WO2006070941A1

WO2006070941A1 - Novel screening method for substance using fluorescent molecular probe

Info

Publication number: WO2006070941A1
Application number: PCT/JP2005/024274
Authority: WO
Inventors: Seiichi Tanuma; Atsushi Yoshimori
Original assignee: Tokyo University Of Science
Priority date: 2004-12-28
Filing date: 2005-12-28
Publication date: 2006-07-06
Also published as: JPWO2006070941A1; JP5099675B2

Abstract

Disclosed are a method for analyzing the interaction between a target protein and a compound using a compound capable of interacting with the target protein and capable of emitting fluorescent light natively which has been selected previously; an apparatus for use in the analysis; and a kit for use in the analysis. A method for screening a compound capable of interacting with a target molecule comprising the steps of: performing the competitive reaction between a fluorescent molecular probe and candidate compounds for binding to the target molecule, in which the probe used is a ligand which is capable of binding to the target molecule and is capable of emitting fluorescent light natively without the need of fluorescent labeling; and selecting a compound which has competed with the fluorescent molecular probe by means of fluorescence analysis.

Description

Description New drug screening method using fluorescent molecular probe Technical Field

The present invention relates to a fluorescence analysis method using a fluorescent molecular probe having intrinsic fluorescence, and further relates to a drug discovery screening system using the probe. Background art

As a method for analyzing the interaction between a protein such as a receptor and the protein and a ligand, a ligand labeled with a fluorescent compound has been conventionally used. For example, in the case of screening a compound that can be a drug candidate that interacts with a specific receptor protein, a compound that is known to interact with the protein is labeled with a fluorescent substance, and the fluorescent substance The drug candidate compound competed for binding to the target protein, and the drug candidate compound that interacts with the protein was screened. In this case, for example, the target substance protein is immobilized on a plate or the like, a fluorescent label ligand is bound to the immobilized protein, and the interaction is studied. However, by solidifying the protein, the three-dimensional structure of the protein changes from the structure in the natural state, so that interactions occurring in the living body could not be correctly analyzed.

Therefore, single-molecule fluorescence analysis methods such as fluorescence correlation spectroscopy, fluorescence intensity distribution analysis, or fluorescence polarization analysis have been developed as methods for analyzing interactions in solution without solidifying proteins (W02002). / 048693 International Publication Pamphlet and JP

2003-275GG0 publication).

However, in the analysis of protein-compound interactions using conventional fluorescent compounds, including the single-molecule fluorescence analysis method described above, the compounds were labeled with a fluorescent compound such as TAMRA. This is because there was no compound that could bind to a specific target protein and emit intrinsic fluorescence. Various methods for labeling other compounds using fluorescent substances have also been reported. It has been established as a standard method to use fluorescently labeled compounds capable of interacting with proteins in the interaction analysis between proteins and ligands. Disclosure of the invention

As described above, in the conventional analysis of the interaction between a protein and a compound, the compound is labeled with a fluorescent substance and used. However, although various methods for labeling compounds with fluorescent substances have been established, it takes time and money to label, and it has not been easy to analyze the interaction of many target proteins. In addition, fluorescent substances are bonded to the compounds by covalent bonds using the functional groups of the compounds. However, in low molecular weight compounds, the three-dimensional structure of the compounds changes and is not labeled by binding the fluorescent substances. Compared to the case, there was a problem that the binding affinity with the protein was lowered.

In order to solve the above conventional problems, the present invention searches for a compound that interacts with a target protein and has intrinsic fluorescence, and uses the compound to determine the interaction between the target protein and the compound. The purpose is to provide an analysis method, an analysis device, and an analysis kit.

As a result of diligent investigations to solve the above-mentioned problems in protein and compound fluorescence analysis using a fluorescent probe labeled with a fluorescent substance such as a conventional fluorescent dye, the present inventors have found several hundred. A compound that can interact with a target molecule by screening a fluorescent molecular probe, which is a compound that has unique fluorescence from a compound library containing a wide variety of compounds and can bind to a specific target molecule. It was found that this screening can be performed without causing the above problems. That is, the present inventors select a compound having intrinsic fluorescence from one compound library, construct a fluorescence probe library, and then perform a target by performing docking studies on a computer from the fluorescence probe library. By selecting a compound that has a binding property to a molecule, it has been found that a compound that binds to an arbitrary target molecule and has intrinsic fluorescence can be obtained, and screening a drug candidate compound using the compound The present invention has been completed. That is, the aspect of this invention is as follows.

[1] A screening method for a compound capable of interacting with a target molecule, wherein a ligand capable of binding to the target molecule and having intrinsic fluorescence without being fluorescently labeled is used as a fluorescent molecular probe, and A method for screening a compound capable of interacting with a target molecule, comprising competitively reacting a candidate compound in binding to the target molecule, and selecting a compound that competes with the fluorescent molecular probe by fluorescence analysis.

[2] The method for screening a compound capable of interacting with the target molecule of [1], wherein the target molecule is a protein.

[3] Measure the competition in the binding of the fluorescent molecular probe and the candidate compound to the target molecule using the change in the fluorescent signal due to the binding of the fluorescent molecular probe and the target molecule in solution as an index [1] or [2] A method for screening compounds capable of interacting with a target molecule.

[4] Measure the competition in binding of the fluorescent molecular probe and the candidate compound to the target molecule using fluorescence correlation spectroscopy, fluorescence intensity distribution analysis, or fluorescence ellipsometry [3] A compound that can interact with the target molecule Screening method.

[5] A method for screening a compound capable of interacting with any one of the target molecules according to any one of [1] to [4], wherein the fluorescent molecular probe is selected from a compound library using the binding property to the target molecule as an index.

[6] Fluorescent molecular probes are selected from a docking study with a target molecule from a fluorescent probe library constructed by selecting molecules with intrinsic fluorescence from a compound library, or further structurally optimized. [1] ~

[5] A screening method for a compound capable of interacting with any of the target molecules.

[7] A method for screening a compound capable of interacting with a target molecule according to [6], wherein the construction of a fluorescent probe library from one compound library is performed by at least one of the following methods (a) to (c): .

(a) A compound having a fluorophore as a partial structure is selected from the structural formula of the compound

(b) Select fluorescent compounds using molecular orbital calculation software, and

(c) Selecting fluorescent compounds by actual measurement using a light absorption analyzer [8] The method for screening a compound capable of interacting with a target molecule according to [6] or [7], wherein the structure of the fluorescent molecular probe is optimized by introducing a substituent.

[9] A desired binding constant between the target molecule and the fluorescent molecular probe is set in advance. If the binding constant between the target molecule and the fluorescent molecular probe is larger than the set desired binding constant, the fluorescent molecular probe is targeted. A method for screening a compound capable of interacting with the target molecule of any one of [5:] to [8], which is selected as a fluorescent molecular probe of the molecule.

[10] Preliminarily measure the optimum excitation wavelength and the optimum fluorescence wavelength of the fluorescent molecular probe, excite the fluorescent molecular probe at a wavelength near the optimum excitation wavelength, and detect fluorescence at a wavelength near the optimum fluorescence wavelength. [1] A screening method for a compound capable of interacting with any of the target molecules of [9].

[1 1] Screening for a compound capable of interacting with a target molecule, wherein a ligand that is capable of binding to the target molecule and has intrinsic fluorescence without being fluorescently labeled is used as the target molecule-specific fluorescent molecule probe. A fluorescence analyzer for screening, a light irradiation means capable of continuously irradiating light of the wavelength of the excitation light of the target molecule-specific fluorescent molecule probe to be used; Photodetection means capable of continuously detecting the wavelength of the fluorescence light of the specific fluorescent molecular probe, photodetection means capable of continuously changing the wavelength of the detection light, reaction means for causing a competitive reaction between the fluorescent molecular probe and the candidate compound in the binding of the target molecule Including a fluorescence analyzer.

[12] The fluorescence analyzer according to [1 1], wherein the target molecule is a protein.

[1 3] The wavelength of the irradiation light and the wavelength of the detection light can be changed according to the excitation wavelength and fluorescence wavelength of the target molecule-specific fluorescent molecular probe measured in advance. [1 1] or [12] Fluorescence analyzer.

[14] The fluorescence analysis apparatus according to any one of [11] to [13], wherein the reaction means is a reaction vessel that causes a competitive reaction between a fluorescent molecular probe and a candidate compound in a solution in a solution.

[1 5] Measure the competition in the binding of the fluorescent molecular probe and the candidate compound to the target molecule using fluorescence correlation spectroscopy, fluorescence intensity distribution analysis, or fluorescence ellipsometry [1 1]-[4] Fluorescence analyzer.

[1 6] Able to bind to the target molecule A kit for screening for a compound capable of interacting with a target molecule, comprising a target molecule-specific fluorescent molecular probe which is a fluorescent ligand.

[1 7] The screening kit according to [1 6], wherein the target molecule is a protein.

[18] A kit for screening a compound capable of interacting with a target molecule according to [16] or [17], wherein a fluorescent molecular probe is selected from a compound library using the binding property to the target molecule as an index.

[1 9] Fluorescent molecular probes are selected from a fluorescent probe library constructed by selecting a molecule with intrinsic fluorescence from a compound library by docking studies with the target molecule, or further structural optimization A kit for screening for a compound capable of interacting with the target molecule of any one of [16] to [18].

[20] Construction of a fluorescent probe library from a compound library is performed by at least one of the following methods (a) to (c): [19] Kit for screening for compounds that can interact with the target molecule .

(b) Select fluorescent compounds with molecular orbital calculation software, and

(c) Selecting fluorescent compounds by actual measurement using a light absorption analyzer

[2 1] A kit for screening a compound capable of interacting with a target molecule according to [19] or [20], wherein the structure of the fluorescent molecular probe is optimized by introducing a substituent.

This specification includes the contents described in the specification and Z or drawings of Japanese Patent Application No. 2004-381671, which is the basis of the priority of the present application. Brief Description of Drawings

Figure 1 shows a conceptual diagram of the construction of a fluorescent probe library.

Figure 2 shows a conceptual diagram of how to select the optimal fluorescent molecular probe.

FIG. 3 is a conceptual diagram of a method for selecting an optimal fluorescent molecular probe, and shows that a fluorescent molecular probe having a unique excitation and fluorescent wavelength exists for each target molecule. FIG. 4 shows a conceptual diagram of a method for selecting a compound that can interact with a target molecule.

FIG. 5 is a diagram showing the configuration of the apparatus of the present invention.

FIG. 6 shows the three-dimensional structure of DNase r.

FIG. 7 shows the structure of R396842, the optimal fluorescent molecular probe for DNase r. FIG. 8 is a diagram showing the binding of DNase r and the optimal fluorescent probe.

FIG. 9 is a diagram showing the dissociation of DNase and the optimal fluorescent probe DR396842 complex by ATA (Aut in Tricarboxyl Acid). Explanation of symbols

1 Light irradiation means

2 Filter

3 Light detection means

4 Filter

5 Mirror

6 Lens

7 Reaction means Best mode for carrying out the invention

Hereinafter, the present invention will be described in detail.

In the method for screening a compound that interacts with a target molecule of the present invention, the target molecule is not limited, and includes molecules that interact with all other substances existing in the living body. The target molecule is preferably DNA, RNA, or protein, more preferably a protein, and particularly preferably a receptor protein that can interact with a specific ligand. Receptor proteins interact with specific ligands, transmit receptor signals, and cause various reactions in vivo. Desirably, the receptor protein signal is associated with various diseases in the living body. The compound that interacts with the receptor protein can function as a prophylactic or therapeutic agent for diseases by controlling the signal transduction of the receptor protein in vivo. In the present invention, as an example of a protein as a target molecule DNase, G protein coupled receptor etc.

The compound that can interact with the target molecule is, for example, a ligand compound that can interact with the target molecule. A ligand compound refers to a low molecular weight compound that binds to a biopolymer such as a protein. A ligand compound that can interact with a target molecule refers to a ligand compound that can bind to a target molecule and activate or inhibit the function of the target molecule. The ligand compound can act as an agonist or antagonist. An agonist refers to a substance that binds to a receptor and exhibits various physiological functions, and an antagonist refers to a substance that binds to a receptor and inhibits the effect of agonis. The type of compound, molecular weight, etc. are not limited. According to the method of the present invention, a compound to be screened interacts with a specific protein such as a receptor, exhibits a physiological effect, and can be used as a prophylactic or therapeutic agent for a disease associated with the receptor. It is expected. The compound can also be used as a lead compound in drug discovery.

In the screening method of the present invention, a ligand that is inherently fluorescent and capable of interacting with a target molecule without using a fluorescent label is used. This ligand is referred to as a fluorescent molecular probe in the present invention. Fluorescent molecular probes are prepared for each target molecule as being capable of interacting specifically with the target molecule. The fluorescent molecular probe used in the present invention is 450! A compound having an excitation / fluorescence wavelength within a wavelength range of ˜650 mn. The fluorescent molecular probe is prepared as follows.

First, a compound having intrinsic fluorescence is selected from the compound library. A compound library is a library that includes chemical formula structures and three-dimensional structure information of compounds with known structures. The compound library includes not only a collection of compounds containing commercially available reagents and natural substances, but also a database constructed on a computer storing compound information. A compound library contains information on millions of compounds. As a compound library, various compounds stored in existing databases can be used. One huge library of millions of compounds is also commercially available, and one of these commercially available libraries may be used.

From the compound library, by selecting a partial structure search, fluorescence / absorption spectrum prediction or actual measurement using a light absorption analyzer, select a fluorescent compound that emits fluorescence uniquely, Build a fluorescent probe library. In the partial structure search, a compound having a fluorophore as a partial structure is searched. Examples of the fluorophore include a xanthene skeleton, an azobenzene skeleton, a benzofuran skeleton, an indole skeleton, and an anthracene skeleton. The partial structure search may be performed by using a chemical structure as an index from a catalog of known compounds, literature, etc., not only using a database storing a compound library.

The fluorescence / absorption spectrum is predicted from the molecular structure information of the above compound library. The prediction can be performed by, for example, molecular orbital calculation. Molecular orbital means the existence probability distribution of electrons in a molecule. By calculating the molecular orbitals, it is possible to know how much energy electrons are present in the molecule. By examining the energy distribution, it is possible to predict optical properties such as light absorption. The For example, by using the M0S-F program, it is possible to predict ultraviolet, visible absorption, and fluorescence spectra. The spectrum calculation by the M0S-F program is described, for example, by L Abe and Y. Shirai, J. Am. Chem. So, 118, 4705 (1996), J. Abe,

Y. Shi rai, N. Nemo to and Y. Nagase, J. Phys. Chem. B, 101, 1910 (1997),

J. Abe, Y. Shi rai, N. Nemo to and Y. Nagase, J. Phys. Chem. A, 101, 1 (1997),

J. Abe, Y. Shirai, N. Nemo to, Y. Nagase and T. Iyoda, J. Phys. Chem. B, 101,

145 (1997), S. Tatsuura, W. Sotoyama, K. Motoyoshi, A. Mat suura, T. Hayano and T. Yos imura, Appl. Phys. Let t., 62, 2182 (1993) K. Hiruta, S Toki ta and K. Nishimoto, J. Chem. So Perkin Trans. 2, 1443 (1995). Commercially available M0S-F program software can also be used, for example, Fujitsu's WinMOPAC 3.9.

In the actual measurement using the optical absorption analyzer, the compounds contained in the compound library are obtained, and the fluorescence / excitation wavelength of the compound is actually measured using a commercially available optical absorption analyzer, and the fluorescent compound is selected.

Figure 1 shows a conceptual diagram of the construction of a fluorescent molecular probe library.

From the fluorescent probe library thus obtained, a fluorescent molecule having affinity with the target molecule is selected for each target molecule. Here, a fluorescent molecular probe having affinity for a target molecule is called a target molecule-specific fluorescent molecular probe, and has a high affinity for the finally selected target molecule, and is inherently fluorescent. Have The fluorescent molecular probe is called an optimal fluorescent molecular probe. To select the optimal fluorescent probe from the fluorescent probe library, first select a binding fluorescent molecular probe that can bind to the target molecule from the fluorescent probe library, and construct a binding fluorescent probe library. Whether or not the fluorescent molecular probe contained in the fluorescent probe library binds to the target molecule may be determined by a docking study, for example. Docking study 1 refers to a method of predicting the complex structure by virtually binding a target molecule to a drug discovery target and a ligand on a computer, calculating its binding affinity score. The calculation of the binding affinity score includes a force field-based scoring function, an empirical scoring function, and a knowledge-based scoring function ( Golke H et al., Angew Chem. Int. Ed. Engl. (2002) 41, 2644-2676) _{0 In} addition, genetic algorithms, annealing methods, and the search for complex structures of target molecules and ligand molecules, Evening search is used (Taylor RD et al., J. Co Immediate ut. Aided Mol. Des. (2002) 16, 15 卜 166). Several docking programs using score functions and search methods have been developed (Taylor RD et al., J. Comput. Aided Mol. Des. (2002) 16, 151-166). And used for design. So far, HIV-1 protease inhibitors (DesJarlais L et al., Proc. Natl. Acad. Sci. USA (1990) 87, 6644-6648), kinesin inhibitors (Hopkins SC et al. al., Biochemistry (2000) 39, 2805-2814), Bcl-2 inhibitors (Wang JL et al., Proc. Natl. Acad. Sci. USA (1990) 97, 7124-7129) Has been successfully identified and designed. In the present invention, a docking study can be performed based on the description of these documents, and AutoDock3.0 is well known.

(Goodsell DS et al., Proteins (1990) 8, 195-202; Goodsell DS et al., J Mol Design (1996) 9, 1-5; Morris GM et al., J Comp Aided Mol Design (1996) 10 , 293-304; Morris GM et al., J Comput Chem (1998) 19, 1639-1662), can be suitably used. Also, a commercially available docking study (docking simulation) software program may be used. Commercially available docking study software programs include, for example, BioPackage (MolSoit), LigandFit

(Accelrys Inc.) To perform a docking study, first obtain the three-dimensional structure of the target molecule. 3D structure information can be obtained from 3D structure databases such as Protein Data Bank (tt: //www.rcsb.org/pd. The 3D structure of the target molecule is not registered in the 3D structure database. In this case, the predicted three-dimensional structure can be constructed using the homology modeling method based on the three-dimensional structure of the related molecule, where homology modeling is based on the three-dimensional structure of proteins with sequence homology. In the homology modeling, the amino acid sequence of the protein to be modeled is determined, and the portion having the secondary structure or the loop portion is predicted, and then the homology search is performed. A homology search searches for homology with a known domain, and if the sequence is short, it is performed with the same length, and if the sequence is long, the appropriate length is selected. Next, identify the protein structure to be cocoon-shaped, and select rational ones that have high sequence homology and low homology. In addition, the type information used for homology modeling is organized, the three-dimensional structure of the protein is displayed, and the amino acid sequence is aligned with the type 3 on the modeling software. Homology modeling can be performed using the above docking study software program, and there are various homology modeling software programs (for example, , PDFAMS Pro,

PDFAMS Ligand), etc., can also be used. To determine the target site to which the fluorescent molecule probe is bound on the target molecule, any site such as an active site or interaction site may be specified. Next, a docking study is performed using the fluorescent compound in the fluorescent probe library and the three-dimensional structure of the target site, and a binding compound is selected and stored in the binding fluorescent probe library. Various algorithms can be used in the docking day. For example, the shape-oriented docking algorithm compares the shape of the binding site with the generated multiple ligand conformations one by one using a shape comparison filter, and finds the optimal ligand position that matches each other. Explore. The poses identified by Phil Yuichi are then minimized by evaluating the interaction energy between the protein and the ligand at the binding site using a dalid-based approach. Energy grid The error caused by using is greatly reduced by non-linear interpolation. If a small ligand candidate is desired in comparison with the binding site, site binding may be performed by dividing the binding site into smaller sections and docking. When actually selecting a fluorescent molecular probe for a specific target molecule, set an arbitrary binding constant X [M " ¹ ] that is considered to be the minimum necessary in advance, and calculate the binding constant Y of the compound in the fluorescent probe library. Measure the above or by actual measurement, and store those satisfying the condition KY / X <10 in the binding fluorescent probe library.The binding constant of the normal fluorescent molecular probe is 1 X 10 M— 〜 1 Χ Μ Μ ^-1 ] is desirable.

As described above, the steps of constructing a compound library, a fluorescent probe library, and a binding fluorescent probe library are performed, and the structure optimization described later is performed appropriately, so that a fluorescent molecular probe for an arbitrary target library can be obtained. Can be selected.

Next, the actual compound selected from the fluorescent probe library is obtained, and a fluorescent molecular probe that binds to the target molecule is identified using an in vitro assay corresponding to the target molecule. When binding with sufficient affinity, the fluorescent molecular probe is used as the optimal fluorescent molecular probe. If stronger affinity is required, the selected fluorescent probe is used as a parent, introducing a wide variety of substituents into the mother, diversifying the mother, and further conducting a docking study And determine the substituents that enhance the affinity. Synthesize fluorescent molecular probe with high affinity and perform in vitro assay again. Until a compound with the required affinity is obtained, diversification of the personality, docking study and in vitro assembly are repeated, and finally an optimal fluorescent molecular probe is obtained. This series of processes is called structure optimization. For the obtained optimal fluorescent molecular probe, the optimal excitation wavelength and the optimal fluorescent wavelength are measured using a molecular orbital calculation program such as an optical absorption analyzer or M0S-F, and used for subsequent screening. An optimal fluorescent molecular probe is a compound that emits sufficient fluorescence to study the interaction between a target molecule and a compound, and can bind to the target molecule with an affinity greater than the set affinity. Fig. 2 and Fig. 3 show a conceptual diagram of the method for selecting the optimal fluorescent molecular probe. Fig. 3 shows that each target molecule (A, B, C, etc. in Fig. 3) has a fluorescent molecular probe (a, b, c, etc. in Fig. 3) having a unique excitation and fluorescence wavelength. Fluorescent molecule pro It is shown that the optimum excitation wavelength / fluorescence wavelength (n no m _a , n _b / m _b , n _c / ni _c etc. in FIG. 3) is determined for There can be multiple optimal fluorescent molecular probes for one target molecule.

A compound that can bind to the target molecule selected by the above method and interacts with the target molecule using a fluorescent molecule probe as a fluorescent molecule probe without fluorescent labeling. To screen for compounds that can exert drug action. This screening is performed by utilizing a phenomenon in which a fluorescent molecular probe and a candidate compound that is a drug candidate compete and bind to a target site of a target molecule. That is, when the compound of interest binds to the target site, the fluorescent molecular probe cannot be bound to the target site and is released. Therefore, the degree of binding of the compound to the target molecule can be quantitatively measured based on the abundance ratio between the target molecule fluorescent molecule probe complex and the free fluorescent molecule probe. Actually, it is only necessary to measure the fluctuation of the fluorescence signal when the fluorescent molecule probe is bound to the target molecule. Here, the fluctuation of the fluorescence signal refers to the change in the signal that can be detected by the binding of the fluorescent molecule probe and the target molecule. As described later, the fluorescence intensity by the single molecule fluorescence analysis method or the fluorescence polarization analysis method is used. As well as changes in fluorescence intensity detected after separating a complex of a fluorescent molecular probe and a target molecule and a free fluorescent molecular probe. In the method of the present invention, a target molecule is solid-phased on a solid phase carrier such as 96 well plate, and a fluorescent molecule probe that is an optimal fluorescent molecular probe for the target molecule and a fixed amount of candidate compound on the solid-phased target molecule. And the fluorescent molecular probe and the candidate compound are allowed to compete for binding to the target molecule, and the free fluorescent molecular probe and the target molecule Z fluorescent molecular probe complex are separated and bound to the immobilized target molecule. By measuring the fluorescence of a fluorescent molecular probe or the fluorescence of a free fluorescent molecular probe that is not immobilized, the excitation and fluorescence wavelengths characteristic of the fluorescent molecule are used to determine the interaction between the candidate compound and the target molecule. Can be measured. At this time, a standard curve may be prepared by measuring in advance fluorescence when a certain amount of fluorescent molecular probe is bound to the target molecule. However, when the target molecule is solid-phased, the three-dimensional structure of the target molecule is often different from the natural three-dimensional structure when it exists in the living body, and in vitro between the solid-phased target molecule and the candidate compound. Interactions are not limited to reflecting interactions in vivo. Not. In addition, fluorescent molecular probes are low-molecular compounds, and the molecular weights of target molecules and fluorescent molecular probes are often very different, and it is often difficult to separate the complex as described above from free fluorescent molecular probes. . Furthermore, when using a solid-phased target molecule, it is necessary to use a certain amount of each of the target molecule, fluorescent molecular probe, and candidate compound, which is not suitable for microanalysis. Therefore, it is desirable to measure the reaction of the target molecule with the candidate compound and the fluorescent molecular probe in a solution in which the molecule can move freely without immobilizing the target molecule. One method for measuring the binding between a target molecule and a fluorescent molecular probe in solution is single-molecule fluorescence analysis. Single-molecule fluorescence analysis is a method that can detect fluorescent molecules at the single molecule level, and measures fluorescence fluctuations when fluorescent molecular probes and target molecules bind in solution. Capturing the binding between a molecular probe and a target molecule. Single-molecule fluorescence analysis is performed by measuring the fluctuation motion of fluorescent molecules entering and exiting a small confocal region and analyzing the data obtained by this measurement using functions. Single molecule fluorescence analysis includes fluorescence correlation spectroscopy

(Fluorescence Correlat ion Spectroscopy) analysis, fluorescence intensity distribution analysis

(Fluorescence Intensity Distribution Analyzes) and Fluorescence Intensity Multidistribution Analysis

Analys is).

Fluorescence correlation spectroscopy is a method for measuring the micromotion of individual probe molecules by measuring the fluctuation motion of a fluorescent molecular probe in a solution and using an autocorrelation function (D. Magde et al., Biopolymers 1974 13 (1)

29-61). Fluorescence correlation spectroscopy uses a laser confocal microscope to capture the Brownian motion of a fluorescent molecular probe in a solution in a very small area, thereby analyzing the diffusion time from the fluctuation of fluorescence intensity and measuring physical quantities (number of molecules and size). Is done. In fluorescence correlation spectroscopy, it is only necessary to detect and quantify a fluorescent signal generated from a small visual field in a sample. Since the fluorescently labeled target molecules in the medium are always in motion (Brownian motion), the detected fluorescence intensity depends on the frequency with which the target molecules enter the micro-field region and the time for which they remain in the region. Changes. For example, when a fluorescent molecular probe binds to a target molecule, the molecular weight of the complex that emits fluorescence is large, so the movement of the molecule becomes slow and the apparent number of molecules decreases. As a result, it falls within the micro field of view. The frequency of coming in decreases, and as a result, the fluorescence intensity changes. By monitoring the change in fluorescence intensity, the binding between the target molecule and the fluorescent molecular probe can be detected. In the fluorescence intensity distribution analysis, the sample is irradiated with laser, the excitation light of the fluorescent molecules emitted from the sample is measured with a high-sensitivity photo detector, and the measured fluorescence signal is resolved in a very short time of 40 xs. This is a technique to analyze the double-Poisson distribution function and statistical processing analysis of the photon count (P Kask, et al; PNAS 23, 96, 13756-13761, 1999, W098 / 16814). By doing so, the fluorescence intensity per molecule (brightness; an) and the number of fluorescent molecules (cn) are calculated. At this time, even if there are multiple types of molecules, they can be identified by distribution analysis, and for each molecular type, a value obtained by multiplying the brightness and the number of molecules can be calculated as the total fluorescence.

Fluorescence intensity multi-distribution analysis is the simultaneous analysis of fluorescence correlation spectroscopy and fluorescence intensity distribution analysis, and provides data on the translational diffusion time, number of molecules, and fluorescence intensity per molecule (brightness) at once. (K Palo, Biophysical Journal, 79, 2858-2866, 2000). Single-molecule fluorescence analysis can be performed according to the description in JP 2003-275000 A, JP 2003-279566 A, W02002 / 048693 International Publications, etc. Further, it can be carried out using a commercially available single molecule fluorescence analyzer such as MF20 / MF10S manufactured by Olympus. At this time, the method of the present invention uses fluorescent molecular probes having various excitation / fluorescence wavelengths that can bind to various target molecules, and therefore the excitation wavelength and the fluorescence wavelength must be continuously variable. There is.

In addition, compounds can be screened by fluorescence ellipsometry. In the fluorescence polarization analysis method, when a fluorescent molecule in a liquid is excited by plane-polarized light, it emits fluorescence in the same polarization plane when the fluorophore in the fluorescent molecule is in an excited state and a steady state. This is an analysis method using the fact that when a fluorescent molecule moves, such as rotating, during the excited state of the group, fluorescence is emitted to a plane different from the excitation plane and the fluorescence polarization is canceled (J. Horinaka et al., Polym. L, 31, 172 (1999); J. Hor inaka et al., Co immediately.

Theor. Polym. Sci., 10, 365 (2000); H. Aoki et al., Polym. J., 33, 464 (2001)). The movement of the molecule is affected by the molecular weight, and if the fluorescent molecule is a small molecule, the movement speed is Since it is fast, the polarization of fluorescence is eliminated and the degree of fluorescence polarization is small. On the other hand, in the case of macromolecules, the movement of molecules during the excited state decreases, so that the fluorescence cannot be depolarized and exhibits a large degree of fluorescence polarization. Therefore, the binding between the fluorescent molecular probe and the target molecule can be detected by measuring the fluorescence polarization when the fluorescent molecular probe is bound to the target molecule. In order to measure the fluorescence polarization, the excitation light is irradiated through a polarizing filter, and the fluorescence emitted by the fluorescent molecular probe is emitted on both the vertical polarization plane perpendicular to the polarization plane of the excitation light and the horizontal polarization plane horizontal. taking measurement. The degree of fluorescence polarization can be determined by the formula (fluorescence intensity of parallel polarization plane vs. fluorescence intensity of vertical polarization plane) / (fluorescence intensity of parallel polarization plane + fluorescence intensity of vertical polarization plane).

When the competitive reaction between the candidate compound and the fluorescent molecular probe in the target molecule is performed in a solution, the solution may be performed in a solution capable of binding the candidate compound and the target molecule, such as physiological saline or a phosphate buffer near neutrality. Can be used. Reaction conditions such as temperature, pH, and reaction time at this time may be set as appropriate according to the type of target molecule.

In order to measure competitive reactions in solution using a fluorescent molecular probe, in addition to the single molecule fluorescence analyzer described above, for example, LS55 manufactured by PerkinElmer, Spectra Max manufactured by Molecular Devices, etc. should be used. Can do.

In carrying out the method of the present invention, in order to examine the interaction between the target compound and the candidate compound, it is desirable to react the target compound, the candidate compound and the fluorescent molecular probe at various concentrations. For this reason, it is desirable to conduct a large number of assemblies at once. For this purpose, the apparatus of the present invention is preferably an apparatus capable of performing multi-well assay using a 96 well plate or the like and performing high-throughput screening.

The amount of target molecule, fluorescent molecular probe, and candidate compound in the case of performing a competitive reaction may be set as appropriate depending on the type of target molecule, the affinity of the fluorescent molecular probe to the target molecule, etc. The target molecule has a final concentration of 0 . O lnM lOO ^ M degree, preferably 0.1

About 10 AM. In addition, candidate compounds to be added to the solution have a final concentration of 0.01 nM

It is about I OO M, preferably about 0.1 to 10 M, more preferably about 0.01 to 100 nM, and still more preferably about 0.1 to 50 ιιΜ. The fluorescent molecular probe added to the solution is

About 0.01 to 100 nM, preferably about 0.1 · 50 ηΜ. The reaction of the target molecule, the fluorescent molecular probe and the candidate compound may be performed in an appropriate container such as a test tube, a well, or a cell. Although the container size is not limited, since the reaction is carried out in several tens of il to several mL, it is sufficient to use a container that can accommodate an amount in that range. In the method of the present invention, a fluorescent molecular probe is used for each target molecule. Since the excitation wavelength and the fluorescence wavelength are different, the fluorescence is detected by changing the excitation wavelength and the detection wavelength for each fluorescent molecular probe. As excitation light, 450nn! Use light that can excite fluorescent materials such as argon ion laser, helium-neon laser, krypton, xenon, and helium In order to change the wavelength, it is sufficient to use a filter such as a bandpass filter that passes only light of a desired wavelength or a beam split filter. You can also change the wavelength using a spectroscope. As the spectrometer, any of a spectrometer using an optical filter, a dispersion spectrometer, and a Fourier transform spectrometer can be used. Further, a wavelength tunable laser such as an optical parametric laser (0P0 laser) may be used. The fluorescence emitted by the fluorescent molecular probe may be detected by a photodetector through a band-pass filter that passes only light of the wavelength of the fluorescence. At this time, the excitation wavelength and the detection wavelength of the light used need not be completely the same as the optimum excitation wavelength and the fluorescence wavelength of the fluorescent molecular probe, and may be a wavelength in the vicinity of the optimum wavelength. The excitation wavelength and the detection wavelength to be used may be within the range of ± 30 nm, preferably ± 20 nm, more preferably ± 10 nm of the optimum excitation wavelength.

By the above method, the abundance ratio between the candidate compound bound to the target molecule and the candidate compound existing in a free state in the reaction solution can be measured, and the binding of the candidate compound to the target molecule is facilitated. (Affinity) can be determined as a binding constant. In the present invention, when the binding constant between the candidate compound and the target molecule is z DT, and the binding constant between the target molecule and the fluorescent molecule probe is y — ¹ ], the candidate compound satisfying the formula z / y> l is obtained. Select as drug candidate compound.

Further, the IC _5D value obtained by the competitive inhibition experiment of the compound capable of interacting with the target molecule screened by the method of the present invention is about the following.

FIG. 4 shows a conceptual diagram of a method for selecting a compound that can interact with a target molecule in the present invention. The drug candidate compound selected from the candidate compounds may be subjected to further structural optimization. In this case, the selected drug compound is used as a lead compound for drug discovery.

The lead compound structure is optimized by substituting and modifying the functional group of the lead compound, creating compounds having various structures, and measuring the binding ability of these compounds to the target molecule. Structural optimization can be performed by combining docking simulation and actual measurement as described above.

The present invention is a screening for a compound capable of interacting with a target molecule, wherein a ligand that is capable of binding to the target molecule and has intrinsic fluorescence without being fluorescently labeled is used as the target molecule-specific fluorescent molecule probe. It also includes a fluorescence analyzer for screening. The apparatus of the present invention comprises at least a light irradiation means capable of continuously irradiating light having the wavelength of the excitation light of a target molecule-specific fluorescent molecular probe to be used, and capable of continuously changing the wavelength of the irradiated light, and a target molecule-specific fluorescent molecule to be used Photodetection means capable of continuously detecting the wavelength of the detection light that can detect the light having the fluorescence wavelength of the probe, and reaction means for causing a competitive reaction between the fluorescent molecular probe and the candidate compound in binding to the target molecule. The light irradiation means has a light source that can irradiate excitation light, and 450 nn! A light source that can excite a fluorescent material such as an argon ion laser, a helium neon laser, krypton, xenon, helium and a force dome laser, which has a wavelength range of ˜650 mn may be used. In order to change the wavelength of the irradiation light, it is only necessary to have a filter such as a bandpass filter that allows only light of the desired wavelength to pass through, and a beam splitter. It is also possible to change the wavelength using a spectroscope. As the spectroscope, any spectroscope using an optical filter, a dispersive spectroscope, or a Fourier transform spectroscope can be used. In addition, a wavelength tunable laser such as an optical parametric laser (0P0 laser) may be used. In this case as well, the wavelength of the irradiation light that can be irradiated with the light of the excitation light wavelength of the target molecule-specific fluorescent molecular probe is continuously changed. Included in the light irradiation means to obtain. The light detection means is a means capable of detecting fluorescence of a specific wavelength, and includes a light detector such as a photodiode. The fluorescence emitted by the fluorescent molecular probe may be detected by a photodetector through a pass-pass filter that passes only light of the fluorescence wavelength. In addition, a reaction means for competitively reacting a fluorescent molecule probe and a candidate compound in binding to a target molecule is a reaction volume. Test tubes, wells, cells, etc. may be used. The reaction means is preferably capable of multi-assembling such as a multi-well plate so that multiple samples can be assayed at once. The reaction means also includes a reaction vessel table for containing the reaction vessel. Furthermore, the apparatus of the present invention includes data processing means, and can process the data of the above-described single molecule fluorescence analysis and fluorescence polarization analysis. Furthermore, the apparatus of the present invention may be an automated apparatus so that a large number of specimens can be measured. In this case, the reaction vessel, reagent dispensing means, data processing means, etc. are controlled by a computer, and a large amount of samples can be processed automatically. When the method of the present invention is performed by single-molecule fluorescence analysis, in order to measure the fluorescence of a minute region in the reaction means, a confocal region is set in the region where the fluorescent molecular probe exists in the reaction means, and the region is excited. It is necessary to irradiate light and measure the fluorescence of the region. In this case, means such as a confocal microscope may be used. Furthermore, the apparatus of the present invention may be provided with a mirror and a lens as appropriate on the optical path. FIG. 5 shows an example of the configuration of the apparatus of the present invention, but the apparatus of the present invention is not limited by the figure. The apparatus shown in Fig. 5 includes a light irradiation means (light source) 1 that irradiates excitation light, a filter 2 that changes the wavelength of the excitation light, a light detection means 3 that detects fluorescence emitted from a fluorescent molecular probe, and a wavelength of fluorescence. It includes a changing filter 4, a mirror 5 for changing the optical path, a lens 6 for collecting light, and a reaction means 7. The arrow in Fig. 5 indicates the optical path. When the apparatus of the present invention is an apparatus for single molecule fluorescence analysis, for example, a confocal microscope may be installed at the lens 6 portion.

Furthermore, the present invention includes a screening kit for compounds capable of interacting with a target molecule. The kit includes a target molecule and a target molecule-specific fluorescent molecule probe that is capable of binding to the target molecule and is a ligand that is intrinsically fluorescent without fluorescent labeling. The target molecule is not limited and includes biopolymers such as DNA, RNA, and protein. A target molecule-specific fluorescent molecule probe that can bind to the target molecule and has intrinsic fluorescence without fluorescent labeling constructs a fluorescent molecule probe library from a compound library as described above, and It can be obtained by measuring the binding affinity with the target molecule. The kit of the present invention includes a reaction buffer and the like. In addition, a plurality of target molecules and target molecule-specific fluorescent molecular probes for each target molecule may be included. In addition, the same binding site for one target molecule. Alternatively, a plurality of target molecule-specific fluorescent molecular probes that can bind to different binding sites may be included.

The present invention will be specifically described by the following examples, but the present invention is not limited to these examples.

Example 1 Construction of DNase r inhibitor screening system

DNase r is an endonuclease with a molecular weight of 33 kDa and an optimum pH of 7.2. The DNasel family to which DNase belongs is currently known in four types: DNaseI, DNaseX, DNasea, and DNASIL2. Furthermore, it has been shown that only DNase r can be activated by inducing apoptosis in cells and causing DNA fragmentation in units of nucleosomes. However, it is known that DNA fragmentation at the nucleosome unit in apoptosis is catalyzed by multiple DNases such as CAD and endonuc leaseG in addition to DNase r. There are many unclear points about how they are properly used depending on the type and state of separation. Thus, DNase r inhibitors that specifically inhibit DNase T and suppress DNA fragmentation during apoptosis are important tools for elucidating the mechanism of action and physiological functions of DNase r in vivo. In addition, it is expected to act as a DNA protective agent in apoptosis that is enhanced by disease.

In this example, DNase r inhibitors were screened using the method of the present invention.

The compound library was constructed by collecting libraries provided by suppliers of screening compounds and general reagents.

From this compound library, a fluorescent molecular probe library containing about 2000 kinds of compounds was constructed by partial structure search, fluorescence 'absorption spectrum prediction or actual measurement using a light absorption analyzer.

The fluorescent molecule probe library is used for docking on a computer, and as a DNaser-specific binding fluorescent molecule probe, R396842 (obtained from Sigma Aldricli) with xanthene skeleton is the optimal fluorescent molecule. Identified as a professional. At this time, 1 X 10 M— was set in advance as a desirable coupling constant. chosen

The binding constant of R396842 was 3. 13 X 10 ⁵ [M-li. Figure 7 shows the structural formula of 39684. The program used for docking simulation was AutoDock3.0. When the optimum excitation wavelength and fluorescence wavelength of R396842 were measured using Gemini manufactured by Molecular Devices, they were 468.5 nm and 514.5 mn, respectively. From this value, fluorescence was detected using a 488 nm Ar laser as the excitation light and a 510-560 nm band filter. As a fluorescence analyzer, Olympus MF20, which is a single molecule fluorescence analyzer, was used.

The binding of both was measured using 2 nM R396842 and 0, 3 and IOM DNase. The buffer used was 50 mM Mops-NaOH (pH 7.2). The abundance was calculated using the two-component fitting analysis in the MF20 software.

As a result of this study, the DNaser concentration was set to 3 x M, the R396842 concentration was set to 2 nM, and ATA, a known inhibitor of DNaser (obtained from Aut in Tricarboxylic Acid, Wako Pure Chemical Industries, Ltd.) Were added at concentrations of 0, 3 and 10 / M and a competition experiment was performed. Figure 9 shows the results. The abundance of DNase r and the optimal fluorescent molecular probe complex decreased depending on the ATA concentration. This result indicates that ATA binds to the target site of DNase r competitively with the optimal fluorescent molecular probe. Industrial applicability

According to the present invention, a compound that has intrinsic fluorescence and can interact with a target molecule without using a fluorescent label for a ligand that binds to the target molecule can be used as a molecular probe. Can be screened. Depending on the compound, fluorescent labeling of ligands is difficult to fluorescently label, and there is a problem of reduced cost and ligand reactivity. Depending on the target molecule, it is difficult to screen for compounds that can interact with each other. there were. Since the present invention uses a fluorescent molecule compound that has a binding property to a target molecule and has intrinsic fluorescence, the problems of the conventional methods can be solved.

All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.

Claims

The scope of the claims

1. A screening method for a compound capable of interacting with a target molecule, wherein the fluorescent molecule probe is capable of binding to the target molecule, and uses a ligand having intrinsic fluorescence without being fluorescently labeled. And a candidate compound in a binding to a target molecule, and a method for screening a compound capable of interacting with a target molecule, comprising selecting a compound that competes with the fluorescent molecular probe by fluorescence analysis.

2. The method for screening a compound capable of interacting with a target molecule according to claim 1, wherein the target molecule is a protein.

3. The target according to claim 1 or 2, wherein competition in binding of the fluorescent molecular probe and the candidate compound to the target molecule is measured by using, as an index, a change in fluorescent signal due to binding of the fluorescent molecular probe and the target molecule in the solution. A screening method for compounds that can interact with molecules.

4. The competition in binding of the fluorescent molecular probe and the candidate compound to the target molecule can be detected by fluorescence correlation spectroscopy, fluorescence intensity distribution analysis, or fluorescence ellipsometry, and can interact with the target molecule according to claim 3. Compound screening method.

5. The method for screening a compound capable of interacting with a target molecule according to any one of claims 1 to 4, wherein the fluorescent molecular probe is selected from a compound library using the binding property to the target molecule as an index. .

6. Fluorescent molecular probes are selected from a fluorescent probe library constructed by selecting molecules that are inherently fluorescent from the compound library by docking studies with target molecules, or further structurally optimized. The method for screening a compound capable of interacting with a target molecule according to any one of claims 1 to 5.

7. The construction of the fluorescent probe library from the compound library is performed by at least one of the following methods (a) to (c): One ning method.

(a) A compound having a fluorophore as a partial structure is selected from the structural formula of the compound (b) Select fluorescent compounds with molecular orbital calculation software, and

8. The method for screening a compound capable of interacting with a target molecule according to claim 6 or 7, wherein the structure of the fluorescent molecular probe is optimized by introducing a substituent.

9. A desired binding constant between the target molecule and the fluorescent molecular probe is set in advance, and when the binding constant between the target molecule and the fluorescent molecular probe is larger than the set desired binding constant, the fluorescent molecular probe is targeted. The method for screening a compound capable of interacting with a target molecule according to any one of claims 5 to 8, which is selected as a fluorescent molecular probe of the molecule.

10. Measuring the optimum excitation wavelength and the optimum fluorescence wavelength of the fluorescent molecular probe in advance, exciting the fluorescent molecular probe at a wavelength near the optimum excitation wavelength, and detecting fluorescence at a wavelength near the optimum fluorescence wavelength. The screening method of the compound which can interact with the target molecule of any one of -9.

1 1. Screening of a compound capable of interacting with a target molecule, wherein a ligand capable of binding to the target molecule and having intrinsic fluorescence without being fluorescently labeled is used as the target molecule-specific fluorescent molecule probe. For analyzing the target molecule-specific fluorescent molecular probe to be used, which can irradiate light having the wavelength of the excitation light of the target molecule-specific light irradiation means capable of continuously changing the wavelength of the irradiated light, and target molecule-specific fluorescence used Photodetection means capable of continuously detecting the wavelength of the fluorescence light of the molecular probe, capable of continuously changing the wavelength of the detection light, and reaction means for competitively reacting the fluorescent molecular probe and the candidate compound in binding to the target molecule Fluorescence analyzer.

1 2. The fluorescence analyzer according to claim 11, wherein the target molecule is a protein.

1 3. According to claim 1 1 or 1 2, the wavelength of the irradiation light and the wavelength of the detection light can be changed in accordance with the excitation wavelength and the fluorescence wavelength of the target molecule-specific fluorescent molecular probe measured in advance. Fluorescence analyzer.

14. The fluorescence analysis apparatus according to any one of claims 11 to 13, wherein the reaction means is a reaction vessel that causes a fluorescent molecular probe and a candidate compound to undergo a competitive reaction in binding with a target molecule in a solution.

1 5. The competition in the binding of the fluorescent molecular probe and the candidate compound to the target molecule is measured by fluorescence correlation spectroscopy, fluorescence intensity distribution analysis, or fluorescence polarization analysis. The fluorescence analyzer described in 1.

1 6. Screening kit for a target molecule and a compound capable of binding to the target molecule, including a target molecule-specific fluorescent molecule probe that is a ligand that is intrinsically fluorescent without being fluorescently labeled.卜.

17. The screening kit according to claim 16, wherein the target molecule is a protein.

18. The kit for screening a compound capable of interacting with a target molecule according to claim 16 or 17, wherein the fluorescent molecular probe is selected from the compound library based on the binding property to the target molecule.

1 9. Fluorescent molecular probes are selected from a fluorescent probe library constructed by selecting molecules that are inherently fluorescent from the compound library, or by further screening optimization with the target molecule. The kit for screening a compound capable of interacting with a target molecule according to any one of claims 16 to 18.

20. Screening of a compound capable of interacting with the target molecule according to claim 19, wherein the construction of the fluorescent probe library from the compound library is performed by at least one of the following methods (a) to (c): For kit.

21. The screening kit for a compound capable of interacting with a target molecule according to claim 19 or 20, wherein the structure of the fluorescent molecular probe is optimized by introducing a substituent.