to the invention x^p is any hydrophobic amino acid residue, however residues M, L or F in position x^p are preferred. In some embodiments it may be preferred that the amino acid sequence comprises a three-amino-acid motif K/R-x^p-(x)₀-K/R , wherein x^p is F or L, in another embodiments it may be preferred that the sequence comprises a four-amino-acid motif K/R-x^p-(x)_rK/R, wherein x^p is M. Most prefered a motif which corresponds to an amino acid sequence selected from the following amino acid sequences:

KMEK (SEQ ID NO: 26)

RMDK (SEQ ID NO: 27)

RMEK (SEQ ID NO: 28)

KMRR (SEQ ID NO: 29)

KFK (SEQ ID NO: 30)

KFR (SEQ ID NO: 31)

RFR (SEQ ID NO: 32)

RLK (SEQ ID NO: 33). The invention relates to SEQ ID NOs: 1-17 and SEQ ID NOs: 18-33 as preferred amino acid sequences comprised by the peptide of the invention. However, it is to be understood that any amino acid sequence that comprises the binding motif Q/E- (x)₃-x^p, wherein x^p is a hydrophobic amino acid residue and (x)₃ is an amino acid sequence of three amino acid resides, wherein x is any amino acid residue, or the binding motif K/R-x^p-(x)_0-rK/R, wherein x^p is a hydrophobic amino acid residue and (x) is a charged amino acid residue, may be comprised by the peptide of the invention, and therefore any amino acid sequence which comprises any of these amino acid motifs is within the scope of the invention as an amino acid sequence which is capable of interacting with the FGFR Ig1-to-lg2 reciprocal binding site. Accordingly, SEQ ID NOs:1-17 represent non-limites examples of amino acid sequences which are in the scope of the invention.

In the present context the standard one-letter code for amino acid residues as well as the standard three-letter code are applied. Abbreviations for amino acids are in accordance with the recommendations in the IUPAC-IUB Joint Commission on Biochemical Nomenclature Eur. J. Biochem, 1984, vol. 184, pp 9-37. Throughout the description and claims either the three letter code or the one letter code for natural amino acids are used. Where the L or D form has not been specified it is to be un- derstood that the amino acid in question has the natural L form, cf. Pure & Appl. Chem. Vol. (56(5) pp 595-624 (1984) or the D form, so that the peptides formed may be constituted of amino acids of L form, D form, or a sequence of mixed L forms and D forms.

Where nothing is specified it is to be understood that the C-terminal amino acid of a peptide of the invention exists as the free carboxylic acid, this may also be specified as "-OH". However, the C-terminal amino acid of a compound of the invention may be the amidated derivative, which is indicated as "-NH₂". Where nothing else is stated the N-terminal amino acid of a polypeptide comprise a free amino-group, this may also be specified as "H-".

Where nothing else is specified amino acid can be selected from any amino acid, whether naturally occurring or not, such as alfa amino acids, beta amino acids, and/or gamma amino acids. Accordingly, the group comprises but are not limited to: A, V, L, I₁ P₁ F, W, M, G, S₁ T, C, Y, N, Q, D, E₁ K, R₁ H Aib, NaI, Sar, Orn, Lysine analogues, DAP, DAPA and 4Hyp.

Also, according to the invention modifications of the amino acid sequences may be performed, such as for example glycosylation and/or acetylation of the amino acids.

Basic amino acid residues are according to invention represented by the residues of amino acids H, K and R; acidic amino acid residues - by the residues of amino acids E and D; hydrophobic amino acid residues by the residues of amino acids A, L, I, V₁ M, F, Y and W; neutral, weakly hydrophobic - by P, A and G; neutral hydrophilic - by amino acid residues Q, N, S and T; cross-link forming by amino acid resudue C.

A preferred peptide according to the invention is an isolated contigous peptide sequence which comprises at most 25 amino acid residues. In one embodiment the length of the amino acid sequence of a peptide may be from 15 to 25 amino acid residues, such as, for example 16, 17, 18, 19, 20, 21, 22, 23 or 24 amino acid residues. In another embodiment, the length of the amino acid sequence of a peptide may be from 3 to 15 amino acid residues, such as for example 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 amino acid residues. The peptides which amino acid sequence has the length in the range of 5 to 15 amino acid residues, such as from 6 to 14, for example 7, 8, 9, 10 ,11 12 or 13, are preferred. It is understood that all peptides of the invention comprise at least one amino acid sequence selected from any of the sequences SEQ ID NOs: 1-17 or at least one fragment of any of these sequences.

Thus, in some embodiments of the invention may relate to a peptide comprising a fragment of an sequence selected from SEQ ID NOs:1-17.ln another embodiments, the invention may relates to variants of SEQ ID NOs:1-17.

According to the invention, a variant of an amino acid sequence selected from the sequences SEQ ID NOs: 1-17 may be i) an amino acid sequence which has at least 60% identity with a selected sequence, such as 61-65% identity, for example 66-70% identity, such as 71-75% identity, for example 76-80% identity, such as 81-85 % identity, for example 86-90% identity, such as 91-95% identity, for example 96- 99% identity, wherein the identity is defined as a percentage of identical amino acids in said sequence when it is collated with the selected sequence. The identity between amino acid sequences may be calculated using well known algorithms such as BLOSUM 30, BLOSUM 40, BLO-

SUM 45, BLOSUM 50, BLOSUM 55, BLOSUM 60, BLOSUM 62, BLO- SUM 65, BLOSUM 70, BLOSUM 75, BLOSUM 80, BLOSUM 85, or BLO- SUM 9;

ii) an amino acid sequence which has at least 60% positive amino acid matches with a selected sequence, such as 61-65% positive amino acid matches, for example 66-70% positive amino acid matches, such as 71- 75% positive amino acid matches, for example 76-80% positive amino acid matches, such as 81-85 % positive amino acid matches, for example 86-90% positive amino acid matches, such as 91-95% positive amino acid matches, for example 96-99% positive amino acid matches, wherein the positive amino acid match is defined as the presence at the same position in two compared sequences of amino acid residues which has similar of physical and/or chemical properties. Preferred positive amino acid matches of the present invention are K to R, E to D, L to M, Q to E, I to V,

I to L, A to S, Y to W, K to Q, S to T, N to S and Q to R;

iii) an amino acid sequence which is identical to a selected sequence, or it ■ has at least 60% identity with said sequence such as 61-65% identity, for example 66-70% identity, such as 71-75% identity, for example 76-80% identity, such as 81-85 % identity, for example 86-90% identity, such as 91-95% identity, for example 96-99% identity, or has at least 60% positive amino acid matches with the selected sequence, such as 61-65% positive amino acid matches, for example 66-70% positive amino acid matches, such as 71-75% positive amino acid matches, for example 76-

80% positive amino acid matches, such as 81-85 % positive amino acid matches, for example 86-90% positive amino acid matches, such as 91- 95% positive amino acid matches, for example 96-99% positive amino acid matches and comprises other chemical moieties, e. g. phosphoryl, sulphur, acetyl, glycosyl moieties. The term "variant of a peptide sequence" also means that the peptide sequence may be modified, for example by substitution of one or more of the amino acid residues. Both L-amino acids and D-amino acids may be used. Other modification may comprise derivatives such as esters, sugars, etc., for example methyl and acetyl esters.

In another aspect, variants of the amino acid sequences according to the invention may comprise, within the same variant, or fragments thereof or among different variants, or fragments thereof, at least one substitution, such as a plurality of substitutions introduced independently of one another. Variants of the complex, or fragments thereof may thus comprise conservative substitutions independently of one another, wherein at least one glycine (GIy) of said variant, or fragments thereof is substituted with an amino acid selected from the group of amino acids consisting of Ala, VaI, Leu, and lie, and independently thereof, variants, or fragments thereof, wherein at least one alanine (Ala) of said variants, or fragments thereof is substituted with an amino acid selected from the group of amino acids consisting of GIy, VaI, Leu, and lie, and independently thereof, variants, or fragments thereof, wherein at least one valine (VaI) of said variant, or fragments thereof is substituted with an amino acid selected from the group of amino acids consisting of GIy, Ala, Leu, and lie, and independently thereof, variants, or fragments thereof, wherein at least one leucine (Leu) of said variant, or fragments thereof is substituted with an amino acid selected from the group of amino acids consisting of GIy, Ala, VaI, and lie, and independently thereof, variants, or fragments thereof, wherein at least one isoleucine (lie) of said variants, or fragments thereof is substituted with an amino acid selected from the group of amino acids consisting of GIy, Ala, VaI and Leu, and independently thereof, variants, or fragments thereof wherein at least one aspartic acids (Asp) of said variant, or fragments thereof is substituted with an amino acid selected from the group of amino acids consisting of GIu, Asn, and GIn, and independently thereof, variants, or fragments thereof, wherein at least one aspargine (Asn) of said variants, or fragments thereof is substituted with an amino acid selected from the group of amino acids consisting of Asp, GIu, and GIn, and independently thereof, variants, or fragments thereof, wherein at least one glutamine (GIn) of said variants, or fragments thereof is substituted with an amino acid selected from the group of amino acids consisting of Asp, GIu, and Asn, and wherein at least one phenyla- lanine (Phe) of said variants, or fragments thereof is substituted with an amino acid selected from the group of amino acids consisting of Tyr, Trp, His, Pro, and preferably selected from the group of amino acids consisting of Tyr and Trp, and independently thereof, variants, or fragments thereof, wherein at least one tyrosine (Tyr) of said variants, or fragments thereof is substituted with an amino acid selected from the group of amino acids consisting of Phe, Trp, His, Pro, preferably an amino acid selected from the group of amino acids consisting of Phe and Trp, and independently thereof, variants, or fragments thereof, wherein at least one arginine (Arg) of said fragment is substituted with an amino acid selected from the group of amino acids consisting of Lys and His, and independently thereof, variants, or fragments thereof, wherein at least one lysine (Lys) of said variants, or fragments thereof is substituted with an amino acid selected from the group of amino acids consisting of Arg and His, and independently thereof, variants, or fragments thereof, and independently thereof, variants, or fragments thereof, and wherein at least one proline (Pro) of said variants, or fragments thereof is substituted with an amino acid selected from the group of amino acids consisting of Phe, Tyr, Trp, and His, and independently thereof, variants, or fragments thereof, wherein at least one cysteine (Cys) of said variants, or fragments thereof is substituted with an amino acid selected from the group of amino acids consisting of Asp, GIu, Lys, Arg, His, Asn, GIn, Ser, Thr, and Tyr.

It thus follows from the above that the same functional equivalent of a peptide fragment, or fragment of said functional equivalent may comprise more than one conservative amino acid substitution from more than one group of conservative amino acids as defined herein above. The term "conservative amino acid substitution" is used synonymously herein with the term "homologous amino acid substitution". The groups of conservative amino acids are as the following: A, G (neutral, weakly hydrophobic), Q, N, S, T (hydrophilic, non-charged) E, D (hydrophilic, acidic) H, K, R (hydrophilic, basic) L, P, I, V, M, F, Y, W (hydrophobic, aromatic) C (cross-link forming) Conservative substitutions may be introduced in any position of a preferred predetermined peptide of the invention or fragment thereof. It may however also be desirable to introduce non-conservative substitutions, particularly, but not limited to, a non-conservative substitution in any one or more positions.

A non-conservative substitution leading to the formation of a functionally equivalent fragment of the peptide of the invention would for example differ substantially in polarity, for example a residue with a non-polar side chain (Ala, Leu, Pro, Trp, VaI, He, Leu, Phe or Met) substituted for a residue with a polar side chain such as GIy, Ser, Thr, Cys, Tyr, Asn, or GIn or a charged amino acid such as Asp, GIu, Arg, or Lys, or substituting a charged or a polar residue for a non-polar one; and/or ii) differ substantially in its effect on peptide backbone orientation such as substitution of or for Pro or GIy by another residue; and/or iii) differ substantially in electric charge, for example substitution of a negatively charged residue such as GIu or Asp for a posi- tively charged residue such as Lys, His or Arg (and vice versa); and/or iv) differ substantially in steric bulk, for example substitution of a bulky residue such as His, Trp, Phe or Tyr for one having a minor side chain, e.g. Ala, GIy or Ser (and vice versa).

Substitution of amino acids may in one embodiment be made based upon their hy- drophobicity and hydrophilicity values and the relative similarity of the amino acid side-chain substituents, including charge, size, and the like

According to the invention a fragment of a selected amino acid sequence of the invention, for a example a fragment of a sequence selected from SEQ ID NOs:1-17, may be an amino acid sequence, which has about 25 - 99 % of the length of the selected amino acid sequence. Preferably, a fragment according to the invention comprises at least 3 contigous amino acid residues of any of the seaquences SEQ ID NOs:1-17, such as for example a sequence selected from SEQ ID NOs:18-33.

Both fragments and variants of amino acid sequences of the invention according to the invention are the functional equivalents of said sequences.

By the term "functional equivalent" of an amino acid sequence is in the present context meant a molecule which meets the criteria for a variant or a fragment of said amino acid sequence described above and which is capable of one or more func- tional activities of said sequence or a compound comprising said sequence, in a preferred embodiment the functional equivalent of an amino acid sequence of the invention is capable of binding and modulating activity of a FGFR.

The invention relates both to isolated peptides of the invention and fusion proteins comprising peptides of the invention.

In one embodiment, the peptide is an isolated peptide. By the term "isolated peptide" is meant that the peptide of the invention is an individual compound and not a part of another compound, such as for example a polypeptide comprising more then 25 amino acid residues. The isolated peptide may be produced by use of any recombinant technology methods or chemical synthesis and separated from other compounds, or it was separated from a longer polypeptide or protein by a method of enzymatic or chemical cleavage and further separated from other protein fragments.

An isolated peptide of the invention may in one embodiment comprise one or more of the sequences SEQ ID NOs: 1-33. In another embodiment the isolated peptided may consist of one or more of the sequences SEQ ID Nos:1-33.

Thus, in one embodiment, the peptide comprises a sequence selected from SEQ ID NOs: 1-8 or 18-28, preferably a sequence selected from SEQ ID NOs: 1-8, or a functional equivalent thereof. In another embodiment the peptide may consist of a sequence selected from SEQ ID NOs: 1-8 or a functional equivalent thereof.

In another embodiment, the peptide may comprise a sequence selected from SEQ ID NOs: 9-17 or 26-33, preferably a sequence selected from SEQ ID NOs: 9-17, or a functional homologue thereof.

A preferred amino acid sequence may be chosen depending on which type of modu- lating FGFR activity is nessesary. Thus, in one embodiment it may be nessesary to activate FGFR, in another embodiment it may be nessesary to attenuate activity of

FGFR or inhibit the receptor. Sometimes the chose of a peferred amino acid se- quennce sequence may depend on which receptor of the FGFRs is concerned. For example, in one embodiment FGFR1 may be concerned, in another it may be con- cerned FGFR2 or FGFR3. In still another embodiment it may be nessesary to modu- late activity of FGFR4. However, the choice of a preferred amino acid sequence depending on which of the FGFRs is concerned is sometimes of less importance, as the amino acid sequences described above according to the invention are capable of interacting with the reciprocal Ig1-to-lg2 module binding site of any FGFR.

Multimeric compound

An isolated peptide sequence of the invention may be connected to another isolated peptide sequence by a chemical bond in a fusion protein or the amino acid se- quences may be connected to each other through a linker grouping. In some embodiments a peptide sequence of the invention may be formulated as an oligomer (multimer) of monomers, wherein each monomer is as a peptide seqience defined above. Particularly, multimeric peptides such as dendrimers may form conformational determinants or clusters due to the presence of multiple flexible peptide monomers. In one embodiment the compound is a dimer. In a more preferred embodiment the compound is a dendrimer, such as four peptides linked to a lysine backbone, or coupled to a polymer carrier, for example a protein carrier, such as BSA. Polymerisation such as repetitive sequences or attachment to various carriers are well-known in the art, e.g. lysine backbones, such as lysine dendrimers carrying 4 peptides, 8 peptides, 16 peptides, or 32 peptides. Other carriers may be lipophilic dendrimers, or micelle-like carriers formed by lipophilic derivatives, or starburst (star-like) carbon chain polymer conjugates.

Thus, according to the invention a multimeric compound may be a polymer compris- ing two or more identical or different peptide sequences of the invention, wherein in a preferred embodiment, at least one of the two or more amino acid sequences is selected from SEQ ID NOs: 1-15, or fragments or variants of said sequences.

In some embodiments, the compound may comprise two identical amino acid se- quences selected from SEQ ID NOs: 1-15, or two identical fragments or variants of the selected sequence, wherein said amino acid sequences, fragments or variants. Or₁ the compound may comprise four identical copies of an amino acid sequence selected from SEQ ID NOs: 1-15, or four identical fragments or variants of the selected sequence. In other embodiments, the compound may comprise two or more different amino acid sequences, wherein at least one of the two amino acid sequences is a sequence selected from SEQ ID NOs: 1-15, or fragments or variants thereof.

In yet other embodiments, the compound may comprise two or more different amino acid sequences, wherein said two or more amino acid sequences are selected from SEQ ID NO: 1-15, or fragments or variants thereof.

A preferred multimeric compound of the invention is a compound wherein the amino acid sequences are connected to each other through a linker or a linker grouping.

A linker is according to the invention may be any molecule or chemical moiety capable of cross-linking two or more peptide sequences, for example it may be an achiral di-, tri- or tetracarboxylic acid of the general formula

X[(A)nCOOH][(B)mCOOH]

wherein n and m independently are an integer of from 1 to 20, X is HN, H₂N(CR₂)PCR, RHN(CR₂)pCR, HO(CR₂)pCR, HS(CR₂)pCR, halogen- (CR₂)PCR, HOOC(CR₂)pCR, ROOC(CR₂)pCR, HCO(CR₂)pCR, RCO(CR₂)pCR, [HOOC(A)n][HOOC(B)m]CR(CR₂)pCR, H₂N(CR₂)P, RHN(CR₂)p, HO(CR₂)p, HS(CR₂)P, halogen-(CR₂)p, HOOC(CR₂)p, ROOC(CR₂)P, HCO(CR₂)P, RCO(CR₂)p, or [HOOC(A)n][HOOC(B)m](CR₂)p , wherein p is 0 or integer of from 1 to 20, A and B independently are a substituted or unsubstituted C_1-10 alkyl, a substituted or unsubstituted C_2-i₀ alkenyl, a substituted or unsubstituted cyclic moiety, a substituted or unsubstituted heterocyclic moiety, a substituted or unsubstituted aromatic moiety, or A and B together form a substituted or unsubstituted cyclic moiety, substituted or unsubstituted heterocyclic moiety, substituted or unsubstituted aromatic moiety.

Under the term C_1-10 alkyl is meant straight or branched chain alkyl groups having 1- 10 carbon atoms, e.g. methyl, ethyl, isopropyl, butyl, and tertbutyl.

Under the term C_2-10 alkenyl is meant straight or branched chain alkenyl groups hav- ing 2-10 carbon atoms, e.g. ethynyl, propenyl, isopropenyl, butenyl, and tert-butenyl. Under the term cyclic moiety is meant cyclohexan, and cyclopentane.

Under the term aromatic moiety is meant phenyl.

The wording "A and B forms a cyclic, heterocyclic or aromatic moiety" denotes cyclohexan, piperidine, benzene, and pyridine.

In those embodiments where a multimeric compound of the invention comprises the linker of above, the compound is preferably obtained by the LPA method (a ligand presentation assembly method) as described in WO0018791 and WO2005014623.

Another example of a preferred linker of the invention may be amino acid lysine. Individual peptide sequences may be attached to a core molecule such as lysine forming thereby a dendritic multimer (dendrimer) of an individual peptide sequence(s). Production of dendrimers is also well known in the art (PCT/US90/02039, Lu et al., (1991) MoI Immunol. 28:623-630; Defoort et al., (1992) lnt J Pept Prot Res. 40:214-221 ; Drijfhout et al. (1991) lnt J Pept Prot Res. 37:27- 32), and dedrimers are at present widely used in research and in medical applications.

Still, in some embodiments, amino acid cystein may be preferred a linker molecule.

One of the referred embodiments of the invention concernes a compound comprising four individual amino acid sequences attached to the lysine core molecule, a dendritic tetramer/dendrimer of a peptide sequence of the invention.

Multimeric compounds of the invention, such as LPA-dimers or dendrimers, are most preferred compounds of the invention. However, other types of multimeric compounds comprising two or more individual sequences of the invention are also in the scope of the invention. These compounds may be produced using thechnologies known in the art. The peptide sequences may be covalently bound to the linker through their amino- or carboxy-groups, preferably through the N- or C terminal amino- or carboxy- groups.

Biological activity

According to the invention, compounds described above are functionally active compounds. The term "compound" relates in the present context both to isolated peptide sequences of the invention and compounds comprising said sequences.

The compounds are capable of binding to a functional cell-surface receptor and modulating the activity of said receptor The receptor is according to the invention is FGFR and may be selected from any of the FGFRs, for example it may be FGFR1, FGFR2, FGFR3, FGFR4 or FGFR5. The compound according to the invention is capable of binding to any of the latter FGFRs at the reciprocal Ig1-to-lg2 binding site located in the Ig1 and Ig2 modules of FGFR.

The invention preferably concerns FGFR1 and FG FR1 -associated signalling. The modulating FGFR1 signalling by the compound of the invention results in a change in the receptor activation status which is reflected by FGFR1 tyrosine phosphorylation, or it may be reflected by the status of activation of one or more of the intracellular proteins involved in FGFR1 -associated signal transduction, such as for example the activation status of STAT1 , JNK, PI_Cγ, ERK, STAT5, PI3K, PKC, FRS2 and/or GRB2 proteins. The result of modulating of FGFR1 signalling by a compound of the invention may also be related to a cell differentiation-related effect.

When the FGFR signalling is measured as a level of phosphorylation of FGFR, the degree of phoshorylation is estimated as at least 20% above the control value, such as at least 20-200 %, for example at least 50-200%. The control value in the present content is meant the degree of phosphorylation of FGFR in the medium where a compound capable of activation of FGFR is absent.

When estimating an efficient concentration of a compound with respect to modulating of the FGFR signalling, said concentration may be between 0.1-1000 μM, 1- 1000 μM, for example 1-200 μM, for example 10-200 μM, such as 20-180 μM, for example 30-160 μM, such as 40-140 μM, for example 50-130 μM, such as 60-120 μM, for example 70-110 μM, such as 80-100 μM.

When estimating the downstream FGFR signaling effect such as a cell differentia- tion-related effect the invention preferably relates to cell aggregation, the formation of nodules, formation of cartilage, or two or more of said effects (Listrum, G. P. et al. J. Histochem. Cytochem. 1999, 47:1-6), such effect being detectable by light microscopy, turbidimetry, or flow cytometry. The cell differentiation-related effect may also be measured as a change in expression at RNA or protein levels of bone sialo- protein (J. Bone Miner. Res. 1998, 13:1852-61; Genomics 1998, 53: 391-4), or type X collagen (Cell Tissue Res. 1998, 293: 357-64), the human ILA gene (Osteoarthritis Gartilage 1997, 5: 394-406), or type Il collagen/or MGP (J Miner Res. 1997: 1815.23), and the like.

The FGFR tyrosine phosphorylation or activation of any of the molecules of FGFR- associated downstream signaling, such as for example STST1 , JNK, PLCy, ERK, STAT5, PI3K, PKC, FRS2 and/or GRB2 proteins, may be estimated by any conventional methods, such as for example immunocytochemistry, immunoblotting or im- munoprecipitation, using commercially available antibody against the activated pro- teins. The degree of activation is estimated as at least 20% above/below the control value, such as at least 20-200 %, for example at least 50-200%. The control value is estimated as a degree of phosphorylation of the protein of interest in the medium where a compound capable of activation of FGFR is absent.

In another embodiment the invention relates to a signaling associated with a protein involved in interaction with FGFR, for example the protein being an FGFR1 ligand, preferably a receptor-like ligand FGFR1. Preferred embodiments of such ligands of FGFR1 are neural cell adhesion molecules NCAM and L1. It is understood that receptor-like FGFR ligands may comprise other proteins, which are capable of inter- acting with FGFR and associated with any signal transduction cascade.

The most preferred a receptor-like ligand of FGFR1 of the invention is NCAM. Therefore, the invention concerns biological effects which are associated with FGFR-NCAM interaction, for example such as neurite outgrowth or neural cell dif- ferentiation. NCAM-dependent signal transduction involves the variety of downstream molecules, activation of which upon the signalling may be measured. The invention in particular concerns the assessment of activation of focal adhesion kinase FAK, tyrosine kinase Fyn and/or cyclic-AMP response-binding element protein CREB. The degree of phoshorylation is estimated as at least 20% above/below the control value, such as at least 20-200 %, for example at least 50-200%. The control value is estimated as above.

Activation or inhibition of NCAM-dependent signal transduction may also be measured by evaluating the cellular responses on morphological level, in particular cell differentiation-related effects. Accordingly, the assay concerns in another particular embodiment evaluation of the effect of a candidate compound on NCAM-dependent cellular aggregation, cell motility, neuritogenesis, survival, plasticity associated with memory and learning.

A skilled artisan may select from a number of assays have developed in the art to evaluate the above cellular responses. Cellular aggregation and neuritogenesis may for example be evaluated as described by Skladchikova et al. J. Neurosci. Res 1999, 57: 207-18. Proliferation and apoptosis may be evaluated by using any commercially available assays and kits according to the manufacturer procedure.

Thus, compounds of the invention are capable of activating FGFR directly by binding to the reciprocal binding site described herein, or they may attenuate FGFR acti- vating dependent on other ligands. Thus, the compounds of the invention may modulate receptor signalling dependent on another ligand binding.

It is known that a cellular response to the activation of a receptor depends on the strength of receptor stimulation, which may, for example, be characterised by the value of affinity of interaction of a ligand with the receptor, and/or by the duration of such interaction. Thus, both affinity and duration of interaction of FGF and the receptor may be affected by a compound of the invention. Accordingly, it is another embodiment of the invention to provide a compound, which is capable of modulating the receptor signalling induced by another receptor ligand, for example by a FGF or a cell adhesion molecule. In the present context the term "interacting" is used interchangeably with the term "binding" and refers to a direct or indirect contact between a compound of the invention and FGFR, preferably a direct interaction. The term "direct interaction" means that the compound in question binds directly to the receptor.

The binding affinity of the compound according to the invention preferably has Kd value in the range of 10^"3 to 10^"10 M, such as preferably in the range of 10^"4 to 10^"8 M. According to the present invention the binding affinity may be determined by any available assayes suitable for this purpose, such as for example surface plasmon resonance (SPR) analysis or nuclear magnetic resonance (NMR) spectroscopy.

Binding of the compound of the invention to FGFR leads to a series of cellular re- sponces mediated by FGFR. Thus, the compound which is capable of binding to FGFR and activating/inhibiting FGFR is also capable of inducing differentiation of FGFR presenting cells, modulating of proliferation of FGFR presenting cells, stimulating survival of FGFR presenting cells, and /or stimulating morphological plasticity of FGFR presenting cells.

By the term "cells presenting FGFR" is meant cells expressing FGFR on the external membrane of the cells, these cells are for example neurons, glial cells, all types of muscle cells, neuroendocrine cells, gonadal cells and kidney cells, endothelial cells fibroblasts, osteoblasts, cancer, stem and embryonic cells. Activation of FGFRs has been shown to be associated with growth, differentiation and survival of cells ex- pressing the receptors.

Thus, FGFRs have been shown to be important determinants of neuronal survival both during development and during adulthood (Haspel et al. (2000) J Neurobiol 15:287-302; Roonprapurt et al. (2003) J Neurotrauma 20:871-882; Wiencken-Barger et al. Cereb Cortex (2004) 14:121-131; Loers er al. (2005) J Neurochem 92:1463- 1476; Reuss and von Bohlen und Halbach (2003) Cell tissue Res, 313:139-57), muscle and cancer cells (Ozen et al. (2001) J Nat Cancer Inst. 93:1783-90; Miyamoto et al. (1998) J Cell Physiol. 177:58-67; Detilliux et al. (2003) Cardiovasc Res. 57:8-19). Activation of FGF receptors is involved in normal, as well as in pathologic angiogenesis (Slavin, Cell Biol lnt 1995, 19:431-44). It is important for development, proliferation, functioning and survival skeletal muscle cells, cardiomyocytes and neurons (Merle at al., J Biol Chem 1995, 270:17361-7; Cheng and Mattson, Neuron 1991, 7:1031-41; Zhu et al., Mech Ageing Dev 1999, 108:77-85). Alterations in FGFR signalling have been associated with development different pathologic condi- tions, e.g. diabetes (Hart et al., Nature 2000, 408:864-8). The receptors play a role in maintenance of normal kidney structure (Cancilla et al., Kidney lnt 2001 , 60:147- 55), and it is implicated in mound healing and cancer disease (Powers et al., Endocr Relat Cancer. 2000, 7:165-97). Activation of FGFRs is nessesary for neurite outgrowth (Anderson et al., J Neurochem. 2005 95(2):570-83; Neiindam et al., J Neu- rochem. 2004 91(4):920-35; Niethammer et al., J Cell Biol. 2002 157(3):521-32). The receptors have been shown to be involved in the processes associated with learning and memory (Cambon et al,. J Neurosci. 2004, 24(17):4197-204; Reuss and von Bohlen und Halbach (2003) Cell tissue Res, 313:139-57).

Accordingly, substances with the potential to stimulate FGFR signalling have the potential to promote neurite outgrowth as well as stimulate regeneration and/or differentiation of neuronal cells, to stimulate cell survival, in particular neuronal cell survival, to stimulate stem or premature cell differentiation, such as neuronal cell differentiation, to stimulate neuronal plasticity associated with memory and learning, are prime targets in the search for compounds that facilitate for example neuronal regeneration and other forms of neuronal plasticity.

Compounds of the present invention are shown to promote neurite outgrowth and are therefore considered to be good promoters of regeneration of neuronal connec- tions, and thereby of functional recovery after damages as well as promoters of neuronal function in other conditions where such effect is required. Furthermore, compounds of the present invention are capable of stimulating neuronal progenitor cell differention into marture neurons. Compounds of the present invention are also potent stimulators of morphological plasticity of neurons associated with learning and memory.

In the present context "differentiation" is related both to the processes of initiation of differentiation of neuronal precursor cells/stem, maturation of immature neurons, such as neurite outgrowth which take place after the last cell division of said neurons, and morphological plasticity of mature neurons, such as takes place in the brain in connection with learning and memory. Thus, the compounds of the present invention may be capable of stopping neural precursor and immature neural cell division and initiating maturation said cells, such as initiating extension of neurites. Otherwise, "differentiation" is related to initiation of the process of genetic, biochemical, morphological and physiological transformation of neuronal progenitor cells, immature neural cells or embryonic stem cells leading to formation of cells having functional characteristics of normal neuronal cell as such characteristics are defined in the art. The invention defines "immature neural cell" as a cell that has at least one feature of neural cell accepted in the art as a feature characteristic for the neural cell.

According to the present invention a compound comprising at least one of the above peptide sequences is capable of stimulating neurite outgrowth. The invention concerns the neurite outgrowth improvement/stimulation such as about 75% improvement/stimulation above the value of neurite outgrowth of control/non- stimulated cells, for example 50%, such as about 150%, for example 100%, such as about 250, for example 200%, such as about 350 %, for example 300%, such as about 450%, for example 400%, such as about 500%.

Estimation of capability of a candidate compound to stimulate neurite outgrowth may be done by using any known method or assay for estimation of neurite outgrowth, such as for example as the described in Examples.

According to the invention a compound has neuritogenic activity both as an insoluble immobile component of cell growth substrate and as a soluble component of cell growth media. In the present context "immobile" means that the compound is bound/attached to a substance which is insoluble in water or a water solution and thereby it becomes insoluble in such solution as well. For medical applications both insoluble and soluble compounds are considered by the application, however soluble compounds are preferred. Under "soluble compound" is understood a compound, which is soluble in water or a water solution.

In still another preferred embodiment the compound of the invention is capable of stimulating synaptic plasticity. Accordingly, the compound is capable of stimulating lerning and memory as well. In one embodiment the peptide sequences of the invention may stimulate spine formation, in another embodiment the sequences may promote synaptic efficacy. Thus, the invention further provides a method for stimulating memory and/or learning comprising using a peptide sequence of the invention and/or compound comprising said sequence. The invention relates to both short-term memory and long-term memory.

In another preferred embodiment the compound of the invention is capable of stimulating cell survival. The compounds according to the invention are capable to prevent of cell death, in particular neuronal cell death, for example cell death due to trauma or disease. In the present context the wording "stimulate/promote survival" is used synonymously with the wording "preventing cell death". By stimulating/promoting cell survival it is possible to prevent diseases or prevent further degeneration of the nervous system in individuals suffering from a degenerative disorder. "Survival" refers to the process, wherein a cell has been traumatised and would under normal circumstances, with a high probability die, if not a compound of the invention was used to prevent said cell from degenerating, and thus promoting or stimulating survival of said traumatised cell.

Peripheral nerve cells possess to a limited extent a potential to regenerate and re- establish functional connections with their targets after various injuries. However, functional recovery is rarely complete and peripheral nerve cell damage remains a considerable problem. In the central nervous system, the potential for regeneration is even more limited. Therefore, the identification of substances with the ability to prevent neuronal cell death in the peripheral and the central nervous system is of great interest.

The invention also relates to compounds that are capable of attenuating FGFR function, such as inhibiting FGFR activity. Pathologic activity of FGFRs (FGFR1-FGFR3) have been shown associated with distinct clinical entities, including achondroplasia, hypochondroplasia, platyspondylic lethal skeletal dysplasia, thanatophoric dysplasia, Antley-Bixler syndrome, Apert syndrome, Beare-Stevenson syndrome, Crouzon syndrome, Jackson-Weiss syndrome, Pfeiffer syndrome, and Saethre-Chotzen syndrome (Passos-Bueno et al., 1999Hι/m. Mutat. 14: 115-125). The abnormal appearance and age-dependent loss of resident FGFR2 and gain of activity of FGFR1 in epithelial cells is a hallmark of the slow progression to malignancy in some types of prostate cancer (Wu et al., Cancer Res. 2001 61 (13):5295-302).

Thus, in some embodiments of the invention the compound described above is ca- pable of inhibiting FGFR.

Production of peptide sequences

The peptide sequences of the present invention may be prepared by any conven- tional synthetic methods, recombinant DNA technologies, enzymatic cleavage of full-length proteins which the peptide sequences are derived from, or a combination of said methods.

Recombinant preparation

Thus, in one embodiment the peptides of the invention are produced by use of recombinant DNA technologies.

The DNA sequence encoding a peptide or the corresponding full-length protein the peptide originates from may be prepared synthetically by established standard methods, e.g. the phosphoamidine method described by Beaucage and Caruthers, 1981 , Tetrahedron Lett. 22:1859-1869, or the method described by Matthes et al., 1984, EMBO J. 3:801-805. According to the phosphoamidine method, oligonucleotides are synthesised, e.g. in an automatic DNA synthesiser, purified, annealed, ligated and cloned in suitable vectors.

The DNA sequence encoding a peptide may also be prepared by fragmentation of the DNA sequences encoding the corresponding full-length protein of peptide origin, using DNAase I according to a standard protocol (Sambrook et al., Molecular clon- ing: A Laboratory manual. 2 rd ed., CSHL Press, Cold Spring Harbor, NY, 1989). The present invention relates to full-length proteins selected from the groups of proteins identified above. The DNA encoding the full-length proteins of the invention may alternatively be fragmented using specific restriction endonucleases. The fragments of DNA are further purified using standard procedures described in Sambrook et al., Molecular cloning: A Laboratory manual. 2 rd ed., CSHL Press, Cold Spring Harbor, NY, 1989. The DNA sequence encoding a full-length protein may also be of genomic or cDNA origin, for instance obtained by preparing a genomic or cDNA library and screening for DNA sequences coding for all or part of the full-length protein by hybridisation using synthetic oligonucleotide probes in accordance with standard techniques (cf. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor, 1989). The DNA sequence may also be prepared by polymerase chain reaction using specific primers, for instance as described in US 4,683,202 or Saiki et al., 1988, Science 239:487-491.

The DNA sequence is then inserted into a recombinant expression vector, which may be any vector, which may conveniently be subjected to recombinant DNA procedures. The choice of vector will often depend on the host cell into which it is to be introduced. Thus, the vector may be an autonomously replicating vector, i.e. a vec- tor that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g. a plasmid. Alternatively, the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome(s) into which it has been integrated.

In the vector, the DNA sequence encoding a peptide or a full-length protein should be operably connected to a suitable promoter sequence. The promoter may be any DNA sequence, which shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell. Examples of suitable promoters for directing the transcription of the coding DNA sequence in mammalian cells are the SV 40 promoter (Subramani et al., 1981, MoI. Cell Biol. 1:854-864), the MT-1 (metallothionein gene) promoter (Palmiter et al., 1983, Science 222: 809-814) or the adenovirus 2 major late promoter. A suitable promoter for use in insect cells is the polyhedrin promoter (Vasu- vedan et al., 1992, FEBS Lett. 311:7-11). Suitable promoters for use in yeast host cells include promoters from yeast glycolytic genes (Hitzeman et al., 1980, J. Biol. Chem. 255:12073-12080; Alber and Kawasaki, 1982, J. MoI. Appl. Gen. 1 : 419-434) or alcohol dehydrogenase genes (Young et al., 1982, in Genetic Engineering of Microorganisms for Chemicals, Hollaender et al, eds., Plenum Press, New York), or the TPH (US 4,599,311) or ADH2-4c (Russell et at., 1983, Nature 304:652-654) promoters. Suitable promoters for use in filamentous fungus host cells are, for in- stance, the ADH3 promoter (McKnight et al., 1985, EMBO J. 4:2093-2099) or the tpiA promoter.

The coding DNA sequence may also be operably connected to a suitable terminator, such as the human growth hormone terminator (Palmiter et al., op. cit.) or (for fungal hosts) the TPH (Alber and Kawasaki, op. cit.) or ADH3 (McKnight et al., op. cit.) promoters. The vector may further comprise elements such as polyadenylation signals (e.g. from SV 40 or the adenovirus 5 EIb region), transcriptional enhancer sequences (e.g. the SV 40 enhancer) and translational enhancer sequences (e.g. the ones encoding adenovirus VA RNAs).

The recombinant expression vector may further comprise a DNA sequence enabling the vector to replicate in the host cell in question. An example of such a sequence (when the host cell is a mammalian cell) is the SV 40 origin of replication. The vector may also comprise a selectable marker, e.g. a gene the product of which complements a defect in the host cell, such as the gene coding for dihydrofolate reductase (DHFR) or one which confers resistance to a drug, e.g. neomycin, hydromycin or methotrexate.

The procedures used to ligate the DNA sequences coding the peptides or full-length proteins, the promoter and the terminator, respectively, and to insert them into suitable vectors containing the information necessary for replication, are well known to persons skilled in the art (cf., for instance, Sambrook et al., op.cit).

To obtain recombinant peptides of the invention the coding DNA sequences may be usefully fused with a second peptide coding sequence and a protease cleavage site coding sequence, giving a DNA construct encoding the fusion protein, wherein the protease cleavage site coding sequence positioned between the HBP fragment and second peptide coding DNA, inserted into a recombinant expression vector, and expressed in recombinant host cells. In one embodiment, said second peptide selected from, but not limited by the group comprising glutathion-S-reductase, calf thymosin, bacterial thioredoxin or human ubiquitin natural or synthetic variants, or peptides thereof. In another embodiment, a peptide sequence comprising a protease cleavage site may be the Factor Xa, with the amino acid sequence IEGR, en- terokinase, with the amino acid sequence DDDDK,- thrombin, with the amino acid sequence LVPR/GS, or Acharombacter lyticus, with the amino acid sequence XKX, cleavage site.

The host cell into which the expression vector is introduced may be any cell which is capable of expression of the peptides or full-length proteins, and is preferably a eu- karyotic cell, such as invertebrate (insect) cells or vertebrate cells, e.g. Xenopus laevis oocytes or mammalian cells, in particular insect and mammalian cells. Examples of suitable mammalian cell lines are the HEK293 (ATCC CRL-1573), COS (ATCC CRL-1650), BHK (ATCC CRL-1632, ATCC CCL-10) or CHO (ATCC CCL- 61) cell lines. Methods of transfecting mammalian cells and expressing DNA sequences introduced in the cells are described in e.g. Kaufman and Sharp, J. MoI. Biol. 159, 1982, pp. 601-621 ; Southern and Berg, 1982, J. MoI. Appl. Genet. 1:327- 341 ; Loyter et al., 1982, Proc. Natl. Acad. Sci. USA 79: 422-426; Wigler et al., 1978, Cell 14:725; Corsaro and Pearson, 1981 , in Somatic Cell Genetics 7, p. 603; Gra- ham and van der Eb, 1973, Virol. 52:456; and Neumann et al., 1982, EMBO J. 1:841-845.

Alternatively, fungal cells (including yeast cells) may be used as host cells. Examples of suitable yeast cells include cells of Saccharomyces spp. or Schizosaccharo- myces spp., in particular strains of Saccharomyces cerevisiae. Examples of other fungal cells are cells of filamentous fungi, e.g. Aspergillus spp. or Neurospora spp., in particular strains of Aspergillus oryzae or Aspergillus niger. The use of Aspergillus spp. for the expression of proteins is described in, e.g., EP 238 023.

The medium used to culture the cells may be any conventional medium suitable for growing mammalian cells, such as a serum-containing or serum-free medium containing appropriate supplements, or a suitable medium for growing insect, yeast or fungal cells. Suitable media are available from commercial suppliers or may be prepared according to published recipes (e.g. in catalogues of the American Type CuI- ture Collection).

The peptides or full-length proteins recombinantly produced by the cells may then be recovered from the culture medium by conventional procedures including separating the host cells from the medium by centrifugation or filtration, precipitating the proteinaceous components of the supernatant or filtrate by means of a salt, e.g. ammonium sulphate, purification by a variety of chromatographic procedures, e.g.

HPLC, ion exchange chromatography, affinity chromatography, or the like.

Synthetic preparation

The methods for synthetic production of peptides are well known in the art. Detailed descriptions as well as practical^" advice for producing synthetic peptides may be found in Synthetic Peptides: A User's Guide (Advances in Molecular Biology), Grant G. A. ed., Oxford University Press, 2002, or in: Pharmaceutical Formulation: Devel- opment of Peptides and Proteins, Frokjaer and Hovgaard eds., Taylor and Francis, 1999.

Peptides may for example be synthesised by using Fmoc chemistry and with Acm- protected cysteins. After purification by reversed phase HPLC, peptides may be further processed to obtain for example cyclic or C- or N-terminal modified isoforms. The methods for cyclization and terminal modification are well-known in the art and described in detail in the above-cited manuals.

In a preferred embodiment the peptide sequences of the invention are produced synthetically, in particular, by the Sequence Assisted Peptide Synthesis (SAPS) method.

Peptides may be synthesised either batchwise in a polyethylene vessel equipped with a polypropylene filter for filtration or in the continuous-flow version of the polyamide solid-phase method (Dryland, A. and Sheppard, R.C., (1986) J.Chem. Soc. Perkin Trans. I₁ 125 - 137.) on a fully automated peptide synthesiser using 9- fluorenylmethyloxycarbonyl (Fmoc) or tert. -Butyloxycarbonyl, (Boc) as N-a-amino protecting group and suitable common protection groups for side-chain functionality's.

Medicament

FGFRs are known to be involved in a number of body processes in normal conditions and in disease, in particular in the neural system. These proceses include dif- ferentiation, proliferation, survival, plasticity and motility of cells. Cell death plays a key role in normal neuronal development, where 50% of the developing neurons are eliminated through programmed cell death, and in the pathophysiology of neurodegenerative conditions, such as Alzheimer's and Parkinson's diseases. FGFRs have been shown to be important determinants of neuronal sur- vival both during development and during adulthood (Haspel et al. (2000) J Neurobiol 15:287-302; Roonprapurt et al. (2003) J Neurotrauma 20:871-882; Wiencken-Barger et al. Cereb Cortex (2004) 14:121-131 ; Loers er al. (2005) J Neu- rochem 92:1463-1476; Reuss and von Bohlen und Halbach (2003) Cell tissue Res, 313:139-57). Therefore, a compound, which is capable to promote neuronal cell survival by binding and activation FGFRs is highly desirable. Thus, in one aspect the invention features compounds that promote survival of neural cells and can be used as medicaments for the treatment of conditions involving neural cell death. However, a compound of the invention may also be used as a medicament for promotion of survival of another type of cells, e.g. different type of muscle cells, or, alternatively, for promotion of cell death of still another type of cells, e.g. cancer cells, as the FGFR signalling has been shown to be a survival factor for both muscle and cancer cells (Ozen et al. (2001) J Nat Cancer Inst. 93:1783-90; Miyamoto et al. (1998) J Cell Physiol. 177:58-67; Detilliux et al. (2003) Cardiovasc Res. 57:8-19).

Activity of cell-surface receptors is strictly regulated in a healthy organism. Mutations, abnormal expression or processing of a receptor or the receptor ligands lead to abnormalities in activity of the receptor and therefore lead to dysfunction of the receptor. The dysfunction of the receptor is in turn a reason for dysfunction of the cells which use the receptor for induction and/or maintenance of various cellular processes. The latter is the manifestation of a disease. It has also been shown that attenuation of FGFR signalling leads to development of a number of different pathologic conditions, e.g. diabetes (Hart et al., Nature 2000, 408:864-8). Activation of FGF receptors is involved in normal, as well as in pathologic angiogenesis (Slavin, Cell Biol lnt 1995, 19:431-44). It is important for development, proliferation, function- ing and survival skeletal muscle cells, cardiomyocytes and neurons (Merle at al., J Biol Chem 1995, 270:17361-7; Cheng and Mattson, Neuron 1991 , 7:1031-41; Zhu et al., Mech Ageing Dev 1999, 108:77-85). It plays a role in maintenance of normal kidney structure (Cancilla et al., Kidney lnt 2001 , 60:147-55), and it is implicated in mound healing and cancer disease (Powers et al., Endocr Relat Cancer. 2000, 7:165-97). The present invention provides compounds capable of modulating the activity of FGFRs. Consequently, said compounds are concerned by the invention as medicament for the treatment of diseases, wherein modulation of FGFR activity may be concsemed as essential for curing.

Thus, the medicament of the invention is in one embodiment for prevention and/or treatment of

1) diseases and conditions of the central and peripheral nervous system, or of the muscles or of various organs, and/or

2) diseases or conditions of the central and peripheral nervous system, such as postoperative nerve damage, traumatic nerve damage, impaired myelination of nerve fibers, postischaemic damage, e.g. resulting from a stroke, Parkinson's disease, Alzheimer's disease, Huntington's disease, dementias such as multiin- farct dementia, sclerosis, nerve degeneration associated with diabetes mellitus, disorders affecting the circadian clock or neuro-muscular transmission, and schizophrenia, mood disorders, such as manic depression;

3) for treatment of diseases or conditions of the muscles including conditions with impaired function of neuro-muscular connections, such as after organ transplan- tation, or such as genetic or traumatic atrophic muscle disorders; or for treatment of diseases or conditions of various organs, such as degenerative conditions of the gonads, of the pancreas such as diabetes mellitus type I and II, of the kidney such as nephrosis and of the heart, liver and bowel, and/or

4) cancer disease, and/or 5) prion diseases.

The invention concerns cancer being any type of solid tumors requiring neoangio- genesis.

The invention concerns prion diseases selected from the group consisting of scrapie, Creutzfeldt-Jakob disease. It has been shown that FGFRs plays a distinct role in prion diseases (Castelnau et al. (1994) Exp Neurobiol. 130:407-10; Ye and Carp (2002) J MoI Neurosci. 18:179-88).

In another embodiment a compound of the invention is for the manufacture of a medicament for 1) promotion of wound-healing, and/or

2) prevention of cell death of heart muscle cells, such as after acute myocardial infarction, or after angiogenesis, and/or

3) revascularsation.

In yet another embodiments a compound of the invention is for the manufacture of a medicament for the

1 ) prevention of cell death due to ischemia;

2) prevention of body damages due to alcohol consumption;

The invention concerns the medicament for treating normal, degenerated or damaged FGFR presenting cells or cells presenting an FGFR ligand. By the term "cells presenting an FGFR ligand" is meant cells expressing a receptor or ligand whereto FGFR and/or parts of FGFR may bind (i.e. so-called counter receptor). Examples of FGFR ligands are FGFs (fibroblast growth factors), NCAM, L1 or proteoglycans, such as heparin, heparan sulphateproteoglycans, and chondroitin sulphatepro- ^"' teoglycans.

The medicament of the invention comprises an effective amount of one or more compounds as defined above, or a pharmaceutical composition comprising one or more compounds and pharmaceutically acceptable additives.

Thus, the invention in another aspect also concerns a pharmaceutical composition comprising at least one compound of the invention.

A further aspect of the invention is a process of producing a pharmaceutical composition, comprising mixing an effective amount of one or more of the compounds of the invention, or a pharmaceutical composition according to the invention with one or more pharmaceutically acceptable additives or carriers. In one embodiment the compounds are used in combination with a prosthetic device, wherein the device is a prosthetic nerve guide. Thus, in a further aspect, the present invention relates to a prosthetic nerve guide, characterised in that it comprises one or more of the compounds or the pharmaceutical composition as defined above. Nerve guides are known in the art.

The invention relates to use of a medicament and/or pharmaceutical composition comprising the compound of invention for the treatment or prophylaxis of any of the diseases and conditions mentioned below.

Such medicament and/or pharmaceutical composition may suitably be formulated for oral, percutaneous, intramuscular, intravenous, intracranial, intrathecal, in- tracerebroventricular, intranasal or pulmonal administration.

Strategies in formulation development of medicaments and compositions based on the compounds of the present invention generally correspond to formulation strate- gies for any other protein-based drug product. Potential problems and the guidance required to overcome these problems are dealt with in several textbooks, e.g. "Therapeutic Peptides and Protein Formulation. Processing and Delivery Systems", Ed. A.K. Banga, Technomic Publishing AG, Basel, 1995.

Injectables are usually prepared either as liquid solutions or suspensions, solid forms suitable for solution in, or suspension in, liquid prior to injection. The preparation may also be emulsified. The active ingredient is often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol or the like, and combinations thereof. In addition, if desired, the preparation may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH- buffering agents, or which enhance the effectiveness or transportation of the preparation.

Formulations of the compounds of the invention can be prepared by techniques known to the person skilled in the art. The formulations may contain pharmaceutically acceptable carriers and excipients including microspheres, liposomes, microcapsules, nanoparticles or the like.

The preparation may suitably be administered by injection, optionally at the site, where the active ingredient is to exert its effect. Additional formulations which are suitable for other modes of administration include suppositories, nasal, pulmonal and, in some cases, oral formulations. For suppositories, traditional binders and carriers include polyalkylene glycols or triglycerides. Such suppositories may be formed from mixtures containing the active ingredient(s) in the range of from 0.5% to 10%, preferably 1-2%. Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sus- tained release formulations or powders and generally contain 10-95% of the active ingredient(s), preferably 25-70%.

Other formulations are such suitable for nasal and pulmonal administration, e.g. inhalators and aerosols.

The active compound may be formulated as neutral or salt forms. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the peptide compound) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic acid, oxalic acid, tartaric acid, mandelic acid, and the like. Salts formed with the free carboxyl group may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as iso- propylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

The preparations are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective. The quantity to be administered depends on the subject to be treated, including, e.g. the weight and age of the subject, the disease to be treated and the stage of disease. Suitable dosage ranges are per kilo body weight normally of the order of several hundred μg active ingredient per administration with a preferred range of from about 0.1 μg to 5000 μg per kilo body weight. Using monomeric forms of the compounds, the suitable dosages are often in the range of from 0.1 μg to 5000 μg per kilo body weight, such as » in the range of from about 0.1 μg to 3000 μg per kilo body weight, and especially in the range of from about 0.1 μg to 1000 μg per kilo body weight. Using multimeric forms of the compounds, the suitable dosages are often in the range of from 0.1 μg to 1000 μg per kilo body weight, such as in the range of from about 0.1 μg to 750 μg per kilo body weight, and especially in the range of from about 0.1 μg to 500 μg per kilo body weight such as in the range of from about 0.1 μg to 250 μg per kilo body weight. In particular, when administering nasally smaller dosages are used than when administering by other routes. Administration may be performed once or may be followed by subsequent administrations. The dosage will also depend on the route of administration and will vary with the age and weight of the subject to be treated. A preferred dosage of multimeric forms would be in the interval 1 mg to 70 mg per 70 kg body weight.

For some indications a localised or substantially localised application is preferred.

For other indications, intranasal application is preferred.

Some of the compounds of the present invention are sufficiently active, but for some of the others, the effect will be enhanced if the preparation further comprises pharmaceutically acceptable additives and/or carriers. Such additives and carriers will be known in the art. In some cases, it will be advantageous to include a compound, which promotes delivery of the active substance to its target.

In many instances, it will be necessary to administrate the formulation multiple times. Administration may be a continuous infusion, such as intraventricular infusion or administration in more doses such as more times a day, daily, more times a week, weekly, etc. It is preferred that administration of the medicament is initiated before or shortly after the individual has been subjected to the factor(s) that may lead to cell death. Preferably the medicament is administered within 8 hours from the factor onset, such as within 5 hours from the factor onset. Many of the compounds exhibit a long term effect whereby administration of the compounds may be conducted with long intervals, such as 1 week or 2 weeks.

In connection with the use in nerve guides, the administration may be continuous or in small portions based upon controlled release of the active compound(s). Furthermore, precursors may be used to control the rate of release and/or site of release. Other kinds of implants and well as oral administration may similarly be based upon controlled release and/or the use of precursors.

Treatment

Treatment by the use of the compounds/compositions according to the invention is in one embodiment useful for inducing differentiation, modulating proliferation, stimulate regeneration, neuronal plasticity and survival of cells, for example cells being implanted or transplanted. This is particularly useful when using compounds having a long term effect.

In further embodiment the treatment may be for stimulation of survival of cells which are at risk of dying due to a variety of factors, such as traumas and injuries, acute diseases, chronic diseases and/or disorders, in particular degenerative diseases normally leading to cell death, other external factors, such as medical and/or surgical treatments and/or diagnostic methods that may cause formation of free radicals or otherwise have cytotoxic effects, such as X-rays and chemotherapy. In relation to chemotherapy the FGFR binding compounds according to the invention are useful in cancer treatment.

Thus, the treatment comprises treatment and/or prophylaxis of cell death in relation to diseases or conditions of the central and peripheral nervous system, such as postoperative nerve damage, traumatic nerve damage, e.g. resulting from spinal cord injury, impaired myelination of nerve fibers, postischaemic damage, e.g. resulting from a stroke, multiinfarct dementia, multiple sclerosis, nerve degeneration associated with diabetes mellitus, neuro-muscular degeneration, schizophrenia, AIz- heimer's disease, Parkinson's disease, or Huntington's disease.

Also, in relation to diseases or conditions of the muscles including conditions with impaired function of neuro-muscular connections, such as genetic or traumatic atrophic muscle disorders; or for the treatment of diseases or conditions of various or- gans, such as degenerative conditions of the gonads, of the pancreas, such as diabetes mellitus type I and II, of the kidney, such as nephrosis the compounds according to the invention may be used for inducing differentiation, modulating proliferation, stimulate regeneration, neuronal plasticity and survival , i.e. stimulating survival.

Furthermore, the compound and/or pharmaceutical composition may be for preventing cell death of heart muscle cells, such as after acute myocardial infarction, in order to induce angiogenesis. Furthermore, in one embodiment the compound and/or pharmaceutical composition is for the stimulation of the survival of heart muscle cells, such as survival after acute myocardial infarction. In another aspect the com- pound and/or pharmaceutical composition is for revascularisation, such as after inju- ries.

It is also within the scope of the invention a use of the compound and/or pharmaceutical composition for the promotion of wound-healing. The present compounds are capable of stimulating angiogenesis and thereby they can promote the wound healing process.

The invention further discloses a use of the compound and/or pharmaceutical composition in the treatment of cancer. Regulation of activation of FGFR is important for tumor agiogenesis, proliferation and spreading.

In yet a further embodiment a use of the compound and/or pharmaceutical composition is for the stimulation of the ability to learn and/or of the short and/or long term memory, as FGFR activity is important for differentiation of neural cells.

In still another embodiment a compound and/or pharmaceutical composition of the invention is for the treatment of body damages due to alcohol consumption. Developmental malformations of foetuses, long-term neurobehavioral alterations, alcoholic liver disease are particularly concerned.

Therapeutic treatment of prion diseases including using a compound and/or pharmaceutical composition is still another embodiment of the invention.

In particular the compound and/or pharmaceutical composition of the invention may be used in the treatment of clinical conditions, such as Neoplasms such as malignant neoplasms, benign neoplasms, carcinoma in situ and neoplasms of uncertain behavior, cancer in breast, thyroidal, pancreas, brain, lung, kidney, prostate, liver, heart, skin, blood organ, muscles (sarcoma), cancers with dysfunction and/or over- or under-expression of specific receptors and/or expression of mutated receptors or associated with soluble receptors, such as but not limited to Erb-receptors and FGF- receptors, diseases of endocrine glands, such as diabetes mellitus I and II, pituitary gland tumor, psychoses, such as senile and presenile organic psychotic conditions, alcoholic psychoses, drug psychoses, transient organic psychotic conditions, Alzheimer's disease, cerebral lipidoses, epilepsy, general paresis [syphilis], hepatolen- ticular degeneration, Huntington's chorea, Jakob-Creutzfeldt disease, multiple scle- rosis, Pick's disease of the brain, polyareriti nodosa, syphilis.Schizophrenic disorders, affective psychoses, neurotic disorders, personality disorders, including character neurosis, nonpsychotic personality disorder associated with organic brain syndromes, paranoid personality disorder, fanatic personality, paranoid personality (disorder), paranoid traits, sexual deviations and disorders or dysfunctions (including reduced sexual drive for what ever reason), mental retardation, disease in the nervesystem and sense organs, such as affecting sight, hearing smell, feeling, tasting, cognitive anomalies after disease, injury (e.g. after trauma, surgical procedure, and violence), inflammatory disease of the central nervous system, such as menin- gitis, encephalitis, Cerebral degenerations such as Alzheimer's disease, Pick's disease, senile degeneration of brain, senility NOS, communicating hydrocephalus, obstructive hydrocephalus, Parkinson's disease including other extra pyramidal disease and abnormal movement disorders, spinocerebellar disease, cerebellar ataxia, Marie's Sanger-Brown, Dyssynergia cerebellaris myoclonica, primary cerebellar de- generation, such as spinal muscular atrophy, familial, juvenile, adult spinal muscular atrophy, motor neuron disease, amyotrophic lateral sclerosis, motor neuron disease, progressive bulbar palsy, pseudobulbar palsy, primary lateral sclerosis, other anterior horn cell diseases, anterior horn cell disease, unspecified, other diseases of spinal cord, syringomyelia and syringobulbia, vascular myelopathies, acute infarc- tion of spinal cord (embolic) (nonembolic), arterial thrombosis of spinal cord, edema of spinal cord, hematomyelia, subacute necrotic myelopathy, subacute combined degeneration of spinal cord in diseases classified elsewhere, myelopathy, drug- induced, radiation-induced myelitis, disorders of the autonomic nervous system, disorders of peripheral autonomic, sympathetic, parasympathetic, or vegetative sys- tern, familial dysautonomia [Riley-Day syndrome], idiopathic peripheral autonomic neuropathy, carotid sinus syncope or syndrome, cervical sympathetic dystrophy or paralysis, peripheral autonomic neuropathy in disorders classified elsewhere, amyloidosis, diseases of the peripheral nerve system, brachial plexus lesions, cervical rib syndrome, costoclavicular syndrome, scalenus anticus syndrome, thoracic outlet syndrome, brachial neuritis or radiculitis NOS, including in newborn. Inflammatory and toxic neuropathy, including acute infective polyneuritis, Guillain-Barre syndrome, Postinfectious polyneuritis, polyneuropathy in collagen vascular disease, disorders of the globe including disorders affecting multiple structures of eye, such as purulent endophthalmitis, diseases of the ear and mastoid process, chronic rheumatic heart disease, ischaemic heart disease, arrhythmia, diseases in the pul- monary system, respiratory system, sensoring e.g. oxygene, astma, abnormality of organs and soft tissues in newborn, including in the nerve system, complications of the administration of anesthetic or other sedation in labor and delivery, diseases in the skin including infection, insufficient circulation problem, burn injury and other mechanic and/or physical injuries. Injuries, including after surgery, crushing injury, burns. Injuries to nerves and spinal cord, including division of nerve, lesion in continuity (with or without open wound), traumatic neuroma (with or without open wound), traumatic transient paralysis (with or without open wound), accidental puncture or laceration during medical procedure, injury to optic nerve and pathways, op- tic nerve injury, second cranial nerve, injury to optic chiasm, injury to optic pathways, injury to visual cortex, unspecified blindness, injury to other cranial nerve(s), injury to other and unspecified nerves. Poisoning by drugs, medicinal and biological substances, genetic or traumatic atrophic muscle disorders; or for the treatment of diseases or conditions of various organs, such as degenerative conditions of the go- nads, of the pancreas, such as diabetes mellitus type I and II, of the kidney, such as nephrosis. Scrapie, Creutzfeldt-Jakob disease, Gerstmann-Straussler-Sheinker (GSS) disease; pain syndrome, encephalitis, drug/alcohol abuse, anxiety, postoperative nerve damage, peri-operative ischemia, inflammatory disorders with tissue damage, either by affecting the infections agent or protecting the tissue, HIV, hepati- tis, and following symptoms, autoimmune disorders, such as rheumatoid arthritis, SLE, ALS, and MS. Anti-inflammatory effects, asthma and other allergic reactions, acute myocardial infarction, and other related disorders or sequel from AMI, metabolic disorders, such as obscenity lipid disorders (e.g. hyper cholestorolamia, artheslerosis, disorders of amino-acid transport and metabolism, disorders of purine and pyrimidine metabolism and gout, bone disorders, such as fracture, osteoporosis, osteo arthritis (OA), Atrophic dermatitis, psoriasis, infection cased disorders, stem cell protection or maturation in vivo or in vitro.

Compounds of the invention may also be used for the prevention and treatment of achondroplasia, hypochondroplasia, platyspondylic lethal skeletal dysplasia, thanatophoric dysplasia, Antley-Bixler syndrome, Apert syndrome, Beare-Stevenson syndrome, Crouzon syndrome, Jackson-Weiss syndrome, Pfeiffer syndrome, and Saethre-Chotzen syndrome. According to the invention a method for treatment and/or prevention of the above conditions and symptoms comprises a step of administering an effective amount of a compound and/or pharmaceutical composition to an individual in need.

Examples

1. The reciprocal Ig1-to-lg2 binding site of FGFR.

Methods

To study the structure and binding properties of the FGFR1 Ig1 module, we used the following recombinant proteins: the individual first and second Ig modules (Ig 1 and Ig2), and the combined second and third Ig modules (lg2-3). All recombinant proteins were properly folded as judged by NMR analysis.

Production of recombinant proteins

The Ig1 and Ig2 modules of mouse FGFR1 consist of a His-tag, AGHHHHHH, and amino acids 23-119 and 140-251 , respectively (swissprot p16092). The combined lg2-3 modules of mouse FGFR1 (3c isoform) consist of a His-tag, RSHHHHHH, and amino acids 141-365 (swissprot p 16092). The Ig2 and lg2-3 modules were produced as previously described (Kiselyov et al., Structure 2003 11 : 691-701). The Ig1 module was expressed in the KM71 strain of yeast P. pastoris (Invitrogen, USA) according to the manufacturer's instructions. All the proteins were purified by affinity chromatography using Ni²⁺-NTA resin (Qiagen, USA) and/or ion exchange chromatography and gel filtration.

SPf? Analysis Binding analysis was performed using a BIAcoreX instrument (Biosensor AB, Sweden) at 25 ⁰C using 10 mM sodium phosphate pH 7.4, 150 mM NaCI as running buffer. The flow-rate was 5 μl/min. The lg2-3 modules of FGFR1 were immobilized on the sensor chip CM5 (Biosensor AB, Sweden) as previously described (Kiselyov et al., Structure 2003 11 : 691-701). Binding of the Ig1 module to the immobilized lg2-3 modules was studied in the following way: A protein was injected at a specified concentration simultaneously into a flow-cell with the immobilized FGFR1 modules (Fd cell) and a control flow-cell with nothing immobilized (Fc2-cell). The curve representing unspecific binding of the protein to the surface of the Fc2-cell was subtracted from the curve representing binding of the same protein to the immobilized lg2-3 modules and the surface of the Fd -cell. The resulting curve was used for analysis.

NMR measurements The following samples were used for recording of NMR spectra: 2 mM Ig1 or Ig2 modules (in H₂O or D₂O), 2 mM ¹⁵N-labeled Ig1 or Ig2 modules (in H₂O), 0.5 mM ¹⁵N₁ ¹³C(50%)-labeled Ig2 module (in H₂O). The buffer was 10 mM sodium phosphate pH 7.4, 150 mM NaCI, except for the double-labeled sample, where 10 mM sodium phosphate pH 7.4, 30 mM NaCI was used. The following NMR spectra were recorded and used for assignment of the Ig1 and Ig2 modules: TOCSY in H₂O or D₂O (45 and 70 ms mixing time), NOESY in H₂O or D₂O (80 and 200 ms mixing time), DQFCOSY, ¹⁵N-HSQC, ¹⁵N-TOCSY-HSQC (70 ms mixing time), and ¹⁵N- NOESY-HSQC (125 ms mixing time). For the assignment of the Ig2 module, HNCACB, CBCA(CO)NH, HNCO, HN(CA)CO, HNCA, HN(CO)CA were also used. All spectra were recorded using the standard set-up provided by ProteinPack. The spectra were processed by NMRPipe (Delaglio et al., J Biomol NMR 1995 6: 277- 93) and analysed by Pronto3D (Kjεer et al., Methods Enzymology 1994 239: 288- 307). The NMR experiments were performed using Varian Unity Inova 750 and 800 MHz spectrometers. All spectra were recorded at 298 K.

Structure calculation

A simulated annealing protocol using the X-PLOR program (Brunger, X-PLOR Software Manual, version 3.1. 1992, Yale University, New Haven, CT) was used for structure calculation. 1360 NOE restraints were derived from 80/200 ms NOESY and 125ms ¹⁵N-NOESY-HSQC spectra with upper bounds of 2.7, 3.3 and 6.0 A increased by 0.5 A if the restraint included a methyl group. 83 φ angles restraints with bounds of -120±40° and -57±40° (derived from the ³JnN_Ha coupling constants) were used. After inspection of hydrogen bond energies and the rate of hydrogen exchange, 94 hydrogen bond restraints were applied as NOE restraints (in the final structure calculations) with upper bounds of 2 A and 3 A for the NH-O and N-O distances, respectively. Of 100 structures calculated, 100 were accepted by X-PLOR, discriminating any structure with an NOE restraint violation >0.5A or an angle violation >5°. The structures were analysed and checked using MOLMOL (Koradi et al., J MoI Graph 1996 14: 51-5, 29-32) and PROCHECK_NMR (Laskowski et al., J MoI Biol 1993 231: 1049-67) programs. From these 100 structures, 20 structures with the smallest energies were chosen to represent the structure of the Ig1 module of

FGFR1.

Phosphorylation assay of FGFR-1 Trex293 cells (Invitrogen) were stably transfected with human FGFR-1 with a C- terminal Strep Il tag (IBA Biotech). The cells were maintained in the DMEM medium with Hygromycin (Invitrogen) 200μg/ml, 10% FCS, 1 % (v/v) glutamax, 100 U/ml penicillin, 100μg/ml streptomycin (all from Gibco, BRL). For phosphorylation assay, 2x1 O^ cells were starved overnight with medium without serum. After stimulation with various compounds at the specified concentrations, the cells were lysed by 300μl lysis buffer with 1%(v/v) NP-40, complete protease inhibitors (Roche, Ger- many)(1 :50), and phosphatase inhibitors (Calbiochem inhibitor cocktail lll)(1 :100) in PBS. Then the protein concentration was determined using bicinchoninic acid assay (Pierce, Rockville, IL), and 500μg proteins of each lysate was incubated with 15μl agarose-coupled anti-phosphotyrosine antibodies (4G10-AC) (Upstate biotechnologies) for 6 hr or overnight at 4 ⁰C. The bound proteins were washed and eluted using 180 mM phenyl phosphate (Sigma) in the chromatography columns (BIO- RAD). 25μl of the purified protein for each sample was separated by SDS-PAGE and transferred to a polyvinylidene fluoride membrane (Millipore, Bedford, MA). Im- munoblotting was performed using rabbit antibodies (diluted 1:2000) against the recombinant Strepll tag (IBA Biotech) and swine anti-rabbit IgG horseradish peroxidase conjugate (diluted 1 :2000) (DakoCytomation, Denmark) in 5% (w/v) nonfat dry milk. The immune complexes were developed by SuperSignal® West Dura extended duration substrate (PIERCE), and visualized with SynGene Gene Snap ver- sion 6.00.21 software (Synoptics Ltd, UK). For all assays, the exposed bands were quantified with SynGene Gene Tool image analysis program (Synoptics Ltd, UK).

Results and Discussion

Structure of the Ig1 module of FGFR1

The solution structure of the Ig1 module was determined by NMR. A total of 1360 non-redundant NOEs was assigned and applied in the structure calculations together with 83 backbone dihedral angle constraints derived from the ³JHNH_O coupling constants. After inspection of hydrogen bond energies and the rate of hydrogen ex- change, 94 hydrogen bond constraints were applied as NOEs in the final structure calculations. A ribbon representation of the structure is shown in Fig. 1 A. The three- dimensional fold of the module belongs to the intermediate Ig subgroup and can be described as a β-barrel consisting of two β-sheets. One sheet is formed by A' (L⁴³- 5 V⁴⁵), G (G¹⁰⁸-V¹⁰⁷), F (S⁹⁶-S¹⁰⁵), C (S⁶³-R⁶⁸) and C (V⁷¹-L⁷³) β-strands, and the other by A (T²⁶-L²⁷), B (D⁴⁹-R⁵⁴), B' (L⁵⁷-R⁵⁸), E (E⁸⁵-D⁹⁰) and D (T⁷⁹-T⁸²) strands. An overlay of 20 superimposed structures for the backbone atoms is shown in Fig. 1B. The root mean square (RMS) deviation from the average is 1.05 A for the backbone atoms and 1.53 A for all heavy atoms. 98.8% of the (φ, ψ) angle combinations of the 10 entire ensemble fall into the allowed regions of the Ramachandran plot. A summary of structural statistics is given in Table I below:

Table I

15 Structural precision

RMS deviation for backbone atoms 1.05 A

RMS deviation for heavy ^r atoms 1.53 A

²Number of constraints i 1537:

20 long range NOE 734 medium range NOE 125 sequential NOE 374 intra NOE 127 dihedral angle 83

25 hydrogen bond 94

³Energies (kcal/mol): bonds 4.59 bond angles 241.46

30 NOE 13.08 dihedral angles 0.23 hydrogen bonds -175 van der Waals -32.05 dihedral bond angles 87.6

35. improper bond angles 9.63 overall 149.59

RMS deviations from idealized geometry bonds 0.0029 A

40 bond angles 0.44 deg. improper bond angles 0.36 deg.

RMS violation of constraints

NOE/hydrogen bonds 0.030 A 45 dihedral angles 0.034 deg. Ramachantran plot statistics

Most favored 70.8 %

Additionally allowed 26.8%

Generously allowed 1.2 % Disallowed 1.2 %

¹RMS deviations from the average for residues 25-120 in 20 structures. ²Number of non-redundant constraints. ³The energies were calculated using the CHARMM force field with force constants for NOE of 10 kcal mol-1 A-2 and for dihedral con- straints of 8 kcal mol-1 rad-2.

The general strand topology of the Ig 1 module is similar to that of the Ig2 and Ig3 modules (Plotnikov et al., Cell 1999 98: 641-50). However, the A/A' loop of the Ig1 module is much longer than that of the Ig2 and Ig3 modules. It contains 8 extra resi- dues compared to the Ig3 module, and 5 extra residues compared to Ig2. In contrast to the Ig2 and Ig3 modules, where the A/A^! loop is in parallel with the β-barrel, the A/A' loop of the Ig1 module is situated perpendicularly to the β-barrel (Fig. 1C). This is noteworthy, because it is this region of the Ig1 module that forms a binding site for the Ig2 module (see below).

Ig1 binds to Ig2 in the area of the FGF1-lg2, heparin-lg2 and Ig2-lg2 binding sites Since Ig1 in FGFR3 binds to lg2-3 with a Kd value of 20 μM (Olsen et al., Proc Natl Acad Sci U S A 2004 101 : 935-40), it was of interest to determine this binding for FGFR1. Therefore, binding of soluble Ig1 to immobilized lg2-3 modules of FGFR1 was studied by SPR analysis. A plot of the equilibrium binding response versus the concentration of Ig1 is shown in Fig. 2A. The time-course of the binding, similarly to that for FGFR3, is characterized by very fast association and dissociation phases (not shown). The calculated Kd value for the binding was 33±6 μM, which is very close to the 20 μM Kd value determined for FGFR3.

In order to identify the residues involved in the interaction between Ig1 and Ig2, NMR analysis was employed. NMR analysis provides a very sensitive method for the study of protein interactions in solution. However, it requires production of ¹⁵N labeled proteins and assignment of their ¹⁵N, ¹H resonance frequencies for the backbone atoms. Thus to study the Ig1-lg2 interaction by NMR, assignment of the ¹⁵N, ¹H resonance frequencies for the backbone atoms of the Ig2 module was performed. The Ig2 module contains binding sites for FGF, heparin and the Ig2 module itself (whereas the Ig3 module contains the binding site only for FGF). Moreover, the Ig 1 module inhibits both the FGF-FGFR and heparin-FGFR interactions (Wang et al., J Biol Chem 1995 270: 10231-5; Olsen et al., Proc Natl Acad Sci U S A 2004 101: 935-40).

In an ¹⁵N-HSQC spectrum of an ¹⁵N-labeled protein, a signal for all amino acids with both a nitrogen and a proton can be observed. The changes in chemical shifts of the signals provide a method for identification in a protein of amino acid residues that are perturbed by the binding of another molecule. 2 mM unlabeled Ig1 module was added to a 0.5 mM ¹⁵N-labeled sample of the Ig2 module, and vice versa, 2 mM unlabeled Ig2 module was added to a 0.5 mM ¹⁵N-labeled sample of the Ig1 module. The recorded changes of chemical shifts are shown in Fig. 2B, C. The residues of the Ig1 module that exhibited significant perturbation (higher than 0.04 ppm) by the Ig2 module were L²⁷, E²⁹, Q³⁰, A³¹, Q³², W³⁴, G³⁵ and V³⁶ (Fig. 2B), and the residues of the Ig2 module that exhibited significant perturbation (higher than 0.025 ppm) by the Ig1 module were T¹⁵⁶, S¹⁵⁷, E¹⁵⁹, K¹⁶⁰, A¹⁶⁷, V¹⁶⁸, A¹⁷¹, K¹⁷², T¹⁷³, V¹⁷⁴, K¹⁷⁵, S²¹⁴, I²¹⁵, I²¹⁶, M²¹⁷ and S²¹⁹ (Fig. 2C). The changes of the chemical shifts of these residues demonstrate that the presence of one module close to the other module alters the chemical environment at the perturbed residues, indicating that the perturbed residues are either a part or in the vicinity of the binding site for the interaction be- tween the two modules. Mapping of the perturbed residues onto the structures of the Ig1 and Ig2 modules is shown in Fig. 3A. Since the NMR structure of the Ig2 module is not known, the crystal structure of the module (Plotnikov et al., Cell 1999 98: 641- 50) was used for mapping. The perturbed residues in the Ig1 module are located in the A/A' loop region and form a single patch, whereas the perturbed residues in the Ig2 module are located in two patches: a larger patch consisting of 12 residues (A¹⁶⁷, V¹⁶⁸, A¹⁷¹, K¹⁷², T¹⁷³, V¹⁷⁴, K¹⁷⁵, S²¹⁴, I²¹⁵, I²¹⁶, M²¹⁷, S²¹⁹) and a smaller patch consisting of four residues (T¹⁵⁶, S¹⁵⁷, E¹⁵⁹, K¹⁶⁰). The two patches are located close to each other. Since the residues from the Ig 1 patch cannot bind at the same time to both Ig2 patches, and since the Ig 1 patch and the larger Ig2 patch are approximately equal in size, whereas the smaller Ig2 patch is substantially smaller than the Ig1 patch, it is very likely that the binding of the Ig2 module to the Ig1 module is mediated only by the residues from the larger Ig2 patch and that the residues from the smaller Ig2 patch are unrelated to the Ig1-lg2 binding (the perturbation in this area may be due to a conformational change induced by the binding). According to the symmetrical model of the FGFR dimerisation (Plotnikov et al., Cell

1999 98: 641-50; Plotnikov et al., 2000 Cell 101: 413-24; Schlessinger et al., MoI Cell 2000 6: 743-50), the primary Ig2 site binding to FGF consists of L¹⁶⁵, A¹⁶⁷, P¹⁶⁹ and V²⁴⁸, the secondary Ig2-FGF binding site consists of P¹⁹⁹, D²⁰⁰, I²⁰³, G²⁰⁴, G²⁰⁵, S²¹⁹ and V²²¹, the Ig2-heparin binding site consists of K¹⁶⁰, K¹⁶³, K¹⁷⁵ and K¹⁷⁷, and the Ig2-lg2 binding site consists of A¹⁷¹, K¹⁷², T¹⁷³ and D²¹⁸. Mapping of these binding sites onto the structure of the Ig2 module is shown in Fig. 3B. A¹⁶⁷ from the primary and S²¹⁹ from the secondary Ig2-FGF binding sites, K¹⁷⁵ from the Ig2-heparin binding site, and A¹⁷¹, K¹⁷², T¹⁷³ from the Ig2-lg2 binding site are among the residues in the Ig2 module which are perturbed by Ig1 binding. As can be seen from Fig. 3A and B, both the primary and secondary Ig2-FGF and the Ig2-heparin binding sites are adjacent to the larger Ig2 patch of perturbed residues, whereas three out of four residues of the Ig2-lg2 binding site are located within this patch. These data indicate that the Ig1 binding to Ig2 interferes with the binding of FGFR to itself, FGF and heparin, and thus that Ig1 functions as a competitive intra-molecular inhibitor of FGF-FGFR, heparin-FGFR and FGFR-FGFR interactions.

Kinetic analysis of the auto-inhibitory effect of the Ig 1 module ofFGFRI

If the Ig 1 module functions as a competitive intra-molecular inhibitor of FGF-FGFR and heparin-FGFR interactions, then given that the affinities of interactions between FGF and FGFR, heparin and FGFR, and the Ig1 and lg2-3 modules are known, it may be possible to describe quantitatively the auto-inhibitory effect of the Ig 1 module, namely: an approx. 8- and 4-fold decrease in the affinity of the FGFR1α-FGF1 and FGFRIα-heparin interactions, respectively, as compared to the FGFR1β-FGF1 and FGFRIβ-heparin interactions (Wang θt al., J Biol Chem 1995 270: 10231-5). In the following, we are going to consider binding of FGF to the lg2-3 module of the triple Ig-module of FGFR in the presence of the intra-molecular binding of the Ig 1 module to the lg2-3 module. The FGF-FGFR binding and the intra-molecular Ig1 to lg2-3 binding are considered to be mutually exclusive.

In order to perform the quantitative analysis, some simplifications and approximations will have to be done. Whether these simplifications are justified can be judged by how well the resulting model agrees with the experimental data. The Ig1-lg2 linker of rat FGFRIα consists of 31 amino acids: DALPSSEDDDDDDDSSSEEKETDNTKPNRRP. Analysis of this sequence by various secondary structure prediction engines predicts a random coil for either all or most of the residues, which indicates that the linker forms a random coil in the FGFRIα molecule. This is corroborated by the fact that in the crystal structure of the homologous molecule, FGFR3α, no electron density was found for either the linker region or the Ig1 module (Olsen et al., Proc Natl Acad Sci U S A 2004 101 : 935-40), which means that the Ig1 module and the linker are completely disordered in the crystal. Thus, it is reasonable to assume that the linker region also forms a random coil in the FGFRIα molecule in solution, in Fig. 4A, the structure of FGFRIα is depicted schematically with various random conformations of the linker (assuming for a moment that there is no interaction between the Ig 1 and Ig2 modules). The random movement of the Ig 1 module around the Ig2 module is obviously restricted by the length of the linker, meaning that only a small volume around the Ig1 module is available for random movement of the Ig 1 module. Thus, for the analysis of the in- tra-molecular interaction, the real concentration of the module in the volume available to it will be used instead of its concentration in the total volume. For estimation of the real concentration, the module's concentration in a sphere (depicted as a circle in Fig. 4A) with a radius corresponding to the average distance between the N- termini of the Ig 1 and Ig2 modules will be used. For calculation of this distance, the average end-to-end distance of the linker region must be estimated. The average end-to-end distance of a random coil of polymers can be calculated by the following empirical formula for self-avoiding random walk: (AR²y² = L-N^0'592 , where

(AR² ) is the root average square end-to-end distance, N is the number of links in the polymer, and L is the length of the link (the angle between the consecutive links is random). Let us now assume for a moment that the angle between the consecutive residues of the FGFRIα linker region is random, then, given that the length of the peptide backbone corresponding to one amino acid is approximately 0.4 nm, the average end-to-end distance of the linker can be estimated as approximately 3.1 nm. However, the angle between consecutive residues of a peptide cannot be completely random due sterical interference of the side-chains. Thus, the real distance will be larger than 3.1 nm, but smaller than 12.4 nm, which is the length of the fully extended polypeptide. Since it is not possible to calculate exactly the average length of the linker, we will consider the whole range of average linker distances, from 3.1 to 12.4 nm.

Interaction between FGFRIα and FGF1 (or heparin) when Ig1 is acting auto- inhibitory can be described by the following formulas (derivation is given in the Appendix): where (D₂₃ )_ois the initial concentration of FGFRIα, (F) is the concentration of FGF1, (D₂₃ • F) is the concentration of the FGF1-

FGFRIα complex, K₁ is the Kd value for the FGF1- FGFRIβ interaction, [D₂^D₁) is the concentration of the FGFRIα molecules in which the Ig1 and Ig2 modules are involved in the intra-molecular interaction. (D₂₃ ^D₁) can be determined from the following equation:

(D₂₃ .D₁) and

4000

K₀ = K₀ - N, π(l² +p²Y²(D₂₃)₀ , K₂ being the Kd of the interaction between

the Ig1 module and FGFRIβ, N_A being Avogadro number, / being the average linker length, and p being length of the Ig1 module.

The reported Kd values for the FGF-FGFR interaction range from 10 pM to 100 nM, depending on the method used. The affinity of heparin-FGFR interaction is of the order of 0.3-1 μM. We will at first perform a detailed kinetic analysis assuming that the Kd value of the FGF-FGFR interaction is 60 pM, and then we will analyze the whole range from 10 pM to 1 μM. Fig. 4B shows estimation of the binding between FGF1 and FGFRIβ or FGFRIα (with linker lengths of 4.5, 6.9 and 11.3 nm) and the intra-molecular binding of the Ig1 module as a function of FGF1 concentration. FGFRIα displays a substantially lower binding to FGF1 as compared to FGFRIβ. In the absence of FGF1, the Ig1 module is involved in intra-molecular binding in approx. 80% FGFRIα molecules, and, in the presence of FGF1, this percentage drops quickly with increasing concentrations of FGF1. In order to determine the apparent Kd values of the FGF1-FGFR1α binding for various linker lengths, the data points from Fig. 4B were fitted to an equation describing binding with a single site (Fig. 4C). For linker lengths ranging from 4.5 to 11.3 nm, the decrease in affinity for the FGFR10C-FGF1 interaction as compared to the FGFR1β-FGF1 interaction ranges from 13.1- to 4.6-fold, and the number of FGFRIoc molecules in which the Ig1 module is involved in intra-molecular binding ranges from 87.4 to 69%. The results of the kinetic analysis are summarized in Table Il below: Table Il

It should be noted that linker lengths close to 3 nm are unlikely for the reasons described above, and linker lengths longer than 9-10 nm also are unlikely, because this is very close to the length of the fully extended conformation of the linker. Thus, the linker length most probably ranges from 6 to 9 nm with a corresponding decrease in affinity of approximately 11- to 6-fold, which is in good agreement with the experimentally determined value of 8-fold. In order to determine how the affinity of the ligand-FGFR interaction affects the estimated inhibitory effect of the Ig1 module, we performed the above described calculations for the whole range of affinities (Kd from 10 pM to 1 μM) using an average linker length of 8 nm (which produces the best correlation between the calculated and experimental data). The inhibitory effect of the Ig1 module on the FGF-FGFRIα interaction (Kd from 10 pM to 100 nM) ranges from 10.5- to 4.2-fold (Fig. 4D). The Kd for the heparin-FGFR interaction is as mentioned above ca. 0.3-1 μM, and thus, the corresponding inhibitory effect of the Ig1 module is approximately 4-fold (Fig. 4D), which is in complete agreement with the experimentally determined value of 3.7-fold. Thus, the quantitative analysis of the inhibitory effect of the Ig1 module presented here is in excellent agreement with the available experimental data on the kinetics of the FGF-FGFR1 and heparin-FGFR1 interactions, thereby corroborating a model in which the Ig1 module functions as a competitive intra-molecular inhibitor of the FGF- FGFR1 and FGFR1 -heparin interactions. It should be noted that since the auto- inhibitory effect of Ig1 does not depend very much on the Ig1-lg2 linker length (in the range from 4.5 to 9 nm), approximately the same auto-inhibition is expected for the other FGFR isoforms in which the Ig1-lg2 linkers are slightly shorter. This has been confirmed for FGFR3 which has an approximately 4- and 5.7-fold decrease in the affinity of the FGFR3α-FGF1 and FGFR3α-heparin interactions, respectively, as compared to the FGFR3β-FGF1 and FGFR3β-heparin interactions (Olsen et al., Proc Natl Acad Sci U S A 2004 101: 935-40).

Furthermore, our analysis predicts that in the absence of FGF, the Ig 1 module is involved in intra-molecular binding in approx. 80% FGFR1 molecules, thus inhibiting a putative direct Ig2-lg2 binding. However, in the presence of FGF, FGF relieves the observed inhibition by Ig1 by a competitive inhibition of the Ig1-lg2 interaction (see Fig. 4B). This indicates that the Ig 1 module not only regulates the FGFR-ligand binding affinity, but also prevents the spontaneous FGFR dimerisation in the absence of FGF. It is noteworthy that a switch from FGFRα to FGFRβ correlates with astrocyte malignancy (Yamaguchi et al., Genes Dev 1994 8: 3032-44). Expression of FGFRβ, which may be under less tight control of activation due to the lack of Ig 1 , may lead to a growth advantage of tumorigenic cells, resulting in the tumor development.

Effect of soluble Ig 1 and Ig2 modules and peptides designed from the two modules on FGFRIα activation .

It is well known that a small fraction of FGFR molecules expressed by cells (in a culture) is in the active/phosphorylated state even though there are no FGFs in the culture medium. This background FGFR activity has been suggested to be due to a direct FGFR-FGFR interaction, arid this is supported by the fact that there is a direct Ig2-lg2 contact in the symmetrical model of the ternary FGF-FGFR-heparin complex. As mentioned above, our calculations predict that in the absence of FGFs the Ig1 and Ig2 modules are interacting in approximately 80% FGFRIα molecules. Since the intra-molecular Ig1-lg2 interaction is expected to inhibit the direct FGFR- FGFR interaction (see Fig. 2), only 20% FGFR molecules are available for spontaneous dimerization. Addition of soluble Ig1 or Ig2 modules to the cells expressing FGFR1 α may either decrease or increase the number of receptor molecules available for spontaneous dimerization, and may thus inhibit or promote the background receptor activation. To test this assumption, TREX-293 cells, stably transfected with a full length FGFR1 containing a C-terminal Strepll-tag, were stimulated with FGF1 (positive control), the Ig1 or Ig2 modules or nothing. After stimulation of cells for 20 min, FGFR1 was immunopurified and analyzed by immunoblotting. As appears from Figure 5A and B, the Ig2 module at concentrations of 20, 100 and 500 μg/ml sub- stantially increased the receptor phosphorylation (approx. 2.5 fold), whereas the Ig1 module did not have any significant effect. The stimulatory effect of the Ig2 module probably is due to inhibition of the intra-molecular Ig1-lg2 binding, which increases the number of receptor molecules in which the Ig2 modules are not blocked by intramolecular binding of Ig1 and thus available for spontaneous dimerization. Addition of the Ig 1 module is expected to increase the number of receptor molecules in which the Ig2 module is bound to the Ig 1 module and, thus, decrease the number of receptor molecules available for spontaneous dimerization. However, even before addition of soluble Ig 1 the number of the receptor molecules in which Ig2 is bound to Ig 1 is approximately 80% (and thus close to maximal binding). Therefore, further addi- tion of soluble Ig1 at the used concentrations is not expected significantly to reduce the number of receptor molecules available for spontaneous dimerization, and this probably explains why the Ig1 module did not have any significant effect.

Since the binding sites for the Ig1-lg2 interaction were identified using NMR titration analysis (see above), it was of interest to test if peptides corresponding to these sites could mimic the effects of the Ig1 and Ig2 modules on the FGFRIα phosphorylation. Therefore, based bn the NMR titration data, we designed a peptide derived from the Ig1 module (FRDIa: TLPEQAQPWGV) and two peptides derived from the Ig2 module (FRD2a: E¹⁵⁹KMEKKLHAV¹⁶⁸ and FRD2b: A¹⁷⁰AKTVKFKC¹⁷⁸), suppos- edly mimicking the binding interactions. As appears from Fig. 5A and B, both peptides derived from Ig2 (FRD2a at 20, 100 and 500 μg/ml concentrations and FRD2b at 1 , 5 and 20 μg/ml concentrations) substantially increased the receptor phosphorylation similar to that of the Ig2 module, whereas the peptide derived from Ig 1 (FRDIa at 1, 5, 20, 100 and 500 μg/ml concentrations), as expected, did not have any effect. These results further support a regulatory role of Ig1 in FGFRIα activation.

Appendix

Let us consider binding of FGF to the lg2-3 module of the triple Ig-module of FGFR in the presence of the intra-molecular binding of the Ig 1 module to the lg2-3 module. The FGF-FGFR binding and the intra-molecular Ig1 to lg2-3 binding are considered to be mutually exclusive. These interactions can be described with the following schemes: D_Ώ +F ^ D₂, . F (1) D₂₃ ^-D₁ O D₂₃ - Z)₁ (2)

D₂₃ stands for the lg2-3 module of the triple Ig-module FGFR (Ig1 is implied to be attached to the lg2-3 module), F - for FGF or other ligand (except Ig1), and D₁- for

Ig1. The sign • designates a complex between two molecules. (1) describes an interaction between two different molecules, whereas (2) describes an intra- molecular interaction between two different parts of the same molecule. (1) can be quantitatively described by the following equation:

⁽A₂HQ ₍₃₎

where (F) means concentration of the molecule F , and K_x stands for the Kd value. For the quantitative description of (2), let us assume that the Ig1 module is attached to the Ig2 module via a flexible linker (as depicted in Fig. 4A) that can assume a random conformation (for justification, see the Results and Discussion). The Ig 1 module appears to float randomly around the Ig2 module and can be imagined as being contained in a sphere (around the Ig2 module) of a radius equal to the average distance between the Ig 1 and Ig2 modules. Thus, the module's concentration in this sphere will be used for analysis. Let us assume that the module's concentration in this sphere can be calculated by multiplying (D₁) with a factor λ . The value of this factor will be calculated later. Then (2) can be described by the following equation:

Let us designate the initial concentration of D₂₃ as (D₂₃)₀ , the equilibrium concentration of D₂₃ *F as x , and the equilibrium concentration of D₂₃ ^D₁ as y , and keep F constant. From (1 ) and (2), we obtain that {D_λ\ = (JD_2i\ ,

(A₃) ^~ (A₃)o -x-y and (D₁) = (D₂₃)₀ - y . Then from (3) and (4), we obtain that _κ _ ((A₃)o -x- y) -(F) ₍₅₎. _g ((Aa)₀ -*-yH(A3)o -JO -Λ ₍₆₎

¹ X ' y

Solving equations (5) and (6), we obtain that

/ _{x =} (A^s)^o y . y = hd -4d² -A(D₂X) , where d = &-(F) + K₂ +2(D₂,)₀ 1 I ^Ai -£ ^i

(F)

. ' K₂ and K₂ = -+ .

In order to calculate the factor λ , we have to estimate the average value of the distance between the N termini of the Ig1 and Ig2 modules (see Fig. 6). As appears from Fig. 6, r² = l² +p² -21-p -cosφ, where r is the distance between the N ter- mini of the Ig1 and Ig2 modules, / is the linker length, and p is the length of the Ig1 module. From this, it follows that Ir²) = Il²) + lp²) -(2I ^■ p.cosφ) = (l²\ + p² , where the sign ( ) means averaging.

For estimation of (l²\ see Results and Discussion. The molar concentration c of the Ig 1 module in the sphere of radius r can be calculated as c = where N₄ is the Avogadro num-

ber. Then the factor λ can be calculated as follows: λ = ^■

(A)₀

2. Stimulation of neurite outgrowth

Methods Cerebellar granule neurons (CGN) are prepared from postnatal day seven Wistar rats largely as previously described by Drejer and Schousboe (1989) Neurochem Res. 14:751-4.

For the experiments cerebellar tissue was dissected in modified Krebs-Ringer solution kept on ice, and treated as described for the hippocampal neurons above. All cell cultures were incubated at 37⁰C in a humidified atmosphere containing 5 % CO₂. All animals were handled in accordance with the national guidelines for animal welfare.

CGN were plated at a density of 10,000 cells/cm² on uncoated 8-well permanox Lab- Tek chamber slides in Neurobasal medium supplemented with 0.4 % (w/v) bovine serum albumin (BSA; Sigma-Aldrich), 2 % (v/v) B27 Neurobasal supplement, 1 % (v/v) glutamax, 100 U/ml penicillin, 100 μg/ml streptomycin and 2 % 1 M HEPES (all from Gibco, BRL). Peptide solutions without or with inhibitors of various signal transduction pathways were added to a total volume of 300 μl/cm², and the slides were incubated at 37^DC. After 24 hours, the neurons were fixed with 4 % (v/v) formalde- hyde for 20 minutes and thereafter immunostained using primary rabbit antibodies against GAP-43 and Alexa Fluor secondary goat anti-rabbit antibodies. Images of at least 200 neurons for each group in each individual experiment were obtained systematically by using computer assisted fluorescence microscopy as previously described (Rønn et al., 2000 op. cit). Briefly, a Nikon Diaphot inverted microscope with a Nikon Plan 2Ox objective (Nikon, Tokyo, Japan) coupled to a video camera (Grun- dig Electronics, Germany) was used for recordings. The same software package as described above for the dopaminergic neurite outgrowth assay was used to process the recorded images.

Results and discussion

Figure 7 demonstrates the stimulation of neurite outgrowth of cerebellar granular neurons in response to treatment with different concentrations of the FRD2a (SEQ ID NO:8 )(A) and FRD2b (SEQ ID NO: 13) (B) peptides derived from the Ig1-to-lg2 reciprocal binding site of FGFR The length of neurites in cultures is presented as a percentage of neurite length in the treated cultures compared to control (untreated cultures).

Both the FRD2a and FRD2b peptides are capable of stimulating neurite outgrowth.

Claims

1. A peptide of at most 25 contigous amino acid residues comprising a fragment of a Fibroblast Growth Factor Receptor (FGFR), wherein said fragment comprises amino acid residues involved in the reciprocal interaction of the Immunoglobulin- like modules 2 (Ig2) and Immunoglobulin-like module 1 (Ig1) of said receptor.

2. The peptide according to claim 1 , wherein the reciprocal interaction occurs between the Ig1 and Ig2 modules of the same FGFR polypeptide.

3. The peptide according to claims 1 and 2, wherein the reciprocal interaction takes place at a reciprocal Ig1-to-lg2 binding site of a FGFR.

4. The peptide according to claimsi to 3, wherein the fragment comprices a contigous amino acid sequence derived from the reciprocal Ig1-to-lg2 binding site.

5. The peptide according to claim 4, wherein the contigious amino acid sequence is derived from a part of the reciprocal Ig1-to-lg2 binding site which comprises amino acid residues of the Ig1 module.

6. The peptide according to claim 4, wherein the contigious amino acid sequence is derived from a part of the reciprocal Ig1-to-lg2 binding site which comprises amino acid residues of the Ig2 module.

7. The peptide according to claims 1-6, wherein said peptide comprises an amino acid sequence selected from

TLPEQAQPWGA (SEQ ID NO: 1)

TKYQISQPEV (SEQ ID NO:2)

EPGQQEQLV (SEQ ID NO:3) SLEQQEQKL (SEQ ID NO: 4)

SLVEDTTLEPEEP (SEQ ID NO:5)

GTEQRWGRAAEV (SEQ ID NO:6)

EASEEVELEPCLA (SEQ ID NO-.7)

EKMEKKLHAV (SEQ ID NO:8) EKMEKRLHAV (SEQ ID NO:9) ERMDKKLLAV (SEQ ID NO: 10)

QRMEKKLHAV (SEQ ID NO:11 )

SKMRRRVIAR (SEQ ID NO:12)

AAKTVKFKC (SEQ ID NO:13) AANTVKFRC (SEQ ID NO:14)

AANTVRFRC (SEQ ID NO:15)

AGNTVKFRC ^* (SEQ ID NO:16) or

VGSSVRLKC (SEQ ID NO:17), or a fragment thereof.

8. The peptide according to any of the claims 1-4 or 6-13, wherein the peptide is capable of interacting with the Ig1 module of at the reciprocal Ig1-to-lg2 binding site.

9. The peptide according to claim 8, wherein the peptide comprises an amino acid sequence comprising an amino acid motif of the formula

Q/E-(x)₃~x^p, wherein x^p is a hydrophobic amino acid residue and

(x)_c is an amino acid sequence of three amino acid resides, wherein x is any amino acid residue.

10. The peptide according to claim 9, wherein x^p is V, L or P.

11. The peptide according to claim 9 or 10, wherein (x) ₃ comprises at least one residue Q or E.

12. The peptide according to claim 11 , wherein (x)₃ comprises a charged amino acid residue or a phydrophobic amino acid residue.

13. The peptide according to claim 12, wherein the hydrophobic residue is P, I, L or V.

14. The peptide according to any of the preceding claims 1-5, 7 or 14-18 wherein said peptide is capable of interacting with the Ig2 module of at the reciprocal Ig 1- to-lg2 module binding site of FGFR.

15. The peptide according to claim 14, wherein the peptide comprises an amino acid sequence comprising an amino acid motif of the formula K/R-x^p-(x)o-i-K/R, wherein x^p is a hydrophobic amino acid residue, and

(x) is a charged amino acid residue.

16. The peptide according to claim 15, wherein x^p is M, L or F.

17. The peptide according to claim 15, wherein the amino acid motif is a three- amino-acid motif K/R-x^p-(x)₀-K/R , wherein x^p is F or L.

18. The peptide according to claim 15, wherein the amino acid motif is a four-amino- acid motif K/R-x^p-(x)_rK/R, wherein x^p is M.

19. The peptide according to any of the claims 5, 7 or 14-18 wherein the amino acid sequence selected from

TLPEQAQPWGV (SEQ ID NO: 1 )

TKYQISQPEV (SEQ ID NO:2) EPGQQEQLV (SEQ ID NO:3)

SLEQQEQKL (SEQ ID NO: 4)

SLVEDTTLEPEEP (SEQ ID NO:5)

GTEQRWGRAAEV (SEQ ID NO:6)

EASEEVELEPCLA (SEQ ID NO:7) or a fragment thereof.

20. The peptide according to any of the claims 6-13, wherein the amino acid sequence is selected from

EKMEKKLHAV (SEQ ID NO:8) EKMEKRLHAV (SEQ ID NO:9)

ERMDKKLLAV (SEQ !D NO:10)

QRMEKKLHAV (SEQ ID NO:11)

SKMRRRVIAR (SEQ ID NO: 12)

AAKTVKFKC (SEQ ID NO:13) AANTVKFRC (SEQ ID NO:14) AANTVRFRC (SEQ ID NO:15)

AGNTVKFRC (SEQ ID NO:16) or

VGSSVRLKC (SEQ ID NO: 17), or a fragment thereof.

21. The peptide according to any of the preceding claims, wherein FGFR is selected from FGFR 1 , FGFR 2, FGFR 3, FGFR 4 or FGFR5.

22. The peptide according to claim 21 , wherein FGFR is FGFR 1.

23. The peptide according to any of the preceding claims, said peptide is capable of modulating activity of FGFR.

24. The peptide according to claim 23, said peptide is capable of activating FGFR.

25. The peptide according to calim 23, said peptide is capable of inhibiting FGFR.

26. The peptide according to claim 24, wherein FGFR 1 is activated.

27. The peptide according to claim 25, wherein FGFR1 is inhibited.

28. The peptide according to claim 23, wherein the peptide is capable of stimulating neurite outgrowth.

29. The peptide to claim 23, wherein the peptide is capable of stimulating cell survival.

30. The peptide according to claim 23, wherein the compound is capable of stimulating synaptic plasticity.

31. The peptide according to claim 23, wherein the peptide is capable of stimulating differentiation of stem cells.

32. The peptide according to 23, wherein the peptide is capable of stimulating learning and/or memory.

33. The peptide according to claims 7, 19 and 20, wherein the fragment comprises at least 3 contigious amino acid residues of a sequences selected from SEQ ID NOs: 1-17.

34. The peptide according to claim 33, wherein the fragment comprises 5 amino acid residues of a sequence selected from SEQ ID Nos:1-17.

35. The peptide according to claim 33 or 34, wherein the fragment comprises an amino acid motif as defined in claims 9-13 or 14-18.

36. The peptide according to any of the preceding claims, wherein the peptide is a compound comprising two or more contigous amino acid sequences, such as a dimer or tetramer an amino acid sequence.

37. The peptide according to claim 36, wherein the compound is a dimer comprising at least one of any of the sequences SEQ ID NOs:1-17, or a fragment of any of said sequences.

38. The peptide according to claim 37, wherein the dimer comprises two differrent amino acid sequences selected from any of the sequences SEQ ID NOs:1-17, or any two different fragments of said sequences.

39. The peptide according to claim 37, wherein the dimer comprises two identical amino acid sequences, wherein the sequence is selected from any of the sequences SEQ ID NOs:1-17, or two identical fragments of said sequence.

40. The peptide according to claim 36, wherein the compound is a tetramer comprising four identical sequences, wherein the sequence is selected from any of the sequences SEQ ID NOs: 1-17, or four identical fragments of said sequence.

41. Use of a peptide according to any of the claims 1-40 for the manufacture of a medicament for treatment a disease or condition wherein stimulating neurite outgrowth, cell survival, synaptic plasticity, stem cell differentiation and/or learning and memory is beneficial for recovery from said disease of condition.

42. The use according to claim 41 , wherein the medicament is for the treatment of diseases or conditions of the central and peripheral nervous system, postoperative nerve damage, traumatic nerve damage, impaired myelination of nerve fibers, postischaemic damage, multiinfarct dementia, multiple sclerosis, nerve degeneration associated with diabetes mellitus, neuro-muscular degeneration, schizophrenia, mood disorders, manic depressive disorders, Alzheimer's disease, Parkinson's disease, or Huntington's disease.

43. The use according to claim 41 , wherein the medicament is for the treatment of diseases or conditions of the muscles including conditions with impaired function of neuro-muscular connections, or for the treatment of diseases or degenerative conditions of the gonads, pancreas or kidney.

44. The use according to claim 41, wherein the medicament is for preventing cell death of heart muscle cells.

45. The use according to claim 41 , wherein the medicament is for revascularisation.

46. The use according to claim 41, wherein the medicament is for the promotion of wound healing.

47. The use according to claim 41 , wherein the compound and/or pharmaceutical composition is capable of inhibiting angiogenesis.

48. The use according to claims 41 or 47, wherein the medicament is for the treatment of cancer.

49. The use according to claim 41 , wherein the medicament is for the stimulation of the ability to learn and/or of the short and/or long term memory.

50. The use according to claim 41, wherein the medicament is capable of modulating proliferation and/or differentiation and/or regeneration and/or morphological plasticity of cells.

51. A pharmaceutical composition comprising a peptide according to any of the claims 1-41.

52. The pharmaceutical composition according to claim 51, said composition is formulated for oral, percutaneous, intramuscular, intravenous, intracranial, intrathecal, intracerebroventricular, intranasal or pulmonal administration.

53. The pharmaceutical composition according to claim 52, wherein the administration is continuous.

54. A method of treating a condition or disease wherein activating FGFR is beneficial for the treatment, said method comprising administering to an individual in need a peptide according to any of the claims 1-40 or a pharmaceutical composition according to claims 51-53.

55. The method according to claims 54, wherein the condition or disease is as defined in any of the claims 41-50.