U.S. patent application number 12/937189 was filed with the patent office on 2011-05-12 for molecules inhibiting a metabolic pathway involving the syk protein tyrosine kinase and method for identifying said molecules.
Invention is credited to Piona Dariavach, Pierre Emile Ulysse Martineau, Bruno Villoutreix.
Application Number | 20110112098 12/937189 |
Document ID | / |
Family ID | 40344607 |
Filed Date | 2011-05-12 |
United States Patent
Application |
20110112098 |
Kind Code |
A1 |
Dariavach; Piona ; et
al. |
May 12, 2011 |
MOLECULES INHIBITING A METABOLIC PATHWAY INVOLVING THE SYK PROTEIN
TYROSINE KINASE AND METHOD FOR IDENTIFYING SAID MOLECULES
Abstract
The present invention relates to the C-13 molecule (methyl
2-{5-[(3-benzyl-4-oxo-2-thioxo-1,3-thiazolidin-5-ylidene)methyl]-2-furyl}-
-benzoate) and to organic molecules functionally equivalent to the
C-13 molecule, capable of inhibiting the binding of an antibody or
antibody fragment with the human Syk protein tyrosine kinase, to
the use of these molecules for the production of medicaments for
the prevention or treatment of diseases dependent on metabolic
pathways involving Syk, and also to a method for identifying such
molecules.
Inventors: |
Dariavach; Piona;
(Montpellier, FR) ; Martineau; Pierre Emile Ulysse;
(Saint Gely Du Fesc, FR) ; Villoutreix; Bruno;
(Paris, FR) |
Family ID: |
40344607 |
Appl. No.: |
12/937189 |
Filed: |
April 8, 2009 |
PCT Filed: |
April 8, 2009 |
PCT NO: |
PCT/FR09/00414 |
371 Date: |
January 3, 2011 |
Current U.S.
Class: |
514/237.2 ;
506/9; 514/255.01; 514/290; 514/312; 514/342; 514/369; 544/141;
544/391; 546/111; 546/156; 546/270.4; 548/183 |
Current CPC
Class: |
A61K 31/5377 20130101;
A61K 31/4439 20130101; A61P 37/08 20180101; A61K 31/41 20130101;
A61K 31/517 20130101; A61P 11/06 20180101; A61P 37/06 20180101;
A61K 31/427 20130101; A61K 45/06 20130101; A61P 19/02 20180101;
A61K 31/351 20130101; A61K 31/4196 20130101; A61K 31/4704 20130101;
A61K 31/4015 20130101; A61K 31/4155 20130101; A61P 29/00 20180101;
G01N 2333/186 20130101; A61K 31/451 20130101; A61K 31/404 20130101;
A61K 31/4178 20130101; A61P 37/02 20180101; A61P 7/10 20180101;
A61P 35/00 20180101; G01N 2333/9121 20130101; A61P 11/02 20180101;
A61P 37/00 20180101; A61P 43/00 20180101; A61K 31/445 20130101;
A61K 31/4525 20130101; A61K 31/343 20130101; A61K 31/502 20130101;
A61K 31/506 20130101; A61K 31/33 20130101; A61K 31/4025 20130101;
A61K 31/435 20130101; A61K 31/18 20130101; A61K 31/495 20130101;
A61K 31/53 20130101; A61K 31/4162 20130101; A61K 31/4035 20130101;
A61K 31/426 20130101; A61K 31/553 20130101; A61K 31/167 20130101;
A61P 27/14 20180101 |
Class at
Publication: |
514/237.2 ;
548/183; 506/9; 546/270.4; 546/111; 544/141; 546/156; 544/391;
514/369; 514/342; 514/290; 514/312; 514/255.01 |
International
Class: |
A61K 31/5377 20060101
A61K031/5377; C07D 277/36 20060101 C07D277/36; C07D 417/14 20060101
C07D417/14; C07D 221/16 20060101 C07D221/16; C07D 413/06 20060101
C07D413/06; C07D 215/227 20060101 C07D215/227; C07D 241/04 20060101
C07D241/04; A61K 31/427 20060101 A61K031/427; A61K 31/4439 20060101
A61K031/4439; A61K 31/435 20060101 A61K031/435; A61K 31/4709
20060101 A61K031/4709; A61K 31/4965 20060101 A61K031/4965; A61P
37/06 20060101 A61P037/06 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 9, 2008 |
FR |
08/01959 |
Claims
1. A compound which binds with Syk tyrosine kinase protein on a
three-dimensional cavity comprising the Arginine residue situated
in position 68 and the two glutamic acid residues situated in
positions 121 and 155 of human Syk protein, the sequence of which
is set forth in SEQ ID No. 1, and capable of inhibiting by at least
10% in vitro binding of (i) antibody fragment G4G11 (SEQ ID No. 2),
or (ii) an antibody or antibody fragment which binds with human Syk
tyrosine kinase protein on an epitope comprising at least one of
residues 65 to 74 of the amino acid sequence of human Syk tyrosine
kinase protein represented by the sequence SEQ ID No. 1, or (iii)
an antibody or antibody fragment which binds with human Syk
tyrosine kinase protein and inhibiting by at least 10% the binding
of antibody fragments G4G11 with human Syk tyrosine kinase protein
(SEQ ID No. 1), with human Syk tyrosine kinase protein or with any
of the variants thereof in animals, for the prevention or treatment
of a condition dependent on a metabolic pathway involving Syk in
humans or animals, where said compound (C-13) has the formula
##STR00102## or an organic compound functionally equivalent to
molecule C-13 having a molecular weight between 50 and 2500 Dalton,
selected from compounds having any of the following formulae:
##STR00103## where R1 is an unsubstituted or substituted aromatic
group, or an unsubstituted or substituted heterocycle comprising at
least one S, O or N atom; R2 is an unsubstituted or substituted
aromatic group, an unsubstituted or substituted heterocycle, an
unsaturated or saturated carbon chain comprising an amine group, an
unsaturated or saturated carbon chain comprising an unsubstituted
or substituted aromatic group or an unsaturated or saturated carbon
chain comprising an unsubstituted or substituted heterocycle
comprising at least one S, O or N atom; R3 is an unsubstituted or
substituted phenyl, 2-pyridinyl, 3-pyridinyl or 4-pyridinyl group;
or ##STR00104## where n=0 or 1; n'=0 or 1; R4 is an unsaturated or
saturated carbon chain comprising 1 to 5 carbon atoms,
unsubstituted or substituted with an aromatic group; R5 is an
unsubstituted or substituted aromatic group or an unsubstituted or
substituted amine group; R6 is a hydrogen atom, an alkoxy group, an
alkyl group or a halogen; R7 is a hydrogen atom, an alkoxy group,
an alkyl group or a halogen; R8 is a hydrogen atom, an alkoxy
group, an alkyl group or a halogen; or ##STR00105## where m=0, 1 or
2; R9 is a hydrogen atom and R10 is an unsubstituted or substituted
phenyl group, or R9 and R10 are part of the same unsubstituted or
substituted heterocycle, or R9 and R10 are part of the same
unsubstituted or substituted aromatic group; R11 is a hydrogen
atom, an alkoxy group or an alkyl group; R12 is a hydrogen atom, an
alkoxy group or an alkyl group; R13 is a hydrogen atom or an alkyl
or alkoxy group; R14 is a hydrogen atom or an alkyl or alkoxy
group; ##STR00106## or where A is an oxygen or sulphur atom; R15 is
an unsaturated or saturated carbon chain comprising 1, 2 or 3
carbon atoms, unsubstituted or substituted by an unsubstituted or
substituted aromatic group, an unsubstituted or substituted
heterocycle or an amine group belonging to unsubstituted or
substituted heterocycle; R16 is a hydrogen atom, a halogen or an
alkoxy group; R17 is a hydrogen atom, an alkoxy group or an acetoxy
group; or ##STR00107## ##STR00108## ##STR00109## ##STR00110##
##STR00111## ##STR00112## ##STR00113## ##STR00114## or a
stereo-isomer, racemate or pharmacologically acceptable salt of
C-13 or said equivalent compound.
2. A compound according to claim 1, wherein said compound binds
with Syk tyrosine kinase protein at a site located outside the
catalytic domain thereof.
3. A compound according to claim 1, wherein said antibody or
antibody fragment binds with human Syk tyrosine kinase protein on
an epitope comprising at least 5 of residues 65 to 74 of the amino
acid sequence of human Syk tyrosine kinase protein illustrated by
SEQ ID No. 1.
4. A compound according to claim 1, wherein said three-dimensional
cavity further comprises the Serine residue situated in position 9,
the Glutamine residue situated in position 43, the Phenylalanine
residue situated in position 51, the Isoleucine residue situated in
position 66, the Glutamate residues situated in position 67 and 69,
the Leucine residue situated in position 70, the Asparagine residue
situated in position 71, the Glycine residue situated in position
72, the Threonine residue situated in position 73, the Tyrosine
residue situated in position 74 and the Alanine residue situated in
position 75 of human Syk protein, the sequence of which is
illustrated by SEQ ID No. 1.
5. A compound according to claim 1 wherein said compound is used
for the prevention or treatment of type I hypersensitivity
reactions.
6. A compound according to claim 5, wherein said compound inhibits
IgE-dependent mast cell degranulation.
7. A compound according to claim 6, wherein said compound is
capable of inhibiting, by 50% in vitro, mast cell degranulation at
a concentration (IC50) between 1 ng/ml and 1 mg/ml.
8. A compound according to claim 5, wherein said the metabolic
pathway involving Syk is a mast cell or basophil activation
pathway.
9. Molecule A compound according to claim 5, wherein said condition
is allergic asthma, allergic conjunctivitis, allergic rhinitis,
anaphylaxis, angioedema, urticaria, eosinophilia or an allergy to
an antibiotic.
10. A compound according to claim 5, wherein said compound has no
effect on the metabolic pathways involving human Syk protein (SEQ
ID No. 1) other than those giving rise to mast cell degranulation
and/or type I hypersensitivity reactions.
11. A compound according to claim 10, wherein said compound has no
effect on the antibody response which follows immunisation by a
thymus-dependent antigen or on neutrophil-dependent Syk
recruitment.
12. A compound according to claim 1, wherein said metabolic pathway
involving Syk is a B lymphocyte, T lymphocyte, neutrophil,
eosinophil, NK cell, platelet, erythrocyte, osteoclast, epithelial
cell or cancer cell activation pathway.
13. A compound according to claim 12, wherein said condition is
rheumatoid arthritis, an autoimmune disease, inflammation or
cancer.
14. A compound according to claim 1, wherein said compound is used
in combination with a further therapeutic molecule.
15. (canceled)
16. (canceled)
17. A compound according to claim 1, wherein said compound is
intended to be administered by the oral, sublingual, nasal, ocular,
local, intravenous, intraperitoneal, subcutaneous route, by aerosol
or by inhalation.
18. A compound according to claim 1, wherein said compound is
intended to be administered to adult, child or newborn human
patients.
19. A compound according to claim 1, wherein said compound is
intended to be administered at doses between 0.01 mg/kg and 200
mg/kg.
20. A compound according to claim 19, wherein said equivalent
compound is selected from compounds having any of the following
formulae: formula (I) where R1 is a phenyl group, unsubstituted or
substituted by an F or Cl atom, a methyl or ethyl group, an
N,N-dimethyl-sulphonamide or two groups selected from the methyl,
ethyl, hydroxy, methoxy or ethoxy groups, or a group ##STR00115## a
furan group unsubstituted or substituted by a methyl, ethyl,
hydroxyl, methoxy or ethoxy group, a thiophene group unsubstituted
or substituted by a methyl, ethyl, hydroxy, methoxy or ethoxy
group; R2 a group ##STR00116## where R21 and R22 are carbon atoms
each belonging to an alkyl chain comprising 1, 2 or 3 carbon atoms,
or both belonging with the nitrogen atom with which they are bound
to the same unsaturated or saturated heterocycle also comprising an
oxygen atom or a second nitrogen atom, or a group ##STR00117##
where R23 and R24 are carbon atoms each belonging to an alkyl chain
comprising 1, 2 or 3 carbon atoms, or both belonging with the
nitrogen atom with which they are bound to the same unsaturated or
saturated heterocycle also comprising an oxygen atom or a second
nitrogen atom, or a group ##STR00118## and R3 is a non-substituted
2-pyridinyl, 3-pyridinyl or 4-pyridinyl group, a phenyl group
unsubstituted or substituted by a benzoxy group, and/or by a
hydroxyl group, and/or by a methyl group, and/or by an ethyl group,
and/or by a propyl group, and/or by one or two Br, F or Cl atoms,
and/or by one to three hydroxyl, methoxy or ethoxy groups. or
formula (II) where R4 is an unsaturated or saturated carbon chain
comprising 1, 2 or 3 carbon atoms; R5 is a phenyl group or a
secondary amine group unsubstitued or substituted by an
unsubstituted or substituted phenyl group, or by a group
##STR00119## R6 is a hydrogen or chlorine atom or a methyl, ethyl,
hydroxyl, methoxy or ethoxy group; R7 is a hydrogen or chlorine
atom or a methyl, ethyl, hydroxy, methoxy or ethoxy group; R8 is a
hydrogen or chlorine atom or a methyl, ethyl, hydroxy, methoxy or
ethoxy group. or the formula (III) where R9 is a hydrogen atom and
the group R10 is an unsubstituted or substituted phenyl group, or
the groups R9 and R10 belong to the same unsubstituted or
substituted heterocycle comprising 2 nitrogen atoms and 4 carbon
atoms; R11 is a hydrogen atom or methyl, ethyl, hydroxy, methoxy or
ethoxy group; R12 is a hydrogen atom or methyl, ethyl, hydroxy,
methoxy or ethoxy group; R13 is a hydrogen atom or methyl, ethyl,
hydroxy, methoxy or ethoxy group; R14 is a hydrogen atom or methyl,
ethyl, hydroxy, methoxy or ethoxy group; or formula (IV) where A is
an Oxygen or Sulphur atom; R15 is a group ##STR00120## R16 is a
hydrogen or chlorine atom or a methyl, ethyl, hydroxy, methoxy or
ethoxy group; R17 is a methyl, ethyl, hydroxy, methoxy, ethoxy,
acetoxy, methoxycarhonyl or ethoxycarbonyl group.
21. A compound according to claim 20, wherein said equivalent
compound is selected from the following: ##STR00121## ##STR00122##
##STR00123## ##STR00124## ##STR00125## ##STR00126## ##STR00127##
##STR00128## ##STR00129## ##STR00130## ##STR00131## ##STR00132##
##STR00133## ##STR00134##
22. A compound according to claim 1, wherein the in vitro affinity
of said compound for Syk protein is less than 25 .mu.M.
23. A composition comprising a compound according to claim 1 and a
pharmacologically acceptable excipient for the prevention or
treatment of a condition dependent on a metabolic pathway involving
Syk in humans or animals.
24. (canceled)
25. A method for identifying an organic compound having a molecular
weight between 50 and 2500 Dalton binding with Syk tyrosine kinase
protein and capable of inhibiting by at least 10% in vitro binding
of (i) antibody fragment G4G11 (SEQ ID No. 2), or (ii) an antibody
or antibody fragment which binds with human Syk tyrosine kinase
protein on an epitope comprising at least one of residues 65 to 74
of the amino acid sequence of human Syk tyrosine kinase protein
represented by the sequence SEQ ID No. 1, or (iii) an antibody or
antibody fragment which binds with human Syk tyrosine kinase
protein and inhibiting by at least 10% the binding of antibody
fragment G4G11 with human Syk tyrosine kinase protein (SEQ ID No.
1), with human Syk tyrosine kinase protein or with any of the
variants thereof in animals, comprising at least the following
steps: a) screening, from a bank of candidate organic compounds
having a molecular weight between 50 and 2500 Da, those liable to
bind with Syk protein on the three-dimensional binding cavity on
the Syk protein of a compound selected from the molecules having
formula C-13, I, II, III, IV or 1 to 87 as illustrated above; b)
selecting from the compounds identified in a) those capable of
inhibiting by at least 10% in vitro the binding of the antibody or
antibody fragment (i) or (ii) with Syk protein.
26. (canceled)
27. (canceled)
28. (canceled)
29. (canceled)
30. (canceled)
Description
[0001] The present invention relates to organic molecules capable
of inhibiting the binding of an antibody or antibody fragment with
human Syk tyrosine kinase protein, the use of said molecules for
producing medicinal products for the prevention and treatment of
conditions dependent on metabolic pathways involving Syk, and a
method for identifying such molecules.
[0002] Syk ("Spleen tyrosine kinase") Tyrosine Kinase protein (PKT)
is a cytoplasmic protein which is a key mediator in
immunoreceptor-dependent signalling in the cells involved in
inflammation such as B lymphocytes, mast cells, macrophages and
neutrophils. In mast cells and basophils, cross-linking of the
Fc.epsilon.RI receptor (receptor with high affinity for
immunoglobulin E) with IgE and antigens induces phosphorylation of
Fc.epsilon.RI ITAM ("Immunoreceptor Tyrosine-based Activation
Motif") motifs so as to form a binding site for Syk which is then
activated. The activated Syk protein in turn phosphorylates
numerous substrates, including LAT ("linker for activation of T
cells"), SLP-76 ("Src homology 2 (SH2) domain-containing leukocyte
protein of 76 kD") and Vav adapter proteins, resulting in the
activation of a plurality of signalling cascades, such as those of
PLC-.gamma. (phospholipase C.gamma.), PI3K ("phosphatidylinositol
3-kinase"), Erk ("extracellular signal-regulated kinase"), JNK
("c-jun N-terminal kinase") and p38 (see FIG. 9). These cascades
eventually give rise to the degranulation, synthesis and release of
lipid mediators and the production and secretion of cytokines,
chemokines and growth factors by mast cells and
basophils.sup.1,2.
[0003] Syk protein is thus recognised as a potential pharmaceutical
target, particularly for the treatment of type I hypersensitivity
reactions including allergic rhinitis, urticaria, asthma and
anaphylaxis due to its critical position upstream from
immunoreceptor signalling complexes regulating the inflammatory
response in leukocytes. The fact that Syk regulates Fc.epsilon.RI
signalling positively.sup.3, particularly suggests that it could be
an excellent target for the treatment of allergic disorders.
Furthermore, due to the central role thereof in
Fc.epsilon.RI-dependent signalling, interacting pharmaceutically
with Syk could prove to be more advantageous than the conventional
use of antihistamines or leukotriene receptor agonists inhibiting a
single step downstream from the complex cascades contributing to
the acute and chronic symptoms associated with allergic
conditions.
[0004] Pharmacological inhibitors of Syk kinase activity having a
therapeutic potential, such as, in particular, Syk-specific
anti-sense oligonucleotides in the form of aerosols or small
molecules interfering with Syk activity such as ER-27139,
BAY-613606, piceatannol and R112 have already been developed.sup.1,
4. However, if multiple types of cells expressing Syk are
considered, potential side effects associated with systemic
exposure of the immune system to medicinal products targeting the
Syk kinase domain need to be taken into consideration. Indeed, Syk
protein is widely distributed in various cell types, it is thus
essential to account for the adverse effects of the inhibition
thereof on varied physiological functions such as cell
differentiation, adhesion and proliferation.sup.5, 6.
[0005] The inventors identified Syk protein inhibitors which act by
preventing the interaction thereof with the natural cellular
partners thereof rather than by targeting the catalytic site
thereof, particularly compound C-13 and compounds 1 to 87 given in
table 1.
[0006] The present invention relates to the molecule C-13 (methyl
2-{5-[(3-benzyl-4-oxo-2-thioxo-1,3-thiazolidin-5-ylidene)methyl]-2-furyl}-
benzoate) having the formula
##STR00001##
as a medicinal product for the prevention or treatment of a
condition dependent on a metabolic pathway involving Syk in humans
or animals.
[0007] The present invention further relates to functionally
equivalent organic molecules to molecule C-13, binding with Syk
tyrosine kinase protein and particularly molecules capable of
inhibiting by at least 5%, preferably at least 10%, for example at
least 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80 or 85%
in vitro binding of [0008] (i) antibody fragment G4G11 (SEQ ID No.
2), or [0009] (ii) antibody fragment G4E4 (SEQ ID No. 3), or [0010]
(iii) an antibody or antibody fragment which binds with human Syk
tyrosine kinase protein on an epitope comprising at least one of
residues 65 to 74 of the amino acid sequence of human Syk tyrosine
kinase protein represented by the sequence SEQ ID No. 1, or [0011]
(iv) an antibody or antibody fragment which binds with human Syk
tyrosine kinase protein and inhibiting by at least 10% the binding
of antibody fragments G4G11 or G4E4 with human Syk tyrosine kinase
protein (SEQ ID No. 1) with human Syk tyrosine kinase protein or
with any of the variants thereof in animals, for example murine Syk
tyrosine kinase protein wherein the sequence is illustrated in FIG.
8B (SEQ ID No. 4), as a medicinal product for the prevention or
treatment of a condition dependent on a metabolic pathway involving
Syk in humans or animals.
[0012] The term "functionally equivalent molecule" refers to a
molecule, for example an organic molecule having a molecular weight
between 50 and 2500 Da, capable of resulting in the same effect in
vitro in an intra or extracellular medium or in vivo, optionally
with a different intensity, than a given molecule. In particular,
within the scope of the present invention, the term "functionally
equivalent molecule to molecule C-13" refers to a molecule capable
of producing the same effect in vitro in an intra or extracellular
medium or in vivo, optionally with a different intensity, on human
tyrosine kinase protein as represented by sequence SEQ ID No. 1
(FIG. 8A) as molecule C-13. In particular, it refers to molecules
inhibiting the binding of Syk with another protein produced on the
Syk region comprising the SH2 domains thereof. More specifically,
these molecules do not affect the kinase enzyme activity of Syk.
For example, this consists of molecules capable of inhibiting the
interaction of Syk tyrosine kinase protein with antibody fragment
G4G11 (SEQ ID No. 2), antibody fragment G4E4 (SEQ ID No. 3), or an
antibody or antibody fragment binding with the same epitope as
antibody fragment G4G11 or G4E4 on human Syk protein (SEQ ID No.
1).
[0013] The term "percentage of inhibition" of the binding of an
antibody or antibody fragment with Syk protein, particularly refers
to the ratio [(A-B)/(A.times.100)], where A consists of the
intensity of a signal proportional to the quantity of an antibody
or antibody fragment bound with Syk protein in the absence of a
molecule according to the invention and B the intensity of the same
signal in the presence of a molecule according to the invention
under the same conditions. The inhibition of the binding of an
antibody or antibody fragments with Syk protein may particularly be
demonstrated in vitro by an antibody displacement test based on the
ELISA technique as described for example in international
application WO 2005106481. This test may be performed for example
according to the protocol described in example 3-2)
hereinafter.
[0014] The term "Syk variants in animals" refers to the genes of
various animal species, for example mouse, rat, dog, cat or another
mammal, coding for a protein having a strong sequence homology or
identity with human Syk protein as represented by the sequence SEQ
ID No. 1 (see FIG. 8A), for example a protein having at least 70,
75, 80, 85, 90 or 95% sequence homology or identity with the
sequence SEQ ID No. 1 of human Syk protein, having the same
tyrosine kinase activity and involved in the same functional
cascades as same, particularly in the functional cascade giving
rise to mast cell degranulation. It may particularly refer to
orthologous genes, i.e. genes found in different organisms, having
evolved from the same ancestral gene following speciation
events.
[0015] The present invention also relates to the pharmaceutically
acceptable salts, and if applicable, stereoisomers and racemates of
C-13 or of equivalent molecules according to the invention.
[0016] The term "pharmaceutically acceptable salts" refers to
relatively non-toxic inorganic and organic acid or basic addition
salts preserving the biological activity of the molecules according
to the invention. Examples of pharmaceutically acceptable salts are
particularly described in S. M. Berge et al., "Pharmaceutical
Salts", J. Pharm. Sci, 1977, 66: p. 1-19.sup.40. The
pharmaceutically acceptable addition salts of molecules according
to the invention may for example be hydrobromide, hydrochloride,
sulphate, bisulphate, phosphate, nitrate, acetate, oxalate,
valerate, oleate, palmitate, stearate, laurate, borate, benzoate,
lactate, phosphate, tosylate, citrate, maleate, fumarate,
succinate, tartrate, naphthylate, mesylate, glucoheptanate,
lactobionate, sulphamate, malonate, salicylate, propionate,
methylenebis-b-hydroxynaphthoate, gentisic acid, isethionate,
di-p-toluoyltartrate, methanesulphonate, ethane-sulphonate,
benzenesulphonate, p-toluenesulphonate, cyclohexyl sulphamate and
quinateslaurylsulphonate salts, and equivalents. Other
pharmaceutically acceptable salts which may be suitable include
metal salts, for example pharmaceutically acceptable alkaline metal
or alkaline-earth salts, such as sodium, potassium, calcium or
magnesium salts.
[0017] These pharmaceutically acceptable salts may be prepared in
situ during the final molecule isolation and purification.
Alternatively, the acid or basic addition salts may be prepared by
reacting the purified molecule separately in the acid or basic form
thereof with a base or an organic or inorganic acid and by
isolating the salt formed. For example, a pharmaceutically
acceptable acid addition salt may be prepared by reacting a
molecule according to the invention with a suitable organic or
inorganic acid (such as for example hydrobromic, hydrochloric,
sulphuric, nitric, phosphoric, succinic, maleic, formic, acetic,
propionic, fumaric, citric, tartaric, lactic, benzoic, salicylic,
glutamic, aspartic, p-toluene-sulphonic, benzene-sulphonic,
methane-sulphonic, ethane-sulphonic, hexanoic or
naphthalene-sulphonic acids such as 2-naphthalene sulphonic acid),
optionally in a suitable solvent such as an organic solvent. A
basic addition salt may, when a suitable acid group is present, be
prepared by reacting a molecule according to the invention with a
suitable organic or inorganic base (for example triethyl-amine,
ethanol-amine, triethanol-amine, choline, arginine, lysine or
histidine), optionally in a suitable solvent such as an organic
solvent. The salts thus generated may then be isolated by means of
crystallisation and filtration.
[0018] In some embodiments, pharmaceutically acceptable salts are
preferred in that they provide the molecules according to the
invention with superior stability or solubility, facilitating the
formulation thereof.
[0019] According to one particularly preferred embodiment, the
organic molecules according to the invention bind with Syk tyrosine
kinase protein at a site located outside the catalytic domain
thereof.
[0020] Also preferably, the organic molecules according to the
invention have a molecular weight between 50 and 2500 Dalton, for
example between 50 and 2000 Da, between 50 and 1500 Da or between
50 and 1000 Da.
[0021] According to one particular embodiment, the molecules
according to the invention are capable of inhibiting, by at least
5%, preferably by at least 10%, the binding of an antibody or
antibody fragment binding with human Syk tyrosine kinase protein on
an epitope comprising at least two, preferably at least 3, 4 or 5,
for example 5, 6, 7, 8, 9 or 10 of residues 65 to 74 of the amino
acid sequence of human Syk tyrosine kinase protein (SEQ ID No. 1),
with human Syk tyrosine kinase. According to one preferred
embodiment, the antibody or antibody fragment binds with human Syk
tyrosine kinase protein on the same epitope as antibody fragment
G4G11 or antibody fragment G4E4 on human Syk tyrosine kinase
protein, the sequence of which is illustrated by SEQ ID No. 1.
[0022] According to a further particular embodiment, the molecules
according to the invention are capable of inhibiting, by at least
5%, preferably by at least 10%, the binding of an antibody or
antibody fragment which binds to with Syk tyrosine kinase protein
and inhibits, by at least 15%, preferably by at least 20, 30, 40,
50, 60, 70 or 80%, the binding of antibody fragments G4G11 or G4E4
with human Syk tyrosine kinase protein (SEQ ID No. 1), with human
Syk tyrosine kinase protein.
[0023] Preferably, the molecules according to the invention bind
with human Syk protein on a three-dimensional cavity comprising the
Arginine residue situated in position 68 and the two glutamic acid
residues situated in positions 121 and 155 of the Syk protein, the
sequence of which is illustrated by SEQ ID No. 1. Preferably still,
the three-dimensional cavity further comprises the Serine residue
situated in position 9, the Glutamine residue situated in position
43, the Phenylalanine residue situated in position 51, the
Isoleucine residue situated in position 66, the Glutamate residues
situated in position 67 and 69, the Leucine residue situated in
position 70, the Asparagine residue situated in position 71, the
Glycine residue situated in position 72, the Threonine residue
situated in position 73, the Tyrosine residue situated in position
74 and the Alanine residue situated in position 75 of human Syk
protein, the sequence of which is illustrated by SEQ ID No. 1.
[0024] More preferably, the in vitro affinity, measured by the
dissociation constant (or Kd), of the molecules according to the
invention for Syk protein, is less than 100 more preferably, less
than 50 and particularly preferably, less than 25 .mu.M. The
affinity of the molecules according to the invention for Syk
protein is, for example, between 0.01 and 100 .mu.M, between 0.1
and 50 .mu.M or between 0.5 and 25 .mu.M. The dissociation constant
of the molecules according to the invention with respect to Syk
protein may particularly be measured in vitro by means of
fluorescence spectroscopy (or spectrofluorometry).
[0025] The present invention further relates to the use of a
molecule according to the invention for producing a medicinal
product for the prevention or treatment of a condition dependent on
a metabolic pathway involving Syk in humans or animals.
[0026] According to one particularly preferred embodiment, the
molecules or salts according to the invention are used for
producing a medicinal product for the prevention or treatment of
type I hypersensitivity reactions.
[0027] The term "hypersensitivity" refers to an unsuitable or
excessive immune response to an allergen, for example pollen, dust,
animal hairs or certain foods, with effects ranging from moderate
allergic reaction (skin rash, rhinitis, conjunctivitis, etc.) to
severe systemic reactions potentially resulting in anaphylactic
shock and potentially life-threatening in some cases. Immediate and
delayed hypersensitivity reactions are classified in types I and IV
respectively of the classification defined by Gell and Coombs (Gell
P G H, Coombs R R A, eds. Clinical Aspects of Immunology. 1st ed.
Oxford, England: Blackwell; 1963.sup.39). According to this
classification, "type I (or atopic or anaphylactic)
hypersensitivity" is an immediate allergic reaction associated with
exposure to a specific antigen or allergen, for example by
swallowing, inhalation, injection or direct contact, and the
triggering of immunoglobulin E (IgE) secretion by plasma cells. The
IgE binds with the Fc receptors found on the surface of tissue mast
cells and blood basophils. Subsequent exposure to the sensitised
mast cells and basophils to the same allergen gives rise to the
degranulation of the cells having the corresponding IgE and the
release of mediators such as histamine, leukotriene or
prostaglandins acting on the surrounding tissues, particularly
giving rise to vasodilation and smooth muscle contraction. The
reactions may be local or systemic and the symptoms vary from
moderate irritation to sudden death due to anaphylactic shock.
Examples of conditions caused by type I hypersensitivity include
allergic asthma, allergic conjunctivitis, allergic rhinitis (hay
fever), anaphylaxis, angioedema, urticaria, eosinophilia, allergies
to antibiotics such as penicillin or cephalosporin. "Type II
hypersensitivity" or "antibody-dependent immune response" is a
reaction generally requiring from a few hours to one day,
associated with interactions between antibodies (IgG, IgM) and an
antigen on the surface of the cells of the patient carrying this
antigen, giving rise to the destruction of these cells and the
proliferation of B lymphocytes, producing antibodies against the
antigen. "Type III hypersensitivity" or "immune complex disease" is
a reaction developing over a number of hours, days or weeks,
associated with the presence of similar quantities of antibodies
and antigens giving rise to the formation of immune complex not
suitable for evacuation circulating in the vessels, the deposition
thereof on the walls of said vessels and giving rise to local or
systemic inflammatory responses. "Type IV hypersensitivity" or
"cell-mediated immunity" or "delayed hypersensitivity reaction" is
an immune reaction generally requiring two to three days to develop
and not associated with an antibody response but with the formation
of a complex between cells which express a major histocompatibility
complex I or II antigen and T lymphocytes giving rise to the
release of lymphokines and/or cytotoxicity mediated by T
lymphocytes.
[0028] Preferably, the molecules or salts according to the
invention are used for producing medicinal products for the
prevention or treatment of type I hypersensitivity reactions which
inhibit IgE-dependent mast cell degranulation. More preferably, the
molecules according to the invention are capable of inhibiting by
50% in vitro mast cell degranulation, at a concentration (IC50)
between 1 ng/ml and 1 mg/ml, for example at a concentration between
1 ng/ml and 500 .mu.g/ml, between 1 ng/ml and 250 .mu.g/ml, between
1 ng/ml and 100 .mu.g/ml, between 1 ng/ml and 50 .mu.g/ml, between
1 ng/ml and 10 .mu.g/ml, between 1 ng/ml and 5 .mu.g/ml or between
1 ng/ml and 2 .mu.g/ml. Also preferably, a quantity between 1 nM
and 1 mM, for example between 1 nM and 100 nM, between 10 nM and
100 nM or between 1 nM and 10 nM, of a molecule according to the
invention is capable of inhibiting mast cell degranulation by 50%
in vitro.
[0029] More preferably, the metabolic pathway involving Syk on
which the molecules or salts according to the invention is a mast
cell or basophil activation pathway.
[0030] More preferably, the condition on which the molecules or
salts according to the invention act is allergic asthma, allergic
conjunctivitis, allergic rhinitis, anaphylaxis, angioedema,
urticaria, eosinophilia or an allergy to an antibiotic.
[0031] According to one preferred embodiment, the molecules or
salts according to the invention have no effect on the metabolic
pathways involving human Syk protein (SECT ID No. 1) other than
those giving rise to mast cell degranulation and/or type I
hypersensitivity reactions. More preferably, the molecules or salts
according to the invention have no effect on the antibody response
following immunisation by a thymus-dependent antigen or on
Syk-dependent neutrophil recruitment.
[0032] Syk tyrosine kinase protein is also found on the surface of
B lymphocytes, T lymphocytes, neutrophils, eosinophils, NK cells,
platelets, erythrocytes, osteoclasts, epithelial cells or cancer
cells. According to one alternative embodiment, the metabolic
pathway involving Syk on which the molecules or salts according to
the invention act is a B lymphocyte, T lymphocyte, neutrophil,
eosinophil, NK cell, platelet, erythrocyte, osteoclast, epithelial
cell or cancer cell activation pathway. According to this
embodiment, the condition on which the molecules or salts according
to the invention act may thus be rheumatoid arthritis, an
autoimmune disease, inflammation or cancer.
[0033] According to one particular embodiment, the molecules or
salts according to the invention may be used in combination with
another therapeutic molecule. For example, it may consist of a
therapeutic molecule also used for the prevention or treatment of a
condition dependent on a metabolic pathway involving Syk or, on the
other hand, a therapeutic molecule used for the prevention or
treatment of a condition not dependent on a metabolic pathway
involving Syk. According to one preferred embodiment, the molecules
or salts according to the invention are used in combination with a
molecule used for the treatment of allergy or type I
hypersensitivity or for the treatment of the symptoms associated
therewith. More preferably, the molecules or salts according to the
invention are used in combination with epinephrine (or adrenaline),
an H1 antihistamine, for example diphenhydramine, meclizine,
fluphenazine, perphenazine, prochlorperazine, trifluoperazine,
acrivastine, astemizole, cetirizine, levocetirizine, fexofenadine,
loratadine, desloratadine, mizolastine, azelastine, levocabastine,
olopatadine, cromoglicate, nedocromil, a non-steroidal
anti-inflammatory drug (NSAID) or a steroidal anti-inflammatory
drug, for example cortisone, hydrocortisone (or cortisol),
cortisone acetate, prednisone, prednisolone, methylprednisolone,
dexamethasone, betamethasone, triamcinolone, beclometasone,
fludrocortisone, deoxycorticosterone acetate or aldosterone.
According to a further preferred embodiment, the molecules or salts
according to the invention are used in combination with an allergic
desensitisation (or anti-allergic vaccination), i.e. a treatment
based on regular increasing doses of an allergen. According to one
particular embodiment, the molecule according to the invention is
not used in combination with a glucocorticoid receptor agonist.
[0034] The molecules or salts according to the invention may be
administered by any administration route, particularly by the oral,
sublingual, nasal, ocular, local, intravenous, intraperitoneal,
subcutaneous routes, by aerosol or by inhalation.
[0035] The molecules or salts according to the invention may
particularly be administered to adult, child or newborn human
patients. The molecules or sales according to the invention may
also be administered to animal patients, particularly mammals such
as dogs, cats, rats, mice.
[0036] In particular, the molecules or salts according to the
invention are administered to a human patient at doses determined
particularly on the basis of the patient's condition, medical
history and age, for example doses between 0.1 mg/kg and 200
mg/kg.
[0037] The present invention also relates to a therapeutic method
for the treatment or prevention of a condition dependent on a
metabolic pathway involving Syk in a human or animal patient
comprising the administration of a molecule according to the
invention to the patient at doses, intervals and periods determined
particularly on the basis of the patients condition, medical
history and age.
[0038] According to one particularly preferred embodiment, the
molecules according to the invention are selected from all the
molecules consisting of C-13, molecules No. 1 to 87 given in table
No. 1 and the molecules having any of the following formulas (I),
(II), (III) or (IV):
##STR00002##
[0039] where [0040] R1 is an optionally substituted aromatic group,
or an optionally substituted heterocycle comprising at least one S,
O or N atom;
[0041] R2 is an optionally substituted aromatic group, an
optionally substituted heterocycle, an optionally saturated carbon
chain, comprising an amine group, an optionally saturated carbon
chain comprising an optionally substituted aromatic group or an
optionally saturated carbon chain comprising an optionally
substituted heterocycle comprising at least one S, O or N atom;
[0042] R3 is an optionally substituted phenyl, 2-pyridinyl,
3-pyridinyl or 4-pyridinyl group;
##STR00003##
[0043] where [0044] n=0 or 1; n'=0 or 1; [0045] R4 is an optionally
saturated carbon chain comprising 1 to 5 carbon atoms, optionally
substituted with an aromatic group; [0046] R5 is an optionally
substituted aromatic group or an optionally substituted amine
group; [0047] R6 is a hydrogen atom, alkoxy group, alkyl group or
halogen; [0048] R7 is a hydrogen atom, alkoxy group, alkyl group or
halogen; [0049] R8 is a hydrogen atom, alkoxy group, alkyl group or
halogen;
##STR00004##
[0050] where [0051] m=0, 1 or 2; [0052] R9 is a hydrogen atom and
R10 is an optionally substituted phenyl group, or R9 and R10 are
part of the same optionally substituted heterocycle, or R9 and R10
are part of the same optionally substituted aromatic group; [0053]
R11 is a hydrogen atom, alkoxy group or alkyl group; [0054] R12 is
a hydrogen atom, alkoxy group or alkyl group; [0055] R13 is a
hydrogen atom or an alkyl or alkoxy group; [0056] R14 is a hydrogen
atom or an alkyl or alkoxy group;
##STR00005##
[0057] where [0058] A is an oxygen or sulphur atom; [0059] R15 is
an optionally saturated carbon chain comprising 1, 2 or 3 carbon
atoms, optionally substituted by an optionally substituted aromatic
group, an optionally substituted heterocycle or an amine group
belonging to optionally substituted heterocycle; [0060] R16 is a
hydrogen atom, halogen or alkoxy group; [0061] R17 is a hydrogen
atom, alkoxy group or acetoxy group.
[0062] According to one particular embodiment, the group R1 of
molecules having formula (I) is selected from the following groups:
[0063] a phenyl group, optionally substituted by an F or Cl atom, a
methyl or ethyl group, an N,N-dimethyl-sulphonamide or two groups
selected from the methyl, ethyl, hydroxy, methoxy or ethoxy groups,
[0064] a group
[0064] ##STR00006## [0065] a furan group optionally substituted by
a methyl, ethyl, hydroxyl, methoxy or ethoxy group, [0066] a
thiophene group optionally substituted by a methyl, ethyl, hydroxy,
methoxy or ethoxy group; the group R2 of molecules having formula
(I) is selected from the following groups: [0067] a group
[0067] ##STR00007## [0068] where R21 and R22 are carbon atoms each
belonging to an alkyl chain comprising 1, 2 or 3 carbon atoms, or
both belonging with the nitrogen atom with which they are bound to
the same optionally saturated heterocycle also comprising an oxygen
atom or a second nitrogen atom, [0069] a or a group
[0069] ##STR00008## [0070] where R23 and R24 are carbon atoms each
belonging to an alkyl chain comprising 1, 2 or 3 carbon atoms, or
both belonging with the nitrogen atom with which they are bound to
the same optionally saturated heterocycle also comprising an oxygen
atom or a second nitrogen atom, [0071] or a group
##STR00009##
[0072] and the group R3 of molecules having formulas (I) is
selected from the following groups: [0073] a non-substituted
2-pyridinyl, 3-pyridinyl or 4-pyridinyl group, [0074] a phenyl
group optionally substituted by a benzoxy group, and/or by a
hydroxyl group, and/or by a methyl group, and/or by an ethyl group,
and/or by a propyl group, and/or by one or two Br, F or Cl atoms,
and/or by one to three hydroxyl, methoxy or ethoxy groups.
[0075] According to one particular embodiment, when the group R3 of
molecules having formula (I) is a phenyl group, the group R2 of
molecules having formula (I) is not an aromatic group or a
heterocycle.
[0076] According to one particular embodiment, the group R4 of
molecules having formula (II) is an optionally saturated carbon
chain comprising 1, 2 or 3 carbon atoms; the group R5 is a phenyl
group or a secondary amine grow substituted by an optionally
substituted phenyl group, or by a group
##STR00010##
the group R6 of molecules having formula (II) is a hydrogen or
chlorine atom or a methyl, ethyl, hydroxyl, methoxy or ethoxy
group; the group R7 of molecules having formula (II) is a hydrogen
or chlorine atom or a methyl, ethyl, hydroxy, methoxy or ethoxy
group; and the group R8 of molecules having formula (II) is a
hydrogen or chlorine atom or a methyl, ethyl, hydroxy, methoxy or
ethoxy group.
[0077] According to one particular embodiment, [0078] the group R9
of molecules having formula (III) is a hydrogen atom and the group
R10 is an optionally substituted phenyl group, or the groups R9 and
R10 belong to the same optionally substituted heterocycle
comprising 2 nitrogen atoms and 4 carbon atoms; [0079] the group
R11 of molecules having formula (III) is a hydrogen atom or methyl,
ethyl, hydroxy, methoxy or ethoxy group; [0080] the group R12 of
molecules having formula (III) is a hydrogen atom or methyl, ethyl,
hydroxy, methoxy or ethoxy group; [0081] the group R13 of molecules
having formula (III) is a hydrogen atom or methyl, ethyl, hydroxy,
methoxy or ethoxy group; [0082] and the group R14 of molecules
having formula (III) is a hydrogen atom or methyl, ethyl, hydroxy,
methoxy or ethoxy group.
[0083] According to a further particular embodiment, the group R15
of molecules having formula (IV) is a group
##STR00011##
the group R16 of molecules having formula (IV) is a hydrogen or
chlorine atom or a methyl, ethyl, hydroxy, methoxy or ethoxy group
and the group R17 of molecules having formula (IV) is a methyl,
ethyl, hydroxy, methoxy, ethoxy, acetoxy, methoxycarbonyl or
ethoxycarbonyl group.
[0084] According to one particular embodiment, the molecule
according to the invention is not
##STR00012## ##STR00013##
or if it is any of these molecules, it is not used in combination
with a glucocorticoid agonist.
[0085] The term carbon chain refers to an organic chain having a
linear or cyclic, optionally branched, chain formation of adjacent
carbon atoms, connected by covalent bonds, as the network thereof.
A carbon chain according to the present invention may for example
be a linear chain formation of one to twenty, preferably 1 to 12, 1
to 10, 1 to 6, 1 to 5 or 1 to 4 carbon atoms. In particular, it may
consist of an alkyl group, i.e. derived from an alkane (linear or
branched saturated hydrocarbon molecule) due to the loss of a
hydrogen atom, for example a methyl group, an ethyl group, a linear
or branched propyl group or a linear or branched butyl group or a
linear or branched unsaturated hydrocarbon chain, for example an
ethenyl or ethynyl group. It is understood that the electrons of
the outer layer (4 in number) of each carbon atom forming the
carbon chain network are not involved in a covalent bond with a
further carbon atom or with a heteroatom are involved in a covalent
bond with a hydrogen atom.
[0086] The term heteroatom refers to a non-metallic atom other than
carbon or hydrogen, for example oxygen, nitrogen, sulphur,
phosphorus or halogens.
[0087] The term aromatic or aryl group refers to an unsaturated
cycle system observing Hackers aromaticity rule. For example, it
may consist of a phenyl group (group derived from a benzene
nucleus).
[0088] The term heterocycle refers to a cycle system wherein one or
a plurality of carbon atoms is replaced by a heteroatom such as,
for example, oxygen, nitrogen or sulphur. It may in particular
consist of aromatic heterocycles, such as pyrrole, thiophene, furan
and pyridine or of saturated heterocycles, such as sugars, or uses.
For example, a heterocycle according to the invention comprises 2
to 8 carbon atoms and 1 to 4 heteroatoms, preferably it comprises
2, 3, 4 or 5 carbon atoms and 1, 2, 3 or 4 heteroatoms.
[0089] Each atom belonging to a carbon chain, aromatic group, cycle
or heterocycle according to the present invention may be
substituted via a covalent bond by one or a plurality of halogens,
for example Fluorine, Chlorine, Iodine or Bromine, and/or by one or
a plurality of organic groups, for example one or a plurality of
aromatic (such as a phenyl group), cyclic, heterocyclic (such as a
furan or thiophene group), alkyl (such as a methyl group, ethyl
group, linear or branched propyl group or a linear or branched
butyl group), alkoxy (such as a methoxy (OCH.sub.3) or ethoxy
(OCH.sub.2CH.sub.3) group), carboxyl (such as a carboxy (COON)
group), carbonyl (such as an acetoxy (OCOCH.sub.3) or
methoxycarbonyl (COOCH.sub.3) or ethoxycarbonyl
(COOCH.sub.2CH.sub.3) group), primary, secondary or tertiary amine,
amide (such as an acetamide group) or sulphonamide (such as an
N,N-dimethyl-sulphonamide group) groups. It is understood that a
saturated, unsaturated or aromatic cycle or heterocycle may be
merged with a further cycle, for example by means of a single or
double bond between two carbon atoms.
[0090] The present invention also relates to a pharmaceutical
composition comprising a molecule according to the invention and a
pharmacologically acceptable excipient.
[0091] According to one embodiment, the pharmaceutical composition
according to the invention also comprises a further therapeutic
molecule. For example, it may consist of a therapeutic molecule
also used for the prevention or treatment of a condition dependent
on a metabolic pathway involving Syk or, on the other hand, a
therapeutic molecule used for the prevention or treatment of a
condition not dependent on a metabolic pathway involving Syk.
According to one particular embodiment, the pharmaceutical
composition according to the invention does not comprise a
glucocorticoid receptor agonist.
[0092] According to a further particular embodiment, the
pharmaceutical composition according to the invention may be used
in combination with one or a plurality of further pharmaceutical
compositions. For example, it may be pharmaceutical compositions
also used for the prevention or treatment of a condition dependent
on a metabolic pathway involving Syk or, on the other hand,
pharmaceutical compositions used for the prevention or treatment of
a condition not dependent on a metabolic pathway involving Syk.
According to this embodiment, the pharmaceutical composition
according to the invention and the further pharmaceutical
composition(s) may be administered simultaneously or in
alternation, by the same administration route or by different
routes. According to one particular embodiment, the pharmaceutical
composition according to the invention is not used in combination
with a glucocorticoid receptor agonist.
[0093] The present invention also relates to a method for
identifying an organic molecule having a molecular weight between
50 and 2500 Dalton binding with Syk tyrosine kinase protein and
capable of inhibiting by at least 5%, preferably at least 10%, for
example at least 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,
75, 80 or 85% in vitro the binding of (i) antibody fragment G4G11
(SEQ ID No. 2), or (ii) antibody fragment G4E4 (SEQ ID No. 3), or
(iii) an antibody or antibody fragment which binds with human Syk
tyrosine kinase protein on an epitope comprising at least one of
residues 65 to 74 of the amino acid sequence of human Syk tyrosine
kinase protein represented by the sequence SEQ ID No. 1, or (iv) an
antibody or antibody fragment which binds with human Syk tyrosine
kinase protein and inhibits by at least 10% the binding of antibody
fragments G4G11 or G4E4 with human Syk tyrosine kinase protein (SEQ
ID No. 1), to human Syk tyrosine kinase protein or to any of the
variants thereof in animals, comprising at least the following
steps: [0094] a) screening, from a bank of candidate organic
molecules having a molecular weight between 50 and 2500 Da, those
liable to bind with Syk protein on the three-dimensional binding
cavity on the Syk protein of a molecule selected from the molecules
having formula C-13, I, II, III, IV or 1 to 87 as illustrated
above; [0095] b) selecting from the molecules identified in a)
those capable of inhibiting by at least 5%, preferably at least
10%, for example at least 15, 20, 25, 30, 35, 40, 45, 50, 55, 60,
65, 70, 75, 80 or 85% in vitro the binding of the antibody or
antibody fragment (i), (ii), (iii) or (iv) with Syk protein.
[0096] According to one particularly preferred embodiment, the
molecule from step a) selected from the molecules having formula
C-13, I, II, III, IV or 1 to 87 is the molecule C-13, molecule (59)
or molecule (61).
[0097] According to one preferred embodiment, the method according
to the invention comprises an additional step prior to step a) for
identifying the three-dimensional binding cavity on the Syk protein
of the molecule selected from the molecules having the formula
C-13, I, II, III, IV or 1 to 87. This prior step may particularly
be performed by means of "in silico docking".
[0098] The term "in silico docking" or "molecular docking" or
"virtual docking" refers to the use of a bioinformatics tool for
predicting and modelling the position of a ligand in a
macromolecule. In particular, in silico docking tools can be used
to calculate the probability that a given chemical compound will be
able to dock with an active target protein, for example on a
previously identified three-dimensional binding cavity.
[0099] According to one preferred embodiment, the method according
to the invention comprises an additional step c) for selecting from
the molecules identified in b) those capable of inhibiting by 50%
in vitro at mast cell degranulation to a concentration (IC50)
between 1 ng/ml and 1 mg/ml, for example at a concentration between
1 ng/ml and 500 .mu.g/ml, between 1 ng/ml and 250 .mu.g/ml, between
1 ng/ml and 100 .mu.g/ml, between 1 ng/ml and 50 .mu.g/ml, between
1 ng/ml and 10 .mu.g/ml, between 1 ng/ml and 5 .mu.g/ml or between
1 ng/ml and 2 .mu.g/ml.
[0100] According to one particularly preferred embodiment of the
uses and methods described above, the molecule according to the
invention is selected from the group consisting of the molecule
C-13 and molecules No. 1 to 87 given in table 1.
[0101] These molecules were identified by the inventors within the
scope of a project following a previous study (Dauvillier et al.,
2002.sup.7), during which they expressed scFv ("single chain
variable domain") (or "intracellular antibodies" or "intrabodies"),
G4G11 (SEC) ID No. 2) and G4E4 (SEC) ID No. 3) antibody fragments
in a mast cell line. This study demonstrated the inhibitory effects
of these "intracellular antibodies" or "intrabodies" on the release
of allergic mediators induced by Fc.epsilon.RI stimulation on the
mast cell membrane. The scFv G4G11 and G4E4 antibody fragments were
isolated from a combinatory bank screened against a recombinant
protein containing the SH2 domains of Syk and inter-domain A region
separating same, i.e. a portion of Syk protein not comprising the
Syk kinase domain.sup.8.
[0102] The ADA ("antibody displacement assay") method is a method
developed by the inventors and described in WO2005106481,
particularly for identifying a ligand capable of selectively
modulating a functional cascade involving a target, comprising a
first step for identifying an intracellular antibody capable of
binding with the target and modulating the functional cascade in
question, a second step for screening from a bank of small organic
molecules, ligands modulating the binding between the target and
the intracellular antibody potentially being performed in vitro in
an extracellular test, and a third step for identifying from the
modulating ligands obtained in step 2, those capable of modulating
the functional cascade in the cell.
[0103] The inventors suggested the theory whereby the antibody
fragments G4G11 and G4E4 bind on a Syk region interacting with one
or more essential partners in the functional cascade giving rise to
degranulation. Taking into consideration the limits of the use of
intracellular antibodies in therapy, such as the effective transfer
of the gene encoding the antibody in target cells.sup.9, the
inventors sought to isolate organic molecules acting as functional
mimics of the intrabody G4G11 and suitable for easier use in
therapy. To this end, they used the ADA method for screening a bank
of 3000 small organic molecules and identifying potential allergic
response inhibitors.
[0104] Among the 3000 small organic molecules tested, the inventors
identified the small molecule C-13 (methyl
2-{5-[(3-benzyl-4-oxo-2-thioxo-1,3-thiazolidin-5-ylidene)methyl]-2-furyl}-
benzoate) and demonstrated the ability thereof to modulate the
interaction of the antibody fragment G4G11 or G4E4 with Syk in
vitro and inhibit mast cell degranulation induced by Fc.epsilon.RI
in vitro.
[0105] The inventors particularly demonstrated the fact that the
compound C-13 inhibits anaphylactic shock when administered orally
and has promising anti-allergic properties, illustrating the strong
therapeutic potential of medicinal product candidates isolated
using the approach described herein.
[0106] The inventors also demonstrated the fact that C-13 binds
with Syk on a newly identified cavity situated between both SH2
domains and inter-domain A of Syk (FIG. 1). The binding cavity of
C-13 forms a unique interaction zone which is specific to Syk, and
does not correspond to a known binding side of physiological
ligands of Syk such as doubly phosphorylated ITAM peptide on
tyrosine residues (FIG. 1A). The results obtained suggest that C-13
inhibits the interaction of Syk with some of the macromolecular
substrates thereof, either directly in that C-13 occupies a surface
whereon a partner of Syk could establish direct contact, and/or by
means of an allosteric effect.
[0107] The biochemical studies conducted on mast cells indeed
demonstrated that C-13 inhibits Fc.epsilon.R1-dependent
phosphorylation of SLP-76 on tyrosine residues contributing to the
adapting function thereof for the binding and/or stabilisation of
Btk, PLC-.gamma. and Vav with the macromolecular signalling complex
formed with LAT.sup.22, 28-30 (FIG. 2). This affects the
phosphorylation and catalytic activity of Btk and PLC-.gamma.
renewal in the vicinity of Syk and/or Btk for the complete
phosphorylation thereof which is required for maintaining calcium
flow and exocytosis.sup.31-35. Indeed, C-13 inhibited early
(.beta.-hexosaminidase release) and delayed (TNF-.alpha. secretion)
mast cell responses induced by aggregation on the Fc.epsilon.RI
receptors with an estimated IC50 of 2 .mu.M (FIG. 3).
[0108] Significantly, the oral administration of a single dose of
C-13 inhibited IgE-induced passive systemic anaphylaxis (PSA) with
an estimated IC50 of 110 mg/kg (FIG. 4), confirming the promising
anti-allergic properties of this compound. On the contrary, a
single oral administration of 100 mg/kg of C-13 did not affect
Syk-dependent neutrophil recruitment induced by thioglycollate in
the peritoneal cavity in the presence of Bordetella pertussis toxin
(FIG. 5A). Furthermore, the inventors demonstrated that, despite
the fact that BCR-dependent B lymphocyte in vitro proliferation was
inhibited in a dose-dependent fashion by C-13 (FIG. 5B), the
antibody responses of mice immunised with a thymus-dependent
antigen were not affected by the oral administration of 150 mg/kg
of C-13 (FIG. 5C). Therefore, the molecule C-13 does not affect
some responses dependent on Syk but not dependent on mast cells in
vivo at administration doses and periods at which it is liable to
inhibit a severe allergic response.
[0109] Taking into consideration the lack of an apparent toxic
effect following the oral or local administration of C-13 over a
period ranging from one hour to 12 days, C-13 may be considered as
the potential first member of a new family of Syk inhibitors
suitable for oral administration and pharmacologically active
molecules having an anti-inflammatory effect. The pharmaceutical
molecule screening approach described herein represents a generic
platform wherein the initial use of antibodies makes it possible to
detect the domains of the target molecule having a therapeutic
potential, thus facilitating the design of chemical molecules (via
in silico and/or in vitro screening) capable acting as functional
antibody mimics and as potential protein-protein interaction
inhibitors. Furthermore, the inventors demonstrated that these
small molecules can induce the desired response in cell and animal
models, supporting the concept in favour of the replacement of
large macromolecules that are difficult to administer by small
organic molecules suitable for oral administration.
[0110] The possible binding site with Syk was predicted in silico,
guided by the location of the epitope of G4G11. One candidate
cavity situated next to the epitope of G4G11, on the interface
situated between the two SH2 domains and the inter-domain binder of
Syk and comprising the residues Ser 9, Gln 43, Phe 51, Ile 66, Glu
67, Arg 68, Glu 69, Leu 70, Asn 71, Gly 72, Thr 73, Tyr 74, Ala 75,
Glu 121 and Glu 155 was thus identified (FIG. 10). Targeted
mutagenesis experiments confirmed that the residues Arg 68, Glu 121
and Glu 155 of human Syk protein (SEQ ID No, 1) play a significant
role in interaction with C-13, the mutation of said residues
suppressing the inhibition caused by C-13, whereas the binding of
scFv G4G11 is maintained (FIG. 1A, 10). These results tend to
confirm the theory whereby the binding cavity of C-13 or Syk is
located in the vicinity of the binding site of the intrabody
G4G11.
[0111] The inventors then performed virtual docking on a molecule
bank to identify candidate molecules having the best binding
properties on said three-dimension cavity, and tested the ability
of said candidate molecules to inhibit the binding of scFv G4G11
with Syk. These molecules are given in table 1 (see example 2). The
ability of these molecules to inhibit mast cell degranulation in
vitro was also tested. With molecules No. 59 and 61 in particular
the concentration inhibiting mast cell degranulation by 50% in
vitro is in the region of 5 .mu.M (see FIG. 6).
[0112] The results given in the experimental part and particularly
in table 1 demonstrate that the molecules or salts according to the
invention inhibit type I hypersensitivity reactions, particularly
IgE-dependent mast cell degranulation, and are also capable of
interfering in vivo with passive cutaneous and systemic anaphylaxis
in BALB/c mice.
BRIEF DESCRIPTION OF THE FIGURES
[0113] FIG. 1. Binding of C-13 with Syk in vitro. A. Chemical
structure of C-13. 3D structure of cavity predicted and validated
for C-13 (or "Compound 13") comprising the residues Ser 9, Gln 43,
Phe 51, Arg 68, Glu 121 and Glu 155 (mesh representation); the
G4G11 epitope comprises the residues 65 to 74 of human Syk protein
represented by SEQ ID No. 1. B. Binding of scFv G4G11 fragment with
Syk measured using ADA method in the presence of C-13
(.box-solid.). Binding of C-13 with Syk measured using fluorescence
spectroscopy (o) (Kd=4.8.+-.0.2 .mu.M). C. Binding of G4G11 with
Syk mutants with ADA method. Significant inhibition with C-13
versus DMF: **P<0.01.
[0114] FIG. 2. C-13 inhibits Fc.epsilon.RI-induced mast cell
activation. A. The immunoblots produced on RBL-2H3 cell lysates
were analysed with immunoblots with the specified antibodies. The
in vitro kinase activities of the Syk, Btk and Lyn immunoblots were
examined. B. The RBL-2H3 cell lysates and C. BMMCs were subjected
to electrophoresis and the proteins were analysed by means of
immunoblot. These data are representative of at least two
experiments.
[0115] FIG. 3. C-13 inhibits Fc.epsilon.RI-dependent calcium
release and degranulation. A. FACS analysis of IgE-dependent
calcium flow in RBL-2H3 cells. B. Release of .beta.-hexosaminidase
in RBL-2H3 cells sensitised with IgE/DNP. C. Release of
.beta.-hexosaminidase and, D. titration of TNF-.alpha. in BMMCs
cells sensitised with IgE/DNP; C-13: 3 .mu.M. E. FACS analysis of
Fc.epsilon.RI surface expression in RBL-2H3 cells. (0=0.25% DMF),
Significant inhibition with C-13 versus DMF: **P<0.01 and
*P<0.05.
[0116] FIG. 4. In vivo studies on BALB/c mice. A-C. PSA response.
A. Temperature progression, B. Evans blue extravasation
quantification, C Photograph representing Evans blue extravasation
according to an oral administration of 130 mg/kg of C-13, a
non-relevant compound (IR) or the vector (T); Untreated animal
(NT). The ears and tail of the mice received the non-relevant
compound (IR) or the vector alone (T) turned a pronounced blue
colour, those of the mice treated with C-13 turned a light blue
colour and those of the untreated mice are normal in colour D. PCA
response: Evans blue extravasation quantification. Significant
inhibition with C-13 versus a non-relevant compound: **P<0.01;
versus the vehicle+DNP-KLH: *P<0.05.
[0117] FIG. 5. In vivo and in vitro effects of C-13 on neutrophils
and B lymphocytes. A. Neutrophil recruitment in the peritoneal
cavity after injecting thioglycollate or the vehicle in the
presence of Bordetella Pertussis toxin (where specified) (n=4). B.
In vitro B lymphocyte proliferation induced by anti-IgM. C. Serum
immunoglobulin concentration 12 days after immunisation with
TNP-KLH (n=4).
[0118] FIG. 6. Effect of molecules No. 59 (ref. Chembridge 7501888)
and 61 (ref. Chembridge 7722851) on RBL-2H3 cell degranulation. The
level of .beta.-hexosaminidase release in RBL-2H3 cells sensitised
with IgE/DNP was measured in the presence A. of molecule No. 59 and
B. of Molecule No. 61 at concentrations ranging from 0 to 40
.mu.M.
[0119] FIG. 7. Amino acid sequences of intrabodies scFv A. G4G11
(SEQ ID No. 2) and B. G4E4 (SEQ ID No. 3).
[0120] FIG. 8. Amino acid sequence of human A. (SEQ ID No. 1)
corresponding to accession No. NP.sub.--003168 (NCBI) and B. murine
Syk protein corresponding to accession No. NP.sub.--035648 (NCBI).
The residues identified as belonging to the three-dimensional
binding cavity of C-13 on human Syk protein (A) and the equivalent
thereof on murine Syk protein (B) are shown in bold type and
underlined.
[0121] FIG. 9. Schematic representation of the metabolic pathways
involving Syk tyrosine kinase in mast cells.
[0122] FIG. 10. Toxicity test of C-13 on BMMC cells. The BMMC cells
were incubated at 37.degree. C. in the presence of 2.5 .mu.M or 5
.mu.M of C-13, 0.25% DMF or Staurosporine. The percentages of live
BMMC cells after 3 hours and after 5 days were detected by means of
double Annexin-V and Propidium Iodide labelling. The viability of
BMMC cells incubated in the presence of 2.5 .mu.M or 5 .mu.M of
C-13 or 0.25% DMF was not affected to noteworthy degree after 3
hours (78%, 74% and 79% respectively) or five days (62, 56 and 57%,
respectively). On the other hand, the viability of BMMC cells
treated under the same conditions with Staurosporine was reduced to
a level of 16% after only 3 hours.
EXAMPLES
Example 1
Identification of Molecule C-13 Potentially Suitable for Oral
Administration to Prevent Anaphylactic Shock
[0123] 1) Identification of Compound 13 (C-13) and the Binding
Cavity thereof on Syk
[0124] The inventors developed the ADA (Antigen Displacement Assay)
method to identify small molecules capable of displacing the
association with scFv G4G11 with Syk. Of the members of a bank of
3000 chemical molecules, 15 small molecules proved to be capable of
competing with the binding of scFv G4G11 with Syk, and of these
compounds, that hereinafter referred to as C-13 (FIG. 1A) displayed
the best inhibition potential with an estimated IC50 of 4 .mu.M
(FIG. 1B). This result is in line with the dissociation constant
value (or Kd) equal to 4.8 .mu.M obtained by measuring by means of
fluorescence spectroscopy (or spectrofluorometry) the in vitro
affinity of C-13 for Syk (FIG. 1B). To understand the mode of
action of C-13, the inventors firstly identified the binding site
of G4G11 using the SPOT method.sup.16. An epitope located on the
N-terminal SH2 domain of Syk, comprising amino acids 65-74 and 100%
preserved in mouse, rat and human sequences, was identified.
[0125] On the basis of this information and the known 3D structure
of the peptide complex formed by the SH2 domains of Syk and ITAM
motifs.sup.17, the inventors used computing approaches to locate a
putative binding site for C-13. A candidate cavity comprising the
residues Ser 9, Gln 43, Phe 51, Ile 66, Glu 67, Arg 68, Glu 69, Leu
70, Asn 71, Gly 72, Thr 73, Tyr 74, Ala 75, Glu 121 and Glu 155 of
human Syk protein (see FIG. 8A, SEQ ID No. 1) and situated in the
vicinity of the G4G11 epitope was identified (FIG. 1C). A
structural analysis specified that the residues Ser 9, Gln 43, Phe
51, Arg 68, Glu 121 and Glu 155 could be involved in the binding of
the ligand, and could be mutated without impairing the 3D structure
of the protein. To validate the cavity in more detail, these six
amino acids were mutated individually and the Syk mutants were
subjected to the ADA test. The residues Arg 68, Glu 121 and Glu 155
proved to have a significant role in the interaction with the small
molecule, given that the mutation thereof cancelled the inhibition
caused by C-13, whereas the binding of scFv G4G11 was maintained
(FIG. 10). These data confirmed the fact that the binding cavity of
C-13 on Syk is located in the vicinity of the binding site of
G4G11.
2) Fc.epsilon.RI-Induced Mast Cell Activation
[0126] To examine the functional similarities with G4G11, the
inventors explored the biological effects of C-13 on mast cell
activation. The incubation of RBL-2H3 cells with 0-13 did not
affect the phosphorylation and Fc.epsilon.RI-induced kinase
activity of Syk (FIG. 2A) and, accordingly, the overall level of
tyrosine phosphorylation of all the cell proteins known as being
essentially Syk-dependent was normal (FIG. 2B, C). Similarly to the
intrabody G4G11, C-13 inhibited the phosphorylation and
Fc.epsilon.RI-induced kinase activity of Btk (FIG. 2A) and the
phosphorylation of PLC-.gamma.1 and PLC-.gamma.2, the two
PLC-.gamma. isoforms expressed in mast cells (FIG. 2A, C). PTK Lyn
phosphorylates both Syk and Btk giving rise to the complete
activation thereof and the subsequent phosphorylation of
PLC-.gamma..sup.18. Given that C-13 did not affect
Fc.epsilon.RI-dependent Lyn activation (FIG. 2A), it can be
concluded that the reduction of the level of Btk and PLC-.gamma.
phosphorylation could be due to a defect with respect to the
correct location thereof in the vicinity of the upstream PTK.
3) Fyn- and Lyn-Dependent Signalling Cascade Analysis
[0127] In mast cells, the signalling cascade Fyn/Gab2/PI3K gives
rise to the activation of PI3K and the generation of PI-3,4,5-P3
recruiting a number of proteins containing a pleckstrin homology
domain (PH), including Btk and PLC-.gamma. on the plasma
membrane.sup.19. The analysis of the phosphorylation of Akt, a PI3K
activity marker, indicated that C-13 did not affect the
Fyn-dependent cascade (FIG. 2B, C), suggesting that the reduced
level of phosphorylation of Btk and PLC-.gamma. was not due to a
defect on the membrane location thereof, known as being an
essential factor in the increase in calcium flows.sup.20.
[0128] Btk and PLC-.gamma. recruitment on the membrane also
requires the canonical signalling cascade Lyn/Syk/LAT/SLP-76. The
phosphorylation of LAT by Syk gives rise to the translocation of
SLP-76 to the complex organised by LAT.sup.21, where SLP-76 is
co-located with Syk.sup.22. This location enables Syk to
phosphorylate N-terminal tyrosines of SLP-76.sup.23 which become
binding sites for Vav, Nck and Btk. LAT and SLP-76 (via the
proline-rich domain thereof recruiting PLC-.gamma.) interact to
locate PLC-.gamma. on said membrane complex, enabling the
phosphorylation and activation of PLC-.gamma. by Btk.sup.24 and/or
Syk.sup.25. The use of phospho-specific antibodies demonstrated
that C-13 inhibits the phosphorylation of SLP-76, but increases the
phosphorylation of LAT in a dose-dependent fashion (FIG. 2A). The
inventors suggested the theory whereby the inhibition of SLP-76
phosphorylation could enable a larger quantity of LAT to interact
with Syk, thus causing an increase in the phosphorylation level
thereof. These results demonstrate that the reduction in SLP-76
phosphorylation was not due to a defect in terms of the recruitment
thereof to LAT, and resulted in a co-location defect of Btk and, to
a lesser extent, that of Vav with SLP-76 (FIG. 2A). Nevertheless,
Vav phosphorylation known to be independent from the recruitment
thereof to SLP-76.sup.26 was not inhibited (FIG. 2A).
4) MAPK Activation
[0129] The association of SLP-76 with Vav and/or Nck plays a role
in optimal MAP kinase activation in mast cells.sup.27. The
inventors demonstrated that C-13 affects MAP kinase activation
slightly (evaluated via the phosphorylation level thereof): a high
C-13 concentration reduces the phosphorylation level ERK1/2,
whereas the phosphorylation levels of p38 and JNK remain normal
(FIG. 2B, C).
5) Calcium Flow and Degranulation
[0130] Binding of Btk and Vav with SLP-76 is critical for
regulating PLC-.gamma. activity on the membrane, calcium
mobilisation and granule exocytosis.sup.27, 28. The inventors
demonstrated that the association of PLC-.gamma. with LAT was
inhibited by C-13 in a dose-dependent fashion (FIG. 2A).
Consistently with the defect in terms of PLC-.gamma.1 and
PLC-.gamma.2 phosphorylation, the mast cells showed a reduced
calcium flow range in response to Fc.epsilon.RI binding (FIG. 3A),
and early and delayed Fc.epsilon.RI-induced allergic responses in
BMMC ("bone marrow derived mast cell") cells and in the RBL-2H3
cell line are also weakened in a dose-dependent fashion, based on
the measurement of .beta.-hexosaminidase release and TNF-.alpha.
secretion (FIG. 3B, C, D). The results also demonstrated that C-13
had no toxic effect on mast cells. Indeed, ionomycin-induced
degranulation (FIG. 3B), or BMMC cell viability (see FIG. 10) were
not detectably affected by treatment with C-13. Furthermore, the
defects observed in terms of mast cell activation are not due to a
reduced level of Fc.epsilon.RI surface expression, flow cytometry
analysis indicating that the cells incubated with C-13 express
similar levels of Fc.epsilon.RI to those of control cells (FIG.
3E).
6) Passive Systemic (PSA) and Cutaneous Anaphylaxis (PCA)
[0131] Finally, to extend these observations to mast cell functions
in vivo, the inventors tested the effects of C-13 on passive
systemic (PSA) and cutaneous anaphylaxis (PCA) induced in BALB/c
mice by administering DNP-specific IgE molecules followed by
intravenous stimulation with DNP-KLH hapten. This mimics systemic
anaphylaxis as demonstrated by the immediate cardiopulmonary
changes and the increase in vascular permeability. The intensity of
systemic anaphylaxis was determined by measuring both the drop in
body temperature and the increase in vascular permeability
following antigen administration. After the oral administration of
C-13 (and prior to antigen stimulation), the animals appeared to be
healthy with no obvious sign of toxicity. The administration of a
single oral dose of 100 mg/kg of C-13 inhibited hypothermia and
accelerated the recovery of the animals (FIG. 4A). On the basis of
the Evans blue extravasation quantification, it was determined that
C-13 inhibits the increase in vascular permeability with an
estimated IC50 of 110 mg/kg (FIGS. 4B and 4C). C-13 also
demonstrated an inhibitory effect on PCA with an estimated IC50 of
25 .mu.M (FIG. 4D).
Example 2
Identification Using Binding Cavity of C-13 of Further Potential
Mast Cell Degranulation Inhibitors
[0132] Using the binding cavity identified in example 1-1), virtual
docking screening was conducted on a set of 350,000 molecules
contained in the ChemBridge Corporation (San Diego, USA) chemical
bank to identify the 1000 molecules displaying the best binding
properties in said cavity.
[0133] The ADA method was then applied to each of these 1000
molecules to measure the ability thereof to inhibit the binding of
scFv G4G11 with Syk. The 87 molecules having the best inhibition
rate (between 11 and 86.5%) are given in table 1.
[0134] These 87 molecules were also tested in vitro to assess the
ability thereof to inhibit RBL-2H3 cell degranulation (see table
1). The two molecules exhibiting the best potential (molecules No.
59 and 61) were tested at various concentrations on RBL-2H3 cells
to assess the concentration inhibiting degranulation by 50% in more
detail (see FIG. 6).
[0135] Finally, the in vitro affinity of the molecule C-13 and some
of the 87 molecules mentioned above for Syk protein was measured by
means of fluorescence spectroscopy (or spectrofluorometry) and is
expressed by the dissociation constant (or Kd) in .mu.mole/litre
(.mu.M).
TABLE-US-00001 TABLE 1 C-13 and the 87 molecules identified using
C-13 and the antibody fragment G4G11 CB In vit. Degran. Structure
ref. Name Rank inhib. IC50 Kd No. Gp ##STR00014## 6197026 methyl
2-{5- [(3-benzyl-4- oxo-2-thioxo- 1,3- thiazolidin-5- ylidene)
methyl]-2- furyl} benzoate -- 81% 1 .mu.g/ml 4.8 .mu.M C-13 --
##STR00015## 6752784 4-(4-chloro benzoyl)-3- hydroxy-5-(3- phenoxy
phenyl)-1-(3- pyridinyl methyl)-1,5- dihydro-2H- pyrrol-2-one 611
86.5 >10 .mu.g/ml 5 .mu.M 1 I ##STR00016## 6670340 5-(2,4-
dimethoxy phenyl)-3- hydroxy-1-[3- (1H-imidazol- 1-yl)propyl]-4-
(2-thienyl carbonyl)-1,5- dihydro-2H- pyrrol-2-one 792 81 >10
.mu.g/ml 16.3 .mu.M 2 I ##STR00017## 6422575 {4-bromo-2- [3-(ethoxy
carbonyl)-2- methyl-5-oxo- 4,5-dihydro- 1H- indeno[1,2-
b]pyridin-4- yl]phenoxy} acetic acid 706 81 >10 .mu.g/ml 6.2
.mu.M 3 -- ##STR00018## 6882059 3-hydroxy-5- (3-methoxy
phenyl)-4-(4- methyl benzoyl)-1-[3- (4- morpholinyl) propyl]-1,5-
dihydro-2H- pyrrol-2-one 243 79.5 >10 .mu.M 9.4 .mu.M 4 I
##STR00019## 7111786 4-benzoyl-5- (2,5- dimethoxy phenyl)-3-
hydroxy-1-[3- (1H-imidazol- 1-yl)propyl]- 1,5-dihydro- 2H-pyrrol-2-
one 557 77.5 >10 .mu.g/ml -- 5 I ##STR00020## 6203863 ethyl
4-[3- benzoyl-2- (2,4- dimethoxy phenyl)-4- hydroxy-5- oxo-2,5-
dihydro-1H- pyrrol-1-yl] benzoate 423 73.5 >10 .mu.g/ml 6.1
.mu.M 6 I ##STR00021## 7347627 4-(2,5- dimethyl benzoyl)-3-
hydroxy-5-(2- methoxy phenyl)-1-[2- (4- morpholinyl) ethyl]-1,5-
dihydro-2H- pyrrol-2-one 775 72 >10 .mu.g/ml -- 7 I ##STR00022##
7489416 5-(3-bromo-4- hydroxy-5- methoxy phenyl)-3- hydroxy-1-(2-
phenylethyl)- 4-(2-thienyl carbonyl)-1,5- dihydro-2H- pyrrol-2-one
648 71 >10 .mu.g/ml -- 8 I ##STR00023## 6719738 4-(4-fluoro
benzoyl)-3- hydroxy-5-(3- phenoxy phenyl)-1-(3- pyridinyl
methyl)-1,5- dihydro-2H- pyrrol-2-one 977 71 >10 .mu.g/ml -- 9 I
##STR00024## 6650234 ethyl 2-[3-(4- fluoro- benzoyl)- 4-hydroxy-2-
(4-methyl phenyl)-5-oxo- 2,5-dihydro- 1H-pyrrol-1- yl]-4-methyl-
1,3-thiazole-5- carboxylate 946 71 ~10 .mu.g/ml 6.3 .mu.M 10 I
##STR00025## 6652639 5-(2,5- dimethoxy phenyl)-3- hydroxy-4-(4-
methyl benzoyl)-1-(3- pyridinyl methyl)-1,5- dihydro-2H-
pyrrol-2-one 829 70.5 >10 .mu.g/ml -- 11 I ##STR00026## 6673225
ethyl 2-[3- benzoyl-4- hydroxy-2-(4- methoxy phenyl)-5-oxo-
2,5-dihydro- 1H-pyrrol-1- yl]-4-methyl- 1,3-thiazole-5- carboxylate
301 69 >10 .mu.g/ml -- 12 I ##STR00027## 6800873 3-[2-(2,4-
dimethoxy phenyl)-2- oxoethyl]-3- hydroxy-1-(1- naphthyl
methyl)-1,3- dihydro-2H- indol-2-one 250 67.5 >10 .mu.g/ml 4.6
.mu.M 13 -- ##STR00028## 6879058 3-hydroxy-4- (4-methoxy-2- methyl
benzoyl)-1-[2- (4- morpholinyl) ethyl]-5-(3- pyridinyl)-1,5-
dihydro-2H- pyrrol-2-one 758 67.5 >10 .mu.g/ml -- 14 I
##STR00029## 6282824 2-methoxy-N- (4-{4-methyl- 5-[(2-oxo-2-
phenylethyl) thio]-4H-1,2, 4-triazol-3- yl}phenyl) benzamide 905
67.5 ~2.5 .mu.g/ml 5.6 .mu.M 15 II ##STR00030## 6750319 methyl
2-[3- benzoyl-4- hydroxy-2-(4- methyl phenyl)-5-oxo- 2,5-dihydro-
1H-pyrrol-1- yl]-4-methyl- 1,3-thiazole-5- carboxyiate 850 67
>10 .mu.g/ml -- 16 I ##STR00031## 6474819 4-[(4-benzyl-
1-piperidinyl) methyl]-N-(2- methoxy-5- methylphenyl) benzamide 954
63 >10 .mu.g/ml 17.9 .mu.M 17 -- ##STR00032## 6498669 4-{[N-[(4-
methoxy phenyl) sulphonyl]-N- (2-phenyl ethyl)glycyl] amino}
benzamide 808 63 >10 .mu.g/ml 0.8 .mu.M 18 III ##STR00033##
6651552 4-(4-fluoro benzoyl)-3- hydroxy-5-(4- isopropyl
phenyl)-1-[3- (4- morpholinyl) propyl]-1,5- dihydro-2H-
pyrrol-2-one 100 60 >10 .mu.g/ml -- 19 I ##STR00034## 6453860
N,N'-1,5- naphthalene- diylbis[2-(3- methyl- phenoxy) acetamide]
145 60 >10 .mu.g/ml -- 20 -- ##STR00035## 6853966 N-[4-({[4-
(acetyl amino) phenyl] sulphonyl} amino)-2,5- dimethoxy phenyl]
benzamide 11 58 >10 .mu.g/ml -- 21 -- ##STR00036## 6866968
7,7-dimethyl- 1-(4-methyl phenyl)-2,5- dioxo-N- (2,2,6,6-
tetramethyl-4- piperidinyl)- 1,2,5,6,7,8- hexahydro-3- quinoline
carboxamide 255 57.5 >10 .mu.g/ml -- 22 -- ##STR00037## 7938324
4-(benzyl{[1- phenyl-3-(2- thienyl)-1H- pyrazol-4- yl]methyl}
amino)-4-oxo butanoic acid 795 57 >10 .mu.g/ml -- 23 _--
##STR00038## 6905988 4-(4-fluoro benzoyl)-3- hydroxy-5-(3- methoxy
phenyl)-1-[3- (4- morpholinyl) propyl]-1,5- dihydro-2H-
pyrrol-2-one 843 57 >10 .mu.g/ml -- 24 I ##STR00039## 6885782
4-(4-chloro benzoyl)-3- hydroxy-1-[3- (4- morpholinyl)
propyl]-5-(3, 4,5-trimethoxy phenyl)-1,5- dihydro-2H- pyrrol-2-one
249 56.5 >10 .mu.g/ml -- 25 I ##STR00040## 6663684 4-benzoyl-3-
hydroxy-5-(4- isopropyl phenyl)-1 -[2- (4- morpholinyl) ethyl]-1,5-
dihydro-2H- pyrrol-2-one 530 56.5 >10 .mu.g/ml -- 26 I
##STR00041## 6672500 4-(4-chloro benzoyl)-5- (3,4- dimethoxy
phenyl)-3- hydroxy-1-[2- (4- morpholinyl) ethyl]-1,5- dihydro-2H-
pyrrol-2-one 194 55 >10 .mu.g/ml -- 27 I ##STR00042## 7721949
1-[2-(diethyl amino)ethyl]- 5-(2,5- dimethoxy phenyl)-3-
hydroxy-4-(4- methyl benzoyl)-1,5- dihydro-2H- pyrrol-2-one 139 54
>10 .mu.g/ml -- 28 I ##STR00043## 7966545 2-fluoro-N- [(5-
{[(4-oxo-3,4- dihydro-2- quinazolinyl) methyl]thio}- 4-phenyl-4H-
1,2,4-triazol- 3-yl)methyl] benzamide 773 53.5 ~10 .mu.g/ml 6.7
.mu.M 29 -- ##STR00044## 7437580 2-{[4-(1,3- dioxo-1,3- dihydro-2H-
isoindol-2- yl)butanoyl] amino}-N- (tetrahydro-2- furanyl
methyl)-5,6- dihydro-4H- cyclopenta[b] thiophene-3- carboxamide 222
51 >10 .mu.g/ml -- 30 -- ##STR00045## 6654239 5-(3,4- dimethoxy
phenyl)-4-(4- fluoro benzoyl)-3- hydroxy-1-[2- (4- morpholinyl)
ethyl]-1,5- dihydro-2H- pyrrol-2-one 343 50 >10 .mu.g/ml -- 31 I
##STR00046## 6670570 4-({4- hydroxy- 1-[2-(4- morpholinyl)
ethyl]-5-oxo- 2-phenyl-2,5- dihydro-1H- pyrrol-3-yl} carbonyl)-
N,N-dimethyl benzene sulphonamide 926 50 >10 .mu.g/ml -- 32 I
##STR00047## 6670673 5-(2,4- dimethoxy phenyl)-4-(4- fluoro-
benzoyl)- 3-hydroxy-1- [2-(4- morpholinyl) ethyl]-1,5- dihydro-2H-
pyrrol-2-one 742 50 >10 .mu.g/ml -- 33 I ##STR00048## 6670747
3-hydroxy-5- (4-methoxy phenyl)-4-(4- methyl benzoyl)-1-[2- (4-
morpholinyl) ethyl]-1,5- dihydro-2H- pyrrol-2-one 413 50 >10
.mu.g/ml -- 34 I ##STR00049## 6671401 5-(3,4- dimethoxy
phenyl)-4-(2- furoyl)-3- hydroxy-1-[2- (4- morpholinyl) ethyl]-1,5-
dihydro-2H- pyrrol-2-one 954 50 >10 .mu.g/ml -- 35 I
##STR00050## 6672500 4-(4-chloro benzoyl)-5- (3,4- dimethoxy
phenyl)-3- hydroxy-1-[2- (4- morpholinyl) ethyl]-1,5- dihydro-2H-
pyrrol-2-one 194 50 >10 .mu.g/ml -- 36 I ##STR00051## 6673225
ethyl 2-[3- benzoyl-4- hydroxy-2-(4- methoxy phenyl)-5-oxo-
2,5-dihydro- 1H-pyrrol-1- yl]-4-methyl- 1,3-thiazole-5- carboxylate
124 50 >10 .mu.g/ml -- 37 I ##STR00052## 6677533 5-(3,4-
dimethoxy phenyl)-3- hydroxy-1-[2- (4- morpholinyl) ethyl]-4-(2-
thienyl carbonyl)-1,5- dihydro-2H- pyrrol-2-one 409 50 >10
.mu.g/ml -- 38 I ##STR00053## 6683618 4-(4-chloro benzoyl)-3-
hydroxy-5-(4- methoxy phenyl)-1-[2- (4- morpholinyl) ethyl]-1,5-
dihydro-2H- pyrrol-2-one 808 50 >10 .mu.g/ml -- 39 I
##STR00054## 6417902 N-[2-(4- benzyl-1- piperazinyl)-2-
oxoethyl]-N- (3,5-dimethyl phenyl) benzene sulphonamide 897 49.5
>10 .mu.g/ml -- 40 III ##STR00055## 6437157 1-methyl-2-(4-
methyl phenyl)-2- oxoethyl 2-(3- chloro-4- methyl phenyl)-1,3-
dioxo-5- isoindoline carboxylate 871 49.5 >10 .mu.g/ml -- 41 --
##STR00056## 6465972 N~2~-[(3,4- dimethoxy phenyl) sulphonyl]-
N~1~-(2- methoxy-5- methyl- phenyl)- N~2~-(4- methylphenyl)
glycinamide 926 49 >10 .mu.g/ml -- 42 III ##STR00057## 7723671
5-(2,5- dimethoxy phenyl)-4-(4- fluoro benzoyl)-3- hydroxy-1-[3-
(4- morpholinyl) propyl]-1,5- dihydro-2H- pyrrol-2-one 34 48.5
>10 .mu.g/ml -- 43 I ##STR00058## 6648368 4-(4- chloro-
benzoyl)- 5-(2- fluorophenyl)- 3-hydroxy-1- [3-(4- morpholinyl)
propyl]-1,5- dihydro-2H- pyrrol-2-one 465 48.5 >10 .mu.g/ml --
44 I ##STR00059## 6656195 4-(4-fluoro benzoyl)-3- hydroxy-5-(4-
isopropyl phenyl)-1-[2- (4- morpholinyl) ethyl]-1,5- dihydro-2H-
pyrrol-2-one 613 48.5 >10 .mu.g/ml -- 45 I ##STR00060## 7778331
4-{[2-(4- morpholinyl) ethyl]amino}- 3-(4- morpholinyl
sulphonyl)-N- phenyl- benzamide 998 48 >10 .mu.g/ml 3 .mu.M 46
-- ##STR00061## 6994060 2-chloro-N- {4-[4-methyl- 5-({2-oxo-2-
[(tetrahydro- 2-furanyl methyl) amino] ethyl}thio)- 4H-1,2,4-
triazol-3-yl] phenyl} benzamide 623 47.5 >10 .mu.g/ml -- 47 II
##STR00062## 6458830 N-[2-(4- benzyl-1- piperazinyl)-2-
oxoethyl]-N- (3,4-dimethyl phenyl)-4- methyl benzene sulphonamide
837 47 >10 .mu.g/ml -- 48 III ##STR00063## 7524107 2-[(4-{[(4-
isopropyl phenoxy) acetyl]amino}- 3-methyl benzoyl) amino] benzoic
acid 789 46.5 ~10 .mu.g/ml 5.5 .mu.M 49 -- ##STR00064## 6661524
4-({2-(3,4- dichloro phenyl)-1-[3- (dimethyl amino)propyl]-
4-hydroxy-5- oxo-2,5- dihydro-1H- pyrrol-3-yl} carbonyl)-
N,N-dimethyl benzene sulphonamide 428 46.5 >10 .mu.g/ml -- 50 I
##STR00065## 7739436 4-(1,3- benzodioxol- 5-yl carbonyl)-
1-[3-(diethyl amino)propyl]- 3-hydroxy-5- (3-pyridinyl)-
1,5-dihydro- 2H-pyrrol-2- one 976 46 >10 .mu.g/ml -- 51 I
##STR00066## 6881804 1-[2-(dimethyl amino)ethyl]- 3-hydroxy-4-
(5-methyl-2- furoyl)-5- (3,4,5- trimethoxy phenyl)-1,5- dihydro-2H-
pyrrol-2-one 230 46 >10 .mu.g/ml -- 52 I ##STR00067## 6907921
3-hydroxy-5- (3-methoxy phenyl)-4-(5- methyl-2- furoyl)-1-[2-(4-
morpholinyl) ethyl]-1,5- dihydro-2H- pyrrol-2-one 609 45.5 >10
.mu.g/ml -- 53 I ##STR00068## 7495334 ethyl 4-({[(5,6- di-2-furyl-
1,2,4-triazin-3- yl)thio]acetyl} amino) benzoate 380 43 ~5 .mu.g/ml
1.6 .mu.M 54 -- ##STR00069## 7509862 ethyl 5-cyano- 4-(2-furyl)-6-
({2-[(3- methoxy phenyl) amino]-2- oxoethyl} thio)-2-phenyl-
1,4-dihydro-3- pyridine carboxylate 170 42.5 ~5 .mu.g/ml -- 55 --
##STR00070## 7325385 5-(2,3- dimethoxy phenyl)-4- (2,5-dimethyl
benzoyl)-3- hydroxy-1-[3- (4- morpholinyl) propyl]-1,5- dihydro-2H-
pyrrol-2-one 65 42 ~10 .mu.g/ml -- 56 I ##STR00071## 6669449
3-(6-amino-5- cyano-3- phenyl-1,4- dihydro pyrano[2,3- c]pyrazol-4-
yl)phenyl 2- furoate 140 42 >10 .mu.g/ml -- 57 -- ##STR00072##
7348779 1,4-bis [(mesityloxy) acetyl] piperazine 209 40 ~10
.mu.g/ml -- 58 -- ##STR00073## 7501888 N-(4-chloro pheny)-2-{[4-
(2-phenyl ethyl)-5- (3,4,5- trimethoxy phenyl)-4H- 1,2,4-triazol-
3-yl]thio} acetamide 544 40 ~2 .mu.g/ml 8.2 .mu.M 59 --
##STR00074## 6946138 {[3-(ethoxy carbonyl)-2- phenyl-1-
benzofuran-5- yl]oxy} (phenyl) acetic acid 298 40 >10 .mu.g/ml
-- 60 -- ##STR00075## 7722851 ethyl 4-({[1- (4-chloro phenyl)-5-
oxo-3-(3- pyridinyl methyl)-2- thioxo-4- imidazolidinyl] acetyl}
amino) benzoate 638 39.5 ~2 .mu.g/ml 21.8 .mu.M 61 IV ##STR00076##
7517583 5,5'-oxybis [2- (tetra hydro-2- furanyl methyl)-1H-
isoindole- 1,3(2H)-dione] 912 38.5 >10 .mu.g/ml -- 62 --
##STR00077## 6634701 7-acetyl-6-[3- (benzyloxy) phenyl]-3-
(methylthio)- 6,7-dihydro [1,2,4] triazino[5,6- d][3,1]
benzoxazepine 934 38.5 ~5 .mu.g/ml -- 63 -- ##STR00078## 7726450
3-hydroxy-1- [3-(1H- imidazol-1- yl)propyl]-4- [(7-methoxy-
1-benzofuran- 2-yl) carbonyl]-5- (2-pyridinyl)- 1,5-dihydro-
2H-pyrrol-2- one 472 37.5 >10 .mu.g/ml -- 64 I ##STR00079##
6662088 4-{[4-hydroxy- 1-[3-(4- morpholinyl) propyl]-5-oxo- 2-
(3-pyridinyl)- 2,5-dihydro- 1H-pyrrol-3- yl]carbonyl}- N,N-dimethyl
benzene sulphonamide 156 37 >10 .mu.g/ml -- 65 I ##STR00080##
7752193 N-{1-[4-allyl- 5-({2-[(3- methoxy phenyl) amino]-2-
oxoethyl} thio)-4H-1,2,4- triazol-3- yl]ethyl} benzamide 248 35 ~5
.mu.g/ml 8 .mu.M 66 -- ##STR00081## 7238569 N-(2-hydroxy-
1,1-dimethyl- ethyl)-5-{4-[(3- hydroxyphenyl) amino]-1-
phthalazinyl}- 2-methyl benzene sulphonamide 749 32 >10 .mu.g/ml
-- 67 -- ##STR00082## 7724000 4-(1,3- benzodioxol- 5-ylcarbonyl)-
5-(2- fluorophenyl)- 3-hydroxy-1- [3-(4- morpholinyl) propyl]-1,5-
dihydro-2H- pyrrol-2-one 303 30 ~10 .mu.g/ml -- 68 I ##STR00083##
7443270 N-(2,4- dimethoxy- phenyl)- 2-{[3-(2- furylmethyl)-4-
oxo-3,4- dihydro-2- quinazolinyl] thio} acetamide 895 30 ~5
.mu.g/ml -- 69 -- ##STR00084## 7245019 N-[(2-hydroxy- 7-methyl-3-
quinolinyl) methyl]-3- methoxy-N-(2- methoxy phenyl) benzamide 608
30 >10 .mu.g/ml -- 70 -- ##STR00085## 6909597 4-(4-
chlorobenzoyl)- 3-hydroxy-5- (3-methoxy phenyl)-1-[2- (4-
morpholinyl) ethyl]-1,5- dihydro-2H- pyrrol-2-one 790 29 >10
.mu.g/ml -- 71 I ##STR00086## 7661882 2-(4-methoxy phenoxy)-N-
[2-methyl-5- (3-methyl-4- oxo-3,4- dihydro-1- phthalazinyl) benzyl]
acetamide 941 28 ~10 .mu.g/ml -- 72 -- ##STR00087## 7667791
N-{4-[({[4-(4- methoxy phenyl) tetrahydro-2H- pyran-4-yl] methyl}
amino) carbonyl] phenyl}-2- furamide 797 28 >10 .mu.g/ml -- 73
-- ##STR00088## 6891745 4-benzoyl-5- (2,3- dimethoxy phenyl)-3-
hydroxy-1 -[3- (4- morpholinyl) propyl]-1,5- dihydro-2H-
pyrrol-2-one 115 27.5 >10 .mu.g/ml -- 74 I ##STR00089## 7783660
isopropyl 3- ({[(4-allyl-5- {[(3-methyl benzoyl) amino] methyl}-4H-
1,2,4-triazol-3- yl)thio]acetyl} amino) benzoate 266 27 ~5 .mu.g/ml
4.8 .mu.M 75 II ##STR00090## 7653478 2-{5-[1-(4- morpholinyl)
cyclohexyl]- 1H-tetrazol-1- yl}ethyl 1- naphthyl carbamate 365 27
>10 .mu.g/ml -- 76 -- ##STR00091## 7723330 methyl 4-({[3- (1,3-
benzodioxol- 5-ylmethyl)- 2,5-dioxo-1- phenyl-4- imidazol
idinyl]acetyl} amino) benzoate 965 26 >10 .mu.g/ml -- 77 IV
##STR00092## 7199725 4-(3,4- dihydro-2(1H)- isoquinolinyl-
methyl)-N-[2- (1-pyrrolidinyl carbonyl) phenyl] benzamide 987 25
>10 .mu.g/ml -- 78 -- ##STR00093## 6987235 9-{3-chloro-4-
[(4-methyl benzyl)oxy] phenyl}-10- ethyl- 3,4,6,7,9,10- hexahydro-
1,8(2H,5H)- acridine dione 680 25 >10 .mu.g/ml -- 79 _--
##STR00094## 7140931 ethyl 1-(4- {[(3,4-dimethyl phenyl) (methyl
sulphonyl) amino] methyl} benzoyl)-4- piperidine carboxylate 14
24.5 >10 .mu.g/ml -- 80 -- ##STR00095## 7787455 methyl 4-{[N-
(3-methoxy phenyl)-N- (phenyl sulphonyl) glycyl] amino} bonzoate
407 20 >10 .mu.g/ml -- 81 III ##STR00096## 7660465
N-{[5-({2-[(4- bromo-2,3- dimethyl phenyl) amino]-2- oxoethyl}
thio)-4-ethyl- 4H-1,2,4- triazol-3- yl]methyl}-4- chloro benzamide
881 20 ~7 .mu.g/ml 3.2 .mu.M 82 II ##STR00097## 7661751 4-(2,3-
dihydro-1,4- benzodioxin- 6-yl carbonyl)- 3-hydroxy-1- [3-(4-
morpholinyl) propyl]-5-(4- pyridinyl)-1,5- dihydro-2H- pyrrol-2-one
951 19 ~10 .mu.g/ml -- 83 I ##STR00098## 7722914 N-(4-ethoxy
phenyl)-2-{1- (4-methoxy phenyl)-3-[2- (4- morpholinyl)
ethyl]-5-oxo-2- thioxo-4- imidazolidinyl} acetamide 806 19 ~7
.mu.g/ml 15.6 .mu.M 84 IV ##STR00099## 7745040 5-(3-bromo
phenyl)-3- hydroxy-4-[(7- methoxy-1- benzo furan- 2-yl)
carbonyl]-1-[2- (4- morpholinyl) ethyl]-1,5- dihydro-2H-
pyrrol-2-one 313 18 ~10 .mu.g/ml -- 85 I ##STR00100## 7735385
1-[3-(diethyl amino)propyl]- 3-hydroxy-4- [(7-methoxy-
1-benzofuran- 2-yl) carbonyl]-5- (2-pyridinyl)- 1,5-dihydro-
2H-pyrrol-2- one 21 17 ~10 .mu.g/ml -- 86 I ##STR00101## 7756003
1-(4-{[(4,6- dimethyl-2- pyrimidinyl) thio]acetyl}-1-
piperazinyl)-4- (4-methyl phenyl) phthalazine 361 11 ~5 .mu.g/ml --
87 --.sub.-- CB ref: ChemBridge reference; Rank: rank of each
molecule in the list of the 1000 best molecules after in silico
docking; In vit. inhib.: mean inhibition % obtained with each
molecule for the displacement of the binding of scFv G4G11 with Syk
in vitro in the ADA method; Degran. IC50: concentration inhibiting
mast cell degranulation by 50%; Kd: dissociation constant with
respect to Syk measured in vitro by spectrofluorometry No.: number
assigned by the inventors Gp: groups to which the molecules
belong.
Example 3
Materials and Methods
[0136] 1) Chemical products and antibodies. A chemical bank of 3000
molecules (a varied subset) was acquired from ChemBridge, Inc. (San
Diego, Calif.). Stocks of solutions of small molecules were
prepared at a rate of 10 mM in DMSO (dimethylsulphoxide), except
for C-13 (methyl
2-{5-[(3-benzyl-4-oxo-2-thioxo-1,3-thiazolidin-5-ylidene)methyl]-2-furyl}-
benzoate, Chem Bridge ID No. 6197026) prepared in DMF
(dimethylformamide). Unless specified otherwise, all the reagents
were supplied by Sigma. Dinitrophenyl (DNP) hapten was acquired
from Calbiochem. Sepharose GammaBind G and all the secondary
antibodies were supplied by GE Health Amersham Biosciences. The
anti-Syk, anti-Lyn, anti-Btk, anti-PLC-.gamma.1, anti-PLC-.gamma.2,
anti-LAT, anti-SLP-76, anti-p38, anti-JNK, anti-Vav, anti-Akt1 and
9E10 antibodies conjugated with HRP were acquired from Santa Cruz
Biotechnology. The anti-phospho-p44/42 MAP Kinase, anti-p44/42 MAP
Kinase, anti-phospho-p38, anti-phospho-JNK, anti-phospho-Akt1
antibodies were acquired from Cell Signaling. The anti-phospho-LAT
and anti-phospho-PLC-.gamma.1 antibodies were supplied by Biosource
and the anti-phospho-SLP-76 antibodies by BD Pharmingen. The 4G10
anti-phosphotyrosine monoclonal antibody was acquired from Upstate
Biotechnology.
[0137] 2) ADA method: ELISA type high-speed molecule screening
test, based on antibody displacement (WO 2005106481). The
recombinant fusion protein GST:Syk 6-242.sup.8 comprising the
residues 6 to 242 of murine Syk tyrosine kinase protein illustrated
in FIG. 8B (SEQ ID No. 4) was immobilised on an ELISA plate at a
final concentration of 10 .mu.g ml.sup.-1. For the screening of the
chemical molecule bank, the small molecules, diluted in PBS at a
final concentration of 10 .mu.M were added to the wells for one
hour at ambient temperature, before adding the fragment scFv G4G11
at a final concentration of 100 nM for an additional hour. The
binding of G4G11 with Syk was assessed by adding the 9E10
monoclonal antibody conjugated with HRP detecting the amino acid
sequence EQKLISEEDLN of human c-myc protein located at the
C-terminal end of the scFv fragment. To generate Syk mutants,
targeted mutagenesis was used on GST:Syk 6-242 protein and the
binding of G4G11 with the mutants was assessed in the presence of 5
.mu.M of C-13.
[0138] 3) Cells, culture conditions and functional tests. Anti-DNP
2682-I mouse monoclonal antibody was used as the culture
supernatant of hybridomas containing 1 .mu.g/ml of IgE. Femoral
bone marrow cells were sampled and cultured in Opti-MEM medium
(Gibco) supplemented with 10% foetal calf serum and 4% X63
transfectant supernatant secreting murine IL-3. RBL-2H3 (ATCC)
leukaemic rat basophil cells were maintained in a single-layer
culture in RPMI 1640 medium supplemented with 10% foetal calf serum
(Gibco). Measurements of .beta.-hexosaminidase released by the
RBL-2H3 cells were performed as described previously.sup.7, except
that, after 12-16 hours of incubation with anti-DNP IgE (0.5
.mu.g/ml), the cells were incubated for 90 min at 37.degree. C. in
RPMI medium supplemented with the specified concentrations of C-13
or DMF (0.25%). The cells were stimulated for 45 min with DNP-BSA
(50 ng ml.sup.-1) or ionomycin (1.5 .mu.M). The BMMC cells were
incubated for one hour at 37.degree. C. with anti-DNP IgE (100
ng/ml). They were then incubated with C-13 (3 .mu.M) or DMF (0.3%)
for 3 hours at 37.degree. C., and stimulated with varied
concentrations of DNP-BSA. The level of .beta.-hexosaminidase
released was measured 10 min later and the TNF-.alpha. titration
was performed by means of a cytotoxicity test on L929 cells as
described previously.sup.10, 3 hours after stimulation. The results
illustrated in FIG. 3 are representative of three independent
experiments.
[0139] 4) Immunoprecipitations, in vitro kinase assays and
immunodetection. All the experiments were conducted as described
previously.sup.7, except that, before stimulation with DNP-BSA (50
ng ml.sup.-1, 3 min), the RBL-2H3 cells were incubated for 90 min
at 37.degree. C. in RPMI medium supplemented with the specified
concentrations of C-13 or DMF. The cells were solubilised in DOC
modified lysis buffer (1% NP-40, 0.25% sodium deoxycholate, 0.1%
SDS in PBS buffer supplemented with protease and phosphatase
inhibitors) and the protein concentration was determined (BCA
Protein Assay, PIERCE). For immunoprecipitations, cell lysates,
non-stimulated and stimulated with IgE/DNP were incubated with
preformed complexes of antibodies and Sepharose GammaBind G, and
the in vitro kinase activity of Syk, Btk and Lyn immunoprecipitates
were examined. Before SDS-PAGE gel separation the lysates or
immunoprecipitates were prepared by adding SDS sample buffer (60 mM
Tris, pH 6.8, 2.3% SDS, 10% glycerol, 0.01% bromophenol blue). The
proteins were transferred onto a nitrocellulose membrane
(Schleicher & Schuell), and detected using suitable antibodies
and the chemoluminescence system improved (ExactaCruz, Santa Cruz
Biotechnology).
[0140] 5) Flow cytometry analysis of level and calcium mobilisation
and Fc.epsilon.RI membrane expression. The intracellular free
calcium concentration was determined by previously charging
1.times.10.sup.6 cells with 5 mM of Fluo-3 AM (Molecular Probes,
Invitrogen) in the presence of 0.2% Pluronic F-127 for 30 min at
ambient temperature. Prior to stimulation with DNP-BSA or
ionomycin, the cells were treated for 90 min at 37.degree. C. in
RPMI medium supplemented with C-13 or DMF (0.25%), and the
intracellular free calcium concentration was measured with a flow
cytometer (Beckton Dickinson). For the Fc.epsilon.RI surface
expression evaluation, the cells were incubated for 2 hours at
37.degree. C. with anti-DNP IgE. Membrane-bound IgE was detected
using biotinylated anti-mouse Ig, and streptavidine conjugated with
Fitc.
[0141] 6) Anaphylaxis induction. Female BALB/c mice (aged 6-8
weeks) were acquired from Charles River and kept at the IRCM animal
house under pathogen-free conditions. The Ig-dependent passive
systemic anaphylaxis (PSA) and passive cutaneous anaphylaxis (PCA)
protocols were conducted as described previously.sup.11. Briefly,
the mice received, by intravenous injection, 100 .mu.g of IgE
(SPE-7, Sigma) in 200 .mu.l of PBS for PSA, or, by intradermal
injection, 25 ng of IgE in 10 .mu.l of PBS for PCA, and were
stimulated 24 hours later by means of an intravenous injection of 1
mg of DNP-KLH in 2% of Evans blue. C-13, a non-relevant chemical
molecule or the vehicle were administered 1 hours before
stimulation, either orally (PSA) in 200 .mu.l of 1%
carboxymethylcellulose, or locally in the ear by means of
intradermal injection (PCA) in an acetone/olive oil mixture (4:1).
The animals were sacrificed 20 min after stimulation. The ears were
removed, ground and Evans blue was extracted after overnight
incubation in formamide at 80.degree. C. For the temperature
measurements in PSA, C-13 (100 mg/kg) or the vehicle were
administered orally, 3 hours prior to stimulation performed in the
absence of Evans blue. The temperature was measured using an
electronic thermometer with a rectal probe (YSI, Yellow Springs,
Ohio) before stimulation and for 60 minutes afterwards, prior to
sacrifice. The absorbance was measured at 610 nm. The experiments
were conducted with 4-5 mice per condition. The data illustrated in
FIG. 4 are representative of three different experiments.
[0142] 7) Structural studies. The three-dimensional cavities liable
to be pharmaceutical targets were predicted using
Q-SiteFinder.sup.12 and ICM.sup.13. The molecule C-13 was docked
using LigandFit.sup.14 and Surflex.sup.15. The first 20 positions
were analysed and a consensus position is given in FIG. 1A. The
images were generated with PyMol.
[0143] 8) Peritonitis (FIG. 5). Syk-dependent peritoneal neutrophil
recruitment was induced in 8-week old female BALB/c mice as
described in.sup.36 by intravenous injection of 4 .mu.g of
Bordetella pertussis toxin (donated by Dr D. Raze, Inserm, Lille,
France) and 2 hours later by intraperitoneal injection of 4%
thioglycollate in sterile water. A peritoneal lavage was performed
with 5 ml of PBS 4 hours later and the total number of neutrophils
was determined after labelling with anti-Gr1 conjugated with APC
(Becton-Dickinson) and flow cytometry analysis. C-13 (100 mg/kg) in
CMC or the vehicle alone was administered orally one hour prior to
injecting Bordetella pertussis toxin.
[0144] 9) In vitro B Lymphocyte purification and proliferation.
Spleen B lymphocyte cells were purified from 8-week old female
BALB/c mice on magnetic beads by negative selection using
micro-beads coated with CD43 and LS columns (Miltenyi Biotec) as
described previously.sup.37. After two hours of incubation with
variable concentrations of C-13 or the vehicle, 50,000 cells were
cultured for 48 hours in 96-well plates in the presence of absence
of 10 .mu.g/ml of donkey anti-mouse IgM F(ab').sub.2 fragment
(Jacson Immunoresearch). Alamar blue (Serotec) was added to the
cultures 24 prior to measuring the reduced versus oxidised forms of
the reagent at 570 and 620 nm, in accordance with the manufacturers
instructions.
[0145] 10) Antibody production (FIG. 5C). This experiment was
conducted as described in.sup.38. Eight-week old female BALB/c mice
received an oral dose of C-13 (150 mg/kg) in 1% CMC or the vehicle
alone, were immunised 3 hours later by an intraperitoneal injection
of 10 .mu.g of trinitophenyl-keyhole limpet haemocyanin (TNP-KLH)
in Rehydragel alum (Reheiss). The serum was collected before
immunisation and on day 12. The antigen-specific immunoglobulin
levels were measured by means of ELISA with 10 .mu.g/ml of
plate-bound TNP-OVA (Biosearch technologies) as the capture agent.
The IgM, IgG1, IgG2a, IgG2b, IgG3 and IgA levels were measured
using samples diluted to 1:5000 using goat antibodies conjugated
with peroxidase and isotype-specific (Southern Biotechnology),
whereas the IgE levels were measured using samples diluted to 1:50
using biotinylated anti-mouse IgE rat antibodies (Becton Dickinson)
and streptavidine conjugated with peroxidase (R&D Systems).
After incubation with TMB substrate, the optical density (OD) was
measured at 450 nm.
[0146] 11) Statistical analyses. The mean numeric data are
expressed as means.+-.standard deviations (SD). Students t test was
used to determine the statistical significance of the differences
between groups.
[0147] 12) C-13 toxicity test on BMMC cells. To assess the
potential toxic effect of C-13 on mast cells, BMMC cells were
incubated for 5 days at 37.degree. C. in the presence of 2.5 .mu.M
or 5 .mu.M of C-13, or 0.25% DMF (corresponding to the DMF
concentration used with 5 .mu.M of C-13) under the same conditions
as for functional tests. Double labelling with Annexin-V and
Propidium Iodide demonstrated the level of BMMC cell viability
after 3 hours and after 5 days. The viability of the BMMC cells
treated under the same conditions with Staurosporine was measured
under the same conditions.
REFERENCES
[0148] 1. Kambayashi T, Koretzky G A. Proximal signaling events in
Fc epsilon R1-mediated mast cell activation. J Allergy Clin Immunol
2007; 119:544-52; quiz 53-4. [0149] 2. Rivera J, Gilfillan A M,
Molecular regulation of mast cell activation. J Allergy Clin
Immunol 2006; 117:1214-25; quiz 26. [0150] 3. Costello P S, Turner
M, Walters A E, Cunningham C N, Bauer P H, Downward J, et al.
Critical role for the tyrosine kinase Syk in signalling through the
high affinity IgE receptor of mast cells. Oncogene 1996;
13:2595-605. [0151] 4. Wong W S, Leong K P. Tyrosine kinase
inhibitors: a new approach for asthma. Biochim Biophys Acta 2004;
1697:53-69. [0152] 5. Turner M, Schweighoffer E, Colucci F, Di
Santo J P, Tybulewicz V L. Tyrosine kinase Syk: essential functions
for immunoreceptor signalling. Immunol Today 2000; 21:148-54.
[0153] 6. Coopman P J, Mueller S C. The Syk tyrosine kinase: a new
negative regulator in tumor growth and progression. Cancer Lett
2006; 241:159-73. [0154] 7. Dauvillier S, Merida P, Visintin M,
Cattaneo A, Bonnerot C, Dariavach P. Intracellular single-chain
variable fragments directed to the Src homology 2 domains of Syk
partially inhibit Fc epsilon R1 signaling in the RBL-2H3 cell line.
J Immunol 2002; 169:2274-83. [0155] 8. Peneff C, Lefranc M P,
Dariavach P. Characterisation and specificity of two single-chain
Fv antibodies directed to the protein tyrosine kinase Syk. J
Immunol Methods 2000; 236:105-15. [0156] 9. Lobato M N, Rabbitts T
H. Intracellular antibodies and challenges facing their use as
therapeutic agents. Trends Mol Med 2003; 9:390-6. [0157] 10. Latour
S, Bonnerot C, Fridman W H, Daeron M. Induction of tumor necrosis
factor-alpha production by mast cells via Fc gamma R. Role of the
Fc gamma RIII gamma subunit. J Immunol 1992; 149:2155-62. [0158]
11. Dombrowicz D, Flamand V, Miyajima I, Ravetch J V, Galli S J,
Kinet J P. Absence of Fc epsilonRI alpha chain results in
upregulation of Fc gamnnaRIII-dependent mast cell degranulation and
anaphylaxis. Evidence of competition between Fc epsilonRI and Fc
gammaRIII for limiting amounts of FcR beta and gamma chains. J Clin
Invest 1997; [0159] 12. Laurie A T, Jackson R M. Q-SiteFinder: an
energy-based method for the prediction of protein-ligand binding
sites. Bioinformatics 2005; 21:1908-16. [0160] 13. An J, Totrov M,
Abagyan R. Comprehensive identification of "druggable" protein
ligand binding sites. Genome Inform 2004; 15:31-41. [0161] 14.
Venkatachalam C M, Jiang X, Oldfield T, Waldman M. LigandFit: a
novel method for the shape-directed rapid docking of ligands to
protein active sites. J Mol Graph Model 2003; 21:289-307. [0162]
15. Jain A N. Surflex: fully automatic flexible molecular docking
using a molecular similarity-based search engine. J Med Chem 2003;
46:499-511. [0163] 16. Frank R, Overwin H. SPOT synthesis. Epitope
analysis with arrays of synthetic peptides prepared on cellulose
membranes. Methods Mol Biol 1996; 66:149-69. [0164] 17. Futterer K,
Wong J, Grucza R A, Chan A C, Waksman G. Structural basis for Syk
tyrosine kinase ubiquity in signal transduction pathways revealed
by the crystal structure of its regulatory SH2 domains bound to a
dually phosphorylated ITAM peptide. J Mol Biol 1998; 281:523-37.
[0165] 18. Nadler M J, Matthews S A, Turner H, Kinet J P. Signal
transduction by the high-affinity immunoglobulin E receptor Fc
epsilon R1: coupling form to function. Adv Immunol 2000; 76:325-55.
[0166] 19. Parravicini V, Gadina M, Kovarova M, Odom S,
Gonzalez-Espinosa C, Furumoto Y, et al. Fyn kinase initiates
complementary signals required for IgE-dependent mast cell
degranulation. Nat Immunol 2002; 3:741-8. [0167] 20. Rameh L E,
Rhee S G, Spokes K, Kazlauskas A, Cantley L C, Cantley L G.
Phosphoinositide 3-kinase regulates phospholipase Cgamma-mediated
calcium signaling. J Biol Chem 1998; 273:23750-7. [0168] 21. Saitoh
S, Odom S, Gomez G, Sommers C L, Young H A, Rivera J, et al. The
four distal tyrosines are required for LAT-dependent signaling in
FcepsilonRI-mediated mast cell activation. J Exp Med 2003;
198:831-43. [0169] 22. Silverman M A, Shoag J, Wu J, Koretzky G A.
Disruption of SLP-76 interaction with Gads inhibits dynamic
clustering of SLP-76 and FcepsilonRI signaling in mast cells. Mol
Cell Biol 2006; 26:1826-38. [0170] 23. Hendricks-Taylor L R, Motto
D G, Zhang J, Siraganian R P, Koretzky G A. SLP-76 is a substrate
of the high affinity IgE receptor-stimulated protein tyrosine
kinases in rat basophilic leukemia cells. J Biol Chem 1997;
272:1363-7. [0171] 24. Takata M, Kurosaki T. A role for Bruton's
tyrosine kinase in B cell antigen receptor-mediated activation of
phospholipase C-gamma 2. J Exp Med 1996; 184:31-40. [0172] 25. Law
C L, Chandran K A, Sidorenko S P, Clark E A. Phospholipase C-gammal
interacts with conserved phosphotyrosyl residues in the linker
region of Syk and is a substrate for Syk. Mol Cell Biol 1996;
16:1305-15. [0173] 26. Pivniouk V I, Martin T R, Lu-Kuo J M, Katz H
R, Oettgen H C, Geha R S. SLP-76 deficiency impairs signaling via
the high-affinity IgE receptor in mast cells. J Clin Invest 1999;
103:1737-43. [0174] 27. Kettner A, Pivniouk V, Kumar L, Falet H,
Lee J S, Mulligan R, et al. Structural requirements of SLP-76 in
signaling via the high-affinity immunoglobulin E receptor (Fc
epsilon R1) in mast cells. Mol Cell Biol 2003; 23:2395-406. [0175]
28. Manetz T S, Gonzalez-Espinosa C, Arudchandran R, Xirasagar S,
Tybulewicz V, Rivera J. Vav1 regulates phospholipase cgamma
activation and calcium responses in mast cells. Mol Cell Biol 2001;
21:3763-74. [0176] 29. Wilson B S, Pfeiffer J R, Oliver J M.
FcepsilonRI signaling observed from the inside of the mast cell
membrane. Mol Immunol 2002; 38:1259-68. [0177] 30. Wu J N, Jordan M
S, Silverman M A, Peterson E J, Koretzky G A. Differential
requirement for adapter proteins Src homology 2 domain-containing
leukocyte phosphoprotein of 76 kDa and adhesion- and
degranulation-promoting adapter protein in FcepsilonRI signaling
and mast cell function. J Immunol 2004; 172:6768-74. [0178] 31.
Barker S A, Caldwell K K, Pfeiffer J R, Wilson B S.
Wortmannin-sensitive phosphorylation, translocation, and activation
of PLCgamma1, but not PLCgamma2, in antigen-stimulated RBL-2H3 mast
cells. Mol Biol Cell 1998; 9:483-96. [0179] 32. Fluckiger A C, Li
Z, Kato R M, Wahl M I, Ochs H D, Longnecker R, et al. Btk/Tec
kinases regulate sustained increases in intracellular Ca2+
following B-cell receptor activation. Embo J 1998; 17:1973-85.
[0180] 33. Scharenberg A M, El-Hillal O, Fruman D A, Beitz L O, Li
Z, Lin S, et al. Phosphatidylinositol-3,4,5-trisphosphate
(PtdIns-3,4,5-P3)/Tec kinase-dependent calcium signaling pathway: a
target for SHIP-mediated inhibitory signals. Embo J 1998;
17:1961-72. [0181] 34. Wen R, Jou S T, Chen Y, Hoffmeyer A, Wang D.
Phospholipase C gamma 2 is essential for specific functions of Fc
epsilon R and Fc gamma R. J Immunol 2002; 169:6743-52. [0182] 35.
Qi Q, August A. Keeping the (Kinase) Party Going: SLP-76 and ITK
Dance to the Beat. Sci STKE 2007; 2007: pe39. [0183] 36. Mocsai A,
Zhou M, Meng F, Tybulewicz V L, Lowell C A. Syk is required for
integrin signaling in neutrophils. Immunity 2002; 16: 547-558.
[0184] 37. Matsumoto T, Guo Y J, Ikejima T, Yamada H. Induction of
cell cycle regulatory proteins by murine B cell proliferating
pectic polysaccharide from the roots of Bupleurum falcatum L.
Immunology letters 2003; 89: 111-118. [0185] 38. Okkenhaug K,
Bilancio A, Farjot G, Priddle H, Sancho S, Peskett E, et al.
Impaired B and T cell antigen receptor signaling in p110delta PI
3-kinase mutant mice. Science 2002; 297: 1031-1034. [0186] 39. Gell
PGH, Coombs RRA, eds. Clinical Aspects of Immunology. 1st ed.
Oxford, England: Blackwell; 1963 [0187] 40. S. M. Berge et al.
Pharmaceutical Salts. J. Pharm. Sci, 1977, 66: p. 1-19
Sequence CWU 1
1
41635PRTHomo sapiens 1Met Ala Ser Ser Gly Met Ala Asp Ser Ala Asn
His Leu Pro Phe Phe1 5 10 15Phe Gly Asn Ile Thr Arg Glu Glu Ala Glu
Asp Tyr Leu Val Gln Gly 20 25 30Gly Met Ser Asp Gly Leu Tyr Leu Leu
Arg Gln Ser Arg Asn Tyr Leu 35 40 45Gly Gly Phe Ala Leu Ser Val Ala
His Gly Arg Lys Ala His His Tyr 50 55 60Thr Ile Glu Arg Glu Leu Asn
Gly Thr Tyr Ala Ile Ala Gly Gly Arg65 70 75 80Thr His Ala Ser Pro
Ala Asp Leu Cys His Tyr His Ser Gln Glu Ser 85 90 95Asp Gly Leu Val
Cys Leu Leu Lys Lys Pro Phe Asn Arg Pro Gln Gly 100 105 110Val Gln
Pro Lys Thr Gly Pro Phe Glu Asp Leu Lys Glu Asn Leu Ile 115 120
125Arg Glu Tyr Val Lys Gln Thr Trp Asn Leu Gln Gly Gln Ala Leu Glu
130 135 140Gln Ala Ile Ile Ser Gln Lys Pro Gln Leu Glu Lys Leu Ile
Ala Thr145 150 155 160Thr Ala His Glu Lys Met Pro Trp Phe His Gly
Lys Ile Ser Arg Glu 165 170 175Glu Ser Glu Gln Ile Val Leu Ile Gly
Ser Lys Thr Asn Gly Lys Phe 180 185 190Leu Ile Arg Ala Arg Asp Asn
Asn Gly Ser Tyr Ala Leu Cys Leu Leu 195 200 205His Glu Gly Lys Val
Leu His Tyr Arg Ile Asp Lys Asp Lys Thr Gly 210 215 220Lys Leu Ser
Ile Pro Glu Gly Lys Lys Phe Asp Thr Leu Trp Gln Leu225 230 235
240Val Glu His Tyr Ser Tyr Lys Ala Asp Gly Leu Leu Arg Val Leu Thr
245 250 255Val Pro Cys Gln Lys Ile Gly Thr Gln Gly Asn Val Asn Phe
Gly Gly 260 265 270Arg Pro Gln Leu Pro Gly Ser His Pro Ala Thr Trp
Ser Ala Gly Gly 275 280 285Ile Ile Ser Arg Ile Lys Ser Tyr Ser Phe
Pro Lys Pro Gly His Arg 290 295 300Lys Ser Ser Pro Ala Gln Gly Asn
Arg Gln Glu Ser Thr Val Ser Phe305 310 315 320Asn Pro Tyr Glu Pro
Glu Leu Ala Pro Trp Ala Ala Asp Lys Gly Pro 325 330 335Gln Arg Glu
Ala Leu Pro Met Asp Thr Glu Val Tyr Glu Ser Pro Tyr 340 345 350Ala
Asp Pro Glu Glu Ile Arg Pro Lys Glu Val Tyr Leu Asp Arg Lys 355 360
365Leu Leu Thr Leu Glu Asp Lys Glu Leu Gly Ser Gly Asn Phe Gly Thr
370 375 380Val Lys Lys Gly Tyr Tyr Gln Met Lys Lys Val Val Lys Thr
Val Ala385 390 395 400Val Lys Ile Leu Lys Asn Glu Ala Asn Asp Pro
Ala Leu Lys Asp Glu 405 410 415Leu Leu Ala Glu Ala Asn Val Met Gln
Gln Leu Asp Asn Pro Tyr Ile 420 425 430Val Arg Met Ile Gly Ile Cys
Glu Ala Glu Ser Trp Met Leu Val Met 435 440 445Glu Met Ala Glu Leu
Gly Pro Leu Asn Lys Tyr Leu Gln Gln Asn Arg 450 455 460His Val Lys
Asp Lys Asn Ile Ile Glu Leu Val His Gln Val Ser Met465 470 475
480Gly Met Lys Tyr Leu Glu Glu Ser Asn Phe Val His Arg Asp Leu Ala
485 490 495Ala Arg Asn Val Leu Leu Val Thr Gln His Tyr Ala Lys Ile
Ser Asp 500 505 510Phe Gly Leu Ser Lys Ala Leu Arg Ala Asp Glu Asn
Tyr Tyr Lys Ala 515 520 525Gln Thr His Gly Lys Trp Pro Val Lys Trp
Tyr Ala Pro Glu Cys Ile 530 535 540Asn Tyr Tyr Lys Phe Ser Ser Lys
Ser Asp Val Trp Ser Phe Gly Val545 550 555 560Leu Met Trp Glu Ala
Phe Ser Tyr Gly Gln Lys Pro Tyr Arg Gly Met 565 570 575Lys Gly Ser
Glu Val Thr Ala Met Leu Glu Lys Gly Glu Arg Met Gly 580 585 590Cys
Pro Ala Gly Cys Pro Arg Glu Met Tyr Asp Leu Met Asn Leu Cys 595 600
605Trp Thr Tyr Asp Val Glu Asn Arg Pro Gly Phe Ala Ala Val Glu Leu
610 615 620Arg Leu Arg Asn Tyr Tyr Tyr Asp Val Val Asn625 630
6352269PRTArtificialantibody fragment G4G11 2Met Ala Gln Val Gln
Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro1 5 10 15Ser Asp Thr Leu
Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser 20 25 30Ser Ser Asn
Trp Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu 35 40 45Glu Trp
Ile Gly Tyr Ile Tyr His Ser Gly Ser Thr Tyr Tyr Asn Pro 50 55 60Ser
Leu Lys Ser Arg Val Thr Ile Ser Val Asp Lys Ser Lys Asn Gln65 70 75
80Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr
85 90 95Tyr Cys Ala Arg Asn Val Gly Phe His Asp Trp Gly Gln Gly Thr
Leu 100 105 110Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser Gly 115 120 125Gly Ser Ala Leu Gln Ser Val Leu Thr Gln Pro
Pro Ser Ala Ser Gly 130 135 140Thr Pro Gly Gln Arg Val Thr Ile Ser
Cys Ser Gly Ser Ser Ser Asn145 150 155 160Ile Gly Ser Asn Tyr Val
Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala 165 170 175Pro Lys Leu Leu
Ile Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro 180 185 190Asp Arg
Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile 195 200
205Ser Gly Leu Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp
210 215 220Asp Asp Ser Leu Ala Ser Pro Val Phe Gly Gly Gly Thr Lys
Leu Thr225 230 235 240Val Leu Gly Ala Ala Ala His His His His His
His Gly Ala Ala Glu 245 250 255Gln Lys Leu Ile Ser Glu Glu Asp Leu
Asn Gly Ala Ala 260 2653269PRTArtificialantibody fragment G4E4 3Met
Ala Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro1 5 10
15Ser Asp Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser
20 25 30Ser Ser Asn Trp Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly
Leu 35 40 45Glu Trp Ile Gly Tyr Ile Tyr His Ser Gly Ser Thr Tyr Tyr
Asn Pro 50 55 60Ser Leu Lys Ser Arg Val Thr Ile Ser Val Asp Lys Ser
Lys Asn Gln65 70 75 80Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala
Asp Thr Ala Val Tyr 85 90 95Tyr Cys Ala Arg Asn Val Gly Phe His Asp
Trp Gly Gln Gly Thr Leu 100 105 110Val Thr Val Ser Ser Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Gly 115 120 125Gly Ser Ala Leu Gln Ser
Val Leu Thr Gln Pro Pro Ser Ala Ser Gly 130 135 140Thr Pro Gly Gln
Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn145 150 155 160Ile
Gly Ser Asn Thr Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala 165 170
175Pro Lys Leu Leu Ile Tyr Ser Asn Asn Gln Arg Pro Ser Gly Val Pro
180 185 190Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu
Ala Ile 195 200 205Ser Gly Leu Gln Ser Glu Asp Glu Ala Asp Tyr Tyr
Cys Ala Ala Trp 210 215 220Asp Asp Ser Leu Phe Gly Ala Val Phe Gly
Gly Gly Thr Lys Leu Thr225 230 235 240Val Leu Gly Ala Ala Ala His
His His His His His Gly Ala Ala Glu 245 250 255Gln Lys Leu Ile Ser
Glu Glu Asp Leu Asn Gly Ala Ala 260 2654629PRTMus musculus 4Met Ala
Gly Ser Ala Val Asp Ser Ala Asn His Leu Thr Tyr Phe Phe1 5 10 15Gly
Asn Ile Thr Arg Glu Glu Ala Glu Asp Tyr Leu Val Gln Gly Gly 20 25
30Met Thr Asp Gly Leu Tyr Leu Leu Arg Gln Ser Arg Asn Tyr Leu Gly
35 40 45Gly Phe Ala Leu Ser Val Ala His Asn Arg Lys Ala His His Tyr
Thr 50 55 60Ile Glu Arg Glu Leu Asn Gly Thr Tyr Ala Ile Ser Gly Gly
Arg Ala65 70 75 80His Ala Ser Pro Ala Asp Leu Cys His Tyr His Ser
Gln Glu Pro Asp 85 90 95Gly Leu Ile Cys Leu Leu Lys Lys Pro Phe Asn
Arg Pro Pro Gly Val 100 105 110Gln Pro Lys Thr Gly Pro Phe Glu Asp
Leu Lys Glu Asn Leu Ile Arg 115 120 125Glu Tyr Val Lys Gln Thr Trp
Asn Leu Gln Gly Gln Ala Leu Glu Gln 130 135 140Ala Ile Ile Ser Gln
Lys Pro Gln Leu Glu Lys Leu Ile Ala Thr Thr145 150 155 160Ala His
Glu Lys Met Pro Trp Phe His Gly Asn Ile Ser Arg Asp Glu 165 170
175Ser Glu Gln Thr Val Leu Ile Gly Ser Lys Thr Asn Gly Lys Phe Leu
180 185 190Ile Arg Ala Arg Asp Asn Ser Gly Ser Tyr Ala Leu Cys Leu
Leu His 195 200 205Glu Gly Lys Val Leu His Tyr Arg Ile Asp Arg Asp
Lys Thr Gly Lys 210 215 220Leu Ser Ile Pro Glu Gly Lys Lys Phe Asp
Thr Leu Trp Gln Leu Val225 230 235 240Glu His Tyr Ser Tyr Lys Pro
Asp Gly Leu Leu Arg Val Leu Thr Val 245 250 255Pro Cys Gln Lys Ile
Gly Ala Gln Met Gly His Pro Gly Ser Pro Asn 260 265 270Ala His Pro
Val Thr Trp Ser Pro Gly Gly Ile Ile Ser Arg Ile Lys 275 280 285Ser
Tyr Ser Phe Pro Lys Pro Gly His Lys Lys Pro Ala Pro Pro Gln 290 295
300Gly Ser Arg Pro Glu Ser Thr Val Ser Phe Asn Pro Tyr Glu Pro
Thr305 310 315 320Gly Gly Pro Trp Gly Pro Asp Arg Gly Leu Gln Arg
Glu Ala Leu Pro 325 330 335Met Asp Thr Glu Val Tyr Glu Ser Pro Tyr
Ala Asp Pro Glu Glu Ile 340 345 350Arg Pro Lys Glu Val Tyr Leu Asp
Arg Ser Leu Leu Thr Leu Glu Asp 355 360 365Asn Glu Leu Gly Ser Gly
Asn Phe Gly Thr Val Lys Lys Gly Tyr Tyr 370 375 380Gln Met Lys Lys
Val Val Lys Thr Val Ala Val Lys Ile Leu Lys Asn385 390 395 400Glu
Ala Asn Asp Pro Ala Leu Lys Asp Glu Leu Leu Ala Glu Ala Asn 405 410
415Val Met Gln Gln Leu Asp Asn Pro Tyr Ile Val Arg Met Ile Gly Ile
420 425 430Cys Glu Ala Glu Ser Trp Met Leu Val Met Glu Met Ala Glu
Leu Gly 435 440 445Pro Leu Asn Lys Tyr Leu Gln Gln Asn Arg His Ile
Lys Asp Lys Asn 450 455 460Ile Ile Glu Leu Val His Gln Val Ser Met
Gly Met Lys Tyr Leu Glu465 470 475 480Glu Ser Asn Phe Val His Arg
Asp Leu Ala Ala Arg Asn Val Leu Leu 485 490 495Val Thr Gln His Tyr
Ala Lys Ile Ser Asp Phe Gly Leu Ser Lys Ala 500 505 510Leu Arg Ala
Asp Glu Asn Tyr Tyr Lys Ala Gln Thr His Gly Lys Trp 515 520 525Pro
Val Lys Trp Tyr Ala Pro Glu Cys Ile Asn Tyr Tyr Lys Phe Ser 530 535
540Ser Lys Ser Asp Val Trp Ser Phe Gly Val Leu Met Trp Glu Ala
Phe545 550 555 560Ser Tyr Gly Gln Lys Pro Tyr Arg Gly Met Lys Gly
Ser Glu Val Thr 565 570 575Ala Met Leu Glu Lys Gly Glu Arg Met Gly
Cys Pro Ala Gly Cys Pro 580 585 590Arg Glu Met Tyr Asp Leu Met Asn
Leu Cys Trp Thr Tyr Asp Val Glu 595 600 605Asn Arg Pro Gly Phe Thr
Ala Val Glu Leu Arg Leu Arg Asn Tyr Tyr 610 615 620Tyr Asp Val Val
Asn625
* * * * *