JP2003509070A - Assay - Google Patents

Abstract

(57) SUMMARY The present invention relates to assays that measure the binding interaction between glucokinase (GLK), the regulatory protein GLKRP, and fructose-6-phosphate. The method is useful for identifying compounds that modulate glucokinase. Such compounds may have utility in treating non-insulin dependent diabetes.

Description

Detailed Description of the Invention

The present invention relates to an assay method suitable for identifying compounds that stimulate glucokinase and lead to a decreased threshold of insulin secretion in pancreatic β cells. Furthermore, the compounds are expected to lower blood glucose by increasing hepatic glucose uptake. Such compounds are known to have non-insulin dependent diabetes (N
It may have utility in the treatment of IDDM).

In pancreatic β cells and liver parenchymal cells, the major plasma membrane glucose transporters are
It is GLUT2. Under physiological glucose concentrations, GLUT2 is not rate limiting and in these cells the rate of glucose uptake is glucokinase (GL).
K) has the effect limited by the rate of glucose-catalyzed phosphorylation of glucose to glucose-6-phosphate (G-6-P) (Pilkis and Granner).
1992, Malaise 1993). GLK has a high (6-10 mM) Km for glucose and is not inhibited by physiological concentrations of G-6-P,
And is mainly expressed by liver, kidney and pancreatic β cells (Pilkis et al.
1994 and Caro et al. 1995).

Recent developments suggest that GLK may play an important role in the development of NIDDM (Glasser et al. 1998). First, GLK mutations are thought to be the major defects responsible for several forms of adult-onset diabetes mellitus (MODY), a rare form of NIDDM, in young people (Froguel).
1993, Bell et al. 1996 and Shiota et al. 1998). Second, constitutive expression of yeast hexokinase in transgenic mice has been shown to cause increased insulin secretion and hypoglycemia. This data provides new evidence to support the crucial role of GLK in determining glucose phosphorylation and insulin secretion rates in β cells.

GLK binds to a regulatory protein (GLKRP) that binds to GLK and inactivates it in the presence of fructose-6-phosphate (F-6-P). Fructose-1-phosphate (F-1-P) and inorganic phosphate interfere with the binding of F-6-P, thereby preventing the binding of GLKRP to GLK (see Figure 1). G
Compounds that also interfere with the interaction between LK and GLKRP are effectively GLK.
And may offer opportunities for the development of new therapeutic agents for the treatment of NIDDM (Veiga-da-Cunhe et al. 1996).

In hepatocytes, GLK acts as the rate-limiting step in glucose uptake and utilization. Compounds that stimulate GLK by interfering with the interaction between GLK and GLKRP will lower blood glucose by increasing hepatic glucose uptake. In pancreatic β-cells, increased glucose metabolism leads to ATP-dependent K ⁺ channel closure through increased β-cell [ATP] / [ADP] concentration ratios, hence membrane depolarization and voltage-gated Ca ²⁺ channels. Guide the release of. Subsequent increase in β-cell Ca ²⁺ induces glucose-sensitive insulin release (Matschinsky et al. 1993). Therefore, in β cells,
Compounds that stimulate GLK by interfering with the interaction between GLK and GLKRP will also lead to a decrease in the glucose threshold for insulin secretion. Both effects would be beneficial in the treatment of NIDDM.

The enzymatic activity of GLK can be measured by incubating GLK, ATP and glucose, as shown in FIG. The rate of product formation can be determined by coupling the assay to the G-6-P dehydrogenase, NADP / NADPH system, and measuring the increase in optical density at 340 nm (Matschinsky et al. 1993). .

However, this type of assay has some disadvantages. First, the use of such an assay to screen compounds will identify all compounds that modulate enzyme activity, regardless of mechanism of action.
For example, the assay may involve compounds that directly activate glucokinase, compounds that interfere with F-6-P binding, or F-1-P mimetics in the absence of GLKRP.
will detect compounds that act as ics). In addition, the spectrophotometric assay will detect compounds that inhibit protein-protein interactions between GLK and GLKRP equally well.

Second, the assay may identify false positives, ie, those that apparently increase glucokinase activity but do not. For example,
A compound capable of stimulating a coupling enzyme (s), or 340 n
Those with a significant absorbance at m would not be distinguished in this assay from compounds that act by stimulating glucokinase itself.

Therefore, there is a need for new assays that can provide information regarding the precise mechanism of action of compounds that regulate GLK activity. In particular, the assay method is GL
There is a need to identify compounds that stimulate GLK activity by interfering with the binding interaction between K and GLKRP. Such assays are disclosed in the present invention,
Provided by GLK / GLKRP binding assay and F-6-P / GLKRP binding assay.

In the present invention, we have developed a novel method for identifying compounds that regulate GLK. In particular, we have developed an assay system that identifies compounds that regulate GLK by interfering with the interaction between GLK and GLKRP. It should be understood that whenever reference is made to an assay system as described herein, methods and / or processes utilizing said assay system are also contemplated.

The assay system comprises two related assay methods; the GLK / GLKRP binding assay and the F-6-P / GLKRP binding assay. The GLK / GLKRP binding assay provides an assay method for measuring the binding interaction between GLK and GLKRP. GLK and GLKRP using the method
By modulating the interaction between and it is possible to identify compounds that regulate GLK.

[0012] GLKRP and GLK are treated with an inhibitory concentration of F, optionally in the presence of a test compound.
Incubate with -6-P and measure the degree of interaction between GLK and GLKRP. Replace F-6-P, or otherwise use GLK / GL
Compounds that reduce KRP interaction will be detected by a decrease in the amount of GLK / GLKRP complex formed. Compounds that promote F-6-P binding or otherwise enhance GLKRP interactions are formed GLK / GLK.
Increased amounts of RP complex will be detected. (See Figure 3).

The F-6-P / GLKRP binding assay provides an assay method for measuring the binding interaction between GLKRP and F-6-P. This method can be used to provide additional information regarding the mechanism of action of the compound.

The compounds identified in the GLK / GLKRP binding assay have the interaction of GLK and GLKRP by substituting F-6-P or by modifying the GLK / GLKRP interaction in some other way. It is possible to adjust. For example, protein-protein interactions are generally known to occur through interactions through multiple binding sites. Thus, compounds that modify the interaction between GLK and GLKRP may act by binding to one or more of several different binding sites.

The F-6-P / GLKRP binding assay identifies only compounds that modulate the interaction of GLK and GLKRP by displacing F-6-P from its binding site on GLKRP.

GLKRP is incubated with a test compound and an inhibitory concentration of F-6-P in the absence of GLK, and the degree of interaction between F-6-P and GLKRP is measured. Compounds that displace the binding of F-6-P to GLKRP can be detected by a change in the amount of GLKRP / F-6-P complex formed. (See Figures 4 and 5).

Accordingly, in accordance with one aspect of the present invention, we provide an assay method that includes measuring the binding interaction between GLKRP and either GLK or F-6-P.

In a preferred embodiment, the assay method comprises: (i) (a) GLKRP or a homologue or fragment thereof, and (b) GLK or a homologue or fragment thereof, and / or (c) an inhibitory concentration. F-6-P is contacted in the presence and absence of the test compound; and (ii) the binding interaction between (a) and (b) or (c) is measured; And (iii) comprising determining whether the test compound modulates the binding interaction measured in (ii).

Assay Method 1 A particularly preferred embodiment of the assay method of the present invention is a scintillation proximity assay (
SPA).

SPA is a fluorescent microsphere coated with an acceptor molecule (such as an enzyme or a receptor) whose ligands will selectively bind in a reversible manner.
with the use of rosphere (Bosworth and Towers, 19
89). The technique requires the use of isotopically labeled ligands that emit low energy radiation that is easily dissipated in aqueous media. At any point during the assay, the bound labeled ligand will be in close proximity to the fluorescent microspheres, allowing the energy released to activate fluorescence and produce light. In contrast, the majority of unbound labeled ligand will be too far from the fluorescent microspheres to allow energy transfer. The bound ligand produces light, but not free ligand, allowing the degree of ligand binding to be measured without the need to separate bound and free ligand.

The scintillation proximity assay is based on the GLK / GLKRP binding assay and the F
It will be appreciated that one or both of the -6-P / GLKRP binding assays may be used.

Therefore, in a further aspect of the invention, we have one of (a), (b) or (c) radiolabeled as defined in Assay 1 and defined in Assay 1. A scintillation proximity assay for measuring the interaction between GLK and GLKRP, wherein another of (a), (b) or (c) is bound to a fluorescent microsphere.

In a particularly preferred embodiment, the radiolabeled ligand is [ ³ H] F-6-P. In another particularly preferred embodiment, the GLK is biotinylated and the fluorescent microspheres are coated with streptavidin. Biotinylated GLK binds to streptavidin-coated fluorescent microspheres, as shown in FIG. GLK, GLKR
Complex formation between P and [ ³ H] F-6-P can be detected by emission of light from the fluorescent microspheres. Compounds that modulate the interaction between GLK and GLKRP can be identified by changes in light emission from fluorescent microspheres.

In a further aspect of the invention, we define as defined in Assay 1 (
One of a) or (c) is radiolabeled and as defined in Assay 1 the other of (a) or (c) is attached to a fluorescent microsphere and as defined in Assay 1 (B) is omitted, providing scintillation proximity to measure the interaction between F-6-P and GLKRP.

In a preferred embodiment of the scintillation proximity assay for measuring the interaction between F-6-P and GLKRP, the radiolabeled ligand is [ ³ H] F-6-.
P. In a particularly preferred embodiment, (a) as defined in Assay Method 1.
Are labeled with FLAG tags, and fluorescent microspheres are coated with anti-FLAG antibody.

In a further particularly preferred embodiment, (a) as defined in Assay Method 1.
Are biotinylated and the fluorescent microspheres are coated with streptavidin.

The binding of F-6-P to GLKRP can be detected by the emission of light from the fluorescent microspheres. Compounds that displace F-6-P from its binding site on GLKRP can be identified by changes in light emission from fluorescent microspheres.

Compounds that may be tested in the assay include simple organic molecules commonly known as "small molecules," such as those having a molecular weight of less than 2000 daltons.
The assay may also be used to screen compound libraries, such as peptide libraries, including synthetic peptide libraries and peptide phage libraries. Other suitable molecules include antibodies, nucleotide sequences and any other molecule that stimulates GLK.

Modulation of activity includes either activation or inhibition. Thus, a compound that regulates GLK is a compound that stimulates or inhibits GLK. The term "GLK
"Regulator of" and "GLK modulator" are also used herein to refer to compounds that stimulate or inhibit GLK. The compounds of the present invention are NI
It has utility in the treatment of DDM, and generally this will be caused by stimulation of GLK.

The inhibitory concentration of F-6-P reduces the GLK enzyme reaction rate. It is preferred that the speed be reduced by more than 85%. This reduction is typically 25
Achieved using F-6-P concentrations of μM (when used with rat GLKRP) and 10 μM (when used with human GLKRP).

In a preferred embodiment, step (i) of assay method 1 is performed in the presence of glucose. Any suitable concentration of glucose may be used, eg 1, 2, 5, 10, 50 or 100 mM. In a particularly preferred embodiment, the glucose concentration is 5 mM.

A particular advantage of the assays of the invention is that they are very suitable for use. The assay can be performed in 96 well microtiter plates, allowing large numbers of compounds to be tested simultaneously.

Preferably, the GLK and GLKRP used in the assay of the present invention are human proteins. By homologue we mean a protein having an amino acid sequence similar to the GLK protein sequence or the GLKRP protein sequence (Tanizawa et al. 1
991 and Bonthron, D.M. T. Et al., 1994). A homologue may be a protein from the same species, ie a homologous protein family member. Alternatively, the homologue may be a similar protein from a different species, such as rat or mouse, useful in providing an animal model for NIDDM. Suitable homologues include Tanizawa et al. 1991 and Bo.
nthron, D.N. T. Et al., 1994, respectively, GLK or GL
Those that share 70% or more sequence similarity with the KRP sequence are included. Preferred sequence similarities include 75% and 80% identity, other preferred sequence similarities include 85% and 90% identity, and more preferred sequence similarity is 95%.
Includes identity.

Fragments as used herein include peptides comprising 6 or more contiguous amino acid sequences of the GLK and GLKRP sequences (shown in Tanizawa et al. 1991 and Bonthron, DT et al. 1994, respectively). Preferably, the fragment has the same or essentially the same biological activity as the full length molecule from which it is derived. The fragment may correspond, for example, to 1% or more or 5% or more or 10% or more or 50% or more or 90% or more of the full length molecule from which it is derived.

In the method of the present invention, the GLK / GLKRP binding interaction and F-6-P /
It would be expected that there are several alternative methods that may be used to measure GLKRP binding interactions. Such methods include rapid filtration of equilibrium binding mixtures, enzyme linked immunosorbent assays (ELISA), radioimmunoassays (RIA) and fluorescence resonance energy transfer assays (FRET). For more information on FRET, see International Patent Application WO 94/28166 (Zeneca).
). Methods for identifying potential drug candidates are described by Bevan et al., 199.
5 is outlined.

Once the compound is identified, medicinal chemistry techniques are applied to further refine its properties,
For example, it is possible to increase efficacy and / or reduce side effects. Therefore, in a further aspect of the invention, we have determined that in assay 1 (a) and (
Provided are compounds that modulate the interaction with either b) or (c).

In a further aspect of the invention we provide a novel modulator of GLK or a pharmaceutically acceptable salt thereof for use in the treatment of NIDDM in the human or animal body by therapy.

In a further aspect of the invention we provide a pharmaceutical composition comprising a novel modulator of GLK or a pharmaceutically acceptable salt thereof together with a pharmaceutically acceptable diluent or carrier. .

The composition may be in a form suitable for oral use, eg tablets, capsules, aqueous or oily solutions, suspensions or emulsions; suitable for topical use, eg creams, ointments, gels or aqueous or oily solutions or Suspensions; forms suitable for nasal use, such as olfactory, nasal sprays or nasal drops; forms suitable for vaginal or rectal use, such as suppositories; Separated powders, microcrystalline forms or liquid aerosols; forms suitable for sublingual or buccal use, eg tablets or capsules; or suitable for parenteral use (including intravenous, subcutaneous, intramuscular, intravascular or infusion) Type,
It may be, for example, a sterile aqueous or oily solution or suspension. In general, the compositions described above can be prepared in a conventional manner with conventional excipients.

The present invention is also a method of treating a medical condition mediated alone or in part by NIDDM or GLK, as defined above in a warm-blooded animal in need of such treatment. Also included are methods that include administering an effective amount of a different GLK modulator.

The present invention also provides the use of GLK modulators in the manufacture of a medicament for use in treating NIDDM. The dose size of the GLK modulator for therapeutic or prophylactic purposes will, of course, be in accordance with known principles of medicine, depending on the nature and severity of NIDDM, the age and sex of the patient,
And will depend on the route of administration.

When using a novel GLK modulator for therapeutic or prophylactic purposes, the factor is generally given in individual doses, if necessary, eg 0.5 per kg body weight.
It will be administered as a daily dose in the range of mg to 75 mg is administered. Generally, lower doses will be administered when the parenteral route is used.
Thus, for intravenous administration, for example, 0.5 m / kg body weight is generally used.
A dose in the range g to 30 mg will be used. Similarly, for administration by inhalation, a dose in the range, for example, 0.5 mg to 25 mg per kg body weight will be used.

The present invention will now be illustrated, but will not be limited by reference to the following examples and figures. References

[0044]

[Chemical 1]

[0045]

【Example】

Example 1 Preparation of recombinant GLK and GLKRP production mRNA Human liver total mRNA was obtained from Sambrook J, Fritsch EF &.
Maniatis T, 1989, 4 M guanidine isothiocyanate, 2.5 mM citric acid, 0.5% sarcosyl, 100.
After polytron homogenization in mM β-mercaptoethanol, 135,
Prepared by centrifugation through 5.7 M CsCl, 25 mM sodium acetate at 000 g (max).

PolyA ⁺ mRNA is available in the FastTrack ^™ mRNA Isolation Kit (Inv.
It was prepared directly using itrogen). PCR Amplification of GLK and GLKRP cDNA Sequences Human GLK and GLKRP cDNA are available from Sambrook, Frits.
ch. & Maniatis, 1989 using the established techniques
Obtained by PCR from human liver mRNA. PCR primers are Taniza
wa et al. 1991 and Bonthron, D .; T. Designed according to the GLK and GLKRP cDNA sequences shown in et al.

Cloning GLK and GLKRP cDNA in Bluescript II vector is a recombinant cloning vector system similar to that used in Yanisch-Perron C et al. (1985),
Flanked by bacteriophage T3 and T7 promoter sequences and carrying a polylinker DNA fragment containing multiple unique restriction sites, c
pBluescript II (Short et al. 1) containing the olEI-based replicon; the filamentous phage origin of replication and the ampicillin drug resistance marker gene.
998) and cloned into E. coli.

Transformation E. coli transformation was generally performed by electroporation. Share D
A 400 ml culture of H5α or BL21 (DE3) was given an OD 600 of 0.5.
Until grown in L-broth and harvested by centrifugation at 2,000 g. Cells were washed twice in ice cold deionized water, resuspended in 1 ml 10% glycerol and aliquoted and stored at -70 ° C. The ligation mixture is Milli
Desalted using pore V series ^™ membrane (0.0025 mm pore size).
40 μl cells with 1 μl ligation mixture or plasmid DNA, 0.2 c
m electroporation cuvette, incubated for 10 min on ice, and then pulsed with a Gene Pulser ^™ instrument (BioRad) at 0.5 kVcm ⁻¹ , 250 μF, 250 1/2. Transformants were selected on L-agar supplemented with 10 μg / ml tetracycline or 100 μg / ml ampicillin.

Expression GLK was expressed in E. coli BL21 cells from the vector pTB375NBSE, yielding a recombinant protein containing a 6-His tag immediately adjacent to the N-terminal methionine. Alternatively, another suitable vector is pET21 (+) DNA, Nova
gen, catalog number 697703. Using 6-His tag, Qiage
Nickel-nitrilotriacetate agarose purchased from N. (Cat. No. 30250
) Allowed the purification of the recombinant protein on a column.

GLKRP is the vector pFLAG CTC (in E. coli BL21 cells.
IBI Kodak) resulted in a recombinant protein containing a C-terminal FLAG tag. The protein was first isolated by DEAE Sepharose ion exchange,
It was then purified by utilizing the FLAG tag for final purification on a M2 anti-FLAG immunoaffinity column (catalog number A1205) purchased from Sigma-Aldrich.

Example 2 Biotinylation of GLK GLK was biotinylated by reaction with biotinamide caproate N-hydroxysuccinimide ester (biotin-NHS) (catalog number B2643) purchased from Sigma-Aldrich. Briefly, the unbound amino groups of the target protein (GLK) are reacted with biotin-NHS in the stated molar ratio to form a stable amide bond, yielding a product containing covalently bound biotin. Excess unconjugated biotin-NHS is removed from the product by dialysis. Specifically, 7.5 mg of GLK was added to 4 ml of 25 mM HEPES pH.
7.3, 0.15 M KCl, 1 mM dithiothreitol, 1 mM E
DTA, 0.31 mg biotin-N in 1 mM MgCl ₂ (buffer A)
Added to HS. The reaction mixture was dialyzed against 100 ml Buffer A containing an additional 22 mg biotin-NHS. Excess biotin-N after 4 hours
HS was removed by exhaustive dialysis against buffer A.

Example 3 GLK / GLKRP Scintillation Proximity Assay "Mix and measure" 96 well S using recombinant human GLK and GLKRP.
PA (Scintillation Proximity Assay) was developed. (A schematic of the assay is
(Shown in Figure 3). As shown in FIG. 3, GLK (biotinylated) and GLK
RP is an inhibitory concentration of radiolabeled [3H] F-6-P (Amersham Cust).
om Synthesis TRQ8689) in the presence of Streptavidin-linked SPA beads (Amersham) and shows signal. A compound that replaces F-6-P or otherwise disrupts the GLK / GLKRP binding interaction will abolish this signal.

The binding assay was performed at room temperature for 2 hours. The reaction mixture was 50 mM Tris.
-HCl (pH 7.5), 2 mM ATP, 5 mM MgCl 2, 0.5
mM DTT, recombinant biotinylated GLK (0.1 μg), recombinant GLKRP (
0.1 μg), 0.05 mCi [3H] F-6-P (Amersham), resulting in a final volume of 100 μl. After incubation, GLK / GLKR
The degree of P complex formation was 0.1 mg / well of avidin-linked SPA beads (
Amersham) and add Packard TopCount NX
Determined by scintillation counting on T.

Example 4 F-6-P / GLKRP Scintillation Proximity Assay A "mix and measure" 96-well scintillation proximity assay was developed using recombinant human GLKRP. (A schematic of the assay is shown in Figure 4). FL
AG-tagged GLKRP was prepared in the presence of an inhibitory concentration of radiolabeled [3H] F-6-P,
Incubate with Protein A coated SPA beads (Amersham) and anti-FLAG antibody. As shown in Figure 4, a signal is generated. F-6
Compounds that replace -P will abolish this signal. This assay, combined with the GLK / GLKRP binding assay, would allow the observer to identify compounds that disrupt the GLK / GLKRP binding interaction by substituting F-6-P.

The binding assay was performed at room temperature for 2 hours. The reaction mixture was 50 mM Tris.
-HCl (pH 7.5), 2 mM ATP, 5 mM MgCl 2, 0.5
mM DTT, recombinant FLAG-tagged GLKRP (0.1 μg), anti-Flag
M2 antibody (0.2 μg) (IBI Kodak), 0.05 mCi [3H]
F-6-P (Amersham) was included resulting in a final volume of 100 μl. After the incubation, the degree of F-6-P / GLKRP complex formation was 0.1 m.
Determined by adding g / well of Protein A-coupled SPA beads (Amersham) and scintillation counting on a Packard TopCount NXT.

Example 5 Production of Human Anti-GLKRP Antibody Purified recombinant human FLAG-tagged GLKRP protein was used in combination with Freund's adjuvant (Freund, 1956) to produce antibodies in New Zealand white rabbits. did. Methods for producing antibodies are known in the art and are described in Harlow and Lane 1988.

GLKRP protein antibodies or fragments thereof can be used to reduce inappropriately enhanced inhibition of GLK by GLKRP that occurs in certain pathological abnormalities including diabetes.

[Brief description of drawings]

FIG. 1 shows intracellular glucose uptake by the plasma membrane glucose transporter GLUT2, and glucose-6-by glucokinase (GLK).
Figure 7 shows its conversion to phosphoric acid (G-6-P). Enzyme activity is downregulated by the glucokinase regulatory protein (GLKRP). The binding interaction between GLK and GLKRP is due to fructose-6-phosphate (F
-6-P) results in decreased glucokinase activity and decreased glucose metabolism. Fructose-6-phosphate is displaced from its binding site on GLKRP by fructose-1-phosphate (F-1-P), leading to dissociation of GLK and GLKRP, and increased glucokinase activity and glucose metabolism. Cause irritation.

FIG. 2 shows a reaction pathway in which glucose is phosphorylated to G-6-P by glucokinase in the presence of ATP. When the reaction is coupled to G-6-P dehydrogenase, it is possible to determine the reaction rate by measuring the reduction of NADP to NADPH, which is detected as a change in optical density at 340 nm.

FIG. 3 shows a preferred embodiment of the GLK / GLKRP binding assay. The figure illustrates the emission of light from streptavidin-coated fluorescent microspheres upon formation of a binding complex between biotinylated GLK, GLKRP and [ ³ H] F-6-P.

FIG. 4 shows a preferred embodiment of the F-6-P / GLKRP binding assay. The figure illustrates light emission from fluorescent microspheres coated with Protein A and anti-FLAG antibody on formation of a binding complex between FLAG-tagged GLKRP and [ ³ H] F-6-P.

FIG. 5 is a titration curve showing displacement of F-1-P by F-6-P, obtained using the GLK / GLKRP scintillation proximity assay of the present invention.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) // C12N 15/09 C12N 15/00 A (81) Designated country EP (AT, BE, CH, CY, DE) , DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML) , MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, BZ, CA, CH, CN, CR, C , CZ, DE, DK, DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, PL, PT, RO, RU, SD, SE, SG , SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW

Claims (11)

[Claims] 1. An assay method comprising measuring the binding interaction between GLKRP and either GLK or F-6-P. 2. The assay method according to claim 1, wherein: (i) (a) GLKRP or a homologue or fragment thereof, and (b) GLK or a homologue or fragment thereof, and / or (c) an inhibitory concentration. F-6-P are contacted in the presence and absence of the test compound; and (ii) the binding interaction between (a) and either (b) or (c) is measured. And (iii) determining whether the test compound modulates the binding interaction measured in (ii). 3. Any one of (a), (b) or (c) is radiolabeled and another of (a), (b) or (c) is attached to a fluorescent microsphere. The method according to claim 2. 4. The method of claim 3, wherein (c) is [ ³ H] F-6-P. 5. The method according to claim 3 or 4, wherein (b) is biotinylated and the fluorescent microspheres are coated with streptavidin. 6. The method according to claim 2, wherein (b) is omitted. 7. The method of claim 3, wherein (a) is labeled with a FLAG tag, (b) is omitted and the fluorescent microspheres are coated with anti-FLAG antibody. 8. The method of claim 3, wherein (a) is biotinylated, (b) is omitted, and the fluorescent microspheres are coated with streptavidin. 9. The method according to any one of claims 2 to 8, wherein step (i) is performed in the presence of glucose. 10. The method according to claim 2, wherein (a) and (b) or (c).
A compound that regulates the interaction with either one of. 11. Use of a compound according to claim 10 in the manufacture of a medicament for use in the treatment of NIDDM.