U.S. patent application number 10/539440 was filed with the patent office on 2006-12-14 for metod of modulation of interaction between receptor and ligand.
Invention is credited to Morten Albrechtsen, Vladimir Berezin, Elisabeth Bock.
Application Number | 20060281130 10/539440 |
Document ID | / |
Family ID | 32683717 |
Filed Date | 2006-12-14 |
United States Patent
Application |
20060281130 |
Kind Code |
A1 |
Bock; Elisabeth ; et
al. |
December 14, 2006 |
Metod of modulation of interaction between receptor and ligand
Abstract
The present invention relates to a method for modulating the
interaction between at least two proteins, wherein at least one of
the two proteins is a functional cell-surface receptor and the
other protein is the receptor ligand. The invention features a
binding site of said functional cell-surface receptor on the
receptor ligand and discloses a series of amino acid sequences,
which are part of the structure of said binding site and/or
involved in the interaction between the receptor and the ligand.
Moreover, the present invention features methods for molecular
design and screening of a candidate compound capable of modulating
the interaction between the functional cell-surface receptor and
receptor ligand through the described binding site, and provides a
screening assay for identification of such a compound. The
invention further describes an antibody capable of binding to the
above binding site and/or to an epitope comprising an amino acid
sequence essential for executing the receptor ligand interaction
through said binding site. The invention also concerns a variety of
uses of the disclosed methods, peptide sequences and antibodies.
The invention in preferred embodiments concerns the binding site of
the fibroblast growth factor receptor (FGFR) on FGFR ligands,
compounds capable of modulating the receptor ligand interaction
through said binding site, and antibody capable of recognition of
said binding site.
Inventors: |
Bock; Elisabeth;
(Copenhagen, DK) ; Berezin; Vladimir; (Copenhagen,
DK) ; Albrechtsen; Morten; (Charlottenlund,
DK) |
Correspondence
Address: |
BROWDY AND NEIMARK, P.L.L.C.;624 NINTH STREET, NW
SUITE 300
WASHINGTON
DC
20001-5303
US
|
Family ID: |
32683717 |
Appl. No.: |
10/539440 |
Filed: |
December 18, 2003 |
PCT Filed: |
December 18, 2003 |
PCT NO: |
PCT/DK03/00901 |
371 Date: |
June 20, 2005 |
Current U.S.
Class: |
435/7.1 ;
424/144.1; 435/7.23; 514/13.3; 514/17.2; 514/17.7; 514/19.1;
514/19.3; 514/56; 514/8.3; 514/9.1; 514/9.3; 514/9.4; 530/326;
530/388.22 |
Current CPC
Class: |
C07K 7/06 20130101; A61P
39/00 20180101; A61P 13/12 20180101; A61P 1/16 20180101; A61P 7/02
20180101; A61P 25/00 20180101; G01N 33/74 20130101; A61P 25/30
20180101; A61P 25/16 20180101; A61P 43/00 20180101; A61K 38/00
20130101; A61P 9/10 20180101; A61P 25/08 20180101; A61P 15/04
20180101; A61P 17/02 20180101; A61P 31/00 20180101; A61P 41/00
20180101; A61P 25/28 20180101; A61P 19/00 20180101; A61P 25/18
20180101; A61P 9/06 20180101; A61P 27/02 20180101; A61P 31/04
20180101; C07K 16/22 20130101; A61P 9/00 20180101; A61P 35/00
20180101; A61P 25/14 20180101; A61P 3/10 20180101; A61P 21/00
20180101; G01N 33/566 20130101; A61P 11/00 20180101; G01N 2333/50
20130101; A61P 25/02 20180101; A61P 17/00 20180101; A61P 9/14
20180101; C07K 7/08 20130101 |
Class at
Publication: |
435/007.1 ;
514/012; 514/056; 435/007.23; 514/013; 514/014; 530/326;
530/388.22; 424/144.1 |
International
Class: |
G01N 33/53 20060101
G01N033/53; G01N 33/574 20060101 G01N033/574; A61K 38/17 20060101
A61K038/17; A61K 38/10 20060101 A61K038/10; A61K 39/395 20060101
A61K039/395; A61K 31/727 20060101 A61K031/727; C07K 7/08 20060101
C07K007/08 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 20, 2002 |
DK |
PA 2002 01982 |
Mar 3, 2003 |
DK |
PA 2003 00330 |
Claims
1. A method of modulating the interaction between a functional
cell-surface fibroblast growth factor receptor, or a variant
thereof, and a polypeptide having a binding site to said receptor,
wherein said binding site comprises at least one of the sequences
set forth in SEQ ID NOS: 2-146, said method comprising i) providing
a compound capable of interacting with the receptor at the binding
site of the receptor for the polypeptide; ii) presenting the
compound of step (i) to the the receptor and the polypeptide.
2-3. (canceled)
4. The method according to claim 1, wherein the cell-surface
receptor is selected from the family of fibroblast growth factor
receptors (FGFRs) comprising FGFR1, FGFR2, FGFR3 and FGFR4
5. The method according to claim 1, wherein the receptor is
FGFR1.
6-7. (canceled)
8. The method according to claim 1, wherein the polypeptide is a
cell adhesion molecule which is selected from the group consisting
of Neural Cell Adhesion Molecule (NCAM), Neural cell adhesion
molecule L1, Neural Cell Adhesion Molecule-2 (NCAM-2) Neuron-glia
Cell Adhesion Molecule (Ng-CAM), Neural cell adhesion molecule
CALL, Neuroglian, Nr-CAM (HBRAVO, NRCAM, NR-CAM 12) Axonin-1/TAG-1,
Myelin-Associated Glycoprotein (MAG), Neural cell adhesion molecule
BIG-1, Neural cell adhesion molecule BIG-2, Fasciclin (FAS-2)
Neural cell adhesion molecule HNB-3/NB-3 Neural cell adhesion
molecule HNB-2/NB-2, Cadherin, Junctional Adhesion Molecule-1
(JAM-1), Neural cell adhesion F3/F11(Contactin), Neurofascin,
B-lymphocyte cell adhesion molecule CD22, Neogenin (NEO1),
Intercellular Cell Adhesion Molecule-5 (ICAM-5/telencephalin)
Galactose binding lectin-12 (galectin-12), Galactose binding
lectin-4 (galectin-4)
9. The method according to claim 1, wherein the polypeptide is a
functional cell-surface receptor which is selected from the group
consisting of Neurotrophin Tyrosin Kinase Type-2 (NTRKT-2),
Leukocyte Antigen Related Protein-Tyrosine Phosphatase (LAR-PTPRF)
Nephrin, Protein-Tyrosine Phosphatase Receptor type S (PTPRS)
Protein-Tyrosine Phosphatase Receptor type kappa (R-PTP-kappa),
Protein-Tyrosine Phosphatase Receptor type D (PTPRD), Ephrin type-A
receptor 8 (EPHA8/Tyrosine-Protein Kinase Receptor EEK) Ephrin
type-A receptor 3 (EPHA8/Tyrosine-Protein Kinase Receptor
ETK-1/CEK4), Ephrin type-A receptor 2 Insulin Receptor (IR)
Insulin-like Growth Factor-1 Receptor (IGF-1) Insulin-related
Receptor (IRR), Tyrosine-Protein Kinase Receptor Tie-1, Roundabout
receptor-1 (robo-1), Neuronal nicotinic acetylcholine receptor
alpha 3 subunit (CHRNA3) Neuronal acetylcholine receptor alpha 6
subunit Platelet-Derived Growth Factor Receptor Beta (PDGFRB)
Interleukin-6 Receptor (IL-6R), Interleukin-23 Receptor (IL-23R),
Beta-common cytokine receptor of IL-3, IL5 and GmCsf Cytokine
Receptor-Like molecule 3 (CRLF1), Class I Cytokine Receptor
(ZCYTOR5) Netrin-1 receptor DCC, Leukocyte Fc Receptor-like Protein
(IFGP2), Macrophage Scavenger Receptor 2 (MSR2) and Granulocyte
Colony Stimulating Factor Receptor (G-CSF-R).
10. The method according to claim 1, wherein the polypeptide is
perlecan.
11. The method according to, claim 1, wherein the polypeptide is a
metalloprotease is selected from the group consisting of A
disintegrin and metalloprotease-8 (ADAM-8) A disintegrin and
metalloprotease-19 (ADAM-19) A disintegrin and metalloprotease-12
(ADAM-12) A disintegrin and metalloprotease-28 (ADAM-28) A
disintegrin and metalloprotease-33 (ADAM-33) precursor, A
disintegrin and metalloprotease-9 (ADAM-9), A disintegrin and
metalloprotease-7 (ADAM-7), A disintegrin and metalloprotease-1A
(ADAM-1A) Fertilin alpha, A disintegrin and metalloprotease-15
(ADAM-15) Metalloproteinase-desintegrin domain containing protein
(TECAM), and Metalloproteinase 1.
12. The method according to claim 1, wherein the polypeptide is an
extracellular matrix molecule which is selected from the group
consisting of Collagen type VII, Fibronectin and Tenascin-R.
13. The method according to claim 1, wherein the polypeptide is
Cytokine-like factor-1 (CLF-1).
14. The method according to claim 1, wherein the interaction
between the receptor and polypeptide is a low affinity
interaction.
15. The method according to claim 14, wherein the affinity of
interaction is within the range of Kd 10.sup.-3-10.sup.-11 M, such
as within the range Kd 10.sup.-5-10.sup.-8.
16. (canceled)
17. The method according to claim 1, wherein the compound is a
peptide.
18. The method of claim 17, wherein the peptide comprises 6 to 16
contiguous amino acid residues and consists of a sequence selected
from any of the amino acid sequences set forth on SEQ ID NOS: 1-10,
100, or 125, or comprises a fragment of said sequence.
19. (canceled)
20. The method of claim 17, wherein the peptide comprises 6 to 16
contiguous amino acid residues and consists of a sequence selected
from any of the amino acid sequences set forth in SEQ ID NOS:
11-99, 101-124, or 126-146, or comprises a fragment of said
sequence.
21-24. (canceled)
25. A screening method for a compound capable of modulating
interaction between a functional cell-surface fibroblast growth
factor receptor or a variant thereof, and a polypeptide having a
binding site to said receptor, wherein said binding site comprises
at least one of the sequences set forth in SEQ ID NOS: 2-146, said
method comprising i) providing the polypeptides of a fibroblast
growth factor receptor (FGFR) and the neural cell adhesion molecule
(NCAM); ii) providing a candidate compound which is designed by
using structural data on the binding site of NCAM with FGFR; iii)
presenting the candidate compound of (ii) to the polypeptides of
(i); iv) determining the interaction between NCAM and FGFR before
and after the presenting of the candidate compound to said
polypeptides; v) determining whether the interaction between FGFR
and NCAM is modulated by the candidate compound, vi) selecting a
compound capable of modulating the interaction between FGFR and a
polypeptide having a binding site to said receptor, wherein said
binding site comprises at least one of the sequences set forth in
SEQ ID NOS:2-146.
26. The screening method according to claim 25, wherein FGFR is
selected from FGFR1, FGFR2, FGFR3 or FGFR4.
27. The screening method according to claim 25, wherein the
polypeptide having a binding site for the cell-surface receptor,
wherein said binding site comprises at least one of the sequences
set forth in SEQ ID NOS:2-146, is selected from the group
consisting of Neural cell adhesion molecule L1, Neural Cell
Adhesion Molecule-2 (NCAM-2), Neuron-glia Cell Adhesion Molecule
(Ng-CAM), Neural cell adhesion molecule CALL, Neuroglian, Nr-CAM,
Axonin-1/TAG-1, Axonal-associated Cell Adhesion Molecule,
Myelin-Associated Glycoprotein (MAG), Neural cell adhesion molecule
BIG-1, Neural cell adhesion molecule BIG-2, Fasciclin, Neural cell
adhesion molecule HNB-3/NB-3, Neural cell adhesion molecule
HNB-2/NB-2, Cadherin, Junctional Adhesion Molecule-1 (JAM-1) Neural
cell adhesion F3/F11(Contactin), Neurofascin, B-lymphocyte cell
adhesion molecule CD22, Neogenin (NEO1), Intercellular Cell
Adhesion Molecule-5 (ICAM-5/telencephalin), Galactose binding
lectin-12 (galectin-12), Galactose binding lectin-4 (galectin-4),
Neurotrophin Tyrosin Kinase Type-2 (NTRKT-2), Leukocyte Antigen
Related Protein-Tyrosine Phosphatase (LAR-PTPRF), Nephrin,
Protein-Tyrosine Phosphatase Receptor type S (PTPRS),
Protein-Tyrosine Phosphatase Receptor type kappa (R-PTP-kappa),
Protein-Tyrosine Phosphatase Receptor type D (PTPRD), Ephrin type-A
receptor 8, Ephrin type-A receptor 3, Ephrin type-A receptor 2,
Insulin Receptor (IR), Insulin-like Growth Factor-1 Receptor
(IGF-1), Insulin-related Receptor (IRR), Tyrosine-Protein Kinase
Receptor Tie-1 Roundabout receptor-1 (robo-1), Neuronal nicotinic
acetylcholine receptor alpha 3 subunit (CHRNA3), Neuronal
acetylcholine receptor alpha 6 subunit, Platelet-Derived Growth
Factor Receptor Beta (PDGFRB), Interleukin-6 Receptor (IL-6R),
Interleukin-23 Receptor (IL-23R), Beta-common cytokine receptor of
IL-3, IL5 and GmCsf, Cytokine Receptor-Like molecule 3 (CRLF1),
Class I Cytokine Receptor (ZCYTOR5), Netrin-1 receptor DCC,
Leukocyte Fc Receptor-like Protein (IFGP2), Macrophage Scavenger
Receptor 2 (MSR2), Granulocyte Colony Stimulating Factor Receptor,
Perlecan, A disintegrin and metalloprotease-8 (ADAM-8), A
disintegrin and metalloprotease-19 (ADAM-19), A disintegrin and
metalloprotease-12 (ADAM-12), A disintegrin and metalloprotease-28
(ADAM-28), A disintegrin and metalloprotease-33 (ADAM-33)
precursor, A disintegrin and metalloprotease-9 (ADAM-9), A
disintegrin and metalloprotease-7 (ADAM-7), A disintegrin and
metalloprotease-1A (ADAM-1A Fertilin alpha), A disintegrin and
metalloprotease-15 (ADAM-15), Metalloproteinase-desintegrin domain
containing protein (TECAM), Metalloproteinase 1, Collagen type VII,
Fibronectin, Tenascin-R, or Cytokine-like factor-1 (CLF-1).
28. The screening method according to claim 25, wherein the
polypeptide, is NCAM.
29. The screening method according to claim 25, wherein the
compound is for the manufacture of a medicament for the treatment
of normal, degenerated or damaged NCAM presenting cells.
30. The screening method according to claim 25, wherein the
compound is for the manufacture of a medicament treatment of
diseases and conditions of the central and peripheral nervous
system, or of the muscles or of various organs.
31. The screening method according to claim 25, wherein the
compound is for the manufacture of a medicament for the treatment
of diseases or conditions of the central and peripheral nervous
system, such as postoperative nerve damage, traumatic nerve damage,
impaired myelination of nerve fibers, postischaemic damage, e.g.
resulting from a stroke, Parkinson's disease, Alzheimer's disease,
Huntington's disease, dementias such as multiinfarct dementia,
sclerosis, nerve degeneration associated with diabetes mellitus,
disorders affecting the circadian clock or neuro-muscular
transmission, and schizophrenia, mood disorders, such as manic
depression; for treatment of diseases or conditions of the muscles
including conditions with impaired function of neuro-muscular
connections, such as after organ transplantation, or such as
genetic or traumatic atrophic muscle disorders; or for treatment of
diseases or conditions of various organs, such as degenerative
conditions of the gonads, of the pancreas such as diabetes mellitus
type I and II, of the kidney such as nephrosis and of the heart,
liver and bowel.
32. The screening method according to claim 25, wherein the
compound is for the manufacture of a medicament for the treatment
of postoperative nerve damage, traumatic nerve damage, impaired
myelination of nerve fibers, postischaemic, e.g. resulting from a
stroke, Parkinson's disease, Alzheimer's disease, Huntington's
disease, dementias such as multiinfarct dementia, sclerosis, nerve
degeneration associated with diabetes mellitus, disorders affecting
the circadian clock or neuro-muscular transmission, and
schizophrenia, mood disorders, such as manic depression.
33. The screening method according to claim 25, wherein the
compound is for the manufacture of a medicament for the promotion
of wound-healing.
34. The screening method according to claim 25, wherein the
compound is for the manufacture of a medicament for the treatment
of cancer.
35. The screening method according to claim 31, wherein the cancer
is any type of solid tumors requiring neoangiogenesis.
36. The screening method according to claim 25, wherein the first
compound is for the manufacture of a medicament for the prevention
of cell death of heart muscle cells, such as after acute myocardial
infarction, or after angiogenesis.
37. The screening method according to claim 25, wherein the
compound is for the manufacture of a medicament for
revascularsation.
38. The screening method according to claim 25, wherein the
compound is for the manufacture of a medicament for the stimulation
of the ability to learn and/or the short and/or long-term
memory.
39. An assay for sequential screening of a candidate compound for
the capability of modulating the interaction between a functional
cell-surface fibroblast growth factor receptor (FGFR) or a variant
thereof, and a polypeptide having a binding site to said receptor,
wherein said binding site comprises at least one of the sequences
set forth in SEQ ID NOS: 2-146, said method comprising i) providing
at least one functional FGFR molecule, and a molecule of at least
one polypeptide having a binding site to said receptor, wherein
said binding site comprises at least one of the sequences set forth
in SEQ ID NOS: 2-146, ii) presenting the at least one receptor
molecule of step (i) to the at least one polypeptide of step (i),
or presenting the at least one polypeptide of step (i) to the at
least one receptor molecule of step (i) and permitting the
interaction between the said receptor and said polypeptide, iii)
recording the interaction between the molecules of step (ii), iv)
presenting a candidate compound to the molecules of step (ii); v)
recording the interaction between the molecules of step (iv), vi)
assessing at least one effect of the candidate compound on the
interaction between the molecules of step (iv), vii) selecting a
compound capable of modulating interaction between receptor and
polypeptide of step (i).
40. The assay according to claim 39, wherein step (vii) is followed
by the steps of viii) presenting the selected on step (vii)
candidate compound to at least one cell presenting at least one
FGFR molecule or a variant thereof, and the at least one
polypeptide having a binding site to said receptor, wherein said
binding site comprises at least one of the sequences set forth in
SEQ ID NOS: 2-146, and ix) assessing at least one effect of the
compound on the cell of step (viii).
41. The assay according to claim 39, wherein the recording of
interaction between the molecules on step (iii) or step (v), and
the assessment of the at least one effect of the candidate compound
on step (vi) is achieved by using a method selected from surface
plasmon resonance analysis, nucleic magnetic resonance
spectroscopy, sedimentation, immunoprecipitation, two-hybrid
system, or resonance energy transfer analysis.
42. The assay according to claim 40, wherein the at least one
effect of step (ix) is selected from stimulation/inhibition of
receptor phosphorylation, intracellular signal transduction, gene
expression, cellular adhesion, cell motility, neuritogenesis,
apoptosis, cell proliferation or synaptic plasticity.
43. A method for molecular design for a compound capable of
modulating the interaction between fibroblast growth factor
receptor and a polypeptide having a binding site for said receptor,
wherein said binding site comprises at least one of the sequences
set forth in SEQ ID NOS: 2-146, comprising using structural data on
the binding site of NCAM with FGFR.
44. (canceled)
45. A peptide fragment consisting of an amino acid sequence
selected from the group consisting of the following amino acid
sequences: TABLE-US-00002 NIEVWVEAENALGKKV, (SEQ ID NO: 2)
ATNRQGKVKAFAHL, (SEQ ID NO: 3) RYVELYVVADSQEFQK, (SEQ ID NO: 4)
VAENSRGKNVAKG, (SEQ ID NO: 5) GEYWCVAENQYGQR, (SEQ ID NO: 6)
RLAALNGKGLGEIS, (SEQ ID NO: 7) KYIAENMKAQNVAKEI, (SEQ ID NO: 8)
TIMGLKPETRYAVR, (SEQ ID NO: 9) KGLGEISAATEFKT, (SEQ ID NO: 10)
NMGIWVQAENALG, (SEQ ID NO: 11) IWVQAENMLG, (SEQ ID NO: 12)
EIWVEATNRLG, (SEQ ID NO: 13) VWVQAANALG, (SEQ ID NO: 14)
EVWIEKDPAKGRI, (SEQ ID NO: 15) ATNKGGEVKKNGHL, (SEQ ID NO: 16)
KYVELYLVADYLEFQK, (SEQ ID NO: 17) RYVELYVVVDNAEFQ, (SEQ ID NO: 18)
KYVELVIVADNREFQR, (SEQ ID NO: 19) KYIEYYLVLDNGEFKR, (SEQ ID NO: 20)
RYLELYIVADHTLF, (SEQ ID NO: 21) KYVEMFVVVNHQRFQ, (SEQ ID NO: 22)
RYVELFIVVDKERY, (SEQ ID NO: 23) KYVELFIVADDTVYRR, (SEQ ID NO: 24)
KFIELFVVADEYVYRR, (SEQ ID NO: 25) KIVEKVIVADNSEVRK, (SEQ ID NO: 26)
VELVIVADHSEAQK, (SEQ ID NO: 27) VAENSRGKNIAKG, (SEQ ID NO: 28)
IAENSRGKNVARG, (SEQ ID NO: 29) AENSRGKNSFRG, (SEQ ID NO: 30)
IASNLRGRNLAKG, (SEQ ID NO: 31) IPENSLGKTYAKG, (SEQ ID NO: 32)
IAENMKAQNEAK, (SEQ ID NO: 33) QFIAENMKSHNETKEV, (SEQ ID NO: 34)
GEYWCVAKNRVGQ, (SEQ ID NO: 35) GSYTCVAENMVGK, (SEQ ID NO: 36)
GKYVCVGTNMVGER, (SEQ ID NO: 37) GNYTCVVENEYG, (SEQ ID NO: 38)
GEYTCLAGNSIG, (SEQ ID NO: 39) QYYCVAENGYG, (SEQ ID NO: 40)
GEYYQEAEQNGYG, (SEQ ID NO: 41) GNYTCLVENEYG, (SEQ ID NO: 42)
GMYQCLAENAYG, (SEQ ID NO: 43) GMYQCAENTHG, (SEQ ID NO: 44)
GIYYCLASNNYG, (SEQ ID NO: 45) GGYYCTADNSYG, (SEQ ID NO: 46)
GEYQCFARNDYG, (SEQ ID NO: 47) GEYFCLASNKMG, (SEQ ID NO: 48)
GEYQCFARNKFG, (SEQ ID NO: 49) GEYFCLASNKMG, (SEQ ID NO: 50)
GGYYCTADNNYG, (SEQ ID NO: 51) GNYSCEAENAWGTK, (SEQ ID NO: 52)
GEYTCLAENSLG, (SEQ ID NO: 53) GEYECVAENGRLG, (SEQ ID NO: 54)
GNYTCVVENKFGR, (SEQ ID NO: 55) GEYTCLAGNSIG, (SEQ ID NO: 56)
GEYFCVASNPIG, (SEQ ID NO: 57) EYTCIANNQAGE, (SEQ ID NO: 58)
GMYQCVAENKHLG, (SEQ ID NO: 59) GEYMCTASNTIGQ, (SEQ ID NO: 60)
EYVCIAENKAGEQ, (SEQ ID NO: 61) GDYTLIAKNEYGK, (SEQ ID NO: 62)
GFYQCVAENEAG, (SEQ ID NO: 63) GKYECVATNSAGTR, (SEQ ID NO: 64)
GEYFCVYNNSLG, (SEQ ID NO: 65) GEYECAATNAHGR, (SEQ ID NO: 66)
GAYWCQGTNSVGK, (SEQ ID NO: 67) GTYSCVAENILG, (SEQ ID NO: 68)
RVAAVNGKGQGDYS, (SEQ ID NO: 69) RVAAINGCGIGPFS, (SEQ ID NO: 70)
AVLNGKGLG, (SEQ ID NO: 71) ALNGQGLGATS, (SEQ ID NO: 72)
RLAAKNRAGLGE, (SEQ ID NO: 73) RLGVVTGKDLGEI, (SEQ ID NO: 74)
TVTGLKPETSYMVK, (SEQ ID NO: 75) TLTGLKPSTRYRI, (SEQ ID NO: 76)
TLTGLQPSTRYRV, (SEQ ID NO: 77) TLLGLKPDTTYDIK, (SEQ ID NO: 78)
TLQGLRPETAYELR, (SEQ ID NO: 79) TLRGLRPETAYELR, (SEQ ID NO: 80)
TLMNLRPKTGYSVR, (SEQ ID NO: 81) TVSGLKPGTRY, (SEQ ID NO: 82)
TISGLKPDTTY, (SEQ ID NO: 83) TLQGLKPDTAY, (SEQ ID NO: 84)
LRGLKPWTQYAV, (SEQ ID NO: 85) IDGLEPDTEYIVR, (SEQ ID NO: 86)
LQGLKPWTQYAI, (SEQ ID NO: 87) TITGLEPGTEYTIQ, (SEQ ID NO: 88)
GLKPWTQYAV, (SEQ ID NO: 89) TLASLKPWTQYAV, (SEQ ID NO: 90)
LMGLQPATEYIV, (SEQ ID NO: 91) KGMGPMSEAVQFRT, (SEQ ID NO: 92)
TLTGLKPDTTYDVK, (SEQ ID NO: 93) ISGLQPETSYSL, (SEQ ID NO: 94)
TLLGLKPDTTYDIK, (SEQ ID NO: 95) TISGLTPETTYSI, (SEQ ID NO: 96)
GNYSCLAENRLGR, (SEQ ID NO: 97) GNYTCVVENRVG, (SEQ ID NO: 98)
GTYHCVATNAHG, (SEQ ID NO: 99) LSHNGVLTGYLLSY, (SEQ ID NO: 100)
NGVLTGYVLRY, (SEQ ID NO: 101) NGVLTGYNLRY, (SEQ ID NO: 102)
NGNLTGYLLQY, (SEQ ID NO: 103) VDENGVLTGYKIYY, (SEQ ID NO: 104)
THNGALVGYSVRY, (SEQ ID NO: 105) NGILTEYILKY, (SEQ ID NO: 106)
NGILIGYTLRY, (SEQ ID NO: 107) THSGQITGYKIRY, (SEQ ID NO: 108)
NGKITGYIIYY, (SEQ ID NO: 109) LSHNGIFTLY, (SEQ ID NO: 110)
NGILTEYTLKY, (SEQ ID NO: 111) LDPNGIITQYEISY, (SEQ ID NO: 112)
NGKITGYIIYY, (SEQ ID NO: 113) HLEVQAFNGRGSGPA, (SEQ ID NO: 114)
HLTVRAYNGAGYGP, (SEQ ID NO: 115) HLSVKAYNSAGTGPS, (SEQ ID NO: 116)
HLAVKAYNSAGTGPS, (SEQ ID NO: 117) NLEVRAFNSAGDGP, (SEQ ID NO: 118)
HLTVLAYNSKGAGP, (SEQ ID NO: 119) LRVLVFNGRGDGP, (SEQ ID NO: 120)
HIDVSAFNSAGYGP, (SEQ ID NO: 121) HLAVELFNGR, (SEQ ID NO: 122)
LELQSINFLGGQPA, (SEQ ID NO: 123) HFTVRAYNGAGYGP, (SEQ ID NO: 124)
HLEVQAFNGRGSQPA, (SEQ ID NO: 125)
VIADQPTFVKYLIK, (SEQ ID NO: 126) TIKGLRPGVVYEGQ, (SEQ ID NO: 127)
TLTELSPSTQYTVK, (SEQ ID NO: 128) TLDDLAPDTTYLVQ, (SEQ ID NO: 129)
TVSDVTPHAIYTVR, (SEQ ID NO: 130) IIRGLNASTRYLFR, (SEQ ID NO: 131)
TLMNLRPKTGYSVR, (SEQ ID NO: 132) TLTGLKPGTEYEVR, (SEQ ID NO: 133)
GPEHLMPSSTYVAR, (SEQ ID NO: 134) RVTGLTPKKTYEFR, (SEQ ID NO: 135)
LTGLKPGTEYEFR, (SEQ ID NO: 136) EVRVQAVNGGGNGPP, (SEQ ID NO: 137)
LIKVVAINDRGE, (SEQ ID NO: 138) VVSIIAVNGREE, (SEQ ID NO: 139)
VVSVYAQNQNGE, (SEQ ID NO: 140) TISLVAEKGRHK, (SEQ ID NO: 141)
HLEVQAFNGRGSGPA, (SEQ ID NO: 142) HVEVQAFNGRGLGPA, (SEQ ID NO: 143)
HVEVQAFNGRGLGPA, (SEQ ID NO: 144) EFRVPAVNGAGEG, (SEQ ID NO: 145)
or VARVRTRLAPGSRLS, (SEQ ID NO: 146)
or a fragment, or variant of said sequence, herein said amino acid
sequence is isolated by a method according to claim 25 or a method
of claim 43.
46. A compound comprising at least one peptide fragment consisting
of at least one of the sequences set forth in SEQ ID NO: 2-146 or a
fragment, or variant, or homologue of said sequences.
47. (canceled)
48. An antibody capable of binding to an epitope comprising a
binding site to a cell surface receptor, wherein said binding site
comprises at least one of the sequences set forth in SEQ ID NOS:
1-146, or a fragment or a variant of said antibody.
49. An antibody capable of binding to an epitope comprising at
least one of the sequences set forth in SEQ ID NOS: 1-146, or a
fragment, or a variant of said antibody.
50-54. (canceled)
55. A method for treating an individual in need, wherein said
treatment comprising using a peptide fragment as defined in claim
45.
56. A method for treatment an individual in need, wherein said
treatment comprising using an antibody capable of binding to an
epitope comprising at least one of the sequences set forth in SEQ
ID NOS: 1-146, or a fragment, or a variant of said antibody.
57. A method for determining in a sample the presence of a
substance comprising an epitope comprising at least one of the
sequences set forth in SEQ ID NOS: 1-146.
Description
FIELD OF INVENTION
[0001] The present invention relates to a method for modulating the
interaction between at least two proteins, wherein at least one of
the two proteins is a functional cell-surface receptor and the
other protein is the receptor ligand. The invention preferably
concerns interaction of the fibroblast growth factor receptor
(FGFR) and FGFR ligands. The invention further relates to a series
of amino acid sequences involved in forming a binding site for FGFR
in a FGFR ligand. Moreover, the present invention features the
methods for molecular design and screening of a candidate compound
capable of modulating the interaction between FGFR and a protein
having the above binding site, and provides a screening assay for
identification of such a compound.
BACKGROUND OF INVENTION
[0002] Neural cell adhesion molecules (CAMs) of the immunoglobulin
superfamily nucleate and maintain groups of cells at key sites
during early development and in the adult. In addition to their
adhesive properties, CAMs homophylic and heterophylic interactions
can affect intracellular signaling. Their ability to influence
developmental events, including cell migration, proliferation, and
differentiation may therefore result both from their adhesive as
well as their signaling properties.
[0003] The neural cell adhesion molecule, NCAM, was the first
discovered neural CAM. Since the discovery NCAM has been
intensively studied and now it is well characterised.
[0004] NCAM belongs to the immunoglobulin (Ig) superfamily. Its
extracellular part consists of five Ig-like and two fibronectin
type III (F3) modules (Berezin et al., 2000). NCAM assists both the
cell-cell and cell-substratum interactions. NCAM binds to various
extracellular matrix components such as heparin/heparan sulfate,
chondroitin sulfate proteoglycans, and different types of collagen.
Cell-cell interactions are mostly assisted by the NCAM homophilic
interaction.
[0005] The different modules of NCAM have been shown to perform
distinct functions. Thus, NCAM homophilic binding is believed now
to depend on the first three Ig modules. The heparin binding
sequence is localized to the Ig2 module. NCAM also binds to the
neural cell adhesion molecule L1. This interaction is believed to
take place between the fourth Ig module of NCAM and an
oligomannosidic moiety expressed on L1. The two membrane-proximal
F3 modules of NCAM has been suggested to be involved in FGFR
binding. Among a number of ligands of NCAM the most intriguing
remain to be ATP. NCAM has been demonstrated to hydrolyze
extracellularly adenosine triphosphate (ATP) (Dzhandzhugazyan and
Bock, 1993 and 1997), and the proximal F3 modules have been
suggested to be involved in this activity of NCAM. Recently, ATP
has been demonstrated to be a modulator of NCAM induced
neuritogenesis (Skladchikova et al., 1999). However, the role
extracellular ATP, which is one of the most abundant
neurotransmitters in the brain, in relation to known biological
functions of NCAM is not well understood.
[0006] A number of research groups has now accumulated a large body
of evidence indicating that intracellular signaling cascades
underlying the NCAM-mediated axonal outgrowth are similar to signal
transduction cascades which are activated due to stimulation of the
fibtoblast growth factor receptor (FGFR).
[0007] Fibroblast growth factor receptors (FGFRs) are a family of
four closely related receptor protein tyrosine kinases consisting
extracellularly of three Ig-like modules and intracellularly of a
split tyrosine-kinase module (Powers et al., 2000). The receptors
are known as key regulators of morphogenesis, development,
angiogenesis, and wound healing. FGFR activation and signaling are
dependent on dimerization of the receptor which is induced by a
high affinity binding of its ligand, fibroblast growth factor
(FGF), and it also requires participation of cell surface heparin
or heparan sulphate proteoglycans.
[0008] The major neural CAMs, NCAM, L1 and N-cadherin, all have
been regarded as a new class of putative alternative ligands of
FGFR, although there have not been so far obtained any evidence for
a direct interaction between these CAMs and the receptor so far.
The identification of a common structural motif which might be a
prerequisite for the interaction with FGFR seems to be very
advantageous in view of the development of new drugs for the
treatment of a variety of pathologic disorders where the regulation
of activity of FGFR may play the key role.
SUMMARY OF INVENTION
[0009] The present invention is directed to a method of modulating
the interaction between at least two different proteins, wherein
one of the at least two different proteins is represented by a
functional cell-surface receptor, or a fragment, or a variant
thereof, and the other of the at least two different proteins is
represented by a polypeptide having a binding site to said
receptor, wherein at least a part of said binding site comprises at
least one of the sequences set forth in SEQ ID NOS: 1-146 or
fragments, or variants, or homologues of said sequences, or
fragments or variants of said homologues, comprising [0010] i)
providing a compound capable of interacting with the receptor
and/or the polypeptide thereby interfering with said receptor and
said polypeptide interaction, [0011] ii) presenting the compound of
step (i) to the at least two different proteins.
[0012] The invention is also directed to a method of modulating the
interaction between at least two different proteins, wherein one of
the at least two different proteins is represented by a functional
cell-surface receptor, or a fragment, or a variant thereof, and
another of the at least two different proteins is represented by a
polypeptide having a binding site to said receptor, wherein said
binding site is essentially consisting of one or more
"strand-loop-strand" structural motifs, comprising [0013] i)
providing a compound capable of interacting with the receptor
and/or polypeptide thereby interfering with said receptor and said
polypeptide interaction, [0014] ii) presenting the compound of step
(i) to the at least two different proteins.
[0015] An another aspect of the invention concerns a screening
method for a candidate compound capable of modulating the
interaction between at least two different proteins, wherein one of
the at least two different proteins is represented by a functional
cell-surface receptor, or a fragment, or a variant thereof, and the
other of the at least two different proteins is represented by a
polypeptide having a binding site to said receptor, wherein at
least a part of said binding site comprises at least one of the
sequences set forth in SEQ ID NOS: 1-146 or fragments, or variants,
or homologues of said sequences, or fragments or variants of said
homologues, comprising [0016] i) providing the at least two
different proteins; [0017] ii) providing a compound; [0018] iii)
presenting the compound of (ii) to the at least two different
proteins of (i); [0019] iv) determining the interaction between the
at least two different proteins before and after the presenting the
compound to said proteins; [0020] v) determining whether the
interaction between the at least two different proteins has been
modulated by the presented compound, [0021] vi) selecting a
candidate compound capable of modulating the interaction between
the at least two different proteins.
[0022] Furthermore, the invention discloses an assay for sequential
screening of a candidate compound capable of modulating the
interaction between at least two different proteins, wherein one of
the least two different proteins is represented by a functional
cell-surface receptor, or a fragment, or a variant thereof, and the
other of the at least two different proteins is represented by a
polypeptide having a binding site to said receptor, wherein at
least a part of said binding site comprises at least one of the
sequences set forth in SEQ ID NOS: 1-146 or fragments, or variants,
or homologues of said sequences, or fragments or variants of said
homologues, comprising the steps of [0023] i) providing the at
least one functional cell-surface receptor molecule, or a fragment,
or a variant thereof, and the at least one polypeptide having a
binding site to said receptor, wherein at least a part of said
binding site comprises at least one of the sequences set forth in
SEQ ID NOS: 1-146 or fragments, or variants, or homologues of said
sequences, or a fragments or a variants of said homologues, [0024]
ii) presenting the at least one receptor molecule of step (i) to
the at least one polypeptide of step (i), or presenting the at
least one polypeptide of step (i) to the at least one receptor
molecule of step (i) and permitting the interaction between the
said receptor and said polypeptide, followed by the [0025] iii)
recording the interaction between the molecules of step (ii),
[0026] iv) presenting the candidate compound to the molecules of
step (ii); [0027] v) recording the interaction between the
molecules of step (iv), followed by the [0028] vi) assessment of at
least one effect of the candidate compound on the interaction
between the molecules of step (iv), followed by the [0029] vii)
selection of a compound capable of modulating the interaction
between the at least one functional cell-surface receptor molecule
and the at least one polypeptide of step (i).
[0030] The method(s) and the assay of the invention are all
preferably designated to the modulation of interaction between FGFR
and a protein comprising the FGFR binding site, said site
comprising at least one of the sequences set forth in SEQ ID NOS:
1-146 or fragments, or variants, or homologues of said sequences,
by a compound capable of this modulation. Therefore, it is an
important aspect of the invention to provide a method for molecular
design of such a compound, said method comprising the structural
data on the binding FGFR with a protein comprising the above
binding site, said data comprising a molecular model of interaction
between FGFR and NCAM.
[0031] Furthermore, it is another important aspect of the invention
to provide a method A method for isolating a candidate compound
capable of modulating the interaction between at least two
different proteins, wherein one of the least two different proteins
is represented by a functional cell-surface receptor, or a
fragment, or a variant thereof, and the other of the at least two
different proteins is represented by a polypeptide having a binding
site to said receptor, wherein at least a part of said binding site
comprises at least one of the sequences set forth in SEQ ID NOS:
1-146 or fragments, or variants, or homologues of said sequences,
or fragments or variants of said homologues, comprising the steps
of [0032] i) providing a method for sequential screening the
candidate compound as the defined above and/or [0033] ii) providing
a method for molecular design of the candidate compound as the
defined above, [0034] i) isolating the candidate compound.
[0035] The invention further discloses a series of peptide
fragments having the sequences set forth in SEQ ID NOS: 2-146, said
fragments i) being involved in a direct interaction with FGFR, or
being representing a part of an FGFR binding site on a FGFR ligand,
and/or ii) being capable of modulating the interaction between at
least two different proteins, wherein one of the least two
different proteins is represented by a functional cell-surface
receptor, or a fragment, or a variant thereof, and the other of the
at least two different proteins is represented by a polypeptide
having a binding site to said receptor, wherein at least a part of
said binding site comprises at least one of the sequences set forth
in SEQ ID NOS: 1-146, or fragments, or variants, or homologues of
said sequences, or fragments or variants of said homologues.
[0036] The invention also relates to a compound comprising the
above peptide fragments and use of said compound for the
preparation of a medicament.
[0037] Furthermore, the invention relates to [0038] i) an antibody
capable of binding to an epitope comprising a binding site for a
cell surface receptor, wherein at least a part of said binding site
comprises at least one of the sequences set forth in SEQ ID NOS:
1-146, or fragments, or variants, or homologues of said sequences,
or a fragment or a variant of said antibody; [0039] ii) an antibody
capable of binding to an epitope comprising at least one of the
sequences set forth in SEQ ID NOS: 1-146, or a fragment, or a
variant of said antibody.
[0040] The invention also provides a method for the production of
the above antibody. Furthermore, the invention also concerns the
use of an antibody as the defined above for [0041] 1) the
modulation of interaction between a cell surface receptor, or a
fragment or variant thereof, and a polypeptide having a binding
site to said receptor, wherein at least a part of said binding site
comprises at least one of the sequences set forth in SEQ ID NOS:
1-146, or fragments, or variants, or homologues of said sequences,
or fragments or variants of said homologues, [0042] 2) for the
manufacture of a medicament [0043] 3) for determining the presence
of a substance comprising an epitope comprising at least one of the
sequences set forth in SEQ ID NOS: 1-146, or fragments, or
variants, or homologues of said sequences, or fragments or variants
of said homologues in a sample.
[0044] Finally, the invention relates to a method for producing a
pharmaceutical composition comprising the steps of identifying of a
candidate compound and further the step of formulating the compound
capable of modulating modulation of interaction between a cell
surface receptor, or a fragment or variant thereof, and a
polypeptide having a binding site to said receptor, wherein at
least a part of said binding site comprises at least one of the
sequences set forth in SEQ ID NOS: 1-146, or fragments, or
variants, or homologues of said sequences, or fragments or variants
of said homologues with pharmaceutically acceptable carrier or
solvent.
DESCRIPTION OF DRAWINGS
[0045] Table 1 presents a summary of NOE statistics, energy terms
and deviations from the idealized geometry calculated from the NMR
measurements.
[0046] FIG. 1 shows the structure of the NCAM F3 module 2 and
demonstrates the regions of the module involved in interaction with
the FGFR1 Ig module 3 or AMP-PCP. a) Stereo view of an overlay of
the backbone atoms of 30 superimposed structures. b) Ribbon
representation of the structure. The N-terminal is located at the
top of the picture. c and d) Changes in the chemical shifts of
.sup.1H and .sup.15N atoms of 0.05 mM .sup.15N labeled sample of
the NCAM F3 module 2 after addition of 1 mM unlabeled sample of the
FGFR1 Ig module 3 (c) or 5 mM AMP-PCP (d). The chemical shifts were
determined from the .sup.15N-HSQC spectra. The data are presented
as averages from two independent experiments. e) Mapping of the
residues of the NCAM F3 module 2 perturbed by the FGFR Ig module 3
(N.sup.79, Q.sup.81, G.sup.82, K.sup.83, black) onto the structure
of the module. f) Mapping of the residues of the NCAM F3 module 2
perturbed by AMP-PCP, Y.sup.74 and V.sup.75 (black), and the
residues of the ATP binding Walker motif A, A.sup.77ENQQGKS4 (grey)
and K.sup.85 (dark grey, outlined) to the structure of the module;
all other residues are colored light grey. g) Arrangement of the
complex of the NCAM F3 module 2 with ATP. K.sup.83, K.sup.85 and
Y.sup.74 are indicated on the ribbon representation of the backbone
atoms.
[0047] FIG. 2 demonstrates the binding of the combined NCAM F3
modules 1 and 2 to the combined FGFR1 Ig modules 2 and 3 studied by
means of surface plasmon resonance (SPR) analysis. The binding is
given as a response difference (Resp. Diff.) between the binding to
the sensor chip with the immobilized FGFR modules and a blank
sensor chip (unspecific binding). Four independent experiments were
performed using two different preparations of all proteins
used.
[0048] FIG. 3 demonstrates the inhibiting effect of ATP on binding
between the FGFR Ig modules 2, 3 and the NCAM F3 modules 1, 2 or
the dendrimeric FG loop peptide (SEQ ID NO: 1). The binding was
studied by SPR analysis. Approx. 2000 resonance units (RU) of the
FGFR modules were immobilized on the sensor chip. Three independent
experiments were performed. A) Binding of 30 .mu.M F3 modules 1, 2
to the FGFR modules in the presence or absence of 10 mM ATP. B) A
plot of % inhibition by ATP of the binding between 30 .mu.M F3
modules 1, 2 and the immobilized FGFR modules versus the ATP
concentration used for inhibition, and the calculated Ki value. C)
Binding of the dendrimeric FG loop peptide (34, 17 and 8 .mu.M
concentrations) to the immobilized FGFR modules and binding of 34
.mu.M peptide in the presence of 10 mM ATP.
[0049] FIG. 4 demonstrates the binding between the dendrimeric EF
loop peptide (SEQ ID NO: 9) and the FGFR Ig modules 2, 3 recorded
by SPR. The binding is given as a response difference (Resp. Diff.)
between the binding to the sensor chip with the immobilized FGFR
modules and a blank sensor chip (unspecific binding). Four
independent experiments were performed using two different
preparations of all proteins used.
[0050] FIG. 5 shows the effect of the NCAM F3 module 2 and the FG
loop peptide on phosphorylation of FGFR and demonstrates the
immunoprecipitation of NCAM by FGFR. A) TREX-293 cells, stably
transfected with FGFR containing a C-terminal Strepll-tag, were
stimulated for 20 min with either 10 ng/ml FGF2, 5 .mu.M F3 module
2 or 2.5 .mu.M dendrimeric FG loop peptide. After stimulation, FGFR
was immunopurified using anti-phosphotyrosine antibodies and then
analyzed by immunoblotting using antibodies against the
Strepll-tag. B) HEK293 cells, transiently transfected with a
His-tagged version of FGFR1, were stimulated for 20 min with either
5 .mu.M F3 module 2 or 25 .mu.M FG loop peptide. The total amount
of FGFR1 and the amount of FGFR phosphorylation was estimated by
immunoblotting using anti-pentahis (anti-His) and
anti-phosphotyrosine (anti-pY) antibodies, respectively.
Quantification of FGFR phosphorylation was performed by
densitometric analysis of the band intensity. Phosphorylation was
estimated relative to the control (untreated cells), which has been
normalized to 1.0. Error bar represents one standard error of the
mean. P<0.05 by paired t-test when comparing treated cells with
controls (the t-test was performed on six independent sets of
non-normalized data). Ctl stands for the control, F3-F3 module 2,
FGL-FG loop peptide. C) TREX-293 cells, stably transfected with
FGFR containing a C-terminal Strepll-tag, were transiently
transfected with a control vector or NCAM. FGFR was purified from
the cell lysate via the Strepll-tag and analyzed by immunoblotting
using antibodies against NCAM.
[0051] FIG. 6 shows the effect of the NCAM F3 module 2 and its FGFR
binding part (the FG loop peptide (SEQ ID NO: 1)) on neurite
outgrowth from hippocampal neurons. a) Phase contrast micrograph of
control (untreated) neurons. b) Phase contrast micrograph of
neurons treated with 5 .mu.M F3 module. c) Neurite length versus
the concentration of the F3 module, the FG-loop peptide and a
truncated version of the peptide. d) Effect of an anti-FGFR
antibody on neurite outgrowth induced by 5 .mu.M F3 module or 50
.mu.M FG loop peptide. e) Effect of substitutions of the various
amino acids with Ala in the FG loop peptide or truncated versions
of the peptide on neurite outgrowth from hippocampal neurons. The
concentration of the various peptides was in all cases 50 .mu.M.
Four independent experiments were performed. Error bar represents
one standard error of the mean. * and ** stand for statistical
significance of p<0.05 and p<0.01, respectively (by
t-test).
[0052] FIG. 7 shows the effect of the EF loop peptide (EFL-peptide)
(SEQ ID NO: 9) on neurite outgrowth from hippocampal neurons.
SU5402 is a specific inhibitor of FGFR1.
[0053] FIG. 8 displays a sequential, structural and functional
similarity between peptides derived from the FGFR binding part of
NCAM and FGF2. a) Sequential alignment of the peptides, in which
the signs |, , :, * indicate the level of similarity in decreasing
order from strong to low similarity; and structural alignment of
the backbone atoms of the peptides from NCAM (1) and FGF2 (2). b)
Effect of the FGF2 derived peptide and various Ala substituted
forms on neurite outgrowth from hippocampal neurons including a
comparison to the corresponding NCAM derived peptide. Four
independent experiments were performed. Error bar represents one
standard error of the mean. * stands for statistical significance
of p<0.05 (by t-test).
[0054] FIG. 9 exhibits the effect of ATP (a) and AMP-PCP (b) on the
potency of the NCAM F3 module 2, the monomeric FG loop peptide and
a modified version of the peptide to stimulate neurite outgrowth
from hippocampal neurons. Neurons were stimulated with either 5
.mu.M second F3 module or 50 .mu.M peptide in the presence of
various concentrations of ATP or AMP-PCP (0, 0.4, 1.0 mM). cnt
stands for control, F3--the F3 module, FGL--the FG loop peptide,
and YKK--the FG loop peptide in which Y.sup.74, K.sup.83 and
K.sup.85 were substituted with alanine. Four independent
experiments were performed. Error bar represents one standard error
of the mean. * and ** stand for statistical significance of
p<0.05 and p<0.01, respectively (by t-test).
[0055] FIG. 10 shows immunoreactivity of the intact FGL-peptide
(SEQ ID NO: 1) (open bar) and various truncated variants thereof
(t1-t6, cross-hatched bars) with polyclonal antibody raised against
the intact FGL-peptide measured by competitive ELISA.
[0056] FIG. 11 presents the results of in vitro stimulation the
neurite outgrowth of rat hippocampal neurons by a fragment of the
sequence set forth in SEQ ID NO: 5 derived from the
axonal-associated cell adhesion molecule. Treated cells are
compared with cell received no treatment
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DETAILED DESCRIPTION OF THE INVENTION
[0073] Method for Modulation of Interaction Between Two
Proteins
[0074] Cells of a multicellular organism communicate to each other
and respond to a variety of signals of the cellular environment
using an elaborate system of communication. This system enables a
single cell to respond adequately to arrays of the signals by
employing a finely tuned network of interactions between molecules
of cell-surface receptors receiving the signals and the many
cell-surface and intracellular proteins adapting and transmitting
the signal into the cell. A minor intervention into a single
interaction of this network may often lead to a major change in a
specific cellular response to the extracellular signal.
[0075] Accordingly, the present invention relates in one aspect to
a method of modulating the interaction between at least two
different proteins, wherein one of the at least two different
proteins is represented by a functional cell-surface receptor, or a
fragment, or a variant thereof, and the other of the at least two
different proteins is represented by a polypeptide having a binding
site to said receptor, wherein at least a part of said binding site
comprises at least one of the sequences set forth in SEQ ID NOS:
1-146, or fragments, or variants, or homologues of said sequences,
or fragments or variants of said homologues, comprising [0076] i)
providing a compound capable of interacting with the receptor
and/or polypeptide thereby interfering with the interaction of said
receptor and said polypeptide, [0077] ii) presenting the compound
of step (i) to the at least two different proteins.
[0078] Thus, modulation of interaction between a functional
cell-surface receptor and another protein is the first objective of
the method of the invention.
[0079] In the present context by the term "interaction" is meant
that a functional cell-surface receptor has a transient or
permanent direct or indirect contact with another protein.
Functional Cell Surface Receptor
[0080] A cell-surface receptor is defined in the content of the
present invention as any molecule comprising at least one
polypeptide chain, said molecule being associated with the plasma
membrane of a cell in such a way that enables said molecule to
receive a signal at the outer side of the membrane, transduce the
signal through the membrane, and convey said signal further by
inducing a certain action on molecular level inside the cell, for
example by inducing association or dissociation of molecular
complexes, or initiating a biochemical reaction, for example
auto-phosphorylation of the receptor, or proteolytic cleavage of
the receptor leading to initiation of intracellular signal
transduction.
[0081] The invention designates to a "functional cell-surface
receptor". "Functional cell-surface receptor" is meant that the
cell-surface receptor of the invention has an identifiable group of
ligands, the binding of these ligands to the receptor induces
intracellular signal transduction, which results in a physiological
response of the cell. The physiological response, such as for
example differentiation, proliferation, survival, apoptosis or
motility of a cell, depends on the ligand that is involved in the
interaction with the receptor, and/or characteristics of said
interaction, such as the affinity or duration, and/or a species of
the cell which expresses the receptor. By the term "ligand" is
defined a compound which is capable to bind to the receptor and
thereby to activate said receptor. "Activation" of a receptor is
meant that after extracellular binding of a ligand the receptor
became capable to transmit the effect of "ligand binding" into a
cascade of biochemical reactions collectively termed "receptor
signaling" or "signal transduction" inside the cell resulting in
one of the above mentioned physiological responses of the cell.
[0082] A capability of a cell-surface receptor to induce and/or
maintain a cellular process upon the ligand binding is herein
termed "biological function" of the receptor.
[0083] The functional cell-surface receptor of the invention, which
recognises and interacts with another protein, is, in a preferred
embodiment, a receptor of the family of fibroblast growth factor
receptors (FGFRs) comprising FGFR1, FGFR2, FGFR3 and FGFR4. In the
most preferred embodiment a cell-surface receptor of the invention
is FGFR1 or a functional homologue thereof.
[0084] By the term "functional homologue of the receptor" is meant
a molecule which is capable of [0085] i) extracellular binding of a
ligand of FGFR and thereby activating the receptor dependent signal
transduction cascade in the cell, and/or [0086] ii) intracellular
binding of an adaptor molecule of FGFR and thereby activating the
receptor dependent signal transduction cascade in the cell.
[0087] The term "binding" refers to a direct or indirect
interaction between an FGFR homologue and a counter molecule having
a binding site for FGFR. In the present context the counter
molecule is an extracellular ligand of FGFR, or an intracellular
adaptor molecule. An "adaptor molecule" is defined in the content
of the invention as a molecule, which is capable to recognize an
"active state" of the receptor, selectively bind to such receptor
and convey the signal of "activatated receptor" in the cell by
inducing a cascade of reactions of signal transduction. An
intracellular adaptor molecule may be represented by for example
STN, FRS, Grb, SHP2, PLC.gamma., or PIP3.
[0088] The invention relates to ligands having a relatively low
affinity to FGFR. The affinity means the strength of attraction
between molecules. The ligand of FGFR in the present context is
determined as a molecule, which comprises at least one binding site
to FGFR and is capable of low affinity binding to the receptor.
Thus, according to the invention, the other protein, which is
involved in interaction with the cell-surface receptor is in one
aspect a ligand of FGFR.
Binding Site
[0089] According to the invention a cell-surface receptor interacts
with another protein through a receptor binding site, said binding
site being located on said another protein. The binding site of a
molecule may be defined as a specific part of said molecule, which
is involved in interaction with another molecule. In the present
context "binding site" is defined as a fragment of a protein,
specific features of which confer on the protein a capacity to
interact directly and selectively with another molecule, for
example a receptor molecule. Under "specific features" in the
present context is understood a particular amino acid sequence or
the three-dimensional structure of that fragment of a protein which
is referred as the binding site. By the term "selectively" in the
present context is meant that the binding site is involved in
interaction with the certain molecule or certain group of
molecules, said molecule(s) having a specific structure which makes
said molecule(s) capable to recognise the binding site and interact
with it.
[0090] In one embodiment the present invention features a binding
site to a cell-surface receptor, wherein at least a part of said
binding site comprises at least one of the sequences set forth in
SEQ ID NOS: 1-146, or fragments, or variants, or homologues of said
sequences, or fragments or variants of said homologues, such as the
fragments, variants and homologues defined below.
[0091] In another embodiment the binding site comprises, or at
least part of said binding site comprises at least one of the
sequences set forth in SEQ ID NOS: 1-10, 100, 125, such as for
example SEQ ID NOS: 2-10, 100, 125, or fragments or variants, or
homologues said sequences, or fragments or variants of said
homologues. The present invention relates to fragments having at
least 40%, more preferably at least 50%, more preferably at least
60%, more preferably at least 70%, more preferably at least 80%,
more preferably at least 90%, more preferably at least 95% of the
length of a predetermined sequence set forth in SEQ ID NOS: 1-10,
100, 125, or SEQ ID NOS: 2-10, 100, 125, wherein an amino acid
sequence homology between a fragment and the predetermined sequence
is 100%. A variant in the present context is defined as an amino
acid sequence having at least 60%, more preferably at least 70%,
more preferably at least 80%, more preferably at least 90%, more
preferably 95% homology to a sequence selected from SEQ ID NOS:
1-10, 100, 125, or SEQ ID NOS: 2-10, 100, 125, or an amino acid
sequence having at least 60%, more preferably at least 70%, more
preferably at least 80%, more preferably at least 90%, more
preferably 95% positive amino acid matches compared to a sequence
selected from SEQ ID NOS: 1-10, 100, 125, or SEQ ID NOS: 2-10, 100,
125. A positive amino acid match is defined as an identity or
similarity defined by physical and/or chemical properties of the
amino acids having the same position in two compared sequences.
Preferred positive amino acid matches of the present invention are
K to R, E to D, L to M, Q to E, I to V, I to L, A to S, Y to W, K
to Q, S to T, N to S and Q to R. The homology of one amino acid
sequence with another amino acid is defined as a percentage of
identical amino acids in the two collated sequences. A homologue in
the present context is defined as an amino acid sequence which has
less then 60% and more then 19%, such as 50-59%, for example 55%,
such as 40-49%, for example 45%, such as 30-39%, for example 35%,
such as 20-29%, for example 25% homology to any of the sequences
set forth in SEQ ID NOS: 1-10, 100, 125, or SEQ ID NOS: 2-10, 100,
125 having remained some of the physical properties of the
predetermined sequences, such as for example the three dimensional
structure or some of the functional properties, such as for example
a capability to interact with another molecule, in particular with
a receptor molecule. A variant of a homologue in the present
context is defined as an amino acid sequence having at least 60%,
more preferably at least 70%, more preferably at least 80%, more
preferably at least 90%, more preferably 95% positive amino acid
matches compared to a homologue of any of the sequences selected
from SEQ ID NOS: 1-10, 100, 125, or SEQ ID NOS: 2-10, 100, 125.
Preferred embodiments of the positive amino acid matches is as the
defined above. The invention concerns the fragments, variants and
homologues, which remain a capability of the predetermined
sequences to interact with the cell-surface receptor defined
above.
[0092] In still another embodiment the invention features a binding
site comprising at least one variant, or fragment, or homologue of
any of the sequences set forth in SEQ ID NOS: 1-10, 100, 125, or
SEQ ID NOS: 2-10, 100, 125, said variant, or fragment, or homologue
being preferably selected from the sequences identified in SEQ ID
NOS: 11-99, 101-124, 126-146.
[0093] The described above binding site is according to invention
characterised by specific structural features. The invention
preferably features a binding site, which essentially consists of
one or more "strand-loop-strand" structural motifs.
[0094] Side chains of the amino acids in a contiguous amino acid
sequence interact to each other. The numerous interactions lead to
folding the amino acid sequence into a specific structural pattern,
such as .alpha.-helix and .beta.-strand, which is usually
determined by the amino acids of the sequence. Interactions between
amino acids of the individual patterns hold the long amino acid
sequence of a polypeptide folded in a compact tertiary structure.
In a tertiary structure the amino acid sequence of a polypeptide
tends to wind its way back and forth across the entire structure,
either as an .alpha.-helix or .beta.-strand, reversing its
direction suddenly by making a turn and creating thus the loop
regions. The loop regions, which may vary in length and have
irregular shape, often form biding sites for other molecules. The
present invention concerns a binding site, which comprises at least
one loop region. It is preferred, if the at least one loop region
in the binding site connects two interacting 13-strands forming
thereby a "strand-loop-strand" structural motif. By the term
"motif" in the present content is meant a unit of the polypeptide
chain of a protein having the defined sequence of amino acids of
certain length and/or characterised by specific structural features
and/or (optionally) having certain functional capabilities common
for a group of proteins.
Polypeptide Comprising a Binding Site
[0095] As it has been mentioned above, certain combinations of
.alpha.-helices and .beta.-strands pack together and form a
compactly folded tertiary structure of the protein. If the protein
comprises a long amino acid sequence, such as for example more then
100 amino acids, its tertiary structure may consist of several
modular units, each of which is called a protein domain. Domains
are usually constructed from a section of polypeptide chain that
contains between 50 and 350 amino acids. Because there is only a
limited number of ways of combining .alpha.-helices and
.beta.-strands to make a globular structure, certain combinations
of these elements may occur repeatedly in the core of related and
also unrelated proteins bestowing some common structural features
on these proteins. The present invention contemplates both the
proteins having common structural characteristics, structurally
related proteins, and structurally unrelated proteins. In a
preferred embodiment the invention concerns at least two different
proteins capable of interacting to each other, wherein at least one
of the at least two proteins comprises at least two immunoglobulin
(Ig)-like domains and/or at least two fibronectin type 3 (F3)
domains, or at least one Ig-like domain and at least one F3
domain.
[0096] According to the invention a functional cell-surface
receptor interacts with another protein. The invention preferably
relates to the interaction between a cell-surface receptor and a
heterologous protein. By the term "heterologous" in the present
context is meant a protein the amino acid sequence of which has at
most 40% homology and/or at most 50% of positive amino acid matches
with the amino acid sequence of a polypeptide chain of the
receptor. Preferably said heterologous protein itself is not
capable to execute any biological function of the receptor. A
heterologous protein is preferably an extracellular protein or a
protein, at least part of which is exposed to the extracellular
space. However, any heterologous protein which has more then 40%
homology and/or more then 50% of positive amino acid matches with
the amino acid sequence of a polypeptide chain of the receptor is
in the scope of the invention, if said protein comprises a binding
site to the receptor described above.
[0097] The present invention relates to a heterologous protein
comprising the above described binding site, which is preferably
selected from the group comprising transmembrane, cell-surface
associated, extracellular matrix associated or soluble
proteins.
[0098] A protein is in the present context defined as
"transmembrane" if at least one part of the polypeptide chain of
said protein is positioned extracellularly, at least one part of
the polypeptide chain of said protein is represented by the plasma
membrane spanning domain, and at least one part of the polypeptide
chain of said protein is positioned intracellularly, wherein the at
least one extracellular part is connected with the at least one
intracellular part via the at least one membrane spanning
domain.
[0099] In the present context "plasma membrane associated protein"
is defined as a protein having a direct or indirect contact with
the plasma membrane. The "direct" contact is meant that the protein
has an immediate connection with the membrane assisted by a moiety
covalently attached to said protein, such as for example a lipid
moiety, for example glycosylphosphatidylinositol or a fatty acid
moiety. The "indirect" contact is meant that the protein has a
connection with the membrane through another molecule which said
protein is associated with in a complex, for example another
protein, lipopolysaccharide or proteoglycan molecule.
[0100] The extracellular matrix associated protein in the present
context is defined as a protein, or a molecule comprising a
polypeptide chain, said protein or said molecule being attached to
a component of the extracellular matrix through a chemical bond, or
said protein or said molecule itself being an immediate component
of the extracellular matrix.
[0101] The soluble protein in the present context is meant a
protein, which is present free in solution in the extracellular
space, such as for example a secreted protein, or a shed protein
produced due to extracellular proteolysis of the membrane
associated protein.
[0102] Furthermore, the invention features particular polypeptides
from the above groups, which are preferably selected from cell
adhesion molecules, cell-surface receptors, proteoglycans,
membrane-anchored cell-surface proteolytic enzymes, extracellular
matrix molecules, or growth factors.
[0103] Thus, in one embodiment the polypeptide of the invention is
selected from the group of cell adhesion molecules comprising
[0104] Neural Cell Adhesion Molecule (NCAM) (Swiss-Prot Ass. Nos:
P13591, P13595-01, P13595), [0105] Neural cell adhesion molecule L1
(Swiss-Prot Ass. Nos: Q9QYQ7, Q9QY38, P11627, Q05695, P32004),
[0106] Neural Cell Adhesion Molecule-2 (NCAM-2) (Swiss-Prot Ass.
No: P36335) [0107] Neuron-glia Cell Adhesion Molecule (Ng-CAM)
(Swiss-Prot Ass. No: Q03696; Q90933), [0108] Neural cell adhesion
molecule CALL (Swiss-Prot Ass. No: 000533), [0109] Neuroglian
(Swiss-Prot Ass. No: P91767, P20241), [0110] Nr-CAM (HBRAVO, NRCAM,
NR-CAM 12) (Swiss-Prot Ass. Nos: Q92823, O15179, Q9QVN3 [0111]
Axonin-1/fAG-1 (Swiss-Prot Ass. Nos: Q02246, P22063, P28685),
[0112] Axonal-associated Cell Adhesion Molecule (AXCAM) (NCBI Ass.
No: NP.sub.--031544.1; Swiss-Prot Ass. No: Q8TC35), [0113]
Myelin-Associated Glycoprotein (MAG) (Swiss-Prot Ass. No: P20917),
[0114] Neural cell adhesion molecule BIG-1 (Swiss-Prot Ass. No:
Q62682), [0115] Neural cell adhesion molecule BIG-2 (Swiss-Prot
Ass. No: Q62845), [0116] Fasciclin (FAS-2) (Swiss-Prot Ass. No:
P22648), [0117] Neural cell adhesion molecule HNB-3/NB-3
(Swiss-Prot Ass. Nos: Q9UQ52, P97528, Q9JMB8) [0118] Neural cell
adhesion molecule HNB-2/NB-2 (Swiss-Prot Ass. Nos: O94779, P07409,
P97527), [0119] Cadherin (Swiss-Prot Ass. No: Q9VW71), [0120]
Junctional Adhesion Molecule-1 (JAM-1) (Swiss-Prot Ass. Nos:
Q9JKD5, O88792), [0121] Neural cell adhesion F3/F11 (Contactin)
(Swiss-Prot Ass. Nos: Q63198, P1260, Q12860, Q28106, P14781,
O93250), [0122] Neurofascin (Swiss-Prot Ass. Nos: Q90924, Q91Z60;
O042414), [0123] B-lymphocyte cell adhesion molecule CD22
(Swiss-Prot Ass. Nos: Q9R094, P20273), [0124] Neogenin (NEO1)
(Swiss-Prot Ass. Nos: Q92859, P97603, Q90610, P97798), [0125]
Intercellular Cell Adhesion Molecule-5 (ICAM-5/telencephalin)
(Swiss-Prot Ass. Nos: Q8TAM9, Q60625) or [0126] Galactose binding
lectin-12 (galectin-12) (Swiss-Prot Ass. Nos: Q91VD1, Q9JKX2,
Q9NZ03), [0127] Galactose binding lectin-4 (galectin-4) (Swiss-Prot
Ass. No: Q8K419; P38552) or fragments, or variants thereof.
[0128] In another embodiment the polypeptide is selected from the
group of functional cell-surface receptors comprising [0129]
Fibroblast Growth Factor Receptor 1 (FGFR1) (Swiss-Prot Ass. Nos:
Q9QZM7, Q99AW7, Q9UD50, Q63827), [0130] Fibroblast Growth Factor
Receptor 2 (FGFR2) (Swiss-Prot Ass. Nos: Q96KM2, P21802, Q63241),
[0131] Fibroblast Growth Factor Receptor 3 (FGFR3) (Swiss-Prot Ass.
Nos: Q95M13, AF487554, Q99052), [0132] Fibroblast Growth Factor
Receptor 4 (FGFR4) (Swiss-Prot Ass. No: Q91742), [0133]
Neurotrophin Tyrosin Kinase Type-2 (NTRKT-2) (Swiss-Prot Ass. No:
Q8WXJ5), [0134] Leukocyte Antigen Related Protein-Tyrosine
Phosphatase (LAR-PTPRF) (Swiss-Prot Ass. Nos: Q9EQ17, Q64605,
Q64604, Q9QW67, Q9VIS8 P10586), [0135] Nephrin (Swiss-Prot Ass.
Nos: Q925S5, Q9JIX2, Q9ET59, Q9R044, Q9QZS7, Q06500), [0136]
Protein-Tyrosine Phosphatase Receptor type S (PTPRS) (Swiss-Prot
Ass. [0137] Nos: Q64699, Q13332, O75870), [0138] Protein-Tyrosine
Phosphatase Receptor type kappa (R-PTP-kappa) (Swiss-Prot Ass. No:
Q15262), [0139] Protein-Tyrosine Phosphatase Receptor type D
(PTPRD) (Swiss-Prot Ass. Nos: Q8WX65, Q91AJ1, P23468, Q64487),
[0140] Ephrin type-A receptor 8 (EPHA8/Tyrosine-Protein Kinase
Receptor EEK) (Swiss-Prot Ass. Nos: O09127, P29322), [0141] Ephrin
type-A receptor 3 (EPHA8/Tyrosine-Protein Kinase Receptor
ETK1/CEK4) (Swiss-Prot Ass. No: P29318), [0142] Ephrin type-A
receptor 2 (Swiss-Prot Ass. No: Q8N3Z2) [0143] Insulin Receptor
(IR) (Swiss-Prot Ass. No: Q9PWN6) [0144] Insulin-like Growth
Factor-1 Receptor (IGF-1) (Swiss-Prot Ass. Nos: Q9QVW4, P08069,
P24062, Q60751, P15127, P15208) [0145] Insulin-related Receptor
(IRR) (Swiss-Prot Ass. No: P14616), [0146] Tyrosine-Protein Kinase
Receptor Tie-1 (Swiss-Prot Ass. Nos: 06805, P35590, Q06806), [0147]
Roundabout receptor-1 (robo-1) (Swiss-Prot Ass. Nos: O44924,
AF041082, Q9Y6N7), [0148] Neuronal nicotinic acetylcholine receptor
alpha 3 subunit (CHRNA3) (Swiss-Prot Ass. Nos: Q8VHH6, P04757,
Q8R4G9, P32297) [0149] Neuronal acetylcholine receptor alpha 6
subunit (Swiss-Prot Ass. Nos: Q15825, Q9R0W9) [0150]
Platelet-Derived Growth Factor Receptor Beta (PDGFRB) (Swiss-Prot
Ass. Nos: Q8R406, Q05030), [0151] Interleukin-6 Receptor (IL-R)
(Swiss-Prot Ass. No: Q00560), [0152] Interleukin-23 Receptor
(IL-23R) (Swiss-Prot Ass. No: AF461422), [0153] Beta-common
cytokine receptor of IL-3, IL5 and GmCsf (Swiss-Prot Ass. No:
P32927) [0154] Cytokine Receptor-Like molecule 3 (CRLF1)
(Swiss-Prot Ass. No: Q9JM58), [0155] Class I Cytokine Receptor
(ZCYTOR5) (Swiss-Prot Ass. No: Q9UHH5) [0156] Netrin-1 receptor DCC
(Swiss-Prot Ass. No: P43146), [0157] Leukocyte Fc Receptor-like
Protein (IFGP2) (Swiss-Prot Ass. Nos: Q96PJ6, Q96KM2), [0158]
Macrophage Scavenger Receptor 2 (MSR2) (Swiss-Prot Ass. No:
Q91YK7), or [0159] Granulocyte Colony Stimulating Factor Receptor
(G-CSF-R) (Swiss-Prot Ass. No: Q99062), [0160] or fragments, or
variants thereof.
[0161] In still another embodiment the polypeptide is selected from
the group of proteoglycans. More preferably the proteoglycan is
selected from the group comprising heparan sulphate proteoglycans.
In the most preferres embodiment the proteoglycan is perlecan
(Swiss-Prot Ass. No: P98160), or a fragment, or a variant
thereof.
[0162] It is also another embodiment to select the polypeptide from
the group of membrane-anchored cell-surface proteolytic enzymes.
Preferably the polypeptide is selected from the group comprising
the pitrilysin family of metalloproteinases or the family of
desintegrin and metalloproteases (ADAMs) comprising [0163] ADAM-8
(Swiss-Prot Ass. No: Q05910), [0164] ADAM-19 (Swiss-Prot Ass. Nos:
Q9H013, O35674), [0165] ADAM-8 (Swiss-Prot Ass. No: P78325), [0166]
ADAM-12 (Swiss-Prot Ass. Nos: O43184, Q61824), [0167] ADAM-28
(Swiss-Prot Ass. Nos: Q9JLN6, Q61824, Q9XSL6, Q9UKQ2), [0168]
ADAM-33 precursor (Swiss-Prot Ass. Nos: Q8R533, Q923W9), [0169]
ADAM-9 (Swiss-Prot Ass. Nos: Q13433, Q61072), [0170] ADAM-7
(Swiss-Prot Ass. NoS: Q9H2U9, O35227, Q63180), [0171] ADAM-1A
Fertilin alpha (Swiss-Prot Ass. No: Q8R533), [0172] ADAM-15
(Swiss-Prot Ass. Nos: Q9QYV9, O88839, Q13444), [0173]
Metalloproteinase-desintegrin domain containing protein (TECAM)
(Swiss-Prot Ass. No: AF163291), [0174] Metalloproteinase 1
(Swiss-Prot Ass. Nos: O95204, Q9BSI6), [0175] or fragments, or
variants thereof.
[0176] In yet another embodiment, the polypeptide is selected from
the group of extracellular matrix molecules comprising [0177]
Collagen type VII (Swiss-Prot Ass. No: Q63870), [0178] Fibronectin
(Swiss-Prot Ass. Nos: Q95 KV4, Q95 KV5, P07589, Q28377, U42594,
O95609, P07589, P11276), or [0179] Tenascin (Swiss-Prot Ass. Nos:
Q15568, O00531, P10039, Q90995), [0180] or fragments, or variants
thereof.
[0181] In still yet another embodiment the polypeptide is selected
from the group of growth factors. In the most preferred embodiment
the growth factor is Cytokine-like factor-1 (CLF-1) (Swiss-Prot
Ass. No:O75462), or a fragment, or a variant thereof.
[0182] In further another embodiment the polypeptide is selected
from the group of soluble proteins. In the most preferred
embodiment the soluble protein is SPLIT (Swiss-Prot Ass. No:
Q9XW4).
[0183] In the most preferred embodiment, the polypeptide is the
neural cell adhesion molecule, NCAM, having the sequence identified
in Swiss-Prot Ass. Nos: P13591, P13595-01 or P13595.
[0184] By the wording "fragments, or variants" of the above
polypeptides is meant [0185] (i) polypeptide(s) essentially
consisting of the amino acid sequence of the predetermined
polypeptide, wherein the length of amino acid sequence of said
polypeptide(s) being shorter or longer then the length of said
predetermined polypeptide, said polypeptide(s) being capable to
interact with the functional cell-surface receptor according to
invention and/or [0186] (ii) polypeptide(s) comprising at least a
fragment of the amino acid sequence of the predetermined
polypeptide, or a fragment of a sequence having at least 60%
homology to the sequence of said predetermined polypeptide, wherein
the length of said fragment consists of at least 25% of the length
of the predetermined polypeptide, said polypeptides being capable
of interacting with the functional cell-surface receptor according
to invention, and/or [0187] (iii) polypeptides essentially having
the features described in prior art for the predetermined
polypeptide, said features being essential for the interaction of
said polypeptides with the cell-surface receptor according to
invention, and/or [0188] (iv) polypeptides lacking one or more
features described in prior art for the predetermined polypeptide,
said polypeptides being capable of interacting with the functional
cell-surface receptor according to invention.
[0189] Such fragments or variants may be selected from, but not
limited by examples of naturally occurring isoforms of the above
polypeptides, pro-polypeptides, proteolytic fragments of the above
polypeptides, corresponding recombinant polypeptides or fusion
proteins containing amino acid sequences derived from the above
polypeptides.
Affinity of Interaction
[0190] Any interaction between two molecules may be characterised
by its affinity, which means the strength of attraction between two
molecules. The affinity of interaction is commonly expressed by a
value of Kd, a dissociation equilibrium constant. Kd reflects a
ratio between the rate of dissociation and the rate of binding
between two molecules and thus represents a measure of the strength
of binding between two molecules. The stronger the binding, the
lower is the value of Kd. The invention relates to a low affinity
interaction between a cell-surface receptor and another protein,
such as the defined above. The low affinity interaction of the
invention is characterised by Kd having a value in the range of
10.sup.-3 to 10.sup.-10, such for example in the range of 10.sup.-4
to 10.sup.-8.
[0191] According to the invention affinity of the interaction is
determined by surface plasmon resonance analysis (SPR) or nuclear
magnetic resonance spectroscopy (NMR).
Modulation of Interaction
[0192] By the term "modulation" in relation to interaction between
two molecules is meant a change in the strength of interaction,
such as either a decrease or increase in the strength of
interaction. Modulation of interaction takes place when it is
important to adjust the strength of interaction between molecules
adequately to a change in the situation dependent on said
interaction. For example, modulating the strength of interaction
between a receptor and receptor ligand makes possible the
modulating the receptor dependent signal transduction and thereby
the physiological status of the cell.
[0193] By "modulating receptor signalling" is meant activation or
inhibition of the production of a cascade of messenger molecules,
which normally takes place in the cell in response to activation of
the receptor by an extracellular stimulus.
[0194] The receptor signaling leads to a physiological response of
the cell. Therefore, modulating the strength of interaction between
a receptor and the receptor ligand, otherwise termed "strength of
receptor stimulation", makes possible to modulate the receptor
signaling, and thereby a physiological response of the cell. It has
been shown that the cellular response to stimulation of a receptor
depends on the strength of receptor stimulation, which may, for
example, be characterised by the value of affinity of interaction
of the receptor with a ligand, and/or by the duration of such
interaction. The both affinity and duration of interaction may be
affected if the interacting molecules are exposed to a compound
capable of modulating the interaction.
Compound
[0195] The present invention relates to a compound, which is
capable of [0196] i) interacting with a cell-surface receptor
thereby mimicking the interaction of the receptor with a ligand,
and/or [0197] ii) interacting with a cell-surface receptor thereby
interfering with the interaction of the receptor and the receptor
ligand, and/or [0198] iii) interacting with a cell-surface receptor
ligand thereby interfering with the interaction of the receptor
ligand and the receptor, and/or [0199] iv) simultaneous interacting
with a cell-surface receptor and the receptor ligand thereby
interfering with the interaction of the receptor and the receptor
ligand, and/or [0200] v) interacting with a molecule which is
involved in assistance of the interaction between a cell-surface
receptor and the receptor ligand thereby interfering with the
interaction the receptor and the receptor ligand.
[0201] The invention in one aspect relates to compounds that are
capable to interact with a site on the receptor, which is involved
in interaction of the receptor with a ligand, or are capable to
interact with a receptor binding site in the ligand molecule. In
another aspect the invention relates to compounds that are capable
to bind to another sites in the receptor or ligand presuming that
such interaction will interfere with the interaction of the
receptor and the ligand.
[0202] The invention features any compounds capable of the above
bindings. However, preferred compounds of the invention are
selected from the group comprising peptides, carbohydrates, lipids
or nucleotides. Among the preferred compounds the most preferred
are peptide or nucleotide compounds.
[0203] Accordingly, in one embodiment, the invention relates to a
compound selected from the group comprising nucleotides, nucleotide
analogues, nucleotide derivatives, di- or oligomers of nucleotides,
or nucleotide comprising substances. Preferably a nucleotide is
selected from the group comprising nucleotide triphosphates. More
preferably a nucleotide triphosphate is selected from the group
comprising UTP, GTP, CTP, TTP and ATP or analogues, derivatives,
di- or oligomers thereof, or substances comprising thereof. The
most preferred nucleotide of the invention is ATP or an analogue
thereof. A nucleotide analogue is defined as a molecule comprising
a nucleotide base or a modified nucleotide base, a sugar residue or
a modified sugar residue and a mono-, di-, tri-, quadra-, or
penta-ester group. It is one of preferred embodiments of the
invention if a nucleotide analogue possesses an increased stability
in an aquatic solution in vitro and/or an increased stability in an
ezymatic system comprising an enzyme capable of utilising a
nucleotide as a substrate. The content of a cell is an example of
such ezymatic system. The invention preferably relates to stable
nucleotide triphosphate analogues, wherein at least one phosphorus
group in the triphosphate ester is substituted for another chemical
group, such for example CH--, S--, or NH-- group.
[0204] In another embodiment, the invention relates to a compound,
which comprises a nucleotide. In a more preferred embodiment the
compound comprises ATP, or an analogue thereof.
[0205] A peptide compound of the invention may be any contiguous
amino acid sequence, that meets the requirements for a compound
defined above. Preferably a peptide compound comprises at least one
of the sequences identified in SEQ ID NOS: 2-10, 100, 125, or a
peptide fragment derived from any of said sequences. By the term
"derived" in the present context is meant that the peptide compound
may be represented by an amino acid sequence comprising a variant,
or a fragment, or a combination of any of the sequences identified
in SEQ ID NOS: 2-10, 100, 125, or a combination of variants, or
fragments thereof. The present invention relates to fragments
having at least 40%, more preferably at least 50%, more preferably
at least 60%, more preferably at least 70%, more preferably at
least 80%, more preferably at least 90%, more preferably at least
95% of the length of a predetermined sequence set forth in SEQ ID
NOS: 2-10, 100, 125, wherein an amino acid sequence homology
between a fragment and the predetermined sequence is 100%. A
variant in the present context is defined as an amino acid sequence
having at least 60%, more preferably at least 70%, more preferably
at least 80%, more preferably at least 90%, more preferably 95%
homology to a sequence selected from SEQ ID NOS: 2-10, 100, 125, or
an amino acid sequence having at least 60%, more preferably at
least 70%, more preferably at least 80%, more preferably at least
90%, more preferably 95% positive amino acid matches compared to a
sequence selected from SEQ ID NOS: 2-10, 100, 125. A homology of
one amino acid sequence with another amino acid is defined as a
percentage of identical amino acids in the two collated sequences.
A positive amino acid match is defined as an identity or similarity
defined by physical and/or chemical properties of the amino acids
having the same position in two collated sequences. Preferred
positive amino acid matches of the present invention are K to R, E
to D, L to M, Q to E, I to V, I to L, A to S, Y to W. K to Q, S to
T, N to S and Q to R. A combination of amino acid sequences may
either be represented by a homopolymer or a heteropolymer of said
sequences, wherein said homopolymer being represented a sequence
comprising one or more repeats of any of the sequences selected
from SEQ ID NOS: 2-10, 100, 125, and said homopolymer being
represented by a sequence comprising one or more repeats of at
least two different sequences selected from SEQ ID NOS: 2-10, 100,
125. A number of repeats in the sequences in a homopolymer, or a
heteropolymer may vary from 2 to 20, for example 15, such as 2 to
15, for example 10, such as 2 to 10, for example 3, 4, 5, 6, 7, 8,
or 9.
[0206] Another preferred peptide compounds of the invention are
those that comprise at least one of the sequences selected from SEQ
ID NOS: 11-99, 101-124, 126-146, a fragment, variant or homologue
thereof.
[0207] According to the invention sequences identified as SEQ ID
NOS: 1-146 are derived from the sequences of FGFR receptor ligands
and involved in interaction of the receptor with said ligands.
Therefore, any of the sequences selected from SEQ ID NOS: 1-146 may
anticipated being capable of modulating the interaction between
FGFR and FGFR ligand, said ligand comprising the binding site of
the invention.
[0208] The amino acid sequence of the peptide compound of the
invention may be of any suitable length, in that the length of the
amino acid sequence is dictated by the functonality of the peptide
and the formulation of the compound into a pharmaceutical
composition. Thus, the compound normally comprises amino acid
residues in the range of from 3-100 amino acid residues, such as
from 10-90 amino acid residues, for example from 15-85 amino acid
residues, such as from 20-80 amino acid residues, for example from
25-75 amino acid residues, such as from 30-70 amino acid residues,
for example from 35-65 amino acid residues, such as from 40-60
amino acid residues, for example from 45-55 amino acid
residues.
[0209] In another aspect the peptide compound comprises amino acid
residues in the range of from 3 to 20 amino acid residues, such as
from 3-19 amino acid residues, for example from 3-18 amino acid
residues, such as from 3-17 amino acid residues, for example from
3-16 amino acid residues, such as from 3-15 amino acid residues,
for example from 3-14 amino acid residues, such as from 3-13 amino
acid residues.
[0210] In still another aspect the peptide compound encompasses a
sequence of at least 6 to 16 contiguous amino acids, such as for
example 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids.
[0211] In yet another aspect the amino acid sequence of a peptide
compound is capable of forming a strand-loop-strand fold analogous
to the strand-loop-strand motif of the described above binding site
of the invention.
[0212] A compound of the present invention may preferably be in the
form of an oligomer (multimer) of monomers, wherein each monomer is
as a peptide compound defined above. Particularly, multimeric
peptides such as dendrimers may form conformational determinants or
clusters due to the presence of multiple flexible peptide monomers.
In one embodiment the compound is a dimer. In a more preferred
embodiment the compound is a dendrimer, such as four peptides
linked to a lysine backbone, or coupled to a polymer carrier, for
example a protein carrier, such as BSA. Polymerisation such as
repetitive sequences or attachment to various carriers are
well-known in the art, e.g. lysine backbones, such as lysine
dendrimers carrying 4 peptides, 8 peptides, 16 peptides, or 32
peptides. Other carriers may be lipophilic dendrimers, or
micelle-like carriers formed by lipophilic derivatives, or
starburst (star-like) carbon chain polymer conjugates.
[0213] The individual monomers may be homologous, i.e. identical to
one another, or the individual monomers may be heterologous, i.e.
different from one another. The latter type of monomers may
comprise at least two different monomers. In general dimers and
multimers may comprise two or more identical monomers, or two or
more monomers different from one another.
Screening Method
[0214] It is an important aspect of the invention to provide a
screening method for a candidate compound capable of modulating the
interaction between at least two different proteins, wherein one of
the at least two different proteins is represented by a functional
cell-surface receptor, or a fragment, or a variant thereof, and
another of the at least two different proteins is represented by a
polypeptide having a binding site to said receptor, wherein at
least a part of said binding site comprises at least one of the
sequences set forth in SEQ ID NOS: 1-146, or fragments, or
variants, or homologues of said sequences, or fragments or variants
of said homologues, comprising [0215] i) providing the at least two
different proteins; [0216] ii) providing a compound; [0217] iii)
presenting the compound of (ii) to the at least two different
proteins of (i); [0218] iv) determining the interaction between the
at least two different proteins before and after the presenting the
candidate compound to said proteins; [0219] v) determining whether
the interaction between the at least two different proteins has
been modulated by the presented compound; [0220] vi) selecting a
candidate compound capable of modulating the interaction between
the at least two different proteins.
[0221] The screening method according to invention concerns in a
preferred embodiment identification of a candidate compound capable
of modulating the interaction between a cell-surface receptor,
wherein the receptor is FGFR, and a polypeptide, wherein the
polypeptide is a FGFR ligand. More preferably FGFR is FGFR1 having
the amino acid sequence identified in Swiss-Prot Seq Nos: Q9QZM7,
Q99AW7, Q9UD50 or Q63827, or fragments, or variants thereof, or a
functional homologue of said receptor. The FGFR ligand is
preferably selected from the FGFR1 ligands, polypeptide chain of
which comprises the described above binding site. Examples of
preferred polypeptides are described above. The most preferred
polypeptide for the screening method of the invention is the neural
cell adhesion molecule, NCAM, having the amino acid sequence
identified as Swiss-Prot Ass Nos: P13591, P13595-01 or P13595, or
fragments, or variants thereof, a functional homologue thereof.
Screening Assay
[0222] Modulation of a receptor-ligand interaction may be assessed
by using a number of in vitro assays developed in the art. However,
there are no any universal approach in selection of these assays
for estimation of the capability of candidate compounds to modulate
the interaction between different receptors and their ligands. The
present invention provides an assay for sequential screening of a
candidate compound capable of modulating the interaction between at
least two different proteins, wherein one of the least two
different proteins is represented by a functional cell-surface
receptor, or a fragment, or a variant thereof, and another of the
at least two different proteins is represented by a polypeptide
having a binding site to said receptor, wherein at least a part of
said binding site comprises at least one of the sequences set forth
in SEQ ID NOS: 1-146, or fragments, or variants, or homologues of
said sequences, or fragments or variants of said homologues,
comprising the steps of [0223] i) providing the at least one
functional cell-surface receptor molecule, or a fragment, or a
variant thereof, and the at least one polypeptide having a binding
site to said receptor, wherein at least a part of said binding site
comprises at least one of the sequences set forth in SEQ ID NOS:
1-146, or fragments, or variants, or homologues of said sequences,
or a fragments or a variants of said homologues, [0224] ii)
presenting the at least one receptor molecule of step (i) to the at
least one polypeptide of step (i), or presenting the at least one
polypeptide of step (i) to the at least one receptor molecule of
step (i) and permitting the interaction between said receptor and
said polypeptide, followed by the [0225] iii) recording the
interaction between the molecules of step (ii), [0226] iv)
presenting the candidate compound to the molecules of step (ii);
[0227] v) recording the interaction between the molecules of step
(iv), followed by the [0228] vi) assessment of at least one effect
of the candidate compound on the interaction between the molecules
of step (iv), followed by the [0229] vii) selection of a compound
capable of modulating the interaction between the at least one
functional cell-surface receptor molecule and the at least one
polypeptide of step (i).
[0230] By the term "permitting the interaction" in the present
content is meant that conditions of step (ii) include a suitable
medium in which the interaction is favoured and long enough time
interval to allow the molecules to interact.
[0231] The steps (ii)-(v) of the above assay may optionally be
performed as the following: (ii) presenting the compound to the at
least one receptor molecule of step (i) or presenting the compound
to the at least one polypeptide of step (i), (iii) presenting at
least one polypeptide of step (i) to the compound and receptor of
step (ii) or presenting the at least one receptor molecule to the
compound and polypeptide of step (i), (iv) recording the
interaction between the molecules of step (iii). If the latter
option is used, a candidate compound is selected when no
interaction between the molecules of step (iii) has been recorded
or the recorded interaction has been modulated compared to the
interaction between said molecules in the absence of said compound.
It is presumed that parameters of interaction between the receptor
and the polypeptide in the absence of the compound have been
assessed using any of the described below methods before performing
the modified assay.
[0232] The described above assay is preferably provided by the
invention for the sequential screening of a candidate compound
capable of modulation of interaction between FGFR1 and the FGFR1
ligand, said ligand being one of the polypeptides described above.
In a preferred embodiment the ligand is NCAM.
[0233] Assessment of the effect of a candidate compound on the
interaction between the molecules fo the assay may be done by using
a conventional method of the art, such as for example surface
plasmon resonance analysis (SPR), nucleic magnetic resonance
spectroscopy (NMR), sedimentation analysis, immunoprecipitation,
two-hybrid system, or resonance energy transfer (BRET or FRET). A
candidate compound capable of modulating the interaction may be
selected from compounds decreasing the strength of interaction
between the receptor and the receptor ligand or from compounds
increasing the strength of the interaction.
[0234] After the selection the candidate compound is according to
the invention to be tested in an in vivo or in an in vitro cell
system to evaluate significance of the recorded modulation for
cellular metabolism. In such a system it may in one embodiment be
measured a cell-surface receptor associated downstream signaling
event, for example activation of a downstream protein. If the
protein involved in interaction with a cell-surface receptor is
represented by a receptor-like molecule, it may in another
embodiment be measured a downstream signaling event associated with
said protein. Therefore, the invention further provides a screening
method for the compound selected on step (vii) of the above assay,
comprising [0235] viii) presenting the selected compound to at
least one cell presenting the at least one functional cell-surface
receptor molecule, or a fragment, or a variant thereof, and the at
least one polypeptide having a binding site to said receptor,
wherein at least a part of said binding site comprises at least one
of the sequences set forth in SEQ ID NOS: 1-146, or fragments, or
variants, or homologues of said sequences, or a fragments or a
variants of said homologues with the compound of step (vii) of the
above assay, [0236] ix) assessing at least one effect of the
compound on the cell of step (viii).
[0237] The invention preferably concerns an FGFR1-associated
downstream signaling event, which may be selected from estimation
of i) the FGFR1 tyrosine phosphorylation; ii) the activation of one
or more intracellular proteins involved in any of the
FGFR-associated signal transduction pathways, such as for example
the STAT1, JNK, PLC.gamma., ERK, STAT5, P13K, PKC, FRS2 and/or GRB2
proteins; and/or iii) a cell differentiation-related effect.
[0238] When the FGFR signalling is measured as a level of
phosphorylation of FGFR, the degree of phoshorylation is estimated
as at least 20% above the control value, such as at least 20-200%,
for example at least 50-200%. The control value in the present
content is meant the degree of phosphorylation of FGFR in the
medium where a compound capable of activation of FGFR is
absent.
[0239] When estimating an efficient concentration of a compound
with respect to modulating of the FGFR signalling, said
concentration may be between 0.1-1000 .mu.M, 1-1000 .mu.M, for
example 1-200 .mu.M, for example 10-200 .mu.M, such as 20-180
.mu.M, for example 30-160 .mu.M, such as 40-140 .mu.M, for example
50-130 .mu.M, such as 60-120 .mu.M, for example 70-110 .mu.M, such
as 80-100 .mu.M.
[0240] When estimating the downstream FGFR signaling effect such as
a cell differentiation-related effect the invention preferably
relates to cell aggregation, the formation of nodules, formation of
cartilage, or two or more of said effects (Listrum, G. P. et al. J.
Histochem. Cytochem. 1999, 47:1-6), such effect being detectable by
light microscopy, turbidimetry, or flow cytometry. The cell
differentiation-related effect may also be measured as a change in
expression at RNA or protein levels of bone sialoprotein (J. Bone
Miner. Res. 1998, 13:1852-61; Genomics 1998, 53: 391-4), or type X
collagen (Cell Tissue Res. 1998, 293: 357-64), the human ILA gene
(Osteoarthritis Gartilage 1997, 5: 394-406), or type 11 collagen/or
MGP (J Miner Res. 1997: 1815.23), and the like.
[0241] The FGFR tyrosine phosphorylation or activation of any of
the molecules of FGFR-associated downstream signaling, such as for
example STST1, JNK, PLC.gamma., ERK, STAT5, P13K, PKC, FRS2 and/or
GRB2 proteins, may be estimated by any conventional methods, such
as for example immunocytochemistry, immunoblotting or
immunoprecipitation, using commercially available antibody against
the activated proteins. The degree of activation is estimated as at
least 20% above/below the control value, such as at least 20-200%,
for example at least 50-200%. The control value is estimated as a
degree of phosphorylation of the protein of interest in the medium
where a compound capable of activation of FGFR is absent.
[0242] In another preferred embodiment the invention concerns the
downstream signaling associated with a protein involved in
interaction with FGFR, said protein being the FGFR1 ligand. Such
downstream signaling is understood to be associated with a
receptor-like ligand FGFR1. Preferred embodiments of such ligands
of FGFR1 are disclosed above. It is understood that such ligands
comprise the group of proteins, which have been associated with any
downstream signal transduction cascade, or can potentially be
associated with a downstream signal transduction cascade.
[0243] The most preferred a receptor-like ligand of FGFR1 of the
invention is NCAM.
[0244] Therefore, the assay further concerns any in vitro or in
vivo cellular systems comprising at least one cell presenting at
least one molecule of functional FGFR1 or a functional homologue
thereof and at least one molecule of NCAM or a functional homologue
thereof.
[0245] NCAM-dependent signal transduction involves the variety of
downstream molecules, activation of which upon the signaling may be
measured. The invention in particular concerns the assessment of
activation of focal adhesion kinase FAK, tyrosine kinase Fyn and/or
cyclic-AMP response-binding element proteiri CREB. The degree of
phoshorylation is estimated as at least 20% above/below the control
value, such as at least 20-200%, for example at least 50-200%. The
control value is estimated as above.
[0246] Activation or inhibition of NCAM-dependent signal
transduction may also be measured by evaluating the cellular
responses on morphological level, in particular cell
differentiation-related effects. Accordingly, the assay concerns in
another particular embodiment evaluation of the effect of a
candidate compound on NCAM-dependent cellular aggregation, cell
motility, neuritogenesis, proliferation or survival.
[0247] A skilled artisan may select from a number of assays have
developed in the art to evaluate the above cellular responses.
Cellular aggregation and neuritogenesis may for example be
evaluated as described by Skladchikova et al. J. Neurosci. Res
1999, 57: 207-18. Proliferation and apoptosis may be evaluated by
using any commercially available assays and kits according to the
manufacturer procedure.
[0248] It is a preferred embodiment of the invention selecting a
compound capable of modulating NCAM-FGFR1 interaction by evaluating
the effect of the compound on neuritogenesis. Therefore, cells of
neural origin, which are capable of neuronal differentiation, are
preferred by the invention. Such cell may be selected from primary
cells, which have been extracted from selected areas of the neural
system of an organisml and maintained in culture according to
standard methods, or the cells may be clonal cells, which have been
transformed to immortality from different neural tumor or embryonic
neural cells. Examples of such cells can be rat pheochromocytoma PC
12, mouse neuroblastoma N2A, human teratocarcinoma NT-2 or mouse
carcinoma F9.
[0249] In the present invention a compound is considered promising
when it is capable of doubling the neurite outgrowth of cultured
cells when compared to control cells, such as improving neurite
outgrowth three-fold, such as four-fold, for example five fold,
such as six-fold.
[0250] Further, the present assay in another preferred embodiment
concerns the selection of a compound, which is capable of
stimulating/promoting survival of cells by modulating the
FGFR1--NCAM interaction. In the present context the wording
"stimulating/promoting survival" is used synonymously with the
wording "preventing cell death" or "neuro-protection". By
stimulating/promoting survival it is possible to prevent diseases
or prevent further degeneration of the nervous system in
individuals suffering from a neuro-degenerative disorder.
"Survival" refers to the process, wherein a cell has been
traumatised and would under normal circumstances, with a high
probability die, if not the compound of the invention was used to
prevent said cell from degenerating, and thus promoting or
stimulating survival of said traumatised cell. In particular the
invention concerns promoting or stimulating the survival of cell of
neural origin.
[0251] A cell culture of the assay may further comprise mammalian
cells of non-neural origin. Examples of such cells may for example
include HEK293, COS7, CHO or TREX293. These cells may desirably be
transformed to express different variants or homologues of FGFR or
NCAM by using the methods well known in art, such as for example
methods of stable or transient transfection of cells with DNA
constructs encoding different variants or homologues of FGFR and/or
NCAM and/or other preferred ligands of FGFR of the invention. Such
cells may be useful when using the evaluation of activation of the
downstream molecules mentioned above for selection of a candidate
compound.
Method for Molecular Design of a Candidate Compound
[0252] It is a further aspect of the invention to provide a method
for molecular design for a compound capable of modulating the
interaction between at least two different proteins, wherein one of
the least two different proteins is represented by a functional
cell-surface receptor, or a fragment, or a variant thereof, and
another of the at least two different proteins is represented by a
polypeptide having a binding site to said receptor, wherein at
least a part of said binding site comprises at least one of the
sequences set forth in SEQ ID NOS: 1-146, or fragments, or
variants, or homologues of said sequences, or fragments or variants
of said homologues comprising using structural data on the binding
site of NCAM with FGFR.
[0253] Based on structural data on the binding site of NCAM with
FGFR the invention provides a molecular model of interaction of
FGFR1 and NCAM. The structural data provided by the invention
disclose an amino acid sequence motif and a particular structural
motif involved in the interaction these two molecules.
[0254] Accordingly, the peptide fragments [0255] i) encompassing a
sequence of at least 6 to 16 contiguous amino acids and [0256] ii)
having at least 60%, more preferably at least 70%, more preferably
at least 80%, more preferably at least 90%, more preferably 95%
sequence homology to the SEQ ID NO: 1 and/or [0257] iii) peptide
fragments having the three-dimensional characteristics, such as for
example strand-loop-strand fold, similar to the structural
characteristics of the peptide fragment corresponding to SEQ ID NO:
1, wherein the sequences homology between the aligned peptide
fragments is at least 40% and/or the positive amino acid match is
at least 50%, are considered by the invention as candidate
compounds capable of modulation of interaction between FGFR and
NCAM. Furthermore, a peptide fragment having at least 60%, more
preferably at least 70%, more preferably at least 80%, more
preferably at least 90%, more preferably 95% sequence homology to
any of the sequences identified in SEQ ID NOS: 2-146 is considered
by the invention as a potential candidate compound and designated
by the invention for the screening in the screening assay for a
capability of modulation of interaction between FGFR and a protein
having the described above FGFR binding site. Method for
Identifying a Candidate Compound
[0258] According to the above described methods for screening and
molecular design the candidate compound capable of modulating the
interaction between at least two different proteins, wherein one of
the least two different proteins is represented by a functional
cell-surface receptor, or a fragment, or a variant thereof, and the
other of the at least two different proteins is represented by a
polypeptide having a binding site to said receptor, wherein at
least a part of said binding site comprises at least one of the
sequences set forth in SEQ ID NOS: 1-146, or fragments, or
variants, or homologues of said sequences, or fragments or variants
of said homologues can be isolated and used for the formulation of
a pharmaceutical composition.
[0259] Thus, the invention provides
[0260] a method for isolating a candidate compound capable of
modulating the interaction between at least two different proteins,
wherein one of the least two different proteins is represented by a
functional cell-surface receptor, or a fragment, or a variant
thereof, and the other of the at least two different proteins is
represented by a polypeptide having a binding site to said
receptor, wherein at least a part of said binding site comprises at
least one of the sequences set forth in SEQ ID NOS: 1-146, or
fragments, or variants, or homologues of said sequences, or
fragments or variants of said homologues, comprising the steps of
[0261] i) providing a method for sequential screening the candidate
compound as the defined above and/or [0262] ii) providing a method
for molecular design of the candidate compound as the defined
above, [0263] iii) isolating the candidate compound; a method for
producing a pharmaceutical composition comprising the steps of
identifying of a candidate compound and further the step of
formulating of the compound capable of modulating the interaction
between a cell surface receptor, or a fragment or variant thereof,
and a polypeptide having a binding site to said receptor, wherein
at least a part of said binding site comprises at least one of the
sequences set forth in SEQ ID NOS: 1-146, or fragments, or
variants, or homologues of said sequences, or fragments or variants
of said homologues with pharmaceutically acceptable carrier or
solvent. Peptide Fragments
[0264] Using the described above method(s) and/or assay of the
invention the peptide fragments which are capable of modulating the
interaction between FGFR and a protein having the described above
FGFR binding site may be identified. According the invention these
peptide fragments in one embodiment are having at most about 100
amino acids comprising at least one of the amino acid sequences
which are numbered from 2 to 146 and disclosed in the following
sequence list: TABLE-US-00001 NIEVWVEAENALGKKV, (SEQ ID NO: 2)
ATNRQGKVKAFAHL, (SEQ ID NO: 3) RYVELYVVADSQEFQK, (SEQ ID NO: 4)
VAENSRGKNVAKG, (SEQ ID NO: 5) GEYWCVAENQYGQR, (SEQ ID NO: 6)
RLAALNGKGLGEIS, (SEQ ID NO: 7) KYIAENMKAQNVAKEI, (SEQ ID NO: 8)
TIMGLKPETRYAVR, (SEQ ID NO: 9) KGLGEISAATEFKT, (SEQ ID NO: 10)
NMGIWVQAENALG, (SEQ ID NO: 11) IWVQAENMLG, (SEQ ID NO: 12)
EIWVEATNRLG, (SEQ ID NO: 13) VWVQAANALG, (SEQ ID NO: 14)
EVWIEKDPAKGRI, (SEQ ID NO: 15) ATNKGGEVKKNGHL, (SEQ ID NO: 16)
KYVELYLVADYLEFQK, (SEQ ID NO: 17) RYVELYVVVDNAEFQ, (SEQ ID NO: 18)
KYVELVIVADNREFQR, (SEQ ID NO: 19) KYIEYYLVLDNGEFKR, (SEQ ID NO: 20)
RYLELYIVADHTLF, (SEQ ID NO: 21) KYVEMFVVVNHQRFQ, (SEQ ID NO: 22)
RYVELFIVVDKERY, (SEQ ID NO: 23) KYVELFIVADDTVYRR, (SEQ ID NO: 24)
KFIELFVVADEYVYRR, (SEQ ID NO: 25) KIVEKVIVADNSEVRK, (SEQ ID NO: 26)
VELVIVADHSEAQK, (SEQ ID NO: 27) VAENSRGKNIAKG, (SEQ ID NO: 28)
IAENSRGKNVARG, (SEQ ID NO: 29) AENSRGKNSFRG, (SEQ ID NO: 30)
IASNLRGRNLAKG, (SEQ ID NO: 31) IPENSLGKTYAKG, (SEQ ID NO: 32)
IAENMKAQNEAK, (SEQ ID NO: 33) QFIAENMKSHNETKEV, (SEQ ID NO: 34)
GEYWCVAKNRVGQ, (SEQ ID NO: 35) GSYTCVAENMVGK, (SEQ ID NO: 36)
GKYVCVGTNMVGER, (SEQ ID NO: 37) GNYTCVVENEYG, (SEQ ID NO: 38)
GEYTCLAGNSIG, (SEQ ID NO: 39) QYYCVAENGYG, (SEQ ID NO: 40)
GEYYQEAEQNGYG, (SEQ ID NO: 41) GNYTCLVENEYG, (SEQ ID NO: 42)
GMYQCLAENAYG, (SEQ ID NO: 43) GMYQCAENTHG, (SEQ ID NO: 44)
GIYYCLASNNYG, (SEQ ID NO: 45) GGYYCTADNSYG, (SEQ ID NO: 46)
GEYQCFARNDYG, (SEQ ID NO: 47) GEYFCLASNKMG, (SEQ ID NO: 48)
GEYQCFARNKFG, (SEQ ID NO: 49) GEYFCLASNKMG, (SEQ ID NO: 50)
GGYYCTADNNYG, (SEQ ID NO: 51) GNYSCEAENAWGTK, (SEQ ID NO: 52)
GEYTCLAENSLG, (SEQ ID NO: 53) GEYECVAENGRLG, (SEQ ID NO: 54)
GNYTCVVENKFGR, (SEQ ID NO: 55) GEYTCLAGNSIG, (SEQ ID NO: 56)
GEYFCVASNPIG, (SEQ ID NO: 57) EYTCIANNQAGE, (SEQ ID NO: 58)
GMYQCVAENKHLG, (SEQ ID NO: 59) GEYMCTASNTIGQ, (SEQ ID NO: 60)
EYVCIAENKAGEQ, (SEQ ID NO: 61) GDYTLIAKNEYGK, (SEQ ID NO: 62)
GFYQCVAENEAG, (SEQ ID NO: 63) GKYECVATNSAGTR, (SEQ ID NO: 64)
GEYFCVYNNSLG, (SEQ ID NO: 65) GEYECAATNAHGR, (SEQ ID NO: 66)
GAYWCQGTNSVGK, (SEQ ID NO: 67) GTYSCVAENILG, (SEQ ID NO: 68)
RVAAVNGKGQGDYS, (SEQ ID NO: 69) RVAAINGCGIGPFS, (SEQ ID NO: 70)
AVLNGKGLG, (SEQ ID NO: 71) ALNGQGLGATS, (SEQ ID NO: 72)
RLAAKNRAGLGE, (SEQ ID NO: 73) RLGVVTGKDLGEI, (SEQ ID NO: 74)
TVTGLKPETSYMVK, (SEQ ID NO: 75) TLTGLKPSTRYRI, (SEQ ID NO: 76)
TLTGLQPSTRYRV, (SEQ ID NO: 77) TLLGLKPDTTYDIK, (SEQ ID NO: 78)
TLQGLRPETAYELR, (SEQ ID NO: 79) TLRGLRPETAYELR, (SEQ ID NO: 80)
TLMNLRPKTGYSVR, (SEQ ID NO: 81) TVSGLKPGTRY, (SEQ ID NO: 82)
TISGLKPDTTY, (SEQ ID NO: 83) TLQGLKPDTAY, (SEQ ID NO: 84)
LRGLKPWTQYAV, (SEQ ID NO: 85) IDGLEPDTEYIVR, (SEQ ID NO: 86)
LQGLKPWTQYAI, (SEQ ID NO: 87) TITGLEPGTEYTIQ, (SEQ ID NO: 88)
GLKPWTQYAV, (SEQ ID NO: 89) TLASLKPWTQYAV, (SEQ ID NO: 90)
LMGLQPATEYIV, (SEQ ID NO: 91) KGMGPMSEAVQFRT, (SEQ ID NO: 92)
TLTGLKPDTTYDVK, (SEQ ID NO: 93) ISGLQPETSYSL, (SEQ ID NO: 94)
TLLGLKPDTTYDIK, (SEQ ID NO: 95) TISGLTPETTYSI, (SEQ ID NO: 96)
GNYSCLAENRLGR, (SEQ ID NO: 97) GNYTCVVENRVG, (SEQ ID NO: 98)
GTYHCVATNAHG, (SEQ ID NO: 99) LSHNGVLTGYLLSY, (SEQ ID NO: 100)
NGVLTGYVLRY, (SEQ ID NO: 101) NGVLTGYNLRY, (SEQ ID NO: 102)
NGNLTGYLLQY, (SEQ ID NO: 103) VDENGVLTGYKIYY, (SEQ ID NO: 104)
THNGALVGYSVRY, (SEQ ID NO: 105) NGILTEYILKY, (SEQ ID NO: 106)
NGILIGYTLRY, (SEQ ID NO: 107) THSGQITGYKIRY, (SEQ ID NO: 108)
NGKITGYIIYY, (SEQ ID NO: 109) LSHNGIFTLY, (SEQ ID NO: 110)
NGILTEYTLKY, (SEQ ID NO: 111) LDPNGIITQYEISY, (SEQ ID NO: 112)
NGKITGYIIYY, (SEQ ID NO: 113) HLEVQAFNGRGSGPA, (SEQ ID NO: 114)
HLTVRAYNGAGYGP, (SEQ ID NO: 115) HLSVKAYNSAGTGPS, (SEQ ID NO: 116)
HLAVKAYNSAGTGPS, (SEQ ID NO: 117) NLEVRAFNSAGDGP, (SEQ ID NO: 118)
HLTVLAYNSKGAGP, (SEQ ID NO: 119) LRVLVFNGRGDGP, (SEQ ID NO: 120)
HIDVSAFNSAGYGP, (SEQ ID NO: 121) HLAVELFNGR, (SEQ ID NO: 122)
LELQSINFLGGQPA, (SEQ ID NO: 123)
HFTVRAYNGAGYGP, (SEQ ID NO: 124) HLEVQAFNGRGSQPA, (SEQ ID NO: 125)
VIADQPTFVKYLIK, (SEQ ID NO: 126) TIKGLRPGVVYEGQ, (SEQ ID NO: 127)
TLTELSPSTQYTVK, (SEQ ID NO: 128) TLDDLAPDTTYLVQ, (SEQ ID NO: 129)
TVSDVTPHAIYTVR, (SEQ ID NO: 130) IIRGLNASTRYLFR, (SEQ ID NO: 131)
TLMNLRPKTGYSVR, (SEQ ID NO: 132) TLTGLKPGTEYEVR, (SEQ ID NO: 133)
GPEHLMPSSTYVAR, (SEQ ID NO: 134) RVTGLTPKKTYEFR, (SEQ ID NO: 135)
LTGLKPGTEYEFR, (SEQ ID NO: 136) EVRVQAVNGGGNGPP, (SEQ ID NO: 137)
LIKVVAINDRGE, (SEQ ID NO: 138) VVSIIAVNGREE, (SEQ ID NO: 139)
VVSVYAQNQNGE, (SEQ ID NO: 140) TISLVAEKGRHK, (SEQ ID NO: 141)
HLEVQAFNGRGSGPA, (SEQ ID NO: 142) HVEVQAFNGRGLGPA, (SEQ ID NO: 143)
HVEVQAFNGRGLGPA, (SEQ ID NO: 144) EFRVRAVNGAGEG, (SEQ ID NO: 145)
VARVRTRLAPGSRLS (SEQ ID NO: 146)
or a variant, or a fragment, or a homoloque thereof.
[0265] According to the invention a peptide compound in one
embodiment comprises at least one of the above sequences. In
another embodiment, the compound is essentially comprising at least
one of the above sequences. In still another embodiment, the
compound is consisting of at least one of the above sequences.
[0266] The fragments, variants and homologues of the above
sequences are defined according to the criteria for fragments,
variants and homologues of a peptide compound described above.
[0267] It is an objective of the present invention to provide one
or more the above amino acid sequences for the manufacture of a
medicament.
[0268] Another objective of the invention is to use the above
sequences as antigenic epitopes for the production of
antibodies.
Antibodies
[0269] It an objective of the present invention to provide an
antibody capable of selectively binding to an epitope comprising
the binding site of the invention. Therefore, the invention relates
to any antibody capable of selectively binding to an epitope
comprising [0270] 1) any of the sequences set forth in SEQ ID NOS:
1-10, 100, 125, or fragments or variants, or homologues said
sequences, or fragments or variants of said homologues, wherein
[0271] i) a fragment is defined as an amino acid sequence having at
least 40%, more preferably at least 50%, more preferably at least
60%, more preferably at least 70%, more preferably at least 80%,
more preferably at least 90%, more preferably at least 95% of the
length of a predetermined sequence set forth in SEQ ID NOS: 1-10,
100, 125, wherein an amino acid sequence homology between a
fragment and the predetermined sequence is 100%, [0272] ii) a
variant is defined as an amino acid sequence having at least 60%,
more preferably at least 70%, more preferably at least 80%, more
preferably at least 90%, more preferably 95% homology to a sequence
selected from SEQ ID NOS: 1-10, 100, 125 or an amino acid sequence
having at least 60%, more preferably at least 70%, more preferably
at least 80%, more preferably at least 90%, more preferably 95%
positive amino acid matches compared to a sequence selected from
SEQ ID NOS: 1-10, 100, 125, wherein a positive amino acid match is
defined as an identity or similarity defined by physical and/or
chemical properties of the amino acids having the same position in
two compared sequences. Preferred positive amino acid matches of
the present invention are K to R, E to D, L to M, Q to E, I to V, I
to L, A to S, Y to W, K to Q, S to T. N to S and Q to R. The
homology of one amino acid sequence with another amino acid is
defined as a percentage of identical amino acids in the two
collated sequences, [0273] iii) a homologue is defined as an amino
acid sequence which has less then 60% and more then 19%, such as
50-59%, for example 55%, such as 40-49%, for example 45%, such as
30-39%, for example 35%, such as 20-29%, for example 25% homology
to any of the sequences set forth in SEQ ID NOS: 1-10, 100, 125
having remained some of the physical properties of the
predetermined sequences, such as for example the three-dimensional
structure or some of the functional properties, such as for example
a capability to interact with another molecule, particularly with a
receptor molecule, [0274] iv) a variant of a homologue is defined
as an amino acid sequence having at least 60%, more preferably at
least 70%, more preferably at least 80%, more preferably at least
90%, more preferably 95% positive amino acid matches compared to a
homologue of any of the sequences selected from SEQ ID NO: 1-10,
100, 125 wherein the positive amino acid matches being as the
defined above; and/or [0275] 2) any of the sequences identified as
SEQ ID NOS: 11-99, 101-124, 126-146 or fragments or variants, or
homologues said sequences, or fragments or variants of said
homologues, wherein said fragments or variants, or homologues said
sequences, or fragments or variants of said homologues being
defined as the above, and/or [0276] 3) a peptide fragment having
structural characteristics, such as for example strand-loop-strand
fold, similar to the structural characteristics of a peptide
fragment corresponding to SEQ ID NO: 1, wherein the sequences
homology between the aligned peptide fragments is at least 40%
and/or the positive amino acid match is at least 50%.
[0277] In one embodiment the antibody is capable to modulate the
cell-surface receptor function by decreasing or increasing the
capability of a ligand to activate the receptor by modulating the
binding of said ligand to said receptor. By "modulating binding" in
the present context is meant a capability of an antibody to
increase/decrease the affinity and/or strength of ligand-receptor
interaction. In a more preferred embodiment the antibody is capable
to bind to the described above epitope in a FGFR ligand and thereby
to modulate activating of FGFR. By "modulate" in the present
context is meant to promote or inhibit the activating of the
receptor. Preferred embodiments for these FGFR ligands are
described above.
[0278] In another embodiment the antibody is capable to modulate
the biological function of a cell-surface receptor ligand by
interfering with the binding of said ligand to said receptor. This
embodiment relates to those FGFR ligands that [0279] (i) may be the
receptor molecules independent from FGFR, and/or [0280] (ii) may
use an alternative receptor when the FGFR signaling is inhibited,
and/or [0281] (iii) may have additional biological functions that
are not associated with any downstream signaling.
[0282] Preferred embodiments for such ligands comprising the
epitope of the invention are described above.
[0283] Fragments of the polypeptides comprising the binding site of
the invention can be used to raise antibodies useful in the
invention. It is preferred to use for raising the above described
antibodies the peptide fragments having the sequences set forth in
SEQ ID NOS: 1-146, or fragments or variants, or homologues said
sequences, or fragments or variants of said homologues. In general,
the peptides can be coupled to a carrier protein, such as KLH, as
described in Ausubel et al., supra, mixed with an adjuvant, and
injected into a host mammal. Such polypeptides or peptide fragments
can be produced by recombinant techniques as described the above or
synthesized (see, for example, "Solid Phase Peptide Synthesis,"
supra; Ausubel et al., supra). Also the carrier could be PPD.
Antibodies can be purified by peptide antigen affinity
chromatography.
[0284] Thus, the invention concerns a method for the production of
antibodies comprising administering to an animal a peptide fragment
comprising at least one sequence selected from the sequences set
forth in SEQ ID NOS: 1-146.
[0285] In particular, various host animals can be immunized by
injection with the above peptide fragments. Host animals include
rabbits, mice, guinea pigs, rats, and chickens. Various adjuvants
that can be used to increase the immunological response depend on
the host species and include Freund's adjuvant (complete and
incomplete), mineral gels such as aluminum hydroxide, surface
active substances such as lysolecithin, pluronic polyols,
polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and
dinitrophenol. Potentially useful human adjuvants include BCG
(bacille Calmette-Guerin) and Corynebacterium parvum. Immunizations
may also be carried out by the injection of DNA encoding an
FGFR-ligand or fragments thereof corresponding to the binding site.
Polyclonal antibodies are heterogeneous populations of antibody
molecules that are contained in the sera of the immunized
animals.
[0286] Antibodies within the invention therefore include polyclonal
antibodies and, in addition, monoclonal antibodies, humanized or
chimeric antibodies, single chain antibodies, Fab' fragments,
F(ab').sub.2 fragments, and molecules produced using a Fab
expression library, and antibodies or fragments produced by phage
display techniques.
[0287] Monoclonal antibodies, which are homogeneous populations of
antibodies to a particular antigen, can be prepared using the
peptide fragments comprising the above described binding site and
standard hybridoma technology (see, for example, Kohler et al.,
Nature 256:495, 1975; Kohler et al., Eur. J. Immunol. 6:511, 1976;
Kohler et al., Eur. J. Immunol. 6:292, 1976; Hammerling et al.,
"Monoclonal Antibodies and T Cell Hybridomas," Elsevier, N.Y.,
1981; Ausubel et al., supra).
[0288] In particular, monoclonal antibodies can be obtained by any
technique that provides for the production of antibody molecules by
continuous cell lines in culture such as described in Kohler et
al., Nature 256:495, 1975, and U.S. Pat. No. 4,376,110; the human
B-cell hybridoma technique (Kosbor et al., Immunology Today 4:72,
1983; Cole et al., Proc. Natl. Acad. Sci. USA 80:2026, 1983), and
the EBV-hybridoma technique (Cole et al., "Monoclonal Antibodies
and Cancer Therapy," Alan R. Liss, Inc., pp. 77-96, 1983). Such
antibodies can be of any immunoglobulin class including IgG, IgM,
IgE, IgA, IgD and any subclass thereof. (In the case of chckens,
the immunoglobulin class can also be IgY.) The hybridoma producing
the mAb of this invention may be cultivated in vitro or in vivo.
The ability to produce high titers of mAbs in vivo makes this the
presently preferred method of production, but in some cases, in
vitro production will be preferred to avoid introducing cancer
cells into live animals, for example, in cases where the presence
of normal immunoglobulins coming from the acitis fluids are
unwanted, or in cases involving ethical considerations.
[0289] Once produced, polyclonal, monoclonal, or phage-derived
antibodies are tested for specific recognition of the above
described epitope by Western blot or immunoprecipitation in samples
containing the polypeptides comprising the binding site or
fragments thereof, e.g., as described in Ausubel et al., supra.
Antibodies that specifically recognise and bind to a FGFR ligand
are useful in the invention. For example, such antibodies can be
used in an immunoassay to monitor the level of expression a FGFR
ligand in a sample collected from an individual.
[0290] In some cases it may be desirable to minimize the potential
problems of low affinity or specificity of antisera. In such
circumstances, two or three fusions can be generated for each
protein, and each fusion can be injected into at least two animals.
Antisera can be raised by injections in a series, preferably
including at least three booster injections. Spleen cells from the
immunised animals may be used for generating monoclonal
antibodies.
[0291] An antibody capable of binding to an epitope as defined
above may be used for determining a substance comprising an epitope
comprising at least one of the sequences set forth in SEQ ID NOS:
1-146, or fragments, or variants, or homologues of said sequences,
or fragments or variants of said homologues in a sample. The sample
may be any sample comprising the substance, for example a solution
of the substance or a biological sample. The substance comprising
an epitope may be of natural or synthetic origin. The examples of
such a substance include, but not limited a cell comprising a
compound comprising an epitope, a compound comprising an epitope, a
compound consisting of an epitope. The cell comprising a compound
comprising an epitope may be for example a cell expressing a ligand
of a cell surface receptor of the invention. Examples of such
ligands are described above. The compound comprising the epitope
may be any compound described above.
[0292] The above antibodies may for example be used in the
detection of an FGFR ligand in a biological sample. The antibody
may also be used in a screening assay for measuring the activity of
FGFR, for example as a part of a diagnostic assay. Depending on the
detection technique the antibody may be coupled to a compound
comprising a detectable marker. The markers or labels may be
selected from any markers and labels known in the art. The antibody
may also be used for determining the concentration of a substance
comprising an epitope or epitope in a solution of said substance or
said epitope. A wide spectrum of detection and labelling techniques
is available now in the art and the techniques may therefore be
selected depending on skills of the artisan practising the
antibodies or on the purpose of using thereof.
[0293] In addition, techniques developed for the production of
"chimeric antibodies" (Morrison et al., Proc. Natl. Acad. Sci. USA,
81:6851, 1984; Neuberger et al., Nature, 312:604, 1984; Takeda et
al., Nature, 314:452, 1984) by splicing the genes from a mouse
antibody molecule of appropriate antigen specificity together with
genes from a human antibody molecule of appropriate biological
activity can be used. A chimeric antibody is a molecule in which
different portions are derived from different animal species, such
as those having a variable region derived from a murine mAb and a
human immunoglobulin constant region.
[0294] Alternatively, techniques described for the production of
single chain antibodies (U.S. Pat. Nos. 4,946,778, 4,946,778, and
4,704,692) can be adapted to produce single chain antibodies
against an FGFR-ligand or a fragment thereof. Single chain
antibodies are formed by linking the heavy and light chain
fragments of the Fv region via an amino acid bridge, resulting in a
single chain polypeptide.
[0295] Antibody fragments that recognise and bind to specific
epitopes can be generated by known techniques. For example, such
fragments include but are not limited to F(ab').sub.2 fragments
that can be produced by pepsin digestion of the antibody molecule,
and Fab' fragments that can be generated by reducing the disulfide
bridges of F(ab').sub.2 fragments. Alternatively, Fab' expression
libraries can be constructed (Huse et al., Science, 246:1275, 1989)
to allow rapid and easy identification of monoclonal Fab' fragments
with the desired specificity.
[0296] Antibodies can be humanized by methods known in the art. For
example, monoclonal antibodies with a desired binding specificity
can be commercially humanized (Scotgene, Scotland; Oxford
Molecular, Palo Alto, Calif.). Fully human antibodies, such as
those expressed in transgenic animals are also features of the
invention (Green et al., Nature Genetics 7:13-21, 1994; see also
U.S. Pat. Nos. 5,545,806 and 5,569,825, both of which are hereby
incorporated by reference).
Production of Polypeptides and Peptide Fragments
[0297] Polypeptides and peptide fragments of the invention can be
provided by any suitable conventional method known in the art. The
peptide fragments and polypeptides of the invention can be for
example chemically synthesized (for example, see Creighton,
"Proteins: Structures and Molecular Principles," W.H. Freeman &
Co., NY, 1983), or, perhaps more advantageously, produced by
recombinant DNA technology as described herein. For additional
guidance, skilled artisans may consult Ausubel et al. (supra),
Sambrook et al. ("Molecular Cloning, A Laboratory Manual," Cold
Spring Harbor Press, Cold Spring Harbor, N.Y., 1989), and,
particularly for examples of chemical synthesis Gait, M. J. Ed.
("Oligonucleotide Synthesis," IRL Press, Oxford, 1984).
Recombinant Molecules
[0298] The methods and the assay described above all include a step
of providing at least two interacting proteins.
[0299] The two interacting proteins of the methods and the assay of
the invention are preferably recombinant proteins. Therefore, the
invention in additional embodiment encompasses expression vectors
that contain the coding DNA sequences for the interacting proteins
and genetically engineered host cells that contain any of the
foregoing expression vectors and thereby express the nucleic acid
molecules of the invention in the host cell.
[0300] Preferred embodiments for the expression vectors are: (a)
expression vectors that contain any of the foregoing FGFRs,
including FGFR1, FGFR2, FGFR3 and FGFR4, or FGFR-ligand related
coding sequences, said FGFR-ligand being selected from the
preferred polypeptides of the invention described above, and/or
their complements (that is, "antisense" sequence); (b) expression
vectors that contain any of the foregoing FGFRs or FGFR-ligand
related coding sequences comprising the above mutation operatively
associated with a regulatory element (examples of which are given
below) that directs the expression of the coding sequences; (c)
expression vectors containing, in addition to sequences encoding
mutated FGFRs or FGFR-ligands, nucleic acid sequences that are
unrelated to nucleic acid sequences encoding the FGFRs or
FGFR-ligands, such as molecules encoding a reporter or marker.
[0301] Recombinant nucleic acid molecule may contain a sequence
encoding mutated FGFRs or FGFR-ligands, soluble FGFRs or
FGFR-ligands, truncated FGFRs or FGFR-ligands, or functional
domains of FGFRs, such as for example Ig 2 or Ig 3 domain of FGFR1,
or the domains of a FGFR ligand, such as for example the domains of
NCAM, for example the Ig-like or F3 type domains. The full-length
mutated polypeptides, domains of FGFRs or FGFR-ligands, or
fragments thereof may be fused to additional polypeptides, as
described below.
[0302] The regulatory elements referred to above include, but are
not limited to, inducible and non-inducible promoters, enhancers,
operators and other elements, which are known to those skilled in
the art, and which drive or otherwise regulate gene expression.
Such regulatory elements include but are not limited to the
cytomegalovirus hCMV immediate early gene, the early or late
promoters of SV40 adenovirus, the lac system, the trD system, the
TAC system, the TRC system, the major operator and promoter regions
of phage A, the control regions of fd coat protein, the promoter
for 3-phosphoglycerate kinase, the promoters of acid phosphatase,
and the promoters of the yeast--mating factors.
[0303] Similarly, the nucleic acid can form part of a hybrid gene
encoding additional polypeptide sequences, for example, sequences
that function as a marker or reporter. Examples of marker or
reporter genes include -lactamase, chloramphenicol
acetyltransferase (CAT), adenosine deaminase (ADA), aminoglycoside
phosphotransferase (neo.sup.r, G418.sup.r), dihydrofolate reductase
(DHFR), hygromycin-B-phosphotransferase (HPH), thymidine kinase
(TK), lacZ (encoding galactosidase), green fluorescent protein
(GFP), and xanthine guanine phosphoribosyltransferase (XGPRT). As
with many of the standard procedures associated with the practice
of the invention, skilled artisans will be aware of additional
useful reagents, for example, of additional sequences that can
serve the function of a marker or reporter. Generally, the hybrid
polypeptide will include a first portion and a second portion; the
first portion being, for example, a portion of the FGFR1 or NCAM
amino acid sequence and the second portion being, for example, the
reporter described above or an immunoglobulin heavy chain.
[0304] The expression systems that may be used for purposes of the
invention include, but are not limited to, microorganisms such as
bacteria (for example, E. coli and B. subtilis) transformed with
recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA
expression vectors containing the nucleic acid molecules of the
invention; yeast (for example, Saccharomyces and Pichia)
transformed with recombinant yeast expression vectors containing
the nucleic acid molecules encoding FGFR1 or the nucleic acid
sequence encoding NCAM; insect cell systems infected with
recombinant virus expression vectors (for example, baculovirus)
containing the nucleic acid molecules of the invention; plant cell
systems infected with recombinant virus expression vectors (for
example, cauliflower mosaic virus (CaMV) and tobacco mosaic virus
(TMV)) or transformed with recombinant plasmid expression vectors
(for example, Ti plasmid) containing FGFR nucleotide sequences; or
mammalian cell systems (for example, COS, CHO, BHK, 293, VERO,
HeLa, MDCK, W138, and NIH 3T3 cells) harbouring recombinant
expression constructs containing promoters derived from the genome
of mammalian cells (for example, the metallothionein promoter) or
from mammalian viruses (for example, the adenovirus late promoter
and the vaccinia virus 7.5K promoter).
[0305] In bacterial systems, a number of expression vectors may be
advantageously selected depending upon the use intended for the
gene product being expressed. Such vectors include, but are not
limited to, the E. coli expression vector pUR278 (Ruther et al.,
EMBO J. 2:1791, 1983), in which the coding sequence of the insert
may be ligated individually into the vector in frame with the lacZ
coding region so that a fusion protein is produced; pIN vectors
(Inouye and Inouye, Nucleic Acids Res. 13:3101-3109, 1985; Van
Heeke and Schuster, J. Biol. Chem. 264:5503-5509, 1989); and the
like. pGEX vectors may also be used to express foreign polypeptides
as fusion proteins with glutathione S-transferase (GST). In
general, such fusion proteins are soluble and can easily be
purified from lysed cells by adsorption to glutathione-agarose
beads followed by elution in the presence of free glutathione. The
pGEX vectors are designed to include thrombin or factor Xa protease
cleavage sites so that the cloned target gene product can be
released from the GST moiety.
[0306] In an insect system, Autographa californica nuclear
polyhidrosis virus (AcNPV) can be used as a vector to express
foreign genes. The virus grows in Spodoptera frugiperda cells. The
coding sequence of the insert may be cloned individually into
non-essential regions (for example the polyhedrin gene) of the
virus and placed under control of an AcNPV promoter (for example
the polyhedrin promoter). Successful insertion of the coding
sequence will result in inactivation of the polyhedrin gene and
production of non-occluded recombinant virus (i.e., virus lacking
the proteinaceous coat coded for by the polyhedrin gene). These
recombinant viruses are then used to infect Spodoptera frugiperda
cells in which the inserted gene is expressed. (for example, see
Smith et al., J. Virol. 46:584, 1983; Smith, U.S. Pat. No.
4,215,051).
[0307] In mammalian host cells, a number of viral-based expression
systems may be utilized. In cases where an adenovirus is used as an
expression vector, the nucleic acid molecule of the invention may
be ligated to an adenovirus transcription/translation control
complex, for example, the late promoter and tripartite leader
sequence. This chimeric gene may then be inserted in the adenovirus
genome by in vitro or in vivo recombination. Insertion in a
non-essential region of the viral genome (for example, region E1 or
E3) will result in a recombinant virus that is viable and capable
of expressing a FGFR gene product in infected hosts (for example,
see Logan and Shenk, Proc. Nat. Acad. Sci. USA 81:3655-3659, 1984).
Specific initiation signals may also be required for efficient
translation of inserted nucleic acid molecules. These signals
include the ATG initiation codon and adjacent sequences. In cases
where an entire gene or cDNA, including its own initiation codon
and adjacent sequences, is inserted into the appropriate expression
vector, no additional translational control signals may be needed.
However, in cases where only a portion of the coding sequence is
inserted, exogenous translational control signals, including,
perhaps, the ATG initiation codon, must be provided. Furthermore,
the initiation codon must be in phase with the reading frame of the
desired coding sequence to ensure translation of the entire insert.
These exogenous translational control signals and initiation codons
can be of a variety of origins, both natural and synthetic. The
efficiency of expression may be enhanced by the inclusion of
appropriate transcription enhancer elements, transcription
terminators, etc. (see Bittner et al., Methods in Enzymol.
153:516-544, 1987).
[0308] In addition, a host cell strain may be chosen, which
modulates the expression of the inserted sequences, or modifies and
processes the gene product in the specific fashion desired. Such
modifications (for example, glycosylation) and processing (for
example, cleavage) of protein products may be important for the
function of the protein. Different host cells have characteristic
and specific mechanisms for the post-translational processing and
modification of proteins and gene products. Appropriate cell lines
or host systems can be chosen to ensure the correct modification
and processing of the foreign protein expressed. To this end,
eukaryotic host cells which possess the cellular machinery for
proper processing of the primary transcript, glycosylation, and
phosphorylation of the gene product may be used. The mammalian cell
types listed above are among those that could serve as suitable
host cells.
[0309] For long-term, high-yield production of recombinant
proteins, stable expression is preferred. For example, cell lines,
which stably express the FGFR sequences or any FGFR ligand
sequences described above may be engineered. Rather than using
expression vectors which contain viral origins of replication, host
cells can be transformed with DNA controlled by appropriate
expression control elements (for example, promoter, enhancer
sequences, transcription terminators, polyadenylation sites, etc.),
and a selectable marker. Following the introduction of the foreign
DNA, engineered cells may be allowed to grow for 1-2 days in an
enriched media, and then switched to a selective media. The
selectable marker in the recombinant plasmid confers resistance to
the selection and allows cells to stably integrate the plasmid into
their chromosomes and grow to form foci which in turn can be cloned
and expanded into cell lines. Such engineered cell lines may be
particularly useful in the screening assay and evaluation of
candidatecompounds that affect the interaction between FGFR and an
FGFR ligand.
[0310] A number of selection systems can be used. For example, the
herpes simplex virus thymidine kinase (Wigler, et al., Cell 11:223,
1977), hypoxanthine-guanine phosphoribosyltransferase (Szybalska
and Szybalski, Proc. Natl. Acad. Sci. USA 48:2026, 1962), and
adenine phosphoribosyltransferase (Lowy, et al., Cell 22:817, 1980)
genes can be employed in tk.sup.-, hgprt.sup.- or aprt.sup.- cells,
respectively. Also, antimetabolite resistance can be used as the
basis of selection for the following genes: dhfr, which confers
resistance to methotrexate (Wigler et al., Proc. Natl. Acad. Sci.
USA 77:3567, 1980; O'Hare et al., Proc. Natl. Acad. Sci. USA
78:1527, 1981); gpt, which confers resistance to mycophenolic acid
(Mulligan and Berg, Proc. Natl. Acad. Sci. USA 78:2072, 1981); neo,
which confers resistance to the aminoglycoside G-418
(Colberre-Garapin et al., J. Mol. Biol. 150:1, 1981); and hygro,
which confers resistance to hygromycin (Santerre et al., Gene
30:147, 1984).
[0311] Alternatively, any fusion protein may be readily purified by
utilizing an antibody specific for the fusion protein being
expressed. For example, a system described by Janknecht et al.
allows for the ready purification of non-denatured fusion proteins
expressed in human cell lines (Proc. Nail. Acad. Sci. USA 88:
8972-8976, 1991). In this system, the gene of interest is subcloned
into a vaccinia recombination plasmid such that the gene's open
reading frame is translationally fused to an amino-terminal tag
consisting of six histidine residues. Extracts from cells infected
with recombinant vaccinia virus are loaded onto Ni.sup.2+.
nitriloacetic acid-agarose columns and histidine-tagged proteins
are selectively eluted with imidazole-containing buffers.
[0312] It is presumed that molecules of one group of the
recombinant proteins of the invention comprise the described above
binding site, and molecules of another group of the recombinant
proteins comprise a site for recognition of said binding site.
Medicament
[0313] Activation of cell-surface receptors upon the ligand binding
is strictly regulated in a healthy organism. Mutations, abnormal
expression or processing of a receptor or the receptor ligands lead
to abnormalities in activity of the receptor and therefore lead to
dysfunction of the receptor. The dysfunction of the receptor is in
turn a reason for dysfunction of the cells which use the receptor
for maintenance of various cellular processes. The latter is a
manifestation of a disease. FGFRs are expressed by a wide variety
of cell species during embryonic development and in the adult.
Dysfunction of these receptors has been associated with a number of
diseases.
[0314] Accordingly, it is an objective of the invention to provide
a compound capable of modulation of the activity of FGFRs, said
compound being concerned by the invention as a medicament for the
treatment of diseases, wherein modulation of the activity of FGFRs
may be considered as an essential condition for the curing.
[0315] Thus, the medicament of the invention is in one embodiment
for the treatment of [0316] 1) normal, degenerated or damaged NCAM
presenting cells, and/or [0317] 2) diseases and conditions of the
central and peripheral nervous system, or of the muscles or of
various organs, and/or [0318] 3) diseases or conditions of the
central and peripheral nervous system, such as postoperative nerve
damage, traumatic nerve damage, impaired myelination of nerve
fibers, postischaemic damage, e.g. resulting from a stroke,
Parkinson's disease, Alzheimer's disease, Huntington's disease,
dementias such as multiinfarct dementia, sclerosis, nerve
degeneration associated with diabetes mellitus, disorders affecting
the circadian clock or neuro-muscular transmission, and
schizophrenia, mood disorders, such as manic depression; for
treatment of diseases or conditions of the muscles including
conditions with impaired function of neuro-muscular connections,
such as after organ transplantation, or such as genetic or
traumatic atrophic muscle disorders; or for treatment of diseases
or conditions of various organs, such as degenerative conditions of
the gonads, of the pancreas such as diabetes mellitus type I and
II, of the kidney such as nephrosis and of the heart, liver and
bowel, and/or [0319] 4) postoperative nerve damage, traumatic nerve
damage, impaired myelination of nerve fibers, postischaemic, e.g.
resulting from a stroke, Parkinson's disease, Alzheimer's disease,
Huntington's disease, dementias such as multiinfarct dementia,
sclerosis, nerve degeneration associated with diabetes mellitus,
disorders affecting the circadian clock or neuro-muscular
transmission, and schizophrenia, mood disorders, such as manic
depression, and/or [0320] 5) cancer disease, wherein the cancer is
any type of solid tumors requiring neoangiogenesis.
[0321] In another embodiment the medicament of the invention is for
the manufacture of a medicament for [0322] 1) promotion of
wound-healing, and/or [0323] 2) prevention of cell death of heart
muscle cells, such as after acute myocardial infarction, or after
angiogenesis, and/or [0324] 3) revascularsation, and/or [0325] 4)
stimulation of the ability to learn and/or of the short and/or
long-term memory.
[0326] In one embodiment the medicament of the invention comprises
at least one the amino acid sequences set forth in SEQ ID NOS:
2-146, or fragments or variants, or homologues said sequences, or
fragments or variants of said homologues. In another embodiment the
medicament of the invention comprises an antibody capable of
binding to an epitope comprising the binding site of the invention,
or a fragment, or a variant of said antibody.
[0327] The medicament of the invention comprises an effective
amount of one or more of the compounds as defined above, or a
composition as defined above in combination with pharmaceutically
acceptable additives. Such medicament may suitably be formulated
for oral, percutaneous, intramuscular, intravenous, intracranial,
intrathecal, intracerebroventricular, intranasal or pulmonal
administration.
[0328] Strategies in formulation development of medicaments and
compositions based on the compounds of the present invention
generally correspond to formulation strategies for any other
protein-based drug product. Potential problems and the guidance
required to overcome these problems are dealt with in several
textbooks, e.g. "Therapeutic Peptides and Protein Formulation.
Processing and Delivery Systems", Ed. A. K. Banga, Technomic
Publishing AG, Basel, 1995.
[0329] Injectables are usually prepared either as liquid solutions
or suspensions, solid forms suitable for solution in, or suspension
in, liquid prior to injection. The preparation may also be
emulsified. The active ingredient is often mixed with excipients
which are pharmaceutically acceptable and compatible with the
active ingredient. Suitable excipients are, for example, water,
saline, dextrose, glycerol, ethanol or the like, and combinations
thereof. In addition, if desired, the preparation may contain minor
amounts of auxiliary substances such as wetting or emulsifying
agents, pH buffering agents, or which enhance the effectiveness or
transportation of the preparation.
[0330] Formulations of the compounds of the invention can be
prepared by techniques known to the person skilled in the art. The
formulations may contain pharmaceutically acceptable carriers and
excipients including microspheres, liposomes, microcapsules,
nanoparticles or the like.
[0331] The preparation may suitably be administered by injection,
optionally at the site, where the active ingredient is to exert its
effect. Additional formulations which are suitable for other modes
of administration include suppositories, nasal, pulmonal and, in
some cases, oral formulations. For suppositories, traditional
binders and carriers include polyalkylene glycols or triglycerides.
Such suppositories may be formed from mixtures containing the
active ingredient(s) in the range of from 0.5% to 10%, preferably
1-2%. Oral formulations include such normally employed excipients
as, for example, pharmaceutical grades of mannitol, lactose,
starch, magnesium stearate, sodium saccharine, cellulose, magnesium
carbonate, and the like. These compositions take the form of
solutions, suspensions, tablets, pills, capsules, sustained release
formulations or powders and generally contain 10-95% of the active
ingredient(s), preferably 25-70%.
[0332] Other formulations are such suitable for nasal and pulmonal
administration, e.g. inhalators and aerosols.
[0333] The active compound may be formulated as neutral or salt
forms. Pharmaceutically acceptable salts include acid addition
salts (formed with the free amino groups of the peptide compound)
and which are formed with inorganic acids such as, for example,
hydrochloric or phosphoric acids, or such organic acids as acetic
acid, oxalic acid, tartaric acid, mandelic acid, and the like.
Salts formed with the free carboxyl group may also be derived from
inorganic bases such as, for example, sodium, potassium, ammonium,
calcium, or ferric hydroxides, and such organic bases as
isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine,
procaine, and the like.
[0334] The preparations are administered in a manner compatible
with the dosage formulation, and in such amount as will be
therapeutically effective. The quantity to be administered depends
on the subject to be treated, including, e.g. the weight and age of
the subject, the disease to be treated and the stage of disease.
Suitable dosage ranges are per kilo body weight normally of the
order of several hundred .mu.g active ingredient per administration
with a preferred range of from about 0.1 .mu.g to 5000 .mu.g per
kilo body weight. Using monomeric forms of the compounds, the
suitable dosages are often in the range of from 0.1 .mu.g to 5000
.mu.g per kilo body weight, such as in the range of from about 0.1
.mu.g to 3000 .mu.g per kilo body weight, and especially in the
range of from about 0.1 .mu.g to 1000 .mu.g per kilo body weight.
Using multimeric forms of the compounds, the suitable dosages are
often in the range of from 0.1 .mu.g to 1000 .mu.g per kilo body
weight, such as in the range of from about 0.1 .mu.g to 750 .mu.g
per kilo body weight, and especially in the range of from about 0.1
.mu.g to 500 .mu.g per kilo body weight such as in the range of
from about 0.1 .mu.g to 250 .mu.g per kilo body weight. In
particular when administering nasally smaller dosages are used than
when administering by other routes. Administration may be performed
once or may be followed by subsequent administrations. The dosage
will also depend on the route of administration and will vary with
the age and weight of the subject to be treated. A preferred dosage
of multimeric forms would be in the interval 1 mg to 70 mg per 70
kg body weight.
[0335] For most indications a localised or substantially localised
application is preferred.
[0336] Some of the compounds of the present invention are
sufficiently active, but for some of the others, the effect will be
enhanced if the preparation further comprises pharmaceutically
acceptable additives and/or carriers. Such additives and carriers
will be known in the art. In some cases, it will be advantageous to
include a compound, which promote delivery of the active substance
to its target.
[0337] In many instances, it will be necessary to administrate the
formulation multiple times. Administration may be a continuous
infusion, such as intraventricular infusion or administration in
more doses such as more times a day, daily, more times a week,
weekly, etc. It is preferred that administration of the medicament
is initiated before or shortly after the individual has been
subjected to the factor(s) that may lead to cell death. Preferably
the medicament is administered within 8 hours from the factor
onset, such as within 5 hours from the factor onset. Many of the
compounds exhibit a long term effect whereby administration of the
compounds may be conducted with long intervals, such as 1 week or 2
weeks.
[0338] In connection with the use in nerve guides, the
administration may be continuous or in small portions based upon
controlled release of the active compound(s). Furthermore,
precursors may be used to control the rate of release and/or site
of release. Other kinds of implants and well as oral administration
may similarly be based upon controlled release and/or the use of
precursors.
Treatment
[0339] Treatment by the use of the compounds/compositions according
to the invention is in one embodiment useful for inducing
differentiation, modulating proliferation, stimulate regeneration,
neuronal plasticity and survival of cells being implanted or
transplanted. This is particularly useful when using compounds
having a long term effect.
[0340] In further embodiment the treatment may be for stimulation
of survival of cells which are at risk of dying due to a variety of
factors, such as traumas and injuries, acute diseases, chronic
diseases and/or disorders, in particular degenerative diseases
normally leading to cell death, other external factors, such as
medical and/or surgical treatments and/or diagnostic methods that
may cause formation of free radicals or otherwise have cytotoxic
effects, such as X-rays and chemotherapy. In relation to
chemotherapy the FGFR binding compounds according to the invention
are useful in cancer treatment of all cancer cells presenting
FGFRs.
[0341] Thus, the treatment comprises treatment and/or prophylaxis
of cell death in relation to diseases or conditions of the central
and peripheral nervous system, such as postoperative nerve damage,
traumatic nerve damage, e.g. resulting from spinal cord injury,
impaired myelination of nerve fibers, postischaemic damage, e.g.
resulting from a stroke, multiinfarct dementia, multiple sclerosis,
nerve degeneration associated with diabetes mellitus,
neuro-muscular degeneration, schizophrenia, Alzheimer's disease,
Parkinson's disease, or Huntington's disease.
[0342] Also, in relation to diseases or conditions of the muscles
including conditions with impaired function of neuro-muscular
connections, such as genetic or traumatic atrophic muscle
disorders; or for the treatment of diseases or conditions of
various organs, such as degenerative conditions of the gonads, of
the pancreas, such as diabetes mellitus type I and II, of the
kidney, such as nephrosis the compounds according to the invention
may be used for inducing differentiation, modulating proliferation,
stimulate regeneration, neuronal plasticity and survival, i.e.
stimulating survival.
[0343] Furthermore, the compound and/or pharmaceutical composition
may be for preventing cell death of heart muscle cells, such as
after acute myocardial infarction, in order to induce angiogenesis.
Furthermore, in one embodiment the compound and/or pharmaceutical
composition is for the stimulation of the survival of heart muscle
cells, such as survival after acute myocardial infarction. In
another aspect the compound and/or pharmaceutical composition is
for revascularisation, such as after injuries.
[0344] It is also within the scope of the invention to use the
compound and/or pharmaceutical composition for the promotion of
wound-healing. The present compounds are capable of stimulating
angiogenesis and thereby promote the wound healing process.
[0345] The invention further discloses the use of the compound
and/or pharmaceutical composition in the treatment of cancer.
Regulation of activation of FGFR is important for tumor
agiogenesis, proliferation and spreading.
[0346] In yet a further embodiment the use of the compound and/or
pharmaceutical composition is for the stimulation of the ability to
learn and/or of the short and/or long term memory, as FGFR activity
is important for differentiation of neural cells.
[0347] In particular the compound and/or pharmaceutical composition
of the invention may be used in the treatment of clinical
conditions, such as Neoplasms such as malignant neoplasms, benign
neoplasms, carcinoma in situ and neoplasms of uncertain behavior,
diseases of endocrine glands, such as diabetes mellitus, psychoses,
such as senile and presenile organic psychotic conditions,
alcoholic psychoses, drug psychoses, transient organic psychotic
conditions, Alzheimer's disease, cerebral lipidoses, epilepsy,
general paresis [syphilis], hepatolenticular degeneration,
Huntington's chorea, Jakob-Creutzfeldt disease, multiple sclerosis,
Pick's disease of the brain, syphilis, Schizophrenic disorders,
affective psychoses, neurotic disorders, personality disorders,
including character neurosis, nonpsychotic personality disorder
associated with organic brain syndromes, paranoid personality
disorder, fanatic personality, paranoid personality (disorder),
paranoid traits, sexual deviations and disorders, mental
retardation, disease in the nervesystem and sense organs, cognitive
anomalies, inflammatory disease of the central nervous system, such
as meningitis, encephalitis, Cerebral degenerations such as
Alzheimers disease, Pick's disease, senile degeneration of brain,
communicating hydrocephalus, obstructive hydrocephalus, Parkinson's
disease including other extra pyramidal disease and abnormal
movement disorders, spinocerebellar disease, cerebellar ataxia,
Marie's, Sanger-Brown, Dyssynergia cerebellaris myoclonica, primary
cerebellar degeneration, such as spinal muscular atrophy, familial,
juvenile, adult spinal muscular atrophy, motor neuron disease,
amyotrophic lateral sclerosis, motor neuron disease, progressive
bulbar palsy, pseudobulbar palsy, primary lateral sclerosis, other
anterior horn cell diseases, anterior horn cell disease,
unspecified, other diseases of spinal cord, syringomyelia and
syringobulbia, vascular myelopathies, acute infarction of spinal
cord (embolic) (nonembolic), arterial thrombosis of spinal cord,
edema of spinal cord, subacute necrotic myelopathy, subacute
combined degeneration of spinal cord in diseases classified
elsewhere, myelopathy, drug-induced, radiation-induced myelitis,
disorders of the autonomic nervous system, disorders of peripheral
autonomic, sympathetic, parasympathetic, or vegetative system,
familial dysautonomia [Riley-Day syndrome], idiopathic peripheral
autonomic neuropathy, carotid sinus syncope or syndrome, cervical
sympathetic dystrophy or paralysis. peripheral autonomic neuropathy
in disorders classified elsewhere, amyloidosis, diseases of the
peripheral nerve system, brachial plexus lesions, cervical rib
syndrome, costoclavicular syndrome, scalenus anterior syndrome,
thoracic outlet syndrome, brachial neuritis or radiculitis,
including in newborn. Inflammatory and toxic neuropathy, including
acute infective polyneuritis, Guillain-Barre syndrome,
Postinfectious polyneurits, polyneuropathy in collagen vascular
disease, disorders affecting multiple structures of eye, purulent
endophthalmitis, diseases of the ear and mastoid process, chronic
rheumatic heart disease, ischaemic heart disease, arrhythmia,
diseases in the pulmonary system, abnormality of organs and soft
tissues in newborn, including in the nerve system, complications of
the administration of anesthetic or other sedation in labor and
delivery, diseases in the skin including infection, insufficient
circulation problem, injuries, including after surgery, crushing
injury, burns. Injuries to nerves and spinal cord, including
division of nerve, lesion in continuity (with or without open
wound), traumatic neuroma (with or without open wound), traumatic
transient paralysis (with or without open wound), accidental
puncture or laceration during medical procedure, injury to optic
nerve and pathways, optic nerve injury, second cranial nerve,
injury to optic chiasm, injury to optic pathways, injury to visual
cortex, unspecified blindness, injury to other cranial nerve(s),
injury to other and unspecified nerves. Poisoning by drugs,
medicinal and biological substances, genetic or traumatic atrophic
muscle disorders; or for the treatment of diseases or conditions of
various organs, such as degenerative conditions of the gonads, of
the pancreas, such as diabetes mellitus type I and II, of the
kidney, such as nephrosis.
[0348] A further aspect of the invention is a process of producing
a pharmaceutical composition, comprising mixing an effective amount
of one or more of the compounds of the invention, or a
pharmaceutical composition according to the invention with one or
more pharmaceutically acceptable additives or carriers, and
administer an effective amount of at least one of said compound, or
said pharmaceutical composition to a subject.
[0349] In one embodiment of the process as mentioned above, the
compounds are used in combination with a prosthetic device, wherein
the device is a prosthetic nerve guide. Thus, in a further aspect,
the present invention relates to a prosthetic nerve guide,
characterised in that it comprises one or more of the compounds or
the pharmaceutical composition as defined above. Nerve guides are
known in the art.
[0350] The invention relates to the use a pharmaceutical
composition comprising the compound of invention for the treatment
or prophylaxis of any of the diseases and conditions mentioned
above.
[0351] In yet a further aspect the invention relates to a method of
treating a disease or condition as discussed above by administering
a compound as defined herein.
EXAMPLES
Production of Recombinant Proteins
[0352] The NCAM F3 modules 1, 2 (without expression of exons a,
MG), and FGFR1 Ig modules 2, 3 were produced using rat NCAM cDNA
and mouse FGFR1 (IIIC isoform) cDNA. The F3 module 1 and the
combined F3 modules 1, 2 consist of AGHHHHHH and amino acids
507-611 and 507-705 of NCAM (swissprot p13596), respectively. The
F3 module 2 consists of AG and amino acids 612-705 of NCAM
(swissprot p13596), and is sequentially numbered from 1 to 96, A
being numbered 1. The FGFR Ig module 2 consists of AGHHHHHH and
amino acids 140-251 of FGFR (swissprot p16092). The FGFR Ig module
3 and the combined Ig modules 2, 3 consist of RSHHHHHH and amino
acids 249-365 and 141-365 of FGFR (swissprot p16092), respectively.
The F3 modules and the FGFR Ig module 2 were expressed in a KM71
strain of yeast P. pastoris (Invitrogen, USA) as described (Thomsen
et al., 1996). The FGFR Ig module 3 and modules 2, 3 were expressed
in Drosophila S2 cells (Invitrogen, USA) according to the
manufacturer's instructions. All the proteins were purified by
affinity chromatography using Ni.sup.2+-NTA resin (Qiagen, USA)
and/or ion exchange chromatography and gel filtration.
.sup.15N-labelled F3 module 2 was produced as described (Thomsen et
al., 1996). The NCAM Ig modules 1, 2 and 3 (RV and amino acids
20-308 of rat NCAM, swissprot p13596) was produced as described
(Soroka et. al., 2002).
NMR Analysis: the Structure Calculations of the NCAM F3 Module 2
and Identification of the Residues Involved in Binding with FGFR
and ATP
[0353] The following samples were used for the structure
determination of the NCAM F3 module 2: 2 mM module in H.sub.2O or
D.sub.2O and 1 mM .sup.15N-labelled module in H.sub.2O. The buffer
was 30 mM NaCl, 10 mM sodium phosphate buffer, pH 7.27. The
following NMR spectra were recorded and used for assignment: TOCSY
in H.sub.2O or D.sub.2O (45 and 70 ms mixing time), NOESY in
H.sub.2O or D.sub.2O (80 and 200 ms mixing time), DQFCOSY,
.sup.15N-HSQC, .sup.15N-TOCSY-HSQC (70 ms mixing time), and
.sup.15N-NOESY-HSQC (125 ms mixing time) (Bodenhausen and Ruben,
1980; Braunsweiler and Ernst, 1983; Kumar et al., 1981; Piantini et
al., 1982; Zhang et al., 1994). The NMR experiments were performed
on a Bruker AMX-600 MHz and Varian Unity Inova 500, 750 and 800 MHz
spectrometers. All spectra were recorded at 298 K. The assignment
of the .sup.1H and .sup.15N resonances were performed using the
program PRONTO (Kjosr et al., 1994).
Structure Calculation
[0354] A distance geometry/simulated annealing protocol using the
X-PLOR program was used for structure calculation. The NOE
restraints were derived from 80/200 ms NOESY and 125 ms
.sup.15N-NOESY-HSQC spectra with upper bounds of 2.7, 3.3 and 6.0
.ANG. increased by 0.5 .ANG. if the restraint included a methyl
group. 40.phi. angles restraints with bounds of -120.+-.40.degree.
and -57.+-.40.degree. (derived from the .sup.3J.sub.HNH.alpha.
coupling constants) and 4 .chi..sup.1 angles were applied. After
inspection of hydrogen bond energies, 80 hydrogen bond restraints
were applied as NOE restraints with upper bounds of 2 .ANG. and 3
.ANG. for the NH--O and N--O distances, respectively. Of 96
structures calculated, 96 were accepted by X-PLOR, discriminating
any structure with an NOE restraint violation >0.5 .ANG. or an
angle violation >5.degree.. The structures were examined with
MOLMOL, PROCHECK_NMR and WHATCHECK programs. 78 structures had the
absolute values of the Z-scores for the 2.sup.nd generation packing
quality, the Ramachandran plot, the .chi..sub.1/.chi..sub.2 plot
and the backbone conformation less than 3.0. From these 78
structures, 30 structures with the absolute values of the Z-scores
less than 2.4 were chosen to represent the structure of the F3
module 2.
Determination of the Structure of the NCAM F3 Module 2 by NMR
[0355] An overlay of 30 superimposed structures for the backbone
atoms is shown in FIG. 1A (accession number 1LWR, Protein Data
Bank). A ribbon representation of the structure labeling the
7-strands is shown in FIG. 1B. A summary of the structural
statistics is given in Table 1.
[0356] The structure consists of 7 antiparallel .beta.-strands
arranged in a sandwich of two .beta. sheets, one containing three
strands (ABE) and the other four strands (GFCD). Both of the .beta.
sheets have a right-handed twist. The triple-stranded .beta. sheet
consists of residues K.sup.7-G.sup.13 (A), S.sup.18-I.sup.24 (B),
H.sup.59-S.sup.63 (E), and the four-stranded .beta. sheet consists
of residues I.sup.33-A.sup.42 (C), I.sup.51, A.sup.52 (D),
E.sup.70-N.sup.79 (F) and G.sup.82-R.sup.92 (G). There are two wide
type .beta.-bulges (Chan et al., 1993) involving residues K.sup.85,
A8.sup.6 and V.sup.76 (G and F .beta.-stands), and residues
A.sup.77 and H.sup.35, Y.sup.35 (F and C .beta.-strands). The two
.beta.-bulges contribute to the right-handed twist conformation of
the four-stranded .beta.-sheet. Analysis of the characteristic
.beta.-sheet inter-strand NOEs shows unambiguously that this module
has the fibronectin type III fold.
Identification of the Residues of the NCAM F3 module 2 Interacting
with FGFR and ATP
[0357] In the .sup.15N-HSQC spectrum of an .sup.15N-labeled
protein, a signal for each amino acid with both a nitrogen and
proton can be observed. The changes in the chemical shifts of the
signals provide a method for the identification of residues in a
protein that are perturbed by the binding of another molecule. To a
50 .mu.M .sup.15N-labeled sample of the NCAM F3 module 2, 1 mM
unlabeled FGFR Ig modules 2 or 3, or 5 mM AMP-PCP (a
non-hydrolysable analogue of ATP) were added. No significant
changes of the chemical shifts were found in the presence of the Ig
module 2 (data not shown). The recorded changes of the chemical
shifts in the presence of the Ig module 3 or AMP-PCP are shown in
FIG. 1C,D. The residues of the F3 module that exhibited
perturbation by the Ig module 3 were N.sup.79, Q.sup.81, G.sup.82
and K.sup.83 (FIG. 1C). The changes of the chemical shifts of these
residues demonstrate that the presence of the Ig module 3 close to
the F3 module 2 alters the chemical environment at the perturbed
residues of the F3 module, indicating that the perturbed residues
are either a part or in the vicinity of the binding site for the
interaction between the two modules. These residues are located in
the turn region between the F and G .beta.-strands, and as can be
seen from FIG. 2E, they are close to the N-terminus of the module
and, thus, close to the C-terminus of the F3 module 1 in the NCAM
molecule. Since the recombinant protein consisting of the combined
F3 modules 1, 2 was found to bind to FGFR with a much higher
affinity than any of the two individual F3 modules, it is possible
that the perturbed residues at the N-terminus of the F3 module 2
together with residues at the C-terminus of the F3 module 1 form a
single binding site which is destroyed when the F3 modules are
separated, thus greatly reducing their affinity to FGFR compared to
the double module protein.
[0358] The residues of the F3 module perturbed by AMP-PCP were
Y.sup.74 and V.sup.75. The side chain of Y.sup.74 is exposed on the
surface of the module and located in the close vicinity of the
nucleotide binding motif: A.sup.77ENQQGKS.sup.84 and K.sup.85 (FIG.
1E). Both K.sup.83 and K.sup.85 are exposed on the surface of the
module, and presumably the positively charged side chains of
K.sup.83 and K.sup.85 interact with the negatively charged
triphosphate moiety of ATP, whereas the side chain of Y.sup.74 is
involved in a hydrophobic interaction with the adenosine moiety of
ATP. A possible arrangement of the complex of ATP and the F3 module
2 is depicted in FIG. 1G. The residues perturbed by the FGFR Ig
module 3 (N.sup.79, Q.sup.81, G.sup.82 and K.sup.83) are also a
part of the nucleotide binding motif, indicating that the ATP
binding site and the FGFR binding site are overlapping.
[0359] Thus, these data indicate that N.sup.79, Q.sup.81, G.sup.82
and K.sup.83 of the NCAM F3 module 2 are located in a site binding
to FGFR, and that the FGFR binding site overlaps the ATP binding
site.
SPR Analysis of Interaction Between FGFR and NCAM
[0360] Binding analysis was performed using a BIAcoreX instrument
(Biosensor AB) at 25.degree. C. using 10 mM sodium phosphate pH
7.4, 150 mM NaCl as running buffer. The flow-rate was 5 .mu.l/min.
Data were analysed by non-linear curve-fitting using the
manufacture's software. The FGFR Ig modules 2, 3 were immobilized
on a sensor chip CM5 using amine coupling kit (Biosensor AB) as
follows: 1) the two halves of the chip (designated Fc1 and Fc2)
were activated by 20 .mu.l activation solution; 2) the protein was
immobilized on Fc1 using 12 .mu.l 20 .mu.g/ml protein in 10 mM
sodium phosphate buffer pH 6.0; 3) Fc1 and Fc2 were blocked by 35
.mu.l blocking solution. Binding of various compounds to the
immobilized FGFR modules was studied as follows: A compound was
injected simultaneously into Fc1 (with the immobilized FGFR
modules) and Fc2 (with nothing immobilized). The curve representing
unspecific binding of the compound to the surface of Fc2 was
subtracted from the curve representing binding of the protein to
the immobilized FGFR modules and the surface of Fc1. The resulting
curve was used for analysis. For ATP competition experiments, the
specified compounds were preincubated for 10 min with ATP at a
specified concentration. The KI of ATP for the interaction between
the NCAM F3 modules 1, 2 and the FGFR modules 2, 3 was estimated as
previously described (Kiselyov et al, 1997): The initial binding
rates of 30 .mu.M F3 modules, V.sub.0, and 30 .mu.M F3 modules
preincubated with ATP at a specified concentration, VATP, were
determined. The % inhibition, 1.sub.%, was calculated using the
formula: I.sub.%=(1-V.sub.ATP/V.sub.0).times.100. I.sub.% was
plotted against ATP concentration and Ki was calculated by
non-linear fitting of the theoretical curve to the experimental
data.
Demonstration of a Direct Interaction Between NCAM and FGFR
[0361] From FIG. 2 it appears that a protein consisting of the NCAM
F3 modules 1, 2 binds to an immobilized protein comprising the FGFR
modules 2, 3, whereas a control protein consisting of the NCAM Ig
modules 1, 2 and 3 does not bind to the FGFR fragment. The
dissociation constant (Kd) and the coefficients of association and
dissociation were: 9.97.+-.0.37 .mu.M, 889.+-.332 M.sup.-1s.sup.-1
and 5.56.+-.0.07.times.10.sup.-3s.sup.-1 (mean.+-.standard
deviation), respectively.
[0362] Thus, these data show that the NCAM F3 modules bind directly
to FGFR
Binding of NCAM to FGFR Can be Inhibited by ATP
[0363] As the NCAM sites binding to FGFR and ATP overlap, then ATP
might be expected to interfere with the NCAM-FGFR binding. To test
this assumption, SPR analysis was used. As can be seen from FIG.
3A, adding 10 mM ATP to 30 .mu.M NCAM F3 modules 1, 2 completely
inhibited binding of the F3 modules to the FGFR Ig modules 2, 3. To
determine the inhibition constant (Ki), the inhibition effect was
measured at various ATP concentrations (FIG. 3B). The calculated Ki
was 0.37.+-.0.05 mM. Since the NMR experiments indicate that the
turn region between the F and G .beta.-strands of the F3 module 2
is involved in binding to FGFR, it was of interest to test if a
peptide fragment spanning these residues could bind to FGFR.
Moreover, another peptide fragment located in the F3 module 1 was
of interest to test, as the turn region between the E and F strands
of this module may constitute an N-terminal part of the binding
site in NCAM interacting with the Ig2-Ig3 module of FGFR. For this
purpose, a peptide corresponding to residues E.sup.72-A.sup.86 of
the F3 module 2 (SEQ ID NO: 1) (termed the FG loop peptide) and a
peptide corresponding to residues T.sup.573-R.sup.586 of the F3
module 1 (SEQ ID NO: 9) (termed the EF loop peptide). However,
binding of monomeric forms of these peptides cannot be detected by
SPR. Therefore, there were synthesized dendrimeric versions of the
peptides in which four peptide sequences of one type (such as SEQ
ID NO: 1 or SEQ ID NO: 9) were connected to a three-lysine backbone
through their C-termini. This made it possible for the peptide to
bind to several molecules of FGFR simultaneously, thus greatly
increasing the apparent affinity of the peptides, and, at the same
time, increasing the sensitivity of the SPR analysis four fold
(since the molecular weight of each peptide was increased four
times).
[0364] As appears from FIG. 3C, the dendrimeric FG loop peptide
binds to FGFR with an apparent Kd of 2.58.+-.2.06 .mu.M (the
coefficients of association and dissociation being
2.07.+-.1.08.times.10.sup.3 M.sup.-1s.sup.-1 and
3.97.+-.2.29.times.10.sup.-3 s.sup.-1, respectively). Since binding
of the F3 modules 1, 2 to FGFR could be inhibited by ATP and since
the peptide contains the entire nucleotide binding motif of NCAM,
It was also tested if ATP could interfere with binding of the
peptide to FGFR. Indeed, adding 10 mM ATP to 34 .mu.M peptide
inhibited the binding by approx. 70% (FIG. 3C). FIG. 4 demonstrates
the real-time binding of the dendromeric form of the EF loop
peptide to FGFR.
[0365] These experiments demonstrate that NCAM binding to FGFR can
be inhibited by ATP and support the notion that the turn regions
between the F and G .beta.-strands of the F3 module 2 and the E and
F .beta.-strands of the F3 module 1 are involved in binding to
FGFR, and that NCAM sites for FGFR and ATP overlap.
Assays for Determination of FGFR1 Phosphorylation and NCAM
Immunoprecipitation
Activation of FGFR by the NCAM F3 Module 2 and the FG Loop
Peptide
[0366] Since the NCAM F3 module 2 and the dendrimeric FG loop
peptide bind to FGFR, they may be also expected to induce FGFR
activation in living cells. To test this assumption, two assays
have been used.
[0367] Assay 1: TREX-293 cells (Invitrogen) were stably transfected
with human FGFR1 having a C-terminal Strepll-tag (IBA biotech),
using the Flp-In system (Invitrogen). For the study of
phosphorylation: .about.2.times.10.sup.7 cells were starved
overnight before stimulation for 20 min with the specified
compounds. Cells were lysed in PBS with 1% NP-40 and phosphatase
inhibitors cocktail set 11 (Calbiochem). The cleared cell lysates
were incubated with 50 .mu.l agarose-coupled anti-phosphotyrosine
antibodies (4G10-AC, Upstate Biotechnologies) for 3 hrs at
4.degree. C. Care was taken to calibrate the amount of cells
employed, so that considerable increases in phosphorylated FGFR
could be detected. The bound protein was washed, eluted using 150
mM phenyl phosphate (Sigma), precipitated by 12% trichloroacetic
acid, washed in cold acetone and dissolved in SDS-PAGE sample
buffer. Immunoblotting was performed using antibodies against the
recombinant Strepll-tag (IBA biotech). For immunoprecipitation:
.about.1.times.10.sup.7 cells were transiently transfected with the
180 kDa NCAM isoform or a control vector. Cells were incubated for
24 h and then starved overnight. After lysis, FGFR was purified on
a StrepTactin Minicolumn (IBA Biotech) according to the
manufacturer's instructions, and analyzed by immunoblotting with
polyclonal antibodies against human NCAM.
[0368] As appears from FIG. 5A, both 5 .mu.M F3 module and 2.5
.mu.M dendrimeric FG loop peptide substantially increase FGFR
phosphorylation compared to the non-stimulated cells.
[0369] Assay 2: The cDNA for the rat FGFR1 (IIIC isoform) was
cloned by RT-PCR using RNA isolated from the rat PC12 cell line and
inserted into a pcDNA3.1(+) plasmid (Invitrogen), which allows
expression of FGFR1 fused to the N-terminal of hexahistidine.
.about.8.times.10.sup.5 HEK293 cells were cultured for 24 h in 60
mm plates in full medium (DMEM 1965 supplemented with 10% FCS, 100
U/ml penicillin, 100 .mu.g/ml streptomycin and 58.4 g/l Glutamax)
and then transfected with 0.2 .mu.g plasmid (with FGFR) using the
LipofectAMIN PLUS.TM. reagent kit according to the manufacturer's
instructions (Gibco BRL). Cells were grown for another 24 hrs in
full medium, and then shifted to starvation media (DMEM 1965)
overnight. FGFR transfected cells were stimulated for 20 min with
the specified compounds, lysed in 8M urea, 1 mM or thovanadate (in
PBS) and purified from the lysate via the His-tag as follows: The
lysate was loaded on Ni.sup.2+/NTA-sepharose (Qiagen), washed with
lysis buffer plus 10 mM imidazole, and the FGFR was eluted with
lysis buffer plus 250 mM imidazole. The purified FGFR was analysed
by immunoblotting using anti-pentahis (Qiagen) or
anti-phosphotyrosine (PY20, Transduction Laboratories) antibodies.
The bands were visualised by chemilumiscense and the band density
was measured using a GeneGnome apparatus (SynGene).
[0370] From FIG. 5B, it appears that addition of 5 .mu.M F3 module
2 or 25 .mu.M monomeric FG loop peptide increase FGFR
phosphorylation by approx. 150% and 100%, respectively, compared to
control cells.
Co-Immunoprecipitation of NCAM and FGFR
[0371] Since the binding between the NCAM and FGFR fragments could
be demonstrated in vitro by SPR and NMR analyses it was of interest
to confirm that native NCAM binds to FGFR in the living cells.
Therefore, TREX-293 cells, stably transfected with FGFR containing
a C-terminal Strepll-tag, were transiently transfected with the 180
kDa NCAM isoform. After lysing the cells, FGFR was affinity
purified via the Strepll-tag and analyzed by immunoblotting using
antibodies against NCAM. As appears from FIG. 5C, NCAM is indeed
precipitated by FGFR, thus supporting our SPR and NMR
experiments.
Stimulation of Neurite Length of Hippocampal Neurons by the NCAM F3
Module 2, FG Loop (FGL-Peptide, SEQ ID NO: 1), EF Loop Peptide
(EFL-Peptide, SEQ ID NO: 9) and a Peptide Derived from the
Axonal-Associated Cell Adhesion Molecule.
[0372] Dissociated neurons from embryonic rat hippocampus
(embryonic day 19), prepared as described (Skladchikova et al.,
1999), were grown on Permanox plastic (Nunc) for 24 h at a density
of 6000 cells/cm.sup.2, at 37.degree. C., 5% CO.sub.2 in Neurobasal
medium containing 20 mM Hepes, 100 U/ml penicillin, 100 .mu.g/ml
streptomycin, 0.4% BSA supplemented with B27 (Gibco BRL) and the
below specified compounds. After 24 h, cells were fixed with
paraformaldehyde, stained with Coomassie Brilliant Blue R250 and
the length of neurites was measured as described (Skladchikova et
al., 1999).
[0373] As can be seen from FIG. 6A,B, 5 .mu.M F3 module 2
substantially increased the length of neurites compared to the
non-stimulated neurons. The effect was quantified in a
dose-response study (FIG. 6C) demonstrating that the F3 module, the
FG loop peptide and a truncated version of the peptide
(A.sup.77-K.sup.83) all induced neurite outgrowth with a
bell-shaped curve typical of growth factor induced neuritogenesis
(Hatten et al., 1988). The potency of the peptides was lower than
that of the module, since a 10 times higher concentration was
required for maximum effect, and the truncated form was less
efficient than the extended form. The stimulatory effect of the F3
module and the FG loop peptide could be completely abrogated by an
inhibitor of NCAM-stimulated neurite outgrowth, an antibody against
FGFR (Williams et al., 1994) (FIG. 6D), further supporting the
notion that the module and the FG loop peptide interact with FGFR.
The EF loop peptide of the F3 module 2 was also capable to
stimulate the neurite outgrowth significantly. As it appears from
FIG. 7 the neurite outgrowth stimulation by the peptide was
specifically blocked by an inhibitor of FGFR1, SU54402.
[0374] To determine the functionally important amino acids of the
FG loop peptide, it was analyzed by truncations and Ala
substitutions of various amino acids. Two truncated versions (from
the N- and C-terminal) of the FG loop peptide were produced: the
nonamer V.sup.78-S.sup.84 and the heptamer Ala.sup.77-K.sup.83.
Both of the truncated peptides retained approximately 50% of the
stimulatory effect of the entire FG loop peptide (FIG. 6E),
indicating that the turn region between the F and G .beta.-strands
is important for the NCAM-FGFR interaction. As can be seen from
FIG. 6E, substitution of any amino acid in the heptameric peptide
with Ala resulted in a decrease of the neuritogenic potency and a
complete loss of function was achieved if E.sup.78, N.sup.79,
Q.sup.80, G.sup.82, K.sup.83 were substituted, indicating that
these residues are important for interaction with FGFR.
Substitution of both Q.sup.80 and Q.sup.81 from the turn region of
the FG loop with Ala in the entire FG loop peptide also completely
inactivated the peptide (FIG. 6E). These findings corroborate the
NMR experiments showing that N.sup.79, Q.sup.81, G.sup.82 and
K.sup.83 were perturbed in the F3 module 2 by binding to the FGFR
Ig module 3. However, when the residues which seem to be important
for interaction with ATP (Y.sup.74, K.sup.83 and K.sup.85) were
substituted with Ala in the FG loop peptide, the peptide retained
about 60% of the stimulatory effect of the non-mutated peptide
(FIG. 6E).
[0375] Furthermore, the structure of the heptameric peptide in the
F3 module was compared to the known structure of a natural ligand
of FGFR, FGF2 (PDB code: 4FGF, Eriksson et al., 1993), and it was
found that the peptide exhibited a striking structure and sequence
similarity to a loop region in FGF2, A.sup.42-R.sup.48 (FIG. 8A).
The conformations of the backbone atoms of the two peptides are
virtually identical, with the side chains also having similar
conformations. The only exception is R.sup.48 (in FGF2) whose side
chain has a somewhat different conformation from that of K.sup.83.
As can be seen from FIG. 8B, the peptide derived from FGF2 induced
neurite outgrowth to the same extent as the similar NCAM peptide,
and substitution of any amino acid with Ala resulted in a complete
loss of function. The close sequence and structure similarity
between the FG loop of the NCAM F3 module 2 and a part of FGF2
further corroborate the present contention that the former is
directly interacting with FGFR.
[0376] Since ATP can inhibit the NCAM-FGFR binding and interfere
with the FGFR activation by the F3 module 2 and, it may
consequently affect the neuritogenic activity of the module. To
test this assumption, neurons were stimulated with the below
described compounds in the presence of ATP or AMP-PCP. As can be
seen from FIG. 9, both ATP and AMP-PCP substantially reduced the
neuritogenic effect induced by the F3 module 2 and the FG loop
peptide. A complete inhibition of the effect of both the F3 module
and the FG loop peptide was achieved when AMP-PCP was added,
whereas only the effect of the FG loop peptide was completely
inhibited when ATP was added, indicating that ATP is a less potent
inhibitor than its non-hydrolysable analogue. Most significantly,
when the amino acid residues of the FG loop peptide presumed to be
of importance for ATP binding (Y.sup.74, K.sup.83 and K.sup.85)
were substituted with Ala, the peptide retained its neuritogenic
potency, but the stimulatory effect of the peptide could no longer
be inhibited by ATP (FIG. 9), supporting the notion that ATP
binding regulates the interaction between the F3 module and
FGFR.
[0377] These results shows that activation of FGFR in neurons by
the NCAM F3 module 2, the FG loop and EF loop peptides induce
neuritogenesis and that this effect can be inhibited by ATP or a
ATP analogue.
[0378] To investigate a neuritogenic potency of other FGL-like
peptides, which according to invention constitute at least a part
of the binding site of the FGFR in a FGFR ligand, a peptide
consisting of the sequence derived from the axonal-associated cell
adhesion molecule [NCBI: NP.sub.--031544.1] was used in the assay
described above. The selected peptide was an 11-amino acid fragment
of the sequence set forth in SEQ ID NO: 5 having two amino acids
truncated from the C-terminus. FIG. 11 demonstrates that likewise
the FG-loop and EF-loop peptides the peptide was capable of
stimulation of neurite outgrowth of hippocampal neurons. The
maximal effect was observed at concentration of the peptide of
about 0.3 .mu.g/ml.
Epitope Specific Antibodies
[0379] Antibody specifically recognising an epitope comprising SEQ
ID NO: 1 (FGL-peptide) were raised in rabbits according the
standard procedure (Gill B M, Barbosa J A, Dinh T Q, Garrod S,
O'Connor D T: Chromogranin B: isolation from pheochromocytoma,
N-terminal sequence, tissue distribution and secretory vesicle
processing. Regul Peptides 33:223-35, 1991). Synthetic FGL-peptide
was coupled to a carrier protein, KLH (Keyhole Limpets Hemocyanin).
The peptide was mixed with Freunds incomplete adjuvant and the
mixture was injected into rabbits. 208 .mu.g peptide was used per
immunisation (per rabbit).
[0380] Antibodies (serum) were tested for specificity by
competitive ELISA using a series FGL-peptide truncated variants
according to the protocol described in R. J. Jenny, T. L. Messier,
L. A. Ouellette, and W. R. Church, Methods Enzymol. 222, 400
(1993). The results of the test are presented in FIG. 10. 100%
immunoreactivity was observed only with the intact FGL-peptide
consisting of SEQ ID NO: 1. However, two truncated variants of the
FGL peptide comprising the motif WAENQQGK did demonstrate a
decreased, but distinct immunoreactivity: peptide t4 lacking the
three C terminal amino acids of full-length FGL-peptide (80%
immunoreactivity), and peptide t1 lacking the three N-terminal
amino acids of full-length FGL-peptide (40% immunoreactivity).
Sequence CWU 0
0
SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 146 <210>
SEQ ID NO 1 <211> LENGTH: 15 <212> TYPE: PRT
<213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: NCAM Fn III, 2 [Swiss-Prot: P13591]:
FGFR binding motif <400> SEQUENCE: 1 Glu Val Tyr Val Val Ala
Glu Asn Gln Gln Gly Lys Ser Lys Ala 1 5 10 15 <210> SEQ ID NO
2 <211> LENGTH: 16 <212> TYPE: PRT <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Interleukin-6 receptor beta chain [Swiss- Prot:
Q00560]: FGFR binding motif <400> SEQUENCE: 2 Asn Ile Glu Val
Trp Val Glu Ala Glu Asn Ala Leu Gly Lys Lys Val 1 5 10 15
<210> SEQ ID NO 3 <211> LENGTH: 14 <212> TYPE:
PRT <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Heparan sulfate proteoglycan
perlecan [Swiss- Prot: P98160]: FGFR binding motif <400>
SEQUENCE: 3 Ala Thr Asn Arg Gln Gly Lys Val Lys Ala Phe Ala His Leu
1 5 10 <210> SEQ ID NO 4 <211> LENGTH: 16 <212>
TYPE: PRT <213> ORGANISM: Artificial sequence <220>
FEATURE: <223> OTHER INFORMATION: Disintegrin and
metalloprotease domain 8 (ADAM-8) [Swiss-Prot: Q0 5910]: FGFR
binding motif <400> SEQUENCE: 4 Arg Tyr Val Glu Leu Tyr Val
Val Ala Asp Ser Gln Glu Phe Gln Lys 1 5 10 15 <210> SEQ ID NO
5 <211> LENGTH: 13 <212> TYPE: PRT <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Axonal-associated cell adhesion molecule [NCBI:
NP_446331]: FGFR binding motif <400> SEQUENCE: 5 Val Ala Glu
Asn Ser Arg Gly Lys Asn Val Ala Lys Gly 1 5 10 <210> SEQ ID
NO 6 <211> LENGTH: 14 <212> TYPE: PRT <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Myelin-associated glycoprotein (MAG)
[Swiss-Prot: P20917]: FGFR binding motif <400> SEQUENCE: 6
Gly Glu Tyr Trp Cys Val Ala Glu Asn Gln Tyr Gly Gln Arg 1 5 10
<210> SEQ ID NO 7 <211> LENGTH: 14 <212> TYPE:
PRT <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: FIII,1 domain of NCAM [Swiss-Prot:
P13591]: FGFR binding motif <400> SEQUENCE: 7 Arg Leu Ala Ala
Leu Asn Gly Lys Gly Leu Gly Glu Ile Ser 1 5 10 <210> SEQ ID
NO 8 <211> LENGTH: 16 <212> TYPE: PRT <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Neuronal nicotinic acetylcholine receptor alpha
3 subunit (CHRNA 3) [Swiss-Prot: Q8VHH6/P04757:/Q8R4G9/ P32297]:
FGFR binding motif <400> SEQUENCE: 8 Lys Tyr Ile Ala Glu Asn
Met Lys Ala Gln Asn Val Ala Lys Glu Ile 1 5 10 15 <210> SEQ
ID NO 9 <211> LENGTH: 14 <212> TYPE: PRT <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: FIII,1 domain of NCAM (Swiss-Prot: P13591): FGFR
binding motif <400> SEQUENCE: 9 Thr Ile Met Gly Leu Lys Pro
Glu Thr Arg Tyr Ala Val Arg 1 5 10 <210> SEQ ID NO 10
<211> LENGTH: 14 <212> TYPE: PRT <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Granulocyte colony stimulating factor receptor
precursor (G-CSF-R; CD114 antigen)[ Swiss-Prot: Q99062]: FGFR
binding motif <400> SEQUENCE: 10 Lys Gly Leu Gly Glu Ile Ser
Ala Ala Thr Glu Phe Lys Thr 1 5 10 <210> SEQ ID NO 11
<211> LENGTH: 13 <212> TYPE: PRT <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: NCAM Fn III, 1 [Swiss-Prot: P13591]: FGFR binding
motif <400> SEQUENCE: 11 Asn Met Gly Ile Trp Val Gln Ala Glu
Asn Ala Leu Gly 1 5 10 <210> SEQ ID NO 12 <211> LENGTH:
10 <212> TYPE: PRT <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Granulocyte
colony stimulating factor receptor precursor (G-CSF-R; CD114
antigen) [Swiss-Prot: P40223]: FGFR binding motif <400>
SEQUENCE: 12 Ile Trp Val Gln Ala Glu Asn Met Leu Gly 1 5 10
<210> SEQ ID NO 13 <211> LENGTH: 11 <212> TYPE:
PRT <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Cytokine-like factor-1 precursor
(CLF-1) [Swiss-Prot: O75462]: FGFR binding motif <400>
SEQUENCE: 13 Glu Ile Trp Val Glu Ala Thr Asn Arg Leu Gly 1 5 10
<210> SEQ ID NO 14 <211> LENGTH: 10 <212> TYPE:
PRT <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Interleukin-23 receptor (IL-23R)
[Q8NFQ9]: FGFR binding motif <400> SEQUENCE: 14 Val Trp Val
Gln Ala Ala Asn Ala Leu Gly 1 5 10 <210> SEQ ID NO 15
<211> LENGTH: 13 <212> TYPE: PRT <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Complement factor 1 q , alpha polypeptide (C1QA)
[Swiss-Prot: Q9D CM6]: FGFR binding motif <400> SEQUENCE: 15
Glu Val Trp Ile Glu Lys Asp Pro Ala Lys Gly Arg Ile 1 5 10
<210> SEQ ID NO 16 <211> LENGTH: 14 <212> TYPE:
PRT <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Fasciclin II precursor (FAS2)
[Swiss-Prot: P22648]: FGFR binding motif <400> SEQUENCE: 16
Ala Thr Asn Lys Gly Gly Glu Val Lys Lys Asn Gly His Leu 1 5 10
<210> SEQ ID NO 17 <211> LENGTH: 16 <212> TYPE:
PRT <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: ADAM-19 precursor (EC 3.4.24.-)
[Swiss-Prot: Q9H013/O35674]: FGFR binding motif
<400> SEQUENCE: 17 Lys Tyr Val Glu Leu Tyr Leu Val Ala Asp
Tyr Leu Glu Phe Gln Lys 1 5 10 15 <210> SEQ ID NO 18
<211> LENGTH: 15 <212> TYPE: PRT <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: ADAM-8 precursor (EC 3.4.24.-) [Swiss-Prot: P78325]:
FGFR binding motif <400> SEQUENCE: 18 Arg Tyr Val Glu Leu Tyr
Val Val Val Asp Asn Ala Glu Phe Gln 1 5 10 15 <210> SEQ ID NO
19 <211> LENGTH: 16 <212> TYPE: PRT <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: ADAM-12 precursor (EC 3.4.24.-)[Swiss-Prot:
O43184; Q61824]: FGFR binding motif <400> SEQUENCE: 19 Lys
Tyr Val Glu Leu Val Ile Val Ala Asp Asn Arg Glu Phe Gln Arg 1 5 10
15 <210> SEQ ID NO 20 <211> LENGTH: 16 <212>
TYPE: PRT <213> ORGANISM: Artificial sequence <220>
FEATURE: <223> OTHER INFORMATION:
Metalloproteinase-disintegrin domain containing protein TECADAM
[AF163291] : FGFR binding motif <400> SEQUENCE: 20 Lys Tyr
Ile Glu Tyr Tyr Val Val Leu Asp Asn Gly Glu Phe Lys Lys 1 5 10 15
<210> SEQ ID NO 21 <211> LENGTH: 14 <212> TYPE:
PRT <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: ADAM-33 precursor (EC
3.4.24.-)[Swiss-Prot: Q9BZ11/Q923W9]: FGFR binding motif
<400> SEQUENCE: 21 Arg Tyr Leu Glu Leu Tyr Ile Val Ala Asp
His Thr Leu Phe 1 5 10 <210> SEQ ID NO 22 <211> LENGTH:
15 <212> TYPE: PRT <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: ADAM-1A
Fertilin alpha [Swiss-Prot: Q8R533]: FGFR binding motif <400>
SEQUENCE: 22 Lys Tyr Val Glu Met Phe Val Val Val Asn His Gln Arg
Phe Gln 1 5 10 15 <210> SEQ ID NO 23 <211> LENGTH: 14
<212> TYPE: PRT <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: ADAM-9
[Swiss-Prot: Q13433; Q61072]: FGFR binding motif <400>
SEQUENCE: 23 Arg Tyr Val Glu Leu Phe Ile Val Val Asp Lys Glu Arg
Tyr 1 5 10 <210> SEQ ID NO 24 <211> LENGTH: 16
<212> TYPE: PRT <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: ADAM-7
precursor [Swiss-Prot: Q9H2U9]: FGFR binding motif <400>
SEQUENCE: 24 Lys Tyr Val Glu Leu Phe Ile Val Ala Asp Asp Thr Val
Tyr Arg Arg 1 5 10 15 <210> SEQ ID NO 25 <211> LENGTH:
16 <212> TYPE: PRT <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: ADAM-7
precursor [Swiss-Prot: O35227; Q63180]: FGFR binding motif
<400> SEQUENCE: 25 Lys Phe Ile Glu Leu Phe Val Val Ala Asp
Glu Tyr Val Tyr Arg Arg 1 5 10 15 <210> SEQ ID NO 26
<211> LENGTH: 16 <212> TYPE: PRT <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: ADAM-15 precursor [Swiss-Prot: Q9QYV0; O88839]: FGFR
binding motif <400> SEQUENCE: 26 Lys Ile Val Glu Lys Val Ile
Val Ala Asp Asn Ser Glu Val Arg Lys 1 5 10 15 <210> SEQ ID NO
27 <211> LENGTH: 14 <212> TYPE: PRT <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: ADAM-15 precursor [Swiss-Prot: Q13444]: FGFR
binding motif <400> SEQUENCE: 27 Val Glu Leu Val Ile Val Ala
Asp His Ser Glu Ala Gln Lys 1 5 10 <210> SEQ ID NO 28
<211> LENGTH: 13 <212> TYPE: PRT <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Neural cell adhesion protein BIG-2 precursor
[Swiss-Prot: Q62845]: FGFR binding motif <400> SEQUENCE: 28
Val Ala Glu Asn Ser Arg Gly Lys Asn Ile Ala Lys Gly 1 5 10
<210> SEQ ID NO 29 <211> LENGTH: 13 <212> TYPE:
PRT <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Neuronal glycoprotein CNTN3
[Swiss-Prot: Q07409]: FGFR binding motif <400> SEQUENCE: 29
Ile Ala Glu Asn Ser Arg Gly Lys Asn Val Ala Arg Gly 1 5 10
<210> SEQ ID NO 30 <211> LENGTH: 12 <212> TYPE:
PRT <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: NB-2(HNB-2/NB-2), a neural cell
recognition molecule of the contactin/F3 subgroup [Swiss-Prot:
O94779/ P97527]: FGFR binding motif <400> SEQUENCE: 30 Ala
Glu Asn Ser Arg Gly Lys Asn Ser Phe Arg Gly 1 5 10 <210> SEQ
ID NO 31 <211> LENGTH: 13 <212> TYPE: PRT <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: HNB-3/NB-3 [Swiss-Prot: Q9UQ52/P97528/ Q9JMB8]:
FGFR binding motif <400> SEQUENCE: 31 Ile Ala Ser Asn Leu Arg
Gly Arg Asn Leu Ala Lys Gly 1 5 10 <210> SEQ ID NO 32
<211> LENGTH: 13 <212> TYPE: PRT <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Putative fat-like cadherin precursor (Drosiphila)
[Swiss-Prot: Q9VW71]: FGFR binding motif <400> SEQUENCE: 32
Ile Pro Glu Asn Ser Leu Gly Lys Thr Tyr Ala Lys Gly 1 5 10
<210> SEQ ID NO 33 <211> LENGTH: 12 <212> TYPE:
PRT <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Neuronal nicotinic acetylcholine
receptor alpha 3 subunit (CHRNA3) [Swiss-Prot:
Q8VHH6/P04757/Q8R4G9/ P32297]: FGFR binding motif <400>
SEQUENCE: 33 Ile Ala Glu Asn Met Lys Ala Gln Asn Glu Ala Lys 1 5 10
<210> SEQ ID NO 34 <211> LENGTH: 16 <212> TYPE:
PRT <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Neuronal acetylcholine receptor
protein, alpha-6 chain precursor (CHRNA6) [Swiss-prot:Q15825 ]:
FGFR binding motif <400> SEQUENCE: 34
Gln Phe Ile Ala Glu Asn Met Lys Ser His Asn Glu Thr Lys Glu Val 1 5
10 15 <210> SEQ ID NO 35 <211> LENGTH: 13 <212>
TYPE: PRT <213> ORGANISM: Artificial sequence <220>
FEATURE: <223> OTHER INFORMATION: ROBO-1 [O44924]: FGFR
binding motif <400> SEQUENCE: 35 Gly Glu Tyr Trp Cys Val Ala
Lys Asn Arg Val Gly Gln 1 5 10 <210> SEQ ID NO 36 <211>
LENGTH: 13 <212> TYPE: PRT <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
ROBO-1[AF041082; Q9Y6N7]: FGFR binding motif <400> SEQUENCE:
36 Gly Ser Tyr Thr Cys Val Ala Glu Asn Met Val Gly Lys 1 5 10
<210> SEQ ID NO 37 <211> LENGTH: 14 <212> TYPE:
PRT <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: ROBO-1[AF041082; Q9Y6N7]: FGFR
binding motif <400> SEQUENCE: 37 Gly Lys Tyr Val Cys Val Gly
Thr Asn Met Val Gly Glu Arg 1 5 10 <210> SEQ ID NO 38
<211> LENGTH: 11 <212> TYPE: PRT <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: FGFR2 [Q96KM2; P21802]: FGFR binding motif <400>
SEQUENCE: 38 Asn Tyr Thr Cys Val Val Glu Asn Glu Tyr Gly 1 5 10
<210> SEQ ID NO 39 <211> LENGTH: 12 <212> TYPE:
PRT <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: FGFR2[Q63241]: FGFR binding site
<400> SEQUENCE: 39 Gly Glu Tyr Thr Cys Leu Ala Gly Asn Ser
Ile Gly 1 5 10 <210> SEQ ID NO 40 <211> LENGTH: 11
<212> TYPE: PRT <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Fc
receptor-like protein 1[Q96KM2] / fragment of IFGP 1 [Q96PJ6]: FGFR
binding motif <400> SEQUENCE: 40 Gln Tyr Tyr Cys Val Ala Glu
Asn Gly Tyr Gly 1 5 10 <210> SEQ ID NO 41 <211> LENGTH:
13 <212> TYPE: PRT <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Junctional
adhesion molecule (JAM-1) [Q9JKD5/ O88792]: FGFR binding motif
<400> SEQUENCE: 41 Gly Glu Tyr Tyr Gln Glu Ala Glu Gln Asn
Gly Tyr Gly 1 5 10 <210> SEQ ID NO 42 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: FGFR2 [Q96KM2;
P21802]: FGFR binding motif <400> SEQUENCE: 42 Gly Asn Tyr
Thr Cys Leu Val Glu Asn Glu Tyr Gly 1 5 10 <210> SEQ ID NO 43
<211> LENGTH: 12 <212> TYPE: PRT <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Contactin precursor (Neural adhesion molecule
F3)[Q63198;/P1260; Q12860]: FGFR binding motif <400>
SEQUENCE: 43 Gly Met Tyr Gln Cys Leu Ala Glu Asn Ala Tyr Gly 1 5 10
<210> SEQ ID NO 44 <211> LENGTH: 11 <212> TYPE:
PRT <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Contactin precursor (Neural adhesion
molecule F3/F11) [Q28106]: FGFR binding motif <400> SEQUENCE:
44 Gly Met Tyr Gln Cys Ala Glu Asn Thr His Gly 1 5 10 <210>
SEQ ID NO 45 <211> LENGTH: 12 <212> TYPE: PRT
<213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Contactin precursor (Neural adhesion
molecule F3/F11) [Q28106]: FGFR binding motif <400> SEQUENCE:
45 Gly Ile Tyr Tyr Cys Leu Ala Ser Asn Asn Tyr Gly 1 5 10
<210> SEQ ID NO 46 <211> LENGTH: 12 <212> TYPE:
PRT <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: IFGP2[Q96PJ5]: FGFR binding motif
<400> SEQUENCE: 46 Gly Gly Tyr Tyr Cys Thr Ala Asp Asn Ser
Tyr Gly 1 5 10 <210> SEQ ID NO 47 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Neurofascin
precursor [Q90924]: FGFR binding motif <400> SEQUENCE: 47 Gly
Glu Tyr Gln Cys Phe Ala Arg Asn Asp Tyr Gly 1 5 10 <210> SEQ
ID NO 48 <211> LENGTH: 12 <212> TYPE: PRT <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Neurofascin [Q90924]: FGFR binding motif
<400> SEQUENCE: 48 Gly Glu Tyr Phe Cys Leu Ala Ser Asn Lys
Met Gly 1 5 10 <210> SEQ ID NO 49 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Neurofascin 155
Da isoform [Q91Z60]: FGFR binding motif <400> SEQUENCE: 49
Gly Glu Tyr Gln Cys Phe Ala Arg Asn Lys Phe Gly 1 5 10 <210>
SEQ ID NO 50 <211> LENGTH: 12 <212> TYPE: PRT
<213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Neurofascin 155 Da isoform [Q91Z60]:
FGFR binding motif <400> SEQUENCE: 50 Gly Glu Tyr Phe Cys Leu
Ala Ser Asn Lys Met Gly 1 5 10 <210> SEQ ID NO 51 <211>
LENGTH: 12 <212> TYPE: PRT <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Macrophage scavenger receptor 2 (MSR2) [Q91YK7]:FGFR binding motif
<400> SEQUENCE: 51 Gly Gly Tyr Tyr Cys Thr Ala Asp Asn Asn
Tyr Gly 1 5 10 <210> SEQ ID NO 52 <211> LENGTH: 14
<212> TYPE: PRT <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Macrophage
scavenger receptor 2 (MSR2) [Q91YK7]: FGFR binding motif
<400> SEQUENCE: 52 Gly Asn Tyr Ser Cys Glu Ala Glu Asn Ala
Trp Gly Thr Lys 1 5 10 <210> SEQ ID NO 53 <211> LENGTH:
12 <212> TYPE: PRT <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Neural cell
adhesion molecule L1[Q9QYQ7; Q9QY38; P11627; Q05695;P32004]: FGFR
binding motif <400> SEQUENCE: 53 Gly Glu Tyr Thr Cys Leu Ala
Glu Asn Ser Leu Gly 1 5 10 <210> SEQ ID NO 54 <211>
LENGTH: 13 <212> TYPE: PRT <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Neural-glial cell adhesion molecule Ng-CAM [Q03696]: FGFR binding
motif <400> SEQUENCE: 54 Gly Glu Tyr Glu Cys Val Ala Glu Asn
Gly Arg Leu Gly 1 5 10 <210> SEQ ID NO 55 <211> LENGTH:
13 <212> TYPE: PRT <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: FGFR3 [Q95M13;
AF487554; Q99052]: FGFR binding motif <400> SEQUENCE: 55 Gly
Asn Tyr Thr Cys Val Val Glu Asn Lys Phe Gly Arg 1 5 10 <210>
SEQ ID NO 56 <211> LENGTH: 12 <212> TYPE: PRT
<213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: FGFR3 [Q95M13; Q99052]: FGFR binding
motif <400> SEQUENCE: 56 Gly Glu Tyr Thr Cys Leu Ala Gly Asn
Ser Ile Gly 1 5 10 <210> SEQ ID NO 57 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Neural cell
adhesion molecule 2 (NCAM2) [P36335]: FGFR binding motif
<400> SEQUENCE: 57 Gly Glu Tyr Phe Cys Val Ala Ser Asn Pro
Ile Gly 1 5 10 <210> SEQ ID NO 58 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Neural cell
adhesion molecule 2 (NCAM2) [P36335]: FGFR binding motif
<400> SEQUENCE: 58 Glu Tyr Thr Cys Ile Ala Asn Asn Gln Ala
Gly Glu 1 5 10 <210> SEQ ID NO 59 <211> LENGTH: 13
<212> TYPE: PRT <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Axonin-1
(TAG-1) [Q02246;P22063; P28685]: FGFR binding motif<400>
SEQUENCE: 59 Gly Met Tyr Gln Cys Val Ala Glu Asn Lys His Leu Gly 1
5 10 <210> SEQ ID NO 60 <211> LENGTH: 13 <212>
TYPE: PRT <213> ORGANISM: Artificial sequence <220>
FEATURE: <223> OTHER INFORMATION: Neural cell adhesion
molecule NCAM-140 AND ncam-140 [P13595]: FGFR binding motif
<400> SEQUENCE: 60 Gly Glu Tyr Met Cys Thr Ala Ser Asn Thr
Ile Gly Gln 1 5 10 <210> SEQ ID NO 61 <211> LENGTH: 13
<212> TYPE: PRT <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Neural cell
adhesion molecule NCAM-140 AND ncam-140 [P13595]: FGFR binding
motif <400> SEQUENCE: 61 Glu Tyr Val Cys Ile Ala Glu Asn Lys
Ala Gly Glu Gln 1 5 10 <210> SEQ ID NO 62 <211> LENGTH:
13 <212> TYPE: PRT <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Neurotrophin
receptor tyrosin kinase type 2 (NTRKT) [Q8WXJ5]:FGFR binding motif
<400> SEQUENCE: 62 Gly Asp Tyr Thr Leu Ile Ala Lys Asn Glu
Tyr Gly Lys 1 5 10 <210> SEQ ID NO 63 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Colorectal
cancer suppressor DCC [P43146]: FGFR binding motif <400>
SEQUENCE: 63 Gly Phe Tyr Gln Cys Val Ala Glu Asn Glu Ala Gly 1 5 10
<210> SEQ ID NO 64 <211> LENGTH: 14 <212> TYPE:
PRT <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Tyrosine phosphatase LAR (ptprf)
[Q9EQ17; Q64604; P23468]: FGFR binding motif <400> SEQUENCE:
64 Gly Lys Tyr Glu Cys Val Ala Thr Asn Ser Ala Gly Thr Arg 1 5 10
<210> SEQ ID NO 65 <211> LENGTH: 12 <212> TYPE:
PRT <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Platelet-derived growth factor
receptor beta (PDGFRB) [Q8R406; Q05030]: FGFR binding motif
<400> SEQUENCE: 65 Gly Glu Tyr Phe Cys Val Tyr Asn Asn Ser
Leu Gly 1 5 10 <210> SEQ ID NO 66 <211> LENGTH: 13
<212> TYPE: PRT <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Intercellular
adhesion molecule-5 (ICAM-5, telencephalin) [Q8TAM9; Q60625]: FGFR
binding motif <400> SEQUENCE: 66 Gly Glu Tyr Glu Cys Ala Ala
Thr Asn Ala His Gly Arg 1 5 10 <210> SEQ ID NO 67 <211>
LENGTH: 13 <212> TYPE: PRT <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION: B-cell
receptor CD22 precursor (Leu-14; B- lymphocyte cell adhesion
molecule) [P20273]: FGFR binding motif <400> SEQUENCE: 67 Gly
Ala Tyr Trp Cys Gln Gly Thr Asn Ser Val Gly Lys 1 5 10 <210>
SEQ ID NO 68 <211> LENGTH: 12 <212> TYPE: PRT
<213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: B-cell receptor CD22 precursor
(Leu-14; B- lymphocyte cell adhesion molecule) [P20273]: FGFR
binding motif <400> SEQUENCE: 68 Gly Thr Tyr Ser Cys Val Ala
Glu Asn Ile Leu Gly 1 5 10 <210> SEQ ID NO 69 <211>
LENGTH: 14 <212> TYPE: PRT <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION: NCAM-2
[Swiss-Prot: O15394; O35136]: FGFR binding motif <400>
SEQUENCE: 69 Arg Val Ala Ala Val Asn Gly Lys Gly Gln Gly Asp Tyr
Ser 1 5 10
<210> SEQ ID NO 70 <211> LENGTH: 14 <212> TYPE:
PRT <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: HCF-2 (Host cell factor 2)
[Swiss-Prot: Q9Y5Z7]: FGFR binding motif: FGFR binding motif
<400> SEQUENCE: 70 Arg Val Ala Ala Ile Asn Gly Cys Gly Ile
Gly Pro Phe Ser 1 5 10 <210> SEQ ID NO 71 <211> LENGTH:
9 <212> TYPE: PRT <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: ICLN (Chloride
channel regulator, inducer) [Swiss-Prot: P97506; Q9NRD2; Q61189;
P54105]: FGFR binding motif <400> SEQUENCE: 71 Ala Val Leu
Asn Gly Lys Gly Leu Gly 1 5 <210> SEQ ID NO 72 <211>
LENGTH: 11 <212> TYPE: PRT <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Galectin-12 [Swiss-Prot: Q91VD1; Q9JKX2; Q9NZ03]: FGFR binding
motif <400> SEQUENCE: 72 Ala Leu Asn Gly Gln Gly Leu Gly Ala
Thr Ser 1 5 10 <210> SEQ ID NO 73 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Human
receptor-like protein tyrosine phosphatase leukocyte common
antigen-related molecule (PTPRF) [Swiss-Prot: P10586]: FGFR bind
ing motif <400> SEQUENCE: 73 Arg Leu Ala Ala Lys Asn Arg Ala
Gly Leu Gly Glu 1 5 10 <210> SEQ ID NO 74 <211> LENGTH:
13 <212> TYPE: PRT <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Natural
resistance-associated macrophage protein 1(NRAMP-1, SLC11A1)
[Swiss-Prot: O77741]: FGFR binding motif <400> SEQUENCE: 74
Arg Leu Gly Val Val Thr Gly Lys Asp Leu Gly Glu Ile 1 5 10
<210> SEQ ID NO 75 <211> LENGTH: 14 <212> TYPE:
PRT <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: NCAM2 (180 kDa isoform precursor )
[Swiss- Prot: P36335]: FGFR binding motif <400> SEQUENCE: 75
Thr Val Thr Gly Leu Lys Pro Glu Thr Ser Tyr Met Val Lys 1 5 10
<210> SEQ ID NO 76 <211> LENGTH: 13 <212> TYPE:
PRT <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Nephrin [Swiss-Prot: Q925S5; Q9JIX2;
Q9ET59; Q9R044; Q9QZS7]: FGFR binding motif <400> SEQUENCE:
76 Thr Leu Thr Gly Leu Lys Pro Ser Thr Arg Tyr Arg Ile 1 5 10
<210> SEQ ID NO 77 <211> LENGTH: 13 <212> TYPE:
PRT <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Nephrin [Swiss-Prot: O60500]: FGFR
binding motif <400> SEQUENCE: 77 Thr Leu Thr Gly Leu Gln Pro
Ser Thr Arg Tyr Arg Val 1 5 10 <210> SEQ ID NO 78 <211>
LENGTH: 14 <212> TYPE: PRT <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Tyrosine phosphatase LAR (PTPRF) [Swiss- Prot : Q9EQ17]: FGFR
binding motif <400> SEQUENCE: 78 Thr Leu Leu Gly Leu Lys Pro
Asp Thr Thr Tyr Asp Ile Lys 1 5 10 <210> SEQ ID NO 79
<211> LENGTH: 14 <212> TYPE: PRT <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Leukocyte common antigen-related phosphatase ptp2
precursor (LAR-PTP2) [Swiss-Prot: Q64605]: FGFR binding motif
<400> SEQUENCE: 79 Thr Leu Gln Gly Leu Arg Pro Glu Thr Ala
Tyr Glu Leu Arg 1 5 10 <210> SEQ ID NO 80 <211> LENGTH:
14 <212> TYPE: PRT <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION:
Protein-tyrosine phosphatase, receptor-type, S precursor (EC
3.1.3.48) (Protein-tyrosine phosphatase sigma) (RPTP-sigma)
[Swiss-Prot: Q64699]: FGFR binding motif <400> SEQUENCE: 80
Thr Leu Arg Gly Leu Arg Pro Glu Thr Ala Tyr Glu Leu Arg 1 5 10
<210> SEQ ID NO 81 <211> LENGTH: 14 <212> TYPE:
PRT <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Tyrosine-protein kinase receptor
Tie-1 precursor (TIE1.) (EC 2.7.1.112) [Swiss-Prot: Q06805;
P35590]: FGFR binding motif <400> SEQUENCE: 81 Thr Leu Met
Asn Leu Arg Pro Lys Thr Gly Tyr Ser Val Arg 1 5 10 <210> SEQ
ID NO 82 <211> LENGTH: 11 <212> TYPE: PRT <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Ephrin type-A receptor 8 precursor to (EPHA8..)
(EC 2.7.1.112)(Tyrosine-protein kinase receptor EEK) (EPH-and
ELK-related kinase)]: [Swiss-Prot: O09127; O09127; P29322];FGFR
binding motif <400> SEQUENCE: 82 Thr Val Ser Gly Leu Lys Pro
Gly Thr Arg Tyr 1 5 10 <210> SEQ ID NO 83 <211> LENGTH:
11 <212> TYPE: PRT <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Ephrin type-A
receptor 3 precursor (EC 2.7.1.112) (Tyrosine-prote in kinase
receptor ETK1) (CEK4) (EPHA3..) [tn: P29318]: FGFR binding motif
<400> SEQUENCE: 83 Thr Ile Ser Gly Leu Lys Pro Asp Thr Thr
Tyr 1 5 10 <210> SEQ ID NO 84 <211> LENGTH: 11
<212> TYPE: PRT <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION:
Protein-tyrosine phosphatase receptor-type S precursor (EC
3.1.3.48) (Protein-tyrosine phosphatase sigma, PTPRS) [Swiss-Prot:
Q13332]: FGFR binding motif <400> SEQUENCE: 84 Thr Leu Gln
Gly Leu Lys Pro Asp Thr Ala Tyr 1 5 10 <210> SEQ ID NO 85
<211> LENGTH: 12 <212> TYPE: PRT <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Insulin receptor [Swiss-Prot: Q9PWN6]: FGFR binding
motif <400> SEQUENCE: 85 Leu Arg Gly Leu Lys Pro Trp Thr Gln
Tyr Ala Val 1 5 10 <210> SEQ ID NO 86 <211> LENGTH: 13
<212> TYPE: PRT <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Type VII
collagen [Swiss-Prot: Q63870]: FGFR binding motif
<400> SEQUENCE: 86 Ile Asp Gly Leu Glu Pro Asp Thr Glu Tyr
Ile Val Arg 1 5 10 <210> SEQ ID NO 87 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Insulin-like
growth factor-1 receptor precursor [Swiss-Prot: O73798]: FGFR
binding motif <400> SEQUENCE: 87 Leu Gln Gly Leu Lys Pro Trp
Thr Gln Tyr Ala Ile 1 5 10 <210> SEQ ID NO 88 <211>
LENGTH: 14 <212> TYPE: PRT <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Fibronectin [Swiss-Prot: Q95KV4; Q95KV5; P07589; Q28377;
U42594;O95609]: FGFR binding motif <400> SEQUENCE: 88 Thr Ile
Thr Gly Leu Glu Pro Gly Thr Glu Tyr Thr Ile Gln 1 5 10 <210>
SEQ ID NO 89 <211> LENGTH: 10 <212> TYPE: PRT
<213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Insulin-like growth factor I
receptor (IGF I receptor beta-subun it, IGF I receptor
alpha-subunit) [Swiss- Prot: Q9QVW4; P08069; P24062; Q60751;
P15127; P15208]: FGFR binding motif <400> SEQUENCE: 89 Gly
Leu Lys Pro Trp Thr Gln Tyr Ala Val 1 5 10 <210> SEQ ID NO 90
<211> LENGTH: 13 <212> TYPE: PRT <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Insulin receptor-related protein precursor (EC
2.7.1.112) (IRR) (IR-related receptor) [Swiss-Prot: P14616]: FGFR
binding motif <400> SEQUENCE: 90 Thr Leu Ala Ser Leu Lys Pro
Trp Thr Gln Tyr Ala Val 1 5 10 <210> SEQ ID NO 91 <211>
LENGTH: 12 <212> TYPE: PRT <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Tenascin-R (restrictin) [Swiss-Prot: Q15568; O00531]: FGFR binding
motif <400> SEQUENCE: 91 Leu Met Gly Leu Gln Pro Ala Thr Glu
Tyr Ile Val 1 5 10 <210> SEQ ID NO 92 <211> LENGTH: 14
<212> TYPE: PRT <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Neogenin
precursor (NEO1..) [Swiss-Prot: Q92859; P97603; Q90610;P97798]:
FGFR binding motif <400> SEQUENCE: 92 Lys Gly Met Gly Pro Met
Ser Glu Ala Val Gln Phe Arg Thr 1 5 10 <210> SEQ ID NO 93
<211> LENGTH: 14 <212> TYPE: PRT <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Protein tyrosine phosphatase receptor type D (PTPRD,
BA175E13.1)[Swiss-Prot: Q8WX65; Q9IAJ1; P23468; Q64487]: FGFR
binding motif <400> SEQUENCE: 93 Thr Leu Thr Gly Leu Lys Pro
Asp Thr Thr Tyr Asp Val Lys 1 5 10 <210> SEQ ID NO 94
<211> LENGTH: 12 <212> TYPE: PRT <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Protein tyrosine phosphatase receptor type D (PTPRD,
BA175E13.1 )[Swiss-Prot: Q8WX65; Q9IAJ1; P23468; Q64487]: FGFR
binding motif <400> SEQUENCE: 94 Ile Ser Gly Leu Gln Pro Glu
Thr Ser Tyr Ser Leu 1 5 10 <210> SEQ ID NO 95 <211>
LENGTH: 14 <212> TYPE: PRT <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Protein-tyrosine phosphatase receptor-type F precursor (EC
3.1.3.48) (LAR protein) (Leukocyte antigen related) [Swiss-Prot:
Q64604; Q9QW67; P10586]: FGFR binding motif <400> SEQUENCE:
95 Thr Leu Leu Gly Leu Lys Pro Asp Thr Thr Tyr Asp Ile Lys 1 5 10
<210> SEQ ID NO 96 <211> LENGTH: 13 <212> TYPE:
PRT <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Protein-tyrosine phosphatase
receptor-type F precursor (EC 3.1.3.48) (Leukocyte antigen related)
[Swiss-Prot: Q64604; Q9QW67; P10586]: FGFR binding motif
<400> SEQUENCE: 96 Thr Ile Ser Gly Leu Thr Pro Glu Thr Thr
Tyr Ser Ile 1 5 10 <210> SEQ ID NO 97 <211> LENGTH: 13
<212> TYPE: PRT <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: CD22 [Q9R094]:
FGFR binding motif <400> SEQUENCE: 97 Gly Asn Tyr Ser Cys Leu
Ala Glu Asn Arg Leu Gly Arg 1 5 10 <210> SEQ ID NO 98
<211> LENGTH: 12 <212> TYPE: PRT <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: FGFR-4 [Q91742]: FGFR binding motif <400>
SEQUENCE: 98 Gly Asn Tyr Thr Cys Val Val Glu Asn Arg Val Gly 1 5 10
<210> SEQ ID NO 99 <211> LENGTH: 12 <212> TYPE:
PRT <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: ICAM-5 [Q8TAM9]: FGFR binding motif
<400> SEQUENCE: 99 Gly Thr Tyr His Cys Val Ala Thr Asn Ala
His Gly 1 5 10 <210> SEQ ID NO 100 <211> LENGTH: 14
<212> TYPE: PRT <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: FIII,4 domain
of L1: FGFR binding motif [Swiss-Prot: Q9QY38] <400>
SEQUENCE: 100 Leu Ser His Asn Gly Val Leu Thr Gly Tyr Leu Leu Ser
Tyr 1 5 10 <210> SEQ ID NO 101 <211> LENGTH: 11
<212> TYPE: PRT <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Neuron-glia
cell adhesion molecule (Ng-CaM) precursor .[Gallus gallus];
[Swiss-Prot: Q90933]: FGFR binding motif <400> SEQUENCE: 101
Asn Gly Val Leu Thr Gly Tyr Val Leu Arg Tyr 1 5 10 <210> SEQ
ID NO 102 <211> LENGTH: 11 <212> TYPE: PRT <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Neurofascin precursor .[Gallus gallus];
[Swiss-Prot: O42414]: FGFR binding motif <400> SEQUENCE: 102
Asn Gly Val Leu Thr Gly Tyr Asn Leu Arg Tyr 1 5 10 <210> SEQ
ID NO 103 <211> LENGTH: 11 <212> TYPE: PRT <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: (CALL) Neural cell adhesion molecule. [Homo
sapiens] .[ Swiss-Prot: O00533]: FGFR binding motif
<400> SEQUENCE: 103 Asn Gly Asn Leu Thr Gly Tyr Leu Leu Gln
Tyr 1 5 10 <210> SEQ ID NO 104 <211> LENGTH: 14
<212> TYPE: PRT <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: f
Neuroglian.[Manduca sexta] .[ Swiss-Prot: P91767]: FGFR binding
motif <400> SEQUENCE: 104 Val Asp Glu Asn Gly Val Leu Thr Gly
Tyr Lys Ile Tyr Tyr 1 5 10 <210> SEQ ID NO 105 <211>
LENGTH: 13 <212> TYPE: PRT <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Protein-tyrosine phosphotase sigma [Swiss- Prot: O75870]; and
[Swiss-Prot: Q13332] [Homo sapiens] :FGFR binding motif <400>
SEQUENCE: 105 Thr His Asn Gly Ala Leu Val Gly Tyr Ser Val Arg Tyr 1
5 10 <210> SEQ ID NO 106 <211> LENGTH: 11 <212>
TYPE: PRT <213> ORGANISM: Artificial sequence <220>
FEATURE: <223> OTHER INFORMATION: NR-CaM 12 [Rattus sp] ,
[Swiss-Prot: Q9QVN3]: FGFR binding motif <400> SEQUENCE: 106
Asn Gly Ile Leu Thr Glu Tyr Ile Leu Lys Tyr 1 5 10 <210> SEQ
ID NO 107 <211> LENGTH: 11 <212> TYPE: PRT <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Neurofascin 155 kDa isoform.[Rattus
norvegicus],[ Swiss-Prot: Q91Z60]: FGFR binding motif <400>
SEQUENCE: 107 Asn Gly Ile Leu Ile Gly Tyr Thr Leu Arg Tyr 1 5 10
<210> SEQ ID NO 108 <211> LENGTH: 13 <212> TYPE:
PRT <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Neogenin (Fragment).[Gallus gallus],
[Swiss-Prot: Q90610]: FGFR binding motif <400> SEQUENCE: 108
Thr His Ser Gly Gln Ile Thr Gly Tyr Lys Ile Arg Tyr 1 5 10
<210> SEQ ID NO 109 <211> LENGTH: 11 <212> TYPE:
PRT <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Neogenin (Fragment).[Gallus gallus],
[Swiss- Prot: Q90610]:FGFR binding motif <400> SEQUENCE: 109
Asn Gly Lys Ile Thr Gly Tyr Ile Ile Tyr Tyr 1 5 10 <210> SEQ
ID NO 110 <211> LENGTH: 10 <212> TYPE: PRT <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Metalloprotease 1 (pitrilysin family).[Homo
sapiens] [ Swiss-Prot: Q9BSI6]:FGFR binding motif <400>
SEQUENCE: 110 Leu Ser His Asn Gly Ile Phe Thr Leu Tyr 1 5 10
<210> SEQ ID NO 111 <211> LENGTH: 11 <212> TYPE:
PRT <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: HBRAVO/Nr-CaM.[Homo
sapiens].[Swiss-Prot: Q92823; O15179]: FGFR binding motif
<400> SEQUENCE: 111 Asn Gly Ile Leu Thr Glu Tyr Thr Leu Lys
Tyr 1 5 10 <210> SEQ ID NO 112 <211> LENGTH: 14
<212> TYPE: PRT <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION:
Protein-tyrosine phosphatase kappa precursor (EC 3.1.3.48)
(R-PTP-kappa).[Homo sapiens].[Swiss-Prot: Q15262]: FGFR binding
motif <400> SEQUENCE: 112 Leu Asp Pro Asn Gly Ile Ile Thr Gln
Tyr Glu Ile Ser Tyr 1 5 10 <210> SEQ ID NO 113 <211>
LENGTH: 11 <212> TYPE: PRT <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Neogenin precursor (NEO1..).[Homo sapiens and Mus
musculus][Swiss-Prot: Q92859; P97798]: FGFR binding motif
<400> SEQUENCE: 113 Asn Gly Lys Ile Thr Gly Tyr Ile Ile Tyr
Tyr 1 5 10 <210> SEQ ID NO 114 <211> LENGTH: 15
<212> TYPE: PRT <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Neural cell
adhesion L1( SPLICE ISOFORM 2 ) [Homo sapiens [Swiss-Prot: P32004
]; [Mus musculus Swiss-Prot: Q9QY38]: FGFR binding motif
<400> SEQUENCE: 114 His Leu Glu Val Gln Ala Phe Asn Gly Arg
Gly Ser Gly Pro Ala 1 5 10 15 <210> SEQ ID NO 115 <211>
LENGTH: 14 <212> TYPE: PRT <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
NB-2.[Rattus norvegicus] [Swiss-Prot: P97527]:FGFR binding motif
<400> SEQUENCE: 115 His Leu Thr Val Arg Ala Tyr Asn Gly Ala
Gly Tyr Gly Pro 1 5 10 <210> SEQ ID NO 116 <211>
LENGTH: 15 <212> TYPE: PRT <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION: Neural
cell adhesion protein BIG-2 precursor. [Rattus
norvegicus][Swiss-Prot: Q62845]: FGFR binding motif <400>
SEQUENCE: 116 His Leu Ser Val Lys Ala Tyr Asn Ser Ala Gly Thr Gly
Pro Ser 1 5 10 15 <210> SEQ ID NO 117 <211> LENGTH: 15
<212> TYPE: PRT <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION:
Axonal-associated cell adhesion molecule. [Homo sapiens].
[Swiss-Prot: Q8TC35]:FGFR binding motif <400> SEQUENCE: 117
His Leu Ala Val Lys Ala Tyr Asn Ser Ala Gly Thr Gly Pro Ser 1 5 10
15 <210> SEQ ID NO 118 <211> LENGTH: 14 <212>
TYPE: PRT <213> ORGANISM: Artificial sequence <220>
FEATURE: <223> OTHER INFORMATION: Contactin A/F3/F11.[Xenopus
laevis] [Swiss- Prot: O93250]: FGFR binding motif <400>
SEQUENCE: 118 Asn Leu Glu Val Arg Ala Phe Asn Ser Ala Gly Asp Gly
Pro 1 5 10 <210> SEQ ID NO 119 <211> LENGTH: 14
<212> TYPE: PRT <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Neural cell
adhesion molecule CALL.[Homo sapiens][Swiss-Prot: O00533]:FGFR
binding motif <400> SEQUENCE: 119 His Leu Thr Val Leu Ala Tyr
Asn Ser Lys Gly Ala Gly Pro 1 5 10 <210> SEQ ID NO 120
<211> LENGTH: 13 <212> TYPE: PRT <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Neuron-glia cell adhesion molecule (Ng-CaM)
precursor.[Gallus gallus][Swiss-Prot: Q909339]: FGFR binding motif
<400> SEQUENCE: 120 Leu Arg Val Leu Val Phe Asn Gly Arg Gly
Asp Gly Pro 1 5 10 <210> SEQ ID NO 121 <211> LENGTH: 14
<212> TYPE: PRT <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Contactin
precursor (Neural cell recognition molecule F11).[Gallus
gallus][Swiss-Prot: P14781]: FGFR binding motif <400>
SEQUENCE: 121 His Ile Asp Val Ser Ala Phe Asn Ser Ala Gly Tyr Gly
Pro 1 5 10 <210> SEQ ID NO 122 <211> LENGTH: 10
<212> TYPE: PRT <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: SLIT
[Drosophila melanogaster][Swiss-Prot: Q9XYV4]: FGFR binding motif
<400> SEQUENCE: 122 His Leu Ala Val Glu Leu Phe Asn Gly Arg 1
5 10 <210> SEQ ID NO 123 <211> LENGTH: 14 <212>
TYPE: PRT <213> ORGANISM: Artificial sequence <220>
FEATURE: <223> OTHER INFORMATION: Galectin-4.[Mus
musculus][Swiss-Prot: Q8K419, P38552]: FGFR binding motif
<400> SEQUENCE: 123 Leu Glu Leu Gln Ser Ile Asn Phe Leu Gly
Gly Gln Pro Ala 1 5 10 <210> SEQ ID NO 124 <211>
LENGTH: 14 <212> TYPE: PRT <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
HNB-2.[Homo sapiens]Swiss-Prot: O94779: FGFR binding motif
<400> SEQUENCE: 124 His Phe Thr Val Arg Ala Tyr Asn Gly Ala
Gly Tyr Gly Pro 1 5 10 <210> SEQ ID NO 125 <211>
LENGTH: 15 <212> TYPE: PRT <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION: The
EFL peptide (from the FIII,3 domain of L1) [Swiss-Prot: P32004]:
FGFR binding motif <400> SEQUENCE: 125 His Leu Glu Val Gln
Ala Phe Asn Gly Arg Gly Ser Gln Pro Ala 1 5 10 15 <210> SEQ
ID NO 126 <211> LENGTH: 14 <212> TYPE: PRT <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Fragment of Neuroglian (Drosophila)[ Swiss-
prot: P202419]: FGFR binding motif <400> SEQUENCE: 126 Val
Ile Ala Asp Gln Pro Thr Phe Val Lys Tyr Leu Ile Lys 1 5 10
<210> SEQ ID NO 127 <211> LENGTH: 14 <212> TYPE:
PRT <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Fragment of Fibronectin (bovine)
[Swiss-prot: P07589]: FGFR binding motif <400> SEQUENCE: 127
Thr Ile Lys Gly Leu Arg Pro Gly Val Val Tyr Glu Gly Gln 1 5 10
<210> SEQ ID NO 128 <211> LENGTH: 14 <212> TYPE:
PRT <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Tenascin (chick) [Swiss-prot:
P10039]: FGFR binding motif <400> SEQUENCE: 128 Thr Leu Thr
Glu Leu Ser Pro Ser Thr Gln Tyr Thr Val Lys 1 5 10 <210> SEQ
ID NO 129 <211> LENGTH: 14 <212> TYPE: PRT <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: Ephrin type A receptor2 [Swiss-prot: Q8N3Z2]:
FGFR binding motif <400> SEQUENCE: 129 Thr Leu Asp Asp Leu
Ala Pro Asp Thr Thr Tyr Leu Val Gln 1 5 10 <210> SEQ ID NO
130 <211> LENGTH: 14 <212> TYPE: PRT <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: LAR [Swiss-prot Q9VIS8]: FGFR binding motif
<400> SEQUENCE: 130 Thr Val Ser Asp Val Thr Pro His Ala Ile
Tyr Thr Val Arg 1 5 10 <210> SEQ ID NO 131 <211>
LENGTH: 14 <212> TYPE: PRT <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION: RTK
(Tie-1,hu) [Swiss-prot P35590]: FGFR binding motif <400>
SEQUENCE: 131 Ile Ile Arg Gly Leu Asn Ala Ser Thr Arg Tyr Leu Phe
Arg 1 5 10 <210> SEQ ID NO 132 <211> LENGTH: 14
<212> TYPE: PRT <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: RTK (Tie-1,hu)
[Swiss-prot P35590]: FGFR binding motif <400> SEQUENCE: 132
Thr Leu Met Asn Leu Arg Pro Lys Thr Gly Tyr Ser Val Arg 1 5 10
<210> SEQ ID NO 133 <211> LENGTH: 14 <212> TYPE:
PRT <213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Consensus sequence (conserved domain
database) : FGFR binding motif <400> SEQUENCE: 133 Thr Leu
Thr Gly Leu Lys Pro Gly Thr Glu Tyr Glu Val Arg 1 5 10 <210>
SEQ ID NO 134 <211> LENGTH: 14 <212> TYPE: PRT
<213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: The beta-common cytokine receptor of
IL-3. Il-5 and GmCsf [Swiss-prot P32927]: FGFR binding motif
<400> SEQUENCE: 134 Gly Pro Glu His Leu Met Pro Ser Ser Thr
Tyr Val Ala Arg 1 5 10 <210> SEQ ID NO 135 <211>
LENGTH: 14 <212> TYPE: PRT <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION: Unc-22
(C. Elegance) [Swiss-prot: Q23550]: FGFR binding motif <400>
SEQUENCE: 135 Arg Val Thr Gly Leu Thr Pro Lys Lys Thr Tyr Glu Phe
Arg 1 5 10 <210> SEQ ID NO 136 <211> LENGTH: 14
<212> TYPE: PRT <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Consensus
sequence (conserved domain database): FGFR binding motif
<400> SEQUENCE: 136 Thr Leu Thr Gly Leu Lys Pro Gly Thr Glu
Tyr Glu Phe Arg 1 5 10 <210> SEQ ID NO 137 <211>
LENGTH: 15 <212> TYPE: PRT <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Consensus sequence (conserved domain database):FGFR binding
motif
<400> SEQUENCE: 137 Glu Val Arg Val Gln Ala Val Asn Gly Gly
Gly Asn Gly Pro Pro 1 5 10 15 <210> SEQ ID NO 138 <211>
LENGTH: 12 <212> TYPE: PRT <213> ORGANISM: Artificial
sequence <220> FEATURE: <223> OTHER INFORMATION:
Drosophila Neuroglian [Swiss-prot: P20241]: FGFR binding motif
<400> SEQUENCE: 138 Leu Ile Lys Val Val Ala Ile Asn Asp Arg
Gly Glu 1 5 10 <210> SEQ ID NO 139 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Artificial sequence
<220> FEATURE: <223> OTHER INFORMATION: Fibronectin
(mouse) [Swiss-prot: P11276]: FGFR binding motif <400>
SEQUENCE: 139 Val Val Ser Ile Ile Ala Val Asn Gly Arg Glu Glu 1 5
10 <210> SEQ ID NO 140 <211> LENGTH: 12 <212>
TYPE: PRT <213> ORGANISM: Artificial sequence <220>
FEATURE: <223> OTHER INFORMATION: Fibronectin (bovine)
[Swiss-prot: P07589]: FGFR binding motif <400> SEQUENCE: 140
Val Val Ser Val Tyr Ala Gln Asn Gln Asn Gly Glu 1 5 10 <210>
SEQ ID NO 141 <211> LENGTH: 12 <212> TYPE: PRT
<213> ORGANISM: Artificial sequence <220> FEATURE:
<223> OTHER INFORMATION: Tenascine (chick) [Swiss-prot:
Q90995]: FGFR binding motif <400> SEQUENCE: 141 Thr Ile Ser
Leu Val Ala Glu Lys Gly Arg His Lys 1 5 10 <210> SEQ ID NO
142 <211> LENGTH: 15 <212> TYPE: PRT <213>
ORGANISM: Artificial sequence <220> FEATURE: <223>
OTHER INFORMATION: L1 (human, F3,EFL) [Swiss-prot: P32004]: FGFR
binding motif <400> SEQUENCE: 142 His Leu Glu Val Gln Ala Phe
Asn Gly Arg Gly Ser Gly Pro Ala 1 5 10 15 <210> SEQ ID NO 143
<211> LENGTH: 15 <212> TYPE: PRT <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: L1 (mouse, F3,EFL) [Swiss-prot: P11627]: FGFR binding
motif <400> SEQUENCE: 143 His Val Glu Val Gln Ala Phe Asn Gly
Arg Gly Leu Gly Pro Ala 1 5 10 15 <210> SEQ ID NO 144
<211> LENGTH: 15 <212> TYPE: PRT <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: L1 (rat, F3,EFL) [Swiss-prot: Q05695]: FGFR binding
motif <400> SEQUENCE: 144 His Val Glu Val Gln Ala Phe Asn Gly
Arg Gly Leu Gly Pro Ala 1 5 10 15 <210> SEQ ID NO 145
<211> LENGTH: 13 <212> TYPE: PRT <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: Consensus sequence (conserved domain database): FGFR
binding motif <400> SEQUENCE: 145 Glu Phe Arg Val Arg Ala Val
Asn Gly Ala Gly Glu Gly 1 5 10 <210> SEQ ID NO 146
<211> LENGTH: 15 <212> TYPE: PRT <213> ORGANISM:
Artificial sequence <220> FEATURE: <223> OTHER
INFORMATION: The beta-common cytokine receptor of IL-3. Il-5 and
GmCsf [Swiss-prot: P32927]: FGFR binding motif <400>
SEQUENCE: 146 Val Ala Arg Val Arg Thr Arg Leu Ala Pro Gly Ser Arg
Leu Ser 1 5 10 15
* * * * *