WO2006070941A1 - Nouveau procede de criblage de substance au moyen d'une sonde moleculaire fluorescente - Google Patents

Nouveau procede de criblage de substance au moyen d'une sonde moleculaire fluorescente Download PDF

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WO2006070941A1
WO2006070941A1 PCT/JP2005/024274 JP2005024274W WO2006070941A1 WO 2006070941 A1 WO2006070941 A1 WO 2006070941A1 JP 2005024274 W JP2005024274 W JP 2005024274W WO 2006070941 A1 WO2006070941 A1 WO 2006070941A1
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target molecule
fluorescent
compound
fluorescence
molecular probe
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Japanese (ja)
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Seiichi Tanuma
Atsushi Yoshimori
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Tokyo University of Science
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Tokyo University of Science
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"

Definitions

  • 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.
  • a ligand labeled with a fluorescent compound 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.
  • 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
  • the compound is labeled with a fluorescent substance and used.
  • 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.
  • fluorescent substances are bonded to the compounds by covalent bonds using the functional groups of the 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.
  • 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.
  • 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.
  • the aspect of this invention is as follows.
  • 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
  • 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.
  • [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.
  • 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 compound having a fluorophore as a partial structure is selected from the structural formula of the compound
  • 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.
  • [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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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].
  • 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 .
  • a compound having a fluorophore as a partial structure is selected from the structural formula of the compound
  • [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.
  • 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
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a compound having a fluorophore as a partial structure is searched.
  • 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 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,
  • 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.
  • a fluorescent molecule having affinity with the target molecule is selected for each target molecule.
  • 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.
  • 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
  • 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.
  • 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.
  • 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 can also be used.
  • any site such as an active site or interaction site may be specified.
  • 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.
  • site binding may be performed by dividing the binding site into smaller sections and docking.
  • 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.
  • 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.
  • the fluorescent molecular probe is used as the optimal fluorescent molecular probe.
  • 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.
  • 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.
  • 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.
  • 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.
  • the fluorescence intensity by the single molecule fluorescence analysis method or the fluorescence polarization analysis method is used.
  • changes in fluorescence intensity detected after separating a complex of a fluorescent molecular probe and a target molecule and a free fluorescent molecular probe is used.
  • 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.
  • 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.
  • the excitation and fluorescence wavelengths characteristic of the fluorescent molecule are used to determine the interaction between the candidate compound and the target molecule.
  • 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.
  • 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.
  • 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. .
  • 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 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)
  • 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.
  • 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.
  • the binding between the target molecule and the fluorescent molecular probe can be detected.
  • 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.
  • 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.
  • 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.
  • compounds can be screened by fluorescence ellipsometry.
  • 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.
  • 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.
  • the movement of molecules during the excited state decreases, so that the fluorescence cannot be depolarized and exhibits a large degree of fluorescence polarization.
  • 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.
  • 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).
  • 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.
  • 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
  • candidate compounds to be added to the solution have a final concentration of 0.01 nM
  • the fluorescent molecular probe added to the solution 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 ⁇ .
  • the fluorescent molecular probe added to the solution is
  • 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.
  • 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.
  • 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!
  • 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.
  • a spectrometer any of a spectrometer using an optical filter, a dispersion spectrometer, and a Fourier transform spectrometer can be used.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • any spectroscope using an optical filter, a dispersive spectroscope, or a Fourier transform spectroscope can be used.
  • a wavelength tunable laser such as an optical parametric laser (0P0 laser) may be used.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a confocal microscope may be installed at the lens 6 portion.
  • 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.
  • a plurality of target molecules and target molecule-specific fluorescent molecular probes for each target molecule may be included.
  • the same binding site for one target molecule may be included.
  • a plurality of target molecule-specific fluorescent molecular probes that can bind to different binding sites may be included.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the DNaser concentration was set to 3 x M
  • the R396842 concentration was set to 2 nM
  • 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.
  • 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.
  • fluorescent labeling of ligands is difficult to fluorescently label, and there is a problem of reduced cost and ligand reactivity.

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Abstract

L'invention concerne un procédé d'analyse de l'interaction entre une protéine cible et un composé, utilisant un composé capable d'interagir avec la protéine cible et capable d'émettre une lumière fluorescente native, après sélection au préalable ; un appareil pour utilisation dans l'analyse ; et un kit pour utilisation dans l'analyse. Elle porte sur un procédé de criblage d'un composé capable d'interagir avec une molécule cible, comprenant les phases de : réalisation de la réaction compétitive entre une sonde moléculaire fluorescente et des composés candidats pour une liaison avec la molécule cible, la sonde utilisée étant un ligand capable de se lier à la molécule cible et capable d'émettre une lumière fluorescente native sans exiger d'étiquetage fluorescent ; et sélection d'un composé ayant concouru avec la sonde moléculaire fluorescente au moyen d'une analyse de fluorescence.
PCT/JP2005/024274 2004-12-28 2005-12-28 Nouveau procede de criblage de substance au moyen d'une sonde moleculaire fluorescente Ceased WO2006070941A1 (fr)

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008249433A (ja) * 2007-03-29 2008-10-16 Kansai Bunri Sogo Gakuen 被験物質に対するプローブの結合親和性を測定する方法及びその利用
WO2015125851A1 (fr) * 2014-02-19 2015-08-27 国立大学法人京都大学 Développement de système de criblage de ligand pour des récepteurs de neurotransmetteur
CN113189068A (zh) * 2021-04-27 2021-07-30 华南农业大学 一种基于荧光分析的农药检测方法
CN113406045A (zh) * 2020-03-16 2021-09-17 广州创瑞健康科技有限公司 一种疟原虫检测的荧光染色方法
JP2022532661A (ja) * 2019-05-15 2022-07-15 ホワイトヘッド・インスティテュート・フォー・バイオメディカル・リサーチ 薬剤－凝縮物相互作用を特徴付け及び利用する方法
US12174198B2 (en) 2019-02-08 2024-12-24 Dewpoint Therapeutics, Inc. Methods of characterizing condensate-associated characteristics of compounds and uses thereof
US12422427B2 (en) 2018-10-15 2025-09-23 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Compounds for treatment of diseases and methods of screening therefor
US12422437B2 (en) 2019-09-18 2025-09-23 Dewpoint Therapeutics, Inc. Methods of screening for condensate-associated specificity and uses thereof

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0228542A (ja) * 1988-04-26 1990-01-30 Olympus Optical Co Ltd 蛍光顕微鏡装置
JP2001272404A (ja) * 2000-03-27 2001-10-05 Olympus Optical Co Ltd 蛍光相関分光法による抗原抗体反応
JP2003508788A (ja) * 1999-09-09 2003-03-04 マックス−プランク−ゲゼルシャフト ツール フォルデルング デル ヴィッセンシャフテン エー．ファウ． ペアードヘリカルフィラメントの核形成のための最小タウペプチド
JP2004110262A (ja) * 2002-09-17 2004-04-08 Enkaku Iryo Kenkyusho:Kk 計算機上の仮想プロテインアレイによる蛋白質−リガンド相互作用の解析方法
JP2004123568A (ja) * 2002-09-30 2004-04-22 Sanyo Boku ヘモグロビン・リガンド分子複合体の結晶、その製造方法および物質の探索方法
JP2004530125A (ja) * 2001-04-02 2004-09-30 ツェプトゼンス　アクチエンゲゼルシャフト 多光子励起のための光学構造及びその使用

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FI98765C (fi) * 1995-01-16 1997-08-11 Erkki Soini Virtaussytometrinen menetelmä ja laite
DE10038382A1 (de) * 2000-08-07 2002-02-21 Direvo Biotech Ag Zweifarbiges fluorimetrisches Proteaseassay

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0228542A (ja) * 1988-04-26 1990-01-30 Olympus Optical Co Ltd 蛍光顕微鏡装置
JP2003508788A (ja) * 1999-09-09 2003-03-04 マックス−プランク−ゲゼルシャフト ツール フォルデルング デル ヴィッセンシャフテン エー．ファウ． ペアードヘリカルフィラメントの核形成のための最小タウペプチド
JP2001272404A (ja) * 2000-03-27 2001-10-05 Olympus Optical Co Ltd 蛍光相関分光法による抗原抗体反応
JP2004530125A (ja) * 2001-04-02 2004-09-30 ツェプトゼンス　アクチエンゲゼルシャフト 多光子励起のための光学構造及びその使用
JP2004110262A (ja) * 2002-09-17 2004-04-08 Enkaku Iryo Kenkyusho:Kk 計算機上の仮想プロテインアレイによる蛋白質−リガンド相互作用の解析方法
JP2004123568A (ja) * 2002-09-30 2004-04-22 Sanyo Boku ヘモグロビン・リガンド分子複合体の結晶、その製造方法および物質の探索方法

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008249433A (ja) * 2007-03-29 2008-10-16 Kansai Bunri Sogo Gakuen 被験物質に対するプローブの結合親和性を測定する方法及びその利用
WO2015125851A1 (fr) * 2014-02-19 2015-08-27 国立大学法人京都大学 Développement de système de criblage de ligand pour des récepteurs de neurotransmetteur
JPWO2015125851A1 (ja) * 2014-02-19 2017-03-30 国立大学法人京都大学 神経伝達物質受容体のリガンドスクリーニングシステムの開発
US12422427B2 (en) 2018-10-15 2025-09-23 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Compounds for treatment of diseases and methods of screening therefor
US12174198B2 (en) 2019-02-08 2024-12-24 Dewpoint Therapeutics, Inc. Methods of characterizing condensate-associated characteristics of compounds and uses thereof
JP2022532661A (ja) * 2019-05-15 2022-07-15 ホワイトヘッド・インスティテュート・フォー・バイオメディカル・リサーチ 薬剤－凝縮物相互作用を特徴付け及び利用する方法
US12422437B2 (en) 2019-09-18 2025-09-23 Dewpoint Therapeutics, Inc. Methods of screening for condensate-associated specificity and uses thereof
CN113406045A (zh) * 2020-03-16 2021-09-17 广州创瑞健康科技有限公司 一种疟原虫检测的荧光染色方法
CN113189068A (zh) * 2021-04-27 2021-07-30 华南农业大学 一种基于荧光分析的农药检测方法

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