JP2001333778A - Glucokinase activating substance, therapeutic agent for diabetes and antiobese agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a glucokinase activating substance, especially capable of activating the glucokinase by binding to the glucokinase, and further a therapeutic agent and an antiadiposis agent characterized by activating the glucokinase. SOLUTION: This method for activating the glucokinase is characterized by making the glucokinase activating substance act on the glucokinase. The glucokinase activating substance is capable of acting on the glucokinase and activating the glucokinase.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to an activator of glucokinase (hereinafter sometimes abbreviated as GK),
In particular, it relates to a glucokinase activator that activates glucokinase by binding to glucokinase.
The present invention also relates to a therapeutic agent for diabetes and an anti-obesity agent characterized by activating glucokinase. Furthermore, the present invention also relates to a method for activating glucokinase and a gene encoding a protein having a glucokinase activating action.

[0002]

BACKGROUND OF THE INVENTION Glucokinase (GK) is the rate-limiting enzyme in sugar utilization in pancreatic β cells and is regarded as a glucose sensor that controls insulin secretion. So far, a substance called glucokinase regulator (GKRP) has been shown to suppress glucokinase activity in hepatocytes, but no glucokinase activator has been found.

[0003] The ability to activate glucokinase in pancreatic β-cells will help prevent and treat diabetes via increased insulin secretion.

Further, it has been suggested that glucokinase may stimulate the satiety center in the pituitary gland. If glucokinase can be activated, an anti-obesity drug through suppression of appetite can be provided. An object of the present invention is to provide an activator of glucokinase, and to provide a preventive or therapeutic agent for diabetes and an antiobesity agent comprising the activator as an active ingredient. Another object of the present invention is to provide a method for activating glucokinase, a protein having glucokinase activating action, and a gene encoding the protein.

[0005]

[Means for Solving the Problems] It is known that in hepatocytes, a glucokinase regulator (GKRP) forms a complex with glucokinase (GK) in the presence of F6P and reduces GK activity. I have. However, the presence of GKRP and its equivalent has not been reported in pancreatic β cells. Therefore, the present inventor tried to find a GKRP equivalent in pancreatic β cells. The present inventor used the yeast two hybrid system to perform HI using rat pancreatic β-cell-specific glucokinase cDNA.
Screening was performed using a cDNA library made from T-T15 cells. As a result, the present inventor
cDNA of oA carboxylase β subunit precursor (pβPCCase) was detected. Furthermore, the binding of this pβPCCase to GK was confirmed at the protein level by GST-Pull Down assay. When the effect of pβPCCase on GK enzyme activity was examined, pβPCCase increased the activity of GK. Substances that bind to GK and increase GK activity (particularly proteins)
Was first discovered by the present invention. From the above, pβPCCa
By using a GK activator such as se, it is possible to provide an anti-obesity drug that prevents or treats diabetes and suppresses appetite by stimulating satiety center based on glucokinase.

[0006] The present invention provides the following items 1 to 14. Item 1. A method for activating glucokinase, comprising causing a glucokinase activator to act on glucokinase. Item 2. A glucokinase activator that acts on glucokinase to activate glucokinase. Item 3. Item 3. The glucokinase-activating substance according to Item 2, which activates glucokinase by binding to glucokinase. Item 4. The glucokinase activator according to claim 2 or 3, comprising the following protein (a) or (b): (a) a protein comprising the amino acid sequence represented by SEQ ID NO: 1 or 2, (b) A protein comprising an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the amino acid sequence of a), and having a glucokinase activating action. Item 5. Item 5. An anti-obesity drug comprising the glucokinase activator according to any one of Items 2 to 4 as an active ingredient. Item 6. Item 6. The antiobesity agent according to Item 5, which acts by activating glucokinase to stimulate the satiety center. Item 7. An agent for preventing or treating diabetes, comprising the glucokinase activator according to any one of Items 2 to 4 as an active ingredient. Item 8. Item 8. The preventive or therapeutic drug for diabetes according to Item 7, which acts by activating glucokinase to promote insulin secretion. Item 9. Item 10. A protein comprising an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 1 or 2, and having a glucokinase activating action. Item 10. A composition comprising the glucokinase activator according to any one of Items 2 to 4, or the protein according to Item 9, and a pharmaceutically acceptable carrier. Item 11. Use of the glucokinase activator according to any one of Items 2 to 4 or the protein according to Item 9, for preparing a therapeutic agent for diabetes. Item 12. Use of the glucokinase activator according to any one of claims 2 to 4 or the protein according to claim 9 for preparing an antiobesity drug. Item 13. One or more bases of the polynucleotide represented by SEQ ID NO: 1 are substituted, deleted or added, and have a homology of 70% or more, preferably 80% or more, more preferably 90% or more, particularly 95% or more. And a gene encoding a protein having a glucokinase activating action. Item 14. A gene that hybridizes with the base sequence represented by SEQ ID NO: 1 or a complementary strand thereof under a stringent condition and encodes a protein having a glucokinase activating action (Rat pbPCCase and Hum
an pbPCCAse gene is excluded).

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION The gene encoding the glucokinase activating protein of the present invention was obtained in the process of finding a glucokinase regulatory protein, and pβPC
It turned out to be the same gene as Case. According to the provision of the gene of the present invention, prevention and treatment of diabetes and treatment of obesity are possible. In addition, all or a part of the genetically modified gene encoding the glucokinase-activating protein of the present invention can be used as a probe, and the use can be used as a part of a diagnostic kit for diabetes and a diagnostic kit.

[0008] Human pbPCCAse protein and gene represented by SEQ ID NO: 1, Rat pbPCCA represented by SEQ ID NO: 2
The proteins and genes of se are known. However, it is not known that a precursor of propionyl-CoA carboxylase β subunit (pβPCCase) activates glucokinase. Therefore, the present invention provides SEQ ID NO: 1,
2 (a protein having one or more amino acids or bases substituted / deleted / added and having a glucokinase activating effect or a gene encoding the protein) and human The pbPCCAse protein or gene of an organism other than rat is included in the scope of the present invention as long as it has a glucokinase activating effect. Furthermore, substances that bind to glucokinase and suppress glucokinase activity are known,
No substance that binds to glucokinase and increases glucokinase activity is known. Accordingly, the present invention also relates to substances that act on glucokinase to activate glucokinase.

In the present specification, abbreviations for amino acids, peptides, base sequences, nucleic acids and the like are used in IUPAC, IUPAC,
The rules of UB, “Guidelines for preparation of specifications including base sequence or amino acid sequence” (Japan Patent Office), and commonly used symbols in the art shall be followed. In the present invention, a gene (DNA) refers to not only double-stranded DNA,
It is intended to include each single-stranded DNA such as a sense strand and an antisense strand constituting the strand, and the length is not limited at all. As a specific example of the gene encoding the glucokinase-activating protein of the present invention, a modified version of the pβPCCase gene is exemplified. pβPCCa
A gene derived from an organism whose se gene is unknown is included in the gene of the present invention as long as the gene encodes a protein that activates glucokinase.

[0010] Examples of the gene of the present invention include a gene which substitutes, adds or deletes at least one base in the nucleotide sequence shown in SEQ ID NO: 1 or 2, and which encodes a glucokinase activating protein. It is possible, but not limited to, to include homologues of the gene.

As used herein, the term "homologous gene" means that it has sequence homology to the gene of the present invention, has the above-mentioned structural characteristics, commonality in gene expression patterns, and similarity in the above-mentioned biological functions. Means a series of related genes recognized as one gene family, and includes alleles (alleles) of the gene except for the sequences of SEQ ID NOS: 1 and 2.

[0012] The gene of the present invention comprises a DNA comprising a base sequence encoding a protein consisting of an amino acid sequence in which one or several or a plurality of amino acids are deleted, substituted or added in the amino acid sequences shown in SEQ ID NOS: 1 and 2. Molecules are also included.

Here, the degree of "deletion, substitution or addition of amino acids" and the positions thereof are such that the modified protein has the same glucokinase activity as the protein having the amino acid sequence shown in SEQ ID NOS: 1 and 2. There is no particular limitation as long as it has the chemical action. In the present invention, “glucokinase activating action” means an activity and a function of a protein having the amino acid sequence represented by SEQ ID NO: 1.

The above-mentioned modification (mutation) of the amino acid sequence
Although it may occur in nature due to, for example, mutation or post-translational modification, it can also be artificially modified based on a naturally-occurring gene. The present invention encompasses all modified genes having the above-mentioned properties, irrespective of the cause and means of such modification / mutation.

As the above-mentioned artificial means, for example, site-specific mutagenesis [Methods in Enzym
ology, 154, 350, 367-382 (1987);
3); Nucleic Acids Res., 12, 9441 (1984); Lectures on Sequent Chemistry Experiment 1, Genetic Research Method II, edited by The Biochemical Society of Japan, p105
(1986)) and chemical synthesis means such as the phosphate triester method and the phosphate amidite method (J. Am.
Chem. Soc., 89, 4801 (1967); id. 91, 3350 (1969); S
cience, 150, 178 (1968); Tetrahedron Lett., 22, 185
9 (1981); ibid. 24, 245 (1983)] and methods of combining them. More specifically, the synthesis of DNA can be performed by a chemical synthesis using a phosphoramidite method or a triester method, or can be performed on a commercially available automatic oligonucleotide synthesizer. The double-stranded fragment is synthesized chemically to produce a single-stranded fragment, either by synthesizing the complementary strand and annealing the strands together under appropriate conditions, or adding the complementary strand using a DNA polymerase with the appropriate primer sequences. It can also be obtained from things.

As a specific embodiment of the gene of the present invention, a modified gene having the nucleotide sequence shown in SEQ ID NO: 1 or 2 can be exemplified. The coding region in this base sequence shows an example of one combination of codons indicating each amino acid residue in the amino acid sequence shown in SEQ ID NOs: 1 and 2. The gene of the present invention is not limited to a gene having such a specific base sequence, and may have a base sequence selected by combining arbitrary codons for each amino acid residue. Codons can be selected according to a conventional method, for example, considering the codon usage of a host to be used [Ncleic Acids Res., 9, 43 (1981)].

As described above, the gene of the present invention also includes a gene comprising a nucleotide sequence having a certain homology with the nucleotide sequence shown in SEQ ID NOS: 1 or 2.

The above homology is at least 70% identity, preferably at least 80%, to a polynucleotide encoding a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1 or the base sequence shown in SEQ ID NO: 1. Identity, more preferably at least 90%, even more preferably at least 95%, especially at least 97%
And homologous polynucleotides thereof (excluding 100% homology).

As such a gene, for example, 0.1%
50 ° C. or 0.1% SD in 0.2 × SSC containing SDS
A gene having a nucleotide sequence that hybridizes with a DNA consisting of the nucleotide sequence shown in SEQ ID NO: 1 under stringent conditions of 60 ° C. in 1 × SSC containing S can also be exemplified.

The gene of the present invention can be prepared based on sequence information on specific examples of the gene of the present invention disclosed by the present invention.
It can be easily produced and obtained by general genetic engineering techniques [Molecular Cloning 2d Ed, Cold Spring H
arbor Lab. Press (1989); see Seismic Chemistry Laboratory Course "Gene Research Methods I, II, III", edited by The Biochemical Society of Japan (1986), etc.)

Specifically, a cDNA library is prepared from an appropriate source in which the gene of the present invention is expressed by a conventional method, and the desired library is prepared from the library using an appropriate probe or antibody specific to the gene of the present invention. Acad. Sci., USA., Proc. Natl. Acad.
8, 6613 (1981); Science, 222, 778 (1983) and the like].

In the above, the origin of cDNA is as follows:
Examples include various cells and tissues expressing the gene of the present invention, and cultured cells derived therefrom. Separation of total RNA therefrom, separation and purification of mRNA, acquisition of cDNA, and cloning thereof can all be carried out according to conventional methods. In addition, cDNA libraries are commercially available, and in the present invention, such cDNA libraries, for example, Clontech Lab.
Various cDNA libraries commercially available from c.) can also be used.

The method for screening the gene of the present invention from a cDNA library is not particularly limited, either, and any conventional method can be used.

More specifically, for example, a method for selecting a corresponding cDNA clone from a protein produced by cDNA by immunoscreening using an antibody specific to the protein, and selectively binding to a target DNA sequence Examples include plaque hybridization and colony hybridization using a probe, and combinations thereof.

As the probe used herein, DNA or the like chemically synthesized based on the information on the nucleotide sequence of the gene of the present invention can be generally used, and the gene of the present invention or a fragment thereof which has already been obtained can also be used. Good use. In addition, sense primers and antisense primers set based on the nucleotide sequence information of the gene of the present invention can be used as screening probes.

The nucleotide sequence used as the probe is a partial nucleotide sequence corresponding to SEQ ID NOS: 1 and 2, wherein at least 15 consecutive nucleotides,
Preferably 20 contiguous bases, more preferably 30
Also included are those having a number of contiguous bases, most preferably 50 contiguous bases, or the positive clone itself having the above sequence can be used as a probe.

In obtaining the gene of the present invention, PCR
DNA / RN by the method [Science, 230, 1350 (1985)]
The A amplification method can be suitably used. Particularly, when it is difficult to obtain a full-length cDNA from the library, RACE
Method [Rapid amplification of cDNA ends; Experimental Medicine, 12
(6), 35 (1994)], especially the 5′-RACE method [MA Frohma
n, et al., Proc. Natl. Acad. Sci., USA., 8, 8998
(1988)].

The primers used when employing the PCR method can be appropriately set based on the sequence information of the gene of the present invention revealed by the present invention, and can be synthesized according to a conventional method. The isolation and purification of the amplified DNA / RNA fragment can be carried out according to a conventional method as described above, for example, by gel electrophoresis.

The gene of the present invention or various DNA fragments obtained above can be obtained by a conventional method, for example, the dideoxy method [Proc.
Natl. Acad. Sci., USA., 74, 5463 (1977)) and the Maxam-Gilbert method (Methods in Enzymology, 65, 499).
(1980)], or simply using a commercially available sequencing kit or the like.

According to the gene of the present invention thus obtained, for example, by utilizing a part or the entire nucleotide sequence of the gene, the presence or absence of the expression of the gene of the present invention in an individual or various tissues can be specifically determined. Can be detected.

Such detection can be carried out according to a conventional method. For example, RT-PCR [Reverse transcribed-Polyme
rase chain reaction; ES Kawasaki, et al., Amplif
ication of RNA.In PCR Protocol, A Guide to method
s and applications, AcademicPress, Inc., SanDiego, 2
RNA amplification and Northern blotting analysis [Molecular Cloning, Cold Spring Harbor La.
b. (1989)], in situ RT-PCR [Nucl.
s., 21, 3159-3166 (1993)] and cell-level measurements using in situ hybridization, NAS
BA method (Nucleic acid sequence-based amplification,
Nature, 350, 91-92 (1991)] and various other methods. Preferably, a detection method by RT-PCR can be mentioned.

The primer used in the case of employing the PCR method is not particularly limited as long as it is specific to the gene of the present invention and can specifically amplify only the gene of the present invention. Can be set appropriately based on Usually, primers having a partial sequence of the gene of the present invention having a length of about 10 to 35 nucleotides, preferably about 15 to 30 nucleotides can be mentioned.

As described above, the gene of the present invention includes a specific primer and / or a specific primer for detecting the gene of the present invention.
Alternatively, a DNA fragment used as a specific probe is also included.

The DNA fragment can be defined as a DNA characterized in that it hybridizes under stringent conditions to a DNA consisting of the nucleotide sequence shown in SEQ ID NO: 1. Here, examples of the stringent conditions include ordinary conditions used as a primer or a probe, and are not particularly limited. For example, 0.2 × S containing 0.1% SDS as described above may be used.
1 × SS containing 50% in SC or 0.1% SDS in SC
The condition of 60 ° C. in C can be exemplified.

According to the gene of the present invention, the gene product or a protein containing the gene product can be easily and stably produced in a large amount by using ordinary genetic engineering techniques.

The protein of the present invention also includes mammals such as horses, sheep, cows, dogs, monkeys, cats, and the like, which have the same activity as the protein having the amino acid sequence shown in SEQ ID NO: 1.
Variants of proteins of rodents such as bears and rabbits are also included.

The protein of the present invention can be prepared by a conventional gene recombination technique [for example, Science, 224, 1431 (1984)] based on the sequence information of the gene variant of the present invention provided by the present invention.
Biochem. Biophys. Res. Comm., 130, 692 (1985);
Proc. Natl. Acad. Sci., USA., 80, 5990 (1983), etc.].

More specifically, the production of the protein is carried out by preparing a recombinant DNA (expression vector) capable of expressing the gene encoding the desired variant protein in a host cell, and introducing it into the host cell. Transforming, culturing the transformant,
It is then performed by recovery from the resulting culture.

As the host cell, any of prokaryote and eukaryote can be used. For example, prokaryote hosts include a wide variety of commonly used hosts such as Escherichia coli and Bacillus subtilis. Escherichia coli, especially those contained in the Escherichia coli K12 strain can be exemplified. Eukaryotic host cells also include
Cells such as vertebrates and yeast are included, and the former includes, for example, COS cells [Cell, 23: 175 (198
1)] and Chinese hamster ovary cells and their dihydrofolate reductase-deficient strains (Proc. Natl. Acad. Sc.
i., USA., 77: 4216 (1980)]. As the latter, yeast cells of the genus Saccharomyces are preferably used. Of course, it is not limited to these.

When a prokaryotic cell is used as a host, a promoter and an SD (Shine & Sand) are used upstream of the gene so that the gene of the present invention can be expressed in the vector using a vector that can be replicated in the host cell.・ Dargano)
An expression plasmid to which a base sequence and an initiation codon (for example, ATG) necessary for initiation of protein synthesis are added can be suitably used. As the above vector, generally, a plasmid derived from Escherichia coli, for example, pBR322, pBR325, pUC1
2, pUC13 and the like are often used, but are not limited thereto, and various known vectors can be used.
Commercially available products of the above-mentioned vectors used in an expression system using Escherichia coli include, for example, pGEX-4T (Amersham Pharm
acia Biotech), pMAL-C2, pMAl-P2
(New England Biolabs), pET21, pET21
/ Lacq (Invitrogen), pBAD / His (Invitrogen)
trogen).

When a vertebrate cell is used as a host, the expression vector usually has a promoter, an RNA splice site, a polyadenylation site and a transcription termination sequence located upstream of the gene of the present invention to be expressed. Which may optionally have a replication origin. Specific examples of the expression vector include, for example, pS having the early promoter of SV40.
V2 dhfr [Mol. Cell. Biol., 1: 854 (1981)] and the like. In addition to the above, various known commercial vectors can be used. Commercially available products of such a vector used in an expression system using animal cells include, for example, pEGF
PN, pEGFP-C (Clontrech), pIND (I
nvitrogen), pcDNA3.1 / His (Invitroge
n) and animal cells such as pFastBac
HT (GibciBRL), pAcGHLT (PharMi
ngen), pAc5 / V5-His, pMT / V5-H
is, pMT / Bip / V5-his (Invitrogen
And insect cell vectors.

As a specific example of an expression vector when a yeast cell is used as a host, for example, pAM82 [Proc.
Natl. Acad. Sci., USA., 80: 1 (1983)]. Commercially available expression vectors for yeast cells include, for example, pP
ICZ (Invitrogen), pPICZα (Invitrogen
And the like.

There is no particular limitation on the promoter. When Escherichia is used as a host, for example, tryptophan (trp) promoter, lpp promoter, lac promoter, recA promoter, PL / PR promoter and the like can be preferably used. When the host is a bacterium belonging to the genus Bacillus, SP01 promoter, SP02 promoter, penP promoter and the like are preferable. When yeast is used as a host, for example, a pH05 promoter, a PGK promoter, a GAP promoter, an ADH promoter and the like can be suitably used. When an animal cell is used as a host, preferable promoters include an SV40-derived promoter, a retrovirus promoter, a metallothionein promoter, a heat shock promoter, a cytomegalovirus promoter, and an SRα promoter. In addition, as the expression vector of the gene of the present invention, an ordinary fusion protein expression vector can also be preferably used. Specific examples of the vector include glutathione-S-
Examples include pGEX (Promega) for expression as a fusion protein with transferase (GST).

A method for introducing a desired recombinant DNA (expression vector) into a host cell and a transformation method using the same include:
There is no particular limitation, and various general methods can be employed.

The obtained transformant can be cultured according to a conventional method, and the target protein of the present invention encoded by a gene designed as desired by the culture can be cultured in the cell, extracellularly or on the cell membrane of the transformant. Expression, production (accumulation, secretion)
Is done.

As the medium used for the culturing, various media commonly used depending on the host cells used can be appropriately selected and used, and the culturing can be carried out under conditions suitable for the growth of the host cells.

The recombinant protein of the present invention thus obtained can be subjected to various separation operations (eg, “Biochemical Data Book II”,
1175-1259, 1st edition, 1st printing, June 23, 1980, published by Tokyo Chemical Co., Ltd .; Biochemistry, 25 (25), 8274 (198
6); Eur. J. Biochem., 163, 313 (1987)].

Specific examples of the method include ordinary reconstitution treatment, treatment with a protein precipitant (salting out method), centrifugation,
Osmotic shock method, sonication, ultrafiltration, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, high-performance liquid chromatography (HPL
Various types of liquid chromatography such as C), dialysis, and combinations thereof can be exemplified. Particularly preferred methods include affinity chromatography using a column to which a specific antibody against the protein of the present invention is bound. it can.

In designing a desired gene encoding the protein of the present invention, the nucleotide sequence of the gene shown in SEQ ID NO: 1 can be used favorably. The gene is
If desired, codons indicating each amino acid residue can be appropriately selected and changed for use.

The protein of the present invention can be produced by modifying the amino acid sequence shown in SEQ ID NO: 1 or 2 by a general chemical synthesis method. The method includes a conventional liquid phase method and a solid phase method for peptide synthesis.

The method for peptide synthesis is described in more detail below.
Based on the amino acid sequence information, a so-called stepwise elongation method in which each amino acid is sequentially linked one by one to extend the chain, a fragment consisting of several amino acids is synthesized in advance, and then each fragment is subjected to a coupling reaction. And the fragment condensation method, and the synthesis of the protein of the present invention may be carried out by any of them.

The condensation method employed in the above peptide synthesis also includes
Conventional methods can be followed, for example, azide method, mixed acid anhydride method, DCC method, active ester method, redox method, DPP
A (diphenylphosphoryl azide) method, DCC + additive (1-hydroxybenzotriazole, N-hydroxysuccinamide, N-hydroxy-5-norbornene-
2,3-dicarboximide) method and Woodward method.

The protein of the present invention thus obtained is appropriately purified according to the various methods described above, for example, methods widely used in the field of peptide chemistry such as ion exchange resin, partition chromatography, gel chromatography, and countercurrent partitioning. It can be carried out.

Accordingly, the present invention also provides a pharmaceutical composition containing the protein of the present invention as an active ingredient. The protein as an active ingredient in the pharmaceutical composition also includes a pharmaceutically acceptable salt thereof. Such salts include non-toxic alkali metal salts, such as, for example, sodium, potassium, lithium, calcium, magnesium, barium, ammonium, alkaline earth metal salts, ammonium salts and the like, prepared by methods well known in the art. You. Further, the above salts also include non-toxic acid addition salts obtained by reacting the protein of the present invention with a suitable organic or inorganic acid.

Representative non-toxic acid addition salts include, for example, hydrochloride, hydrochloride, hydrobromide, sulfate, bisulfate, acetate, oxalate, valerate, oleate, lauric acid Acid salt, borate, benzoate, lactate, phosphate, p-
Toluenesulfonate (tosylate), citrate, maleate, fumarate, succinate, tartrate, sulfonate, glycolate, maleate, ascorbate, benzenesulfonate and napsilate Is exemplified.

The above-mentioned pharmaceutical compositions include those containing the glucokinase activator of the present invention as an active ingredient and a pharmaceutically effective amount thereof together with a suitable pharmaceutically acceptable carrier or diluent.

Pharmaceutically acceptable carriers which can be used in the above-mentioned pharmaceutical composition (pharmaceutical preparation) include fillers, extenders, binders, humectants, disintegrants which are usually used depending on the use form of the preparation. And diluents or excipients such as surfactants and lubricants, which are appropriately selected and used according to the dosage unit form of the resulting preparation.

Particularly preferred pharmaceutical preparations of the present invention include various components which can be used in ordinary protein preparations, such as stabilizers, bactericides, buffers, isotonic agents, chelating agents, pH adjusters, surfactants It is prepared by using such as appropriate.

Examples of the stabilizer include human serum albumin, ordinary L-amino acids, saccharides, and cellulose derivatives. These can be used alone or in combination with a surfactant. In particular, according to this combination, there are cases where the stability of the active ingredient can be further improved.

The L-amino acid is not particularly limited, and may be, for example, any of glycine, cysteine, glutamic acid and the like.

The above-mentioned sugars are not particularly limited. For example, monosaccharides such as glucose, mannose, galactose and fructose, sugar alcohols such as mannitol, inositol and xylitol, disaccharides such as sucrose, maltose and lactose, dextran, hydroxypropyl Polysaccharides such as starch, chondroitin sulfate, and hyaluronic acid and derivatives thereof can be used.

The surfactant is not particularly limited, and any of ionic and nonionic surfactants can be used. For example, polyoxyethylene glycol sorbitan alkyl ester type, polyoxyethylene alkyl ether type,
Sorbitan monoacyl esters, fatty acid glycerides and the like can be used.

The cellulose derivative is not particularly limited, and methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose and the like can be used.

The amount of the saccharide to be added is about 0.0001 mg or more, preferably about 0.01 to 0.01 mg / μg of the active ingredient.
Suitably, the range is about 10 mg. The amount of the surfactant added is about 0.00001 mg per 1 μg of the active ingredient.
It is appropriate that the amount is not less than about, preferably about 0.0001 to 0.01 mg. The amount of human serum albumin to be added is about 0.0001 mg or more, preferably about 0.001 to 0.1 mg per 1 μg of the active ingredient. The amino acid is suitably used in an amount of about 0.001 to 10 mg per 1 μg of the active ingredient.
The amount of the cellulose derivative to be added is about 0.00001 mg or more per 1 μg of the active ingredient, preferably about 0.00001 mg.
It is appropriate to set the range of about 01 to 0.1 mg.

The amount of the active ingredient contained in the pharmaceutical preparation of the present invention is appropriately selected from a wide range, but is usually about 0.00000.
It is suitable that the content is in the range of 1 to 70% by weight, preferably about 0.0001 to 5% by weight.

In the pharmaceutical preparation of the present invention, various additives,
For example, a buffer, an isotonic agent, a chelating agent and the like can be added. Here, examples of the buffer include boric acid, phosphoric acid, acetic acid, citric acid, ε-aminocaproic acid, glutamic acid, and / or a salt corresponding thereto (eg, an alkali such as a sodium salt, a potassium salt, a calcium salt, and a magnesium salt thereof). Metal salts and alkaline earth metal salts). Examples of the tonicity agent include sodium chloride, potassium chloride, sugars, glycerin and the like. Examples of the chelating agent include sodium edetate and citric acid.

The pharmaceutical preparation of the present invention can be used as a solution preparation. Alternatively, the preparation can be lyophilized to a state that can be stored, and then dissolved in a buffer solution containing water for use, a saline solution, or the like, for example. It is also possible to use it after adjusting it to an appropriate concentration.

As the dosage unit form of the pharmaceutical preparation of the present invention, various forms can be selected according to the purpose of treatment, and typical examples are tablets, pills, powders, powders, granules, capsules Solid dosage forms, such as solutions, suspensions,
Includes liquid dosage forms such as emulsions, syrups and elixirs which, depending on the route of administration, may further be oral, parenteral,
They are classified into nasal preparations, vaginal preparations, suppositories, sublingual preparations, ointments and the like, and can be prepared, molded or prepared according to the usual methods. For example, when formed into a tablet form, for example, lactose, sucrose, sodium chloride, glucose, urea, starch, calcium carbonate,
Excipients such as kaolin, microcrystalline cellulose, silicic acid, potassium phosphate, water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethyl cellulose, hydroxypropyl cellulose,
Methylcellulose, binders such as polyvinylpyrrolidone, carboxymethylcellulose sodium, carboxymethylcellulose calcium, low-substituted hydroxypropylcellulose, dried starch, sodium alginate, agar powder, laminaran powder, sodium hydrogen carbonate,
Disintegrating agents such as calcium carbonate, polyoxyethylene sorbitan fatty acid esters, surfactants such as sodium lauryl sulfate and stearic acid monoglyceride, disintegrating inhibitors such as sucrose, stearin, cocoa butter, and hydrogenated oil; quaternary ammonium bases; Absorption enhancers such as sodium lauryl sulfate, humectants such as glycerin and starch, adsorbents such as starch, lactose, kaolin, bentonite, and colloidal silicic acid, and lubricants such as purified talc, stearates, boric acid powder, and polyethylene glycol. Sauces and the like can be used. Further tablets are tablets coated with a normal coating as necessary, such as sugar-coated tablets, gelatin-coated tablets, enteric-coated tablets,
It can be a film-coated tablet or a double or multilayer tablet.

In the case of molding into the form of pills, pharmaceutical carriers such as glucose, lactose, starch, cocoa butter,
Excipients such as hardened vegetable oil, kaolin, and talc, gum arabic powder, tragacanth powder, binders such as gelatin and ethanol, and disintegrants such as laminaran and agar can be used.

Capsules are prepared by mixing the active ingredient of the present invention with the various pharmaceutical carriers exemplified above and filling the mixture into hard gelatin capsules, soft capsules, and the like, according to a conventional method.

Liquid dosage forms for oral administration include pharmaceutically acceptable solutions, emulsions, suspensions, syrups, elixirs and the like containing conventional inert diluents, for example water, and further include wetting agents, Auxiliaries such as emulsions and suspensions can be included, which are prepared according to conventional methods.

In preparing liquid dosage forms for parenteral administration, such as sterile aqueous to non-aqueous solutions, emulsions and suspensions, diluents such as water, ethyl alcohol, propylene glycol, polyethylene glycol,
Vegetable oils such as ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, polyoxyethylene sorbitan fatty acid esters and olive oil can be used, and injectable organic esters such as ethyl oleate can be blended. These further include ordinary solubilizers, buffers, wetting agents, emulsifiers, suspending agents, preservatives,
A dispersant or the like can be added. Sterilization can be performed by, for example, a filtration operation of passing through a bacteria retaining filter, blending of a bactericide, irradiation treatment, heat treatment, and the like. They can also be prepared in the form of sterile solid compositions which can be dissolved in sterile water or a suitable sterilizable medium immediately before use.

For shaping in the form of a suppository or a preparation for vaginal administration, for example, polyethylene glycol, cocoa butter, higher alcohols, esters of higher alcohols, gelatin, semisynthetic glyceride and the like can be used as the preparation carrier.

When shaping into the form of an ointment such as paste, cream, gel and the like, diluents such as white petrolatum, paraffin, glycerin, cellulose derivatives, propylene glycol, polyethylene glycol,
Vegetable oils such as silicon, bentonite and olive oil can be used.

The composition for nasal or sublingual administration can be prepared according to a conventional method using well-known standard excipients.

The drug of the present invention may contain a coloring agent, a preservative, a fragrance, a flavoring agent, a sweetening agent, and other pharmaceuticals, if necessary.

The method of administration of the above pharmaceutical preparation is not particularly limited, and is determined according to various preparation forms, age, sex and other conditions of the patient, degree of disease, and the like. For example, tablets, pills,
Solutions, suspensions, emulsions, granules and capsules are administered orally, injections are administered alone or mixed with normal replenishers such as glucose and amino acids, and administered intravenously. Intradermal, subcutaneous or intraperitoneal administration, suppositories are administered rectally, vaginal agents are administered intravaginally, nasal agents are administered intranasally, sublingual agents are administered intraorally, ointments are transdermal Is administered locally.

The amount of the active ingredient to be contained in the pharmaceutical preparation and the dose thereof are not particularly limited, and may be selected according to the desired therapeutic effect, administration method, treatment period, age of the patient, sex and other conditions. It is appropriately selected from a wide range. In general,
The dose is usually about 0.0 / kg of body weight per day.
About 1 μg to 1000 mg, preferably about 0.1 μg to
The dosage is preferably about 100 mg, and the preparation can be administered once or several times a day.

[0079]

According to the present invention, a glucokinase activator useful as an agent for preventing or treating diabetes and an antiobesity agent can be provided. Further, by confirming the presence or absence of glucokinase activation, a diabetic drug or an antiobesity drug can be screened, and the present invention can be applied to search for a compound having a lower molecular weight that induces a glucokinase activator.

It is known that glucokinase acts as a glucose sensor in hepatocytes and pancreatic β cells and as a rate-limiting enzyme in glucose metabolism. In particular, it is known that pancreatic β cells are enzymes that regulate glucose-responsive insulin secretion, and it is known that they secrete insulin in response to increased blood glucose and lower blood glucose (Matschinsky FM
`` Glucokinase as glucose sensor and metabolic sign
al generator in pancreatic beta-cells and hepatocy
tes. "Diabetes. 1990 Jun; 39 (6): 647-52. Review; Mat.
schinsky F, Liang Y, Kesavan P, Wang L, Froguel P,
Velho G, Cohen D, Permutt MA, Tanizawa Y, Jetton
TL, et al .; “Glucokinase as pancreaticbeta cell g
lucose sensor and diabetes gene. "J Clin Invest. 1
993 Nov; 92 (5): 2092-8. Review) Further, it has been experimentally revealed that glucokinase activity and insulin secretion cooperatively change in pancreatic β cells. That is,
Physiological experiments have shown that increasing glucokinase activity also increases insulin secretion.
In addition, it is thought that the theoretical mechanism of action is mediated by an increase in glucokinase activity causing an increase in ATP production (Matschinsky FM, Glaser B, Magnus
on MA.``Pancreatic beta-cell glucokinase: closing
the gap between theoretical concepts and experimen
tal realities. "Diabetes. 1998 Mar; 47 (3): 307-15.R
eview). Furthermore, in an experimental system that overexpresses glucokinase in a human pancreatic β-cell-derived cell line, it has been experimentally revealed that increasing the amount of glucokinase increases the insulin secretory response to glucose accordingly. ing. From this, it is considered that increasing the activity of glucokinase improves glucose-responsive insulin secretion. Therefore, substances that increase the activity of glucokinase, such as pβPCCase, help to enhance the insulin secretory response, and may be an effective therapeutic agent for diabetic patients whose function is thought to be reduced (Wang H, Iynedjian PB. `` Modulation of glucose res
ponsiveness of insulinoma beta-cells by graded ove
rexpression of glucokinase. "Proc Natl Acad Sci U
S A. 1997 Apr 29; 94 (9): 4372-7).

In recent years, against the above background, methods for increasing the activity of glucokinase have been studied. As a representative example, among some reported glucokinase gene abnormalities, a mutant with higher glucokinase activity than the normal type has been found, but the cause has been investigated in detail to increase glucokinase activity (Mahalingam)
B, Cuesta-Munoz A, Davis EA, Matschinsky FM, Harri
son RW, Weber IT.``Structural model of human gluco
kinase in complex with glucose and ATP: implicatio
ns for the mutants that cause hypo- and hyperglyce
mia. "Diabetes. 1999 Sep; 48 (9): 1698-705).

As described above, a glucokinase activator is extremely promising as a therapeutic agent for diabetes, and researchers around the world are currently competing for development. Under these circumstances, pβPCCase, a glucokinase activator we have identified, is extremely unique, and its application is not only useful for the treatment of diabetes, but also its gene abnormalities and pβPCCase dysfunction due to other causes. It can be widely applied to the diagnosis of suspected glucose intolerance.

It is known that the hypothalamus pituitary has a satiety center. Recent studies have shown that pituitary gland expresses glucokinase in the pituitary gland, and that pituitary nerve cells generate signals in response to glucose concentration. Glucokinase acts as a glucose sensor in pituitary nerve cells as well as in pancreatic β cells, and it may sense the rise in blood glucose and send satiety stimuli to the center to play a role in suppressing eating behavior. Is suggested. That is, the activator of glucokinase may be used as an anti-obesity drug by increasing the activity of glucokinase to promote satiety stimulation and suppressing eating behavior. From these viewpoints, pβPCCase, a glucokinase activator, is considered to be useful for the development of antiobesity drugs (Yang XJ, Kow LM, F
unabashi T, Mobbs CV `` Hypothalamic glucose sensor:
similarities to and differences from pancreatic b
eta-cell mechanisms. "Diabetes. 1999 Sep; 48 (9): 176
3-72).

[0084]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in more detail with reference to the drawings. (I) cDNA cloning The full-length cDNA of rat pancreatic islet glucokinase was amplified by PCR using oligonucleotides specific to pancreatic β cells (Magnuson, MA, and Shelton, KD (1989) J. Bio.
l. Chem. 264, 15936-15942). This was incorporated into two types of plasmids. One is pBTM116, yeast two hybrid
Used as 'bait' for screen (pBTM116-GK). The other is pGEX4T-1 (Amersham Pharmacia Biotech)
And was used to synthesize glutathione S-transferase (GST) fusion glucokinase (GST-GK). In the same manner, the full-length cDNA of pβPCCase from pancreatic islet is
It was synthesized using βPCCase oligonucleotide (Kr
aus, JP, Firgaira, F., Novotny, J., Kalousek, F., Wi
lliams, KR, Williamson, C., Ohura, T., and Rosenber
g, LE (1986) Proc. Natl. Acad. Sci. USA 83, 8049-8
053). (II) Genes of pancreatic GK and pβPCCase from artificially synthesized protein rats were incorporated into a plasmid called pGEX4T-1 and transformed into M15 E. coli. Derivation and purification of GST fusion protein using isopropylthio-bD-galactosidase is described in the manufacturer's instructions.
(Amersham Pharmacia Biotech) (Inada, A., Someya,
Y., Yamada, Y., Ihara, Y., Kubota, A., Ban, N., Watana
be, R., Tsuda, K., and Seino, Y. (1999) J. Biol. Chem. 27
4,21095-21103). The produced protein was dissolved in 50 mM Tris-HCl (pH 8.0). Recombinant GK without GST tag (rGK) was performed using a protease called thrombin, also according to the manufacturer's instructions. (III) Pancreatic β cell specific by Yeast Two-hybrid System
Screening for glucokinase Yeast two hybrid screen was performed by the method according to the relevant literature (Kotake, K., Ozaki, N., Mizuta, M., Sekiya, S.,
Inagaki, N., and Seino S. (1997) J. Biol. Chem. 272,
29407-29410). pBTM116-GK as bait plasmid
Was used. This is a pancreatic β-cell GK fused to a LexA DNA-binding domain. Yeast strain L40 (MATa
trp1 leu2 his3 LYS2 :: lexA-HIS3 URA3 :: lexA-LacZ) was transformed into pBTM116-GK using the lithium acetate method (Vojtek, AB, Hollenberg, SM, and Coop
er, JA (1993) Cell. 74, 205-214). The L40 strain containing pBTM116-GK is HIT-T15 (hamster pancreatic β cell line (A
shcroft, SJ, Hammonds, P., and Harrison, DE (198
6) Diabetologia. 29, 727-733)) It was transformed together with a cell-derived cDNA library. About 1.0 ×
The transformant of 10 ⁶ contains 0.5 mM 3-amino-1,2,4-triazole but does not contain tryptophan, histidine, uracil and leucine in a Yc plate medium (2% glucose, 0.
5% ammonium sulfate, 1% succinic acid, and 0.12% yeast
Growed for screening on nitrogen base). His + colonies were transplanted onto paper filters and stained with 5-bromo-4-chloro-3-indolyl-bD-galactopyranoside to determine β-galactosidase activity (Vojtek, AB, Hollenberg, SM, and
Cooper, JA (1993) Cell. 74, 205-214). This screening resulted in two positive clones of HIS + & LacZ +. Plasmids are extracted from yeast and Escherichia coli
And then isolated. (IV) Preparation of rat pancreatic islets and liver extract Pancreatic islets were obtained from rats by collagenase digestion (Ka
jikawa, M., Ishida, H., Fujimoto, S., Mukai, E., Nishi
mura, M., Fujita, J., Tsuura, Y., Okamoto, Y., Norman,
AW, Seino, Y. (1999) Endocrinology.140,4706-471
2). Then, RIPA (150 mM NaCl, 1.0% Nonidet P-40, 50
mM Tris-HCl (pH 8.0)) and centrifuged at 10,000 xg for 15 minutes at 4 ° C. The supernatant was then collected as an islet extract. Rat liver tissue was similarly extracted. (V) Confirmation of protein interaction in vitro GST pull-down assay was performed by the method of the relevant literature (Inada, A., Someya, Y., Yamada, Y., Ihara, Y., Kubota,
A., Ban, N., Watanabe, R., Tsuda, K., and Seino, Y. (19
99) J. Biol. Chem. 274, 21095-21103). Glutathione-Sepharose beads (Amersham Ph
armacia Biotech) to which a GST fusion protein was immobilized was used. Sample containing GK (rGK, rat islet and liver extract) is GST or GST-pβPCCase and binding buffer (20 mM Tris-HCl (pH 7.6), 0.1 mM EDTA, 20% glycerol, 300 mM KCl, 0.1% Nonidet P-40 , 0.5 mM phenylmethylsulfonyl fluoride, 2 mM MgCl ₂ , 5 mM 2-
(Mercaptoethanol) for 1 hour at 4 ° C. After the incubation, beads 1
Washed 5 times with 0 volumes of binding buffer. Then, the cells were resuspended in an elution buffer (binding buffer + 10 mM reduced glutathione). The protein bound to the beads
Extracted in this way, 10% SDS-PAGE and rabbit anti-GK
The analysis was performed by Western blotting using an antibody (diluted to 1: 150; Toyoda, Miwa et al.).

Although no data are shown, a similar test was also performed in the immunoprecipitation method, and the same as in the GST pull-down assay,
The binding between glucokinase and pbPCCase was shown. (VI) Measurement of glucokinase activity The enzyme activity of glucokinase was measured by a fluorometric assay (Mahalingam, B., Cuesta-Munoz,
A., Davis, EA, Matschinsky, FM, Harrison, RW, a
nd Weber, IT (1999) Diabetes. 48, 1698-1705). Glucokinase activity was measured as the fluorescence of NADPH. Excitation and emission wavelengths are 50 mM HEPES-NaOH (pH 7.4), 100 mM KC
l, 8 mM MgCl ₂ , 0.5 mM NADP, 1 mM DTT, 1 U / ml G6PDH, various concentrations of ATP, 0.2 mg / ml GST-GK, and various concentrations of D-glucose at 340 nm and 450 nm, respectively. Yes, then incubated at 37 ° C. for 30 minutes. The reaction was stopped by adding a stop solution (300 mM NaH ₂ PO ₄ and 0.46 mM sodium dodecyl sulfate, pH 8.0). Glucokinase activity was determined by subtracting from the value of the complete reaction solution containing ATP as a control without ATP. Km, Vmax and Hill coefficient are fitted to Hill equation using nonlinear least squares method.
It was determined using software called Sigma plot (Matschin
sky, FM, Glaser, B., and Magnuson, MA (1998) Diabe
tes. 47, 307-315; and Liang, Zhong. Xu., Robert, WH, I
rene, TW, and Simon, JP (1995) J. Biol. Chem. 270, 9
939-9946). (VII) Statistical processing All data were expressed as mean + standard error. The value of "n" is the number of experiments, that is, the number of samples. Statistical processing is unpa
Performed using ired Student's t-test. (VIII) Results pBTM116-GK by Yeast Two Hybrid System
The beit plasmid was used to screen cDNA against the HIT-T15 cell line cDNA library. The resulting cDNA obtained by nutrient selection and β-galactosidase activity values is based on rat liver pβPCC
Sequence analysis revealed that it corresponded to 68% of ase.
The full-length cDNA of pβPCCase was obtained by PCR from cDNA of pancreatic islets. Pancreatic islet-specific GK and pancreatic islet pβPCCase
Was examined in vitro. GST fusion pβPCCa
se (GST-pβPCCase) is derived from artificially synthesized GK (rGK),
(Islet extract) and liver cell extract (Liver extract)
Was shown to bind to GK. (FIGS. 1-3). Thus, pβPCCase was shown to bind to GK at the protein level. The effect of GST-pβPCCase on GK enzyme activity was examined. GK enzyme activity is determined by fluorometric assay
It was measured using ST fusion GK (GST-GK) (FIGS. 3 to 6). GST-
The GK activity was significantly higher in the group to which GST-pβPCCase was added than in the control group (FIG. 4). Furthermore, GST tag produced in the same manner as GST-pβPCCase did not affect GST-GK activity. GST-GK activity is 10 mM with mannoheptulose
In the presence of glucose and 5 mM ATP, GST-pβPCCase group
It was suppressed to 7% and 60% in the control group. Vmax and K
m is 57.17 ± 4.0 U / mg, 5.31 ± in GST-pβPCCase group
0.76 mM, 47.46 ± 3.36 U / mg, 6.25 ± in control group
0.43 mM (Table 1). Thus, in the GST-pβPCCase group, Vmax was increased by 20% and Km was decreased by 18% as compared with the control group. Hill coefficient was 1.8 in the GST-pβPCCase group and 1.7 in the control group (Table 1). The Km for ATP was calculated and compared between the two groups, but there was no significant difference (see FIG. 5, Table 1). GST-
When the relationship between GK activity and GST-pβPCCase concentration was examined,
ST-GK activity was activated depending on GST-pβPCCase concentration
(FIG. 6)

[0086]

[Table 1]

In Table 1, h is the Hill coefficient number. Data are means ± SE, Km, Vmax and h were calculated from 5 independent experiments and KmATP was calculated from 4 independent experiments.

Thus, GST-pβPCCase was found to increase GST-GK activity on glucose. The present inventors have concluded in vitro that pβPCCase has an effect as an activator of pancreatic islet-specific GK (GKA). (IX) Discussion It has been reported that the activity of GST-GK is equivalent to that of GK without GST tag (Mahalingam, B., Cuesta-Munoz, A., Davi
s, EA, Matschinsky, FM, Harrison, RW, and Webe
r, IT (1999) Diabetes. 48, 1698-1705). It was found that GST tag alone did not affect GST-GK activity, so GST-GK
It was revealed that pST was activated by pβPCCase in GST-pβPCCase. pβPCCase is propionyl
CoA carboxylase (PCCase) is a subunit of a biotin-dependent enzyme present in the mitochondrial matrix (Browner, MF, Taroni, F., Sztul, E., an
d Rosenberg, LE (1989) J. Biol. Chem. 264, 12680-1268
5; and Moss, J., and Lane, MD (1971) Adv. Enzymol. 35, 3
21-34222, 23). pβPCCase is a 61.5KDa protein,
It is synthesized by cytoplasmic ribosomes and has an absorption signal to mitochondria (MacDonald, MJ, Tang, J., and Polon
sky, KS (1996) Diabetes. 45, 1626-1630). After being incorporated into mitochondria, it becomes a mature βPCCase, 7.5 KDa smaller than pβPCCase, due to a zinc-dependent proteolytic mechanism. And it becomes a part of PCCase which forms oligomer of α4β4 (Kraus, JP, Kalousek, F., and Rosenberg, L.
E. (1983) J. Biol. Chem. 258, 7245-7248). Thus, pβP
CCase is synthesized in the cytoplasm and incorporated into mitochondria to become a mature βPCCase. Since GK is a cytoplasmic enzyme, pβP
It is likely to interact with CCase in the cytoplasm.

The activity of glucokinase has been studied in various situations, such as diabetes and lipotoxic conditions (Liu,
YQ, Tornheim, K., and Leahy, JL (1998) J. Clin. Inves
t. 101, 1870-1875). It has been reported that pancreatic islets increase glucose utilization at low Km and increase basal insulin secretion in a lipotoxic situation. (Liu, YQ, Tornheim,
K., and Leahy, JL (1998) J. Clin. Invest. 101, 1870-1
875; and Unger, RH, (1995) Diabetes. 44, 863-87.
0). Low Km hexokinase affects glucose sensitivity and lowers the threshold for glucose-dependent insulin secretion (Schuit, F., Moens, K., Heimberg, H., Pipel
eers, D. (1999) J. Biol. Chem. 274, 32803-32809), K of GK
The decrease in m is of particular interest in considering the mechanism of glucose sensitivity of pancreatic β cells.

[0090]

[Sequence List] SEQUENCE LISTING <110> KANSAI TLO Co., ltd. <120> Glucokinase Binding Protein <130> 14500JP <160> 2 <170> PatentIn Ver. 2.0 <210> 1 <211> 1630 <212> DNA < 213> Human pbPCCAse <400> 1 atg gcg gcg gca tta cgg gtg gcg gcg gtc ggg gca agg ctc agc gtt 48 Met Ala Ala Ala Ala Leu Arg Val Ala Ala Val Gly Ala Arg Leu Ser Val 1 5 10 15 ctg gcg agc ggt cgc gcc gcg gtc cgc agc ctt tgc agc cag gcc 96 Leu Ala Ser Gly Leu Arg Ala Ala Val Arg Ser Leu Cys Ser Gln Ala 20 25 30 acc tct gtt aac gaa cgc atc gaa aac aag cgc cgg acc gcg Serctg Val Asn Glu Arg Ile Glu Asn Lys Arg Arg Thr Ala Leu Leu 35 40 45 gga ggg ggc caa cgc cgt att gac gcg cac gac aag cga gga aag cta 192 Gly Gly Gly Gln Arg Arg Ile Asp Ala His Asp Lys Arg Gly Lys Leu 50 55 60 aca gcc agg gag agg atc agt ctc ttg ctg gac cct ggc agc ttt gtt 240 Thr Ala Arg Glu Arg Ile Ser Leu Leu Leu Asp Pro Gly Ser Phe Val 65 70 75 80 gag agc gac atg ttt gtg gaa cac aga tgt gca gat ttt gga atg gct 288 Glu Ser Asp Met Phe Val Glu His Arg Cys Ala Asp Phe Gly Met Ala 85 90 95 gct gat aag aat aag ttt cct gga gac agc gtg gtc act gga cga ggc 336 Ala Asp Lys Asn Lys Phe Pro Gly Asp Ser Val Val Thr Gly Arg Gly 100 105 110 cga atc aat gga aga ttg gtt tat gtc ttc agt cag gat ttt aca gtt 384 Arg Ile Asn Gly Arg Leu Val Tyr Val Phe Ser Gln Asp Phe Thr Val 115 120 125 ttt gga ggc agt ctg tca gga gca cat gcc caa aag atc tgc aaa atc 432 Phe Gly Gly Ser Leu Ser Gly Ala His Ala Gln Lys Ile Cys Lys Ile 130 135 140 atg gac cag gcc ata acg gtg ggg gct cca gtg att ggg ctg aat gac 480 Met Asp Gln Ala Ile Thr Val Gly Ala Pro Val Ile Gly Leu Asn Asp 145 150 155 160 tct ggg gga gca cgg atc caa gaa gga gtg gag tct ttg gct ggc tat 528 Ser Gly Gly Ala Arg Ile Gln Glu Gly Val Glu Ser Leu Ala Gly Tyr 165 170 175 gca gac atc ttt ctg agg gtt acg gca tcc gga gtc atc cct cag 576 Ala Asp Ile Phe Leu Arg Asn Val Thr Ala Ser Gly Val Ile Pro Gln 180 185 190 att tct ctg atc atg ggc cca tgt gct ggt ggg gcc gtc tac tcc cca 624 Ile Ser Leu Ile Me t Gly Pro Cys Ala Gly Gly Ala Val Tyr Ser Pro 195 200 205 gcc cta aca gac ttc acg ttc atg gta aag gac acc tcc tac ctg ttc 672 Ala Leu Thr Asp Phe Thr Phe Met Val Lys Asp Thr Ser Tyr Leu Phe 210 215 220 atc act ggc cct gat gtt gtg aag tct gtc acc aat gag gat gtt acc 720 Ile Thr Gly Pro Asp Val Val Lys Ser Val Thr Asn Glu Asp Val Thr 225 230 235 240 cag gag gag ctc ggt ggt gcc aag acc cac acc acc atg tca ggt gtg 768 Gln Glu Glu Leu Gly Gly Ala Lys Thr His Thr Thr Met Ser Gly Val 245 250 255 gcc cac aga gct ttt gaa aat gat gtt gat gcc ttg tgt aat ctc cgg 816 Ala His Arg Ala Phe Glu Asn Asp Val Asp Ala Leu Cys Asn Leu Arg 260 265 270 gat ttc ttc aac tac ctg ccc ctg agc agt cag gac ccg gct ccc gtc 864 Asp Phe Phe Asn Tyr Leu Pro Leu Ser Ser Gln Asp Pro Ala Pro Val 275 280 280 cgt gag t gat ccc agt gac cgt ctg gtt cct gag ctt gac aca 912 Arg Glu Cys His Asp Pro Ser Asp Arg Leu Val Pro Glu Leu Asp Thr 290 295 300 att gtc cct ttg gaa tca acc aaa gcc tac aac atg gtg gac atc ata 960 Ile Va l Pro Leu Glu Ser Thr Lys Ala Tyr Asn Met Val Asp Ile Ile 305 310 315 320 cac tct gtt gtt gat gag cgt gaa ttt ttt gag atc atg ccc aat tat 1008 His Ser Val Val Asp Glu Arg Glu Phe Phe Glu Ile Met Pro Asn Tyr 325 330 335 gcc aag aac atc att gtt ggt ttt gca aga atg aat ggg agg act gtt 1056 Ala Lys Asn Ile Ile Val Gly Phe Ala Arg Met Asn Gly Arg Thr Val 340 345 350 gga att gtt ggc aac caa cct aag gtg gcc tca gga tgc ttg gat att 1104 Gly Ile Val Gly Asn Gln Pro Lys Val Ala Ser Gly Cys Leu Asp Ile 355 360 365 aat tca tct gtg aaa ggg gct cgt ttt gtc aga ttc tgt gat gca ttc 1152 Asn Ser Ser Val Lys Gly Ala Arg Phe Val Arg Phe Cys Asp Ala Phe 370 375 380 aat att cca ctc atc act ttt gtt gat gtc cct ggc ttt cta cct ggc 1200 Asn Ile Pro Leu Ile Thr Phe Val Asp Val Pro Gly Phe Leu Pro Gly 385 390 395 400 aca gca cag gaa tac ggg ggc atc atc cgg cat ggt gcc aag ctt ctc 1248 Thr Ala Gln Glu Tyr Gly Gly Ile Ile Arg His Gly Ala Lys Leu Leu 405 410 415 tac gca ttt gct gag gca act gta ccc aaa gtc acagt atc acc agg 1296 Tyr Ala Phe Ala Glu Ala Thr Val Pro Lys Val Thr Val Ile Thr Arg 420 425 430 aag gcc tat gga ggt gcc tat gat gtc atg agc tct aag cac ctt tgt 1344 Lys Ala Tyr Gly Gly Ala Tyr Asp Val Met Ser Ser Lys His Leu Cys 435 440 445 ggt gat acc aac tat gcc tgg ccc acc gca gag att gca gtc atg gga 1392 Gly Asp Thr Asn Tyr Ala Trp Pro Thr Ala Glu Ile Ala Val Met Gly 450 455 460 gca aag ggc gct gtg gag atc atc ttc aaa ggg cat gag aat gtg gaa 1440 Ala Lys Gly Ala Val Glu Ile Ile Phe Lys Gly His Glu Asn Val Glu 465 470 475 480 gct gct cag gca gag tac atc gag aag ttt gcc aac cct ttc cct gca 1498 Ala Gln Ala Glu Tyr Ile Glu Lys Phe Ala Asn Pro Phe Pro Ala 485 490 495 gca gtg cga ggg ttt gtg gat gac atc atc caa cct tct tcc aca cgt 1546 Ala Val Arg Gly Phe Val Asp Asp Ile Ile Gln Pro Ser Ser Thr Arg 500 505 510 gcc cga atc tgc tgt gac ctg gat gtc ttg gcc agc aag aag gta caa 1594 Ala Arg Ile Cys Cys Asp Leu Asp Val Leu Ala Ser Lys Lys Val Gln 515 520 525 cgt cct tgg aga aaa cat gca aat att cca ttg taa 1630 Arg Pro Trp Arg Lys His Ala Asn Ile Pro Leu Glx 530 535 540 <210> 2 <211> 1626 <212> DNA <213> Rat pbPCCase <400> 2 atg gcg gcg gtg att cgg att cgg gcg atg gca gct gga acg agg ctc 48 Met Ala Ala Val Ile Arg Ile Arg Ala Met Ala Ala Gly Thr Arg Leu 1 5 10 15 aga gtt ttg aac tgc ggc ctc ggc acc aca atc cgc agc ctt tgc agc 96 Arg Val Leu Asn Gly Leu Gly Thr Thr Ile Arg Ser Leu Cys Ser 20 25 30 cag ccg gtc tca gtt aat gag cgc atc gaa aac aag cgc cat gcc gcg 144 Gln Pro Val Ser Val Asn Glu Arg Ile Glu Asn Lys Arg His Ala Ala 35 40 45 ctg ctg gga gga ggc cag cga cgc atc gac gca cag cac aag cga gga 192 Leu Leu Gly Gly Gly Gly Gln Arg Arg Ile Asp Ala Gln His Lys Arg Gly 50 55 60 aag ctg aca gcc agg gag cgg atc agt ctc gct ggc agc 240 Lys Leu Thr Ala Arg Glu Arg Ile Ser Leu Leu Leu Asp Pro Gly Ser 65 70 75 80 ttc ctg gag agc gac atg ttt gtg gaa cac agg tgt gcc gac ttc gga 288 Phe Leu Glu Ser Asp Met Phe Val Glu His Arg Cys Ala Asp Phe Gly 85 90 95 atg gct gcc ga g aag aat aag ttt cct gga gat agt gtg gtc act gga 336 Met Ala Ala Glu Lys Asn Lys Phe Pro Gly Asp Ser Val Val Thr Gly 100 105 110 cgg ggc aga atc aac gga cga ttg gtt tat gtc ttc agt cag gac ttt 384 Arg Gly Arg Ile Asn Gly Arg Leu Val Tyr Val Phe Ser Gln Asp Phe 115 120 125 aca gtt ttt gga ggc agt ctg tca ggc gca cat gct cag aag atc tgc 432 Thr Val Phe Gly Gly Ser Leu Ser Gly Ala His Ala Gln Lys Ile Cys 130 135 140 aaa atc atg gac cag gcc ata aca gtg ggg gct cca gtg atc gga ctc 480 Lys Ile Met Asp Gln Ala Ile Thr Val Gly Ala Pro Val Ile Gly Leu 145 150 155 160 aat gac tcc ggg ggc gcc aga atc cag gag ggg gtg gag tcc ttg gct 528 Asn Asp Ser Gly Gly Ala Arg Ile Gln Glu Gly Val Glu Ser Leu Ala 165 170 175 ggc tat gca gac atc ttc ctg agg aat gtc aca gcg tcc gga gtc atc Ala Tly Phe Leu Arg Asn Val Thr Ala Ser Gly Val Ile 180 185 190 cct cag att tct ctg atc atg ggc cca tgc gct ggt ggg gcc gtc tac 624 Pro Gln Ile Ser Leu Ile Met Gly Pro Cys Ala Gly Gly Ala Val Tyr 195 200 205 tc c ccc gcc ctg aca gac ttc aca ttc atg gtg aag gac acg tcc tac 672 Ser Pro Ala Leu Thr Asp Phe Thr Phe Met Val Lys Asp Thr Ser Tyr 210 215 220 ctg ttt atc act ggc cct gaa ttt gtg aag tct gtt acc aat gaa gac 720 Leu Phe Ile Thr Gly Pro Glu Phe Val Lys Ser Val Thr Asn Glu Asp 225 230 235 240 gtg act cag gag cag ctg ggt ggt gcc aag acc cac acc act gtg tca 768 Val Thr Gln Glu Gln Leu Gly Gly Ala Lys Thr His Thr Thr Val Ser 245 250 255 ggt gtg gcc cac aga gct ttc gac aat gac gtc gat gct ctg tgt aac 816 Gly Val Ala His Arg Ala Phe Asp Asn Asp Val Asp Ala Leu Cys Asn 260 265 270 ctg cgt gaa ttc tta aac ttt ctg ccc ctc agc aac cag gac ccg gct 864 Leu Arg Glu Phe Leu Asn Phe Leu Pro Leu Ser Asn Gln Asp Pro Ala 275 280 285 tcc atc cga gag tgc cat gac ccc agt gac cgt ctg gtt cct gag ctg Arg Glu Cys His Asp Pro Ser Asp Arg Leu Val Pro Glu Leu 290 295 300 gat aca gtt gtc cct ttg gag tca agc aaa gcc tat aac atg ctg gac 960 Asp Thr Val Val Pro Leu Glu Ser Ser Lys Ala Tyr Asn Met Leu Asp 30 5 310 315 320 atc ata cat gca gtg att gac gag aga gag ttt ttt gag ata atg ccc 1008 Ile Ile His Ala Val Ile Asp Glu Arg Glu Phe Phe Glu Ile Met Pro 325 330 335 aat tat gca aaa aac att gtc att ggc ttt gca aga atg aat ggg agg 1056 Asn Tyr Ala Lys Asn Ile Val Ile Gly Phe Ala Arg Met Asn Gly Arg 340 345 350 acc gtt gga att gtt ggc aac cag ccc aac gtg gct tca ggg tgc ctg 1104 Thr Val Gly Ile Val Gly As Gln Pro Asn Val Ala Ser Gly Cys Leu 355 360 365 gac att aat tca tct gtg aag ggg gct cgc ttt gtc aga ttc tgt gat 1152 Asp Ile Asn Ser Ser Val Lys Gly Ala Arg Phe Val Arg Phe Cys Asp 370 375 380 gct ttc agc att cct ctc atc act ttc gtt gac gtc cct ggc ttc ctg 1200 Ala Phe Ser Ile Pro Leu Ile Thr Phe Val Asp Val Pro Gly Phe Leu 385 390 395 400 400 cct ggc aca gcg cag gaa tat gga ggc atc atc cgg cat aag 1248 Pro Gly Thr Ala Gln Glu Tyr Gly Gly Ile Ile Arg His Gly Ala Lys 405 410 415 ctg ctc tac gcc ttt gcc gag gcc acc gtg ccc aaa atc acg gtc atc 1296 Leu Leu Tyr Ala Phe Ala Glu Ala Thr Val Pro Lys Ile Thr Val Ile 420 425 430 acc agg aag gcc tat gga ggt gcc tat gat gtc atg agc tcc aaa cac 1344 Thr Arg Lys Ala Tyr Gly Gly Ala Tyr Asp Val Met Ser Ser Lys His 435 440 445 ctt ctt ggt gat acc aac tat gcc tgg ccc aca gct gag att gca gtc 1392 Leu Leu Gly Asp Thr Asn Tyr Ala Trp Pro Thr Ala Glu Ile Ala Val 450 455 460 atg ggt gca aag ggt gcc gtg gag atc atc ttc aaa gga cac gaa gac 1440 Met Gly Lys Gly Ala Val Glu Ile Ile Phe Lys Gly His Glu Asp 465 470 475 480 gtg gaa gcc gcc cag gca gag tat gtg gag aag ttt gcc aat cct ttc 1488 Val Glu Ala Ala Gln Ala Glu Tyr Val Glu Lys Phe Ala Asn Pro 485 490 495 cct gca gcc gtg aga ggg ttc gtg gat gac atc atc cag ccg tcc tct 1536 Pro Ala Ala Val Arg Gly Phe Val Asp Asp Ile Ile Gln Pro Ser Ser 500 505 510 act cgt gct cgg ata tgc tgt gac ctg gaa ctg gcc agc aag aag 1584 Thr Arg Ala Arg Ile Cys Cys Asp Leu Glu Val Leu Ala Ser Lys Lys 515 520 525 gtc cat cgt cct tgg agg agg aag cat gcc aat gtc cca ctg tga 1626 Val His Arg Pro Trp Arg Lys His Ala Asn Val Pro Leu Glx 530 535 540 542

[Brief description of the drawings]

FIG. 1 is a photograph substituted for a drawing, showing the interaction between pβPCCase and GK. Interaction with artificially synthesized GK.

FIG. 2 is a photograph substituted for a drawing, showing the interaction between pβPCCase and GK. Islet cell extract interaction.

FIG. 3 is a drawing substitute photograph showing the interaction between pβPCCase and GK. Interaction with hepatocyte extract. In FIGS. 1-3, pβPCCase interacts with GK in vitro.
GST or GST-pβPC immobilized on glutathione beads
Cases were incubated with recombinant GK, rat islet and hepatocyte extracts. Bound protein was detected with anti-GK antibody.

FIG. 4. Activity of GK. GST-GK (●) or GST-GK + GS using a glucose concentration of 0.05 mM to 50 mM at 5 mM ATP
The reaction rate of T-pβPCCase (○) is shown (n = 5).

FIG. 5 shows the activity of GK. 0.01 mM in 5 mM glucose
GST-GK (●) or GST-GK + G using ATP concentration of 1010 mM
The reaction rate of ST-pβPCCase (○) is shown (n = 4).

FIG. 6 shows the activity of GK. The reaction rates of GK using various concentrations of pβPCCase at 10 mM glucose and 5 mM ATP are shown (n = 5). 4-6, shown as mean ± SE from 4 or 5 experiments.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 43/00 111 A61K 48/00 C07K 14/47 C12N 15/00 ZNAA // A61K 48/00 A61K 37 / 02 F term (reference) 4B024 BA80 CA04 CA07 DA12 GA11 GA19 GA27 HA11 4C084 AA02 AA03 AA17 BA01 BA22 CA04 CA19 CA53 DC50 NA14 ZC192 ZC352 ZC412 4H045 AA10 AA30 BA10 CA40 EA20 EA50 FA74

Claims (14)

[Claims] 1. A method for activating glucokinase, which comprises causing a glucokinase activator to act on glucokinase. 2. A glucokinase activator which acts on glucokinase to activate glucokinase. 3. The glucokinase-activating substance according to claim 2, which binds to glucokinase to activate glucokinase. 4. The glucokinase activator according to claim 2, which comprises the following protein (a) or (b): (a) a protein comprising an amino acid sequence represented by SEQ ID NO: 1 or 2, (B) a protein comprising an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the amino acid sequence of (a) and having a glucokinase activating action; 5. An anti-obesity drug comprising the glucokinase-activating substance according to any one of claims 2 to 4 as an active ingredient. 6. The anti-obesity drug according to claim 5, which acts by activating glucokinase to stimulate the satiety center. 7. A preventive or therapeutic drug for diabetes comprising the glucokinase activator according to claim 2 as an active ingredient. 8. The preventive or therapeutic agent for diabetes according to claim 7, which acts by activating glucokinase to promote insulin secretion. 9. A protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 1 or 2, and having a glucokinase activating action. 10. A composition comprising the glucokinase activator according to any one of claims 2 to 4, or the protein according to claim 9, and a pharmaceutically acceptable carrier. 11. A method for preparing a drug for treating diabetes.
Use of the glucokinase activator according to any one of claims 4 to 4 or the protein according to claim 9. 12. A method for preparing an anti-obesity drug according to claims 2 to 4.
Use of a glucokinase activator according to any one of claims 1 to 4 or a protein according to claim 9. 13. The polynucleotide of SEQ ID NO: 1 in which one or more bases have been substituted, deleted or added,
A gene encoding a protein having a homology of at least 80%, more preferably at least 90%, especially at least 95%, and having a glucokinase activating action. 14. A gene that hybridizes with the nucleotide sequence of SEQ ID NO: 1 or a complementary strand thereof under a stringent condition and encodes a protein having a glucokinase activating action (Rat pbPCCase and Human pbPCCAse gene is excluded).