JP2006078196A - Method of screening specimen - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method of speedily screening a candidate material having a desired binding characteristic for ligand from many specimens. <P>SOLUTION: In the method of screening the specimens, any one or more of adsorption rate constant (ka) of the specimens and the ligand, separation speed constant (kd), binding constant (KD), and binding amount are measured regarding one group of specimens. Any one or more of the measured adsorption rate constant (ka) of the specimens and the ligand, separation speed constant (kd), binding constant (KD), and binding amount are compared with the physical constant of the specimens, and the correlations are analyzed. A specimen separated from obtained one group of correlations is extracted. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a test substance screening method. More specifically, the present invention relates to a test substance screening method based on a comparison between various rate constants of interaction reaction between a test substance and a ligand and physical constants of the test substance.

  Compounds used as pharmaceuticals often interact with proteins in vivo to exert their functions. For example, an enzyme inhibitor binds to a target enzyme and inhibits the enzyme reaction, thereby changing the biochemical reaction in the living body and expressing its pharmacological action. For this reason, it is important to know the strength of binding between a protein and a drug candidate compound in the search for a drug.

  In recent years, surface plasmon resonance has attracted attention as a technique for analyzing the interaction between proteins and chemical substances. In this method, protein is immobilized on a metal surface such as gold, and the interaction between the protein and the chemical substance is quantified by measuring the change in surface plasmon resonance when the chemical substance is donated to the metal surface.

The analyte molecule, which is a chemical substance, adsorbs over time to molecules that interact with the analyte molecule, such as proteins immobilized on the metal surface. The amount of adsorption at this time varies according to the following equation.
dθ / dt = ka ・ Cs ・ (1 -θ)-kd ・ θ Equation (1)
(In the formula, θ represents the adsorption rate (= adsorption amount / saturated adsorption amount), ka represents the adsorption rate constant, kd represents the separation constant, and Cs represents the concentration of the molecule to be analyzed in the vicinity of the metal surface.)

Here, c _s is constant under ideal conditions in which the metal surface is constantly replaced with fresh liquid, and ka and kd can be obtained from the measurement results by solving a simple differential equation. Since the SPR signal changes in proportion to θ, ka and kd can be obtained from the change in the SPR signal. The binding constant (KD) indicates the ratio between adsorption and desorption and is expressed in ka / kd.

  On the other hand, the interaction between a test substance and a ligand often correlates with the physical constant of the chemical substance. For example, the hydrophobicity parameter (logP) is known to correlate with the uptake into the biological membrane and the binding to the hydrophobic part of the protein with a straight line or a curve, and is utilized as a physical constant representing the function of the drug. The bonds that can be explained by such physical constants are not different in proteins as a whole and show binding properties.

  However, it is ideal that pharmaceuticals interact only with the target protein and have little interaction with other biological substances. This is because having an interaction with a physiologically active substance that is not the target changes an unexpected biochemical reaction and appears as a so-called side effect.

  An object of the present invention is to provide a method for rapidly screening a candidate substance having a desired binding property to a ligand from a large number of test substances.

  As a result of intensive studies to solve the above problems, the present inventors have determined that at least one of the adsorption rate constant (ka), the desorption rate constant (kd), the binding constant (KD), and the binding amount between the test substance and the ligand. And, by comparing the physical constants of the test substance and extracting those that are out of a group of correlations, it has been found that a test substance having a desired binding property can be rapidly screened from a large number of candidate substances. The invention has been completed.

  That is, according to the present invention, for a group of test substances, any one or more of an adsorption rate constant (ka), a release rate constant (kd), a binding constant (KD), or a binding amount between the test substance and a ligand. Any one or more of the adsorption rate constant (ka), desorption rate constant (kd), binding constant (KD), or binding amount between the test substance and the ligand measured above, and the physical properties of the test substance There is provided a test substance screening method comprising analyzing a correlation with a constant and extracting a test substance that is out of the obtained group of correlations.

  Preferably, the physical constant of the test substance is any one or more of a hydrophobic parameter (logP), an acid dissociation constant (pKa), a dielectric constant, a polarizability, a dipole moment, a molecular occupied volume, or a numerical value representing a molecular shape. It is.

Preferably, the correlation is analyzed using 50 or more kinds of test substances.
Preferably, any one or more of the adsorption rate constant (ka), desorption rate constant (kd), binding constant (KD), or binding amount between the test substance and the ligand and the physical constant of the test substance are correlated. Is approximated by either a straight line, a quadratic curve, a normal distribution, or a binomial distribution.

  Preferably, the adsorption rate constant (ka), desorption rate constant (kd), binding constant (KD), or binding amount between the ligand and the test substance can be determined by a surface plasmon resonance measuring apparatus.

  Preferably, a metal film, a light source that generates a light beam, and an optical system that causes the light beam to be incident so as to obtain a total reflection condition at an interface with the metal film and include various incident angle components; Surface plasmon resonance measurement comprising a flow path system including cells formed on the metal film, and light detection means for detecting the state of surface plasmon resonance by measuring the intensity of the light beam totally reflected at the interface. Using the apparatus, the adsorption rate constant between the ligand and the test substance is measured by exchanging the liquid in the channel system from a solution not containing the test substance to a solution containing the test substance and measuring the change in surface plasmon resonance. (ka), separation rate constant (kd), binding constant (KD), or amount of binding can be determined.

  Preferably, the change in surface plasmon resonance can be measured in a state where the flow of the liquid is stopped after the liquid in the flow path system is changed from a solution containing no test substance to a solution containing the test substance.

  According to the present invention, it is possible to rapidly screen a substance having a desired binding property from a large number of test substances.

Hereinafter, embodiments of the present invention will be described.
The screening method for a test substance according to the present invention comprises a group of test substances, any one of an adsorption rate constant (ka), a release rate constant (kd), a binding constant (KD), or a binding amount between the test substance and a ligand. One or more of the adsorption rate constant (ka), desorption rate constant (kd), binding constant (KD), or binding amount between the test substance and the ligand measured above, and the test substance The correlation is analyzed with the physical constants of the above, and a test substance that is out of the obtained group of correlations is extracted.

  The test substance used in the present invention is not particularly limited as long as it is a substance that interacts with a physiologically active substance (ligand) as described below in the present specification, but a low molecular organic compound, a high molecular compound, DNA, etc. And nucleic acids, proteins (including antibodies), peptides or fragments thereof, sugars, amino acids and the like. Although the kind of test substance is not specifically limited, Preferably it is 10 or more types, More preferably, it is 50 or more types, More preferably, it is 100 or more types.

  In the present invention, preferably, the adsorption rate constant (ka), the desorption rate constant (kd), the binding constant (KD), or the binding amount between the ligand and the test substance can be determined by a surface plasmon resonance measuring apparatus. The measurement by the surface plasmon resonance measuring apparatus will be described later in this document.

  The physical constant of the test substance in the present invention is not particularly limited, but preferably represents a hydrophobicity parameter (logP), an acid dissociation constant (pKa), a dielectric constant, a polarizability, a dipole moment, a molecular occupied volume, or a molecular shape. It is one of numerical values. Here, the numerical value representing the molecular shape refers to the major axis or aspect ratio (major axis / minor axis) of the molecule, and any numerical value may be used as long as it shows the characteristics of the molecular shape.

  The physical constant of the test substance in the present invention may be determined by any method. For example, it can be obtained by calculation such as molecular orbital calculation or simulation, and can also be obtained experimentally.

  The group of correlations in the present invention can be expressed with a certain correlation formula for 50% or more of the compounds, and the correlation coefficient is preferably 0.6 or more, more preferably 0.7 or more. preferable. The number of compounds showing a group of correlations is preferably 50 or more, more preferably 100 or more. The correlation of a group of compounds is preferably approximated by a straight line, a quadratic curve, a normal distribution, or a binomial distribution.

  In the present invention, deviating from a group of correlations means that a group of compounds that match the correlation is farther from the one furthest from the correlation function line or curve.

  The screening method of the present invention comprises a step of deriving one or more of an adsorption rate constant (ka), a desorption rate constant (kd), a binding constant (KD), and a binding amount from a change in SPR signal, a physicochemical constant of a test substance And a step of extracting a test substance that deviates from the correlation.

  In the present invention, a test substance that is out of the group of correlations is preferably displayed on the user interface of the apparatus.

  In one embodiment of the present invention, after the liquid in the flow path system of the surface plasmon resonance measuring apparatus is exchanged from a solution not containing the test substance to a solution containing the test substance, the surface of the surface is stopped in a state where the liquid flow is stopped. Changes in plasmon resonance can be measured. By such measurement, it is possible to suppress the noise width of the signal change of the reference cell and the baseline fluctuation within the measurement time, and thereby it is possible to acquire highly reliable combined detection data. The time for stopping the flow of the liquid is not particularly limited, but is, for example, 1 second to 30 minutes, preferably 10 seconds to 20 minutes, and more preferably about 1 minute to 20 minutes.

  In the present invention, preferably, a reference cell that does not bind a substance that interacts with a test substance and a detection cell that binds a substance that interacts with the test substance are connected in series and installed in the flow path system, A change in surface plasmon resonance can be measured by flowing a liquid through the reference cell and the detection cell.

In the present invention, the amount of liquid exchange per measurement (Ve) with respect to the volume (Vs ml) of the cell used for measurement (the total volume of those cells when the reference cell and the detection cell are used). ml) (Ve / Vs) can be, for example, 1 or more and 100 or less. Ve / Vs is more preferably 1 or more and 50 or less, and particularly preferably 1 or more and 20 or less. The volume (Vs ml) of the cell used for the measurement is not particularly limited, but is preferably about 1 × 10 ^{−6 to} 1.0 ml, particularly preferably about 1 × 10 ⁻⁵ to 1 × 10 ⁻¹ ml. Further, the time required for exchanging the liquid is preferably 0.01 seconds or more and 100 seconds or less, and more preferably 0.1 seconds or more and 10 seconds or less.

  The phenomenon of surface plasmon resonance is due to the fact that the intensity of monochromatic light reflected from the boundary between an optically transparent substance such as glass and the metal thin film layer depends on the refractive index of the sample on the metal exit side. Yes, so the sample can be analyzed by measuring the intensity of the reflected monochromatic light. Hereinafter, a surface plasmon resonance measuring apparatus that can be used in the present invention will be described.

  A surface plasmon resonance measuring apparatus is an apparatus for analyzing the characteristics of a substance to be measured using a phenomenon in which surface plasmons are excited by light waves. An example of the surface plasmon resonance measuring apparatus used in the present invention is a dielectric block, a metal film formed on one surface of the dielectric block, a light source for generating a light beam, and the light beam to the dielectric block. On the other hand, the optical system that allows the total reflection condition to be obtained at the interface between the dielectric block and the metal film and that includes various incident angle components, and the intensity of the light beam that is totally reflected at the interface Photodetection means for measuring and detecting the surface plasmon resonance state.

  Further, as described above, the dielectric block is formed as one block including all of the light beam incident surface, the light exit surface, and one surface on which the metal film is formed, and the metal film is formed on the dielectric block. It is integrated.

  In the present invention, specifically, the surface plasmon resonance measuring apparatus described in FIGS. 1 to 32 described in JP 2001-330560 A and described in FIGS. 1 to 15 described in JP 2002-296177 A are described. A surface plasmon resonance measuring apparatus which is used can be preferably used. The contents described in Japanese Patent Laid-Open No. 2001-330560 and Japanese Patent Laid-Open No. 2002-296177 are all cited in this specification as part of the disclosure of this specification.

  For example, as a surface plasmon resonance measuring apparatus described in Japanese Patent Application Laid-Open No. 2001-330560, for example, a dielectric block, a thin film layer made of a metal film formed on one surface of the dielectric block, and a surface of the thin film layer A plurality of measurement units each including a sample holding mechanism for holding a sample; a support that supports the plurality of measurement units; a light source that generates a light beam; and the light beam with respect to the dielectric block. An optical system that is incident at various incident angles so that a total reflection condition can be obtained at the interface between the dielectric block and the metal film, and the intensity of the light beam that is totally reflected at the interface are measured, and the total intensity by surface plasmon resonance. The total reflection condition and various incident angles can be obtained sequentially with respect to the light detection means for detecting the reflection attenuation state and each dielectric block of the plurality of measurement units. As described above, the apparatus includes a driving unit that relatively moves the support, the optical system, and the light detection unit, and sequentially arranges each measurement unit at a predetermined position with respect to the optical system and the light detection unit. And a surface plasmon resonance measuring apparatus.

  In the above measurement apparatus, for example, the optical system and the light detection means are kept stationary, and the driving means moves the support.

  In that case, the support body is a turntable that supports the plurality of measurement units on a circumference around a rotation axis, and the driving means rotates the turntable intermittently. It is desirable to be. Further, in this case, the support body is one that supports the plurality of measurement units arranged in a line in a straight line, and the support body is intermittently linear in the arrangement direction of the plurality of measurement units as the driving means. You may apply what is moved.

  On the other hand, contrary to the above, the support may be kept stationary, and the drive means may move the optical system and the light detection means.

  In that case, the support body supports the plurality of measurement units on a circumference, and the driving means places the optical system and the light detection means on the plurality of measurement units supported by the support body. It is desirable to rotate intermittently along. Further, in this case, the support body is one that supports the plurality of measurement units arranged in a line in a line, and the optical system and the light detection means are supported by the support body as the drive means. You may apply what moves linearly intermittently along this measurement unit.

  On the other hand, when the drive means has a rolling bearing that supports the rotation shaft, the drive means rotates the rotation shaft in one direction to perform a series of measurements on the plurality of measurement units. When completed, the rotation shaft is returned to the other direction by the same amount as the rotation amount, and then the rotation shaft is rotated in the one direction for the next series of measurements. Is desirable.

  Further, in the measurement apparatus, the plurality of measurement units are connected in a row by a connecting member to form a unit connection body, and the support body is configured to support the unit connection body. desirable.

  In the measurement apparatus, it is preferable that a means for automatically supplying a predetermined sample is provided to each sample holding mechanism of the plurality of measurement units supported by the support.

  Furthermore, in the measurement apparatus, the dielectric block of the measurement unit is fixed to the support, and the thin film layer of the measurement unit and the sample holding mechanism are integrated to form a measurement chip. It is desirable that the block is formed to be exchangeable.

  When such a measurement chip is applied, a cassette containing a plurality of the measurement chips, and a chip supply means for taking out the measurement chips one by one from the cassette and supplying the measurement chips in combination with the dielectric block are provided. It is desirable to be provided.

  Alternatively, the dielectric block, the thin film layer, and the sample holding mechanism of the measurement unit may be integrated to form a measurement chip, and the measurement chip may be formed to be replaceable with respect to the support.

  When the measurement chip has such a configuration, a cassette in which a plurality of measurement chips are stored and chip supply means for taking out the measurement chips one by one from the cassette and supplying them in a state supported by the support are provided. It is desirable that

  On the other hand, the optical system is configured to make the light beam incident on the dielectric block in the state of convergent light or divergent light, and the light detection means is present in the totally reflected light beam, and is attenuated by total reflection. It is desirable to be configured to detect the position of the dark line.

  The optical system is preferably configured to allow a light beam to enter the interface in a defocused state. In that case, it is desirable that the beam diameter in the moving direction of the support at the interface of the light beam be 10 times or more the mechanical positioning accuracy of the support.

  Furthermore, in the measurement apparatus, the measurement unit is supported above the support, the light source is disposed so as to emit the light beam downward from a position above the support, and the optical system includes It is desirable that a reflection member that reflects the light beam emitted toward the lower side to travel upward toward the interface is preferably provided.

  In the measurement apparatus, the measurement unit is supported on an upper side of the support, and the optical system is configured to cause the light beam to enter the interface from a lower side of the interface. Is a reflecting member that is disposed at a position above the support so that the light detection surface faces downward, reflects the light beam totally reflected at the interface upward, and travels toward the light detection means. It is desirable to be provided.

  On the other hand, in the above-described measuring apparatus, it is preferable that temperature adjusting means for maintaining the measuring unit at a predetermined set temperature before and / or after being supported by the support body is provided.

  In the measurement apparatus, it is preferable that a means for stirring the sample stored in the sample holding mechanism of the measurement unit supported by the support before detecting the total reflection attenuation state is provided.

  In the measurement apparatus, a reference liquid supply unit that supplies a reference liquid having optical characteristics related to the optical characteristics of the sample is provided in at least one of the plurality of measurement units supported by the support. In addition, it is preferable that correction means for correcting the data indicating the total reflection attenuation state relating to the sample obtained by the light detection means based on the data indicating the total reflection attenuation state relating to the reference liquid is preferably provided. .

  In that case, it is desirable that the reference liquid supply means supplies the solvent as a reference liquid if the sample is obtained by dissolving the analyte in the solvent.

  Further, the measuring apparatus includes a mark indicating individual identification information given to each measurement unit, reading means for reading the mark from the measurement unit used for measurement, and a sample relating to a sample supplied to the measurement unit An input means for inputting information, a display means for displaying a measurement result, and connected to the display means, the input means, and the reading means to associate the individual identification information and the sample information for each measurement unit And a control means for displaying the measurement result obtained for the sample held in a certain measurement unit on the display means in association with the individual identification information and the sample information stored for the measurement unit. It is desirable to provide.

  When detecting or measuring a substance that interacts with a physiologically active substance using the measurement apparatus described above, after detecting the state of total reflection attenuation with respect to the sample in one of the measurement units, the support, the optical system, and the light are detected. The detection means is moved relative to each other to detect the state of total reflection attenuation with respect to the sample in another measurement unit, and then the support, the optical system and the light detection means are moved relative to each other in the one measurement unit. Measurement can be performed on the sample by detecting the total reflection attenuation state again.

  A measurement chip used in the present invention is a measurement chip for use in a surface plasmon resonance measurement apparatus having the configuration described in the present specification, and is a dielectric block and a metal film formed on one surface of the dielectric block. The dielectric block is formed as one block including all of the light beam incident surface, the light exit surface, and one surface on which the metal film is formed, and the metal film is integrated with the dielectric block. It has become.

  The metal constituting the metal film is not particularly limited as long as surface plasmon resonance can occur. Preferred examples include free electron metals such as gold, silver, copper, aluminum, and platinum, with gold being particularly preferred. These metals can be used alone or in combination. In consideration of adhesion to the metal film, an intervening layer made of chromium or the like may be provided between the substrate and the layer made of metal.

  Although the thickness of the metal film is arbitrary, for example, when considering use for a surface plasmon resonance measuring apparatus, it is preferably 1 angstrom or more and 5000 angstrom or less, and particularly preferably 10 angstrom or more and 2000 angstrom or less. If it exceeds 5000 angstroms, the surface plasmon phenomenon of the medium cannot be sufficiently detected. When an intervening layer made of chromium or the like is provided, the thickness of the intervening layer is preferably 1 angstrom or more and 100 angstrom or less.

  The metal film may be formed by a conventional method, for example, sputtering, vapor deposition, ion plating, electroplating, electroless plating, or the like.

  The metal film is preferably disposed on the substrate. Here, “arranged on the substrate” means that the metal film is arranged so as to be in direct contact with the substrate, and that the metal film is not directly in contact with the substrate, but through other layers. This also includes the case where they are arranged. As a substrate that can be used in the present invention, for example, when considering use for a surface plasmon resonance measuring apparatus, generally, optical glass such as BK7, or synthetic resin, specifically, polymethyl methacrylate, polyethylene terephthalate, polycarbonate A material made of a material transparent to laser light such as a cycloolefin polymer can be used. Such a substrate is preferably made of a material that does not exhibit anisotropy with respect to polarized light and has excellent processability.

  The metal film preferably has a functional group capable of immobilizing a physiologically active substance (that is, a ligand for the test substance) on the outermost surface. Here, the “outermost surface” means “the side farthest from the metal film”.

Preferred functional groups include —OH, —SH, —COOH, —NR ¹ R ² (wherein R ¹ and R ² independently represent a hydrogen atom or a lower alkyl group), —CHO, —NR ³ NR ^1. R ² (wherein R ¹ , R ² and R ³ each independently represents a hydrogen atom or a lower alkyl group), —NCO, —NCS, an epoxy group, or a vinyl group can be mentioned. Here, the number of carbon atoms in the lower alkyl group is not particularly limited, but is generally about C1 to C10, preferably C1 to C6.

  As a method for introducing those functional groups on the outermost surface, for example, a polymer containing a precursor of those functional groups is coated on a metal surface or metal film, and then a precursor located on the outermost surface by chemical treatment. The method of producing | generating those functional groups from is mentioned.

  In the measurement chip obtained as described above, the physiologically active substance can be immobilized on the metal film by covalently bonding the physiologically active substance via the functional group.

  The physiologically active substance (that is, the ligand) immobilized on the surface of the measurement chip of the present invention is not particularly limited as long as it interacts with the test substance that is the measurement target. For example, immune proteins, enzymes, microorganisms Nucleic acid, low molecular weight organic compound, non-immune protein, immunoglobulin binding protein, sugar binding protein, sugar chain that recognizes sugar, fatty acid or fatty acid ester, or polypeptide or oligopeptide having ligand binding ability .

  Examples of immunity proteins include antibodies and haptens that use the measurement target as an antigen. As the antibody, various immunoglobulins, that is, IgG, IgM, IgA, IgE, IgD can be used. Specifically, when the measurement target is human serum albumin, an anti-human serum albumin antibody can be used as the antibody. In addition, when using pesticides, insecticides, methicillin-resistant Staphylococcus aureus, antibiotics, narcotics, cocaine, heroin, cracks, etc. as antigens, for example, anti-atrazine antibodies, anti-kanamycin antibodies, anti-methamphetamine antibodies, or pathogenic E. coli Among them, antibodies against O antigens 26, 86, 55, 111, 157 and the like can be used.

  The enzyme is not particularly limited as long as it shows activity against the measurement object or a substance metabolized from the measurement object, and various enzymes such as oxidoreductase, hydrolase, isomerase , A desorbing enzyme, a synthesizing enzyme, etc. Specifically, if the measurement object is glucose, glucose oxidase can be used, and if the measurement object is cholesterol, cholesterol oxidase can be used. In addition, when pesticides, insecticides, methicillin-resistant Staphylococcus aureus, antibiotics, narcotics, cocaine, heroin, cracks, etc. are used as measurement objects, they exhibit specific reactions with substances metabolized from them, such as acetylcholinesterase. Enzymes such as catecholamine esterase, noradrenaline esterase and dopamine esterase can be used.

The microorganism is not particularly limited, and various microorganisms including Escherichia coli can be used.
As the nucleic acid, one that hybridizes complementarily with the nucleic acid to be measured can be used. As the nucleic acid, either DNA (including cDNA) or RNA can be used. The type of DNA is not particularly limited, and may be any of naturally derived DNA, recombinant DNA prepared by gene recombination technology, or chemically synthesized DNA.
Examples of the low-molecular organic compound include any compound that can be synthesized by an ordinary organic chemical synthesis method.

The non-immune protein is not particularly limited, and for example, avidin (streptavidin), biotin or a receptor can be used.
As the immunoglobulin-binding protein, for example, protein A or protein G, rheumatoid factor (RF) and the like can be used.
Examples of sugar-binding proteins include lectins.
Examples of the fatty acid or fatty acid ester include stearic acid, arachidic acid, behenic acid, ethyl stearate, ethyl arachidate, and ethyl behenate.

  When the physiologically active substance is a protein or nucleic acid such as an antibody or an enzyme, the immobilization can be performed by covalently bonding to a functional group on the metal surface using the amino group, thiol group or the like of the physiologically active substance. .

The measurement chip on which the physiologically active substance is immobilized as described above can be used for detection and / or measurement of a test substance that interacts with the physiologically active substance.
The following examples further illustrate the present invention, but the scope of the present invention is not limited to these examples.

Example 1
(1) Measurement of binding constant (KD) Ligand fixation (amine coupling) was performed as follows. Biacore3000 (manufactured by Biacore) is used as a measuring device, and 5 mg of human serum albumin (hereinafter abbreviated as HSA) (manufactured by Sigma) as a ligand is 1 ml of phosphate buffer 10 mM pH7.4 (hereinafter abbreviated as PBS7.4). What was melt | dissolved in was prepared. The measurement temperature was 25 ° C. The chip used is CM5 (carboxymethyldextran sensor) (Biacore). The running buffer was PBS7.4). The flow rate is 10 μl / min. As an activation liquid, an equal volume mixture of 1-ethyl-2,3-dimethylaminopropylcarbodiimide (400 mM) and N-hydroxysuccinimide (100 mM) was used, and the activation time was 15 minutes. As the immobilization solution, HSA 5 mg / ml (PBS7.4) diluted 10-fold with acetate buffer pH 5.0 (Biacore) was used, and the immobilization time was 15 minutes. A 1M ethanolamine solution (pH 8.5) (manufactured by Biacore) was used as the inactivation liquid, and the inactivation time was 15 minutes.

  The drug binding was measured as follows. Biacore 3000 (manufactured by Biacore) was used as a measuring device. As the running buffer, a phosphate buffer solution 10 mM (pH 7.4) containing 5 wt% DMSO (hereinafter abbreviated as 5% DMSO / PBS7.4) was used. The compound concentration is 0.05 mM. The dissolution formulation is Compound 10 mM DMSO solution 2.5 μl, PBS7.4 47.5 μl, and 5% DMSO / PBS 7.4 450 μl.

  The compounds used are Furosemide, Digtoxin, Warfarin, 5,5-Diphenylhydantoin, Naproxen, Sulfanilamide, Indoprofen, Naringin, Ibuprofen, Lindpcain, Nicotinic acid, Sulfadimethoxins, Acetazolamide, Chlorothiazide, 5-Chloro-2-rodrothene, 50 species including quinine, 2-Aminobenzenesulfonamide, Hydralazine hydrochloride, Kanamicscin monosulfate, Salicylic acid, Salbutamol, Pyrimethamine, Rifamethamine, Enalapril maleate, Indapamide and the like were evaluated.

  KD value: DMSO correction and KD value calculation were all performed using an application created by Biacore. The obtained KD value was logarithmized and normalized by setting the logarithm of Furosemide KD value to 100.

(2) Calculation of logP '
The relative value of the partition coefficient was determined from the HPLC retention time.
Measuring device: 10ADvp (manufactured by Shimadzu Corporation)
Measurement column: TSKgel ODS-80Ts 4. 6 x 150mm (manufactured by Tosoh Corporation)
Measurement protocol: Elution time (rt) was measured by linearly changing the concentration ratio from 100% water to 100% acetonitrile for 15 minutes, and the dead time (time for the non-retained substance to reach the detection part from the injection) was subtracted. A logarithmic value (log (rt-d)) of time (rt-d) was used as logP ′.

(3) Correlation FIG. 1 shows the correlation between logP ′ and log (KD) determined as described above. As shown in FIG. 1, logP ′ and log (KD) of each drug show a very good correlation (correlation coefficient r ² = 0.98), but some compounds deviate from this correlation. Specifically, Hydrochlorothiazide and Furosemide deviate from this correlation. In the present invention, these compounds can be selected as lead compounds.

(Example 2)
KD was determined by the method described below. The following experiment was performed using the apparatus shown in FIG. 22 of Japanese Patent Laid-Open No. 2001-330560 (hereinafter referred to as the surface plasmon resonance measuring apparatus of the present invention) (shown as FIG. 2 in the present specification), and the diagram of the same publication. 23 (hereinafter referred to as the dielectric block of the present invention) (shown as FIG. 3 in this specification).

  The surface plasmon resonance measuring apparatus shown in FIG. 2 is slidably engaged with two guide rods 400, 400 arranged in parallel with each other as a support for supporting the measurement unit, and an arrow Y in the figure along them. A slide block 401 that is linearly movable in the direction is used. The slide block 401 is screwed with a precision screw 402 arranged in parallel with the guide rods 400, 400 so that the precision screw 402 is rotated forward and backward by a pulse motor 403 constituting the support driving means together with the precision screw 402. It has become.

  The driving of the pulse motor 403 is controlled by a motor controller 404. That is, the motor controller 404 receives an output signal S40 of a linear encoder (not shown) that is incorporated in the slide block 401 and detects the position of the slide block 401 in the longitudinal direction of the guide rods 400, 400. Based on this signal S40, the drive of the pulse motor 403 is controlled.

  A laser light source 31, a condensing lens 32, and a light detector 40 are disposed below the guide rods 400, 400 so that slide blocks 401 that move along the guide rods 400 are sandwiched from the left and right. The condensing lens 32 condenses the light beam 30. In addition, a photodetector 40 is installed.

  Here, in the present embodiment, as an example, a stick-like unit connection body 410 formed by connecting and fixing eight measurement units 10 is used, and the eight measurement units 10 are set on the slide block 401 in a state of being arranged in a row. It has come to be.

  FIG. 3 shows the structure of the unit connector 410 in detail. As shown here, the unit connection body 410 is formed by connecting eight measurement units 10 by connection members 411.

  The measurement unit 10 is formed by integrally molding the dielectric block 11 and the sample holding frame 13 from, for example, a transparent resin, and constitutes a measurement chip that can be exchanged for the turntable. In order to make the exchange possible, for example, the measurement unit 10 may be fitted and held in a through hole formed in the turntable. In this example, the sensing substance 14 is fixed on the metal film 12.

(1) Preparation of dextran measurement chip The dielectric block of the present invention, in which 50 nm gold was deposited as a metal film, was treated with Model-208 UV-ozone cleaning system (TECHNOVISION INC.) For 30 minutes, and then ethanol / water (80 / 20) A 5.0 mM solution of 11-hydroxy-1-undecanethiol was added so as to contact the metal film, and surface treatment was performed at 25 ° C. for 18 hours. Thereafter, washing was performed 5 times with ethanol, once with an ethanol / water mixed solvent, and 5 times with water.

  Next, the surface coated with 11-hydroxy-1-undecanethiol was brought into contact with a 10 wt% epichlorohydrin solution (solvent: 1: 1 mixture of 0.4 M sodium hydroxide and diethylene glycol dimethyl ether), The reaction was allowed to proceed for 4 hours in a shaking incubator. The surface was washed twice with ethanol and five times with water.

  Next, 4.5 ml of 1M sodium hydroxide was added to 40.5 ml of 25% by weight dextran (T500, Pharmacia) aqueous solution and the solution was contacted on the epichlorohydrin treated surface. It was then incubated for 20 hours at 25 ° C. in a shaking incubator. The surface was washed 10 times with 50 ° C. water. Subsequently, a mixture of 3.5 g of bromoacetic acid dissolved in 27 g of 2M sodium hydroxide solution was brought into contact with the dextran-treated surface and incubated for 16 hours in a shaking incubator at 28 ° C. The surface was washed with water and then the above procedure was repeated once.

(2) Preparation of ligand-immobilized chip After removing the solution in the dextran measurement chip prepared in (1) above, 200 mM EDC (N-ethyl-N '-(3-dimethylaminopropyl) carbodiimide hydrochloride) and 50 mM NHS 70 μl of a mixed solution of (N-hydroxysuccinimide) was added and left for 10 minutes. After removing the mixed solution, the plate was washed 3 times with 100 μl of water and 3 times with 100 μl of PBS 7.4 (synonymous with Example 1). This chip was placed in the surface plasmon resonance measuring device of the present invention with 100 μl of PBS7.4, and 5 mg of human serum albumin (hereinafter abbreviated as HSA) made by Sigma was dissolved in 1 ml of PBS7.4. Furthermore, the solution was replaced with 100 μl diluted 10-fold with Acetate 5.0 buffer and allowed to stand for 30 minutes to fix HSA. The inside of the chip was replaced with a 1M ethanolamine solution and left for 10 minutes. The inside of the chip was washed 10 times with 100 μl of PBS7.4.

(3) Creation of reference chip:
After removing the solution in the dextran measurement chip prepared in (1) above, 70 μl of a mixed solution of 200 mM EDC and 50 mM NHS was added and left for 10 minutes. After removing the mixed solution, it was washed 3 times with 100 μl of water and 3 times with 100 μl of PBS7.4. The inside of the chip was replaced with a 1M ethanolamine solution and left for 10 minutes. The inside of the chip was washed 10 times with 100 μl of PBS7.4.

(4) Creation of flow path system A cell having an internal volume of 15 μl was created by covering the dielectric block of the HSA fixed chip of the present invention with silicon rubber. Two channels of 1mm diameter were made in the silicon rubber of the lid, and a Teflon tube with an inner diameter of 0.5mm and an outer diameter of 1mm was passed through to create a flow path. Similarly, a lid and a flow path were created for the reference chip, and the two chips were connected in series to create a flow path system. Two chips of this flow path system were respectively installed in the surface plasmon resonance measuring apparatus of the present invention.

(5) Evaluation of binding performance of test substance The inside of the flow path system was filled with a phosphate buffer solution 10 mM (pH 7.4) containing 5 wt% DMSO (hereinafter abbreviated as 5% DMSO / PBS7.4). The signal change with reference to the time before liquid exchange was measured at 0.5 second intervals. The inside of the channel system was replaced with a test compound (50 compounds same as those used in Example 1 dissolved in 5% DMSO / PBS7.4 to 10 μg / ml) at a rate of 20 μl / sec. . The time required for the replacement was 5 seconds.

  The liquid flow was stopped after 5 seconds from the start of the liquid exchange. Using data from 5 seconds to 2 minutes after liquid replacement, the binding constant (KD) of the test substance was determined. The binding constant was calculated by the following method.

  That is, from the measurement result of the signal change of surface plasmon resonance, the adsorption rate coefficient (ka) and the desorption rate coefficient (kd) are obtained by using the following formulas (1), (2) and (3), and the obtained adsorption rate is obtained. From the coefficient (ka) and the separation rate coefficient (kd), the dissociation constant (KD) was calculated by the equation represented by KD = kd / ka.

_{dθ / dt = ka × c s} × (1-θ) -kd × θ (1)
(In the formula, θ represents the adsorption rate (= adsorption amount / saturated adsorption amount), ka represents the adsorption rate coefficient, kd represents the desorption rate coefficient, and c _s represents the concentration of the molecule to be analyzed in the vicinity of the metal surface.)
^{∂c / ∂t = D × ∂ 2} c / ∂x 2 (2)
(Distance from wherein, x is a metal surface, D is the diffusion coefficient of the analyte molecule, c is represents the concentration of the analyte molecule, and c = c _s when x = 0.)
θ = R / Rmax (3)
(In the formula, θ represents an adsorption rate (= adsorption amount / saturated adsorption amount), R represents a surface plasmon signal, and Rmax represents a signal when the molecule to be analyzed is saturated adsorbed.)

  In Example 2, the same results as in Example 1 were obtained, and the effects of reducing the amount of liquid used and reducing baseline noise were observed.

FIG. 1 is a diagram showing the correlation between logP ′ and log (KD) obtained in the example. FIG. 2 shows the surface plasmon resonance measuring apparatus used in the examples. FIG. 3 shows a dielectric block used in the example.

Explanation of symbols

DESCRIPTION OF SYMBOLS 10 Measurement unit 11 Dielectric block 12 Metal film 13 Sample holding frame 14 Sensing substance 30 Light beam 31 Laser light source 32 Condensing lens 40 Photodetector S40 Output signal 400 Guide rod 401 Slide block 402 Precision screw 403 Pulse motor 404 Motor controller 410 Unit connector 411 connecting member

Claims (7)

For a group of test substances, any one or more of the adsorption rate constant (ka), desorption rate constant (kd), binding constant (KD), or binding amount between the test substance and the ligand was measured and measured as described above. Correlation by comparing one or more of the adsorption rate constant (ka), desorption rate constant (kd), binding constant (KD), or amount of binding between the test substance and the ligand, and the physical constant of the test substance And a test substance screening method comprising: extracting test substances that are out of the obtained group of correlations. The physical constant of the test substance is at least one of a numerical value representing a hydrophobicity parameter (logP), an acid dissociation constant (pKa), a dielectric constant, a polarizability, a dipole moment, a molecular occupied volume, or a molecular shape. The screening method according to claim 1. The screening method according to claim 1 or 2, wherein the correlation is analyzed using 50 or more kinds of test substances. The correlation between any one or more of the adsorption rate constant (ka), desorption rate constant (kd), binding constant (KD), or binding amount between the test substance and the ligand and the physical constant of the test substance is a straight line. The screening method according to claim 1, which is approximated by any one of a quadratic curve, a normal distribution, and a binomial distribution. The screening according to any one of claims 1 to 4, wherein an adsorption rate constant (ka), a desorption rate constant (kd), a binding constant (KD), or a binding amount between the ligand and the test substance is determined by a surface plasmon resonance measuring apparatus. Method. A metal film; a light source that generates a light beam; an optical system that causes the light beam to be incident so that a total reflection condition is obtained at an interface with the metal film and includes various incident angle components; and the metal film Using a surface plasmon resonance measuring apparatus comprising a flow path system including cells formed thereon and light detection means for detecting the state of surface plasmon resonance by measuring the intensity of a light beam totally reflected at the interface The adsorption rate constant (ka) between the ligand and the test substance is measured by exchanging the liquid in the flow path system from a solution containing no test substance to a solution containing the test substance and measuring the change in surface plasmon resonance. The screening method according to claim 1, wherein a withdrawal rate constant (kd), a binding constant (KD), or a binding amount is obtained. The screening according to claim 6, wherein a change in surface plasmon resonance is measured in a state where the flow of the liquid is stopped after the liquid in the flow path system is changed from a solution containing no test substance to a solution containing the test substance. Method.

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