JP2009511613A - Novel FGF receptor binding compound containing peptide fragment of nerve cell adhesion molecule L1 - Google Patents

Abstract

The present invention relates to a novel peptide compound capable of interacting with a fibroblast growth factor receptor (FGFR) comprising an amino acid sequence of 5 to 105 amino acid residues, which has the formula:
x ^'- (x) _n -x ^p- (x) _n -x ^'
[Where:
x ^- is a basic amino acid residue,
x ^p is a hydrophobic amino acid residue, and
(x) _n is any amino acid residue (where n is an integer from 0 to 3)]
Of amino acid motifs. Preferably, the compound of the invention comprises a fragment of the neural adhesion molecule L1. The invention also relates to the use of compounds for the treatment and / or treatment of different medical conditions, in which FGFR and / or L1 play a role in the medical condition and / or recovery from the disease. Accordingly, pharmaceutical compositions comprising the compounds of the invention are also relevant.

Description

The present invention relates to novel peptide compounds that can bind to and activate a fibroblast growth factor receptor (FGFR). The novel compounds of the present invention, FGFR binding motif x ^- - comprise a peptide fragment comprising, _{^{wherein, x - - (x) n}} -x p - (x) n -x is a basic amino acid residue, x ^p is a hydrophobic amino acid residue, and (x) _n is a sequence of any amino acid residue (n is an integer from 0 to 3). Preferably, the compounds of the present invention comprise a fragment of the neural cell adhesion molecule L1 that can bind to FGFR and activate the receptor. The present invention discloses the amino acid sequence of an FGFR binding fragment of L1 and features a pharmaceutical composition comprising the same. The invention also relates to the use of compounds and pharmaceutical compositions for the treatment of different pathological conditions, where FGFR and / or L1 plays a role in the pathology and / or recovery from the disease. Also of interest are antibodies capable of binding to an epitope comprising a peptide sequence of the invention.

BACKGROUND OF THE INVENTION Brain plasticity and the mechanisms that control plasticity are central to learning and memory and the recovery of function after brain injury. It is clear that neurotrophic factor is one of the molecular classes involved in controlling brain plasticity in the adult central nervous system (CNS), but plasticity mechanisms such as long-term potentiation, neural The role of neural adhesion molecules (CAM) in protection and regeneration is less recognized but equally important. Ironically, however, CAM can also cause disturbances that remodel the extracellular space and drive the development of brain conditions in conditions such as Alzheimer's disease and multiple sclerosis. Candidate molecules include amyloid precursor proteins that share many of the properties of classic CAM and beta-amyloid and can impersonate pseudo-CAM. Beta-amyloid can serve as a foci for the formation of senile plaques in Alzheimer's disease, providing an environment for organizing neurotrophic factors and other CAMs, like CAMs. Inflammatory responses can develop in this environment and initiate a vicious circle of sustained nerve damage mediated by microglia, complement and other factors (Cotman et al. (1998) Prog Neurobiol. 55: 659-69) .

  The immunoglobulin superfamily of neural cell adhesion molecules (CAMs) aggregate and maintain a population of cells at key sites during early development and adulthood. In addition to their adhesive properties, homologous and heterologous interactions of CAM can affect intracellular signaling. Thus, the ability to influence their developmental events, including cell migration, proliferation, and differentiation, can arise from their adhesion and signaling properties.

  The neural cell adhesion molecule L1 plays an important role during the development of the nervous system by mediating connections between nerve cells and stimulating axonal stretch and fiber bundle spasm. L1 consists of 6 immunoglobulin (Ig) modules in the amino-terminal region, followed by 5 type III fibronectin (F3) modules, a transmembrane domain, and a highly conserved intracellular tail (Nybroe and Bock, 1990). L1 is known to mediate cell-cell interactions by homologous binding, that is, L1 on one cell binds to L1 on an adjacent cell (Grumet and Edelman, 1984; Lemmon V et al., 1989, Doherty et al., 1995). In addition, neural cell adhesion molecule (NCAM) (Horstkorte et al., 1993), axonal cell adhesion molecule TAG-1 / axonin-1 (Kuhn et al., 1991; Felsenfeld et al., 1994), glycosylphosphatidylinositol Heterogeneous binding to various cell surface molecules is also possible, including the immobilized molecule HAS / CD24 (Sammar et al., 1997) and neural tissue specific chondroitin sulfate proteoglycan neurocan and phosphacan (Margolis et al., 1996) It also binds to components of the extracellular matrix.

  L1-stimulated neurite outgrowth has been shown to involve activation of an intracellular signaling cascade dependent on tyrosine kinase activation of fibroblast growth factor receptor (FGFR) (Williams et al., 1994 ). Based on indirect evidence, L1 was shown to be able to activate FGFR by interacting with the receptor extracellularly, but evidence of interaction has not been proven and interacting molecules No binding site has been identified.

  Fibroblast growth factor receptor (FGFR) is a family of at least four closely related receptor protein tyrosine kinases, FGFR1, FGFR2, FGFR3 and FGFR4, with three extracellular Ig-like modules and intracellular Of tyrosine kinase modules (Powers et al. (2000) Endocr Relat Cancer 7: 165-97). Receptors are known to be important regulators of morphogenesis, development, angiogenesis, and wound healing. FGFR activation and signal transduction is dependent on receptor dimerization induced by high affinity binding of fibroblast growth factor (FGF), the natural ligand of FGFR, which is also cell surface Requires participation of heparin or heparan sulfate proteoglycans. Fibroblast growth factors (FGFs) and their receptors constitute a complex signaling system that participates in many developmental and repair processes in virtually all mammalian tissues, in particular, they are peripheral and central It plays an important role in the functionalization of the nervous system. Thus, 10 of 23 members of the FGF family 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).

  Much evidence accumulated to date indicates that the FGFR receptor is involved in intracellular signaling associated with the neuronal cell adhesion molecules NCAM, L1 and N-cadherin (Williams et al. (1994) Neuron 13: 83-94). NCAM has recently been identified as a new class member of the putative alternative ligand of FGFR, a low affinity ligand (Kiselyov et al. (2005) J Neurochem 94: 1169-1179). There was acquired evidence for a direct interaction between NCAM and the receptor (Kiselyov et al. (2003) Structure (Camb) 11: 691-701). The identified NCAM fragment with the sequence EVYVVAENQQGKSKA (FGL peptide), involved in the direct interaction between NCAM and FGFR, is a novel candidate drug for the treatment of various pathological conditions where FGFR activity plays an important role (WO 03/016351). WO 03/016351 discloses certain biological effects of FGL peptides by binding and activating FGFR.

  L1 has been extensively studied as a neurite outgrowth stimulating and cell survival proting cell adhesion molecule (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). It is also considered important for neuronal precursor proliferation, differentiation and transmitter phenotype subtype production (Dihne et al. (2003) J Neurosci 23: 6638-6650) and is important in synaptic plasticity. It is known to play a role (Saghatelyan et al. (2004) Mol Cell Neurosci 26: 191-203).

  Numerous studies have been conducted to investigate the involvement of different regions of the L1 molecule in the performance of multiple biological activities by L1 and to associate L1-mediated cellular responses with specific intracellular signaling pathways. I came. Thus, the Ig module 1-4 of L1 proved to be important for allogeneic binding and neurite outgrowth (Haspel et al (2000) J Neurobiol 42: 287-302). Antibodies directed against the epitope between F3 domains 2 and 3 have been shown to promote signaling and concomitant neurite outgrowth (Appel et al (1995) J. Neurobiol. 28: 297-312). Holm et al. (1995) demonstrated that certain FN-like domain fragments have the ability to homologous binding, which can have one or more FN-like domains with the potential for self-binding, Presumably, it has been shown that L1 clusters can be generated on the cell surface (such clusters can then be subject to structural constraints imposed by the globular structure of FN-like repeats) (J. Neurosci. Res 42 : 9-20). The entire extracellular domain of L1 as a soluble molecule and substrate is important for promoting neuronal cell survival and stimulating neurite outgrowth (Loers et al. (2005) J Neurochem 92: 1463-1476; Naus et al (2004) J Biol Chem 279: 16083-90).

  The soluble molecule L1 has been shown for use as a neurite outgrowth and survival promoting compound in therapeutic applications (US Pat. No. 6,576,607). However, the use of such molecules for medical applications is limited because L1 extracellular domain polypeptides are very long (form 1100 amino acids (Swissprot P32004)).

  Identification of functionally important regions of L1 polypeptides and the production of these regions as individual peptide fragments that can mimic the biological function of the entire L1 extracellular domain polypeptide, when medical applications of L1 are involved Seems to have solved the problem that occurs. Beneficial provision may also provide identification of L1 binding sites involved in interaction with different L1 binding molecules. Advantageously, novel effective agents may be provided for the treatment of diseases involving molecules that interact with L1, such as FGFR receptors. However, information on the structural need for the functional capacity of the L1 molecule, if available, is not fully disclosed in the prior art.

SUMMARY OF THE INVENTION The present invention relates to identifying L1 FGFR binding sites and providing compounds comprising peptide fragments derived from these binding sites. The novel compounds of the present invention are FGFR binding compounds that can modulate FGFR activity.

  Accordingly, the first object of the present invention is to provide novel compounds comprising peptide sequences that can bind to FGFR and modulate FGFR activity.

The compound of the present invention has an FGFR binding motif:
_{^{x - - (x) n -x}} p - (x) n -x -
[Where:
x ^- is a basic amino acid residue,
x ^p is a hydrophobic amino acid residue, and
(x) _n is a sequence of any amino acid residue (where n is an integer from 0 to 3)]
including. Preferably, the compounds of the invention comprise a fragment of the neural cell adhesion molecule L1, which is at most 105 amino acids long and can bind to FGFR and activate the receptor. The present invention discloses the amino acid sequence of an FGFR binding fragment of L1 and features a pharmaceutical composition comprising the same. The invention also relates to the use of compounds and pharmaceutical compositions comprising them for the treatment of different pathological conditions, wherein FGFR and / or L1 is a recovery from a disease state and / or disease, for example
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) Plays a role in the treatment of cancer.

  Also relevant to the present invention are antibodies capable of binding to an epitope comprising a peptide sequence of the present invention.

Figure Legend Figure 1. Binding of L1 F3 Module IV to FGFR1 Ig Module II-III. Binding was examined by means of SPR analysis. Approximately 3000 resonance units (RU) of the FGFR1 module were fixed to the sensor chip. Combined L1 F3 module IV or combined NCAM Ig module I-II (ctrl) was injected into the sensor chip at specific concentrations. Binding is identified as the response difference between binding to a sensor chip with immobilized FGFR1 module and binding to a blank sensor chip (non-specific binding). Only very low non-specific binding was detected for the above proteins at all concentrations used in this study. 4-5 independent experiments were performed with different preparations of all proteins.

  Figure 2. Demonstration of ATP-induced increase in binding between FGFR1 Ig module II-III and L1 F3 module I-V. Binding was examined by means of SPR analysis. Approximately 3000 resonance units (RU) of the FGFR1 module were fixed to the sensor chip. Three independent experiments were performed for each concentration of ATP (AMP-PCP, AMP or GTP). A, B, C, D) Binding of 14 μM module I-V to FGFR1 module in the presence of different concentrations of ATP (A), AMP-PCP (B), AMP (C) and GTP (D).

Figure 3. Demonstration of the effects of ATP, AMP-PCP, GTP, and AMP on the binding between FGFR1 Ig module II-III and L1 F3 module IV. A) ATP, AMP-PCP, GTP or prior to the addition of AMP (Ctr) and between the F3 modules and FGFR1 after addition equilibrium dissociation constant K _D. Plot of starting binding rate V ₀ versus ATP concentration for binding of F3 modules to B) FGFRl were used to calculate the equilibrium dissociation constant, K _D, of the interaction between F3 modules and ATP.

  Figure 4. Effect of L1 F3 module I-V on FGFR1 phosphorylation and immunoprecipitation of L1 by FGFR1. A) TREX / FLP-IN cells stably transfected with FGFR1 containing a C-terminal StrepII-tag, nothing (lane 1), 4 mM (lane 2), 10 mM (lane 3) and 30 mM ( Stimulated with L1 F3 module IV in lane 4) for 15 minutes. After stimulation, FGFR1 was immunopurified using an anti-phosphotyrosine antibody, and then analyzed by immunoblotting using an antibody against StrepII-tag. B) TREX / FLP-IN cells stably transfected with FGFR1 containing a C-terminal StrepII-tag were treated with 25 mM NCAM Ig module I-II (lane 1 as a control) and 10 mM L1 F3 module IV (lane 2). ) For 25 minutes. A total of nine experiments were performed. All experiments show phosphorylation of FGFR1 upon treatment with L1 F3 module I-V.

  Figure 5. Effect of L1 F3 module I-V on neurite outgrowth from cerebellar neurons. A) Neurite length versus F3 module IV concentration. B) Effect of SU5402, a FGFR1 inhibitor, on neurite outgrowth induced by 4 mM F3 module I-V. C, D) Neurons stimulated with 4 mM F3 module I-V in the presence of various concentrations of ATP (C) or AMP-PCP (D). E, F) Neurons stimulated with 1 mM F3 module I-V in the presence of various concentrations of ATP (E) or AMP-PCP (F). Four independent experiments were performed. Error bars represent one standard error of the mean. * And ** represent statistical significance of p <0.05 and p <0.01, respectively, compared to Ctr. +, ++ and +++ represent statistical significance of p <0.05, p <0.01 and p <0.001, respectively, compared to the maximum response.

  Figure 6. Peptide sequence showing the loop region of L1 F3 domain I-V. Thirty peptides representing all loops of the F3 I-V module were synthesized and used to map the binding site between L1 and FGFR1. The loop region of the F3 I-V module is shown along with the adjacent beta strand portion.

  Figure 7. Evidence of binding between peptides derived from FGFR1 and L1 F3 modules I-V. Binding was examined by SPR analysis. Approximately 3000 resonance units (RU) of the FGFR1 module were fixed to the sensor chip. Three independent experiments were performed. A) Peptides that bind to FGFR1 module II-III (F1CDL- (8 mM), F2BCL- (8 mM), F3ABL- (40 mM), F3CDL- (2 mM), F5BCL- (80 mM), and F5FGL- (80 mM) peptides) . B) Peptides that bind to FGFR1 module II (F1CDL- (16 mM), F2BCL- (16 mM), F3CDL- (8 mM), and F5BCL- (130 mM) peptides).

  Figure 8. Effect of peptides bound to FGFR1 on FGFR1 phosphorylation. Phosphorylation level versus concentration of peptide binding to FGFR1. Four independent experiments were performed. Error bars represent one standard error of the mean. *, ** and *** represent statistical significance of p <0.05, p <0.01 and p <0.001, respectively, compared to PBS. +, ++ and +++ were p <0.05 and p <0.01 respectively compared to the negative control (F1BCL-peptide that did not bind to FGFR1 and did not show concentration-dependent phosphorylation of the receptor peptide) And represents the statistical significance of p <0.001.

  Figure 9. Effect of peptide bound to FGFR1 on neurite outgrowth from cerebellar neurons. Neurite length versus concentration of peptide bound to FGFR1. Four independent experiments were performed. Error bars represent the standard error of the mean. * And ** represent the statistical significance of p <0.05 and p <0.01, respectively, when compared to Ctr (PBS).

  Figure 10. Binding model between L1 and FGFR1. A possible compact structure of the L1 F3 module is proposed, where the marked region corresponds to a peptide that binds to FGFR1 and stimulates neurite outgrowth and receptor phosphorylation. The structure of the dimer of FGFR1 is obtained from the crystal structure of FGFR1 (Pellegrini et al., 2000).

  Figure 11. Gel filtration experiment demonstrating the possibility of a compact structure of L1 F3 domain I-V. A) Gel filtration of L1 F3 domain I-V using a Superdex 200 pg gel filtration column in PBS (blue) and in PBS + 1M NaCl (red). B) Gel filtration of BSA in PBS (blue) and in PBS + 1M NaCl (red).

Detailed Description of the Invention
1. Compound
1.1. Peptide sequences In this specification, the standard one-letter code and the standard three-letter code for 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, compound / peptide modifications may be performed, eg, glycosylation and / or acetylation of amino acids.

  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.

In a first aspect, the invention provides a formula:
_{^{x - - (x) n -x}} p - (x) n -x -
[Where:
x ^- is a basic amino acid residue,
x ^p is a hydrophobic amino acid residue, and
(x) _n is a sequence of any amino acid residue (where n is an integer from 0 to 3)]
The present invention relates to a compound capable of interacting with fibroblast growth factor receptor (FGFR), which comprises an amino acid sequence containing the amino acid motif.

According to the invention, x ⁻ can independently be any basic amino acid residue selected from K, R or H, and x ^p is independently A, V, L, I, Can be any hydrophobic amino acid residue selected from P, F, W, M or Y, and (x) _n is a sequence of any amino acid residue, where n is from 0 to An integer of 3). However, preferred motifs are those in which the residues x ^- and x ^p and the length of the sequence (x) are any of the following motifs:
K- (x) ₃ -L- (x) ₁ -K,
R- (x) ₃ -L- (x) ₁ -K,
K- (x) ₁ -L- (x) ₃ -K,
H- (x) ₁ -L- (x) ₂ -K,
R- (x) ₂ -W- (x) ₀ -R,
K- (x) ₂ -L- (x) ₀ -R,
R- (x) ₁ -V- (x) ₂ -H,
K- (x) ₁ -F- (x) ₃ -K,
R- (x) ₀ -Y- (x) ₂ -K or
R- (x) ₂ -I- (x) ₂ -K.

  Furthermore, it is preferable that at least one residue of the sequence (x) is a basic amino acid residue, a hydrophilic amino acid residue or G. In one preferred embodiment, it can be a basic residue selected from K, R or H, where H is more preferred. In other preferred embodiments, it may be selected from charged or uncharged hydrophilic residues such as D, E, H, K, N, Q, R, S or T, where H is a charged amino acid. More preferred among residues, and Q or S is more preferred among uncharged residues. In yet another preferred embodiment, the residue can be G.

In accordance with the present invention, the amino acid motif described above is an FGFR binding amino acid motif. The present invention provides the following non-limiting examples of such FGFR binding amino acid motifs:
KWFSLGK (SEQ ID NO: 8)
RYQWR (SEQ ID NO: 9)
KGHLR (SEQ ID NO: 10)
RHVHSH (SEQ ID NO: 11)
RFHILFK (SEQ ID NO: 12)
KALPEGK (SEQ ID NO: 13)
HHLAVK (SEQ ID NO: 14).

  In accordance with the present invention, all of the amino acid sequences described above can bind to FGFR, and this ability is conferred on compounds containing the sequence. Therefore, compounds comprising any of the above sequences are implicated as promising compounds for the purpose of activating FGFR. Of such promising compounds of the present invention, peptide sequences containing at most 105 amino acid residues are preferred.

Thus, according to the present invention, one of the preferred compounds is a compound comprising an isolated peptide sequence comprising an FGFR binding motif selected from the motif identified above (SEQ ID NOs: 8-14) and has been isolated. The peptide sequence preferably comprises an amino acid sequence selected from the following amino acid sequences:
APEKWFSLGKV (SEQ ID NO: 1),
DWNAPQIQYRYQWR (SEQ ID NO: 2),
DLAQVKGHLRGYN (SEQ ID NO: 3),
RHVHSHMVVPAN (SEQ ID NO: 4),
RFHILFKALPEGKVSPD (SEQ ID NO: 5) or
LHHLAVKTNGTG (SEQ ID NO: 6).

Essentially preferred are compounds comprising an amino acid sequence selected from SEQ ID NOs: 1-6. The term “essentially comprising” means herein that a sequence selected from SEQ ID NOs: 1-6 constitutes at least 10% compound content, which is expressed as a compound molecular weight or It can be expressed as a range of amino acid residues or amino acid sequences in the compound. A compound is understood to constitute the sequence when the selected amino acid sequence constitutes 100% of the compound content.
Compounds consisting of any of the sequences selected from SEQ ID NOs: 1-6 are also among the preferred compounds of the present invention.

  In one embodiment, a preferred compound consisting of a sequence selected from SEQ ID NOs: 1-6 is a compound consisting of SEQ ID NO: 1. In other embodiments, a preferred compound may consist of SEQ ID NO: 2. In yet other embodiments, a preferred compound may consist of SEQ ID NO: 3. A preferred compound may also consist of SEQ ID NO: 4. In other preferred embodiments, the compound may consist of SEQ ID NO: 5 or SEQ ID NO: 6. In these embodiments, SEQ ID NOs: 1-6 are meant to be isolated peptide sequences.

  An isolated peptide sequence is an amino acid sequence of the desired length according to the present invention, which is produced by the use of any recombinant technique or chemical synthesis, or it can be derived from a longer peptide or protein from an enzyme. They were separated by chemical or chemical cleavage methods.

  Thus, an isolated peptide sequence can be a fragment of a protein separated from other parts of the protein, eg, other fragments of a protein polypeptide. The present invention relates to an isolated peptide fragment of a protein containing the above FGFR motif. According to the present invention, the isolated peptide fragment of the present invention may be derived from any protein, but the preferred isolated protein fragment of the present invention is derived from the neural cell adhesion molecule L1. Thus, a compound of the invention may comprise or consist of a peptide fragment of L1. The present invention relates to all mammalian L1 proteins, such as those identified in the Swiss-Prot database polypeptides P32003, P11627, Q05695. Preferably, L1 is a human having the sequence identified as P32004 in the Swiss-Prot database.

  In particular, the present invention relates to a peptide fragment of L1 derived from a part of the L1 polypeptide, corresponding to type 3 fibronectin modules 1 to 5 (F3, 1-5). Thus, in one embodiment, the peptide fragment of L1 of the present invention comprises the L3 F3,1 module and has the sequence identified as SEQ ID NO: 15. In another embodiment, the L1-derived peptide fragment consists of the L1 F3,2 module and has the sequence identified as SEQ ID NO: 16. In yet another embodiment, the fragment is a F3,3 module of L1 and has the sequence identified as SEQ ID NO: 17. In another embodiment, the fragment is a F3, 4 module of L1 and has the sequence identified as SEQ ID NO: 18. In other embodiments, the fragment may consist of the L3 F3,5 module and has the sequence identified as SEQ ID NO: 19.

The present invention preferably relates to an L1 F3,1-F3,5 module having the following sequence:
F3,1: PGPVPRLVLSDLHLLTQSQVRVSWSPAEDHNAPIEKYDIEFEDKEMAPEKWYSLGKVPGNQTSTTLKLSPYVHYTFRVTAINKYGPGEPSPVSETV
F3,2: PEKNPVDVKGEGNETTNMVITWKPLRWMDWNAPQVQYRVQWRPQGTR GPWQEQIVSD PFLVVSNTSTFVPYEIKVQA VNSQGKGPEP QVTIGYS
F3,3: PQAIPELEGIEILNSSAVLVKWRPVDLAQVKGHLRGYNVTYWREGSQRKHSKRHIHKDHVVVPANTTSVILSGLRPYSSYHLEVQAFNGRGSGPASEFT FST
F3,4: PEGVPGHPEALHLECQSNTSLLLRWQPPLSHNGVLTGYVLSYHPLDEGGKGQLSFNLRDPELRTHNLTDLSPHLRYRFQLQATTKEGPGEAIVREGGT
F3,5: GISDFGNISATAGENYSVVSWVPKEGQCNFRFHILFKALGEEKGGASLSPQYVSYNQSSYTQWDLQPDTDYEIHLFKERMFRHQMAVKTNGTG.

  However, in certain preferred embodiments of the invention it may be advantageous to use shorter peptide sequences. Accordingly, the present invention relates to fragments of the L1 F3 module, eg, F3,1, F3,2, F3,3, F3,4 or F3,5 modules. A fragment of the latter module is a sequence selected from SEQ ID NOs: 1-6 or a fragment of said sequence, for example a fragment having a length of 5-7 amino acid residues and comprising the above FGFR binding motif, for example It is preferable that at least one sequence selected from SEQ ID NOs: 8-14 is included.

1.1 Peptide Sequence Length According to the present invention, the compounds of the present invention comprise an amino acid sequence comprising an FGFR binding motif with at most 105 amino acid residues.

  Thus, in one embodiment, the compound comprises 3-105 amino acid residues, such as 3-100 amino acid residues, such as 3-95 amino acid residues, such as 3-90 amino acid residues. A group such as 3-85 amino acid residues, such as 3-80 amino acid residues, such as 3-75 amino acid residues, such as 3-70 amino acid residues, such as 3- 65 amino acid residues, e.g. 3-60, e.g. 3-55 amino acid residues, e.g. 3-50 amino acid residues, e.g. 3-45 amino acid residues, e.g. 3 -40 amino acid residues, e.g. 3-35 amino acid residues, e.g. 3-30 amino acid residues, e.g. 3-25 amino acid residues, e.g. 3-20 amino acid residues It may comprise a peptide sequence comprising a group, for example 3-15, for example 3-10 amino acid residues, for example 3-5 amino acid residues.

  In other embodiments, the peptide sequence of the compound has 4-100 amino acid residues, such as 4-90 amino acid residues, such as 4-80 amino acid residues, such as 4-70, For example, 4-60, eg, 4-50 amino acid residues, eg, 4-40 amino acid residues, eg, 4-30 amino acid residues, eg, 4-20 amino acid residues For example, it may have a length of between 4-15 amino acid residues or 4-10.

  In a further aspect, the peptide sequence has 5 to 100 amino acid residues, such as 5 to 90 amino acid residues, such as 5 to 80 amino acid residues, such as 5 to 70, such as 5 to 60, e.g. 5 to 50 amino acid residues, e.g. 5 to 40 amino acid residues, e.g. 5 to 30 amino acid residues, e.g. 5 to 20 amino acid residues, e.g. 5 Can have a length between 1 and 15 amino acid residues, for example between 5 and 10 amino acid residues.

  In yet a further aspect, the sequence comprises 6-100 amino acid residues, such as 6-90 amino acid residues, such as 6-80 amino acid residues, such as 6-70 amino acids, such as 6- 60, such as 6-50 amino acid residues, such as 6-40 amino acid residues, such as 6-30 amino acid residues, such as 6-20 amino acid residues, such as 6 There can be -15 amino acid residues, for example 6-10 amino acid residues.

  The present invention also includes 7-100 amino acid residues, such as 7-90 amino acid residues, 7-80 amino acid residues, such as 7-70, such as 7-60, such as 7-50 amino acid residues, e.g. 7-40 amino acid residues, e.g. 7-30 amino acid residues, e.g. 7-20 amino acid residues, e.g. 7-15 amino acids It relates to an amino acid sequence having a length between residues, for example 8, 9, 10, 11, 12, 13, or 14 amino acid residues.

  The length of the isolated sequence may also be 8-80 amino acid residues, such as 8-70, such as 8-60, such as 8-50 amino acid residues, such as 8-40. May be 8-30 amino acid residues, eg, 8-20 amino acid residues. Alternatively, it may be 9-80 amino acid residues, such as 9-70 amino acid residues, such as 9-60 amino acid residues, such as 9-50 amino acid residues, such as 9- There may be 40 amino acid residues, for example 9-30 amino acid residues, for example 9-20 amino acid residues.

  10-80 amino acid residues, such as 10-70 amino acid residues, such as 10-60 amino acid residues, such as 10-50 amino acid residues, such as 10-40 amino acids Compounds comprising an amino acid sequence of residues, eg, 10-30 amino acid residues, eg, 10-20 amino acid residues, are also within the scope of the invention.

  In one preferred embodiment of the invention, the minimum length of the amino acid sequence of the compound is 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 amino acid residues, and a maximum Are 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 amino acid residues. Also preferred are compounds comprising an amino acid sequence whose length is in the range of 25-36 amino acid residues.

  Furthermore, more preferred compounds are those that contain an isolated peptide sequence that is at most 20 amino acid residues in length. Compounds comprising individual peptide fragments having a length of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 amino acid residues are most preferred compounds. Is inside.

  It is understood that all the peptide sequences described above contain the FGFR binding motif of the present invention or the peptide fragment of L1 described above and can bind to FGFR.

1.3. Multimeric compounds The isolated peptide sequences of the present invention can be linked to other isolated peptide sequences by chemical bonds in a fusion protein (although the fusion protein is not an L1 protein). Two or more isolated sequences of the present invention can also be linked to each other via a linker group to provide other types of multimeric presentation of the peptide sequences of the present invention. Both the latter fusion proteins and multimers are relevant as compounds of the invention.

  Accordingly, the peptide sequences of the present invention may be formed as a polymer comprising several copies of the peptide sequence in certain embodiments. A polymer containing two sequences is referred to herein as a dimer. A polymer containing four peptide sequences is referred to herein as a tetramer.

  Thus, in accordance with the present invention, a multimeric compound can be a polymer of two or more identical or different peptide sequences of the present invention, where in a preferred embodiment one of two or more amino acid sequences. Is selected from SEQ ID NOs: 1-6, or fragments or variants of the sequences.

  In some embodiments, the compound comprises two identical amino acid sequences selected from SEQ ID NOs: 1-6, 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: 1-6, or can comprise a fragment or variant of the four selected identical sequences.

  In other embodiments, the compound may comprise two or more different amino acid sequences, wherein at least one of the two amino acid sequences is from SEQ ID NO: 1-6, 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 NOs: 1-6, or fragments or variants 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 group.

A linker can be any molecule or chemical moiety capable of cross-linking two or more peptide sequences according to the present invention, for example, 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 Are aromatic moieties or A and B together form a substituted or unsubstituted cyclic moiety, a substituted or unsubstituted heterocyclic moiety, a substituted or unsubstituted aromatic moiety]
Of achiral di-, tri- or tetracarboxylic acids.

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.

1.4. Fragments and variants of peptide sequences In certain embodiments of the invention, variants or fragments of the FGFR binding sequences of the invention may be included, preferably fragments or variants selected from SEQ ID NOs: 1-6.

In accordance with the present invention, amino acid sequence variants selected from the sequences of SEQ ID NOs: 1-6 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.

  In one aspect, the term “peptide sequence variant” means that the peptide sequence may 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. Examples are methyl and acetyl esters.

  In other aspects, variants of the peptide fragments described in the present invention are introduced into the same variant, or a fragment thereof, or a different variant or fragment thereof, at least one substitution, eg, independently of each other 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)).

  Amino acid substitutions are made in one embodiment based on their hydrophobic and hydrophilic values and the relative similarity of amino acid side chain substituents, including charge, size, and the like.

  In accordance with the present invention, a selected sequence of the present invention, eg, a fragment of a sequence selected from SEQ ID NOs: 1-6, can be an amino acid sequence having about 25-99% of the length of the selected amino acid sequence.

  Fragments and variants of selected sequences of the present invention are functional homologs of the selected sequence according to the present invention.

  The term “functional homologue” of a selected amino acid sequence, as used herein, refers to a molecule or compound containing one or more biological functions of the selected sequence that allows for that biological function. Then, the biological function is performed through a mechanism that binds to and activates FGFR.

1.5 Biological activity In accordance with the present invention, the compounds described above are functionally active compounds. The compound can bind to and activate a functional cell surface receptor. The receptor is FGFR according to the present invention, fibroblast growth factor receptor 1 (FGFR1), fibroblast growth factor receptor 2 (FGFR2), fibroblast growth factor receptor 3 (FGFR3), fiber One can select from a population of FGFR receptors including blast growth factor receptor 4 (FGFR4) or fibroblast growth factor receptor 5 (FGFR5). The compound according to the present invention is a binding site located in the extracellular part of the receptor, for example, a binding site located in the Ig1, Ig2 and / or Ig3 domain of FGFR, preferably Ig2 and / or FGFR. A binding site located in the Ig3 domain that can bind to either of the latter FGFRs. According to the present invention, the latter binding site is different from other described binding sites of FGFR (e.g. Plotnikov AN, Hubbard SR, Schlessinger J, Mohammadi M .. Cell. 2000 May 12; 101 (4): (See 413-24).

  The present invention therefore relates to functional cell surface receptors. “Functional cell surface receptor” means that the receptor is located on the outer cell membrane of a cell and has an identifiable group of extracellular ligands. The binding of these ligands to receptors induces intracellular signaling and results in a physiological response of the cell. Physiological responses, such as changes in cell metabolism induced by ligand binding, induction of cell differentiation, termination or induction of cell proliferation, cell survival or death, changes in cell motility behavior, It depends on the receptor cell and the extracellular environment and / or in particular the ligand receptor interaction, eg the affinity and / or persistence of the interaction. A ligand that binds to a functional receptor usually results in a change in the activation state of the receptor, which, for example, enables the receptor to initiate a cascade of biochemical reactions within the cell, as described above. Produces one of the physiological responses (collectively referred to as “receptor signaling” or “signaling”). Ligand binding can also result in inhibition of receptor activity, which causes the receptor to become “silent” or “inactive” and beyond this, through a cascade of biochemical reactions that normally occur due to ligand binding. Means it cannot start. The present invention relates to a compound capable of activating FGFR and a compound capable of inhibiting FGFR. In one aspect, the present invention relates to a compound capable of activating FGFR, and in another preferred embodiment, the present invention relates to a compound capable of inhibiting FGFR. Preferably, the present invention relates to a compound capable of activating FGFR1 and stimulating FGFR1 signaling.

  Receptor activation is often preceded by receptor dimerization induced by ligand binding. Thus, the ability of a compound to promote receptor dimerization (or multimerization) is relevant to the present invention as an advantageous feature. Thus, in one aspect, the invention relates to compounds that allow FGFR dimerization.

  The compounds of the present invention may be able to reduce the binding of other ligands despite occupying other binding site (s). Thus, the compounds of the present invention may modulate receptor signaling dependent on other ligand binding. The cellular response to receptor activation depends on the intensity of receptor stimulation, eg, is characterized by the affinity value of the ligand-receptor interaction and / or by the persistence of such an interaction Is known. Thus, the affinity and persistence of FGF-receptor interactions can be affected by the compounds of the present invention. Accordingly, another aspect of the present invention is to provide compounds capable of modulating receptor signaling induced by other receptors, for example FGF or FGL-peptides of WO 03/016351.

  As used herein, the term “interact” is used interchangeably with the term “bind” and refers to direct or indirect contact between a compound of the invention and an FGF receptor, Preferably, it is a direct 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 preferably has a Kd value in the range of 10 ⁻³ to 10 ⁻¹⁰ M, such as preferably in the range of 10 ⁻⁴ to 10 ⁻⁸ M. . 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.

  In accordance with the present invention, the binding affinity of a compound to FGFR can be modulated (eg, enhanced or attenuated) in the presence of a nucleotide compound. In one embodiment, the affinity of the interaction can be enhanced. In other embodiments, the affinity of the interaction can be attenuated. According to the present invention, the affinity of interaction is promoted in the presence of trinucleotide phosphates such as ATP, GTP or UTP, and the affinity is dinucleotides in the presence of mononucleotide phosphates such as AMP, GMP. Attenuates in the presence of phosphate, eg, ADP or GDP. Thus, in certain embodiments, the compounds of the invention may be used in combination with nucleotides for purposes of optimally activating or inhibiting FGFR.

  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 (including angiogenesis, antioxidant stress and anti-inflammatory activity).

  The term “FGFR presenting cell” refers to cells that express FGFR on the outer membrane of the cell, such as neurons, glial cells, all types of muscle cells, neuroendocrine cells, gonad cells and kidneys. Cells, endothelial cells, fibroblasts, osteoblasts, cancer, stem cells and embryonic cells.

  In one preferred embodiment, the compound is capable of stimulating neurite outgrowth.

  In other preferred embodiments, the compound can stimulate cell survival.

  In other preferred embodiments, the compound can stimulate synaptic plasticity. Thus, the compounds can stimulate learning and memory as well.

  In yet another preferred embodiment, the compound can stimulate stem cell differentiation.

Stimulation of neuronal differentiation and plasticity Substances that have the potential to promote neurite outgrowth and stimulate neuronal cell regeneration and / or differentiation, such as endogenous trophic factors, for example, other types of neural regeneration and neuroplasticity It is an important target in the search for compounds that promote morphology. To assess the potential of the compounds of the present invention, the ability to stimulate neurite outgrowth related signaling, interfere with cell adhesion, stimulate neurite outgrowth, nerve regeneration may be investigated.

  The compounds of the present invention have been shown to promote neurite outgrowth, and therefore in effective promoters for regeneration of nerve connections and thereby functional recovery after injury and other conditions where such effects are required. It is thought that it is a promoter of neuronal function. Furthermore, the compounds of the present invention can stimulate the differentiation of neural precursor cells into 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 invention relates to neurite outgrowth improvement / stimulation, e.g., greater than about 75%, e.g., greater than 50%, e.g., greater than 150%, e.g., greater than 100%, e.g., control / unstimulated cells. More than 250%, for example more than 200%, for example more than 350%, for example more than 300%, for example more than 450%, for example more than 400%, for example more than 500%.

  Estimation of the ability of a candidate compound to stimulate neurite outgrowth can be done by using any known method or assay, eg, for estimation of neurite outgrowth as described in the Examples.

  In accordance with the present invention, the compounds have neurite generating activity as an insoluble immobilizing component of the cell growth substrate and as a soluble component of the cell growth medium. As used herein, “fixed” means that the compound binds / adheres to a substance that is insoluble in water or an aqueous solution, thereby making it insoluble in such a solution as well. For medical applications, both insoluble and soluble compounds will be applied, but soluble compounds are preferred. A “soluble compound” is understood to be a compound that is soluble in water or an aqueous solution.

Antibodies An object of the present invention is an epitope comprising the FGFR motif of the present invention, for example, an epitope comprising a sequence selected from SEQ ID NO: 8-14 or a sequence selected from SEQ ID NO: 1-6, or a fragment of the sequence, preferably Is to provide an antibody, antigen-binding fragment or recombinant protein thereof capable of recognizing and selectively binding to an epitope of L1 polypeptide.

  The term “epitope” refers to a specific group of atoms (on an antigen molecule) that is recognized by an antibody (of an antigen), thereby producing an immune response. The term “epitope” is equivalent to the term “antigenic determinant”. Epitopes are located in close proximity, for example within a contiguous amino acid sequence, or at a distance from the amino acid sequence of an antigen but close to each other by protein folding, resulting in 3 or more amino acid residues. It may contain groups such as 4, 5, 6, 7, 8 amino acid residues.

  Antibody molecules belong to a family of plasma proteins called immunoglobulins, whose basic components, immunoglobulin folds or domains, are used in many forms by many molecules of the immune system and other biological recognition systems. The A typical immunoglobulin has four polypeptide chains, including an antigen binding region known as the variable region and a non-variable region known as the constant region.

  Natural antibodies and immunoglobulins are usually heterotetrameric glycoproteins of about 150,000 daltons, consisting of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to the heavy chain by one covalent disulfide bond, and the number of disulfide bonds varies between the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has a controlled spatial intrachain disulfide bridge. Each heavy chain has a variable domain (VH) at one end and then a number of constant domains. Each light chain has a variable domain (VL) at one end and a constant domain at the opposite end. The constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain. Certain amino acid residues are believed to form an interface between light and heavy chain variable domains (Novotny J, & Haber E. Proc Natl Acad Sci US A. 82 (14): 4592-6, 1985) .

  Depending on the amino acid sequence of the constant domain of each heavy chain, immunoglobulins are assigned to different classes. There are at least five (5) major immunoglobulins: IgA, IgD, IgE, IgG and IgM, some of which are further subclasses (isotypes), e.g., IgG-1, IgG-2, IgG-3 And IgG-4; can be classified as IgA-1 and IgA-2. The heavy chain constant regions that correspond to the different classes of immunoglobulins are called alpha (α), delta (δ), epsilon (ε), gamma (γ), and micro (μ), respectively. The light chains of antibodies can be assigned to one of two distinct types called kappa (κ) and lambda (λ), based on the amino acid sequence of their constant domains. The subunit structures and three-dimensional structures of different classes of immunoglobulins are known.

  The term “variable” in the context of an antibody variable domain refers to the fact that certain portions of the variable domain are broadly different from the sequences in the antibody. The variable domain is intended for binding and determines the specificity of each particular antibody for its particular antigen. However, variability is not evenly distributed through the variable domains of antibodies. It is concentrated in three parts called complementarity determining regions (CDRs) and is also known as the hypervariable region in light and heavy chain variable domains.

  The more highly conserved portions of variable domains are called the framework (FR). The natural heavy and light chain variable domains each contain mainly four FR regions that employ a β-sheet structure bound to three CDRs, which can be loop-coupled, in some cases β- Form part of the sheet structure. The CDRs of each chain are held together close to the FR region (along with the CDRs from other chains) and contribute to the formation of the antigen binding site of the antibody. The constant domains are not directly involved in the binding of the antibody to the antigen, but indicate antibody participation in various effector functions, such as antibody-dependent cellular cytotoxicity.

  Thus, antibodies intended for use in the present invention include whole immunoglobulins, antibody fragments such as Fv, Fab, and similar fragments, single chain antibodies that contain variable domain complementarity determining regions (CDRs), etc. All of which are broadly under the term “antibody” as used herein. The present invention contemplates the use of any specificity, antibody, polyclonal or monoclonal, and is not limited to antibodies that recognize specific antigens and immunoreact. In a preferred embodiment, an immunospecific antibody or fragment thereof against an antigen or epitope of the invention is used in the context of both the therapeutic and screening methods described below.

The term “antibody fragment” refers to a portion of a full length antibody, generally an antigen binding or variable region. Examples of antibody fragments include Fab, Fab ′, F (ab ′) ₂ and Fv fragments. The papain digestion of antibodies produces two identical antigen-binding fragments, each called a Fab fragment with a single antigen-binding site, and the remaining “Fc” fragments, because of the so-called ability to crystallize easily To do. Pepsin treatment yields an F (ab ′) ₂ fragment and the remaining other fragment (called pFc ′) that have two antigen-binding fragments and can crosslink with the antigen. Additional fragments can include diabodies, linear antibodies, single chain antibody molecules, and multispecific antibodies formed from antibody fragments. As used herein, “functional fragment” with respect to antibodies refers to Fv, F (ab) and F (ab ′) ₂ fragments.

  The term “antibody fragment” is used herein interchangeably with the term “antigen-binding fragment”.

  Antibody fragments are about 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, 9 amino acids, about 12 amino acids, about 15 amino acids, about 17 amino acids, about 18 amino acids. It can be as small as or more than about 1 amino acid, about 20 amino acids, about 25 amino acids, about 30 amino acids. In general, an antibody fragment of the invention can have all the upper size limits as long as it has similar or immunological properties compared to an antibody having specificity for an epitope and is SEQ ID NO: 1. Peptide sequences selected from any of the sequences identified herein as -14, or fragments of the sequences. Thus, in the context of the present invention, the term “antibody fragment” is identical to the term “antigen-binding fragment”.

Antibody fragments retain the ability to selectively bind to its antigen or receptor. One type of antibody fragment is defined as follows:
(1) Fab is a monovalent antigen-binding fragment or a fragment containing an antibody molecule. Fab fragments can be produced by digestion of whole antibodies (the enzyme papain produces an intact light chain and a portion of one heavy chain).

  (2) Fab ′ is a fragment of an antibody molecule that can be obtained by treating whole antibody with pepsin and then reducing it, producing intact light chain and heavy chain portions. Two Fab ′ fragments are obtained per antibody molecule. Fab ′ fragments differ from Fab fragments by the addition of a few residues at the carboxyl terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region.

(3) (Fab ′) ₂ is a fragment of an antibody that can be obtained by treating the whole antibody with the enzyme pepsin without subsequent reduction.

(4) F (ab ′) ₂ is a dimer of two Fab ′ fragments held together by two disulfide bonds. Fv is the smallest antibody fragment that contains a complete antigen recognition and binding site. This region consists of a dimer of one heavy chain and one light chain variable domain in a tight non-covalent bond (V _H -V _L dimer). It is within this structure that the three CDRs of each variable domain interact to define an antigen binding site on the surface of the V _H -V _L dimer. Collectively, the six CDRs provide antigen binding specificity for the antibody. However, even a single variable domain (or half of the Fv that contains only three CDRs specific for the antigen) has the ability to recognize and bind antigen, although with a lower affinity than the entire binding site. Have.

  (5) A single chain antibody ("SCA") defined as a genetically modified molecule comprising a light chain variable region and a heavy chain variable region is a genetically fused single chain molecule, They are linked by a suitable polypeptide linker. Such single chain antibodies are also referred to as “single chain Fv” or “sFv” antibody fragments. In general, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains, allowing the sFv to form the desired structure for antigen binding. For sFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies 113: 269-315 Rosenburg and Moore eds. Springer-Verlag, NY, 1994.

  The term “diabody” refers to a small antibody fragment having two antigen-binding sites, which is a heavy chain bound to a light chain variable domain (VL) on the same polypeptide chain (VH-VL). Contains the chain variable domain (VH). By using a linker that is too short to allow pairing between two domains on the same chain, the domain is forced to pair with the complementary domain of the other chain and bind two antigens Create a site. Diabodies are more fully described, for example, in EP 404,097; WO 93/11161, and Hollinger et al., Proc. Natl. Acad Sci. USA 90: 6444-6448 (1993).

  The present invention contemplates polyclonal and monoclonal antibodies, antigen-binding fragments and recombinant proteins thereof that can bind to the epitopes described in the present invention.

  The production of polyclonal antibodies is known to those skilled in the art. For example, Green et al. 1992. Production of polyclonal Antisera, in: Immunochemical Protocols (Manson, ed.), Pages 1-5 (Humana Press); Coligan, et al., Production of polyclonal Antisera in Rabbits, Rats Mice and Hamsters , in: Current Protocols in Immunology, section 2.4.1, which is hereby incorporated by reference.

  The production of monoclonal antibodies is likewise conventional. See, for example, Kohler & Milstein, Nature, 256: 495-7 (1975); Coligan, et al., Sections 2.5.1-2.6.7; and Harlow, et al., In: antibodies: A Laboratory Manual, page 726, See Cold Spring Harbor Pub. (1988). Monoclonal antibodies can be isolated and purified from hybridoma cultures by a variety of established techniques. Such isolation techniques include affinity chromatography using protein-A sepharose, size exclusion chromatography, and ion exchange chromatography. For example, Coligan, et al., Sections 2.7.1-2.7.12 and sections 2.9.1-2.9.3; Barnes, et al., Purification of Immunoglobulin G (IgG). In: Methods in Molecular Biology, 1992, 10 : 79-104, Humana Press, NY.

  Methods of in vitro and in vivo manipulation of monoclonal antibodies are known to those skilled in the art. For example, monoclonal antibodies for use in accordance with the present invention can be initially produced by the hybridoma method described in Kohler and Milstein, 1975, Nature 256, 495-7, or can be produced by recombinant methods such as those described in US Pat. No. 4,816,567. It can be produced by the method described. Monoclonal antibodies for use in the present invention can also be prepared using the techniques described in Clackson et al., 1991, Nature 352: 624-628 and Marks et al., 1991, J Mol Biol 222: 581-597. Can be used to isolate from a phage antibody library. Other methods involve humanizing monoclonal antibodies by recombinant means to produce antibodies that contain human-specific and recognizable sequences. See Holmes, et al., 1997, J Immunol 158: 2192-2201 and Vaswani, et al., 1998, Annals Allergy, Asthma & Immunol 81: 105-115.

  As used herein, the term “monoclonal antibody” refers to an antibody obtained from a population of substantially homogeneous antibodies, ie, individual antibodies comprising the population can be present in small amounts. Except for naturally occurring mutations that can be considered. Monoclonal antibodies are highly specific and are directed to a single antigenic site. Furthermore, in contrast to conventional polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. It is done. In addition to their specificity, monoclonal antibodies are advantageous in that they are synthesized by hybridoma culture and are not contaminated by other immunoglobulins. The modifier “monoclonal” indicates the character of the antibody as being obtained from a population of substantially homologous antibodies, and should not be construed as requiring production of the antibody by any particular method.

  Monoclonal antibodies herein include in particular “chimeric antibodies” (immunoglobulins), where a portion of the heavy and / or light chain is derived from a particular species so long as they exhibit the desired biological activity. Or the sequence corresponding to an antibody belonging to a particular antibody class or subclass, or is homologous, while the remainder of the chain (s) is derived from other species of antibodies or other Morrison et al., 1984, Proc Natl Acad Sci 81, which is identical or homologous to sequences corresponding to antibodies belonging to the class or subclass of antibodies, and fragments of such antibodies (US Pat. No. 4,816,567); : 6851-6855.

Methods for producing antibody fragments are also known in the art (see, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, NY, 1988, incorporated herein by reference). Do). Antibody fragments of the invention can be produced by proteolytic hydrolysis of the antibody or by expression in E. coli of DNA encoding the fragment. Antibody fragments can be obtained with pepsin or papain by whole antibody conventional methods. For example, antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment represented by F (ab ′) ₂ . This fragment can be further cleaved using a thiol reducing agent, optionally a protecting group for the sulfhydryl groups resulting from cleavage of disulfide bonds, to produce 3.5S Fab ′ monovalent fragments. Alternatively, enzymatic cleavage with pepsin produces two monovalent Fab ′ fragments and an Fc fragment directly. These methods are described, for example, in US Pat. No. 4,036,945 and US Pat. No. 4,331,647, and references contained therein. These patents are hereby incorporated by reference in their entirety.

Other methods of cleaving antibodies, such as heavy chain separation to form monovalent light-heavy chain fragments, further fragment cleavage, or other enzymatic, chemical, or genetic techniques include Alternatively, as long as the fragment binds to the antigen recognized by the intact antibody, it can be used. For example, Fv antibodies comprise V _H and V _L chain binding. This linkage can be non-covalent or the variable chains can be linked by intermolecular disulfide bonds or crosslinked by chemicals such as gratalaldehyde. Preferably, the Fv fragment comprises V _H and V _L chains attached to a peptide linker. These single chain antigen binding proteins (sFv) are produced by constructing a structural gene containing DNA sequences encoding the V _H and V _L domains bound to the oligonucleotide. The structural gene is inserted into an expression vector and then introduced into a host cell, eg, E. coli. Recombinant host cells synthesize a single polypeptide chain with a linker peptide that bridges the two V domains. Methods for producing sFvs are described, for example, in Whitlow, et al., 1991, In: Methods: A Companion to Methods in Enzymology, 2:97; Bird et al., 1988, Science 242: 423-426; US 4,946,778. And Pack, et al., 1993, BioTechnology 11: 1271-77.

  Another form of an antibody fragment is a peptide that encodes a single complementarity determining region (CDR). CDR peptides (“minimal recognition units”) are often involved in antigen recognition and binding. CDR peptides can be obtained by cloning or constructing the gene encoding the CDR of the antibody of interest. Such a gene is preferably obtained by synthesizing a variable region from mRNA of an antibody-producing cell using, for example, a polymerase chain reaction. See, for example, Larrick, et al., Methods: a Companion to Methods in Enzymology, Vol. 2, page 106 (1991).

The present invention contemplates humanized forms of human and non-human (eg, murine) antibodies. Such humanized antibodies include chimeric immunoglobulins, immunoglobulin chains or fragments thereof (e.g., antibody Fv, Fab, Fab ', F (), which contain minimal sequence derived from non-human immunoglobulin, such as epitope recognition sequences. ab ′) ₂ or other antigen-binding subsequence). In many cases, humanized antibodies are non-human species (donor antibodies) in which residues from the complementarity determining regions (CDRs) of the recipient have the desired specificity, affinity and ability, e.g., mouse, rat Or a human immunoglobulin (recipient antibody) that is substituted with a residue from a rabbit CDR. A humanized antibody (s) comprising a minimal sequence (s) of the antibody (s) of the invention, such as a sequence (s) that recognizes the epitope (s) described herein. Is a preferred embodiment of the present invention.

  In certain instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are found neither in the recipient antibody nor in the imported CDR or fragment sequences. These modifications are made to further improve and optimize antibody performance. In general, a humanized antibody comprises substantially all of at least one and typically two variable domains, all or substantially all of the CDR regions corresponding to those of a non-human immunoglobulin. And all or substantially all FR regions are those of the human immunoglobulin consensus sequence. A humanized antibody optimally also comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., 1986, Nature 321, 522-525; Reichmann et al., 1988, Nature 332, 323-329; Presta, 1992, Curr Op Struct Biol 2: 593-596; Holmes et al ., 1997, J Immunol 158: 2192-2201 and Vaswani, et al., 1998, Annals Allergy, Asthma & Immunol 81: 105-115.

The production of antibodies can be any of those in the art for producing polyclonal and monoclonal antibodies using natural or recombinant fragments of L1 as antigens, the fragments comprising a sequence selected from SEQ ID NOs: 1-14. Can be achieved by standard methods. Such antibodies can also be produced using variants, homologues or fragments of the peptide sequence of SEQ ID NOs: 1-14, which variants, homologues and fragments are immunogenic peptide sequences that meet the following criteria: is there:
(i) a contiguous amino acid sequence of at least 6 amino acids;
(ii) including the FGFR motif of the present invention.

  Antibodies can also be produced in vivo by the individual being treated, eg, by administering to the individual an immunogenic fragment according to the present invention. Therefore, the present invention further relates to a vaccine comprising the above immunogenic fragment.

  The application also relates to a method of producing an antibody of the invention, said method comprising providing an immunogenic fragment as described above.

  The present invention is capable of modulating, for example, enhancing or attenuating, and without regulating the biological activity of L1 biological functions, particularly functions related to neuronal differentiation, survival and / or plasticity. Relates to an antibody capable of recognizing and specifically binding to the latter protein.

  The present invention relates to the therapeutic applications of the above antibodies when 1) modulation of L1 or FGFR activity is required, 2) proteins comprising L1 or the above epitopes in vitro and / or in vivo for diagnostic purposes. Detecting and / or monitoring, 3) relating to use for research purposes.

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 present invention are produced through the use of 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, which 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 is 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 of 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.

Sequence-assisted peptide synthesis (SAPS)
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.

  Below is a list of drugs and a description of procedures that may be useful when using SAPS for the synthesis of peptide fragments of the invention.

3. Medicine
FGFR and L1 are known to be involved in many physical processes in normal conditions and diseases, 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 attenuation of 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 involved according to the invention as medicaments for treating diseases, where modulation of the activity of FGFR can be considered as an important condition 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 any type of cancer of solid tumors that require 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.

  L1 has been extensively studied as a neurite outgrowth stimulator. It has also been considered important for neuronal precursor proliferation, differentiation and transmitter phenotype subtype production (Dihne et al. (2003) J Neurosci 23: 6638-6650) and plays a role in synaptic plasticity (Saghatelyan et al. (2004) Mol Cell Neurosci 26: 191-203). FGFRs and their ligands play an important role in the CNS, in particular they are involved in processes related to memory and learning (Reuss and von Bohlen und Halbach (2003) Cell Tissue Res. 313: 139-57) . Thus, in yet other embodiments, the compounds of the invention may be used for the ability to learn and / or for stimulating short-term and / or long-term memory. This embodiment is one of the preferred embodiments of the present invention.

In yet a further aspect, the compound of the invention comprises
1) prevention of cell death due to ischemia;
2) The purpose is to produce a medicine for the prevention of physical damage caused by alcohol consumption.

  In particular, the present invention relates to normal, degenerated or damaged L1 presenting cells. Diseases such as X-chromosome-associated hydrocephalus and MASA syndrome callosal dysplasia, mental retardation, adductional thumb, convulsions and hydrocephalus (CRASH) syndrome are of particular interest in the present invention.

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

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

  A further aspect of the invention is the process of producing a pharmaceutical composition, wherein an effective amount of one or more compounds of the invention, or a pharmaceutical composition according to the invention is added to one or more pharmaceuticals. Together with an acceptable additive or carrier. In one embodiment, the compound is used in combination with a prosthesis, where the device is a nerve guide. Accordingly, in a further aspect, the present invention relates to a nerve guide 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, intranasal or 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, Basel , 1995.

  For injection, they are usually prepared as liquid solutions or suspensions, solids suitable for dissolving or suspending in liquid 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 preparation can be appropriately administered by injection, if desired, to the site where the active ingredient exerts its effect. Additional formulations suitable for other dosage forms 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) within 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 %including.

  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, and such an amount is also a therapeutically effective amount. The amount administered will depend 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 on the order of an effective amount of several hundred μg per kilo body weight for administration, preferably about 0.1 to 5000 μg per kilo body 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, and especially kilo Within the range of about 0.1 μg to 1000 μg per body weight. With multimeric forms of the compound, suitable doses are often in the range of 0.1 μg to 1000 μg per kilo body weight, for example, about 0.1 μg to 750 μg per kilo body weight, and especially about per kilo body weight. Within the range of 0.1 μg to 500 μg, for example within the range of about 0.1 μg to 250 μg per kilo body weight. In particular, when administered nasally, smaller amounts are used than when administered by other routes. Administration can be performed once or the next administration can be performed. The dose will also depend on the route of administration and will vary with the age and weight of the subject to be treated. A preferred dose for the multimeric form is between 1 mg and 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 can be based on controlled release and / or use of precursors, as well as oral administration.

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. 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 wounds); traumatic neuroma (open wounds) Have , Or not), traumatic transient paralysis (with or without open wound), accidental dural puncture or laceration during medical procedures, optic nerve and pathway damage, optic nerve damage, second cranial nerve, Damage to the chiasm, damage to the visual pathway, damage to the visual cortex, nonspecific blindness, damage to the cranial nerve (s), other and nonspecific nerve damage; drug addiction, pharmaceuticals and biological substances, Genetic or traumatic atrophic myopathy; or treatment of various organs, such as degenerative state of the gonad, pancreas, eg, type I and type II diabetes I, kidney, eg, 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, adductional thumb, convulsions and hydrocephalus (CRASH) syndrome, Pain syndrome , Encephalitis, drug / alcohol addiction, anxiety, postoperative nerve injury, intraoperative ischemia, inflammatory disorders with tissue damage by affecting infectious agents or protecting tissues, 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, metabolism Disorders such as obesity lipid disorders (e.g. hypercholesterolemia, atherosclerosis, amino acid transport and metabolism disorders, purine and pyrimidine metabolism disorders and gout, bone disorders such as fractures, osteoporosis, osteoarthritis (OA), atrophic dermatitis, psoriasis, infectious diseases, in vivo or in vitro stem cell protection or maturation.

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

Example
1. L1 fragment of the present invention SEQ ID NO: 1 APEKWFSLGKV F1CDL peptide SEQ ID NO: 2 DWNAPQIQYRVQWR F2BCL peptide SEQ ID NO: 3 DLAQVKGHLRGYN F3ABL peptide SEQ ID NO: 4 RHVHKSHMVVPAN F3CDL peptide SEQ ID NO: 5 RFHILFKALPEGKVSPD F5BCL peptide GL6 GL6

2. Experimental procedure
2. 1. Production of recombinant protein
L1 F3 module IV was produced by RT-PCR using rat brain total RNA. FGFR1 Ig module II-III was produced using mouse FGFR1 (IIIC isoform) cDNA (specialized by Dr. Patrick Doherty, King's College, London). The L1 F3 module consists of RSPWPG, amino acids 608-1109 (swissprot Q05695) of L1, and HHHHHH. FGFR1 Ig module II consists of RSHHHHHH and amino acids 253-365 (swissprot p16092) of FGFR1. The FGFR1-binding Ig module II-III consists of RSHHHHHH and amino acids 141-365 (swissprot p16092) of FGFR1. FGFR1 Ig module II-III and L1 F3 module IV were expressed in Drosophila S2 cells (Invitrogen, USA) according to the manufacturer's instructions. All proteins were purified by affinity chromatography using Ni ²⁺ -NTA resin (Qiagen, USA), ion exchange chromatography, and gel filtration (all chromatographic columns used for purification are Amersham Purchased from Biosciences, Sweden). The FGFR1 IgII module was expressed in the Pichia Pastoris KM71 cell expression system (Invitrogen, USA) according to the manufacturer's instructions. The protein was purified by affinity chromatography using Ni ²⁺ -NTA resin (Qiagen, USA), ion exchange chromatography, and gel filtration (all chromatographic columns used for purification are Amersham Biosciences, Purchased from Sweden). Bound NCAM Ig modules I and II (RV and rat NCAM amino acids 20-208, swissprot p13596) were produced as described above (Jensen et. Al., 1999).

［式中、
k₂およびk₁は、最初の結合速度とATP-F3複合体およびF3モジュールそれぞれの、最初の瞬間での濃度の間の比例定数であり;
Fは、F3モジュールの濃度であり;
aは、ATPの濃度であり;
K_Dは、平衡解離定数である］。V_ATPは、ATP濃度に対してプロットし、そしてK_Dを、実験データに対する理論曲線の非線形フィッティングにより計算した。 2.2 SPR analysis Binding analysis was performed using a BIAcoreX apparatus (Biosensor AB, Sweden) at 25 ° C using 10 mM sodium phosphate pH 7.4, 150 mM NaCl as a running buffer. The flow rate was 5 l / min. Data were analyzed by non-linear curve fitting using the manufacturer's software. FGFR1 Ig module 2-3 was immobilized on sensor chip CM5 using the amine coupling kit (Biosensor AB) as follows: 1) The two parts of the chip (designated as Fc1 and Fc2) were 2) Protein was immobilized on Fc1 with 12 l of 20 g / ml protein in 10 mM sodium phosphate buffer pH 6.0; 3) Fc1 and Fc2 were Blocked with 35 l blocking solution. The binding of various compounds to the immobilized FGFR1 module was investigated as follows: The compounds were injected simultaneously into Fc1 (with the immobilized FGFR1 module) and Fc2 (immobilized, nothing present). The curve showing non-specific binding of the compound to the surface of Fc2 was extracted from the curve showing the binding of the protein to the surface of the immobilized FGFR1 module and Fc1. The resulting curve was used for analysis. For ATP competition experiments, various compounds were preincubated with ATP for 10 minutes at specific concentrations. The affinity between ATP and the L1 F3 module was estimated from the following procedure: the initial binding rate (V ₀ ) of the 14 μM F3 module, and the first of the 14 μM F3 module preincubated with a specific concentration of ATP. The binding rate (V _ATP ) was determined. V _ATP can be calculated from the following formula:

[Where:
k ₂ and k ₁ are the proportionality constant between the initial binding rate and the concentration at the first instant of each of the ATP-F3 complex and F3 module;
F is the concentration of the F3 module;
a is the concentration of ATP;
_The K _D is the equilibrium dissociation constant. V _ATP is plotted against concentration of ATP, and the K _D, was calculated by nonlinear fitting of the theoretical curve to the experimental data.

2.3 Assay for determination of FGFR1 phosphorylation
TREX-293 cells (Invitrogen) were endowed by human FGFR1 (Dr. Lena Claesson-Welsh, Uppsala, Sweden) with a C-terminal StrepII-tag (IBA biotech, Germany) using the Flp-In system (Invitrogen). ) And stably transfected. For phosphorylation studies: ˜1 × 10 ⁶ TREX / FGFR1 cells were starved overnight before stimulation with specific compounds for 15 minutes. Cells were lysed in PBS containing 1% NP-40, Complete protease inhibitor (Roche, Switzerland) and phosphatase inhibitor cocktail set II (Calbiochem, USA). Removed cell lysates were analyzed for total protein content using the Pierce BCA assay (Pierce, USA) and an equal amount of protein was added to 20 μl of agarose-conjugated anti-phosphotyrosine antibody (4G10-AC, Upstate Biotechnologies, USA). And incubated at 4 ° C. for 3 hours. The bound protein was washed with lysis buffer (x1) and PBS (x2) before eluting with 150 mM phenylphosphate (Sigma, Germany). The eluted fraction of protein was precipitated with 12% trichloroacetic acid, washed with cold acetone, and dissolved in SDS-PAGE sample buffer. Immunoblotting was performed using an antibody against StrepII-tag (IBA Biotech).

2.4 Cerebellar granule neuron culture and immunostaining Neurons isolated from rat cerebellum (7-8 days old) were supplemented with 20 mM Hepes, 100 U / ml penicillin / streptomycin supplemented with B27 (Gibco) and certain compounds. 8-well Lab-Tek chamber slides (Nunc, 24%, 37 ° C, 5% CO ₂ in Neurobasal medium (Gibco) containing 1% glutamax, 25 mg / L pyruvate, 0.4% BSA (Sigma) for 24 hours. Germany) (10 ⁴ cells per well). After 24 hours, cells were fixed with paraformaldehyde and immunostained with primary antibody against GAP-43 (1: 1000) (Chemicon, USA), secondary Alexa Flour antibody (1: 700) (Molecular Probes, Netherlands). The length of neurites was measured by steric methods as described (Ronn et al., 2000).

3. Results
To investigate possible interactions between the L1 F3 module and FGFR1, we used the following recombinant proteins: bound NCAM Ig module I-II, bound L1 F3 module IV, FGFR Ig module II, and bound FGFR1 Ig Module II-III. NCAM and FGFR Ig modules are appropriately folded as determined by one-dimensional NMR analysis and can crystallize the extracellular portion of L1 (along with all Ig and F3 modules) expressed in the same procedure, so the L1 F3 module Folded appropriately (Kulahin et al., 2004).

3.1 Proof of direct interaction between L1 and FGFR
To determine whether the L1 F3 module can bind to FGFR1, we used SPR analysis. From FIG. 1, the recombinant protein consisting of L1 F3 module IV bound to the immobilized FGFR1 Ig module II-III, while the control protein consisting of NCAM Ig module I-II did not bind to the FGFR1 fragment (FIG. 1). The equilibrium dissociation constant (Kd) and the coefficients of association and dissociation were 3.246 ± 0.452 μM, 321 ± 65 M ⁻¹ s ⁻¹ and 1.09 ± 0.003 × 10 ⁻³ s ⁻¹ (mean ± standard deviation), respectively. . Thus, these data indicate that the L1 F3 module binds directly to FGFR1.

3.2 Binding of L1 to FGFR1 can be enhanced by ATP, GTP and AMP-PCP
It has been shown that the binding between NCAM and FGFR1 can be inhibited by ATP (Kiselyov et al., 2003). It was therefore of interest to understand whether ATP has the same effect on L1-FGFR1 binding. To investigate this, we used SPR analysis. As can be seen from FIGS. 2A and 3A, addition of ATP to 14 μM L1 F3 module IV promoted binding of the F3 module to FGFR1 Ig module II-III. Whether this effect is specific for ATP, AMP-PCP (non-hydrolyzed analog of ATP) (Figures 2B, 3A), AMP (Figures 2C, 3A), and GTP (Figures 2D, 3A) To investigate, the same setting was tested. GTP and AMP-PCP also increased the affinity of the interaction between the module and FGFR1, binding to the L1 F3 module, while AMP demonstrated insignificant inhibition of the binding (Figure 2C 3A). These results indicated that ATP, GTP and AMP-PCP binding to the L1 F3 module can alter the structure of the protein, thereby increasing the affinity between L1 and FGFR1. The Kd for the interaction between 14 μM L1 F3 module and FGFR1 in the presence of 2 mM ATP (GTP or AMP-PCP) ranges from 3.246 ± 0.452 μM to 1.246 ± 0.129 μM (0.944 ± 0.041 μM or 0.861 ± 0.032, respectively). μM) (FIG. 3A). By fitting the experimental data with a model in which ATP binds to L1F3 and alters the onset rate of binding, we estimated the equilibrium dissociation constant (Kd) of the interaction between the L1 F3 module and ATP (which is It appeared to be 0.388 ± 0.105 mM) (FIG. 3B). These experiments show that only trifphosphate nucleotides can promote L1-FGFR1 interaction.

3.3 FGFR1 is activated by the L1 F3 module
Since L1 F3 modules bind to FGFR1, they can be expected to induce FGFR1 activation in living cells. To investigate this, we used a method for detecting FGFR1 phosphorylation previously described by Kiselyov et al. (2003). TREX-293 cells were stably transfected with FGFR1 containing a C-terminal StrepII-tag and stimulated with L1 F3 modules at different concentrations. After stimulation, FGFR1 was immunopurified using a fixed amount of anti-phoshotyrosine antibody and then analyzed by immunoblotting using an antibody against StrepII-tag. As can be seen from FIG. 4, the L1 F3 module substantially reduced FGFR1 phosphorylation compared to non-stimulated cells and cells treated with bound NCAM Ig I-II module that did not bind to FGFR1 by SPR analysis. Increased (see FIG. 1). Maximum phosphorylation levels were achieved at a concentration of 10 mM L1 F3 module. This indicates that binding of the L1 F3 module to FGFR1 results in receptor activation.

  FGFR1 activation by the L1 F3 module stimulates neurite outgrowth and this stimulatory effect can be regulated by ATP.

  Since the L1 F3 module activates FGFR1, we have shown that they can mimic neuronal differentiation reflected by the characteristic function of L1: neurite outgrowth. To test this hypothesis, isolated cerebellar neurons were seeded on plastic and allowed to grow for 24 hours in the presence of the compounds described below. As can be seen from FIG. 5A, at a concentration of 4 mM, the L1 F3 module substantially increased neurite length compared to unstimulated neurons. The effect was quantified in a concentration response study, demonstrating that the F3 module increased neurite outgrowth with a bell curve typical of growth factor-induced neurite outgrowth (Hatten et al., 1988). The stimulatory effect of the F3 module can be completely abolished by inhibitors of FGFR1, SU5402 (FIG. 5B), further supporting the idea that the module interacts with FGFR1.

  Since ATP promoted L1-FGFR1 binding, we speculated that ATP could interfere with FGFR1 activation by the L1 F3 module and consequently affect the neurite promoting activity of the module. To examine this, neurons were stimulated with the L1 F3 module in the presence of ATP or AMP-PCP. As can be seen from FIGS. 5C and D, ATP and AMP-PCP are substantially activated by the F3 module when using a concentration of L1 F3 module (4 mM) that produces a maximal response in the presence of ATP. Reduced neurite generation effect (FIG. 5A). Complete inhibition of the effect of the F3 module is achieved with AMP-PCP at lower concentrations compared to the concentration of ATP, which is an inhibitor that ATP is less potent than its non-hydrolyzed analog It showed that. However, when the concentration of L1 F3 was lowered to 1 mM, the addition of ATP or AMP-PCP increased the protein's effect on neurite outgrowth (FIGS. 5E and F). It resulted in a higher ratio between bound to L1 and unbound FGFR1, as occurred when adding L1 F3 modules at higher concentrations, by adding ATP which increased the affinity between L1 and FGFR1 (FIG. 5A). Therefore, the concentration of F3 module should be lower than the most effective concentration, demonstrating the promotion of neurite outgrowth by the addition of ATP. These results indicate that activation of FGFR1 in neurons by L1 F3 module-induced neurite generation and this effect can be regulated by ATP.

3.4 Mapping of the binding site between the L1 F3 module and FGFR1 Since all L1 F3 modules comprise a longer protein than FGFR1, we were interested in identifying the portion of L1 involved in binding to FGFR1. It was. The secondary structure of L1 type 3 fibronectin consists of two betas, each containing seven antiparallel beta strands A, C, E, G and B, D, F, folded to form a beta sandwich. It consists of a sheet. Recently, it has been demonstrated that binding between NCAM F3 modules occurs in the region of NCMA corresponding to the loop between F3II F and G beta strands, close to the N-terminus of NCMA F3II module and the C-terminus of F3I module. (Kiselyov et al., 2003). Since NCAM and L1 are structurally homologous proteins, binding between L1 and FGFR1 has also been shown to occur in the loop region of the L1 F3 module, similar to the binding model between NCAM and FGFR1. . Therefore, all 30 regions of the L1 F3 module (FIG. 6) were produced as peptides and the binding between the peptides and FGFR1 was tested using SPR analysis. From FIG. 7A, between the C and D beta strands of the F3I module (F1CDL-peptide, APEKWFSLGKV (SEQ ID NO: 1)), between the B and C beta strands of the F3II module (F2BCL-peptide, DWNAPQIQYRVQWR (SEQ ID NO: 2)), and the F3III module Between A and B beta strands (F3ABL-peptide, DLAQVKGHLRGYN (SEQ ID NO: 3)), between C and D beta strands of F3III module (F3CDL-peptide, RHVHKSHMVVPAN (SEQ ID NO: 4)), B and C beta strands of F3V module The peptide corresponding to the loop region between (F5BCL-peptide, AA: RFHILFKALPEGKVSPD (SEQ ID NO: 5)) and between the F and G beta strands of the F3V module (F5FGL-peptide, AA: LHHLAVKTNGTG (SEQ ID NO: 6)) is FGFR1 It can be seen that it binds to Ig module II-III. Since both the IgII and IgIII modules of FGFR1 can be involved in the binding between L1 and FGFR1, we were interested in examining the binding of peptides to a single module of FGFR1. The same set of SPR experiments was used to demonstrate that 4 out of 6 peptides (F1CDL-, F2BCL-, 3CDL-, and 5BCL-peptide) bind to the IgII module of FGFR1 (Figure 7B). . These data demonstrate that there can be 6 binding sites in the L1 fibronectin module involved in the binding between L1 and FGFR1.

3.5 Peptides that bind to FGFR1 activate the receptor and stimulate neurite outgrowth
Since peptides showing several loop regions of the F3 module bound to FGFR1, it was proposed that they can activate FGFR1. The same method for investigating activation of FGFR1 by F3 was used for peptides to see if it was true. From FIG. 8, F1CDL, F2BCL, F3ABL, F5BCL, and F5FGL peptides are control peptides (F1BCL-peptide, as it did not bind to FGFR1 and did not show concentration-dependent phosphorylation of the receptor. It appears to significantly increase the phosphorylation level of the receptor compared to (used). At the same time, the F3CDL peptide demonstrated a rather small effect on the phosphorylation of FGFR1.

  Since FGFR1 was activated by peptides, we showed that they can mimic the properties of the L3 F3 module described above and stimulate neurite outgrowth. The same method was used to show that the F1CDL, F2BCL, F3ABL, F5BCL and F5FGL peptides (peptides that increase the phosphorylation level of FGFR1) stimulate neurite outgrowth, but the F3CDL and F1BCL peptides (the latter are FGFR1 Did not produce a neurite outgrowth response compared to the control (FIG. 9).

  Based on these data, we proposed a model of binding between the F1 module of L1 and FGFR1 (Figure 10; the dimer of FGFR1 was a model based on the crystal structure of FGFR1; Pellegrini et al., 2000). It may be considered whether the F3 module can have such a compact structure. Therefore, the possible structure of the protein was estimated based on gel filtration chromatography. From Figure 11, the L1 F3 module is slower than when gel filtration is performed with PBS plus 1 M NaCl, eluting from a Superdex 200 pg column with PBS, while the elution time for BSA is the same for both buffers. I understand what happened. These data indicate that the F3 module of L1 can have a relatively compact structure according to the binding model between L1 and FGFR1 described above.

4. Discussion We now provide the first direct evidence of the interaction between the neural cell adhesion molecule L1 and FGFR1. Furthermore, we demonstrate the regulatory role of ATP in this interaction.

  Using SPR analysis, we showed that L1 membrane proximity F3 module I-V binds to FGFR1 membrane proximity Ig module II-III with a Kd of 3.246 ± 0.452 μM. Proximity binding of FGFR1 and L1 was also demonstrated in living cells by immunoprecipitation.

  Stimulation of cells with the L1 F3 module increased FGFR1 phosphorylation. This raises the question of how the module induces FGFR1 activation. One explanation may be that FGFR1 has the ability to dimerize with very low affinity. This results in dynamic equilibrium, where small pieces of FGFR1 molecules temporarily form dimers, resulting in low background levels of FGFR1 phosphorylation. The FGFR1 binding of any ligand can change the dimerization slightly and shift the equilibrium for binding or dissociation. Therefore, we speculate that binding of the F3 module to FGFR1 may shift the equilibrium to binding, resulting in increased FGFR1 phosphorylation.

  In addition to binding and activating FGFR1, the F3 module can mimic the characteristic function of L1: neurite outgrowth. Stimulation of cells by the F3 module can be disrupted by the inhibitor of L1-stimulated neurite outgrowth, the FGFR1 inhibitor SU5402, which further supports the idea that the module interacts with FGFR1.

  Using SPR analysis, we observed that ATP promotes binding of the F3 module to the FGFR1 Ig module II-III. We have also demonstrated that ATP modulates the stimulatory effect of the F3 module on neurite outgrowth. This effect depends on the concentration of the L1 F3 module used to stimulate neurite outgrowth. When the protein concentration corresponds to that having the greatest effect on neurite outgrowth induction (FIG. 5), ATP acts inhibitory. On the other hand, ATP promotes the induction of neurite outgrowth at lower protein concentrations. This may indicate that if the ratio between bound and unbound FGFR1 to L1 is too high, abolishing the stimulatory effect of L1 on neurite outgrowth will occur according to a concentration response study of effects. Based on these data, we therefore show that ATP binding to L1 promotes L1-FGFR1 interaction and results in stimulation or inhibition of neurite outgrowth, depending on the actual concentration of L1. ing.

The experimentally determined affinity value for the L1-FGFR1 interaction (3 μM K _D ) appears to be low, but when the affinity of the interaction increases with the addition of ATP, the L1 F3 module-induced neuron Inhibition of process extension was observed, which means that the low affinity of the interaction between FGFR1 and L1 may be an intrinsic property of the cell adhesion molecules L1 and NCAM (the latter to approximately the same FGFR1 (Kiselyov et al., 2003).

  Although our studies reveal a regulatory role for ATP in previously unproven L1-mediated neurite outgrowth, a series of studies have shown that NCAM and other neural adhesion molecules have external ATPase activity, and ATP It has been shown to appear to inhibit NCAM binding to FGFR1 (Dzhandzhugazyan and Bock, 1997; Kiselyov et al., 2003).

  L1 is a very long molecule with 11 modules in the extracellular part. Using synthetic peptides that mimic the loop region of L1, we have demonstrated that a possible model for the binding between L1 and FGFR1 requires the compact structure of the F3 module. Along with other studies showing the horseshoe shape of the first Ig module of L1 (Su et al. 1998; Freigang et al. 2000), gel filtration experiments show that all 11 L1 modules have a compact form. Makes it possible to estimate

  Homogeneous interaction between L1 in the growth cone and L1 expressed in surrounding cells probably results in L1 clustering in the growth cone and then locally increases the ratio between L1 and FGFR1 concentrations Thus, providing an environment that stimulates axonal outgrowth. ATP is a neurotransmitter that is abundant in the CNS. Thus, when the growth cone reaches the target and a synaptic contact is formed, the release of ATP from the synapse can control the binding between L1 and FGFR1, and thus no longer needed newly formed synaptic contact Can affect, for example, inhibit axonal outgrowth. Conversely, ATP release can cause L1-FGFR1 interaction and subsequent signaling in regions with relatively low density of L1 molecules.

  Thus, our results provide evidence for a direct interaction between L1 and FGFR1, indicating that ATP is a regulator of L1-induced axonal elongation through control of the L1-FGFR1 interaction.

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Binding of L1 F3 module I-V to FGFR1 Ig module II-III. Binding was examined by means of SPR analysis. Approximately 3000 resonance units (RU) of the FGFR1 module were fixed to the sensor chip. Combined L1 F3 module I-V or combined NCAM Ig module I-II (ctrl) was injected into the sensor chip at specific concentrations. Binding is identified as the response difference between binding to a sensor chip with immobilized FGFR1 module and binding to a blank sensor chip (non-specific binding). Only very low non-specific binding was detected for the above proteins at all concentrations used in this study. 4-5 independent experiments were performed with different preparations of all proteins. Evidence of increased ATP-induced binding between FGFR1 Ig module II-III and L1 F3 module I-V. Binding was examined by means of SPR analysis. Approximately 3000 resonance units (RU) of the FGFR1 module were fixed to the sensor chip. Three independent experiments were performed for each concentration of ATP (AMP-PCP, AMP or GTP). A, B, C, D) Binding of 14 μM module I-V to FGFR1 module in the presence of different concentrations of ATP (A), AMP-PCP (B), AMP (C) and GTP (D). Demonstration of the effects of ATP, AMP-PCP, GTP, and AMP on the binding between FGFR1 Ig module II-III and L1 F3 module IV. A) ATP, AMP-PCP, GTP or prior to the addition of AMP (Ctr) and between the F3 modules and FGFR1 after addition equilibrium dissociation constant K _D. Plot of starting binding rate V ₀ versus ATP concentration for binding of F3 modules to B) FGFRl was used to calculate the equilibrium dissociation constant, K _D, of the interaction between F3 modules and ATP. Effect of L1 F3 module I-V on FGFR1 phosphorylation and immunoprecipitation of L1 by FGFR1. A) TREX / FLP-IN cells stably transfected with FGFR1 containing a C-terminal StrepII-tag, nothing (lane 1), 4 mM (lane 2), 10 mM (lane 3) and 30 mM ( Stimulated with L1 F3 module IV in lane 4) for 15 minutes. After stimulation, FGFR1 was immunopurified using an anti-phosphotyrosine antibody, and then analyzed by immunoblotting using an antibody against StrepII-tag. B) TREX / FLP-IN cells stably transfected with FGFR1 containing a C-terminal StrepII-tag were treated with 25 mM NCAM Ig module I-II (lane 1 as a control) and 10 mM L1 F3 module IV (lane 2). ) For 25 minutes. A total of nine experiments were performed. All experiments show phosphorylation of FGFR1 upon treatment with L1 F3 module I-V. Effect of L1 F3 module I-V on neurite outgrowth from cerebellar neurons. A) Neurite length versus F3 module IV concentration. B) Effect of SU5402, a FGFR1 inhibitor, on neurite outgrowth induced by 4 mM F3 module I-V. C, D) Neurons stimulated with 4 mM F3 module I-V in the presence of various concentrations of ATP (C) or AMP-PCP (D). E, F) Neurons stimulated with 1 mM F3 module I-V in the presence of various concentrations of ATP (E) or AMP-PCP (F). Four independent experiments were performed. Error bars represent one standard error of the mean. * And ** represent statistical significance of p <0.05 and p <0.01, respectively, compared to Ctr. +, ++ and +++ represent statistical significance of p <0.05, p <0.01 and p <0.001, respectively, compared to the maximum response. Sequence of peptide showing loop region of L1 F3 domain I-V. Thirty peptides representing all loops of the F3 I-V module were synthesized and used to map the binding site between L1 and FGFR1. The loop region of the F3 I-V module is shown along with the adjacent beta strand portion. Proof of binding between peptides derived from FGFR1 and L1 F3 modules I-V. Binding was examined by SPR analysis. Approximately 3000 resonance units (RU) of the FGFR1 module were fixed to the sensor chip. Three independent experiments were performed. A) Peptides that bind to FGFR1 module II-III (F1CDL- (8 mM), F2BCL- (8 mM), F3ABL- (40 mM), F3CDL- (2 mM), F5BCL- (80 mM), and F5FGL- (80 mM) peptides) . B) Peptides that bind to FGFR1 module II (F1CDL- (16 mM), F2BCL- (16 mM), F3CDL- (8 mM), and F5BCL- (130 mM) peptides). Effect of peptide bound to FGFR1 on phosphorylation of FGFR1. Phosphorylation level versus concentration of peptide binding to FGFR1. Four independent experiments were performed. Error bars represent one standard error of the mean. *, ** and *** represent statistical significance of p <0.05, p <0.01 and p <0.001, respectively, compared to PBS. +, ++ and +++ were p <0.05 and p <0.01 respectively compared to the negative control (F1BCL-peptide that did not bind to FGFR1 and did not show concentration-dependent phosphorylation of the receptor peptide) And represents the statistical significance of p <0.001. Effect of peptide bound to FGFR1 on neurite outgrowth from cerebellar neurons. Neurite length versus concentration of peptide bound to FGFR1. Four independent experiments were performed. Error bars represent the standard error of the mean. * And ** represent the statistical significance of p <0.05 and p <0.01, respectively, when compared to Ctr (PBS). Binding model between L1 and FGFR1. A possible compact structure of the L1 F3 module is proposed, where the marked region corresponds to a peptide that binds to FGFR1 and stimulates neurite outgrowth and receptor phosphorylation. The structure of the dimer of FGFR1 is obtained from the crystal structure of FGFR1 (Pellegrini et al., 2000). Gel filtration experiment demonstrating the possibility of a compact structure of L1 F3 domain I-V. A) Gel filtration of L1 F3 domain I-V using a Superdex 200 pg gel filtration column in PBS (blue) and in PBS + 1M NaCl (red). B) Gel filtration of BSA in PBS (blue) and in PBS + 1M NaCl (red).

Claims (59)

A compound capable of interacting with a fibroblast growth factor receptor (FGFR) comprising an amino acid sequence of 5 to 105 amino acid residues, having the formula:
_{^{x - - (x) n -x}} p - (x) n -x -
[Where:
x ^- is a basic amino acid residue,
x ^p is a hydrophobic amino acid residue, and
(x) _n is a sequence of any amino acid residue (where n is an integer from 0 to 3)]
A compound comprising the amino acid motif of   2. The compound according to claim 1, wherein the amino acid motif is an FGFR binding motif. The amino acid motif is the following amino acid motif:
KxxxLxK, RxxxLxK, KxLxxxK, HxLxxK, RxxWR, KxxLR, RxVxxH
(Where K, L, R, H, W and V correspond to amino acid residues K, L, R, H, W and V, and X is any amino acid residue)
The compound according to claim 1 or 2, which is any one of the following.   4. The compound according to claim 3, wherein at least one x is a basic amino acid residue, a hydrophilic amino acid residue or G.   5. The compound according to claim 4, wherein the basic amino acid residue is H.   5. The compound according to claim 4, wherein the hydrophilic amino acid residue is Q or S. i) the first type III fibronectin (Fn3,1) module of the neural cell adhesion molecule L1 or a fragment thereof, and / or
ii) a second type III fibronectin (Fn3,2) module of the neural cell adhesion molecule L1 or a fragment thereof, and / or
iii) a third type III fibronectin (Fn3,3) module of the neural cell adhesion molecule L1 or a fragment thereof, and / or
iv) the fourth type III fibronectin (Fn3,4) module of the neural cell adhesion molecule L1 or fragment thereof, and / or
The compound according to any one of claims 1 to 6, which comprises v) a fifth type III fibronectin (Fn3,5) module of the neural cell adhesion molecule L1 or a fragment thereof.   A fragment of L1 Fn3,1 and / or L1 Fn3,2 and / or L1 Fn3,3 and / or L1 Fn3,4 and / or L1 Fn3,5, wherein the fragment is claim 1 The compound according to any one of claims 1 to 7, comprising an amino acid sequence of 5 to 7 amino acid residues including the FGFR binding motif according to any one of -6.   9. A compound according to claim 8, wherein the fragment comprises 3 to 90 amino acid residues. APEKWFSLGKV (SEQ ID NO: 1),
DWNAPQIQYRYQWR (SEQ ID NO: 2),
DLAQVKGHLRGYN (SEQ ID NO: 3),
RHVHSHMVVPAN (SEQ ID NO: 4),
RFHILFKALPEGKVSPD (SEQ ID NO: 5) or
LHHLAVKTNGTG (SEQ ID NO: 6)
10. A compound according to any one of claims 1-9, comprising at least one amino acid sequence or fragment selected from   11. The compound according to claim 10, consisting of at least one amino acid sequence selected from the amino acid sequence of SEQ ID NOs: 1-6 or a fragment thereof or a variant thereof.   12. The compound according to claim 11, which consists of the amino acid sequence of SEQ ID NO: 1, or a fragment or variant thereof.   12. The compound according to claim 11, which consists of the amino acid sequence of SEQ ID NO: 2, or a fragment or variant thereof.   12. The compound according to claim 11, which consists of the amino acid sequence of SEQ ID NO: 3, or a fragment or variant thereof.   12. The compound according to claim 11, which consists of the amino acid sequence of column No. 4, or a fragment or variant thereof.   12. The compound according to claim 11, which consists of the amino acid sequence of SEQ ID NO: 5, or a fragment or variant thereof.   12. The compound according to claim 11, which consists of the amino acid sequence of SEQ ID NO: 6, or a fragment or variant thereof.   11. The compound of claim 10, comprising two or more amino acid sequences selected from the sequences of SEQ ID NOs: 1-6, or two or more fragments or variants of the sequences.   Including two identical copies of an amino acid sequence selected from the sequences of SEQ ID NOs: 1-6, or two identical fragments or variants of the selected sequence, wherein said amino acid sequence, fragments or variants 19. A compound according to claim 18.   The compound is four identical copies of an amino acid sequence selected from the sequences of SEQ ID NOs: 1-6, or four identical fragments or variants of the selected sequence, wherein said amino acid sequence, fragment or variant 19. The compound of claim 18 comprising:   Comprising two or more different amino acid sequences, wherein at least one of the two amino acid sequences is a sequence selected from the sequence of SEQ ID NOs: 1-6, or a fragment or variant thereof, Item 10. The compound according to Item 10.   19. A compound according to claim 18, comprising two different amino acid sequences, wherein both amino acid sequences are selected from the sequences SEQ ID NO: 1-6, or fragments or variants thereof.   23. A compound according to any one of claims 18-22, wherein the amino acid sequences are linked to each other via a linker group.   The variant according to any one of claims 10-22, wherein the variant is an amino acid sequence having at least 60% identity with a sequence selected from the sequences of SEQ ID NOs: 1-6 and capable of binding to FGFR. Compound.   The compound according to any one of claims 10-22, wherein the fragment comprises the FGFR-binding motif according to any one of claims 1-6.   FGFR is fibroblast growth factor receptor 1 (FGFR1), fibroblast growth factor receptor 2 (FGFR2), fibroblast growth factor receptor 3 (FGFR3), fibroblast growth factor receptor 4 (FGFR4) Or the compound of any one of claims 1-25, selected from fibroblast growth factor receptor 5 (FGFR5).   27. A compound according to any one of claims 1 to 26, capable of activating FGFR.   28. A compound according to claim 27, capable of stimulating FGFR signaling.   29. The compound of claim 28, wherein the compound stimulates FGFR1 signaling.   30. The compound of any one of claims 1-29, wherein the interaction between the compound and FGFR can be modulated by nucleotides.   32. The compound of claim 30, which promotes an interaction.   32. The compound of claim 31, wherein the nucleotide is a nucleotide triphosphate.   32. The compound of claim 30, which weakens the interaction.   34. The compound of claim 33, wherein the nucleotide is nucleotide monophosphate.   30. The compound of any one of claims 1-29, capable of stimulating neurite outgrowth.   30. The compound of any one of claims 1-29, which can stimulate cell survival.   30. The compound of any one of claims 1-29, capable of stimulating synaptic plasticity.   30. The compound of any one of claims 1-29, which can stimulate stem cell differentiation.   30. A compound according to any one of claims 1-29, capable of stimulating learning and / or memory.   40. A pharmaceutical composition comprising at least one compound as defined in any one of claims 1-39.   41. The pharmaceutical composition of claim 40, wherein the composition is formulated for oral, transdermal, intramuscular, intravenous, intracranial, intrathecal, intraventricular, nasal or intrapulmonary administration.   42. The pharmaceutical composition according to claim 41, wherein administration is continuous.   An effective amount of one or more of the compounds according to any one of claims 1-39 or a pharmaceutical composition according to any one of the claims 40-42 to one or more pharmaceuticals. A process for producing a pharmaceutical composition comprising mixing with a pharmaceutically acceptable additive or carrier.   44. A method according to claim 43, wherein the compound as defined in any one of claims 1-39 is used in combination with a prosthesis.   45. The method of claim 44, wherein the device is a nerve guide.   The nerve guide comprises at least one compound as defined in any one of claims 1-39 or a pharmaceutical composition as defined in any one of claims 40-43. 45. The method according to 45.   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 effective for recovery from the disease or condition, 40. Use of a compound according to any one of claims 1 to 39.   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, 48. Use according to claim 47, 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.   The medicament is intended for the treatment of muscular diseases or conditions, including those with impaired function of nerve-muscle connection, or for the treatment of gonad, pancreatic or renal diseases or degenerative conditions. Use as described in 47.   48. Use according to claim 47, wherein the medicament prevents cell death of cardiomyocytes.   48. Use according to claim 47, wherein the medicament is for revascularization.   48. Use according to claim 47, wherein the medicament is for promoting wound healing.   48. Use according to claim 47, wherein the compound and / or pharmaceutical composition is capable of inhibiting angiogenesis.   54. Use according to claim 47 or 53, wherein the medicament is for the treatment of cancer.   48. Use according to claim 47, wherein the medicament is intended for stimulating the ability to learn and / or short-term and / or long-term memory.   48. Use according to claim 47, wherein the medicament is capable of modulating cell proliferation and / or differentiation and / or regeneration and / or morphological plasticity.   An antibody capable of recognizing and binding to an epitope comprising an amino acid sequence selected from SEQ ID NOs: 1-6.   40. A method of treating a condition or disease in which activating FGFR is effective for treatment to an individual in need thereof, the compound of any one of claims 1-39 or claim 44. A method comprising administering the pharmaceutical composition of any one of 40-43.   59. The method of claim 58, wherein the condition or disease is as defined in any one of claims 47-56.

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