JP2009511614A - Fibroblast growth factor receptor-derived peptide - Google Patents

Abstract

  The present invention relates to a novel peptide capable of binding to the fibroblast growth factor receptor family (FGFR) and regulating the activity of the receptor. The peptide of the present invention is a fragment of FGFR, wherein the fragment comprises amino acid residues involved in the interaction of immunoglobulin-like module 2 (Ig2) and immunoglobulin-like module 1 (Ig1) of FGFR. The present invention discloses amino acid sequences derived from reciprocal Ig1 vs. Ig2 binding sites of FGFR and relates to the use of peptides comprising said amino acid sequences for the treatment of different pathological conditions in which FGFR plays a prominent role. Accordingly, pharmaceutical compositions containing the compounds of the present invention are also relevant.

Description

The present invention relates to novel peptides that can bind to the fibroblast growth factor receptor family (FGFR) and modulate the activity of the receptor. The peptide of the present invention is a peptide fragment of FGFR and is derived from the binding site for mutual binding of immunoglobulin-like modules 1 and 2 of FGFR. The present invention discloses an amino acid sequence derived from the mutual Ig1 vs. Ig2 binding site of FGFR, the use of a peptide comprising said amino acid sequence and a medicament comprising it for the treatment of different pathological conditions in which FGFR plays a prominent role It relates to the use of the composition.

BACKGROUND OF THE INVENTION Fibroblast growth factor receptor (FGFR) is a family of closely related transmembrane tyrosine kinases (FGFR1-FGFR4). FGFR activation and signal transduction depends on receptor dimerization induced by binding of the fibroblast growth factor family (FGF), the natural ligand for FGFR, which is also cell surface heparin or heparan sulfate proteoglycans Requires participation (McKeehan et al., 1998; Itoh and Ornitz, 2004). FGF (FGF1-FGF23) and FGFR constitute a complex signal transduction system that participates in many developmental and repair processes of virtually any mammalian tissue (Bottcher and Niehrs, 2005), in particular, they are adult peripherals. And plays a prominent role in central nervous system functionalization (of the 23 members of the FGF family, 10 were identified in the brain (Jungnickel et al, (2004) Mol Cell Neurosci. 25: 21-9; Reuss and von Bohlen und Halbach (2003) Cell Tissue Res. 313: 139-57)).

  A typical FGFR consists of three immunoglobulin-like modules (Ig1-Ig3), a transmembrane domain and an intracellular tyrosine kinase domain. The linker region between the Ig1 and Ig2 modules is very long, consists of 20-30 amino acid residues, and contains a stretch of acidic amino acids called an acid box. FGFR also binds to heparin / heparan sulfate, which is required for high affinity FGF-FGFR interactions (Yayon et al., 1991; Ornitz et al., 1992). Several FGF binding studies to various FGFR fragments and several FGF crystal structures bound to FGFR fragments consisting of Ig2 and Ig3 modules show that these modules and linker regions are specific for FGF-FGFR interactions. (Wang et al., 1995a; Plotnikov et al., 1999; Pellegrini et al., 2000). FGF-FGFR binding results in FGFR dimerization leading to autophosphorylation of the receptor tyrosine kinase domain. Based on the crystal structure of the three-dimensional FGF-FGFR heparin complex, two models of FGFR dimerization, symmetric and asymmetric models, were proposed. In the symmetric model (Plotnikov et al., 1999; Plotnikov et al., 2000; Schlessinger et al., 2000), the dimer of the two FGF-FGFR complexes is 1) the primary and secondary interaction sites Mediated by bivalent FGF-FGFR interaction, 2) direct FGFR-FGFR interaction, and 3) interaction between heparin-FGF and heparin-FGFR. In the asymmetric model (Pellegrini et al., 2000), there is no protein-protein contact between the two FGF-FGFR complexes, and the three-dimensional complex is stabilized only by the heparin-FGF interaction. Recently, mutations that disrupt interactions at secondary FGF-FGFR interaction sites (observed only in the symmetric model) have been shown to produce reduced FGFR activity without affecting FGF-FGFR binding. This supports the symmetric model (Ibrahimi et al., 2005). Control of the binding specificity of FGF is mainly achieved by alternative splicing of FGFR. Alternative splicing of exons encoding the C-terminal part of the Ig3 module in FGFR1-3 results in two isoforms (3b and 3c) with different FGF binding specificities (Miki et al., 1992; Yayon et al ., 1992). In addition, the following FGFR isoforms exist: those lacking the Ig1 module (FGFR1 and 2), Ig1 module linked to the Ig1-Ig2 linker sequence (FGFR2), or only the Ig1-Ig2 linker (in FGFR3) (McKeehan et al., 1998; Shimizu et al., 2001).

  The structure of the Ig1 module is unknown, and the physiological importance of the module is not well understood. The Ig1 module is not required for FGF-FGFR binding, but the three Ig module types (FGFR1α) are 8 times and FGF1 and heparin, respectively, when compared to the two Ig module types (FGFR1β). It has 3 times lower affinity (Wang et al., 1995b). The deletion of the Ig1-Ig2 linker in FGFR1α destroys the inhibitory effect of the Ig1 module, so the Ig1-Ig2 linker forms a flexible hinge and allows the Ig1 module to interact with the Ig2 / Ig3 module (Wang et al., 1995b), and this assumption was recently supported by Olsen et al. (2004), which showed that the Ig1 module of FGFR3 binds to FGFR3β with a dissociation constant of 20 μM. It was.

  Much evidence accumulated to date indicates that FGFR is involved in intracellular signaling by binding to the major cell adhesion molecules NCAM, L1 and N-cadherin involved in neuronal differentiation, survival and plasticity. (Williams et al. (1994) Neuron 13: 83-94; Doherty and Walsh, 1996; Kiselyov et al., 2003).

  NCAM has recently been identified as a member of a novel class of putative alternative ligands (weak affinity ligands) for FGFR (Kiselyov et al., 2003; Kiselyov et al. (2005) J Neurochem 94: 1169- 1179). There was acquired evidence for direct interaction between NCAM and receptors and stimulation of FGFR by NCAM (Kiselyov et al. (2003) Structure (Camb) 11: 691-701). The identified NCAM fragment involved in the interaction between NCAM and FGFR and having the sequence EVYVVAENQQGKSKA (FGL peptide) has been shown as a novel candidate drug for the treatment of various pathological conditions in which the activity of FGFR may play a role (WO 03/016351). WO 03/016351 discloses some biological activity of FGL peptides for binding and activating FGFR.

  The multi-function of FGFR has been associated with many diseases, such as cancer, which regards the receptor as an important target for therapeutic intervention. However, the large number of biological processes in the control that FGFR plays a prominent role and the presence of large amounts of FGFR ligands make such a search a very challenging task.

  The present invention provides novel compounds that can be advantageously used to modulate FGFR activity and FGFR-dependent biological processes, and thus the present invention provides novel candidate agents for the treatment of FGFR-related diseases. .

SUMMARY OF THE INVENTION The present invention discloses a novel FGFR binding site for the mutual binding of FGFR Ig1 and Ig2 modules of the same FGFR molecule, and a peptide sequence capable of binding to the binding site and thereby modulating the receptor activation. Disclose.

  Accordingly, in a first aspect, the present invention relates to a novel peptide sequence that can bind to FGFR and regulate FGFR activity.

In accordance with the present invention, the peptide sequence that can bind to the mutual Ig1 versus Ig2 binding site is the amino acid motif Q / E- (x) ₃ -x ^p
[Where:
x ^p is a hydrophobic amino acid residue, and
(x) _c is an amino acid sequence of 3 amino acid residues
(Where x is any amino acid residue)]
Or the amino acid motif K / Rx ^p- (x) _0-1 -K / R
[Where:
x ^p is a hydrophobic amino acid residue, and
(x) is a charged amino acid residue]
including.

Preferably, the peptides of the present invention comprise about 25 amino acid residues and comprise fragments of FGFR derived from reciprocal Ig1 vs. Ig2 binding sites. The present invention discloses an amino acid sequence derived from the mutual binding site of the present invention and relates to a pharmaceutical composition comprising the same. The present invention also relates to the use of a peptide and a pharmaceutical composition comprising it for the treatment of different pathological conditions in which FGFR plays a role in the pathology and / or recovery from the disease, Are:
a) Postoperative nerve injury, traumatic nerve injury, myelination of damaged nerve fibers, post-ischemic injury such as that resulting from stroke, Parkinson's disease, Alzheimer's disease, Huntington's disease, dementia, eg, multiple cerebral infarction Neurodegeneration associated with dementia, sclerosis, diabetes, injury or neuro-muscular transmission affecting the circadian clock, and schizophrenia, mood disorders such as central and peripheral nervous system conditions associated with manic depression treatment;
b) for the treatment of conditions having impaired function of nerve-muscle connection, e.g. after organ transplantation or including, for example, hereditary or traumatic atrophic myopathy; or Treatment of various organ diseases or conditions, e.g. degenerative state of the gonad, pancreas, e.g. type I and II diabetes, kidney, e.g. nephrosis and heart, liver and intestinal diseases or conditions;
c) promoting wound healing;
d) prevention of cardiomyocyte death after, for example, acute myocardial infarction;
e) promotion of revascularization;
f) the ability to learn and / or stimulation of short-term and / or long-term memory;
g) prevention of cell death due to ischemia;
h) prevention of physical damage due to alcohol consumption;
i) treatment of prion diseases;
j) Cancer treatment.

Figure Legends Figure 1. Structure of the FGFR1 Ig1 module. A) Ribbon display of Ig1 module structure. B) Overlay of 20 multilayered skeletal atoms of Ig1 module. C) Comparison of the structure of the A / A ′ loop region of Ig1 and Ig3 modules.

Figure 2. Binding of FGFR1 Ig1 module to FGFR Ig2-Ig3 module. A) Plot of equilibrium binding level of Ig1 module to Ig2-3 module vs. concentration of Ig1 module. Binding was examined by means of SPR analysis. Ig1 module was injected into the sensor chip at a specific concentration. Binding is identified as the difference in response between binding to a sensor chip with immobilized Ig2-3 module and a blank sensor chip (non-specific binding). Binding is identified as the average of 6 replicates and error bars indicate standard deviation. The data was fitted to a theoretical curve to calculate the dissociation constant (Kd). B) Change in chemical shift of 0.5 mM ¹⁵ N labeled Ig1 module after addition of 2 mM unlabeled Ig2 module. C) Change in chemical shift of 0.5 mM ¹⁵ N labeled Ig2 module after addition of unlabeled Ig1 module. Chemical shifts were determined from ¹⁵ N-HSQC spectra. Data were identified as the average from two independent experiments. The change in chemical shift was calculated using the following formula:
((5 * ΔH) ^ 2 + (ΔN) ^ 2) ^ 0.5
[Where:
ΔH is the change in ¹ H chemical shift and ΔN is the change in ¹⁵ N chemical shift].

  Figure 3. Mapping of various FGFR1-ligand binding sites in the structure of the FGFR1 Ig1 and Ig2 modules. A) Mapping of Ig2 module residues perturbed by interaction with the Ig1 module on the structure of the Ig2 module; and Ig1 module residues perturbed by interaction with the structure of the Ig1 module on the Ig2 module Mapping. B) Mapping of Ig2 module residues involved in binding to the Ig2 module, heparin, FGF (according to the symmetric and asymmetric models) and secondary interaction sites (according to the symmetric model) via the main interaction sites .

  Figure 4. Quantitative analysis of the autoinhibitory effect of the FGFR1 Ig1 module. A) The structure of FGFR1α (triple Ig type) is described schematically with various random structures of Ig1-Ig2 linker (assuming no interaction between Ig1 and Ig2 modules). The circumference corresponds to the average distance between the N-terminus of the FGFR1 Ig1 and Ig2 modules. B) FGF1-FGFR1β (2 Ig types) binding (blue), FGF1-FGFR1α (triple Ig type) binding (green) and intramolecular Ig1-Ig2 binding (red) for various Ig1-Ig2 linker lengths and FGF1 concentrations ) Irritation. C) Data points for FGF1-FGFR1α binding (from panel B) were fitted to a curve describing single site binding to calculate clear dissociation steady-state values. D) Plot of affinity decrease due to autoinhibitory effect of Ig1 module versus dissociation constant (Kd) of ligand-FGFR1β (2 Ig forms) binding.

  Figure 5. Effect of FGFR1 Ig1 and Ig2 modules and derived peptides on FGFR1 phosphorylation. TREX-293 cells were stably transfected with FGFR1 containing a C-terminal StrepII-tag and stimulated with 100 ng / ml FGF1 or other compounds at a specific concentration for 20 minutes. FRD1 represents the Ig1 module, FRD2 represents the Ig2 module, FRD1a represents the peptide corresponding to the Ig1-Ig2 binding site of the Ig1 module, and FRD2a and FRD2b correspond to the Ig1-Ig2 binding site of the Ig2 module Represents a peptide. After stimulation, FGFR was immunopurified using an anti-phosphotyrosine antibody and then analyzed by immunoblotting using an antibody against StrepII-tag (panel A). Quantification of FGFR1 phosphorylation (panel B) was performed by band intensity concentration analysis. Phosphorylation was estimated relative to control (untreated cells) standardized to 100%. Error bars represent one standard error of the mean. P <0.05 (represented by *) and p <0.01 (represented by **) by paired t-tests when comparing treated cells with controls (t-tests consisted of 6 independent non- Done with standardized data).

  Figure 6. Estimation of the average distance between the N-terminus of the Ig1 and Ig2 modules of FGFR1. r is the distance between the N-terminus of the Ig1 and Ig2 modules, l is the linker length, p is the length of the Ig1 module, and ψ is the long axis of the Ig1 module as well as the N-terminus of the Ig2 module And the angle between the straight lines bound to the C-terminus of the Ig1 module.

  Figure 7. Neurite outgrowth of cerebellar granule neurons in response to treatment with peptides FRD2a (SEQ ID NO: 8) (A) and FRD2b (SEQ ID NO: 13) (B) derived from the Ig1 vs. Ig2 binding site of FGFR . The neurite length in culture is provided as a percentage of the neurite length in the treated culture compared to the control (untreated culture).

Detailed Description of the Invention
1. Receptor “Fibroblast growth factor receptor” or “FGFR” refers to a polypeptide that specifically binds to one or more fibroblast growth factors. FGFR typically contains an extracellular domain consisting of three immunoglobulin-like modules (Ig modules) Ig1, Ig2 and Ig3, a transmembrane domain, and an intracellular tyrosine kinase domain connected to each other via a linker region A single-chain transmembrane polypeptide.

  The present invention relates to a functional cell surface FGFR. “Functional cell surface receptor” means a receptor located on the cell membrane outside the cell and having an identifiable population of extracellular ligands. A “ligand” is any molecule that binds to a specific site on a receptor molecule. Binding of a ligand to a receptor at a specific site usually occurs extracellularly and typically results in a change in the receptor molecule, which is further transmitted through the membrane and induces intracellular signaling This results in a physiological response of the cell. Physiological responses of cells, such as changes in cell metabolism, induction of cell differentiation, termination or induction of cell proliferation, cell survival or death, changes in cell motility behavior, depending on which specific sites of the receptor are affected by the ligand Occupied (may be occupied by multiple ligands), or the intracellular and extracellular environment of the receptor and / or inter alia, ligand-receptor interactions (e.g., affinity and / or interaction) Persistence).

  Ligand binding to the receptor often results in a change in the activation state of the receptor, for example, the receptor initiates a cascade of intracellular biochemical reactions that result in one or more cellular responses as described above. Will be able to produce. Ligand binding can also result in inhibition of receptor activity, meaning that the receptor is unable to initiate a cascade of biochemical reactions normally initiated by ligand binding.

  So far, the population of FGFR ligands described in the art includes fibroblast growth factor (FGF), hairpin and neuronal cell adhesion molecules NCAM, L1 and N-cadherin. The binding sites of the latter ligand are different and are located in the extracellular part of the FGFR molecule. One individual FGFR molecule can also bind extracellularly with other individual FGFR molecules at specific binding sites located in the Ig2 module, thus forming an FGFR dimer. FGFR-to-FGFR, which is important for FGFR activity, occurs in the process of ligand binding, and thus the ligand helps receptor binding / dimerization. Spontaneous dimerization of two individual FGFR molecules rarely occurs and is usually associated with pathological conditions.

  In accordance with the present invention, an individual FGFR molecule comprising Ig1 and Ig2 modules separated by a linker region, the Ig1 and Ig2 module interaction prevents the interaction of the individual FGFR molecule with other individual FGFR molecules, and thus FGFR When interfering with the dimerization, it contains a binding site for interaction. The Ig1 and Ig2 module interaction involves specific amino acid residues of the module. The interacting amino acid residues of the reciprocal binding site include both Ig1 and Ig2 module residues of the same FGFR molecule.

  Thus, according to the present invention, the interaction of Ig1 and Ig2 modules of the same FGFR polypeptide prevents the interaction of the FGFR polypeptide with other FGFR polypeptides, and thus FGFR self-activation in the absence of FGF. Disturb. The mutual binding of Ig1 and Ig2 modules of the same FGFR molecule described in the present invention also reduces the affinity of the FGFR-ligand interaction and / or reduces the stability of the FGFR-ligand receptor complex, Inhibiting FGFR activity reduces activation of FGFR by ligands such as FGF, heparin, and cell adhesion molecules.

  Accordingly, in a first aspect, the present invention relates to a compound capable of modulating the mutual Ig1 versus Ig2 binding site of FGFR and the interaction of Ig1 and Ig2 modules at this binding site.

  In one embodiment of the compounds of the invention is an isolated peptide comprising a fragment of FGFR that contains amino acid residues involved in the interaction of Ig1 and Ig2 modules. It is a preferred embodiment of the present invention that the peptide comprises a fragment of FGFR comprising a contiguous amino acid sequence derived from mutual Ig1 vs. Ig2 binding sites. In one embodiment, the peptide may comprise a contiguous amino acid sequence derived from a portion of a mutual Ig1 vs. Ig2 binding site that includes amino acid residues of an Ig1 module. In other embodiments, the peptides described in the present invention may comprise a contiguous amino acid sequence derived from a portion of a reciprocal Ig1 versus Ig2 binding site that includes amino acid residues of an Ig2 module.

  Thus, in one embodiment, the peptides described in the present invention can interact with the Ig1 module of FGFR at a reciprocal Ig1 vs. Ig2 binding site. In other embodiments, the peptides described in the present invention may interact with the Ig2 module of FGFR at a reciprocal Ig1 versus Ig2 binding site.

  The peptides of the present invention can bind to any FGFR, such as FGFR1, FGFR2, FGFR3, FGFR4 and FGFR5, or any mutant form of FGFR1-5, such as a natural or recombinant FGFR mutant, such as FGFR mutants produced by alternative splicing, such as FGFR1b or FGFR2b, or genetic polymorphisms, or can bind to any form of recombinant FGFR. It should be understood that the FGFRs and variants thereof of the present invention comprise or are at least part of the mutual Ig1 vs. Ig2 module binding sites described herein. Examples of FGFR of the present invention containing the mutual binding site of the present invention are GenBank database 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 described in the present invention is a peptide capable of regulating the activity of FGFR. In one embodiment, the peptide can activate FGFR. In other embodiments, the peptide can inhibit FGFR.

  In a preferred embodiment, the FGFR of the present invention is FGFR1 or a mutant form thereof. Thus, in one embodiment, FGFR1 can be activated by the peptides of the present invention. In other embodiments, FGFR1 can be inhibited by the peptides of the present invention.

Amino acid sequence The peptides described in the present invention comprise a fragment of FGFR comprising a contiguous amino acid sequence derived from a mutual Ig1 vs. Ig2 binding site. Such amino acid sequences described in the present invention may be selected from the following amino acid sequences:
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)
GTEQRVVGRAAEV (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).

In certain preferred embodiments, the peptides described in the present invention may comprise a contiguous amino acid sequence derived from a portion of the mutual Ig1 versus Ig2 binding site and comprising amino acid residues of the Ig1 module of FGFR. Thus, in this aspect, the invention relates to the following sequences:
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)
GTEQRVVGRAAEV (SEQ ID NO: 6)
EASEEVELEPCLA (SEQ ID NO: 7).

In accordance with the present invention, all of the amino acid sequences identified as SEQ ID NOs: 1-7 are binding motifs that are important for the interaction of the sequence with amino acid residues of the Ig1 versus Ig2 mutual binding site of FGFR located in the Ig2 module Wherein the binding motif has the following formula:
Q / E- (x) ₃ -x ^p
[Where:
x ^p is a hydrophobic amino acid residue, and
(x) ₃ is an amino acid sequence of 3 amino acid residues (where x is any amino acid residue)]
Have In accordance with the present invention, residue x ^p can be any hydrophobic amino acid residue, but residues V, L or P are preferred. The amino acid sequence (x) ₃ preferably contains at least one residue Q or at least one residue E, and the (x) ₃ sequence further contains a charged amino acid residue or a hydrophobic amino acid residue. More preferably. Preferred hydrophobic residues can be selected from P, I, L or V. Most preferred are motifs corresponding to any of 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)
EQRVV (SEQ ID NO: 24)
EEVEL (SEQ ID NO: 25).

In other preferred embodiments, the peptides described in the present invention may comprise a contiguous amino acid sequence derived from a portion of the mutual Ig1 vs. Ig2 binding site and comprising amino acid residues of the Ig2 module of FGFR. Thus, in this aspect, 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 ID NO: 13)
AANTVKFRC (SEQ ID NO: 14)
AANTVRFRC (SEQ ID NO: 15)
AGNTVKFRC (SEQ ID NO: 16)
VGSSVRLKC (SEQ ID NO: 17).

In accordance with the present invention, SEQ ID NOs: 8-17 contain a binding motif that is important for the interaction of the sequence with amino acid residues at the Ig1 to Ig2 mutual binding site of FGFR located in the Ig1 module. The binding motif is the formula:
K / Rx ^p- (x) _0-1 -K / R
[Where:
x ^p is a hydrophobic amino acid residue;
(x) is a charged amino acid residue]
Defined by In accordance with the present invention, x ^p is an arbitrary hydrophobic amino acid residues, residues M at the position of x ^p, L or F are preferred. In some embodiments, the amino acid sequence preferably comprises three amino acid motifs K / Rx ^p- (x) ₀ -K / R, where x ^p is F or L, and other embodiments Then, it may be preferred that the sequence comprises four amino acid motifs K / Rx ^p- (x) ₁ -K / R, where x ^p is M. Most preferred are motifs corresponding to amino acid sequences 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 present invention relates to SEQ ID NOs: 1-17 and 18-33 as preferred amino acid sequences comprised by the peptides of the present invention. However, the binding motif
Q / E- (x) ₃ -x ^p
[Where:
x ^p is a hydrophobic amino acid residue, and
(x) ₃ is an amino acid sequence of 3 amino acid residues (where x is any amino acid residue)] or a binding motif
K / Rx ^p- (x) _0-1 -K / R
[Where:
x ^p is a hydrophobic amino acid residue, and
(x) is a charged amino acid residue]
It is to be understood that any amino acid sequence that includes any of these amino acid motifs can be included in the peptides of the present invention, and thus all amino acid sequences that include any of these amino acid motifs are FGFR Ig1 versus Ig2 mutual binding sites and Amino acid sequences that can interact are within the scope of the present invention. Accordingly, SEQ ID NOs: 1-17 represent non-limiting examples of amino acid sequences that are within the scope of the present invention.

  In this specification, standard one-letter code and standard three-letter code of amino acid residues are applied. Abbreviations for amino acids follow the recommendations of the IUPAC-IUB Joint Commission on Biochemical Nomenclature Eur. J. Biochem, 1984, vol. 184, pp 9-37. Throughout this specification and claims, the three-letter or single-letter code for natural amino acids is used. When L-type or D-type is not specified, the amino acid in question has natural L-type (cf. Pure & Appl. Chem. Vol. (56 (5) pp 595-624 (1984)) or D-type. As a result, it should be understood that the peptides formed may constitute amino acids of L-type, D-type, or a mixture of L-type and D-type.

If nothing is specified, it should be understood that the C-terminal amino acid of the peptides of the present invention exists as a free carboxylic acid, which can also be specified as “—OH”. However, the C-terminal amino acid of the compounds of the present invention is an amidated derivative, which is indicated as “—NH ₂ ”. Unless otherwise stated, the N-terminal amino acid of a polypeptide contains a free amino group, which may also be identified as “H-”.

If nothing else is specified, the amino acid may be selected from any amino acid, such as alpha amino acids, beta amino acids, and / or gamma amino acids, whether or not they occur naturally. Thus, the population is not limited to A, V, L, I, P, F, W, M, G, S, T, C, Y, N, Q, D, E, K, R, H Aib , Nal, Sar, Orn, lysine analogues, DAP, DAPA and 4Hyp
including.

  Also, according to the present invention, amino acid sequence modifications such as amino acid glycosylation and / or acetylation may be performed.

  Basic amino acid residues are represented by amino acid H, K and R residues according to the present invention; acidic amino acid residues are represented by amino acid E and D residues; hydrophobic amino acid residues are amino acid residues Represented by residues A, L, I, V, M, F, Y and W; weak neutral hydrophobicity is represented by P, A and G; neutral hydrophilicity is amino acid residue Q, Represented by N, S and T; cross-linking is represented by amino acid residue C.

  A preferred peptide according to the present invention is an isolated contiguous peptide sequence comprising at most 25 amino acid residues. In one embodiment, the length of the amino acid sequence of the peptide is 15 to 25 amino acid residues, for example 16, 17, 18, 19, 20, 21, 22, 23 or 24 amino acid residues. obtain. In other embodiments, the length of the amino acid sequence of the peptide is 3 to 15 amino acid residues, e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 amino acids. Can be a residue. Preference is given to peptides whose amino acid sequence has a length in the range of 5 to 15 amino acid residues, for example 6 to 14, for example 7, 8, 9, 10, 11, 12 or 13. Every peptide of the invention is understood to comprise at least one amino acid sequence selected from any of the sequences of SEQ ID NOS: 1-17 or at least one fragment of any of these sequences.

  Accordingly, certain embodiments of the invention may relate to a peptide comprising a fragment of a sequence selected from SEQ ID NOs: 1-17. In other embodiments, the invention may relate to variants of SEQ ID NOs: 1-17.

In accordance with the present invention, amino acid sequence variants selected from the sequences of SEQ ID NOs: 1-17 may be:
i) at least 60% identity with the selected sequence, e.g. 61-65% identity, e.g. 66-70% identity, e.g. 71-75% identity, e.g. 76-80% An amino acid sequence having identity, e.g. 81-85% identity, e.g. 86-90% identity, e.g. 91-95% identity, e.g. 96-99% identity (where Identity is defined as the percentage of identical amino acids in the sequence when matched to the selected sequence). Identity between amino acid sequences is determined by known algorithms such as BLOSUM 30, BLOSUM 40, BLOSUM 45, BLOSUM 50, BLOSUM 55, BLOSUM 60, BLOSUM 62, BLOSUM 65, BLOSUM 70, BLOSUM 75, BLOSUM 80, BLOSUM 85, Or can be calculated using BLOSUM 9;

  ii) at least 60% positive amino acid match with the selected sequence, e.g. 61-65% positive amino acid match, e.g. 66-70% positive amino acid match, e.g. 71-75% positive amino acid match, e.g. 76 -80% positive amino acid match, e.g. 81-85% positive amino acid match, e.g. 86-90% positive amino acid match, e.g. 91-95% positive amino acid match, e.g. 96-99% positive amino acid Amino acid sequences with a match (where a positive amino acid match is defined as the percentage of amino acid residues that have similar physical and / or chemical properties at the same position of the two compared sequences). Preferred positive amino acid matches of the present invention are K and R, E and D, L and M, Q and E, I and V, I and L, A and S, Y and W, K and Q, S and T, N And S and Q and R;

  iii) an amino acid sequence that is identical to the selected sequence, or it is at least 60% identical, e.g. 61-65% identical, e.g. 66-70% identical, e.g. 71-75% identity, e.g. 76-80% identity, e.g. 81-85% identity, e.g. 86-90% identity, e.g. 91-95% identity, e.g. 96-99% identity or at least 60% positive amino acid identity, such as 61-65% positive amino acid identity, such as 66-70% positive amino acid identity, such as 71- 75% positive amino acid match, e.g. 76-80% positive amino acid match, e.g. 81-85% positive amino acid match, e.g. 86-90% positive amino acid match, e.g. 91-95% positive amino acid match For example, it has an amino acid sequence with 96-99% positive amino acid identity and And other chemical moieties such as phosphoryl, sulfur, acetyl, glycosyl moieties.

  The term “variant form of peptide sequence” also means that the peptide sequence can be modified, for example, by substitution of one or more amino acid residues. Both L-amino acids and D-amino acids can be used. Other modifications may include derivatives such as esters, sugars and the like, eg methyl and acetyl esters.

  In other aspects, variants of the amino acid sequences described in the present invention are introduced independently of one another into the same variant, or a fragment thereof, or a different variant, or fragment thereof, eg Multiple substitutions can be included. Thus, a variant of a complex, or a fragment thereof, can contain conservative substitutions independently of each other, wherein at least one glycine (Gly) of the variant, or a fragment thereof, is Ala, Val, Substituted with an amino acid selected from the group of amino acids consisting of Leu and Ile and each independently a variant, or a fragment thereof, wherein at least one alanine (Ala) of the variant, or a fragment thereof Are substituted with an amino acid selected from the group of amino acids consisting of Gly, Val, Leu, and Ile, and each independently is a variant, or a fragment thereof, wherein the variant, or at least one of the fragments Valine (Val) is substituted with an amino acid selected from the population of amino acids consisting of Gly, Ala, Leu, and Ile, and each independently is a variant, or a fragment thereof, wherein the variant, Also At least one leucine (Leu) of the fragment is replaced with an amino acid selected from the group of amino acids consisting of Gly, Ala, Val, and Ile, and each independently a variant, or a fragment thereof, wherein Wherein at least one isoleucine (Ile) of the variant, or a fragment thereof, is replaced with an amino acid selected from the population of amino acids consisting of Gly, Ala, Val and Leu, and each independently, the variant, or The fragment, wherein the variant, or at least one aspartic acid (Asp) of the fragment, is replaced with an amino acid selected from the group of amino acids consisting of Glu, Asn, and Gln, and each independently , A variant, or a fragment thereof, wherein at least one asparagine (Asn) of the variant, or a fragment thereof, is selected from a population of amino acids consisting of Asp, Glu, and Gln Each of which is substituted with a mino acid and independently of the mutant, or a fragment thereof, wherein at least one glutamine (Gln) of the mutant, or a fragment thereof, is an amino acid consisting of Asp, Glu, and Asn. Substituted with an amino acid selected from the population, and at least one phenylalanine (Phe) of the variant, or a fragment thereof, is replaced with an amino acid selected from the population of amino acids consisting of Tyr, Trp, His, Pro; Preferably, selected from the group of amino acids consisting of Tyr and Trp and each independently a variant, or a fragment thereof, wherein at least one tyrosine (Tyr) of the variant, or a fragment thereof is Phe Substituted with an amino acid selected from the group of amino acids consisting of Trp, His, Pro, and preferably substituted with an amino acid selected from the group of amino acids consisting of Phe and Trp, and Each independently a variant, or a fragment thereof, wherein at least one arginine (Arg) of the fragment is replaced with an amino acid selected from the population of amino acids consisting of Lys and His, and each independently A variant, or fragment thereof, wherein at least one lysine (Lys) of the variant, or fragment thereof, is substituted with an amino acid selected from the group of amino acids consisting of Arg and His, and each independently A variant, or a fragment thereof, and each independently a variant, or a fragment thereof, and wherein at least one proline (Pro) of the variant, or a fragment thereof is Phe, Tyr, Trp And each independently substituted with an amino acid selected from the group of amino acids consisting of His, and His, and at least a variant thereof, or fragment thereof, wherein the variant or fragment thereof One cysteine (Cys) is, Asp, Glu, Lys, Arg, His, Asn, Gln, Ser, is replaced by an amino acid selected Thr, and from a population of amino acids consisting of Tyr.

Thus, from the above, it should be understood that the same functional equivalent of a peptide fragment, or a fragment of the functional equivalent, is not less than two from two or more populations of conservative amino acids as described herein above. Of conservative amino acid substitutions. The term “conservative amino acid substitution” is used herein interchangeably with the term “homologous amino acid substitution”. The conserved amino acid population is:
A, G (neutral, weak hydrophobic),
Q, N, S, T (hydrophilic, uncharged)
E, D (hydrophilic, acidic)
H, K, R (hydrophilic, basic)
L, P, I, V, M, F, Y, W (hydrophobic, aromatic)
C (Crosslink formation).

  Conservative substitutions can be introduced at any position of the preferred predetermined peptides of the invention or fragments thereof. However, it may also be desirable to introduce non-conservative substitutions (particularly, without limitation, non-conservative substitutions at any one or more positions).

  Non-conservative substitutions that result in the formation of functionally equivalent fragments of the peptides of the invention can be substantially different in polarity (e.g., nonpolar side chains (Ala, Leu, Pro, Trp, Val, Ile, Leu, Phe or Met) residues with polar side chains such as Gly, Ser, Thr, Cys, Tyr, Asn, or Gln, or charged amino acids such as Asp, Glu, Arg, or Lys Or substitution of charged or polar residues to non-polar residues); and / or ii) substantially different effects in the peptide backbone direction (e.g. other residues of Pro or Gly) And / or iii) Substantially different charges (e.g., substitution of negatively charged residues such as Glu or Asp with positively charged residues such as Lys, His or Arg) (And vice versa)); and / or iv) substantially the steric bulk may be different (e.g. like His, Trp, Phe or Tyr). Giant residues, Ala, 1 Tsueno substituted with small side chains, such as Gly or Ser (and vice versa)).

  In one embodiment, amino acid substitutions are made based on the relative similarity of amino acid side chain substituents, including their hydrophobicity and hydrophilicity values, and charge, size, and the like.

  In accordance with the present invention, a fragment of a selected amino acid sequence of the present invention, eg, a fragment of a sequence selected from SEQ ID NOs: 1-17 may be an amino acid sequence having a length of about 25-99% of the selected amino acid sequence. . Preferably, a fragment according to the invention comprises at least 3 consecutive amino acid residues of any of the sequences SEQ ID NO: 1-17, for example a sequence selected from the sequences SEQ ID NO: 18-33.

  Both fragments and variants of the amino acid sequences of the invention described in the present invention are functional equivalents of the sequences.

  The term “functional equivalent” of an amino acid sequence, as used herein, meets the criteria for variants or fragments of the amino acid sequence described above, and is one or more of the sequences or compounds comprising the sequence. Means a molecule capable of functional activity of In a preferred embodiment, the functional equivalent of the amino acid sequence of the invention can bind to FGFR and modulate its activity.

  The present invention relates to an isolated peptide of the present invention and a fusion protein comprising the peptide of the present invention.

  In one embodiment, the peptide is an isolated peptide. The term “isolated peptide” means that a peptide of the invention is an individual compound and not a part of another compound, for example a polypeptide comprising more than 25 amino acid residues. To do. Isolated peptides can be produced by any recombinant technique or use of chemical synthesis and separated from other compounds, or separated from longer polypeptides or proteins by enzymatic or chemical cleavage methods. And further separated from other protein fragments.

  In one embodiment, the isolated peptide of the invention can comprise one or more of the sequences of SEQ ID NOs: 1-33. In other embodiments, the isolated peptide can consist of one or more of the sequences of 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 other embodiments, the peptide may consist of a sequence selected from SEQ ID NOs: 1-8 or a functional equivalent thereof.

  In other embodiments, the peptide may comprise a sequence selected from SEQ ID NO: 9-17 or 26-33, preferably a sequence selected from SEQ ID NO: 9-17, or a functional homologue thereof.

  A preferred amino acid sequence may be selected depending on the type required to modulate FGFR activity. Thus, in one embodiment, it may be necessary to activate FGFR and in another embodiment it may be necessary to reduce the activity of FGFR or inhibit the receptor. From time to time, the choice of a preferred amino acid sequence may depend on which receptor of FGFR is involved. For example, in one embodiment, FGFR1 can be involved, and in other embodiments, FGFR2 or FGFR3 can be involved. In other embodiments, it may be necessary to modulate the activity of FGFR4. However, when the amino acid sequences described above can interact with all FGFR reciprocal Ig1 vs. Ig2 module binding sites according to the present invention, the choice of a preferred amino acid sequence depending on which FGFR is involved is sometimes less important. is not.

Multimeric compounds Isolated peptide sequences of the invention can be linked to other isolated peptide sequences by chemical bonds in a fusion protein, or amino acid sequences can be linked to each other via a linker group. In certain embodiments, the peptide sequences of the invention can be formulated as monomeric oligomers (multimers), wherein each monomer is a peptide sequence as defined above. In particular, multimeric peptides such as dendrimers can form conformational determinants or clusters with multiple flexible peptide monomers. In one embodiment, the compound is a dimer. In a more preferred embodiment, the compound is a dendrimer, eg, 4 peptides that are attached to a lysine backbone or attached to a polymer carrier, eg, a protein carrier, eg, BSA. Polymerization such as repetitive sequences or linkage to various carriers is known in the art, e.g. lysine backbone, e.g. 4 peptides, 8 peptides, 16 peptides, or 32 peptides A lysine dendrimer having Other carriers can be fat-soluble dendrimers, or micelle-like carriers formed by fat-soluble derivatives, or star (star-like) carbon chain polymer conjugates.

  Thus, according to the present invention, a multimeric compound can be a polymer comprising two or more identical or different peptide sequences of the present invention, where in a preferred embodiment at least two or more amino acid sequences. Is selected from SEQ ID NOs: 1-15, or fragments or variants of the sequences.

  In certain embodiments, the compound comprises two identical amino acid sequences selected from SEQ ID NOs: 1-15, or two identical fragments or variants of the selected sequence, wherein the amino acid sequences, fragments or variants ). Alternatively, the compound can comprise four identical copies of an amino acid sequence selected from SEQ ID NOs: 1-15, or can comprise a fragment or variant of the four selected identical sequences.

  In other embodiments, the compound can comprise two or more different amino acid sequences, wherein at least one of the two amino acid sequences is from SEQ ID NO: 1-15, or a fragment or variant thereof. The sequence to be selected.

  In yet other embodiments, the compound can comprise two or more different amino acid sequences, wherein the two or more amino acid sequences are from SEQ ID NO: 1-15, or a fragment or variant thereof. Selected.

  Preferred multimeric compounds of the invention are those in which the amino acid sequences are linked to each other via a linker or linker population.

A linker can be any molecule or chemical moiety capable of cross-linking with two or more peptide sequences according to the present invention, for example, it has the general formula;
X [(A) nCOOH] [(B) mCOOH]
[Where:
n and m are each independently an integer from 1 to 20,
X is HN, H ₂ N (CR ₂ ) pCR, RHN (CR ₂ ) pCR, HO (CR ₂ ) pCR, HS (CR ₂ ) pCR, halogen- (CR ₂ ) pCR, HOOC (CR ₂ ) pCR, ROOC (CR ₂ ) pCR, HCO (CR ₂ ) pCR, RCO (CR ₂ ) pCR, [HOOC (A) n] [HOOC (B) m] CR (CR ₂ ) pCR, H ₂ N (CR ₂ ) p , RHN (CR ₂ ) p, HO (CR ₂ ) p, HS (CR ₂ ) p, Halogen- (CR ₂ ) p, HOOC (CR ₂ ) p, ROOC (CR ₂ ) p, HCO (CR ₂ ) p , RCO (CR ₂ ) p, or [HOOC (A) n] [HOOC (B) m] (CR ₂ ) p, where p is 0 or an integer from 1 to 20,
A and B are independently substituted or unsubstituted C _1-10 alkyl, substituted or unsubstituted C _2-10 alkenyl, substituted or unsubstituted cyclic moiety, substituted or unsubstituted heterocyclic moiety, substituted or unsubstituted Is an aromatic moiety or
A and B together form a substituted or unsubstituted cyclic moiety, a substituted or unsubstituted heterocyclic moiety, a substituted or unsubstituted aromatic moiety]
It may be an achiral di-, tri- or tetracarboxylic acid represented by

The term C _1-10 alkyl means a straight or branched alkyl group having 1-10 carbon atoms, for example methyl, ethyl, isopropyl, butyl and tertbutyl.

The term C _2-10 alkenyl means a straight or branched alkenyl group having 2-10 carbon atoms, for example ethynyl, propenyl, isopropenyl, butenyl and tert-butenyl.

  The term cyclic moiety means cyclohexane and cyclopentane.

  The term aromatic moiety means phenyl.

  The expression “A and B form a cyclic, heterocyclic or aromatic moiety” refers to cyclohexane, piperidine, benzene, and pyridine.

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

  Another example of a preferred linker of the present invention may be the amino acid lysine. Individual peptide sequences can bind to a core molecule such as lysine, thereby forming dendritic multimers (dendrimers) of the individual peptide sequence (s). The production of dendrimers is also known to those skilled in the art (PCT / US90 / 02039, Lu et al., (1991) Mol Immunol. 28: 623-630; Defoort et al., (1992) Int J Pept Prot Res. 40: 214-221; Drijfhout et al. (1991) Int J Pept Prot Res. 37: 27-32), dendrimers are now widely used in research and medical applications.

  In some embodiments, the amino acid cysteine may also be a preferred linker molecule.

  One preferred embodiment of the present invention relates to a compound comprising four individual amino acid sequences attached to a lysine core molecule, ie a dendritic tetramer / dendrimer of the peptide sequence of the present invention.

  Multimeric compounds of the present invention, such as LPA-dimers or dendrimers, are the most preferred compounds of the present invention. Other types of multimeric compounds comprising two or more individual sequences of the present invention are also within the scope of the present invention. These compounds can be produced using techniques known to those skilled in the art.

  Peptide sequences can be covalently attached to their linkers via an amino- or carboxy-group, preferably via an N- or C-terminal amino- or carboxy-group.

Biological Activity According to the present invention, the above compounds are functionally active compounds. The term “compound” as used herein relates to an isolated peptide sequence of the invention and a compound comprising the sequence.

  The compound can bind to a functional cell surface receptor and modulate the activity of the receptor. The receptor described in the present invention is FGFR, which can be selected from any of a plurality of FGFRs, and can be, for example, FGFR1, FGFR2, FGFR3, FGFR4, or FGFR5. The compounds described in the present invention can bind to either of the latter FGFRs at the mutual Ig1 vs. Ig2 binding sites located in the Ig1 and Ig2 modules of FGFR.

  The present invention preferably relates to FGFR1 and FGFR1-related signaling. Modulating FGFR1 signaling with the compounds of the present invention results in a change in receptor activation state, which can be reflected by FGFR1 tyrosine phosphorylation, or one or more involved in FGFR1-related signaling. The activation state of the intracellular protein can be reflected by, for example, the activation state of STAT1, JNK, PLCγ, ERK, STAT5, PI3K, PKC, FRS2 and / or GRB2 protein. The consequences of modulation of FGFR1 signaling by the compounds of the present invention may also relate to cell differentiation related effects.

  When FGFR signaling is measured as the level of FGFR phosphorylation, the degree of phosphorylation is at least greater than 20%, such as at least greater than 20-200%, such as at least 50-200, of the control value. Estimated as over%. The control value in the present specification means the degree of phosphorylation of FGFR in a medium in which no compound capable of activating FGFR is present.

  Estimating the effective concentration of a compound from the perspective of modulating FGFR signaling, the concentration is 0.1-1000 μM, 1-1000 μM, such as 1-200 μM, such as 10-200 μM, such as 20-180 μM. For example, 30-160 μM, such as 40-140 μM, such as 50-130 μM, such as 60-120 μM, such as 70-110 μM, such as 80-100 μM.

  Given downstream FGFR signaling effects such as cell differentiation related effects, the present invention preferably relates to nodule formation, cartilage formation, or two or more of the effects (Listrum, GP et al. J Histochem. Cytochem. 1999, 47: 1-6), such effects can be detected by light microscopy, turbidimetry, or flow cytometry. Cell differentiation related effects are also seen in bone sialoprotein (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 expression of RNA such as human ILA gene (Osteoarthritis Gartilage 1997, 5: 394-406), or type II collagen / or MGP (J Miner Res. 1997: 1815.23) or protein level changes. .

  Activation of any of the FGFR tyrosine phosphorylation or FGFR-related downstream signaling molecules, such as STST1, JNK, PLCγ, ERK, STAT5, PI3K, PKC, FRS2 and / or GRB2 proteins are commercially available activated proteins Can be estimated by any conventional method such as immunocytochemistry, immunoblotting or immunoprecipitation. The degree of activation is estimated as at least 20% / less, for example, at least 20-200% / less, for example, at least 50-200% / less of the control value. The control value is estimated as the degree of phosphorylation of the protein of interest in a medium in which no compound capable of activating FGFR is present.

  In another aspect, the invention relates to signaling associated with a protein involved in interaction with FGFR, for example, the protein is an FGFR1 ligand, preferably the receptor-like ligand FGFR1. Preferred embodiments of such ligands for FGFR1 are the neural cell adhesion molecules NCAM and L1. Receptor-like FGFR ligands can include other proteins that can interact with FGFR and are associated with any signaling cascade.

  The most preferred receptor-like ligand for FGFR1 of the present invention is NCAM. Thus, the present invention relates to biological effects associated with FGFR-NCAM interactions such as neurite outgrowth or neuronal differentiation.

  NCAM-dependent signaling involves a variety of downstream molecules and can measure its activity in signaling. The present invention particularly relates to the evaluation of the activity of adhesion plaque kinase FAK, tyrosine kinase Fyn and / or cyclic AMP response binding element protein CREB. The degree of phosphorylation is estimated as at least 20% / less than the control value, eg, at least 20-200% / less, eg, at least 50-200% / less. Control values are estimated as described above.

Activation or inhibition of NCAM-dependent signaling can also be measured by assessing cellular responses at the morphological level, particularly cell differentiation related effects. Thus, the assay, in another particular embodiment, relates to the assessment of the effect of candidate compounds on plasticity associated with NCAM-dependent cell aggregation, cell motility, neurite outgrowth, survival, memory and learning.

  One skilled in the art can choose from a number of assays developed in the art to assess the cellular response. Cell aggregation and neurite outgrowth can be assessed, for example, as described in Skladchikova et al. J. Neurosci. Res 1999, 57: 207-18. Proliferation and apoptosis can be assessed using any commercially available assay and kit according to the manufacturer's procedure.

  Thus, the compounds of the present invention can activate FGFR by binding directly to the reciprocal binding sites described herein, or they activated depending on other ligands. Can reduce FGFR. Thus, the compounds of the present invention may modulate receptor signaling depending on other ligand binding.

  It is known that the cellular response to receptor activation is dependent on the intensity of receptor stimulation, for example, the affinity value of ligand-receptor interaction and / or such interaction Can be characterized by the persistence of Thus, the affinity and persistence of FGF and receptor interactions can be influenced by the compounds of the present invention. Thus, in another aspect of the invention, it is to provide compounds that can modulate receptor signaling induced by other receptor ligands, eg, FGF or cell adhesion molecules.

  As used herein, the term “interact” is used interchangeably with the term “binding” and refers to direct or indirect contact between a compound of the present invention and FGFR, preferably directly. Interaction. The term “direct interaction” means that the compound in question binds directly to the receptor.

The binding affinity of the compounds according to the invention is preferably such that the Kd value is in the range of 10 ⁻³ to 10 ⁻¹⁰ M, for example, preferably in the range of 10 ⁻⁴ to 10 ⁻⁸ M. In accordance with the present invention, binding affinity can be determined by any available assay suitable for this purpose, such as surface plasmon resonance (SPR) analysis or nuclear magnetic resonance (NMR) spectroscopy.

  Binding of the compounds of the present invention to FGFR results in a series of cellular responses mediated by FGFR. Thus, compounds that can bind to and activate / inhibit FGFR also induce differentiation of FGFR-presenting cells, regulate the proliferation of FGFR-presenting cells, stimulate the survival of FGFR-presenting cells, And / or can stimulate morphological plasticity of FGFR-presenting cells.

  The term “cells presenting FGFR” means cells that express FGFR on the outer membrane of the cell, such as nerve cells, glial cells, all types of muscle cells, neuroendocrine cells, genital cells And kidney cells, endothelial cells, fibroblasts, osteoblasts, cancer cells, stem cells and embryonic cells. Activation of FGFR has been shown to be associated with proliferation, differentiation and survival of cells expressing the receptor.

  Thus, FGFR is responsible for neuronal survival during development and 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 cells 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) was shown to be an important determinant. Activation of the FGF receptor has been implicated in normal and pathological angiogenesis (Slavin, Cell Biol Int 1995, 19: 431-44). It is important for development, proliferation, functionalization and survival of 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 signaling have been associated with the development of different pathological conditions, such as diabetes (Hart et al., Nature 2000, 408: 864-8). Receptors play a role in maintaining normal kidney structure (Cancilla et al., Kidney Int 2001, 60: 147-55) and are involved in wound healing and cancer diseases (Powers et al., Endocr Relat Cancer. 2000). 7: 165-97). Activation of FGFR is required for neurite outgrowth (Anderson et al., J Neurochem. 2005 95 (2): 570-83; Neiindam et al., J Neurochem. 2004 91 (4): 920- 35; Niethammer et al., J Cell Biol. 2002 157 (3): 521-32). Receptors have been shown to be involved in processes related to 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).

  Thus, substances with the potential to stimulate FGFR signaling promote neurite outgrowth, stimulate nerve cell regeneration and / or differentiation, stimulate cell survival, in particular nerve cell survival, stem cells or immature A major search for compounds that have the potential to stimulate cell differentiation, eg, neuronal differentiation, and stimulate neural plasticity associated with memory and learning, such as promoting nerve regeneration and other forms of neuroplasticity Is the target.

  The compounds of the present invention have been shown to promote neurite outgrowth and are therefore effective promoters of regeneration of neural connections after injury and thereby functional recovery, as well as other where such effects are required It is considered to be an effective promoter of nerve function in the state. Furthermore, the compounds of the present invention can stimulate neuronal precursor cell differentiation and convert to mature neurons. The compounds of the present invention are also potent stimulators of neuronal morphological plasticity associated with learning and memory.

  As used herein, “differentiation” refers to the process of initiation of neural progenitor cell differentiation, ie, maturation of immature neural cells, eg, neurite outgrowth that occurs after the last cell division of the neural cells, and It relates to both morphological plasticity of mature neurons occurring in the brain in connection with learning and memory. Thus, the compounds of the invention can stop neural progenitor cells and immature neural cell division, initiate maturation of the cells, eg, initiate neurite outgrowth. Alternatively, “differentiation” refers to the initiation of the process of genetic, biochemical, morphological and physiological transformation of neural progenitor cells, immature neural cells or embryonic stem cells, which involves normal neural cells. This results in the formation of cells with functional characteristics, which characteristics are defined in the art. The present invention defines an “immature neuronal cell” as a cell having at least one characteristic of a neuronal cell that is accepted by those skilled in the art as a characteristic characteristic of the neural cell.

  In accordance with the present invention, a compound comprising at least one of the above peptide sequences can stimulate neurite outgrowth. The present invention relates to neurite outgrowth improvement / stimulation, e.g., improvement / stimulation greater than about 75% of the value of neurite outgrowth of control / unstimulated cells, e.g. For example, about 250, eg, 200%, eg, about 350%, eg, 300%, eg, about 450%, eg, 400%, eg, about 500%.

  Estimation of the ability of a candidate compound to stimulate neurite outgrowth can be made, for example, by using known methods or assays for estimation of neurite outgrowth, as described in the Examples.

  In accordance with the present invention, the compound has neurite generating activity as a cell growth substrate insoluble fixed component and as a soluble component of the cell growth medium. As used herein, “fixed” means that the compound binds / adheres to a substrate that is insoluble in water or an aqueous solution, thereby further rendering the compound insoluble in such a solution. For medical applications, both insoluble and soluble compounds are considered for application, but soluble compounds are preferred. Compounds that are soluble in water or aqueous solutions are understood to be under “soluble compounds”.

  In another preferred embodiment, the compound of the present invention can stimulate synaptic plasticity. Thus, the compounds can further stimulate learning and memory. In one embodiment, the peptide sequences of the invention can stimulate spine formation, and in other embodiments, the sequences can promote synaptic efficiency. Thus, the present invention further provides a method for stimulating memory and / or learning comprising using a peptide sequence of the present invention and / or a compound comprising said sequence.

  In other preferred embodiments, the compounds of the invention can stimulate cell survival. The compounds according to the invention can prevent cell death, in particular neuronal cell death, for example cell death due to trauma or disease. As used herein, the expression “stimulate / promote survival” is used synonymously with the expression “prevent cell death”. By stimulating / promoting cell survival, it is possible to prevent disease or prevent further degeneration of the nervous system in an individual suffering from a degenerative disease. “Survival” means that under normal circumstances where a cell is traumatized and die with high probability if the compound of the present invention is not used to prevent the cell from degeneration, the compound Refers to the process of promoting or stimulating the survival of the traumatic cell.

  Peripheral neurons have a limited degree of potential to regenerate and reestablish functional binding with their target after various disorders. However, functional recovery is rarely completed, and peripheral nerve cell damage remains a significant problem. In the central nervous system, the potential for regeneration is even more limited. Therefore, the identification of substances that have the ability to prevent neuronal cell death in the peripheral and central nervous system is of great concern.

  The invention also relates to compounds that reduce FGFR function, eg, can inhibit FGFR activity. The pathological activity of FGFR (FGFR1-FGFR3) is cartilage dysplasia, cardiomyopathy, lethal dysplasia of the spine, lethal dysplasia, Annley-Bixler syndrome, Apert syndrome, Beale-Stevenson syndrome, Cruzon syndrome It has been shown to be associated with different disease types, including Jackson Weiss syndrome, Peifel syndrome, and Setre-Hyozen syndrome (Passos-Bueno et al., 1999 Hum. Mutat. 14: 115-125). Abnormal appearance of resident FGFR2 in epithelial cells and age-dependent deficiency and acquisition of FGFR1 activity are indicators of slow progression in the malignancy of certain types of prostate cancer (Wu et al., Cancer Res. 2001 61 (13): 5295-302).

  Thus, in certain embodiments of the invention, the above compounds can inhibit FGFR.

Production of Peptide Sequences Peptide sequences of the invention can be produced by any conventional synthetic method, recombinant DNA technology, enzymatic cleavage of the full-length protein from which the peptide sequence is derived, or a combination of the methods.

Recombinant Manufacturing Thus, in one embodiment, the peptides of the invention are produced by recombinant DNA technology.

  The DNA sequence encoding the peptide or the corresponding full-length protein from which the peptide is derived can be obtained from established standard methods, such as the phosphoamide method by Beaucage and Caruthers, 1981, Tetrahedron Lett. 22: 1859-1869 or Matthes et al. , 1984, EMBO J. 3: 801-805. According to the phosphoamide method, oligonucleotides are synthesized, for example, on an automated DNA synthesizer, purified, annealed, ligated and cloned into an appropriate vector.

  A DNA sequence encoding a peptide can be produced by fragmentation of a DNA sequence encoding the corresponding full-length protein from which the peptide is derived using DNAase I according to standard protocols (Sambrook et al., Molecular cloning: A Laboratory manual. 2 rd ed., CSHL Press, Cold Spring Harbor, NY, 1989). The present invention relates to full-length proteins selected from the population of proteins identified above. The DNA encoding the full-length protein of the invention can alternatively be fragmented using specific restriction endonucleases. The DNA fragments are further purified using standard protocols as described in Sambrook et al., Molecular cloning: A Laboratory manual. 2 rd ed., CSHL Press, Cold Spring Harbor, NY, 1989.

  The DNA sequence encoding the full-length protein can also be of genomic or cDNA origin, for example, producing a genomic or cDNA library and completing the complete sequence by hybridization with a synthetic oligonucleotide probe according to standard techniques. It can be obtained by screening a DNA sequence encoding all or part of a long protein (cf. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor, 1989). DNA sequences can also be produced by polymerase chain reaction using specific primers, for example as described in US Pat. No. 4,683,202 or Saiki et al., 1988, Science 239: 487-491.

  The DNA sequence can then be any vector and is preferably inserted into a recombinant expression vector that can be subjected to recombinant DNA procedures. The choice of vector often depends on the host cell into which it is introduced. Thus, the vector can be an autonomously replicating vector, ie, a vector that exists as extrachromosomal material, the replication of which is independent of chromosomal replication, eg, a plasmid. Alternatively, a vector can be a vector that, when introduced into a host cell, integrates into the host cell genome and replicates with the integrated chromosome (s).

  In the vector, the DNA sequence encoding the peptide or full-length protein should be operably linked to a suitable promoter sequence. A promoter can be any DNA sequence that exhibits transcriptional activity in a selected host cell and can be derived from a gene encoding a protein that is homologous or heterologous to the host cell. Examples of suitable promoters for directing transcription of the coding DNA sequence in mammalian cells are the SV40 promoter (Subramani et al., 1981, Mol. Cell Biol. 1: 854-864), MT-1 (metallothionein gene) Promoter (Palmiter et al., 1983, Science 222: 809-814) or adenovirus 2 major late promoter. A suitable promoter for use in insect cells is the polyhedrin promoter (Vasuvedan et al., 1992, FEBS Lett. 311: 7-11). Suitable promoters for use in yeast host cells include glycolytic genes (Hitzeman et al., 1980, J. Biol. Chem. 255: 12073-12080; Alber and Kawasaki, 1982, J. Mol. Appl. Gen. 1: 419-434) or a promoter from the alcohol dehydrogenase gene (Young et al., 1982, in Genetic Engineering of Microorganisms for Chemicals, Hollaender et al, eds., Plenum Press, New York), or TPI1 (US 4,599,311 ) Or ADH2-4c (Russell et al., 1983, Nature 304: 652-654) promoter. Suitable promoters for use in filamentous fungal host cells are, for example, the ADH3 promoter (McKnight et al., 1985, EMBO J. 4: 2093-2099) or the tpiA promoter.

  The coding DNA sequence may also be operably adapted to an appropriate terminator, such as human growth hormone terminator (Palmiter et al., Op. Cit.) Or (for fungal hosts) TPI1 (Alber and Kawasaki, op. Cit.). Alternatively, it can bind to the ADH3 (McKnight et al., Op. Cit.) Promoter. The vector further includes elements such as polyadenylation signals (e.g., from SV40 or adenovirus 5 Elb region), transcription enhancer sequences (e.g., SV40 enhancer) and translational enhancer sequences (e.g., sequences encoding adenovirus VA RNA). May be included.

  The recombinant expression vector may further comprise a DNA sequence that allows 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 SV40 origin of replication. Vectors also include selectable markers, such as genes whose gene products complement host cell defects, such as genes encoding dihydrofolate (DHFR) or genes that confer resistance to drugs, such as neomycin, hydromycin or methotrexate. Can be included.

  Procedures known to those skilled in the art for ligating DNA sequences, promoters and terminators encoding peptides or full-length proteins, respectively, and inserting them into the appropriate vector containing the necessary information regarding replication (cf., eg Sambrook et al., op.cit.).

  In order to obtain a recombinant peptide of the invention, the coding DNA sequence can usually be fused with a second peptide coding sequence and a protease cleavage site coding sequence, resulting in a DNA construct that encodes the fusion protein, where the protease cleavage site. The coding sequence is located between the HBP fragment and the second peptide coding DNA, inserted into a recombinant expression vector and expressed in a recombinant host cell). In one embodiment, the second peptide is selected from, but not limited to, glutathione-S-reductase, bovine thymosin, bacterial thioredoxin or human ubiquitin natural or synthetic variants, or a population comprising the peptide. In other embodiments, the peptide sequence comprising the protease cleavage site can be Factor Xa having the amino acid sequence IEGR, enterokinase having the amino acid sequence DDDDK, thrombin having the amino acid sequence LVPR / GS, or Acharombacter lyticus having the amino acid sequence XKX. (Each amino acid sequence is a cleavage site).

  The host cell into which the expression vector is introduced can be any cell that allows expression of the peptide or full-length protein, preferably a eukaryotic cell, such as an invertebrate (insect) cell or vertebrate cell, For example, Xenopus laevis oocytes or mammalian cells, in particular insect and mammalian cells. Examples of suitable mammalian cell lines are HEK293 (ATCC CRL-1573), COS (ATCC CRL-1650), BHK (ATCC CRL-1632, ATCC CCL-10) or CHO (ATCC CCL-61) cell lines. For example, Kaufman and Sharp, J. Mol. Biol. 159, 1982, pp. 601-621; Southern and Berg, 1982, J. Mol. 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; Graham and van der Eb, 1973, Virol. 52: 456; and Neumann et al., 1982, EMBO J. 1: 841-845.

  Alternatively, fungal cells (including yeast cells) can be used as host cells. Examples of suitable yeast cells include cells of Saccharomyces spp. Or Schizosaccharomyces spp., In particular Saccharomyces cerevisiae strains. Examples of other fungal cells are filamentous fungi, for example cells of 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 EP 238 023.

  The medium used to culture the cells can be any conventional medium suitable for growing mammalian cells, such as serum-containing or serum-free medium containing appropriate complements, or growing insects, yeast or It can be a suitable medium for fungal cells. Suitable media are commercially available or can be prepared according to published methods (eg, catalogs from the American Type Culture Collection).

  The peptide or protein produced recombinantly by the cells can then be recovered from the culture medium by conventional procedures, which separate the host cells from the medium by centrifugation or filtration, and the protein-like components of the supernatant Or purification by salt means such as ammonium sulfate filtration, various chromatographic procedures such as HPLC, ion exchange chromatography, affinity chromatography and the like.

The method of synthesis production of the synthetic preparation peptides are known to those skilled in the art. Detailed descriptions and practical advice for producing synthetic peptides can be found in Synthetic Peptides: A User's Guide (Advances in Molecular Biology), Grant GA ed., Oxford University Press, 2002, or Pharmaceutical Formulation: Development of Peptides and Proteins. , Frokjaer and Hovgaard eds., Taylor and Francis, 1999.

  Peptides can be synthesized, for example, by using Fmoc chemistry and Acm protected cysteine. After purification by reverse phase HPLC, the peptide can be further processed, for example, to obtain cyclic or C- or N-terminally modified isoforms. Methods of cyclization and terminal modification are known to those skilled in the art and are described in detail in the manuals cited above.

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

  Peptides were prepared using 9-fluorenylmethyloxycarbonyl (Fmoc) or tert.-butyloxycarbonyl, (Boc) as appropriate conventional protecting groups for Na-amino protecting groups and side chain functionality. In an automatic synthesizer, in a polyethylene container equipped with a polypropylene filter for filtration or in a polyamide solid phase method (Dryland, A. and Sheppard, RC, (1986) J. Chem. Soc. Perkin Trans. I, 125- 137.) can be synthesized in any batch format in a continuous flow version.

Medicine
FGFR is known to be involved in many body processes in normal state and disease, particularly in the nervous system. These processes include differentiation, proliferation, survival, plasticity and cell movement.

  Cell death plays a key role in normal neurogenesis (where 50% of developing neurons are removed by programmed cell death) and pathology of neurodegenerative conditions such as Alzheimer and Parkinson's disease Fulfill. FGFR has been shown to be an important determinant of neuronal survival, both during development and in 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). Therefore, a compound that can promote neuronal survival by binding to and activating FGFR is highly desirable. Accordingly, in one aspect, the invention features a compound that promotes neuronal cell survival and can be used as a medicament for the treatment of conditions involving neuronal cell death. However, the compounds of the present invention have also been shown to promote survival of other types of cells, such as different types of muscle cells, or alternatively, since FGFR signaling has been shown to be a survival factor for muscle and cancer cells. It can be used as a medicament for promoting cell death of other types of cells, for example 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).

  The activity of cell surface receptors is tightly controlled in healthy individuals. Mutations, aberrant expression or processing of receptors or receptor ligands result in abnormalities in receptor activity and thus receptor dysfunction. Receptor dysfunction in turn is responsible for the dysfunction of cells that use the receptor for the induction and / or maintenance of various cellular processes. The latter is a sign of disease. It has also been shown that decreased FGFR signaling results in many different pathological conditions, such as diabetes (Hart et al., Nature 2000, 408: 864-8). Activation of the FGF receptor has been implicated in normal and pathological angiogenesis (Slavin, Cell Biol Int 1995, 19: 431-44). It is important for development, proliferation, functionalization 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 maintaining normal kidney structure (Cancilla et al., Kidney Int 2001, 60: 147-55) and is involved in wound healing and cancer diseases (Powers et al., Endocr Relat Cancer. 2000, 7: 165-97).

  The present invention provides compounds that can modulate the activity of FGFR. As a result, the compounds are implicated according to the present invention as medicaments for treating diseases, where modulation of FGFR activity may be implicated as important for healing.

Accordingly, the medicament of the present invention, in one aspect,
1) diseases and conditions of the central and peripheral nervous system, or muscles or various organs, and / or
2) Diseases or conditions of the central and peripheral nervous system, e.g. postoperative nerve injury, traumatic nerve injury, myelination of damaged nerve fibers, post-ischemic injury resulting from stroke, Parkinson's disease, Alzheimer's disease, Huntington's disease, dementia such as multiple cerebral infarction dementia, sclerosis, neurodegeneration associated with diabetes, injury or neuro-muscular transmission affecting circadian clock, and schizophrenia, mood disorders such as depression disease;
3) for the treatment of conditions having impaired function of nerve-muscle connection, e.g. after organ transplantation or including, for example, hereditary or traumatic atrophic myopathy; or For the treatment of various organ diseases or conditions, e.g. degenerative state of the gonad, pancreas, e.g. type I and II diabetes, renal, e.g. nephrosis and heart, liver and intestinal diseases or conditions, and / Or
4) Cancer diseases and / or
5) To prevent and / or treat prion diseases.

  The present invention relates to those in which the cancer is any type of solid tumor that requires angiogenesis.

  The present invention relates to a prion disease selected from the group consisting of scrapie, Creutzfeldt-Jakob disease. FGFR has been shown to play a different role in prion diseases (Castelnau et al. (1994) Exp Neurobiol. 130: 407-10; Ye and Carp (2002) J Mol Neurosci. 18: 179-88).

In another aspect, the compounds of the present invention are
1) promoting wound healing, and / or
2) prevention of cell death of cardiomyocytes after eg acute myocardial infarction or after angiogenesis, and / or
3) To produce pharmaceuticals for revascularization.

In another embodiment, the compound of the present invention contains
1) prevention of cell death due to ischemia;
2) Prevention of physical damage due to alcohol consumption;
For the manufacture of a medicament for

  The present invention relates to a medicament for treating normal, abnormal or damaged FGFR presenting cells or cells presenting FGFR ligands. The term “cell that presents an FGFR ligand” refers to a cell that expresses a receptor or ligand, to which FGFR and / or a portion of FGFR can bind (ie, a so-called counter-receptor). Examples of FGFR ligands are FGF (fibroblast growth factor), NCAM, L1 or proteoglycans such as heparin, heparan sulfate proteoglycans, and chondroitin sulfate proteoglycans.

  The medicament of the present invention includes a composition comprising an effective amount of one or more compounds as described above, or one or more compounds and a pharmaceutically acceptable additive.

  Accordingly, the present invention in another aspect also relates to a pharmaceutical composition comprising at least one compound of the present invention.

  A further aspect of the present invention is the process of producing a pharmaceutical composition, wherein an effective amount of one or more compounds of the present invention, or a pharmaceutical composition according to the present invention is converted into one or more pharmaceuticals. Mixing with an acceptable additive or carrier. In one embodiment, the compound may be used in combination with a prosthesis, which relates to an artificial nerve guide. Thus, in a further aspect, the present invention relates to an artificial nerve guide characterized by comprising one or more compounds or pharmaceutical compositions as described above. Nerve guides are known to those skilled in the art.

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

  Such medicaments and / or pharmaceutical compositions may suitably be formulated for oral, transdermal, intramuscular, intravenous, intracranial, intrathecal, intraventricular, nasal, pulmonary administration.

  Strategies in formulation development of pharmaceuticals and compositions based on the compounds of the invention generally correspond to formulation strategies for drug products based on any other protein. Potential issues and guidance needed to overcome these issues can be found in several textbooks such as “Therapeutic Peptides and Protein Formulation. Processing and Delivery Systems”, Ed. AK Banga, Technomic Publishing AG, Handled by Basel, 1995.

  For injection, they are usually prepared as liquid solutions or suspensions, solids suitable for liquid solutions or suspensions prior to injection. The formulation can also be emulsified. The active ingredient is often pharmaceutically acceptable and can be mixed with excipients that are compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, and the like, and combinations thereof. In addition, if desired, the formulation may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents or substances that enhance the effectiveness or transport of the formulation.

  Formulations of the compounds of the present invention may be manufactured by techniques known to those skilled in the art. The formulation can include pharmaceutically acceptable carriers and excipients, including microspheres, liposomes, microcapsules, nanoparticles, and the like.

  The formulation may be suitably administered by injection, if desired, where the active ingredient exerts its effect. Additional formulations suitable for other forms of administration include suppositories, nasal, intrapulmonary 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 0.5% to 10%, preferably 1-2%. Oral formulations include such commonly used excipients as pharmaceutical grades such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate and the like. These compositions take the form of solutions, suspensions, tablets, dragees, capsules, sustained release formulations or powders and generally have 10-95% active ingredient (s), preferably 25-70% Contains active ingredients.

  Other formulations are suitable, for example, for nasal and intrapulmonary administration, such as administration by inhaler and aerosol.

  The active compound may be formulated as neutral or salt forms. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino group of the peptide compound), inorganic acids such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, oxalic acid, tartaric acid, Formed with mandelic acid. Salts formed with free carboxyl groups are also inorganic bases such as sodium, potassium, ammonium, calcium, or iron hydroxide, and organic bases such as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, etc. Can be derived from

  The formulation is administered in a manner compatible with the dosage formulation, eg, in a therapeutically effective amount. The amount to be administered depends on the subject being treated, including, for example, the weight and age of the subject, the disease being treated and the stage of the disease. Suitable dosage ranges are usually several hundred μg of active ingredient per kilogram body weight per administration, with a preferred range of about 0.1 μg to 5000 μg per kilobody weight. With monomeric forms of the compound, suitable doses are often within the range of 0.1 μg to 5000 μg per kilo body weight, for example, within the range of about 0.1 μg to 3000 μg per kilo body weight, especially from about 0.1 μg per kilo body weight. It is in the range of 1000 μg. Using multimeric forms of the compound, suitable doses are often within the range of 0.1 μg to 1000 μg per kilo body weight, for example, within the range of about 0.1 μg to 750 μg per kilo body weight, especially about 0.1 μg to 500 μg per kilo body weight. In the range of about 0.1 μg to 250 μg per kilo body weight. In particular, smaller amounts are used when administered nasally than when administered by other routes. Administration can be performed once, or the next administration can be performed thereafter. The dose will also depend on the route of administration and may vary with the age and weight of the subject being treated. A preferred dose in multimeric form will be in the range of 1 mg to 70 mg per 70 kg body weight.

  For certain indications, topical or substantially topical application is preferred.

  For other indications, nasal application is preferred.

  Some of the compounds of the present invention are fully active, while others will be more effective when the preparation further comprises pharmaceutically acceptable additives and / or carriers. Such additives and carriers are known to those skilled in the art. In some cases, it is advantageous to include a compound that facilitates delivery of the active agent to the target.

  In many instances, it will be necessary to administer the formulation multiple times. Administration can be continuous infusion, eg, intracerebroventricular infusion or more, eg, more daily doses, daily, more weekly, weekly, etc. Administration of the medicament is preferably initiated before or immediately after the individual is subjected to the factor (s) that can cause cell death. Preferably, the medicament is administered within 8 hours from the occurrence of the factor, for example, within 5 hours from the occurrence of the factor. Many of the compounds show a long-term effect, whereby administration of the compounds can occur at long intervals, such as, for example, 1 week or 2 weeks.

  In connection with use in a nerve guide, administration can continue or be a small portion based on controlled release of the active ingredient (s). Further, the precursor can be used to control the release rate and / or release site. Other types of implants and oral administration can be based on controlled release and / or use of precursors as well.

Treatment Treatment by use of a compound / composition according to the present invention, in one embodiment, induces differentiation, modulates proliferation, regeneration, neuroplasticity and cells (e.g., cells are those implanted or transplanted). Is useful for stimulating survival). This is particularly useful when using compounds that have long-term effects.

  In a further aspect, the treatment comprises various factors such as trauma and injury, acute diseases, chronic diseases and / or disorders, particularly degenerative diseases that usually result in cell death, other external factors such as free radical formation. It can be a stimulus for the survival of cells that may be at risk of death for medical and / or surgical procedures and / or diagnostics that may occur or have cytotoxic effects such as X-ray and chemotherapy. In connection with chemotherapy, the FGFR binding compounds described in the present invention are useful for the treatment of cancer.

  Thus, treatments are diseases or conditions of the central and peripheral nervous systems, such as post-operative nerve damage resulting from spinal cord injury, traumatic nerve injury, myelination of damaged nerve fibers, post-ischemic damage resulting from stroke , Treatment and / or prevention of cell death associated with multiple cerebral infarction dementia, multiple sclerosis, neurodegeneration associated with diabetes, neuro-muscular degeneration, schizophrenia, Alzheimer's disease, Parkinson's disease, or Huntington's Including.

  It is also associated with a muscular disease or condition, including a condition with impaired function of nerve-muscle connection, such as hereditary or traumatic atrophic myopathy, or various organ diseases or conditions, such as the gonads The compounds described herein for the treatment of pancreatic (e.g., type I and type II diabetes), renal degenerative conditions (e.g., nephrosis) induce differentiation, regulate proliferation, regenerate, It can be used to stimulate neural plasticity and survival, i.e. to stimulate survival.

  Furthermore, the compounds and / or pharmaceutical compositions may be aimed at preventing cell death of cardiomyocytes after, for example, acute myocardial infarction in order to induce angiogenesis. Furthermore, in one embodiment, the compounds and / or pharmaceutical compositions are intended for stimulation of cardiomyocyte survival, eg, survival after acute myocardial infarction. In other aspects, the compounds and / or pharmaceutical compositions are for example for revascularization after injury.

  The use of compounds and / or pharmaceutical compositions for promoting wound healing is also within the scope of the present invention. The compounds of the present invention can stimulate angiogenesis so that they can promote the wound healing process.

  The present invention further discloses the use of compounds and / or pharmaceutical compositions in the treatment of cancer. Control of FGFR activation is important for tumor angiogenesis, growth and expansion.

  Also, in a further aspect, the use of compounds and / or pharmaceutical compositions is aimed at stimulating the ability to learn and / or short-term and / or long-term memory, since FGFR activity is important for neuronal differentiation .

  In another aspect, the compounds and / or pharmaceutical compositions of the present invention are intended for the treatment of physical damage due to alcohol consumption. Fetal developmental malformations, long-term neurobehavioral changes, alcoholic liver disease are particularly relevant.

  Therapeutic treatment of prion diseases involving the use of compounds and / or pharmaceutical compositions is also another aspect of the present invention.

  In particular, the compounds and / or pharmaceutical compositions of the invention may be used in the treatment of clinical conditions such as tumors such as malignant tumors, benign tumors, carcinomas in situ and uncertain behavioral tumors, breasts, thyroid, pancreas, brain, Lung, kidney, prostate, liver, heart, skin, blood, muscle (sarcoma) cancer, dysfunction and / or cancer with specific receptor over- or under-expression and / or mutated receptor expression, or Cancers associated with soluble receptors such as, but not limited to, Erb-receptors and FGF-receptors, endocrine diseases such as type I and type II diabetes, pituitary tumors, psychosis such as old age and Presenile organic psychosis, alcoholic psychosis, drug psychosis, transient organic psychosis, Alzheimer's disease, brain lipidosis, epilepsy, progressive paralysis [syphilis], hepatic lens nuclear degeneration, Huntington's Butoh , Creutzfeldt-Jakob disease, multiple sclerosis, brain pick disease, nodular polyarteritis, syphilis, schizophrenia, emotional psychosis, neurotic disorder, personality disorder including personality disorder, organic brain syndrome and Related nonpsychotic personality disorder, paranoia personality disorder, enthusiastic personality, paranoid personality (disability), paranoid features, sexual perversion and disability or dysfunction (for any reason, reduced libido), mental retardation, nerves Diseases in the system and sensory organs, such as those affecting vision, hearing, olfaction, sensation, taste, cognitive abnormalities after the disease, injury (eg after trauma, surgery, and violence), central nervous system inflammation Sexual diseases such as meningitis, encephalitis, brain degeneration, such as Alzheimer's disease, Pick's disease, senile degeneration of the brain, aging NOS, traffic hydrocephalus, obstructive hydrocephalus, other extrapyramidal diseases and abnormal Including behavioral disorders Parkinson's disease, spinocerebellar disease, cerebellar ataxia, Marie Sanger-Brown, myoclonic cerebellar comotor disorder, early cerebellar degeneration, eg spinal muscular atrophy, familial, juvenile, adult spinal muscular atrophy Motor neuropathy, amyotrophic lateral sclerosis, motor neuropathy, progressive bulbar paralysis, pseudobulbar paralysis, primary lateral sclerosis, other anterior horn cell disease, anterior horn cell disease, nonspecific, spinal cord Other diseases, syringomyelia and medullary medulla, vascular myelopathy, acute infarction of the spinal cord (embolized) (non-embolized), arterial thrombosis of the spinal cord, spinal cord edema, spinal cord bleeding, subacute necrotic myelopathy, etc. Subacute spinal cord combined degeneration, myelopathy, drug induction, radiation-induced myelitis, disorder of the autonomic nervous system, peripheral autonomic disorder, sympathetic, parasympathetic, or plant system in diseases classified in , Familial autonomic nerve Symptoms [Riley-Day Syndrome], idiopathic peripheral autonomic neuropathy, carotid sinus syncope or syndrome, cervical sympathetic dystrophy or paralysis, peripheral autonomic neuropathy in diseases classified elsewhere, Amyloidosis, diseases of the peripheral nervous system, lesions of the brachial plexus, cervical fistula syndrome, rib-clavicular syndrome, anterior oblique angle syndrome, thoracic outlet syndrome, acute brachial neuritis or radiculitis NOS (including neonates);

  Inflammatory and toxic neuropathies, including acute infectious polyneuritis, Guillain-Barre syndrome, post-infectious polyneuritis, polyneuropathy in collagen vascular disease, including ocular changes that affect multiple eye structures Disorders such as purulent endophthalmitis, ear and mastoid disease, chronic rheumatic heart disease, ischemic heart disease, arrhythmia, pulmonary disease, respiratory system, sensory organs such as oxygen, asthma, nerves Neonatal organ and soft tissue abnormalities including the system, complications of administration of anesthesia or other sedatives in labor and parturition, skin diseases including infection, inadequate circulatory problems, burn injury and other mechanical and / or Physical injury; injury including post-surgery, catastrophic injury, burns; nerve and spinal cord injury, including nerve cuts, continuous injury (with or without open wound); traumatic neuroma (with open wound) Or ), Traumatic transient paralysis (with or without open wound), accidental dural puncture or laceration during medical procedure, optic nerve and pathway damage, optic nerve damage, second cranial nerve, vision Crossover damage, visual pathway damage, visual cortex damage, nonspecific blindness, cranial nerve (s) damage, other and nonspecific nerve damage;

  Drug addiction, pharmaceuticals and biologicals, genetic or traumatic atrophic myopathy; or kidneys of various organs, such as the degenerative state of the gonad, pancreas, for example type I and type II diabetes I Treatment of, for example, nephrotic disease or condition; scrapie, Creutzfeldt-Jakob disease, Gersmann-Streisler-Scheinker (GSS) disease; X-chromosome-related hydrocephalus and MASA syndrome corpus callosum dysfunction, mental retardation, adductor Finger, convulsion and hydrocephalus (CRASH) syndrome, pain syndrome, encephalitis, drug / alcohol poisoning, anxiety, postoperative nerve damage, intraoperative ischemia, tissue damage by affecting infectious agents or protecting tissues Inflammatory disorders, HIV, hepatitis, and the following symptoms, autoimmune diseases such as rheumatoid arthritis, SLE, ALS, and MS; anti-inflammatory effects, asthma and other allergic reactions , Acute myocardial infarction, and other related disorders or sequelae from AMI, metabolic disorders such as obesity lipid disorders (e.g. hypercholesterolemia, atherosclerosis, impaired amino acid transport and metabolism, purines and pyrimidines) It can be used in metabolic disorders and gout, bone disorders such as fractures, osteoporosis, osteoarthritis (OA), atrophic dermatitis, psoriasis, infectious diseases, stem cell protection or maturation in vivo or in vitro.

  The compounds of the present invention may also comprise achondroplasia, cardiomyopathy, squamous lethal osteodysplasia, lethal dysplasia, Antley-Bixler syndrome, Apert syndrome, Beale-Stevenson syndrome, Kurson syndrome, Jackson Weiss syndrome, It can be used for the prevention and treatment of Peifel syndrome and Setre-Hyozen syndrome.

  In accordance with the present invention, a method for the treatment and / or prevention of the above conditions and symptoms comprises the step of administering to the individual in need thereof an effective amount of the compound and / or pharmaceutical composition.

Example
1. FGFR mutual Ig1 vs. Ig2 binding sites
Method
To investigate the structure and binding properties of the FGFR1 Ig1 module, we used the following recombinant proteins: each first and second Ig module (Ig1 and Ig2), and the binding second and third Ig modules (Ig2- 3). All recombinant proteins were properly folded as determined by NMR analysis.

Production of recombinant protein 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 binding Ig2-3 module (3c isoform) of mouse FGFR1 consists of a His-tag (RSHHHHHH) and amino acids 141-365 (swissprot p16092). Ig2 and Ig2-3 modules were produced as previously described (Kiselyov et al., Structure 2003 11: 691-701). The Ig1 module was expressed in the yeast P. pastoris strain KM71 (Invitrogen, USA) according to the manufacturer's instructions. All proteins were purified by affinity and / or ion exchange chromatography and gel filtration using Ni ²⁺ -NTA resin (Qiagen, USA).

SPR analysis Binding assays were performed using a BIAcoreX instrument (Biosensor AB, Sweden) at 25 ° C. with 10 mM sodium phosphate pH 7.4, 150 mM NaCl as running buffer. The flow rate was 5 ml / min. The Ig2-3 module of FGFR1 was immobilized on a sensor chip CM5 (Biosensor AB, Sweden) as previously described (Kiselyov et al., Structure 2003 11: 691-701). The binding of the Ig1 module to the immobilized Ig2-3 module was investigated by the following method:
Proteins were injected simultaneously at specific concentrations in suspension cells (Fc1 cells) with immobilized FGFR1 modules and in control suspension cells (Fc2-cells) with nothing immobilized. The curve representing non-specific binding of the protein to the surface of Fc2-cells was removed from the curve representing binding of the same protein to the surface of immobilized Ig2-3 modules and Fc1-cells. The resulting curve was used for analysis.

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

Structural calculation
A simulated annealing protocol using the X-PLOR protocol (Brunger, X-PLOR Software Manual, version 3.1. 1992, Yale University, New Haven, CT) was used for structural calculations. The NOE limit of 1360 was derived from the 80/200 ms NOESY and 125 ms ¹⁵ N-NOESY-HSQC spectra with upper bounds of 2.7, 3.3 and 6.0 増 加 increased to 0.5 と き when the limit included methyl groups . An 83φ angle limit with bonds of −120 ± 40 ° and −57 ± 40 ° (derived from ³ J _HNHα binding constant) was used. After studying the hydrogen bond energy and hydrogen exchange rate, 94 hydrogen bond limits were applied as NOE limits (with final structure calculations), with 2 and 3 upper bounds for NH-O and NO distance, respectively. Of the 100 structures calculated, 100 were accepted by X-PLOR and distinguished all structures with> 0.5 mm NOE restriction violations or> 5 ° angle violations. Structures were analyzed and investigated using the MOLMOL (Koradi et al., J Mol Graph 1996 14: 51-5, 29-32) and PROCHECK_NMR (Laskowski et al., J Mol Biol 1993 231: 1049-67) programs did. From these 100 structures, 20 structures with the lowest energy were selected to represent the structure of the Ig1 module of FGFR1.

FGFR-1 phosphorylation assay
Trex293 cells (Invitrogen) were stably transfected with human FGFR-1 with a C-terminal Strep II tag (IBA Biotech). Cells were maintained in DMEM medium containing Hygromycin (Invitrogen) 200 μg / ml, 10% FCS, 1% (v / v) glutamax, 100 U / ml penicillin, 100 μg / ml streptomycin (all from Gibco, BRL). For the phosphorylation assay, 2 × 10 ⁶ cells were starved overnight in serum-free medium. After stimulation with various compounds at specific concentrations, the cells were treated with 1% (v / v) NP-40, complete protease inhibitor (Roche, Germany) (1:50), and phosphatase inhibitor in PBS. Dissolved in 300 μl lysis buffer containing (Calbiochem inhibitor cocktail III) (1: 100). The protein concentration was then determined using the bicinchoninic acid assay (Pierce, Rockville, IL) and 500 μg of each lysate protein was determined using 15 μl of agarose-conjugated anti-phosphotyrosine antibody (4G10-AC) (Upstate biotechnologies) Incubated for 6 hours or overnight at 4 ° C. The bound protein was washed and eluted with a chromatography column (BIO-RAD) using 180 mM phenylphosphate (Sigma). For each sample, 25 μl of purified protein was separated by SDS-PAGE and transferred to a polyvinylidene fluoride membrane (Millipore, Bedford, Mass.). Immunoblotting was performed in 5% (w / v) nonfat dry milk with a rabbit antibody against recombinant StrepII tag (IBA Biotech) (1: 2000 dilution) and a porcine anti-rabbit IgG horseradish peroxidase conjugate (1: 2000 dilution) (DakoCytomation, Denmark). Immune complexes were developed with SuperSignal® West Dura Extended Sustained Substrate (PIERCE) and visualized with SynGene Gene Snap version 6.00.21 software (Synoptics Ltd, UK). For all assays, the exposed bands were quantified with the SynGene Gene Tool image analysis program (Synoptics Ltd, UK).

Results and Discussion
Structure of Ig1 module of FGFR1
The solution structure of the Ig1 module was determined by NMR. All 1360 non-redundant _NOEs were assigned and applied to the structural calculations, along with 83 skeletal dihedral angle constraints derived from ³ J _HNHα coupling constraints. After examining hydrogen bond energy and hydrogen exchange rate, 94 hydrogen bond constraints were applied as NOE in the final structure calculation. A ribbon representation of the structure is shown in FIG. 1A. The 3D magnification of the module belongs to the intermediate Ig subgroup and can be described as a β-barrel consisting of two β-sheets. Some seats are A '(L ⁴³ -V ⁴⁵ ), G (G ¹⁰⁸ -V ¹⁰⁷ ), F (S ⁹⁶ -S ¹⁰⁵ ), C (S ⁶³ -R ⁶⁸ ) and C' (V ⁷¹ -L ⁷³ ) Formed by β-strands, the other sheets are A (T ²⁶ -L ²⁷ ), B (D ⁴⁹ -R ⁵⁴ ), B '(L ⁵⁷ -R ⁵⁸ ), E (E ⁸⁵ -D ⁹⁰ ) and D (T ⁷⁹ -T ⁸² ) formed by strands. An overlay of 20 superimposed structures with respect to the skeletal atoms is shown in FIG. 1B. The root mean square (RMS) deviation from the mean is 1.05 に 関 し て for the skeletal atoms and 1.53 に 関 し て for all heavy atoms. 98.8% of the (φ, Ψ) angle combinations of the entire population fall into the possible region of the Raman Chandran plot. A summary of the structure based on statistics is provided in Table 1 below.

¹20の構造における25-120個の残基に関する平均からの¹RMS偏差。²非余剰制約の数。³エナジーは、10 kcal mol-1 Å-2のNOEおよび8 kcal mol-1 rad-2の二面角制約に関する力定数を有する、CHARMM力場を用いて計算した。 Table I

¹ RMS deviation from the mean for 25-120 residues in the structure of ¹ 20. ² Number of non-redundant constraints. ³ Energy was calculated using a CHARMM force field with a force constant for a NOE of 10 kcal mol-1 Å-2 and a dihedral angle constraint of 8 kcal mol-1 rad-2.

  The general chain topology of the Ig1 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 excess residues compared to the Ig3 module and 5 excess residues compared to Ig2. In contrast to the Ig2 and Ig3 modules where the A / A ′ loop is parallel to the β barrel, the A / A ′ loop of the Ig1 module is in a state perpendicular to the β barrel (FIG. 1C). This is noteworthy because this region of the Ig1 module forms a binding site for the Ig2 module (see below).

Ig1 binds to the region of FGF1-Ig2, heparin-Ig2 and Ig2-Ig2 binding sites
Since FGFR3 Ig1 binds to Ig2-3 with a Kd value of 20 μM (Olsen et al., Proc Natl Acad Sci USA 2004 101: 935-40), we were interested in determining this binding for FGFR1. . Therefore, the binding of soluble Ig1 to the immobilized Ig2-3 module of FGFR1 was examined by SPR analysis. A plot of the equilibrium binding response versus the concentration of Ig1 is shown in FIG. 2A. Similar to that of FGFR3, the time course of binding was characterized by a very fast binding and dissociation layer (data not shown). The calculated Kd value for binding was 33 ± 6 μM, very close to the 20 μM Kd value determined for FGFR3.

NMR analysis was used to identify residues involved in the interaction between Ig1 and Ig2. NMR analysis provides a very sensitive method for studying protein interactions in solution. However, it requires the production of ¹⁵ N labeled proteins and assignment of their ¹⁵ N, ¹ H resonance frequencies for the backbone atoms. Therefore, to investigate the Ig1-Ig2 interaction by NMR, we assigned their ¹⁵ N, ¹ H resonance frequencies for the skeletal atoms of the Ig2 module. The Ig2 module contains the binding sites for FGF, heparin and the Ig2 module itself (while the Ig3 module contains binding sites only for FGF). Furthermore, the Ig1 module inhibits both FGF-FGFR and heparin-FGFR interactions (Wang et al., J Biol Chem 1995 270: 10231-5; Olsen et al., Proc Natl Acad Sci USA 2004 101: 935 -40)

^In the ¹⁵ N-HSQC spectrum of the ¹⁵ N-labeled protein, the signal of all amino acids having both nitrogen and protons can be observed. Changes in signal chemical shifts provide a method for identification of amino acid residues in proteins that are perturbed by the binding of other molecules. 2 mM unlabeled Ig1 module was added to 0.5 mM ¹⁵ N-labeled sample of Ig2 module (and vice versa), and 2 mM unlabeled Ig2 module was added to 0.5 mM ¹⁵ N-labeled sample of Ig 1 module . The changes in recorded chemical shift are shown in FIGS. 2B and 2C. The residues of the Ig1 module that are more perturbed by the Ig2 module (higher than 0.04 ppm) are L ²⁷ , E ²⁹ , Q ³⁰ , A ³¹ , Q ³² , W ³⁴ , G ³⁵ and V ³⁶ (Figure 2B) Residues of the Ig2 module that are more perturbed by the Ig1 module (higher than 0.025 ppm) are T ¹⁵⁶ , S ¹⁵⁷ , E ¹⁵⁹ , K ¹⁶⁰ , A ¹⁶⁷ , V ¹⁶⁸ , A ¹⁷¹ , K ¹⁷² , T ¹⁷³ , V ¹⁷⁴ , ^K175 , ^S214 , ^I215 , ^I216 , ^M217 and ^S219 (Fig. 2C). Changes in the chemical shifts of these residues prove that the presence of one module in close proximity to the other module changes the chemical environment at the perturbed residue, and the perturbed residue is between two molecules. It is shown that it is part of or near the binding site for the interaction. The mapping of perturbed residues in the structure 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 of the Ig1 module are located in the A / A ′ loop region and form a single patch, while the perturbed residues of the Ig2 module are located in two patches: 12 residues (A ¹⁶⁷ , V ¹⁶⁸ , A ¹⁷¹ , K ¹⁷² , T ¹⁷³ , V ¹⁷⁴ , K ¹⁷⁵ , S ²¹⁴ , I ²¹⁵ , I ²¹⁶ , M ²¹⁷ , S ²¹⁹ ) and four residues (T ¹⁵⁶ , S ¹⁵⁷ , E ¹⁵⁹ , K ¹⁶⁰ ) smaller patches. The two patches are located close to each other. Residues from the Ig1 patch cannot bind to both Ig2 patches at the same time, the Ig1 patch and the larger Ig2 patch are approximately equal in size, while the smaller Ig2 patch is substantially more effective than the Ig1 patch. Because it is smaller, the binding of the Ig2 module to the Ig1 module is mediated only by residues from the larger Ig2 patch, and residues from the smaller Ig2 patch are not associated with Ig1-Ig2 binding (perturbations in this region are It is very possible that this may be due to structural changes induced by binding.

Symmetric model of FGFR dimerization (Plotnikov et al., Cell 1999 98: 641-50; Plotnikov et al., 2000 Cell 101: 413-24; Schlessinger et al., Mol Cell 2000 6: 743-50) Therefore, the first Ig2 site that binds to FGF consists of L ¹⁶⁵ , A ¹⁶⁷ , P ¹⁶⁹ and V ²⁴⁸ , and the second Ig2-FGF binding site is P ¹⁹⁹ , D ²⁰⁰ , I ²⁰³ , G ²⁰⁴ , G ²⁰⁵ , consists S ²¹⁹ and V ^221, Ig2- heparin binding site is composed of K ^160, K ^163, K ¹⁷⁵ and K ^177, and Ig2-Ig2 binding site is composed of A ^171, K ^172, T ¹⁷³ and D ²¹⁸ . The mapping of these binding sites in the Ig2 module structure is shown in FIG. 3B. A ¹⁶⁷ from the first Ig2-FGF binding site and S ²¹⁹ from the second Ig2-FGF binding site, K ¹⁷⁵ from the Ig2-heparin binding site, and A ¹⁷¹ , K ¹⁷² , T ¹⁷³ from the Ig2-Ig2 binding site Are in the residues of the Ig2 module that are perturbed by Ig1 binding. As can be seen from FIGS. 3A and B, the first and second Ig2-FGF and Ig2-heparin binding sites are adjacent to the larger Ig2 patch of perturbed residues, while the Ig2-Ig2 binding site Three of the four residues are located within this patch. These data show that Ig1 binding to Ig2 itself interferes with the binding of FGFR to FGF and heparin, and thus Ig1 is a competitive molecule of FGF-FGFR, heparin-FGFR and FGFR-FGFR interactions. It has been shown to function as an inhibitor.

Dynamic analysis of autoinhibitory effect of Ig1 module of FGFR1
When the Ig1 module functions as a competitive intramolecular inhibitor of FGF-FGFR and heparin-FGFR interactions, and the affinity of the interaction between FGF and FGFR, or between heparin and FGFR is known, the Ig1 module Could be described quantitatively, i.e .: in comparison with the FGFR1β-FGF1 and FGFR1β-heparin interactions, respectively, in the affinity of the FGFR1α-FGF1 and FGFR1α-heparin interactions, approximately 8-fold and 4-fold reduction (Wang et al., J Biol Chem 1995 270: 10231-5). In the following, we examine the binding of FGF to the Ig2-3 module of the triple Ig-module of FGFR in the presence of intramolecular binding of the Ig1 module to the Ig2-3 module. FGF-FGFR binding and intramolecular Ig1, Ig2-3 binding are considered to be mutually exclusive.

  Some simplifications and approximations will have to be made in order to perform a quantitative analysis. Whether these simplifications are valid can be determined by how well the resulting model matches the experimental data.

［式中、

は、実効値両端距離であり、
Nは、ポリマーの結合数であり、そして
Lは、結合の長さである(連続した結合間の角度は、ランダムである)］
。我々は、FGFR1αリンカー領域の連続した残基間の角度が、ランダムであり、次いで、1個のアミノ酸に相当するペプチド骨格の長さが、およそ0.4 nmであるとき、リンカーの平均両端距離は、およそ3.1 nmとして見積もり得ると、現在、少しの間想定している。しかしながら、連続したペプチド残基間の角度は、側鎖の立体障害のために、完全に無秩序ではあり得ない。したがって、実際の距離は、3.1 nmより大きいが、12.4 nmよりは小さく、それは、完全に伸張したポリペプチドの長さである。リンカーの平均長を正確に計算することは不可能なので、我々は、平均リンカー距離の全範囲を3.1から12.4 nmと考える。 The Ig1-Ig2 linker of rat FGFR1α consists of 31 amino acids: DALPSSEDDDDDDDSSSEEKETDNTKPNRRP. Analysis of this sequence by various secondary structure prediction engines predicts random coils for all or most of the residues, indicating that the linker forms random coils within the FGFR1α molecule. This is supported by the fact that in the crystal structure of the homologous molecule FGFR3α, the electron density is not found due to the linker region or Ig1 module (Olsen et al., Proc Natl Acad Sci USA 2004 101: 935-40) That means that the Ig1 module and linker are completely disordered in the crystal. Therefore, it makes sense to assume that the linker region also forms a random coil in FGFR1α in solution. In FIG. 4A, the structure of FGFR1α is shown schematically, along with random structures of various linkers (assuming no interaction between Ig1 and Ig2 modules at that time). The random movement of the Ig1 module around the Ig2 module is clearly limited by the length of the linker, meaning that only a small volume around the Ig1 module is available for random movement of the Ig1 module. Therefore, for the analysis of intramolecular interactions, the actual concentration of the module at the volume available to it is used instead of that concentration at the total volume. To estimate the actual concentration, the concentration of the module in a sphere with a radius corresponding to the average distance between the N-termini of the Ig1 and Ig2 modules (denoted as a circle in FIG. 4A) is used. In order to calculate this distance, the average distance between the ends of the linker region must be estimated. The average end-to-end distance of a polymer random coil can be calculated by the following empirical formula for a self-avoiding random walk:

[Where:

Is the distance between the RMS values,
N is the number of bonds in the polymer, and
L is the length of the bond (the angle between successive bonds is random)]
. We found that when the angle between consecutive residues in the FGFR1α linker region is random, and then 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 is It is currently assumed for a while that it can be estimated as 3.1 nm. However, the angle between consecutive peptide residues cannot be completely disordered due to side chain steric hindrance. Thus, the actual distance is greater than 3.1 nm but less than 12.4 nm, which is the length of the fully extended polypeptide. Since it is impossible to accurately calculate the average length of the linker, we consider the entire range of average linker distance to be 3.1 to 12.4 nm.

［式中、
(D₂₃)₀は、FGFR1αの開始濃度であり、
(F)は、FGF1の濃度であり、
(D₂₃・F)は、FGF1-FGFR1α複合体の濃度であり、
K₁は、FGF1-FGFR1β相互作用のためのKd値であり、
(D₂₃・D₁)は、Ig1およびIg2分子が、分子内相互作用に関与する、FGFR1α分子の濃度である］
。(D₂₃・D₁)は、下記の式から決定し得る:

［式中、

および

であり、
K₂は、Ig1モジュールとFGFR1β間の相互作用のKdであり、
N_Aは、アボガドロ数であり、
lは、平均リンカー長であり、
pは、Ig1モジュールの長さである］。 The interaction between FGFR1α and FGF1 (or heparin) can be described by the following formula when Ig1 is acting in an autoinhibitory manner (differentiation is given in the appendix):

[Where:
(D ₂₃ ) ₀ is the starting concentration of FGFR1α,
(F) is the concentration of FGF1,
(D ₂₃ · F) is the concentration of FGF1-FGFR1α complex,
K ₁ is the Kd value for FGF1-FGFR1β interaction,
(D ₂₃ · D ₁ ) is the concentration of the FGFR1α molecule where Ig1 and Ig2 molecules are involved in intramolecular interactions]
. (D ₂₃ · D ₁₎ may be determined from the following equation:

[Where:

and

And
K ₂ is the Kd of the interaction between Ig1 modules and FGFR1beta,
N _A is Avogadro's number,
l is the average linker length;
p is the length of the Ig1 module].

  Reported Kd values for FGF-FGFR interactions are in the range of 10 pM to 100 nM, depending on the method used. The affinity of the heparin-FGFR interaction is on the order of 0.3-1 μM. We first perform a detailed dynamic analysis assuming that the Kd value of the FGF-FGFR interaction is 60 pM, then we analyze the entire range from 10 pM to 1 μM. FIG. 4B shows intramolecular binding of Ig1 modules as a function of putative estimation (with linker lengths of 4.5, 6.9 and 11.3 nm) and enrichment for FGF1 between FGF1 and FGFR1β or FGFR1α. FGFR1α exhibits substantially lower binding to FGF1 compared to FGFR1β. In the absence of FGF1, the Ig1 module is involved in intramolecular binding with approximately 80% of the FGFR1α molecules, and in the presence of FGF1, this percentage drops off rapidly with increasing concentrations of FGF1. To determine the apparent Kd value for FGF1-FGFR1α binding for various linker lengths, the data points from FIG. 4B were fitted to a formula describing binding at a single site (FIG. 4C). For linker lengths ranging from 4.5 to 11.3 nm, the decrease in affinity for the FGFR1α-FGF1 interaction is in the 13.1--4.6-fold range compared to the FGFR1β-FGF1 interaction, and the Ig1 module is The number of FGFR1α molecules involved in intramolecular binding ranges from 87.4 to 69%. The results of the kinetic analysis are summarized in Table II below:

Table II

  Linker lengths close to 3 nm are unlikely to exist for the above reasons, and linker lengths above 9-10 nm are also unlikely because this is close to the length of the fully extended structure of the linker It should be noted. Thus, the linker is most likely in the 6 to 9 nm range, with a corresponding decrease in affinity of approximately 11- to 6-fold (which is in good agreement with the experimentally determined value 8-fold). is there. To determine how the affinity of the ligand-FGFR interaction affects the estimated inhibitory effect of the Ig1 module, we produced an average linker length of 8 nm (maximum correlation between calculated and experimental data). The above calculation was performed for the full range of affinity (Kd from 10 pM to 1 μM). The inhibitory effect of the Ig1 module on FGF-FGFR1α interaction (Kd from 10 pM to 100 nM) is in the range of 10.5- to 4.2-fold (FIG. 4D). The Kd for the heparin-FGFR interaction is about 0.3-1 μM as described above, thus the corresponding inhibitory effect of the Ig1 module is approximately 4-fold (FIG. 4D), which is determined experimentally The exact value is 3.7-fold.

  Thus, the quantitative analysis of the inhibitory effect of the Ig1 module provided herein is in good agreement with the available experimental data regarding the kinetics of FGF-FGFR1 and heparin-FGFR1 interactions, which Demonstrates a model that functions as a competitive intramolecular inhibitor of FGF-FGFR1 and FGFR1-heparin interactions. The autoinhibitory effect of Ig1 is not very dependent on the Ig1-Ig2 linker length (in the range 4.5 to 9 nm), so approximately the same autoinhibition is expected for other FGFR isoforms with slightly shorter Ig1-Ig2 linkers. It should be noted that. This was confirmed by comparing the FGFR3α-FGF1 and FGFR3α-heparin interactions with FGFR3, which has an approximately 4- and 5.7-fold decrease, respectively, compared to the FGFR3β-FGF1 and FGFR3β-heparin interactions. (Olsen et al., Proc Natl Acad Sci USA 2004 101: 935-40).

  Furthermore, our analysis shows that in the absence of FGF, the Ig1 module is involved in intracellular binding to the FGFR1 molecule in approximately 80%, and therefore inhibits putative direct Ig2-Ig2 binding. Predict. However, in the presence of FGF, FGF reduces the observed inhibition by Ig1 by competitive inhibition of the Ig1-Ig2 interaction (see FIG. 4B). This indicates that the Ig1 module not only controls FGFR-ligand binding affinity but also prevents spontaneous FGFR dimerization in the absence of FGF. It is noteworthy that the conversion of FGFRα to FGFRβ correlates with astrocyte malignancy (Yamaguchi et al., Genes Dev 1994 8: 3032-44). Due to the lack of Ig1, expression of FGFRβ, which may be under control of less robust activity, can give rise to tumor cell growth benefits, resulting in tumor progression.

  Effect of peptides designed from two modules on soluble Ig1 and Ig2 modules and FGFR1α activation

  It is known that small pieces of FGFR molecules expressed in cells (in culture) are in an activated / phosphorylated state even if FGF is not present in the culture medium. This background FGFR activity has been shown to be due to direct FGFR-FGFR interaction, which is the presence of direct Ig2-Ig2 contact in a symmetrical model of the triple FGF-FGFR-heparin complex Supported by the fact that As noted above, our calculations predict that in the absence of FGF, the Ig1 and Ig2 modules interact with approximately 80% of FGFR1α molecules. Since intramolecular Ig1-Ig2 interactions are expected to inhibit direct FGFR-FGFR interactions (see Figure 2), only 20% of FGFR molecules are capable of spontaneous dimerization. Is available for Addition of soluble Ig1 or Ig2 modules to cells expressing FGFR1α can reduce or increase the number of receptor molecules available for spontaneous dimerization and thus background reception Inhibits or promotes body activation. To test this hypothesis, TREX-293 cells stably transfected with full-length FGFR1 containing a C-terminal StrepII-tag were stimulated without FGF1 (positive control), Ig1 or Ig2 module or nothing. After stimulating the cells for 20 minutes, FGFR1 was immunopurified and analyzed by immunoblotting. As seen in FIGS. 5A and B, Ig2, modules at concentrations of 20, 100, and 500 μg / ml substantially increased receptor phosphorylation (approximately 2.5 fold), while Ig1 modules There was no significant effect. This stimulus-inhibitory effect of the Ig2 module is probably due to inhibition of intramolecular Ig1-Ig2 binding, which is not interfered with by intramolecular binding of Ig1 and therefore of spontaneous dimerization. In order to increase the number of receptor molecules that are available. The addition of the Ig1 module is expected to increase the number of receptor molecules, where the Ig2 module binds to the Ig1 module and thus is available for spontaneous dimerization. Decrease the number. However, even before the addition of soluble Ig1, the number of receptor molecules that Ig2 binds to Ig1 is approximately 80% (thus approaching maximum binding). Thus, further addition of soluble Ig1 at the concentration used is less expected to reduce the number of receptor molecules available for spontaneous dimerization, and this is probably the Ig1 module Explain why it did not have any significant effect.

Since binding sites for Ig1-Ig2 interactions could be identified using NMR titration analysis (see above), peptides corresponding to these sites mimic the effects of Ig1 and Ig2 on FGFR1α phosphorylation There was an interest in testing whether it was possible. Therefore, based on NMR titration data, we presumably mimic the binding interaction, a peptide from the Ig1 module (FRD1a: TLPEQAQPWGV) and two peptides from the Ig2 module (FRD2a: E ¹⁵⁹ KMEKKLHAV ¹⁶⁸ and FRD2b : A ¹⁷⁰ AKTVKFKC ¹⁷⁸ ) was designed. As seen in FIGS. 5A and B, both Ig2 derived peptides (FRD2a at concentrations of 20, 100 and 500 μg / ml and FRD2b at concentrations of 1, 5 and 20 μg / ml) are substantially In addition, similar to that of the Ig2 module, it increased receptor phosphorylation, whereas the peptide derived from Ig1 (FRD1a at concentrations of 1, 5, 20, 100 and 500 μg / ml), as expected, There was no effect.

  These results further support the regulatory role of Ig1 in FGFR1α activation.

D₂₃は、トリプルIg-モジュールのIg2-3モジュールを表し(Ig1は、Ig2-3モジュールに結合することを意味する)、F-は、FGFまたは他のリガンド(Ig1を除く)を表し、そしてD₁は、Ig1を表す。記号・は、2個の分子間の複合体を表す。(1)は、2個の異なる分子間の相互作用を示し、一方で、(2)は、同じ分子内の2個の異なる部分間の分子内相互作用を示す。(1)は、量的に、下記の式で記載される:

［式中、
(F)は、分子Fの濃度を意味し、そしてK₁は、Kd値を表す］
。(2)の量的記載に関して、我々は、Ig1が、ランダム構造であると推定し得る柔軟なリンカー(図4Aに記載したとおり)により、Ig2に結合すると想定している(正当性に関しては、結果および考察を参照のこと)。Ig1モジュールは、Ig2モジュールの周りに無秩序に浮いているように思われ、そしてIg1とIg2モジュール間の平均的な距離に等しい半径の球形(Ig2モジュールの周囲)中に含まれると想像し得る。したがって、この球形のモジュール濃度は、解析のために使用される。我々は、この球形のモジュール濃度が、(D₁)に因子λを掛け合わせることにより計算し得ると想定する。この因子の値は、後で計算される。次いで、(2)は、下記の式で記載し得る:

Appendix We examine the binding of FGF to the Ig2-3 module of the triple Ig-module of FGFR in the presence of intramolecular binding of the Ig1 module to the Ig2-3 module. FGF-FGFR binding and intramolecular Ig1, Ig2-3 binding are considered to be mutually exclusive. These interactions can be described in the following scheme:

D ₂₃ represents the Ig2-3 module of the triple Ig-module (Ig1 means to bind to the Ig2-3 module), F- represents FGF or other ligand (except Ig1), and D ₁ represents Ig1. The symbol • represents a complex between two molecules. (1) shows the interaction between two different molecules, while (2) shows the intramolecular interaction between two different parts within the same molecule. (1) is quantitatively described by the following formula:

[Where:
(F) means the concentration of molecule F, and K ₁ represents the Kd value]
. Regarding the quantitative description of (2), we assume that Ig1 binds to Ig2 via a flexible linker (as described in Figure 4A) that can be presumed to be a random structure (for validity, (See Results and Discussion). The Ig1 module appears to float randomly around the Ig2 module and can be imagined to be contained in a sphere of radius equal to the average distance between the Ig1 and Ig2 modules (around the Ig2 module). This spherical module concentration is therefore used for analysis. We assume that this spherical module concentration can be calculated by multiplying (D ₁ ) by the factor λ. The value of this factor will be calculated later. (2) can then be described by the following formula:

を取得する。次いで、(3)および(4)から、我々は、

を取得する。式(5)および(6)を解くとこにより、我々は、

を取得する。 It represents the starting concentration of D ₂₃ as (D _{23) 0} represents the equilibrium concentration of D ₂₃ · F as x, and represents the equilibrium concentration of D ₂₃ · D ₁ as y, and F is kept constant . From (1) and (2), we

To get. Then from (3) and (4) we

To get. By solving equations (5) and (6), we have

To get.

［式中、
rは、Ig1とIg2モジュールのN末端間の距離であり、
pは、Ig1モジュールの長さである]。このことから当然に、

[式中、
記号＜＞は、平均することを意味する]
ということになる。＜l²＞の推定に関しては、結果および考察を参照のこと。半径rの球形中のIg1モジュールのモル濃度cは、

[式中、
N_Aは、アボガドロ数である]
として計算し得る。次いで、因子λは、下記のとおり計算し得る:

To calculate the factor λ, we must estimate the average distance between the N-terminus of the Ig1 and Ig2 modules (see FIG. 6). As can be seen in Figure 6,

[Where:
r is the distance between the N terminus of the Ig1 and Ig2 modules;
p is the length of the Ig1 module]. Naturally from this,

[Where
The symbol <> means to average]
It turns out that. See the results and discussion for estimation of <l ² >. The molar concentration c of the Ig1 module in a sphere of radius r is

[Where
N _A is Avogadro's number]
Can be calculated as The factor λ can then be calculated as follows:

2. Stimulation of neurite outgrowth
Methods Cerebellar granule neurons (CGN) are prepared primarily from 7-day-old Wistar rats as previously described in Drejer and Schousboe (1989) Neurochem Res. 14: 751-4.

For the experiment, cerebellar tissue was dissected in a modified Krebs-Ringer solution maintained on ice and processed as described above for hippocampal neurons. All cell cultures were incubated at 37 ° C. in a humidified atmosphere containing 5% CO ₂ . All animals were handled according to national guidelines on animal protection.

CGN, 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 / Plated at a concentration of 10,000 cells / cm ² on uncoated 8-well permanox Lab-Tek chamber slides in Neurobasal medium supplemented with ml streptomycin and 2% 1 M HEPES (all from Gibco, BRL). Peptide solutions with and without inhibitors of various signaling pathways were added to a total volume of 300 μl / cm ² and the slides were incubated at 37 ° C. After 24 hours, neurons were fixed with 4% (v / v) formaldehyde for 20 minutes and then immunostained with a primary rabbit antibody against GAP-43 and an Alexa Fluor secondary goat anti-rabbit antibody. In each individual experiment, at least 200 images for each population were systematically acquired by using computer-assisted fluorescence microscopy as previously described (Ronn et al., 2000 op. Cit.). Briefly, an Nikon Diaphot inverted microscope with a Nikon Plan 20x objective (Nikon, Tokyo, Japan) connected to a video camera (Grundig Electronics, Germany) was used for recording. The same software package described above for the dopamine neurite outgrowth assay was used to process the recorded images.

Results and Discussion FIG. 7 shows cerebellar granules in response to treatment with different concentrations of FRD2a (SEQ ID NO: 8) (A) and FRD2b (SEQ ID NO: 13) (B) peptides derived from the Ig1 vs. Ig2 mutual binding site of FGFR. It demonstrates the stimulation of neurite outgrowth of neurons. The length of neurites in culture is expressed as a percentage of the length of neurites in treated culture compared to control (untreated culture).

  Both FRD2a and FRD2b peptides can stimulate neurite outgrowth.

FGFR1 Ig1 module structure. A) Ribbon display of Ig1 module structure. B) Overlay of 20 multilayered skeletal atoms of Ig1 module. C) Comparison of the structure of the A / A ′ loop region of Ig1 and Ig3 modules. Binding of FGFR1 Ig1 module to bound FGFR Ig2-Ig3 module. A) Plot of equilibrium binding level of Ig1 module to Ig2-3 module vs. concentration of Ig1 module. Binding was examined by means of SPR analysis. Ig1 module was injected into the sensor chip at a specific concentration. Binding is identified as the difference in response between binding to a sensor chip with immobilized Ig2-3 module and a blank sensor chip (non-specific binding). Binding is identified as the average of 6 replicates and error bars indicate standard deviation. The data was fitted to a theoretical curve to calculate the dissociation constant (Kd). B) Change in chemical shift of 0.5 mM ¹⁵ N labeled Ig1 module after addition of 2 mM unlabeled Ig2 module. C) Change in chemical shift of 0.5 mM ¹⁵ N labeled Ig2 module after addition of unlabeled Ig1 module. Chemical shifts were determined from ¹⁵ N-HSQC spectra. Data were identified as the average from two independent experiments. The change in chemical shift was calculated using the following formula: ((5 * ΔH) ^ 2 + (ΔN) ^ 2) ^ 0.5, where ΔH is the change in ¹ H chemical shift and ΔN is , ¹⁵ N chemical shift change]. Mapping of various FGFR1-ligand binding sites on the structure of FGFR1 Ig1 and Ig2 modules. A) Mapping of Ig2 module residues perturbed by interaction with Ig1 module on the structure of Ig2 module; and Ig1 module perturbed by interaction with Ig2 module on structure of Ig1 module Residue mapping. B) Mapping of Ig2 module residues involved in binding to the Ig2 module, heparin, FGF (according to the symmetric and asymmetric models) and secondary interaction sites (according to the symmetric model) via the main interaction sites . Quantitative analysis of the self-inhibiting effect of the FGFR1 Ig1 module. A) The structure of FGFR1α (triple Ig type) is described schematically with various random structures of Ig1-Ig2 linker (assuming no interaction between Ig1 and Ig2 modules). The circumference corresponds to the average distance between the N-terminus of the FGFR1 Ig1 and Ig2 modules. B) FGF1-FGFR1β (2 Ig types) binding (blue), FGF1-FGFR1α (triple Ig type) binding (green) and intramolecular Ig1-Ig2 binding (red) for various Ig1-Ig2 linker lengths and FGF1 concentrations ) Irritation. C) Data points for FGF1-FGFR1α binding (from panel B) were fitted to a curve describing single site binding in order to calculate clear dissociation steady-state values. D) Plot of affinity decrease due to autoinhibitory effect of Ig1 module versus dissociation constant (Kd) of ligand-FGFR1β (2 Ig forms) binding. Effect of FGFR1 Ig1 and Ig2 modules and derived peptides on phosphorylation of FGFR1. TREX-293 cells were stably transfected with FGFR1 containing a C-terminal StrepII-tag and stimulated with 100 ng / ml FGF1 or other compounds at a specific concentration for 20 minutes. FRD1 represents the Ig1 module, FRD2 represents the Ig2 module, FRD1a represents the peptide corresponding to the Ig1-Ig2 binding site of the Ig1 module, and FRD2a and FRD2b correspond to the Ig1-Ig2 binding site of the Ig2 module Represents a peptide. After stimulation, FGFR was immunopurified using an anti-phosphotyrosine antibody and then analyzed by immunoblotting using an antibody against StrepII-tag (panel A). Quantification of FGFR1 phosphorylation (panel B) was performed by band intensity concentration analysis. Phosphorylation was estimated relative to control (untreated cells) standardized to 100%. Error bars represent one standard error of the mean. P <0.05 (represented by *) and p <0.01 (represented by **) by paired t-tests when comparing treated cells with controls (t-tests consisted of 6 independent non- Done with standardized data). Estimation of the average distance between the N-terminus of the Ig1 and Ig2 modules of FGFR1. r is the distance between the N-terminus of the Ig1 and Ig2 modules, l is the linker length, p is the length of the Ig1 module, and ψ is the long axis of the Ig1 module as well as the N-terminus of the Ig2 module And the angle between the straight lines bound to the C-terminus of the Ig1 module. Neurite outgrowth of cerebellar granule neurons in response to treatment with peptides FRD2a (SEQ ID NO: 8) (A) and FRD2b (SEQ ID NO: 13) (B) derived from the Ig1 vs. Ig2 mutual binding site of FGFR. The neurite length in culture is provided as a percentage of the neurite length in the treated culture compared to the control (untreated culture).

Claims (55)

  A peptide of at most 25 contiguous amino acid residues, comprising a fragment of fibroblast growth factor receptor (FGFR), said fragment comprising immunoglobulin-like module 2 (Ig2) and immunoglobulin of said receptor A peptide comprising amino acid residues involved in the interaction of like module 1 (Ig1).   2. The peptide of claim 1, wherein the interaction occurs between the Ig1 and Ig2 modules of the same FGFR polypeptide.   The peptide according to claims 1 and 2, wherein the interaction takes place at the reciprocal Ig1 to Ig2 binding site of FGFR.   4. The peptide according to any one of claims 1 to 3, wherein the fragment comprises a contiguous amino acid sequence derived from mutual Ig1 versus Ig2 binding sites.   5. The peptide of claim 4, wherein the contiguous amino acid sequence is derived from a portion of the mutual Ig1 vs. Ig2 binding site comprising the amino acid residues of the Ig1 module.   5. The peptide of claim 4, wherein the contiguous amino acid sequence is derived from a portion of the mutual Ig1 vs. Ig2 binding site comprising the amino acid residues of the Ig2 module. 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)
GTEQRVVGRAAEV (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)
The peptide according to any one of claims 1 to 6, which comprises an amino acid sequence selected from a fragment thereof.   The peptide of any one of claims 1-4 or 6-13, capable of interacting with an Ig1 module at a reciprocal Ig1 vs. Ig2 binding site. formula:
Q / E- (x) ₃ -x ^p
[Where:
x ^p is a hydrophobic amino acid residue, and
(x) _c is an amino acid sequence of 3 amino acid residues
(Where x is any amino acid residue)]
9. The peptide according to claim 8, comprising an amino acid sequence comprising an amino acid motif represented by: x ^p is a V, L or P, a peptide according to claim 9. 11. A 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 hydrophobic amino acid residue.   13. A peptide according to claim 12, wherein the hydrophobic residue is P, I, L or V.   The peptide according to any one of claims 1-5, 7 or 14-18, capable of interacting with an Ig2 module at the binding site of reciprocal Ig1 to Ig2 module of FGFR. formula:
K / Rx ^p- (x) _0-1 -K / R
[Where:
x ^p is a hydrophobic amino acid residue, and
(x) is a charged amino acid residue]
15. The peptide according to claim 14, comprising an amino acid sequence comprising an amino acid motif represented by: x ^p is a M, L, or F, a peptide of claim 15. 16. The peptide according to claim 15, wherein the amino acid motif is three amino acid motifs K / Rx ^p- (x) ₀ -K / R (where x ^p is F or L). The peptide according to claim 15, wherein the amino acid motif is four amino acid motifs K / Rx ^p- (x) ₁ -K / R (where x ^p is M). The amino acid sequence is
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)
GTEQRVVGRAAEV (SEQ ID NO: 6)
EASEEVELEPCLA (SEQ ID NO: 7)
Or a peptide according to any one of claims 5, 7 or 14-18, selected from or fragments thereof. The amino acid sequence is
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),
The peptide according to any one of claims 6 to 13, which is selected from or fragments thereof.   The peptide according to any one of claims 1 to 20, wherein FGFR is selected from FGFR1, FGFR2, FGFR3, FGFR4 or FGFR5.   The peptide according to claim 21, wherein the FGFR is FGFR1.   The peptide according to any one of claims 1 to 22, which can regulate the activity of FGFR.   24. A peptide according to claim 23, which is capable of activating FGFR.   24. A peptide according to claim 23, capable of inhibiting FGFR.   25. The peptide of claim 24, wherein FGFR1 is activated.   25. The peptide of claim 24, wherein FGFR1 is inhibited.   24. A peptide according to claim 23, capable of stimulating neurite outgrowth.   24. A peptide according to claim 23, capable of stimulating cell survival.   24. The peptide of claim 23, wherein the compound is capable of stimulating synaptic plasticity.   24. A peptide according to claim 23, capable of stimulating differentiation of stem cells.   24. A peptide according to claim 23, capable of stimulating learning and / or memory.   21. A peptide according to any one of claims 7, 19 and 20, wherein the fragment comprises at least 3 consecutive amino acid residues of a sequence selected from SEQ ID NOs: 1-17.   34. The peptide of claim 33, wherein the fragment comprises 5 amino acid residues of a sequence selected from SEQ ID NOs: 1-17.   35. A peptide according to claim 33 or claim 34, wherein the fragment comprises an amino acid motif as defined in any one of claims 9-13 or 14-18.   36. A peptide according to any one of claims 1 to 35, which is a compound comprising two or more consecutive amino acid sequences, for example a dimeric or tetrameric amino acid sequence.   37. The peptide of claim 36, wherein the compound is a dimer comprising any of the sequences of SEQ ID NOs: 1-17, or at least one fragment of any of the sequences.   38. The peptide of claim 37, wherein the dimer comprises two different amino acid sequences selected from any of the sequences of SEQ ID NOs: 1-17, or any two different fragments of the sequences.   The dimer comprises two identical amino acid sequences, wherein the sequence is selected from any of the sequences of SEQ ID NOs: 1-17, or two identical fragments of the sequences. The peptide according to 37.   The compound is a tetramer comprising four identical sequences, wherein the sequence is selected from any of the sequences of SEQ ID NOS: 1-17, or four identical fragments of the sequence; 37. A peptide according to claim 36.   For the manufacture of a medicament for the treatment of a disease or condition where stimulating neurite outgrowth, cell survival, synaptic plasticity, stem cell differentiation and / or learning and memory is beneficial to recover from the disease state Use of the peptide according to any one of claims 1-40.   The drug is a disease or condition of the central and peripheral nervous system, postoperative nerve injury, traumatic nerve injury, myelination of damaged nerve fibers, postischemic injury, multiple cerebral infarction dementia, multiple sclerosis, 42. Use according to claim 41 for the treatment of neurodegeneration, neuromuscular degeneration, schizophrenia, mood disorders, manic depression, Alzheimer's disease, Parkinson's disease or Huntington's disease associated with diabetes.   41. The medicament is for the treatment of a muscular disease or condition, including a condition with reduced function at the neuromuscular junction, or for the treatment of a gonad, pancreas or kidney disease or degenerative condition. Use as described in.   42. Use according to claim 41, wherein the medicament is intended to prevent cell death of cardiomyocytes.   42. Use according to claim 41, wherein the medicament is for revascularization.   42. Use according to claim 41, wherein the medicament is intended to promote wound healing.   42. Use according to claim 41, wherein the compound and / or pharmaceutical composition is capable of inhibiting angiogenesis.   48. Use according to claim 41 or 47, wherein the medicament is for the treatment of cancer.   42. Use according to claim 41, wherein the medicament is intended to stimulate the ability to learn and / or short-term and / or long-term memory.   42. Use according to claim 41, wherein the medicament is capable of modulating cell proliferation and / or differentiation and / or regeneration and / or morphological plasticity.   42. A pharmaceutical composition comprising the peptide according to any one of claims 1-41.   52. The pharmaceutical composition of claim 51, wherein the composition is formulated for oral, transdermal, intramuscular, intravenous, intracranial, intrathecal, intraventricular, nasal or intrapulmonary administration.   53. A pharmaceutical composition according to claim 52, wherein the administration is continuous.   A method of treating a condition or disease wherein activating FGFR is beneficial for treatment to an individual in need thereof, or a peptide according to any one of claims 1-40 or claim 51- 54. A method comprising administering the pharmaceutical composition according to 53.   55. The method of claim 54, wherein the condition or disease is as defined in any one of claims 41-50.

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