JP2002122602A - 50% inhibition concentration determining method for chemical substance - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for evaluating the hormonic activity (50% inhibition concentration) of a chemical substance easily at a low cost with a small number of steps of operation. SOLUTION: A sample chemical substance is added to a solution containing a bound body of a hormone receptor and a ligand bound to the hormone receptor to emit fluorescence. The fluorescence intensity that changes according to the added concentration of the sample chemical substance is measured, and the concentration of the sample chemical substance showing one half of the difference between the fluorescence intensity of the solution containing the bound body of the hormone receptor and ligand, and the fluorescence intensity of the solution in a ligand separated state in the solution containing the hormone receptor and ligand is made 50% inhibition concentration. The ligand is preferably cumestrol or fluorescein cress-linked ethynil estradiol. As the hormone receptor, an estrogen receptor is given as an example.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for determining the inhibitory concentration of endocrine disrupting substances (so-called chemicals such as environmental hormones), and more particularly, to 50% inhibition of endocrine disrupting chemicals against receptors such as estrogen. It relates to a method for determining the concentration.

[0002]

2. Description of the Related Art Chemical substances derived from industry exist in the natural environment, and some of these chemical substances have hormonal activity, so that they inhabit the natural environment (hereinafter referred to as environmental organisms) and humans. It is suspected that when taken into a living body, it disrupts the endocrine system of environmental organisms and humans, causing reproductive disorders and the like, which is a major social problem. In order to address this problem, a method of assessing the endocrine disrupting activity of chemicals, as well as investigating the actual situation of exposure of environmental suspected chemicals to environmental organisms and humans, elucidating the mechanism of endocrine disrupting effects, Development is urgent.

Conventionally, as a method for evaluating the endocrine substance-like activity of a chemical substance, for example, as a method for evaluating the endocrine substance-like activity for an estrogen receptor which is a kind of hormone receptor, a radioactive ligand such as radiolabeled estradiol is used. There is a binding test method used.

In this binding test method, a chemical substance of a sample is added to a mixture of a ligand and a hormone receptor to release a part of the ligand bound to the hormone receptor. To combine. Here, by measuring the concentration of the free ligand or the concentration of the ligand bound to the hormone receptor, the amount of the sample chemical substance bound to the hormone receptor in place of the ligand can be determined.

[0005] The amount of the sample chemical substance bound to the hormone receptor is proportional to the strength of the binding of the sample chemical substance to the hormone receptor.

[0006] The relationship between the concentration of a chemical substance and the concentration of a ligand bound to a hormone receptor, that is, the "binding curve of a chemical substance in a mixture of a ligand and a hormone receptor" is expressed by:
As shown in FIG. The chemical concentration at which half of the ligand bound to the hormone receptor displaces the chemical is defined as the 50% inhibitory concentration (IC50) of the chemical. Although this IC50 value depends on the test conditions such as the type and concentration of the ligand, it is proportional to the strength of binding between the chemical substance and the hormone receptor under the same test conditions.
From the C50 value, the relative relationship between the binding strength of the chemical substance to the hormone receptor can be evaluated. That is, the degree of the hormone-like activity of the chemical substance can be evaluated from the IC50 value.

[0007] The above radioligand can be detected with high sensitivity. However, in the binding test method using this radioligand, since radioactive substances are handled, facility restrictions,
Safety considerations are required, and in operation, the concentrations of free ligands and sample chemicals, and ligands and sample chemicals bound to hormone receptors are measured after separation. There is a need. for that reason,
There is a problem that the number of operation steps increases.

[0008]

In the above-mentioned binding test method, by changing this radioligand to a non-radioactive ligand, there is a possibility that a simple and inexpensive method for evaluating the degree of hormone-like activity of a chemical substance may be obtained. is there.

While the present inventors have been conducting various studies to solve the above problems, by using a ligand having a specific structure as a non-radioactive ligand, the ligand alone shows low fluorescence intensity, but the hormone receptor Some ligands produce strong fluorescence intensity when bound to the body, and when a ligand having such properties is used, the free ligand and the ligand bound to the hormone receptor are not separated without separation. The inventors have found that the 50% inhibitory concentration of a chemical substance such as an endocrine disrupter can be determined simply and accurately, and have completed the present invention.

Accordingly, it is an object of the present invention to provide a method for determining a 50% inhibitory concentration of a chemical substance, in which the number of operation steps is small, and anyone can easily and inexpensively evaluate the hormone-like activity of the chemical substance. is there.

[0011]

In order to achieve the above object, the present invention provides [1] a sample chemical substance in a solution containing a conjugate of a hormone receptor and a ligand which emits fluorescence by binding to a hormone receptor. Is added, and the fluorescence intensity that changes according to the concentration of the sample chemical substance is measured. The fluorescence intensity of the solution containing the conjugate of the hormone receptor and the ligand, and the conjugate of the receptor and the ligand are included. 1 difference in fluorescence intensity from the solution in which the ligand is released in the solution
A method for determining a 50% inhibitory concentration of a chemical substance, wherein the concentration of the sample chemical substance having a fluorescence intensity of / 2 is a 50% inhibitory concentration, wherein [2] the ligand is cumestrol, 7-nitrobenzofuran-bridged ethinylestradiol, or Being fluorescein cross-linked ethinyl estradiol,
And [3] that the hormone receptor is an estrogen receptor.

Hereinafter, the present invention will be described in detail.

[0013]

BEST MODE FOR CARRYING OUT THE INVENTION In the method for determining the IC50 of a chemical substance of the present invention, the sample chemical substance to be evaluated is not particularly limited.

In the method for determining the IC50 of a chemical substance of the present invention, the hormones to be inhibited include various hormones such as estrogen, androgen, thyroid hormone, and the like.
And their hormone-like chemicals. Also,
Hormone receptors include receptors for the various hormones described above.

Chemicals exhibiting estrogenic activity have received the most attention as chemicals exhibiting a mechanism of action associated with feminization.

The method for preparing the hormone receptor is not particularly limited. For example, the prepared expression vector is introduced into Escherichia coli, and the Escherichia coli is cultured to express a protein which is a hormone receptor. A method of extracting and purifying the expressed protein to prepare the protein is exemplified.

The ligand specifically binds to the hormone receptor of interest, and its binding strength is strong to some extent, and when it is not bound to the receptor, no or no fluorescence is generated. Those which cause a large change in fluorescence intensity and / or fluorescence spectrum when bound to a receptor are preferred. Specifically, the difference in the fluorescence intensity between when the ligand is not bound to the receptor and when the ligand is bound changes by 5 times or more, preferably 20 times or more, the S / N ratio of the fluorescence intensity measurement device. Things are desirable. Usually, those whose fluorescence intensity changes 10 times or more can be used. If the difference between the fluorescence intensities is less than twice the S / N ratio, the measurement error will increase and an accurate IC
50 becomes difficult to determine.

If the IC50 of the sample chemical to be determined is for estrogen, for example, cumestrol (Coom), fluorescein-bridged ethinylestradiol (FEE) and 7-
Nitrobenzofuran cross-linked ethinyl estradiol (N
BD) and the like are mentioned as preferred ligands.

[0019]

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[0020]

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[0021]

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In addition, as for fluorescein-bridged ethinyl estradiol (FEE), the number n of methylene groups in the above structural formula is preferably from 8 to 12. n is 1
When the value is 0, the fluorescence intensity ratio becomes highest and the sensitivity becomes high, which is particularly preferable.

A method for determining the IC50 of a chemical substance using the above-described receptor and ligand will be described below.

In a mixture of a ligand and a hormone receptor,
Upon addition of the sample chemical, a portion of the ligand bound to the hormone receptor is released, and the sample chemical binds to the hormone receptor instead. The fluorescence intensity of the ligand when bound to the receptor is greater than when not bound. Thus, by replacing a portion of the ligand bound to the receptor with the sample chemical, the fluorescence intensity decreases in inverse proportion to the percentage of free ligand.

A sample chemical substance was added to a mixed solution of a ligand and a hormone receptor in different amounts, and the fluorescence intensity at that time was measured. The fluorescence intensity was plotted on the vertical axis, and the sample chemical substance concentration was plotted on the horizontal axis. If the relationship between the two is graphed, a "binding curve of the sample chemical substance in the mixture of the ligand and the hormone receptor" is obtained.

In measuring the fluorescence intensity, it is not necessary to isolate the free ligand, the free sample chemical substance, the ligand or the sample chemical substance bound to the hormone receptor, and measure the concentration. Ligand used in the present invention, compared to when not bound to the receptor,
It produces large fluorescence when bound to the receptor.

As an example of the method for determining the IC50 of the chemical substance of the present invention, the fluorescence intensity of the solution containing the conjugate of the hormone receptor and the ligand and the conjugate of the receptor and the ligand in the above graph are shown. The concentration of the sample chemical substance showing a fluorescence intensity of １ ／ of the fluorescence intensity difference from the solution in a state where the ligand is released in the solution containing the ligand is read from the graph,
There is a method of using this as an IC50 of a chemical substance.

As another example of the method of determining the IC50, there is a method of mathematically obtaining the IC50 by approximating the logarithmic curve. That is, to calculate the IC50 of the sample chemical substance, the following logistic equation is used.

[0029]

(Equation 1)

Here, X is the concentration of the sample chemical substance in the solution (M), Y is the fluorescence intensity of the solution of X (M), Top is the numerical value of the fluorescence intensity of a series of solutions, Bottom is
It is a numerical value of bottoming out in the fluorescence intensity of a series of solutions. To calculate IC50 mathematically.

In any of these methods for calculating the IC50 value, for a chemical substance having a weak binding property to a receptor, the ligand is almost completely released in a solution containing the binding substance between the receptor and the ligand within the concentration range to be measured. Since it cannot be made to be in a state where it was made to run, a bottoming out value cannot be obtained. In this case, using a known compound (for example, estradiol or the like) that is known to have a large binding property to the receptor,
The fluorescence intensity of the solution in which the ligand is released by adding the known compound in an amount sufficient to release the ligand almost completely can be used as the bottom value. For example, when the known compound is estradiol, the ligand is sufficiently released by adding at least 10 times the receptor concentration, usually about 100 times. When a known compound other than estradiol is used as the known compound, the amount of addition may be an amount corresponding to the above-mentioned amount of estradiol.

The wavelength (excitation wavelength) of the excitation light that generates fluorescence having a fluorescence intensity when bound to a hormone receptor is, for example, 3 when cumestrol is used as a ligand.
It is preferably from 20 to 400 nm, more preferably from 340 to 380 nm, particularly preferably from 350 to 370 nm.

When fluorescein-bridged ethinylestradiol is used as the ligand, the excitation wavelength is 420
To 550 nm is preferable, 450 to 520 nm is more preferable, and 475 to 495 nm is particularly preferable.

In determining the IC50, it is necessary to measure the fluorescence intensities of a large number of solutions containing a ligand, a receptor, and a chemical substance with various concentrations of the chemical substance. A measurement method suitable for this purpose is to use a commercially available microplate for multiwell fluorescence measurement.

For example, when a commercially available 96-well microplate is used, three wells are set as one set (measurement repetition unit n =
3), a set in which a solution containing only a predetermined concentration of a ligand was added to each of three wells, a set in which a solution containing a predetermined concentration of a ligand and a receptor were added to the next three wells, respectively, A set is prepared in which a solution containing the body and the first concentration of the chemical substance is added to each of the next three wells. Hereinafter, similarly, only the chemical substance concentration is changed to the second concentration, the third concentration,.
Prepare a set added to the three holes that were sequentially changed to.

Here, the solvent for the solution is preferably a Tris-HCl or phosphate buffer. Specifically, 1 mM
EDTA, 1 mM EGTA, 1 mM NaVO ₃ , 1
A 10 mM Tris-HCl buffer (pH) 7.4 containing 0% by mass glycerol, 0.5 mg / ml γ-globulin, 0.5 mM phenylmethylsulfonyl fluoride, 0.2 mM leupeptin, and the like are exemplified.

The concentration of the ligand in the solution is preferably 1 to 50 nM. The concentration of the receptor in the solution is preferably 2 to 50 nM.

The change range of the concentration of the chemical substance in the solution is, when the sample chemical substance binds to the receptor, a concentration range that is lower than the solubility in the buffer solution. It is halfway between the peak and bottom of the fluorescence intensity caused by binding to the receptor, and it can be determined that the minimum and maximum concentrations of the fluorescence intensity are as close as possible to the peak and bottom values. preferable.

Next, the microplate prepared with each set of the solutions as described above is left for a predetermined time, and the binding relationship between the ligand, the receptor and the chemical substance is brought to equilibrium. Then, using a commercially available fluorescent microplate reader, etc.
The fluorescence intensity of the solution added to each well of the microplate is measured.

In order to reach equilibrium, it is sufficient to keep at 4 to 37 ° C., usually at room temperature for 0.5 to 2 hours.

The measurement results of the fluorescence intensity with respect to each concentration of the chemical substance obtained as described above are graphed as described above, or the IC of the chemical substance is determined by using the logistic equation.
50 can be determined.

As a ligand, cumestrol (Cou)
When m) or fluorescein-bridged ethinyl estradiol is used, the ligand exhibits some fluorescence spectrum even in the free ligand state, although not as much as the expression fluorescence spectrum when bound to the hormone receptor.

FIG. 2 is a fluorescence spectrum chart showing the pH dependence of the fluorescence intensity of free cumestrol (ligand). The maximum fluorescence wavelength was 440 nm in any of the solutions at pH 6.5, pH 7.0, and pH 7.4. It is preferable to use a buffer to adjust the pH of the solution.

In this buffer, in order to prevent the protein which is a hormone receptor from non-specifically adsorbing to the vessel wall or the like,
Preferably, gamma globulin is added.

FIG. 3 is a fluorescence spectrum chart showing a comparison between the fluorescence intensity of a solution containing only free cumestrol and the fluorescence intensity of a solution containing cumestrol bound to an estrogen receptor (ER). The pH of the solution is shown in FIG.
In order to increase the sensitivity of the measurement, pH 6.5 at which the fluorescence intensity of free cumestrol was smallest was used.

As shown in FIG. 3, the maximum fluorescence wavelength was 4 in both the solution containing cumestrol alone and the solution containing cumestrol bound to the hormone receptor.
It was 40 nm. Accordingly, the wavelength at which the fluorescence intensity is measured is most preferably 430 to 450 nm, which is the maximum fluorescence wavelength.
470 nm may be used.

FIG. 4 is a graph showing the change in the fluorescence intensity of the solution when the hormone receptor concentration was changed with the concentration of cumestrol being 10 nM. Hormone receptor concentrations from 5 × 10 ⁻⁹ M (5 nM) to 1 × 10 ⁻⁷ M (100 nM)
M), the fluorescence intensity of the solution also increased.

As shown in FIG. 2, when cumestrol is used as a ligand, the fluorescence spectrum of the solution is pH
It changes greatly in the range of 6.5 to 7.4. With the hormone receptor concentration kept constant at 10 nM and the ligand concentration kept constant at 10 nM, the change in the fluorescence intensity of the solution when the estradiol concentration was changed was examined for each pH. The result is shown in FIG.
Shown in

As shown in FIG. 5, the amount of change in the fluorescence intensity is
There is no significant difference between pH 7.0 and pH 7.4.
The 6.5 solution was about 1.6 times larger than the pH 7.0 and 7.4 solutions. In addition, the solution with a large amount of free ligand, that is, the solution whose log [E2] is around -5, has a fluorescence intensity of pH 6.5
It is much smaller than H7.0 and pH 7.4 solutions. Therefore, the “rate of change in fluorescence intensity”, that is, the “fluorescence intensity of a solution whose log [E2] is around −10 / the fluorescence intensity of a solution whose log [E2] is around −5” is the pH value of the solution at pH 6.5
It is much larger than 7.0 or pH 7.4 solutions. Therefore, in order to evaluate the hormone-like activity from the binding curve in the fluorescence intensity measurement, from the viewpoint of sensitivity, a solution of pH 6.5 requires
More suitable than 7.0 and pH 7.4 solutions.

However, in order to reflect the endocrine disrupting phenomenon in the living body, it is preferable to perform measurement at the pH in the living body. Therefore, when the IC50 is usually determined, the pH of the solution is preferably pH 7.4, which is equivalent to the pH in a living body.

Next, the case where fluorescein-bridged ethinyl estradiol is used as a ligand will be described.

As described above, the excitation wavelength for developing the fluorescence spectrum is preferably from 420 to 550 nm,
To 520 nm is more preferable, and 475 to 495 nm is particularly preferable.

The wavelength at which the fluorescence intensity is measured is most preferably 520 to 530 nm, which is the maximum fluorescence wavelength, but 500 to 570 nm, which is slightly longer, may be used due to restrictions on the apparatus.

The hormone receptor concentration is preferably 1 to 100 nM, more preferably 2 to 50 nM, and more preferably 5 to 20 nM.
Is particularly preferred. The ligand concentration is preferably 0.5 to 50 nM, more preferably 1 to 20 nM, and more preferably 2 to 10 nM.
Is particularly preferred.

The concentration of γ globulin in the buffer is 0.00
1-5 mg / mL is preferable, and 0.01-3 mg / mL.
Is more preferable, and 0.02 to 2 mg / mL is particularly preferable.

When fluorescein-bridged ethinyl estradiol is used as the ligand, the solution when the concentration of the sample chemical is changed when estradiol is used as the sample chemical when the hormone receptor concentration is 10 nM and the ligand concentration is 5 nM. The change in fluorescence intensity was examined for each pH. The result is shown in FIG.

As shown in FIG. 6, the amount of change in the fluorescence intensity is
The solution dropped in the order of pH 7.4, pH 7.0, and pH 6.5. However, the fluorescence intensity of the solution in which the ligand is released, that is, the solution whose log [E2] is around -6,
The pH 6.5 solution is much smaller than the pH 7.0 and pH 7.4 solutions. Accordingly, the “rate of change in fluorescence intensity”, ie, the fluorescence intensity / l of the solution whose log [E2] is around -10
The magnitude of the "fluorescence intensity of the solution whose og [E2] is around -6" is not as large as that of the pH 6.5 solution, but is equivalent to that of the pH 7.0 solution. Therefore, in order to evaluate the hormone-like activity from the binding curve obtained by the fluorescence intensity measurement, a solution of pH 7.4 needs a pH of 6.5 or pH 6.5.
More suitable than the 7.0 solution, but the difference between them is not very large.

As described above, when evaluating the binding of a chemical substance to a receptor in a living body, pH 7.4 is desirable.

[0059]

EXAMPLES The present invention will be described more specifically with reference to the following examples.

In Examples 1 and 2, the estrogen receptor, ligand, buffer, and sample chemical used were as shown in the following AD.

A Estrogen receptor An estrogen receptor was prepared by the following methods.

[0062] After introducing the insertion of the ligand binding domain of the cloned from introducing human uterine library in the preparation of the expression vector and E. coli human estrogen receptor gene into the expression vector multiple cloning site of pGEX-4T ₁ plasmid into E. coli, ampicillin The LB plates were inoculated and grown.

The colony obtained by estrogen receptor expression was inoculated into LB medium (20 mL), and pre-cultured at 37 ° C. overnight. This preculture 20m
L was inoculated into 200 mL LB medium and cultured at 37 ° C. for 2 hours. Then, an expression inducer isopropyl-1-thio-β-D-galactopylanozide (IPTG) was added to a final concentration of 1 mM, and the target protein was added. After induction of expression, 25
Cultured overnight at ℃.

The expressed protein prepared by extraction and purification of the expressed protein was a fusion protein of the ligand binding domain of the estrogen receptor and glutathione-S-transferase (GST). Using the specific binding property between GST and glutathione, the expressed protein was extracted and purified by the following method.

E. coli was collected by centrifuging the culture solution prepared in the above. This Escherichia coli was treated with a sonication buffer (Tris-HCl: 50 mM, pH 8.0).
NaCl 0.50 mM, EDTA 1 mM). Then, the E. coli in this suspension was crushed by an ultrasonic crusher. Glutathione-Sepharose resin was added to this soluble fraction, and the ligand binding domain of the estrogen receptor and glutathione-S-transferase (GS
The expression product fused with T) was specifically bound to glutathione-Sepharose resin. The resin to which the GST fusion expression product is bound is recovered by centrifugation, and the GST fusion expression product is eluted by using excess reduced glutathione. From the eluate, the ligand binding domain of the target estrogen receptor and glutathione- A fusion protein with S-transferase (GST) was obtained.

The eluate for expression protein identification was subjected to SDS-polyacrylamide gel (PAGE) electrophoresis, and the results shown in FIG. 7 were obtained.
As shown in FIG. 7, it was confirmed that the expressed protein had a predetermined molecular weight and was sufficiently purified. That is, the sample before the fractionation treatment contains GST induced expression.
The presence of the fusion protein was confirmed (lane 2, arrow). However, after sonication, the content of this protein in the soluble fraction was quite low (lane 3). This was thought to be because the protein expressed by excessive expression induction formed an insoluble inclusion body in E. coli. Therefore, the soluble fraction after the ultrasonic treatment was concentrated and purified using glutathione-sepharose resin. Lane 4 shows the electrophoresis pattern of the resin sample to which the GST fusion protein was bound, and Lane 5 shows the electrophoresis pattern of the sample obtained by eluting the protein from glutathione-sepharose resin with glutathione. In both samples, a band (arrow) having a molecular weight corresponding to the GST fusion protein was detected. This eluate was used as the final purified product. In addition, the low molecular weight component of the electrophoresis pattern in Lane 5 is considered to be a degradation product due to protease.

The expression protein was determined to be GS by the GST detection kit.
It was confirmed that it had T activity.

The concentration of the expressed protein in the eluate from the measurement of the concentration of the expressed protein was measured by GPC (gel permeation chromatography) using bovine serum albumin as a reference substance. Analytical conditions: column: TOSO G-300
0SW / XL, eluent: 0.1 M phosphate buffer (pH
7.0), Measurement wavelength: 220 nm｝. As a result of the measurement, the expressed protein concentration was 16.9 μmol / L. The eluate of the expressed protein solution at this concentration, that is, the eluate, was used as a stock solution for the evaluation of hormone-like activity in Examples 1 and 2 described below.

Storage of the expressed protein The obtained expressed protein was subdivided and placed in a freezer (-80
° C), and thawed by a necessary amount at the time of use in the evaluation of hormone-like activity in Examples 1 and 2 described later.

B ligand Fluorescein-bridged ethinylestradiol (FE
E) Fluorescein-polymethylenediamine-17α-in which the ethynyl group of 17α-ethynylestradiol is a carboxylethynyl group, polymethylenediamine is bonded to the carboxylethynyl group, and fluorescein is bonded to the terminal amino group of polymethylenediamine. Ethinylestradiol was synthesized. In the analysis of the synthesized fluorescein-polymethylenediamine-17α-ethynylestradiol, a methylene bridged chain having 10 methylenes was used.

Coumestrol (Cum) A product of Fluka, having a purity of 95% was used.

A predetermined amount of each of the above ligands was dissolved in dimethyl sulfoxide (DMSO) to prepare a 10 mM solution. This was diluted with a culture medium at the time of use to prepare an additive solution having a predetermined concentration as an additive stock solution in the evaluation of hormone-like activity in Examples 1 and 2 described below.

C buffer The following two kinds of buffers were used for evaluating the hormone-like activity in Examples 1 and 2 described below.

PH 7.4 buffer EDTA 1 mM, EGTA 1 mM, NaVO ₃ 1 m
M, glycerol 10%, γ-globulin 0.5 mg
/ ML, phenylmethylsulfonyl fluoride 0.
10 mM containing 5 mM and 0.2 mM leupeptin
Tris buffer (pH 7.4) pH 6.5 buffer EDTA 1 mM, EGTA 1 mM, NaVO ₃ 1 m
M, glycerol 10%, γ-globulin 0.5 mg
/ ML, phenylmethylsulfonyl fluoride 0.
10 mM containing 5 mM and 0.2 mM leupeptin
Phosphate buffer (pH 6.5) D Sample chemicals Sample chemicals for measuring the binding curve data of the following natural / synthetic hormones, phenols, phthalates / adipates, and pesticides Used as ○ Natural and synthetic hormones ・ Estradiol (E2) (manufactured by Wako Pure Chemical Industries, Ltd. for biochemistry) ・ Ethynyl estradiol (EE2) (manufactured by Wako Pure Chemical Industries, Ltd., for biochemistry) ・ Estron (E1) (Wako Pure)・ Estriol (E3) (manufactured by Wako Pure Chemical Industries, Ltd., for biochemistry)
・ For biochemistry ・ Biochanin A (Biochanin A, manufactured by Fluka)
○ Phenols ・ Bisphenol A (BPA) (Tokyo Kasei Kogyo Co., Ltd., TCI-EP) ・ Nonylphenol (NP) (Tokyo Kasei Kogyo Co., Ltd.) ・ Octylphenol (OP) (Kanto) Chemical Co., Ltd.
Pentachlorophenol (PCP) (manufactured by Wako Pure Chemical Industries, Ltd., for testing residual agricultural chemicals) ○ Phthalate ester ・ Adipic ester ・ Di (2-ethylhexyl) adipate (DOA) (・ Dibenzylbutyl phthalate (BBP) (manufactured by Wako Pure Chemical Industries, Ltd. for testing phthalate) ・ Dicyclohexyl phthalate (CHP) (manufactured by Wako Pure Chemical Industries, Ltd.) Agrichemicals DDE (manufactured by Wako Pure Chemical Industries, Ltd.) A predetermined amount of each of the sample chemicals was dissolved in dimethyl sulfoxide (DMSO) to prepare a 10 mM solution. This was diluted with a culture medium at the time of use to prepare an additive solution having a predetermined concentration as an additive stock solution in the evaluation of hormone-like activity in Examples 1 and 2 described below.

Example 1 (Evaluation of hormone-like activity) As the ligand, the above-mentioned cumestrol (Cum) was used. Ligand concentration is 1
It was 0 nM.

As the estrogen receptor, a fusion protein of the ligand binding domain of the estrogen receptor and glutathione-S-transferase (GST) prepared by the above method was used. Estrogen receptor concentration is 10
Performed at nM.

As the buffer, buffers of pH 6.5 and 7.4 were used.

The above-mentioned chemical substances were used as sample chemical substances, and the concentrations of the sample chemical substances were adjusted to 8 concentrations having a common ratio of 3 to 10 in accordance with the degree of binding of the sample chemical substances.

200 μL of the solution prepared under the above conditions was added to a microplate for fluorescence measurement, left for 1 hour, and the fluorescence intensity was measured at an excitation wavelength of 360 nm and a measurement wavelength of 465 nm.

As a measuring apparatus, a microplate reader Polarion (manufactured by TECAN) for fluorescence measurement was used.

From the above measurement data, a binding curve was prepared for each sample chemical substance, and an IC50 for evaluating hormone-like activity was calculated from the binding curve. Table 1 shows the results.

[0082]

[Table 1]

Each value of log IC50 in Table 1 is 2 to
It is the average of the measured values obtained by performing four repeated measurements. Of these log IC50, the sample chemical substance was estradiol, a buffer solution of pH 6.5 was used, and the measurement was performed four times under the condition that the fluorescence intensity was measured. FIG. 8 shows a binding curve obtained from each measurement data.

The log IC50 values calculated by applying these binding curves to the logistic equation are-
8.07, -8.13, -8.07 and -8.06 all agreed within the experimental error. Similar results were obtained with other conditions. Therefore, under the conditions of this embodiment, IC5
It can be said that the reproducibility of the measurement of 0 value is good.

The two conditions under which the pH of the buffer solution and the method of measuring the fluorescence spectrum are different, namely, pH 6.5,
And I obtained from two conditions of pH 7.4 and fluorescence intensity.
It can be seen that the C50 values have a high correlation between them.

Example 2 (Evaluation of hormone-like activity) As the ligand, the above-mentioned fluorescein-crosslinked ethinylestradiol (FEE) was used. The ligand concentration was 5 nM.

As the estrogen receptor, a fusion protein of the ligand binding domain of the estrogen receptor and glutathione-S-transferase (GST) prepared by the above method was used. The estrogen receptor concentration was 10 nM.

As the buffer, a buffer having a pH of 6.5 was used.

Each solution was prepared to have a sample chemical substance concentration of 8 with a common ratio of 3 to 10 according to the degree of binding of the sample chemical substance.

200 μL of the solution prepared under the above conditions was added to a microplate for fluorescence measurement, allowed to stand for 1 hour, and the fluorescence intensity was measured at an excitation wavelength of 485 nm and a measurement wavelength of 535 nm.

The measurement apparatus used was a microplate reader Polarion (manufactured by TECAN) for fluorescence measurement.

From the above measurement data, a binding curve was prepared for each sample chemical substance, and an IC50 was calculated from the binding curve. Table 2 shows the results.

[0093]

[Table 2]

Each value of log IC50 in Table 2 is 2 to
It is the average of the measured values obtained by performing four repeated measurements. Of these log IC50s, under the condition that estradiol was used as the sample chemical substance and the fluorescence intensity was measured, the measurement was repeated twice. FIG. 9 shows a binding curve obtained from each measurement data.

The log IC50 values calculated by applying these binding curves to the logistic equation are-
8.19 and -8.25 agreed with the experimental error. Similar results were obtained with other conditions. Therefore, it can be said that the reproducibility of the measurement of the IC50 value is good under the conditions of the present embodiment.

From the results shown in Tables 1 and 2, the IC50 value obtained when Coumestrol (Coom) was used as the ligand and the fluorescein-crosslinked ethinylestradiol (FEE) were used as the ligand when the buffer was pH 6.5, respectively. IC50 obtained when using
Comparison with the values shows that the correlation between them is high.

Example 3 (Evaluation of hormone-like activity) As a ligand, the fluorescein-bridged ethinyl estradiol (FE
A chemical substance (estradiol E) was prepared in the same manner as in Example 2 except that the synthesized 7-nitrobenzofuran-bridged ethinyl estradiol (NBD) was used instead of using E).
The binding property to the receptor of 2) was measured.

The ethyl group of 17α-ethynyl estradiol is changed to a carboxyl ethynyl group, and polymethylene diamine is bonded to the carboxyl ethynyl group. Of 7-nitrobenzofuran-polymethylenediamine-17α-ethynylestradiol were respectively synthesized. The synthesized compound had 2 to 12 methylene groups. FIG.
Shows the relationship between estradiol concentration and fluorescence intensity when using 7-nitrobenzofuran-polymethylenediamine-17α-ethynylestradiol having eight methylene groups in the methylene bridged chain.

As shown in FIG. 10, it was confirmed that NBD can be used in the present method for determining the inhibitory concentration similarly to FEE.

Comparative Example 1 (Evaluation of Hormone-Like Activity) Using the radiolabeled estradiol as a ligand, the IC50 of the sample chemical substance used in Example 1 was determined.

After competitive binding between the sample chemical substance and the radioligand, the free ligand is removed with dextran-coated activated carbon, the radioactivity of the solution is measured.
The amount of ligand bound to the estrogen receptor was calculated.

FIG. 11 is a graph showing the relationship between the IC50 value measured by the binding test method using a radioligand and the IC50 value of the IC50 value of Example 1 measured by a method of measuring fluorescence intensity in a pH 7.4 buffer solution. Show the relationship.

As shown in FIG. 11, there is a good correlation between the IC50 value measured by the method of the present invention and the IC50 value measured by the binding test using a radioligand. Therefore, it is concluded that the method of the present invention can be used instead of the conventional binding test method.

[0104]

According to the method of determining the IC50 of the present invention, since no radioligand is used, it is necessary to set up a control area for generating radiation and to strictly manage the use records of workers and manage health. Without the need for complex equipment and management obligations,
Anyone can easily and inexpensively determine the IC50.

Since the method of the present invention measures the fluorescence intensity without isolating the free ligand and the sample chemical substance and the ligand and the sample chemical substance bound to the hormone receptor, the operation is simple. is there.

[Brief description of the drawings]

FIG. 1 is a graph showing a schematic relationship between a sample concentration and a concentration of a ligand bound to a hormone receptor.

FIG. 2 pH of fluorescence intensity of cumestrol (ligand)
It is a fluorescence spectrum chart which shows dependence.

FIG. 3 is a fluorescence spectrum chart showing a comparison of the fluorescence intensities of free cumestrol (ligand) and cumestrol bound to a hormone receptor.

FIG. 4 is a graph showing a relationship between a change in the fluorescence intensity of a solution and a change in the hormone receptor concentration when the ligand concentration is 10 nM when cumestrol is used as the ligand.

FIG. 5 is a graph showing the relationship between the pH of a solution, the concentration of estradiol as a sample substance, and the fluorescence intensity when using cumestrol as a ligand.

FIG. 6 shows the pH of a solution when fluorescein-bridged ethinylestradiol is used as a ligand,
It is a graph which shows the relationship between the concentration of estradiol of a sample chemical substance, and fluorescence intensity.

FIG. 7: S of the eluate of the fusion expression product of the ligand binding domain and glutathione-S-transferase (GST)
It is a DS-polyacrylamide gel (PAGE) electrophoresis chart.

FIG. 8 is a graph showing binding curves of estradiol measured four times when cumestrol is used as a ligand.

FIG. 9 is a graph showing a binding curve obtained by measuring the fluorescence intensity of twice repeated estradiol when fluorescein-bridged ethinyl estradiol is used as a ligand.

FIG. 10 is a graph showing a binding curve of estradiol when 7-nitrobenzofuran-octamethylenediamine-17α-ethynylestradiol is used as a ligand.

FIG. 11 is a graph showing a relationship between an IC50 value measured by a binding test method using a radioligand and an IC50 determined in Example 1.

Claims (3)

[Claims] 1. A sample chemical substance is added to a solution containing a conjugate of a hormone receptor and a ligand that emits fluorescence by binding to a hormone receptor, and a fluorescence intensity that changes according to the concentration of the sample chemical substance added is determined. The difference between the fluorescence intensity of the solution containing the conjugate of the hormone receptor and the ligand and the fluorescence intensity of the solution in a state where the ligand is released from the solution containing the conjugate of the receptor and the ligand is measured. A method for determining a 50% inhibitory concentration of a chemical substance, wherein the concentration of the sample chemical substance having a fluorescence intensity of / 2 is a 50% inhibitory concentration. 2. The method for determining a 50% inhibitory concentration of a chemical substance according to claim 1, wherein the ligand is cumestrol, 7-nitrobenzofuran-bridged ethinylestradiol, or fluorescein-bridged ethinylestradiol. 3. The method according to claim 1, wherein the hormone receptor is an estrogen receptor.