JP2001183366A - Screening method for agonist - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new agonist screening method, in which the mechanism of the transmission of a signal is used. SOLUTION: In this method for evaluating the agonist selectivity with reference to the receptor of a physiologically active substance, an agonist candidate substance is added to the following elements, i.e., (a) a receptor system, (b) a receptor substrate in which a tyrosine residual group is oxidized by bonding the agonist to the receptor or a peptide fragment which is derived from receptor protein and which contains the tyrosine residual group and (c) a reaction system which contains the target protein of the receptor substrate changed into phosphoric acid. The bond amount of the element (c) to the element (b) which is changed into a solid phase is used as an index, and the selectivity of the candidate substance is evaluated.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The invention of this application relates to a method for evaluating the selectivity of a physiologically active substance for a receptor. More specifically, the invention of this application relates to a method for evaluating agonist selectivity useful for screening various physiologically active substances such as endocrine drugs, growth hormone, and endocrine disrupting chemicals.

[0002]

BACKGROUND OF THE INVENTION Screening agonists for physiologically relevant selectivity is important not only for pharmaceutical needs but also for biological research. However, conventional receptor binding tests may not only provide sufficient information on the activity of agonists, but also may not be able to distinguish between agonists and antagonists.

[0003] Recently, a method for evaluating the chemoselectivity of an agonist based on a physiologically relevant signal transduction mechanism by a receptor protein has been proposed (Reference No. 1-9). Typical examples of such new methods include, for example, those targeting transport proteins embedded in ion channels or lipid bilayer membranes (Reference Nos. 4-7).

The tyrosine kinase signaling system, which is widely present in cells and plays an important role, has also been used to evaluate the agonist selectivity of insulin (Reference No. 2-10).

When insulin binds to its receptor,
The insulin-receptor complex phosphorylates a tyrosine residue of an endogenous substrate protein such as insulin receptor substrate 1 (IRS-1) (Reference Nos. 11 and 12). Therefore,
The inventors of the present application provide a method for evaluating the chemical selectivity of an agonist by measuring the amount of phosphorylated tyrosine of IRS-1 generated by the insulin receptor kinase reaction using an anti-phosphorylated tyrosine monoclonal antibody. (Reference 2).

[0006]

As described above, various methods for screening for an agonist for a receptor have been proposed using a reaction chain of an intracellular signal transduction pathway generated by binding of a physiologically active substance and a receptor. I'm starting. However, in the conventional method, for example, it is necessary to isolate a phosphorylated substrate protein to be measured, or an immunoassay method requires an amplification step. Further improvement has been demanded for automation and automation.

[0007] The invention of this application has been made in view of the circumstances described above, and has as its object to provide a new agonist screening method using a signal transmission mechanism.

[0008]

This application provides the following inventions (1) to (5) to solve the above-mentioned problems. (1) A method for evaluating the selectivity of a physiologically active substance for a receptor, comprising the following elements: (a) a receptor protein; (b) phosphorylation of a tyrosine residue by binding of an agonist to a receptor A receptor substrate, or a tyrosine residue-containing peptide fragment derived from the substrate protein; and (c) an element obtained by adding an agonist candidate substance to a reaction system having a phosphorylated receptor substrate target protein and immobilizing the same.
A method for screening for an agonist, comprising evaluating the selectivity of a candidate substance using the amount of binding of an element (c) to (b) as an index. (2) Element for element (b) fixed to sensor chip
The screening method according to the invention (1), wherein the amount of the binding (c) is measured by a surface plasmon resonance method. (3) The physiologically active substance is insulin, the element (b) is a peptide fragment containing a tyrosine residue derived from insulin receptor substrate 1 (IRS-1), and the element (c) is the src homology-2 (SH2) domain. Containing protein or its SH
The invention (1) or (2), which is a two-domain polypeptide
Screening method. (4) IRS-1 derived tyrosine residue-containing peptide fragment has the amino acid sequence Tyr MetXaa Met, Tyr Xaa Asn Xaa,
Alternatively, the screening method according to the above invention (3), which is a peptide fragment containing Tyr Ile Asp Leu. (5) The screening method according to the invention (4), wherein the peptide fragment is a peptide fragment having the amino acid sequence of SEQ ID NO: 1.

That is, the inventions (1) to (5) of this application
Is a method for evaluating agonist selectivity to a receptor using a reaction downstream of a phosphorylation reaction in a signal transduction reaction chain generated by binding of a physiologically active substance and a receptor. For example, in the case of insulin, insulin
-Receptor complex phosphorylates the tyrosine residue of IRS-1, which is then phosphorylated tyrosine which is then phosphatidylinositol 3-kinase (PI-
3: src homology-2 (SH2) domain such as rasguanine-nucleotide release complex (Grb2-Sos: ref. 14), or src homology-containing phosphatase-2 (SHP2: ref. 15) Serves as a label for binding proteins. Such SH
Proteins containing two domains act as switch units by specifically recognizing phosphorylated tyrosine residues. However, proteins with SH2 domains are
A specific amino acid residue near each phosphotyrosine, rather than recognizing only phosphorylated tyrosine;
PI-3 kinase is "Tyr Met Xaa Met", Grb2-Sos is "Tyr Xaa Asn Xaa", and SHP2 is "Tyr Ile Asp Leu".
Recognize the sequence Therefore, the specific binding of the SH2 domain-containing protein to phosphorylated tyrosine and its neighboring amino acid residues functions as one special signal in insulin signal transduction, and this signal is used as an indicator to determine the selectivity of the agonist for the insulin receptor. It becomes possible to evaluate.

In addition to insulin, epidermal growth factor (EG)
For F), interferon β and the like, an agonist can be screened on the same principle. Hereinafter, the invention
The embodiments (1) to (5) will be described in detail.

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION The invention (1) of the present application comprises the following elements: (a) a receptor protein; (b) a receptor substrate whose tyrosine residue is phosphorylated by binding of an agonist to a receptor; or A tyrosine residue-containing peptide fragment derived from the substrate protein; and (c) an element for the phosphorylated element (b) immobilized by adding an agonist candidate substance to a reaction system having the target protein of the phosphorylated receptor substrate, This is a method characterized by evaluating the selectivity of a candidate substance using the binding amount of c) as an index.

The receptor protein of the element (a) is, for example,
Animal cells and the like in which a receptor protein of a physiologically active substance is expressed in a large amount on its surface can be used. The receptor substrate of the element (b) is IRS-1 when the bioactive substance is insulin, and when the bioactive substance is EGF,
In the case of interferon β, the phosphorylation domain of the EGF receptor is a vav protein or the like. Also elements
(b) may be a peptide fragment derived from a substrate protein.

[0013] The target protein of the element (c) is a protein to which the receptor substrate of the phosphorylated element (b) specifically binds.
SH2 domain-containing proteins such as I-3 kinase, Grb2-Sos, and SHP2, or SH2 domain polypeptides of these proteins. When the physiologically active substance is EGF, for example, the element (c) can be Grb2, and when it is interferon β, it can be Lck protein or the like.

In the present invention (1), after the addition of the agonist candidate substance and the reaction,
By measuring the amount of binding of element (c) to (b),
Evaluate the selectivity of the agonist. That is, element (a),
When a candidate substance is added to the reaction system (liquid phase) composed of (b) and (c), if this substance has excellent selectivity as an agonist, a large amount of a candidate substance-receptor complex is formed to form a complex. Element (b) is phosphorylated. Most of the element (c) binds to the phosphorylated element (b) in the liquid phase, and the amount of binding to the solidified phosphorylated element (b) decreases. on the other hand,
When the candidate substance has low selectivity as an agonist,
Candidate substance-receptor complex is difficult to form
(b) is also not phosphorylated. Therefore, the element (c) that selectively binds to the phosphorylated element (b) binds more to the solid-phased phosphorylation element (b).

As a method for measuring the amount of binding of the element (c) to the phosphorylation element (b), the means of the invention (2),
A method of monitoring the binding amount of the element (c) to the element (b) immobilized on the sensor chip by a surface plasmon resonance (SPR) method can be adopted.

In the method of this application, as described above, a peptide fragment derived from a substrate protein can be used as the element (b). When the physiologically active substance is insulin, this peptide fragment is In particular,
It is a peptide fragment containing the amino acid sequence Tyr Met Xaa Met, Tyr Xaa Asn Xaa, or Tyr Ile Asp Leu. That is, these peptide fragments have, in order, an amino acid sequence that selectively binds to PI-3 kinase, Grb2-Sos and SHP2. Furthermore, when the target protein is PI-
In the case of 3-kinase, examples of the peptide fragment include a peptide having the amino acid sequence of SEQ ID NO: 1 (hereinafter sometimes referred to as Y939 peptide). This Y939
The peptide has a tyrosine phosphorylation site on IRS-1 and an IR
It is composed of the PI-3 kinase binding site of S-1 (Reference
19, 20).

FIG. 1 shows that the physiologically active substance is insulin, the element (b) in the reaction system is Y939 peptide, and the element (c) is PI-
SH2 domain polypeptide of SH3 kinase (SH2
N) is a schematic process diagram in the case where phosphorylated Y939 (pY939 peptide) is immobilized on a sensor chip and the amount of SH2N bound to this pY939 peptide is measured by the SPR method.

Hereinafter, the invention of this application will be described in more detail and specifically with reference to examples, but the invention of this application is not limited to the following examples.

[0019]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Method 1-1. Materials Human insulin was purchased from Peptide Research Institute (Osaka, Japan). Recombinant human insulin-like growth factor-I (IGF-I) was purchased from Funakoshi (Tokyo, Japan).
Recombinant human insulin-like growth factor-II (IGF-II) was provided by Wakunaga (Hiroshima, Japan). Y939 peptide chemically synthesized and purified by HPLC and tyrosine-phosphorylated Y939 (pY939) were both purchased from Sawaday Technology (Tokyo, Japan). The Y939 and pY939 peptides differ from the IRS in that a cysteine residue was added.
-1 differs from the original amino acid sequence. This is because both peptides need to be biotinylated at their C-termini. N-hydroxysuccinimide (NHS) was obtained from Wako Pure Chemical (Osaka, Japan). (±) -5- [4- (6-hydroxy-2,5,7,8-tetramethylchroman-2-ylmethoxy)
[Benzyl] -2,4-thiazolidinedione (CS-045, troglitazone) was provided by Sankyo (Tokyo, Japan). (±)
-5- [p- [2- (5-ethyl-2-pyridyl) ethoxy] benzyl]-
2,4-Thiazolidinedione hydrochloride (AD-4833, pioglitazone) was provided by Takeda Pharmaceutical (Osaka, Japan). 2-
[4- (2-Hydroxy-ethyl) -1-piperazinyl] ethanesulfonic acid (HEPES) and N-ethyl-N '-[(dimethylamino) propyl] carbodiimide (EDC) are available from Dojin Laboratories (Kumamoto, Japan) did. Mercaptoundecanoic acid
(MUA) and biotinylated pY939 were synthesized by the method described in the literature (References 2, 22). The E. coli strain (DH5α) transformed with the SH2 domain was provided by Kobe University (Kobe, Japan). The Chinese hamster egg cell line (CHO-HIR cell) overexpressing the human insulin receptor was provided by the University of Tokyo (Tokyo, Japan). All other salts and solvents were of the highest quality available. Milli-Q level water (> 18.2 MΩ resistance) obtained with a Milli-Q Plus system (Millipore, Bedford, MA) was used for all aqueous solutions. 1-2. SPR measurement All SPR measurements were performed with the SPR sensor system of DKK (Tokyo, Japan). In this sensor system, an SPR is used to detect a change in the refractive index in the flow cell due to the binding of a molecule to a solid-phased ligand.
Is used. The change in the refractive index detected by the SPR sensor system is expressed as an arbitrary unit called a resonance unit (RU) (Reference No. 23). In this example, the flow rate of the solution through the flow cell in contact with the SPR sensor chip was 50 μL at 25 ° C.
/ Min always kept. 1-3. Preparation of SH2 domain polypeptide and human insulin receptor N-terminal SH2N domain of p85 subunit of PI-3 kinase (S
H2N) was obtained by the method described in the literature (literature numbers 21, 2).
Four). SH2 expressed as a glutathione-S-transferase (GST) fusion protein in Escherichia coli strain (DH5α)
N was purified by glutathione-agarose affinity chromatography. The purity of SH2N was evaluated by staining SDS-polyacrylamide electrophoresis (SDS-PAGE) with Coomassie, and a single band of 36 KDa was obtained. Purified SH2
N was stored at -80 ° C until used in experiments.

The insulin receptor has been described in the literature (Ref. No. 24,
25) A Chinese hamster egg cell line overexpressing human insulin receptor (CHO-
HIR cells). The insulin receptor extracted from CHO-HIR cells was purified by WGA-Sepharose affinity column chromatography. The eluate containing the insulin receptor was concentrated by pressure dialysis using a Diaflow ultrafiltration membrane PM30 (Amicon, Burberry, MA). The concentrated insulin receptor solution was dispensed into 200 μl aliquots and stored at −80 ° C. until used in experiments. 1-4. Preparation of sample solution Each sample solution was prepared in a micro test tube. 0.05% Triton X
-100, 150 mM NaCl, 100 (M ATP, 5 mM MnCl ₂ , 10 (g
Insulin receptor (0.16 pmol) 10 mM HEPES / N
The active substance at each concentration was added to a solution of 45 (L) containing aOH (pH 7.4) and reacted at 4 ° C. for 1 hour in a microtube. For phosphorylation of the Y939 peptide by the insulin receptor, a HEPES buffer solution was used. (0.05% Triton X-100, 150 mM NaCl
And 10 (M Y) dissolved in 10 mM HEPES / NaOH, pH 7.4).
Add 30 mL of 939 to a microtube and allow the mixture to
For a time, the mixture was stirred with a mixer (Tomy Seiko, Tokyo, Japan). To stop the kinase reaction by covering Mn ²⁺ with EDTA, HEPES buffer containing EDTA was added to a final concentration of 7.5 mM ²⁴ . When SH2N was added to this solution, it was equilibrated at 4 ° C. for 1 hour prior to SPR measurement. 1-5. Immobilization of pY939 on sensor chip The surface of the gold substrate of the sensor chip is self-assembled by immersing the sensor chip in 1 mM mercaptoundecanoic acid (MUA) dissolved in ethanol at 4 ° C for 10 hours or more. A simple monolayer was formed and modified. After installing the sensor chip modified with MUA on the glass prism of the SPR sensor system, the modified sensor surface is reduced by 0.05%
Triton X-100, 150 mM NaCl and 10 mM HEPES / NaOH,
(PH 7.4). The carboxyl group of MUA on the gold surface is changed to 350 (L
Of NHS / EDC mixture (0.05 M NHS, 0.2 M EDC in Milli-Q aqueous solution) for 7 minutes. 5 mM maleate buffer (p
Coupling of avidin and MUA via free amino groups of avidin was performed by injecting a 125 (g / mL avidin solution in H6.0) onto a 350 mL sensor chip for 7 minutes (Reference No. 26). Is immobilized, then 200 (M
The free carboxyl groups of MUA were masked by injecting 350 ml of an ethanolamine solution (L) onto the sensor chip for 7 minutes. To immobilize pY939 on the gold surface with the avidin-biotin complex, add 7 mM phosphoric acid. Acid buffer
0.15 nM biotinylated pY939 (biotin-pY939) dissolved in (pH 7.4) was injected onto the sensor chip for 5 minutes (Reference No. 2). The gold surface modified with pY939 was washed with 1 mM HCl containing 500 mM NaCl for 3 minutes, and the surface was equilibrated with the prepared solution. 1-6. Correction of SPR signal for binding of pY939 to SH2N RU, which is the observed value of SPR signal, is ΔRU for binding of SH2N to biotinylated pY939 immobilized on a sensor chip.
Indicated by That is, it was obtained as the difference between the SPR signals when SH2N was present and when it was not present in the sample solution.

When the sensor chip on which pY939 is immobilized is repeatedly used for measuring the SPR signal, unexpected experimental changes such as non-selective protein adsorption on the sensor chip and subtle temperature changes in the flow cell may cause ΔRU fluctuated even with the same sample solution. To correct for the possibility of such fluctuations in ΔRU values, two different standard solutions were measured every 10 times as follows: 100 nM SH2N was included in both, 1 (including pY939 of M Things and things not included 2
The ΔRU values measured for each sample solution were corrected for differences in ΔRU values obtained with the two standard solutions, using the two solutions as standards for correction. According to the following formula named Rp.

Rp (%) = (SS ₁ ) / (S ₀ -S ₁ ) × 100 (1) where S is ΔRU of each sample solution, and S ₀ is 1 (ΔRU of a standard solution not containing pY939 of M) , S ₁ is the value of the case containing pY939 of 1 (M. all measurements were carried out in triplicate or more, the results were expressed as mean ± standard deviation. independently to compare the means 2 samples Student P values were determined as statistically significant by P-test; 2. Results 2-1 SPR response to insulin When insulin binds to its receptor, Y939 is phosphorylated. Phosphorylated Y939 (pY939) binds to SH2N by a reaction in which SH2N competitively binds to either biotinylated pY939 on the SPR sensor chip or pY939 in a solution generated by the insulin receptor kinase reaction. , SH2N pY93 for insulin signaling
The binding event to 9 was monitored. A typical temporal trend of the SPR signal is shown in FIG. After equilibrating the sensor chip ((1) in FIG. 2), the SPR signal (RU) observed at this time is represented by 0 RU, but the insulin receptor induced by 100 nM SH2N and insulin was not detected. A sample solution containing pY939 generated by the kinase reaction was injected onto the sensor chip for 5 minutes. For 1.0 x 10 ^-12 M insulin, biotin-pY939 binds to SH2N for SPR
The signal increases (FIG. 2, (2)). At an insulin concentration of 9.8 × 10 ⁻⁸ M, the rate of increase of the SPR signal decreases (data not shown). 0.5% SDS solution 1 to dissociate SH2N from biotin-pY939 on sensor chip
(3) in FIG. 2 and the sensor surface was equilibrated with normal buffer until the SPR signal returned to 0 RU ((4) in FIG. 2). This procedure was repeated for all sample solutions during the measurement.

FIG. 3 shows that the SPR signal is dependent on the concentration of insulin. The Rp value of the Y-axis is given by equation
2 shows the corrected SPR signal calculated by the above method. When the insulin concentration increased, the Rp value decreased. This is due to the fact that as the concentration of insulin increases, pY939 generated by the insulin receptor kinase reaction increases, and SH2N binds to biotin-pY939 on the sensor chip by a competitive reaction.
Is to decrease.

Based on the above results, this detection system is competitive.
Biosynthesis in insulin signaling by combined reactions
Tin-pY939 binding to SH2N can be detected and
Highly sensitive assessment of phosphorus receptor agonists
Was confirmed. 2-2. SPR response to insulin-like growth factor IGF-I and IGF-II have a structure similar to insulin.
It is a peptide hormone and, like insulin, insulin.
Binds to the receptor. Therefore, these growth factors
Like insulin, the insulin receptor itself
Receptor binding, such as oxidation or phosphorylation of endogenous substrates
Induces subsequent insulin signaling. these
Insulin signal for insulin-like growth factor
To assess selectivity as an agonist in transduction
In addition, insulin receptor induced by IGF-I and IGF-II
The amount of pY939 produced by the body's kinase reaction
It was measured as a degree effect. The results are shown in FIG. IGF-I
8.5 x 10 density^-Ten 9.8 x 10 from M^-8 Increase to M
As the Rp value decreased. Its concentration is 8.5 x 10
^-Ten At lower than M, no change in Rp value was observed. I
3.8 x 10 for GF-II ^-Ten 9.8 x 10 from M^-7 At a concentration of M
Rp value decreased. The Rp value in both cases
Decreases are associated with increasing concentrations of these agents.
Caused by the insulin receptor kinase reaction
This is because the amount of 9 increases.

IGF-I for the insulin receptor and
The binding affinity of IGF-II is known (References 27 and 28),
IGF-I and IGF-II have a dissociation constant (Kd) of 11 nM each
And 0.9 nM (Table I). At such a concentration of dissociation constant, each agonist occupies half of all insulin receptors. In this example, the difference in the kinase activity of the insulin receptor bound to the agonist at the Kd value concentration of each agonist was evaluated by comparing the Rp values obtained for each agonist. The results are summarized in Table 1. The Rp value at the Kd value concentration of the agonist is IGF-II <IGF-I <
It was confirmed that the insulin activity increased in the order of insulin, and the kinase activity of the insulin receptor induced by the agonist increased in this order.

[0026]

[Table 1]

2-3. SPR response to antidiabetic drugs It has been reported that the insulin-like effect of vanadate or vanadyl ion stimulates glucose uptake in various cells (Reference Nos. 29-31). Although the potential of such ions for antidiabetic drugs has been investigated (Ref. 32), the exact mechanism why such ions elicit the aforementioned insulin-like effect is not yet clear. Therefore, the effects of vanadate and vanadyl on phosphorylation of Y939 and binding of pY939 to SH2N were examined using the screening method of the present invention.

The results are shown in FIG. The lack of a significant change in Rp values in the presence and absence of vanadyl or vanadate indicates that such ions could also alter the insulin receptor tyrosine kinase activity and subsequently pY93
This shows that 9 does not affect the binding to SH2N.

Vanadate and vanadyl are known as protein phosphotyrosine phosphatase (PTPase), alkaline phosphatase, Ca ²⁺ / Mg ²⁺ ATPase and N ⁺ K ⁺ ATP
It is known to be an inhibitor of ase (Reference No. 33
-37). The exact mechanism why vanadium ions inhibit such phosphate hydrolases is unknown. However, it is widely believed that such a vanadium ion binds to the phosphotyrosine binding site of the enzyme due to its similar structure to phosphate (Reference No. 38). Although SH2N used in this example recognizes phosphorylated tyrosine like PTPase, no effect of vanadate on binding of phosphorylated tyrosine to SH2 domain has been reported. This example confirmed that vanadate and vanadyl did not act as agonists or antagonists for phosphorylated tyrosine.

On the other hand, thiazolidine drugs such as troglitazone and pioglitazone are known as oral hypoglycemic agents for non-insulin-dependent diabetes mellitus (NIDDM) (Reference Nos. 39 and 40). The effect of such drugs on tyrosine kinases at the insulin receptor was assessed by this method. The results obtained are shown in FIG. 10 (Sufficient changes in Rp values were not observed for sample solutions in the presence and absence of troglitazone or pioglitazone at M, indicating that such agents also undergo insulin receptor kinase reactions and subsequent pY939 SH2N It does not affect the binding to.

[0031]

As described in detail above, the present application provides a new agonist screening method utilizing a signal transduction mechanism. This method activates the phosphorylation signal transduction pathway by various physiologically active substances, since it is not necessary to isolate a phosphorylated substrate protein as a specimen, nor does it require an amplification step essential for immunoassay. It can be applied to the rapid screening of such agonists, and therefore, it is an extremely useful method not only in basic biology research but also in pharmaceutical research.

[0032]

[Sequence List] SEQUENCE LOSTING <110> Japan Science and Technology Corporation <120> Agonist screening method <130> NP99419 <160> <210> 1 <211> 12 <212> PRT <213> Artificial Sequence <220> <213 > Synthesized oligopeptide <400> 1 Ser Glu Glu Tyr Met Asn Met Asp Leu Gly Pro Cys 1 5 10

[0033]

[References] 1. J. Pharm. Biomed. Anal. 1999, 19, 205-215. 2. Anal. Chem. 1998, 70, 2345-2352. 3. Anal. Chem. 1997, 69, 3081-3085. 4. Biosens. Bioelectron. 1997, 12, 425-439. 5. Anal. Chem. 1993, 65, 363-369. 6. Sens. Actuators B 1993, 13, 173-175. 7. Anal. Chem. 1991, 63, 2787-2795. 8. Biosens, Bioelectron. 1991, 6, 233-237. 9. Anal. Lett. 1987, 20, 857-870. 10. Anal. Biochem. 1998, 257, 134-138. 11. J. Biol. Chem. 1994, 269, 1-4. 12. Trends Cell Biol. 1994, 4, 115-119. 13. EMBO J. 1992, 11, 3469-3478. 14. Mol. Cell. Biol. 1993 J. Biol. Chem. 1993, 268, 11479-11481. 16. J. Biol. Chem. 1995, 270, 3662-3666. 17. Mol. Cell Biol. 1994, 14, 3577-3587. 18. Science 1997, 278, 2075-2080. 19. Biochemistry 1993, 32, 3197-3202. 20. Biochem. Biophys. Res. Commun. 1992, 183, 280-285. 21. J. Biol. Chem. 1992, 267, 25958-25966. 22. J. Am. Chem. Soc. 1989, 111, 321-325. 23. BioTechnology 1992, 10, 390-393. 24. Anal. Chem., 1999, 71, 3948-3954. 25. J. Med. Chem. 1989, 32, 234. 4-2352. 26. Anal. Biochem 1991, 198, 268-277. 27. J. Biol. Chem. 1993, 268, 1087-1094. 28. J. Biol. Chem. 1991, 266, 20626-20635. 29 Life Sci. 1989, 44, 1491-1497. 30. J. Biol. Chem. 1980, 255, 5306-5312. 31. Nature 1980, 255, 5306-5312. 32. Mol. Cell. Biol. 1998, 188. 33, J. Biol. Chem. 1994, 269, 9521-9527. 34. Biochem. Biophys. Res. Commun. 1982, 107, 1104-1109. 35. Curr. Top. Cell. Regul. 1981. , 20, 247-301. 36. Trends Biochem. Sci. 1980, 5, 92-94. 37. Nature 1978, 272, 552-554. 38. Mol. Cell. Biochem. 1998, 182, 109-119. 39 Arzneimittel-Forschung 1990, 40, 37-42. 40. J. Med. Chem. 1989, 32, 421-428.

[Brief description of the drawings]

FIG. 1 is a schematic view illustrating the process of an insulin screening method as an example of the method of the present invention.

FIG. 2 is a graph showing a typical temporal trend of an absolute angle of an SPR signal.

FIG. 3 is a graph showing a correlation between an Rp value and an insulin concentration. The concentrations of free insulin in the sample solution were 1.0 × 10 ⁻¹² M, 1.0 × 10 ⁻¹¹ M, 1.6 ×
10 ^-10 M, 8.0 x 10 ^-9 M and 9.8 x 10 ^-8 M.

FIG. 4 is a graph showing a correlation between an Rp value and an agonist concentration. The concentrations of free IGF-I in the sample solution were 8.4 x 10 ^-12 M, 8.4 x 10 ^-11 M, and 8.5 x 10 ^{-10, respectively.}
M, 9.1 × 10 ⁻⁹ M, 9.8 × 10 ⁻⁸ M and 9.8 × 10 ⁻⁷ M. The concentrations of free IGF-II in the sample solution were 3.0 x 10 ^-12 M, 3.1 x 10 ^-11 M, 3.8 x 10 ^-10 M, 1.
0 x 10 ^-9 M, 8.1 x 10 ^-9 M, 9.8 x 10 ^-8 M and 9.8 x
10 ^-7 M.

FIG. 5 is a graph showing the relationship between Rp value and agonist concentration. 100 (M) vanadium ions (vanadate and vanadyl) and 10 (M) antidiabetic agents (troglitazone and pioglitazone) were used as candidate agonists.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) // A61K 38/28 A61K 45/00 4H045 45/00 C07K 7/08 ZNA C07K 7/08 ZNA 14/62 14/62 14/72 14/72 17/00 17/00 C12N 1/21 C12N 1/21 C12P 21/02 C 5/10 A61K 37/26 C12P 21/02 C12N 5/00 B (72) Inventor Yoshida Oshi 2-7-7 Sekicho Kita, Nerima-ku, Tokyo F term (reference) 2G045 AA34 AA40 DA36 DA77 FB02 2G059 AA01 BB12 CC16 DD03 DD13 EE02 EE04 JJ12 4B064 AG01 AG20 BH09 CA02 CA10 CA19 CC24 CE06 CE08 CE12 DA13 4B090 AAA BD16 CA24 CA46 4C084 AA30 DB34 ZC021 ZC352 4H045 AA30 BA13 BA16 BA41 BA52 BA60 CA40 DA37 DA50 EA50 FA74 FA81 GA10 GA26

Claims (5)

[Claims] 1. A method for evaluating the agonist selectivity of a physiologically active substance for a receptor, comprising the following elements: (a) a receptor protein; (b) a tyrosine residue is converted to phosphorus by binding of the agonist to the receptor. A receptor substrate to be oxidized, or a peptide fragment containing a tyrosine residue derived from the substrate protein; and (c) phosphorylation in which a candidate agonist is added to a reaction system having a target protein of the phosphorylated receptor substrate and immobilized. A method for screening for an agonist, comprising evaluating the selectivity of a candidate substance using the amount of binding of element (c) to element (b) as an index. 2. The screening method according to claim 1, wherein the amount of binding of the element (c) to the element (b) immobilized on the sensor chip is measured by a surface plasmon resonance method. 3. The physiologically active substance is insulin, the element (b) is a peptide fragment containing a tyrosine residue derived from insulin receptor substrate 1 (IRS-1), and the element (c) is src.
The screening method according to claim 1 or 2, which is a homology-2 (SH2) domain-containing protein or an SH2 domain polypeptide thereof. 4. A tyrosine residue-containing peptide fragment derived from IRS-1 has an amino acid sequence of Tyr Met Xaa Met, Tyr Xaa A
4. The screening method according to claim 3, which is a peptide fragment containing sn Xaa or Tyr Ile Asp Leu. 5. The screening method according to claim 4, wherein the peptide fragment is a peptide fragment having the amino acid sequence of SEQ ID NO: 1.