U.S. patent application number 10/085027 was filed with the patent office on 2002-09-19 for remedies for diseases caused by insulin resistance.
This patent application is currently assigned to DAIICHI PHARMACEUTICAL CO., LTD.. Invention is credited to Asano, Tomoichiro, Kanda, Akira, Kubo, Hideo, Yazaki, Yoshio.
Application Number | 20020132759 10/085027 |
Document ID | / |
Family ID | 17393368 |
Filed Date | 2002-09-19 |
United States Patent
Application |
20020132759 |
Kind Code |
A1 |
Yazaki, Yoshio ; et
al. |
September 19, 2002 |
Remedies for diseases caused by insulin resistance
Abstract
The present invention relates to a remedy for diseases caused by
insulin resistance, which comprises, as an active ingredient, a
substance exhibiting activity for inhibiting the binding of the
full-length insulin receptor substrate-1 (IRS-1) or insulin
receptor substrate-2 (IRS-2) or a portion of the same to the
full-length 14-3-3 protein or a portion of the same; and to a
screening method for a remedy for diseases caused by insulin
resistance, which comprises assaying the activity for inhibiting
the binding. By means of the screening method, there can be
obtained a remedy for diseases caused by insulin resistance, such
as diabetes, diabetic microangiopathies (diabetic nephropathy,
diabetic neuropathy, and diabetic retinopathy), impaired glucose
tolerance, hyperinsulinemia, hyperlipemia, arteriosclerosis,
hypertension, obesity, ischemic heart diseases, ischemic brain
disorders, and peripheral arterial embolism.
Inventors: |
Yazaki, Yoshio; (Tokyo,
JP) ; Asano, Tomoichiro; (Tokyo, JP) ; Kubo,
Hideo; (Tokyo, JP) ; Kanda, Akira; (Tokyo,
JP) |
Correspondence
Address: |
OBLON SPIVAK MCCLELLAND MAIER & NEUSTADT PC
FOURTH FLOOR
1755 JEFFERSON DAVIS HIGHWAY
ARLINGTON
VA
22202
US
|
Assignee: |
DAIICHI PHARMACEUTICAL CO.,
LTD.
14-10, Nihonbashi 3-chome, Chuo-ku
Tokyo
JP
|
Family ID: |
17393368 |
Appl. No.: |
10/085027 |
Filed: |
March 1, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10085027 |
Mar 1, 2002 |
|
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09508691 |
Mar 29, 2000 |
|
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09508691 |
Mar 29, 2000 |
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PCT/JP98/04293 |
Sep 25, 1998 |
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Current U.S.
Class: |
514/1.9 ;
435/7.1; 514/15.7; 514/16.4; 514/17.7; 514/4.8; 514/6.7; 514/6.9;
514/7.4 |
Current CPC
Class: |
A61K 38/17 20130101 |
Class at
Publication: |
514/2 ;
435/7.1 |
International
Class: |
A61K 038/17 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 29, 1997 |
JP |
9-263719 |
Claims
1. A remedy for a disease caused by insulin resistance, which
comprises, as an active ingredient, a substance exhibiting activity
for inhibiting the binding of a full-length or a portion of insulin
receptor substrate-1 (IRS-1) or a full-length or a portion of
insulin receptor substrate-2 (IRS-2) to a full-length or a portion
of 14-3-3 protein.
2. A remedy according to claim 1, wherein the portion of IRS-1 or
IRS-2 is a serine-residue-containing oligopeptide in the amino acid
sequence of IRS-1 or IRS-2, or a phosphorylated product of the
oligopeptide.
3. A remedy according to claim 1 or 2, wherein the disease caused
by insulin resistance is diabetes, diabetic nephropathy, diabetic
neuropathy, diabetic retinopathy, impaired glucose tolerance,
hyperinsulinemia, hyperlipemia, arteriosclerosis, hypertension,
obesity, ischemic heart disease, ischemic brain disorder, or
peripheral arterial embolism.
4. A screening method for a remedy for a disease caused by insulin
resistance, which comprises assaying activity for inhibiting the
binding of a full-length IRS-1 or IRS-2 or a portion of the same to
a full-length 14-3-3 protein or a portion of the same.
5. A screening method according to claim 4, wherein the portion of
IRS-1 or IRS-2 is a serine-residue-containing oligopeptide in the
amino acid sequence of IRS-1 or IRS-2, or a phosphorylated product
of the oligopeptide.
6. A screening method for a remedy for a disease caused by insulin
resistance, which comprises assaying activity for inhibiting the
binding of a protein containing phosphotyrosine binding domain (PTB
domain) of IRS-1 or IRS-2, or a phosphorylated protein to a protein
containing a binding site of 14-3-3 protein to tryptophan
hydroxylase (the box-1 region).
7. A screening method according to any one of claims 4 to 6,
wherein the disease caused by insulin resistance is diabetes,
diabetic nephropathy, diabetic neuropathy, diabetic retinopathy,
impaired glucose tolerance, hyperinsulinemia, hyperlipemia,
arteriosclerosis, hypertension, obesity, ischemic heart disease,
ischemic brain disorder, or peripheral arterial embolism.
8. A remedy for a disease caused by insulin resistance, which
comprises, as an active ingredient, a substance selected by use of
the screening method as described in any one of claims 4 to 7.
9. A pharmaceutical composition for a disease caused by insulin
resistance, which comprises a substance exhibiting activity for
inhibiting the binding of a full-length IRS-1 or IRS-2 or a portion
of the same to a full-length 14-3-3 protein or a portion of the
same, and a pharmaceutically acceptable carrier.
10. A pharmaceutical composition according to claim 9, wherein the
portion of IRS-1 or IRS-2 is a serine-residue-containing
oligopeptide in the amino acid sequence of IRS-1 or IRS-2, or a
phosphorylated product of the oligopeptide.
11. A pharmaceutical composition according to claim 9 or 10,
wherein the disease caused by insulin resistance is diabetes,
diabetic nephropathy, diabetic neuropathy, diabetic retinopathy,
impaired glucose tolerance, hyperinsulinemia, hyperlipemia,
arteriosclerosis, hypertension, obesity, ischemic heart disease,
ischemic brain disorder, or peripheral arterial embolism.
12. Use of a substance exhibiting activity for inhibiting the
binding of a full-length IRS-1 or IRS-2 or a portion of the same to
a full-length 14-3-3 protein or a portion of the same for producing
a remedy for a disease caused by insulin resistance.
13. Use according to claim 12, wherein the portion of IRS-1 or
IRS-2 is a serine-residue-containing oligopeptide in the amino acid
sequence of IRS-1 or IRS-2, or a phosphorylated product of the
oligopeptide.
14. Use according to claim 12 or 13, wherein the disease caused by
insulin resistance is diabetes, diabetic nephropathy, diabetic
neuropathy, diabetic retinopathy, impaired glucose tolerance,
hyperinsulinemia, hyperlipemia, arteriosclerosis, hypertension,
obesity, ischemic heart disease, ischemic brain disorder, or
peripheral arterial embolism.
15. A treatment method for a disease caused by insulin resistance,
which comprises administering to a patient in need thereof an
effective amount of a substance exhibiting activity for inhibiting
the binding of a full-length IRS-1 or IRS-2 or a portion of the
same to a full-length 14-3-3 protein or a portion of the same.
16. A treatment method according to claim 15, wherein the portion
of IRS-1 or IRS-2 is a serine-residue-containing oligopeptide in
the amino acid sequence of IRS-1 or IRS-2, or a phosphorylated
product of the oligopeptide.
17. A treatment method according to claim 15 or 16, wherein the
disease caused by insulin resistance is diabetes, diabetic
nephropathy, diabetic neuropathy, diabetic retinopathy, impaired
glucose tolerance, hyperinsulinemia, hyperlipemia,
arteriosclerosis, hypertension, obesity, ischemic heart disease,
ischemic brain disorder, or peripheral arterial embolism.
18. A pharmaceutical composition for a disease caused by insulin
resistance, which comprises a substance selected by use of the
screening method as described in any one of claims 4 to 7 and a
pharmaceutically acceptable carrier.
19. Use of a substance selected by use of the screening method as
described in any one of claims 4 to 7 for producing a remedy for a
disease caused by insulin resistance.
20. A treatment method for a disease caused by insulin resistance,
which comprises administering to a patient in need thereof an
effective amount of a substance selected by use of the screening
method as described in any one of claims 4 to 7.
Description
TECHNICAL FIELD
[0001] The present invention relates to a drug, particularly a
remedy for diseases caused by insulin resistance, such as diabetes,
as well as to a screening method for the remedy.
BACKGROUND ART
[0002] Insulin is a hormone which regulates the concentration of
blood sugar and blood lipid through the promotion of glucose and
lipid intake into cells and utilization and storage of them.
Insulin resistance indicates the condition in which insulin does
not act normally on cells, and this condition causes elevation of
the concentration of blood sugar or blood lipid. Examples of
diseases caused by insulin resistance include diabetes, diabetic
microangiopathies (diabetic nephropathy, diabetic neuropathy, and
diabetic retinopathy), impaired glucose tolerance,
hyperinsulinemia, hyperlipemia, arteriosclerosis, hypertension,
obesity, ischemic heart diseases, ischemic brain disorders, and
peripheral arterial embolism (Tamio Teramoto, et al., (1995)
Biomedicine & Therapeutics 29, 8-96). The cause of insulin
resistance has not yet been fully elucidated, and causal therapy
thereof has not been developed.
[0003] Recently, abnormality of intracellular signal transduction
induced by insulin has become of interest as a cause of insulin
resistance. In signal transduction of insulin, the first response
induced by insulin is activation of insulin receptor tyrosine
kinase. Subsequently, several intracellular substrates including
insulin receptor substrate-1 (IRS-1) (Sun, X. et al., (1991) Nature
352, 73-77) and insulin receptor substrate-2 (IRS-2) (Sun, X. et
al., (1995) Nature 377, 173-177) are phosphorylated. IRS-1 and
IRS-2 have potential tyrosine-phosphorylated sites in amounts of 21
and 23, respectively, and they function as "docking protein" which
transmits insulin signals to several proteins having Src-homology 2
domains (SH2-protein) (Sun, X. et al., (1993) Mol. Cell. Biol. 13,
7418-7428).
[0004] However, the function of IRS-1 and IRS-2 relating to insulin
signal transduction in the aforementioned action is not necessarily
fully elucidated, and elucidation of novel function thereof and
development of drugs on the basis of the function are demanded.
[0005] An object of the present invention is to elucidate novel
function of IRS-1 and IRS-2, and to provide a drug based on the
function.
DISCLOSURE OF THE INVENTION
[0006] In view of the foregoing, the present inventors have focused
on the relation between IRS-1 or IRS-2 and 14-3-3 protein.
[0007] 14-3-3 Protein is widely distributed in eucaryotes such as
animals, plants, and yeast, and is a protein family which is
supposed to act as a regulatory factor by binding to a particular
target protein in a variety of signal transductions depending on
phosphorylation and dephosphorylation of proteins (Fumiko Shinkai,
et al., (1996) Protein Nucleic Acid Enzyme 41, 313-326). Recently,
it has been reported that 14-3-3 protein binds to
phosphatidylinositol 3-kinase (PI3K) and inhibits its activity in T
lymphocytes (Bonnefoy-Berard, N. et al., (1995) Proc. Natl. Acad.
Sci. 92, 10142-10146). PI3K plays an important role in signal
transduction of insulin (Masato Kasuga, (1996) Saishin-Igaku 51,
1564-1572), and thus 14-3-3 protein has been supposed to effect
some type of regulation against signal transduction of insulin
(Humiko Shinkai, et al., (1996) Protein Nucleic Acid Enzyme 41,
313-326). In addition, very recently, it has been reported that the
.epsilon. isoform of 14-3-3 protein binds to IRS-1, but the
physiological significance has not been elucidated (Craparo, A.
(1997) J. Biol. Chem. 272. 11663-11669).
[0008] The present inventors have performed extensive studies on
the relation between 14-3-3 protein and IRS-1 or IRS-2; have
elucidated that IRS-1 or IRS-2 binds to 14-3-3 protein at a
particular site and that the binding effects negative regulation
against insulin signal transduction; and have found that a
substance inhibiting the binding is useful for a remedy for
diseases caused by insulin resistance. The present invention has
been accomplished on the basis of these findings.
[0009] Accordingly, the present invention provides a remedy for
diseases caused by insulin resistance, which comprises, as an
active ingredient, a substance exhibiting activity for inhibiting
the binding of the full-length IRS-1 or IRS-2 or a portion of the
same to the full-length 14-3-3 protein or a portion of the
same.
[0010] The present invention also provides a screening method for a
remedy for diseases caused by insulin resistance, which comprises
assaying activity for inhibiting the binding of the full-length
IRS-1 or IRS-2 or a portion of the same to the full-length 14-3-3
protein or a portion of the same.
[0011] The present invention also provides a pharmaceutical
composition for diseases caused by insulin resistance, which
comprises a substance exhibiting activity for inhibiting the
binding of the full-length IRS-1 or IRS-2 or a portion of the same
to the full-length 14-3-3 protein or a portion of the same, and a
pharmaceutically acceptable carrier.
[0012] The present invention also provides use of a substance
exhibiting activity for inhibiting the binding of the full-length
IRS-1 or IRS-2 or a portion of the same to the full-length 14-3-3
protein or a portion of the same for producing a remedy for
diseases caused by insulin resistance.
[0013] The present invention also provides a method for treating
diseases caused by insulin resistance, which comprises
administering to a patient in need thereof an effective dose of a
substance exhibiting activity for inhibiting the binding of the
full-length IRS-1 or IRS-2 or a portion of the same to the
full-length 14-3-3 protein or a portion of the same.
BEST MODE FOR CARRYING OUT THE INVENTION
[0014] Active ingredients of the remedy of the present invention
include a substance exhibiting activity for inhibiting the binding
of the full-length IRS-1 or IRS-2 or a portion of the same to the
full-length 14-3-3 protein or a portion of the same in screening
for assaying the inhibiting activity.
[0015] As described below, the present inventors were the first to
elucidate that the binding of 14-3-3 protein to IRS-1 or IRS-2
effects negative regulation against insulin signal
transduction.
[0016] Firstly, in order to identify a unique protein that binds to
IRS-1, the present inventors used .sup.32p -labeled recombinant
IRS-1 as a probe in order to screen a cDNA library derived from
human heart, to thereby obtain two isoforms (.epsilon. and .zeta.)
which belong to a 14-3-3 protein family. In addition, they found
that 14-3-3 protein associates with IRS-1 in L6 muscular cells,
HepG2 hepatoma cells, and Chinese hamster ovary cells, in which
IRS-1 is overexpressed by means of an adenovirus expression system,
as well as in the brain tissue of cow in a natural state.
[0017] The present inventors also elucidated that 14-3-3 protein
associates with IRS-1 or IRS-2 in SF9 cells in which 14-3-3 protein
and IRS-1 or IRS-2 are overexpressed by means of a baculovirus
expression system.
[0018] The present inventors also elucidated, by use of HepG2
hepatoma cells in which IRS-1 is overexpressed in the same manner
as described above, that the amount of 14-3-3 protein binding to
IRS-1 is not changed by insulin stimulation, and that the amount is
significantly increased by okadaic acid, which is an inhibitor of
serine/threonine phosphatase.
[0019] The present inventors also elucidated that IRS-1 has three
putative binding sites (Ser-270, Ser-374, and Ser-641) for 14-3-3
protein, on the basis of the finding that, in a cell lysate of L6
muscular cells, the binding of IRS-1 to 14-3-3 protein fused with
glutathione S-transferase (GST) is inhibited by three types of
15-residue oligopeptide shown in sequence Nos. 2-4 which contains a
serine residue and several amino acid residues in the vicinity of
it corresponding to the amino acid sequence of IRS-1, and the
serine residue is phosphorylated. Of the above three binding sites,
the motif around of Ser-270 are located in the phosphotyrosine
binding domain (PTB domain) of IRS-1, and the domain is known to
play an important role in interaction with insulin receptors (Wolf,
G. (1995) J. Biol. Chem. 270, 27407-27410). The present inventors
elucidated that, in practice, truncated IRS-1 containing the PTB
domain and 205 amino acids adjacent to its C-terminal side
associates with GST-fused 14-3-3 protein, by overexpressing the
IRS-1 in HepG2 hepatoma cells by means of an adenovirus expression
system.
[0020] In addition, the present inventors found that IRS-1 that has
been coprecipitated with an antibody against 14-3-3 protein is
insusceptible to phosphorylation of serine and tyrosine residues by
insulin stimulation as compared with IRS-1 that has been
coprecipitated with an antibody against IRS-1, by analyzing the
effect of the binding of 14-3-3 protein to IRS-1 on phosphorylation
of IRS-1 induced by insulin, and analyzing phosphorylated amino
acids in a HepG2 hepatoma cell in which IRS-1 is overexpressed by
means of an adenovirus expression system. As described above, it
was elucidated that 14-3-3 protein effects negative regulation
against insulin signal transduction by inhibiting the association
of insulin receptors with IRS-1.
[0021] Therefore, abnormal promotion of the binding of 14-3-3
protein to IRS-1 or IRS-2 is a primary cause for insulin
resistance, and thus insulin resistance may be suppressed and
diseases caused by insulin resistance may be treated by inhibiting,
suppressing, and dissociating the binding. In order to inhibit the
binding, direct inhibition may be effected against the binding of
14-3-3 protein to IRS-1 or IRS-2. Alternatively, indirect
inhibition may be effected; for example, phosphorylation of a
particular serine residue in the amino acid sequence of IRS-1 or
IRS-2, which phosphorylation is considered to play an important
role in the binding of 14-3-3 protein to IRS-1 or IRS-2, may be
inhibited, or dephosphorylation may be promoted.
[0022] The full-length IRS-1 or IRS-2 or a portion of the same used
in the present invention may be obtained, for example, by means of
the following procedure: CDNA coding for the full-length IRS-1 or
IRS-2 or a portion of the same is introduced into baculovirus by
means of known methods, and the full-length IRS-1 or IRS-2 or a
portion of the same is isolated from the insect cells infected with
the virus and purified by means of known methods. The amino acid
sequence of IRS-1 is shown in sequence No. 1. When a portion of
IRS-1 or IRS-2 is oligopeptide, the portion may be synthesized by
means of known peptide synthesis methods. A portion of IRS-1 or
IRS-2 may be peptides containing a serine residue in the amino acid
sequence of IRS-1 or IRS-2, or phosphorylated products of the
peptides. Preferably, a portion of IRS-1 or IRS-2 may be peptide
containing the PTB domain (amino acid 161-517 in sequence No. 1 in
the case of IRS-1, the amino acid sequence in sequence No. 5 in the
case of IRS-2 (corresponding to amino acid 196-354 of IRS-2)), more
preferably oligopeptides containing Ser-270, Ser-374, or Ser-641 of
IRS-1, or phosphorylated products of the peptides. The length of
the portion is not limited so long as activity for inhibiting the
binding can be assayed with high sensitivity, and the portion may
be 5-50 amino acids, preferably 10-30 amino acids, more preferably
15 amino acids containing serine which is phosphorylated.
[0023] The full-length 14-3-3 protein or a portion of the same used
in the present invention may be obtained, for example, by means of
the following procedure: cDNA coding for the full-length 14-3-3
protein or a portion of the same is introduced into baculovirus by
means of known methods, and the full-length 14-3-3 protein or a
portion of the same is isolated from the insect cell infected with
the virus and purified by means of known methods. A portion of
14-3-3 protein may be the box-1 region which is the binding site to
tryptophan hydroxylase (Ichimura, T. et al., (1997) FEBS Lett. 413,
273-276), or a peptide containing the region.
[0024] In order to obtain the full-length IRS-1 or IRS-2 or a
portion of the same, and the full-length 14-3-3 protein or a
portion of the same, they may be advantageously expressed as a
fusion protein in a variety of gene expression systems. Fusion
protein expression systems such as those including lactose and
glutathione S-transferase may also be used.
[0025] In order to prepare the aforementioned screening system, the
full-length labeled IRS-1 or labeled IRS-2 or a portion of the
same, or the full-length labeled 14-3-3 protein or a portion of the
same is preferably used. 125I or an enzyme which is often used in
enzyme immunoassay, such as alkaline phosphatase, is appropriately
used for labeling. Such a substance for labeling is bonded to the
protein by means of known methods. When the labeled protein is not
used, a primary antibody specific to the unlabeled protein and a
secondary antibody which is labeled and recognizes the primary
antibody are necessary. The primary and secondary antibodies may be
commercially available ones.
[0026] Next will be described a preferred embodiment for effecting
screening for a substance exhibiting activity for inhibiting the
binding of 14-3-3 protein to IRS-1 or IRS-2 by means of the
above-described system. Firstly, IRS-1 or IRS-2 (the full length or
a portion thereof), or 14-3-3 protein (the full length or a portion
thereof) is prepared by immobilization thereof onto a plastic
material (a microplate or beads) by means of a known method.
Subsequently, the other protein to be bonded which is labeled is
dissolved in an appropriate buffer, and the resultant solution is
added to each well of a microplate (when a microplate is used) or
to test tubes containing the beads (when beads are used). A test
compound is also added thereto. Independently, a solution
containing a very large amount of unlabeled protein is prepared in
order to determine the amount of non-specific binding (NSB). The
solution containing the labeled protein is incubated under
appropriate conditions, and the material (each well of the
microplate, or the beads) is washed with the buffer. The amount of
the labels attached to the protein binding to the well or beads is
measured by means of known methods. When the labeled protein is not
used, the solution containing the unlabeled protein is incubated in
the same manner, an antibody specific to the unlabeled protein (the
primary antibody) is added, and the solution is incubated under
appropriate conditions. Furthermore, the secondary antibody which
is labeled and recognizes the primary antibody is added, and the
solution is incubated under appropriate conditions. Thereafter, the
substrate is washed with the buffer, and the amount of labels
attached to the protein binding to the substrate is measured in the
same manner. When the value of "Bo-NSB"--hich is obtained by
subtracting NSB from the amount of labels attached to the protein
to be bonded in the absence of a binding-inhibitory substance
(Bo)--is regarded as 100%, a test compound providing the value
(amount of specific binding) of 10% or less may be chosen as a
substance exhibiting activity for inhibiting the binding.
[0027] A substance source which is considered to exhibit the
binding inhibitory activity may be tested by means of the
above-described screening system. Examples of such a substance
source include synthetic peptides, low-molecular organic compounds,
and natural products, preferably substances having applicability as
drugs. Specific examples include combinatorial libraries of
different chemical substances and synthetic peptide libraries.
[0028] A substance exhibiting the binding inhibitory activity which
is obtained by means of the above-described screening suppresses
negative regulation against insulin signal transduction in cells.
Therefore, the substance is useful for producing a remedy for
diseases caused by insulin resistance. Examples of such diseases
include diabetes, diabetic microangiopathies (diabetic nephropathy,
diabetic neuropathy, and diabetic retinopathy), impaired glucose
tolerance, hyperinsulinemia, hyperlipemia, arteriosclerosis,
hypertension, obesity, ischemic heart diseases, ischemic brain
disorders, and peripheral arterial embolism.
[0029] A dosage of the remedy of the present invention depends on
the age, sex, and pathological condition of a patient, and is 5
mg-2 g per adult per day, preferably 50-100 mg as reduced to an
active ingredient. The aforementioned dosage per day may be
administered in a single portion once a day, or in divided portions
2-3 times a day. If necessary, a dosage per day may exceed the
aforementioned dosage.
[0030] No particular limitation is imposed on the administration
method and the dosage form of the remedy of the present invention,
and any dosage form suitable for an administration method may be
obtained by means of a conventionally used technique for preparing
products.
[0031] Examples of products for oral administration include
tablets, powders, granules, capsules, solutions, syrups, elixirs,
and oily or aqueous suspensions.
[0032] For preparation of injections, a solution may be stored in a
container and freeze-dried, to thereby provide a solid product, and
the solid product may be prepared into an injection just before
use. If necessary, the product may contain a stabilizer, a
preservative, and a solubilizer. A single dosage of the injection
product may be stored in a container, or a plurality of dosages may
be stored in the same container.
[0033] Examples of external-use products include solutions,
suspensions, emulsions, ointments, gels, creams, lotions, and
sprays.
[0034] Solid products may contain pharmaceutically acceptable
additives together with an active ingredient. If necessary, the
remedy may optionally contain fillers, expanders, binders,
disintegrants, dissolution-promoting agents, humectants, and
lubricants, to thereby prepare products.
[0035] Examples of liquid products include solutions, suspensions,
and emulsions, and the products may contain additives such as
suspending agents and emulsifying agents.
EXAMPLES
[0036] The present invention will next be described in more detail
by way of examples, which should not be construed as limiting the
invention thereto.
Example 1
[0037] (Method for screening inhibitors for the binding of 14-3-3
protein to IRS-1 or IRS-2 characterized by employing the
full-length IRS-1 or IRS-2 or a portion of the same and the
full-length 14-3-3 protein or a portion of the same)
[0038] A solution containing a portion of human IRS-1, i.e., a
portion including the PTB domain (amino acids 161-517) (1 .mu.g/ml,
pH 8.0, 50 mM K.sub.2PO.sub.4) (100 .mu.l) is added to each well of
a 96-well microplate, and the plate is allowed to stand at room
temperature for one hour, to thereby cause the human IRS-1 to be
fixed onto the walls of the wells. The solution in each well is
removed and the well is washed three times with a buffer (50 mM
HEPES, 150 mM NaCl, 0.1% Triton X-100) (300 .mu.l). Subsequently, a
buffer solution containing 0.5% bovine serum albumin (BSA) (300
.mu.l) is added to each well and the plate is allowed to stand at
room temperature for one hour, to thereby effect blocking. The
solution is removed, and each well is washed three times with a
buffer (300 .mu.l). Next, a buffer solution containing full-length
human 14-3-3 protein (1 .mu.g/ml) (50 .mu.l) and a buffer
containing a target sample for screening (50 .mu.l) are
simultaneously added to the well and allowed to stand at room
temperature for two hours. Independently, a buffer (50 .mu.l) not
containing the sample is added to another well which has been
treated in the same manner as described above, and allowed to
stand, in order to measure the amount of maximum binding (Bo). In
order to measure the amount of non-specific binding (NSB), a well
to which a portion including the PTB region has not been fixed is
subjected to the above-described treatment after blocking, and a
buffer solution containing human 14-3-3 protein (50 .mu.l) and a
buffer (50 .mu.l) are added to the well, and allowed to stand.
Solutions in the above wells are removed, and the wells are washed
three times with a buffer (300 .mu.l). A buffer solution containing
an anti-human 14-3-3 rabbit polyclonal antibody (Santa Cruz
Biotechnology) (0.2 .mu.g/ml) (100 .mu.l) is added to each of the
above wells, and the plate is allowed to stand at room temperature
for one hour. The solution is removed and the well is washed three
times with a buffer (300 .mu.l). Subsequently, a buffer solution
containing an alkaline-phosphatase-labeled anti-rabbit IgG goat
polyclonal antibody (Linco) (1 .mu.g/ml) (100 .mu.l) is added to
each of the wells, and allowed to stand at room temperature for one
hour. The solution is removed and the well is washed three times
with a buffer (300 .mu.l). After a p-nitrophenyl phosphate solution
(1 mg/ml, 1 M diethanolamine) (100 .mu.l) is added to each of the
wells, the well is allowed to stand at 37.degree. C. for 30
minutes, and a 5% EDTA aqueous solution (100 .mu.l) is added, to
thereby terminate the reaction. The absorbance of the resultant
product in each well is measured at a wavelength of 405 nm, and the
absorbance is regarded as the amount of binding. The binding amount
when a sample is added is represented by B. A percentage of binding
inhibition by the sample is obtained by the following formula. When
the percentage is 10% or less, the sample is selected as a
candidate for a substance used in the present invention.
[0039] Binding inhibitory percentage
(%)=(1-(B-NSB)/(Bo-NSB)).times.100
Example 2
[0040] Three types of synthetic peptide shown in sequence Nos. 2-4
were obtained by use of reagents for peptide synthesis (for
example, a peptide block, peptides and amino acids with protective
groups, and phosphorylated serine; products of PerkinElmer) by
means of a peptide synthesizer (Model: PerkinElmer 433A, product of
PerkinElmer). Activity of these peptides ware assayed by means of
the binding inhibition screening system described in Example 1.
These synthetic peptides exhibit the binding inhibitory
activity.
[0041] Industrial Applicability
[0042] By means of the screening method of the present invention,
there can be obtained a remedy for diseases caused by insulin
resistance, such as diabetes, diabetic microangiopathies (diabetic
nephropathy, diabetic neuropathy, and diabetic retinopathy),
impaired glucose tolerance, hyperinsulinemia, hyperlipemia,
arteriosclerosis, hypertension, obesity, ischemic heart diseases,
ischemic brain disorders, and peripheral arterial embolism.
Sequence CWU 1
1
5 1 1242 PRT Homo sapiens 1 Met Ala Ser Pro Pro Glu Ser Asp Gly Phe
Ser Asp Val Arg Lys Val 1 5 10 15 Gly Tyr Leu Arg Lys Pro Lys Ser
Met His Lys Arg Phe Phe Val Leu 20 25 30 Arg Ala Ala Ser Glu Ala
Gly Gly Pro Ala Arg Leu Glu Tyr Tyr Glu 35 40 45 Asn Glu Lys Lys
Trp Arg His Lys Ser Ser Ala Pro Lys Arg Ser Ile 50 55 60 Pro Leu
Glu Ser Cys Phe Asn Ile Asn Lys Arg Ala Asp Ser Lys Asn 65 70 75 80
Lys His Leu Val Ala Leu Tyr Thr Arg Asp Glu His Phe Ala Ile Ala 85
90 95 Ala Asp Ser Glu Ala Glu Gln Asp Ser Trp Tyr Gln Ala Leu Leu
Gln 100 105 110 Leu His Asn Arg Ala Lys Gly His His Asp Gly Ala Ala
Ala Leu Gly 115 120 125 Ala Gly Gly Gly Gly Gly Ser Cys Ser Gly Ser
Ser Gly Leu Gly Glu 130 135 140 Ala Gly Glu Asp Leu Ser Tyr Gly Asp
Val Pro Pro Gly Pro Ala Phe 145 150 155 160 Lys Glu Val Trp Gln Val
Ile Leu Lys Pro Lys Gly Leu Gly Gln Thr 165 170 175 Lys Asn Leu Ile
Gly Ile Tyr Arg Leu Cys Leu Thr Ser Lys Thr Ile 180 185 190 Ser Phe
Val Lys Leu Asn Ser Glu Ala Ala Ala Val Val Leu Gln Leu 195 200 205
Met Asn Ile Arg Arg Cys Gly His Ser Glu Asn Phe Phe Phe Ile Glu 210
215 220 Val Gly Arg Ser Ala Val Thr Gly Pro Gly Glu Phe Trp Met Gln
Val 225 230 235 240 Asp Asp Ser Val Val Ala Gln Asn Met His Glu Thr
Ile Leu Glu Ala 245 250 255 Met Arg Ala Met Ser Asp Glu Phe Arg Pro
Arg Ser Lys Ser Gln Ser 260 265 270 Ser Ser Asn Cys Ser Asn Pro Ile
Ser Val Pro Leu Arg Arg His His 275 280 285 Leu Asn Asn Pro Pro Pro
Ser Gln Val Gly Leu Thr Arg Arg Ser Arg 290 295 300 Thr Glu Ser Ile
Thr Ala Thr Ser Pro Ala Ser Met Val Gly Gly Lys 305 310 315 320 Pro
Gly Ser Phe Arg Val Arg Ala Ser Ser Asp Gly Glu Gly Thr Met 325 330
335 Ser Arg Pro Ala Ser Val Asp Gly Ser Pro Val Ser Pro Ser Thr Asn
340 345 350 Arg Thr His Ala His Arg His Arg Gly Ser Ala Arg Leu His
Pro Pro 355 360 365 Leu Asn His Ser Arg Ser Ile Pro Met Pro Ala Ser
Arg Cys Ser Pro 370 375 380 Ser Ala Thr Ser Pro Val Ser Leu Ser Ser
Ser Ser Thr Ser Gly His 385 390 395 400 Gly Ser Thr Ser Asp Cys Leu
Phe Pro Arg Arg Ser Ser Ala Ser Val 405 410 415 Ser Gly Ser Pro Ser
Asp Gly Gly Phe Ile Ser Ser Asp Glu Tyr Gly 420 425 430 Ser Ser Pro
Cys Asp Phe Arg Ser Ser Phe Arg Ser Val Thr Pro Asp 435 440 445 Ser
Leu Gly His Thr Pro Pro Ala Arg Gly Glu Glu Glu Leu Ser Asn 450 455
460 Tyr Ile Cys Met Gly Gly Lys Gly Pro Ser Thr Leu Thr Ala Pro Asn
465 470 475 480 Gly His Tyr Ile Leu Ser Arg Gly Gly Asn Gly His Arg
Cys Thr Pro 485 490 495 Gly Thr Gly Leu Gly Thr Ser Pro Ala Leu Ala
Gly Asp Glu Ala Ala 500 505 510 Ser Ala Ala Asp Leu Asp Asn Arg Phe
Arg Lys Arg Thr His Ser Ala 515 520 525 Gly Thr Ser Pro Thr Ile Thr
His Gln Lys Thr Pro Ser Gln Ser Ser 530 535 540 Val Ala Ser Ile Glu
Glu Tyr Thr Glu Met Met Pro Ala Tyr Pro Pro 545 550 555 560 Gly Gly
Gly Ser Gly Gly Arg Leu Pro Gly His Arg His Ser Ala Phe 565 570 575
Val Pro Thr Arg Ser Tyr Pro Glu Glu Gly Leu Glu Met His Pro Leu 580
585 590 Glu Arg Arg Gly Gly His His Arg Pro Asp Ser Ser Thr Leu His
Thr 595 600 605 Asp Asp Gly Tyr Met Pro Met Ser Pro Gly Val Ala Pro
Val Pro Ser 610 615 620 Gly Arg Lys Gly Ser Gly Asp Tyr Met Pro Met
Ser Pro Lys Ser Val 625 630 635 640 Ser Ala Pro Gln Gln Ile Ile Asn
Pro Ile Arg Arg His Pro Gln Arg 645 650 655 Val Asp Pro Asn Gly Tyr
Met Met Met Ser Pro Ser Gly Gly Cys Ser 660 665 670 Pro Asp Ile Gly
Gly Gly Pro Ser Ser Ser Ser Ser Ser Ser Asn Ala 675 680 685 Val Pro
Ser Gly Thr Ser Tyr Gly Lys Leu Trp Thr Asn Gly Val Gly 690 695 700
Gly His His Ser His Val Leu Pro His Pro Lys Pro Pro Val Glu Ser 705
710 715 720 Ser Gly Gly Lys Leu Leu Pro Cys Thr Gly Asp Tyr Met Asn
Met Ser 725 730 735 Pro Val Gly Asp Ser Asn Thr Ser Ser Pro Ser Asp
Cys Tyr Tyr Gly 740 745 750 Pro Glu Asp Pro Gln His Lys Pro Val Leu
Ser Tyr Tyr Ser Leu Pro 755 760 765 Arg Ser Phe Lys His Thr Gln Arg
Pro Gly Glu Pro Glu Glu Gly Ala 770 775 780 Arg His Gln His Leu Arg
Leu Ser Thr Ser Ser Gly Arg Leu Leu Tyr 785 790 795 800 Ala Ala Thr
Ala Asp Asp Ser Ser Ser Ser Thr Ser Ser Asp Ser Leu 805 810 815 Gly
Gly Gly Tyr Cys Gly Ala Arg Leu Glu Pro Ser Leu Pro His Pro 820 825
830 His His Gln Val Leu Gln Pro His Leu Pro Arg Lys Val Asp Thr Ala
835 840 845 Ala Gln Thr Asn Ser Arg Leu Ala Arg Pro Thr Arg Leu Ser
Leu Gly 850 855 860 Asp Pro Lys Ala Ser Thr Leu Pro Arg Ala Arg Glu
Gln Gln Gln Gln 865 870 875 880 Gln Gln Pro Leu Leu His Pro Pro Glu
Pro Lys Ser Pro Gly Glu Tyr 885 890 895 Val Asn Ile Glu Phe Gly Ser
Asp Gln Ser Gly Tyr Leu Ser Gly Pro 900 905 910 Val Ala Phe His Ser
Ser Pro Ser Val Arg Cys Pro Ser Gln Leu Gln 915 920 925 Pro Ala Pro
Arg Glu Glu Glu Thr Gly Thr Glu Glu Tyr Met Lys Met 930 935 940 Asp
Leu Gly Pro Gly Arg Arg Ala Ala Trp Gln Glu Ser Thr Gly Val 945 950
955 960 Glu Met Gly Arg Leu Gly Pro Ala Pro Pro Gly Ala Ala Ser Ile
Cys 965 970 975 Arg Pro Thr Arg Ala Val Pro Ser Ser Arg Gly Asp Tyr
Met Thr Met 980 985 990 Gln Met Ser Cys Pro Arg Gln Ser Tyr Val Asp
Thr Ser Pro Ala Ala 995 1000 1005 Pro Val Ser Tyr Ala Asp Met Arg
Thr Gly Ile Ala Ala Glu Glu 1010 1015 1020 Val Ser Leu Pro Arg Ala
Thr Met Ala Ala Ala Ser Ser Ser Ser 1025 1030 1035 Ala Ala Ser Ala
Ser Pro Thr Gly Pro Gln Gly Ala Ala Glu Leu 1040 1045 1050 Ala Ala
His Ser Ser Leu Leu Gly Gly Pro Gln Gly Pro Gly Gly 1055 1060 1065
Met Ser Ala Phe Thr Arg Val Asn Leu Ser Pro Asn Arg Asn Gln 1070
1075 1080 Ser Ala Lys Val Ile Arg Ala Asp Pro Gln Gly Cys Arg Arg
Arg 1085 1090 1095 His Ser Ser Glu Thr Phe Ser Ser Thr Pro Ser Ala
Thr Arg Val 1100 1105 1110 Gly Asn Thr Val Pro Phe Gly Ala Gly Ala
Ala Val Gly Gly Gly 1115 1120 1125 Gly Gly Ser Ser Ser Ser Ser Glu
Asp Val Lys Arg His Ser Ser 1130 1135 1140 Ala Ser Phe Glu Asn Val
Trp Leu Arg Pro Gly Glu Leu Gly Gly 1145 1150 1155 Ala Pro Lys Glu
Pro Ala Lys Leu Cys Gly Ala Ala Gly Gly Leu 1160 1165 1170 Glu Asn
Gly Leu Asn Tyr Ile Asp Leu Asp Leu Val Lys Asp Phe 1175 1180 1185
Lys Gln Cys Pro Gln Glu Cys Thr Pro Glu Pro Gln Pro Pro Pro 1190
1195 1200 Pro Pro Pro Pro His Gln Pro Leu Gly Ser Gly Glu Ser Ser
Ser 1205 1210 1215 Thr Arg Arg Ser Ser Glu Asp Leu Ser Ala Tyr Ala
Ser Ile Ser 1220 1225 1230 Phe Gln Lys Gln Pro Glu Asp Arg Gln 1235
1240 2 15 PRT Artificial Sequence misc_feature Description of
Artificial Sequencesynthetic peptide 2 Asp Glu Phe Arg Pro Arg Ser
Lys Ser Gln Ser Ser Ser Asn Cys 1 5 10 15 3 15 PRT Artificial
Sequence misc_feature Description of Artificial Sequencesynthetic
peptide 3 His Pro Pro Leu Asn His Ser Arg Ser Ile Pro Met Pro Ala
Ser 1 5 10 15 4 15 PRT Artificial Sequence misc_feature Description
of Artificial Sequencesynthetic peptide 4 Met Pro Met Ser Pro Lys
Ser Val Ser Ala Pro Gln Gln Ile Ile 1 5 10 15 5 159 PRT Homo
sapiens 5 Glu Val Trp Gln Val Asn Leu Lys Pro Lys Gly Leu Gly Gln
Ser Lys 1 5 10 15 Asn Leu Thr Gly Val Tyr Arg Leu Cys Leu Ser Ala
Arg Thr Ile Gly 20 25 30 Phe Val Lys Leu Asn Cys Glu Gln Pro Ser
Val Thr Leu Gln Leu Met 35 40 45 Asn Ile Arg Arg Cys Gly His Ser
Asp Ser Phe Phe Phe Ile Glu Val 50 55 60 Gly Arg Ser Ala Val Thr
Gly Pro Gly Glu Leu Trp Met Gln Ala Asp 65 70 75 80 Asp Ser Val Val
Ala Gln Asn Ile His Glu Thr Ile Leu Glu Ala Met 85 90 95 Lys Ala
Leu Lys Glu Leu Phe Glu Phe Arg Pro Arg Ser Lys Ser Gln 100 105 110
Ser Ser Gly Ser Ser Ala Thr His Pro Ile Ser Val Pro Gly Ala Arg 115
120 125 Arg His His His Leu Val Asn Leu Pro Pro Ser Gln Thr Gly Leu
Val 130 135 140 Arg Arg Ser Arg Thr Asp Ser Leu Ala Ala Thr Pro Pro
Ala Ala 145 150 155
* * * * *