WO2003070697A1 - Procede de dosage de l'activite de liaison a un recepteur de la vitamine d3, utilisant la polarisation de fluorescence - Google Patents

Procede de dosage de l'activite de liaison a un recepteur de la vitamine d3, utilisant la polarisation de fluorescence Download PDF

Info

Publication number
WO2003070697A1
WO2003070697A1 PCT/JP2003/002045 JP0302045W WO03070697A1 WO 2003070697 A1 WO2003070697 A1 WO 2003070697A1 JP 0302045 W JP0302045 W JP 0302045W WO 03070697 A1 WO03070697 A1 WO 03070697A1
Authority
WO
WIPO (PCT)
Prior art keywords
vitamin
receptor
derivative
compound
fluorescently labeled
Prior art date
Application number
PCT/JP2003/002045
Other languages
English (en)
Japanese (ja)
Inventor
Satoshi Abe
Susumu Ito
Akira Kawase
Takamitsu Kobayashi
Kazutomo Kinoshita
Original Assignee
Chugai Seiyaku Kabushiki Kaisha
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Chugai Seiyaku Kabushiki Kaisha filed Critical Chugai Seiyaku Kabushiki Kaisha
Priority to JP2003569605A priority Critical patent/JP4443933B2/ja
Priority to AU2003207115A priority patent/AU2003207115A1/en
Publication of WO2003070697A1 publication Critical patent/WO2003070697A1/fr

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/59—Compounds containing 9, 10- seco- cyclopenta[a]hydrophenanthrene ring systems
    • A61K31/593—9,10-Secocholestane derivatives, e.g. cholecalciferol, i.e. vitamin D3
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C401/00—Irradiation products of cholesterol or its derivatives; Vitamin D derivatives, 9,10-seco cyclopenta[a]phenanthrene or analogues obtained by chemical preparation without irradiation
    • 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
    • G01N21/6445—Measuring fluorescence polarisation

Definitions

  • the present invention relates to a method for measuring the binding activity of a low-molecular compound to a receptor using fluorescence polarization, and a method for screening a compound that binds to a low-molecular compound using the method.
  • Vitamin D derivative is a vitamin in the nucleus!) Receptor
  • Vitamin D receptor VDR
  • VDR Vitamin D receptor
  • a method using a 3-position fluorescein amide label (US Patent US6291693) describes an ELISA method using a fluorescently labeled vitamin D3 derivative.
  • each of these methods is a method in which the conventional detection method is changed to fluorescence, and these methods are not a method for easily and simply measuring the binding activity between a vitamin D3 derivative and VDR.
  • the polarization filter Excitation light that is linearly polarized is passed through and the linearly polarized fluorescence emitted by the fluorescent molecule is measured.
  • Fluorescence has a lifetime of about nanoseconds, during which fluorescent molecules rotate due to Brownian motion and the like, so that the polarization of the fluorescence gradually disappears. At this time, the speed of the rotating Brownian motion is slow if the fluorescent molecule is large, and fast if the fluorescent molecule is small.
  • the rotational Brownian motion is also limited by the degree of freedom of the fluorescent molecules, the observed polarization of the fluorescent light differs, and it is necessary to detect the environment in which the fluorescent molecules exist, that is, whether or not there is interaction with other molecules. Can be.
  • the fluorescence polarization method measures changes in the polarization of fluorescence caused by the interaction between the fluorescent label and other molecules using the fluorescent label, and can be measured in a homogeneous system.
  • advantages such as being simple and simple, being able to measure while maintaining the equilibrium state, and being miniaturizable because there is no washing step.
  • the use of non-radioactive isotopes has advantages such as easy handling, high sensitivity, and the use of small amounts of receptors and fluorescent labels.
  • the fluorescence polarization method is mainly used for oligonucleotides.
  • Molecules such as oligopeptides have been fluorescently labeled and have been used exclusively for measuring the binding of these to macromolecules such as DNA, protein, and antibodies.
  • the present invention has been made in view of such circumstances, and a method for measuring the binding activity between a low-molecular compound and a receptor using a fluorescence polarization method, and a fluorescent label that can be used in the method.
  • the purpose is to provide low molecular compounds. More specifically, a method for measuring the binding activity of a vitamin D3 receptor using a fluorescence polarization method, a method for screening a compound that binds to a vitamin D3 receptor using the method, and a fluorescent label that can be used in the method Provided vitamin D3 derivatives.
  • the present inventors have conducted intensive studies in order to solve the above-mentioned problems, and as a result, by combining a low-molecular-weight compound, which is a ligand, with a fluorescent substance via a linker, to achieve both fluorescence polarization characteristics and receptor binding properties was found to be possible.
  • the method of using a linker can appropriately adjust the length of the linker, and can easily produce a fluorescent label that can be used for the fluorescence polarization method. That is, it has been found that the low-molecular compound bound to the fluorescent substance changes in the degree of fluorescence polarization when not bound to the receptor of the low-molecular compound and when bound to the receptor.
  • the present inventors have developed a novel method for measuring the binding activity between a low-molecular compound and a receptor using a fluorescence polarization method and completed the present invention.
  • the low molecular weight compound is a vitamin D3 derivative
  • a vitamin D3 derivative in which a fluorescent substance is bonded to the 2nd or 25th position of the vitamin D3 skeleton via an alkyl group of C10 is a ligand and a fluorescent substance
  • the present invention relates to a method for measuring the binding activity of a low-molecular compound to a receptor using a fluorescence polarization method, and a fluorescently labeled low-molecular compound that can be used in the method.
  • X represents a linker
  • Y represents a phosphor
  • X represents a linker
  • Y represents a phosphor
  • Vitamin D comprising a step of measuring the binding activity of a test compound to the vitamin D 3 receptor by the method according to [11] and selecting a compound having a binding activity to the vitamin D 3 receptor. (3) screening method for receptor agonist or antagonist,
  • a therapeutic agent for a bone-related disease comprising as an active ingredient the vitamin D3 receptor agonist or engonist according to (13);
  • the present invention relates to a low molecular compound using a fluorescence polarization method, a method for measuring the binding activity of the compound to a receptor, and a fluorescently labeled low molecular compound that can be used in the method.
  • the present invention first provides a fluorescently labeled low molecular weight compound that can be used in the method.
  • the present invention relates to a compound in which a fluorescent substance and a low-molecular compound form a covalent bond via a linker, wherein the low-molecular compound has a binding activity to a receptor. And the difference between the fluorescence polarization when the low-molecular compound is not bound to the receptor and the fluorescence polarization when the low-molecular compound is bound to the receptor can be detected.
  • Provide a fluorescently labeled small molecule compound is provided.
  • the low molecular compound is an organic compound having a molecular weight of 1000 or less, preferably 50 to 800, more preferably 100 to 700, and most preferably 200 to 500.
  • the receptor of the low molecular weight compound in the present invention (also simply referred to as “receptor” in the present specification) is a protein that naturally exists in a living body and has an activity of binding to a naturally occurring ligand. Protein.
  • a protein in which a part of the protein is artificially or naturally modified is also a receptor as long as it binds to a naturally occurring ligand.
  • the receptor may be a receptor present in blood or a receptor present in cells.
  • the bimin D3 receptor of the present invention When present in a cell, it includes a membrane receptor, a nuclear receptor, and a cytoplasmic receptor.
  • the bimin D3 receptor of the present invention also includes a so-called vitamin D3 receptor-like compound.
  • the naturally-occurring ligand (a molecule having binding activity to a receptor) may be a protein or a low-molecular-weight compound, but is preferably a naturally-occurring, physiologically active substance. Therefore, the low molecular weight compound of the present invention is a compound which exists in nature and binds to a receptor to exert a physiological activity.
  • Examples include dalcocorticoids, mineralocorticoids, vitamin D3, female sex hormones, male sex hormones, thyroid hormones, prostaglandins, leukotrienes, vitamin A, and the like. More specifically, vitamin D3, 1-hydroxyvitamin D3, 1,25-dihydroxyvitamin D3, androgen (testosterone, dihydrotestosterone), estrogens (estradiol, estrone, estrioyl), adrenaline, noradrenaline, histamine , Dopamine, serotonin, progesterone, cortisol, corticosterone, aldosterone, thyroxine, retinol, retinoic acid, prostaglandin E2, leukotriene B4 and the like, and derivatives of these compounds.
  • vitamin D3, 1-hydroxyvitamin D3, 1,25-dihydroxyvitamin D3, androgen testosterone, dihydrotestosterone
  • estrogens estradiol, estrone, estri
  • examples of the low molecular weight compound include a vitamin D3 derivative.
  • Vitamin D3 derivatives are 9, 10-secocholesta 5, 7, 10 (19)-Refers to a compound having a triene structure.
  • Preferred is a compound having a (5Z, 7E) -9,10-secocholes-5,7,10 (19) -triene structure.
  • a compound having a (1 ⁇ !, 5 ⁇ , 7 ⁇ ) -9,10-secocholes-5,7,10 (19) -triene-toluol structure more preferably (1 ⁇ , 5Z, 7E) -9,10-secocholes-a compound having a 5,7,10 (19) -triene-1,25-diol structure, more preferably (1 ⁇ , 3,5 ⁇ ; 7 ⁇ ) -1,9,10-secocholes E-5, 7, 10 (19) -Trien-1,3-diol-containing compound, most preferably (1st, 3j3, 5 ⁇ , 7 ⁇ ) -9, 10-secocholesta-5, 7, 10 ( 19) -Triene A compound having a 1,3,25-triol structure.
  • the vitamin D3 derivative of the present invention is not particularly limited as long as it has the above structure. Therefore, compounds having various substituents on the above compound are also included in the vitamin D3 derivative of the present invention.
  • the vitamin D3 derivative of the present invention also includes an active analog and a structural analog of the above compound.
  • An active analog refers to a compound having the same biological activity as the above-mentioned compound having biological activity. Compounds having the same biological activity regardless of the activity of the active analog are included in the active analog.
  • the structural analog in the present invention refers to a compound in which a specific structure of the compound is variously modified. Structural analogs can be synthesized artificially or can be naturally occurring compounds.
  • the fluorescent substance in the present invention is not particularly limited, and any fluorescent substance can be used.
  • flowresin-tetramethylrhodamine and texalesed can be suitably used.
  • Flow resin is preferred because it is easily available, has an appropriate fluorescence lifetime, and can be measured with a commercially available measuring instrument.
  • flowresin isothiocyanate FITC
  • Covalent bonding of the phosphor and the low molecular compound via a linker can be performed, for example, according to the method described in Examples described later.
  • linker refers to a covalent bond with a low-molecular compound and a phosphor. Refers to a structural part that forms or a compound that is necessary to form the structural part.
  • the linker may have any chemical structure, but preferably does not substantially participate in the binding of the low-molecular compound to the receptor of the compound, and is chemically stable to various solvents. It is desirable that For example, it may be a carbon chain, a peptide chain or a sugar chain. In the case of a carbon chain, it may be an optionally substituted alkyl group or an unsubstituted alkyl group. Further, it may have an oxygen atom, a nitrogen atom and a sulfur atom in the carbon chain.
  • an ethyl group or a propyl group may have a repeating structure plural times via an oxygen atom.
  • the substituent is not particularly limited and includes, for example, an alkyl group, a halogen, a hydroxyl group, an amino group, a carbonyl group, and the like, and is preferably an unsubstituted alkyl because it does not affect the measurement of fluorescence polarization. Group.
  • the terminal of the linker preferably has an appropriate substituent so that a covalent bond can be formed with the phosphor and the low-molecular compound. For example, an amino group, a hydroxyl group, a thiol group and the like can be mentioned.
  • the type of the substituent to be used may be determined in combination with the low molecular compound to be bound and the functional group of the phosphor, and is preferably an amino group or a hydroxyl group.
  • the selection and manufacture of these suitable linkers is a technique known to those skilled in the art.
  • the site of the low-molecular compound forming a covalent bond with the linker is not particularly limited as long as the low-molecular compound bonded to the linker binds to the receptor of the compound.
  • the site where the low molecular compound forms a covalent bond with the linker may be a portion which does not participate in the binding to the receptor or the enzyme in the structure of the low molecular compound.
  • Selection of a site or a functional group to which the linker binds can be determined, for example, by analyzing a mode of interaction with a receptor or an enzyme. Analyzing the three-dimensional structure of the receptor or enzyme all at once, analyzing the binding mode with the desired low-molecular compound, and determining the sites not involved in the binding.
  • the low molecular weight compound when it is a vitamin D3 derivative, it is preferably at a position other than the 1-position and / or the 3-position.
  • the carbon source at positions 2, 20, 21, 22, 23, 24, 25, and 26 of the vitamin D3 skeleton And more preferably a carbon atom at the 2-, 25- or 26-position.
  • the length of the linker does not hinder the binding between the low-molecular compound and the phosphor between the low-molecular compound and the receptor, and the length of the low-molecular compound and the low-molecular compound It is preferable to provide an appropriate distance that can affect the fluorescence polarization degree of the phosphor by binding to the receptor.
  • the appropriate distance between the low-molecular compound and the phosphor means 5 to 25, preferably 5 to 20, more preferably 8 to 15 and most preferably 8 to 12 in terms of carbon chain. It is as follows. Also, if necessary, the optimal binding length of the linker can be determined by analyzing the binding mode with the low molecular weight compound using a computer.
  • the phosphor and the low molecular weight compound form a covalent bond via a linker having an appropriate length.
  • the low molecular weight compound of the present invention has a fluorescence polarization degree when bound to a receptor of the low molecular weight compound and a fluorescence polarization degree when not bound to a receptor.
  • a fluorescence polarization degree when bound to a receptor of the low molecular weight compound and a fluorescence polarization degree when not bound to a receptor.
  • a commercially available measuring instrument can be used for measuring the fluorescence polarization.
  • BEACON trade name
  • PanVera may be used.
  • a fluorescently labeled biluminmin D3 derivative preferably having the following structure can be mentioned.
  • X represents a linker
  • Y represents a phosphor
  • X represents a linker
  • ⁇ represents a phosphor
  • the receptor is preferably purified. Therefore, a recombinant protein is a preferred receptor in the present invention because of its easy purification.
  • Methods for producing a recombinant protein are already known, and can be produced by combining an appropriate vector and a host (Molecular Cloning 2nd Edt., Cold Spring Harbor Laboratory Press, 1989; Basic Methods in Molecular). Biology, Appleton & Lange, 1986). Separation and purification of a recombinant protein or a naturally occurring protein may be performed by any conventional separation and purification methods used for protein purification, and is not limited.
  • the present invention also relates to a method for detecting the binding between a low-molecular compound and the compound receptor using a fluorescence polarization method.
  • the present invention uses a fluorescent polarization method.
  • a method for detecting the binding between the above vitamin D3 derivative and a vitamin D3 receptor is provided.
  • the fluorescently labeled vitamin D3 derivative of the present invention is brought into contact with the vitamin D3 receptor (step (a)), and then the fluorescently labeled bimin D3 derivative is obtained.
  • the degree of fluorescence polarization of the compound that binds to the bimin D3 receptor is measured (step (b)).
  • the presence or absence of binding can be determined by measuring the degree of fluorescence polarization in a state where the fluorescently labeled biminmin D3 derivative is in contact with the biminmin D3 receptor.
  • the change in the degree of fluorescence polarization before and after contact with the vitamin D3 receptor may be measured.
  • the measurement of the degree of fluorescence polarization in the step (b) can be performed by those skilled in the art by using the above-mentioned commercially available measuring instrument.
  • the type of buffer used for the measurement is not particularly limited as long as the fluorescence can be measured appropriately, and those skilled in the art can appropriately determine the type according to the type of the fluorescent substance used.
  • a buffer having a pH of 7.5 or more is preferable as a buffer that stabilizes fluorescence.
  • it is preferably pH 7.5 to 10.0, more preferably pH 7.5 to 9.0, and most preferably pH 7.5 to 8.5.
  • those skilled in the art can appropriately select the type and concentration of the salt, and for example, KC1 can be used.
  • the salt concentration can be appropriately determined according to the measurement. For example, a concentration of 300 to 500 mM can be used. It is preferable to use a stabilizer depending on the properties of the receptor or protein used, such as an enzyme. In the case of a vitamin D3 receptor, EDTA may be added.
  • DMS0 may be used as the organic solvent.
  • vitamin D3 receptor not only vitamin D3 receptor but also vitamin D3 derivatives contained in a sample can be measured.
  • a fixed amount of the vitamin D3 receptor is brought into contact with the fluorescence-labeled vitamin D3 derivative of the present invention simultaneously with or after contact with the sample;
  • the degree of fluorescence polarization resulting from the binding of the derivative to the vitamin D3 receptor is measured.
  • the order of contacting the vitamin D3 receptor, the sample, and the fluorescently labeled vitamin D3 derivative may be any order.
  • the sample is brought into contact with the fluorescently labeled vitamin D3 derivative, and then the vitamin D3 receptor is added. You may.
  • the above “fixed amount” is not particularly limited at the upper or lower limit, but is usually 100 pg to 100 ng.
  • a vitamin D3 derivative contained in a sample is measured by competing a certain amount of vitamin D3 receptor with a fluorescently labeled vitamin D3 derivative and a vitamin D3 derivative present in the sample. can do.
  • a fixed amount of vitamin D3 receptor is contacted with a fluorescently labeled vitamin D3 derivative in the presence of the sample, or, after contact with the sample, a fluorescently labeled vitamin D3 receptor.
  • D3 derivative is inversely proportional to the concentration of the biminmin D3 derivative contained in the sample.
  • the present invention provides a method for measuring the binding activity of a test compound to a vitamin D3 receptor.
  • the fluorescently labeled vitamin D3 derivative of the present invention is brought into contact with a vitamin D3 receptor and a test compound, and then the fluorescently labeled biminmin D3 derivative and the biminmin D3 receptor Measure the fluorescence polarization of the conjugate with the body.
  • the present invention also provides a method for detecting the binding activity of a test compound to a vitamin D3 receptor. And a method for screening a vitamin D3 receptor agonist or antagonist using the same. In this method, the binding activity of a test compound to the vitamin D3 receptor is measured, and a compound having a binding activity to the vitamin D3 receptor is selected.
  • the test compound used in the screening method of the present invention is usually a vitamin D3 derivative, but is not particularly limited thereto.
  • the compound that binds to the vitamin D3 receptor selected by the above method of the present invention is useful as an agonist, an antagonist or an inhibitor or an accelerator for the vitamin D3 receptor.
  • Compounds that bind to the vitamin D3 receptor are also useful as ligands that can detect and purify the bimin D3 receptor.
  • the vitamin D3 receptor used for searching for a compound that binds to the vitamin D3 receptor may be particularly called a target molecule.
  • the formation of a complex resulting from the reaction between the target molecule and the fluorescently labeled test compound is measured based on the change in the degree of fluorescence polarization.
  • a vitamin D3 receptor which interferes with the binding between a vitamin D3 receptor and a bitumin D3 derivative which is known in advance to bind to the vitamin D3 receptor
  • the present invention relates to a method for detecting a compound having a binding activity.
  • the above method is a method for detecting the binding activity of a test compound to a vitamin D3 receptor, and comprises the following steps.
  • the “contact” in the above step (i) can be performed more specifically, for example, as follows.
  • test compound After the test compound is brought into contact with the vitamin D3 receptor, it is brought into contact with a fluorescently labeled vitamin D3 derivative which is already known to form a complex with the vitamin D3 receptor.
  • the above (A) shows that the test compound competes for the binding reaction between the fluorescently labeled vitamin D3 derivative, which is already known to form a complex with the vitamin D3 receptor, and the vitamin D3 receptor (compe This is a method for measuring titi on activity). If no binding between the biminmin D3 receptor and the fluorescently labeled vitamin D3 derivative is observed in the presence of the test compound, the test compound has an activity of binding to the vitamin D3 receptor, It can be identified as a receptor agonist or enhancer, or an antagonist II inhibitor.
  • the above (B) shows the inhibitory activity (inhibition) of the test compound on the binding reaction between the vitamin D3 derivative, which is already known to form a complex with the vitamin D3 receptor, and the vitamin D3 receptor. ac t ivity). Also in this method, when the test compound has a binding activity to the vitamin D3 receptor, the binding of the vitamin D3 receptor to the vitamin D3 derivative is inhibited.
  • the above (C) shows the displacement activity of the test compound on the binding reaction between the benzoin D3 derivative, which is already known to form a complex with the vitamin D3 receptor, and the ubimin D3 receptor ( r is a method for measuring lacement activity (viability).
  • r is a method for measuring lacement activity (viability).
  • the binding activity of the test compound to the vitamin D3 receptor can be detected.
  • a compound that binds to the vitamin D3 receptor can be screened.
  • Compounds selected by this method are useful as agonists and enhancers, or antagonists and inhibitors of the vitamin D3 receptor.
  • the binding to the fluorescently labeled vitamin D3 derivative of the present invention may be measured, or the vitamin D3 receptor and the fluorescently labeled vitamin D3 derivative may be measured once.
  • the test compound is reacted, and the binding of the fluorescently labeled vitamin D3 derivative to the vitamin D3 receptor displaces the vitamin D3 receptor resulting from the displacement of the vitamin D3 receptor. May be detected.
  • a co-binding molecule in addition to the vitamin D3 receptor that directly binds to the fluorescently labeled vitamin D3 derivative, a co-binding molecule (co-binding molecule) can be isolated and identified.
  • a co-binding molecule is also called a co-factor and has an activity of regulating the activity of a bioactive molecule.
  • the co-binding molecule further binds to a complex of a vitamin D3 derivative and a biminmin D3 receptor to enhance bioactivity.
  • the co-binding molecule is referred to as cofactor-1.
  • eo-repressor when the activity is reduced, it is sometimes called a corepressor (eo-repressor).
  • the co-coupling molecule may bind to a complex of a vitamin D3 receptor and a vitamin D3 derivative, may bind only to a vitamin D3 receptor, or may bind only to a vitamin D3 derivative.
  • the vitamin D3 receptor recovered (selected) by the method of the present invention can be analyzed and identified by analytical techniques such as mass spectrometry and amino acid analysis (for example, see US Pat. No. 5,959,529). Using this method, it is possible to identify an unknown vitamin D3 receptor having an activity of binding to a fluorescently labeled vitamin D3 derivative.
  • the new vitamin D3 receptor identified by this method is a drug target Useful as a molecule.
  • the newly identified vitamin D3 receptor can be used to screen for a compound having the same activity as the vitamin D3 receptor used for identification. That is, the binding to the test compound may be measured using the newly identified vitamin D3 receptor, or the competitive reaction with the vitamin D3 derivative used for identification may be measured. These screening methods can be performed by measuring the change in the degree of fluorescence polarization as described above.
  • test compounds and test samples used for screening are not particularly limited, and include, for example, peptides, purified or crudely purified proteins, non-peptide compounds, synthetic compounds, microbial fermentation products, marine organism extracts, plant effluents, A cell extract or the like can be used as appropriate. These test compounds may be novel compounds or known compounds.
  • New compounds such as bimin D3 receptor agonists or antagonists, that can be found by the above methods of the invention are useful as pharmaceuticals.
  • the compound is expected to be a therapeutic agent for bone-related diseases.
  • the bone-related disease include osteoporosis, bone metastasis of cancer, and the like.
  • a biminmin D3 receptor agonist and an engonist obtained by the method of the present invention are also included in the present invention.
  • Compounds obtained using the screening method of the present invention can be used in humans and other mammals, such as mice, rats, guinea pigs, rabbits, chickens, cats, dogs, sheep, pigs, sea lions, monkeys, baboons, and chimpanzees.
  • a pharmaceutical composition containing a compound obtained by using the screening method of the present invention as an active ingredient can be produced by a method known to those skilled in the art, together with pharmaceutically acceptable excipients, stabilizers, and the like.
  • the present invention further provides a measurement kit for use in the above method of the present invention.
  • the kit of the present invention comprises at least the above-described fluorescently labeled vitamin D3 induction of the present invention.
  • the kit includes, for example, a fluorescently labeled vitamin]) 3 derivative, bimin D3 receptor, sample diluent, test tube or microplate for measurement, 1 ⁇ -hydroxyvitamin D3 or 1 ⁇ , 25 -Dihydroxyvitamin D3, and also instructions that describe how to use the kit can be packaged.
  • the present invention also provides the following compounds (i) to (iv) described in the examples described below.
  • FIG. 1 shows a dose response curve for 1.25 (OH) 2 vitamin D3 by ED-532FL.
  • the vertical axis indicates the degree of fluorescence polarization, and the horizontal axis indicates the concentration of the test compound.
  • FIG. 2 is a graph showing dose response curves for various vitamin D3 derivatives (ED-71, OCT, ED-130).
  • the vertical axis indicates the degree of fluorescence polarization, and the horizontal axis indicates the concentration of the test compound.
  • FIG. 3 is a diagram showing the degree of polarization when various vitamin D3 derivatives (ED-7K OCT, ED-130) were added at 1 ⁇ 10 1.
  • the vertical axis indicates the fluorescence polarization degree.
  • FIG. 4 is a diagram showing a dose response curve of ED-533FL for 1.25 (0H) 2 vitamin D3.
  • the vertical axis indicates the degree of fluorescence polarization, and the horizontal axis indicates the concentration of the test compound.
  • FIG. 5 is a diagram showing a scheme up to ED-533 synthesis.
  • FIG. 6 is a diagram showing a scheme up to ED-532 synthesis.
  • FIG. 4 shows a scheme up to the synthesis of ED-532FL and ED-533FL.
  • the obtained filtrate was washed with an aqueous solution of sodium thiosulfate, 0.5N hydrochloric acid, a saturated aqueous solution of sodium bicarbonate, and a saturated saline solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure.
  • the obtained residue was washed with acetonitrile, and (la, 3j3,20S) -l, 3-bis ((1,1-dimethylethyl) dimethylsilyl) oxy) -20-odomethyl-pregna-5-ene (36.63 g, 903 ⁇ 4) as a white solid.
  • the physicochemical properties of this compound are as follows.
  • 1,3_1,3_bis ((1,1-dimethylethyl) dimethylsilyl) oxy) -27-nor-5-cholesten-25-one (16.8 g), N-bromosuccinimide (6.14 g),
  • a colorless oil (3.97 g)
  • tetrahydrofuran 50 ml
  • tetrabutylammonium fluoride 1 M tetrahydrofuran solution, 10 ml
  • the physicochemical properties of this compound are as follows.
  • the flow rate was 1 mL / min, and the injection volume was 2017 times.
  • 0.5 mL of the 265 nm ultraviolet absorption peak portion observed around a retention time of 12 minutes was obtained.
  • ED-533FL in which flow resin was bound to position 25 via a linker was synthesized in the same manner using ED-533 instead of ED-532.
  • the purification was performed using a Varian Bond Elute C18 column, pre-washed sequentially with 2.4 mL of acetonitrile and 4 mL of water, and then 1 mL of a solution obtained by diluting the reaction product to 10-fold volume with water was applied to the column. After washing sequentially with 2.4% of 10% acetonitrile and 2.4 mL of 20% acetonitrile, elution was carried out with 2.4 mL of 30% acetonitrile. A solution obtained by dissolving the eluate in a centrifugal concentrator and drying it in 500 / L ethanol was used for the following experiment.
  • Example 4 Dose-response curve to 1.25 (OH) 2 vitamin D3 by ED-532FL Flow-resin-labeled ED-532 (ED-532FL) obtained in Example 3 (1) 300 mM chloride in 28 zL 7 mL of 50 mM Tris-HCl buffer pH 8.0 containing potassium, 2 mM dithiothreitol, and ImM ethylenediaminetetraacetic acid was added. From this solution, 198 L was transferred to a BEACON test tube 0> 2244 (Panbella), to which 2 L of dimethyl sulfoxide was added to obtain a control sample without vitamin D receptor.
  • FIG. 1 shows the measurement results. The results show the average and standard deviation of three measurements in the same experiment.
  • the leftmost point (VD (-)) is the value measured without the addition of 1,25 (OH) 2 vitamin D3 (ED-532FL). And Vitamin D receptor only), the rightmost point (VDR (-)) indicates the measured value of a control sample without biminmin D receptor.
  • the degree of polarization of fluorescence increases by about 130 millimeters with the addition of vitamin D receptor, and the degree of polarization decreases in a dose-dependent manner with the addition of 1.25 (0H) 2 vitamin D3 with a final concentration of 10 or more. did.
  • ED-71, OCT, ED-130 Various vitamin D3 derivatives (ED-71, OCT, ED-130) dissolved in dimethyl sulfoxide to a predetermined concentration were measured in the same manner as in Example 4, and the respective dose-response curves were compared.
  • the structure of ED-71, OCT, or ED-130 is shown in Chemical Formula 5, 6, or 7, respectively.
  • FIG. 2 shows the measurement results. The results show the average of three measured values in the same experiment.
  • the leftmost point (VD (-)) is the measured value without the addition of various vitamin D3 derivatives
  • the rightmost point (VDR (-)) shows the measured value of the control sample without vitamin D receptor.
  • ED-71, OCT, and ED-130 showed a dose-dependent decrease in the degree of polarization with the addition of 10-3 ⁇ 4 or more as a final concentration slightly higher than 1.25 (OH) 2 vitamin D3. This result indicates that the method we invented can be used for the quantification of various vitamin D3 derivatives, and that the receptor binding ability of various vitamin D derivatives can be compared with each other.
  • Figure 3 shows the measurement results.
  • the results show the average and standard deviation of the values of three experiments on different dates, and each experimental value is calculated as the average of three measurements in the same experiment.
  • the leftmost bar (VD (-)) shows the measured value when no vitamin D3 derivative was added, and the second bar from the leftmost side (VDR (-)) shows the measured value of the control sample without vitamin! Receptor.
  • ED-71, OCT, ED- 130 and the same concentration of polarization is intended to significantly reduced compared with the case without the addition of vitamin D3 derivatives upon 10_ 7 M added as the final concentration 1. 25 (0H) 2 vitamin The degree of the decrease was not remarkable as compared with the case where D3 was added. This result indicates that the receptor binding ability of various vitamin D derivatives can be compared with each other based on the degree of polarization in a single dose.
  • Example 7 Dose-response curve to 1.25 (0H) 2 vitamin D3 by ED-533FL 3.3 L of flow-resin-labeled ED-533 (ED-533FL) obtained in Example 3 (2) To this was added 10.4 mL of 50 mM Tris-HCl buffer pH 8.0 containing 300 mM potassium salt, 2 mM dithiothreitol, and ImM ethylenediaminetetraacetic acid. Transfer this solution to three BEACON test tubes 2244 (Panbella) at 198 / L, add 2L of dimethyl sulfoxide to each, and add a control sample without vitamin D receptor. did.
  • FIG. 4 shows the measurement results. The results show the average and standard deviation of the three measurements in the same experiment.
  • the leftmost point (VD (-)) is the value without 1,25 (0H) 2 vitamin D3 added
  • the rightmost point (VDR (-)) indicates the measured value of the control sample without the pigminin D receptor.
  • the degree of polarization of fluorescence increased by 136 mm with the addition of vitamin D receptor, and the degree of polarization decreased in a dose-dependent manner with the addition of 1.25 (OH) 2 vitamin D3 at a final concentration of 10_3 ⁇ 4 or more. .
  • a novel method capable of easily and accurately detecting the binding activity between a low-molecular compound and a receptor using a fluorescence polarization method.
  • This method can complete the measurement in a short period of time, and is a reliable experiment in which the binding activity of the protein used for the test compound is less inactivated than the binding activity measurement test using radioisotopes. It is expected to get data.
  • this method it became possible to screen a compound that binds to the vitamin D3 receptor or a novel vitamin D3 receptor.
  • the compounds obtained by the screening method of the present invention are highly expected to be therapeutic agents for bone-related diseases.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Physical Education & Sports Medicine (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Orthopedic Medicine & Surgery (AREA)
  • Rheumatology (AREA)
  • Epidemiology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
  • Plural Heterocyclic Compounds (AREA)

Abstract

Selon l'invention, l'activité de liaison peut être détectée par la mesure d'un changement dans le degré de polarisation de fluorescence d'un composé à faible poids moléculaire et à marquage fluorescent. Ledit composé présente en effet une différence entre son degré de polarisation de fluorescence lorsqu'il se lie à un récepteur et son degré de polarisation de fluorescence lorsqu'il ne se lie pas audit récepteur. L'invention concerne également un procédé de criblage de composé se liant à un récepteur de la vitamine D3, ce procédé mettant en oeuvre le procédé de dosage selon l'invention.
PCT/JP2003/002045 2002-02-25 2003-02-25 Procede de dosage de l'activite de liaison a un recepteur de la vitamine d3, utilisant la polarisation de fluorescence WO2003070697A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
JP2003569605A JP4443933B2 (ja) 2002-02-25 2003-02-25 蛍光偏光法を用いたビタミンｄ３受容体結合活性測定方法
AU2003207115A AU2003207115A1 (en) 2002-02-25 2003-02-25 Method of assaying activity of binding to vitamin d3 receptor using fluorescence polarization

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2002048473 2002-02-25
JP2002/48473 2002-02-25

Publications (1)

Publication Number Publication Date
WO2003070697A1 true WO2003070697A1 (fr) 2003-08-28

Family

ID=27750746

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2003/002045 WO2003070697A1 (fr) 2002-02-25 2003-02-25 Procede de dosage de l'activite de liaison a un recepteur de la vitamine d3, utilisant la polarisation de fluorescence

Country Status (3)

Country Link
JP (1) JP4443933B2 (fr)
AU (1) AU2003207115A1 (fr)
WO (1) WO2003070697A1 (fr)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0420102A2 (fr) * 1989-09-28 1991-04-03 Abbott Laboratories Stabilisation d'anticorps monoclonaux pour utiliser dans les techniques de polarisation de fluorescence
EP0597333A2 (fr) * 1992-11-10 1994-05-18 F. Hoffmann-La Roche Ag Composés pour essais immunologiques à polarisation de fluorescence et essais immunologiques les utilisant
WO1997024127A1 (fr) * 1995-12-29 1997-07-10 A And D Assay, Incorporated Composes de vitamine d marques et leur utilisation
EP0867722A2 (fr) * 1997-03-27 1998-09-30 Takara Shuzo Co. Ltd. Méthode pour mesurer l'interaction entre sucre et cible
WO2001016099A1 (fr) * 1999-08-27 2001-03-08 Chugai Seiyaku Kabushiki Kaisha DÉRIVÉS DE VITAMINE D AYANT DES SUBSTITUANTS À LA POSITION 2$g(a)

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0420102A2 (fr) * 1989-09-28 1991-04-03 Abbott Laboratories Stabilisation d'anticorps monoclonaux pour utiliser dans les techniques de polarisation de fluorescence
EP0597333A2 (fr) * 1992-11-10 1994-05-18 F. Hoffmann-La Roche Ag Composés pour essais immunologiques à polarisation de fluorescence et essais immunologiques les utilisant
WO1997024127A1 (fr) * 1995-12-29 1997-07-10 A And D Assay, Incorporated Composes de vitamine d marques et leur utilisation
EP0867722A2 (fr) * 1997-03-27 1998-09-30 Takara Shuzo Co. Ltd. Méthode pour mesurer l'interaction entre sucre et cible
WO2001016099A1 (fr) * 1999-08-27 2001-03-08 Chugai Seiyaku Kabushiki Kaisha DÉRIVÉS DE VITAMINE D AYANT DES SUBSTITUANTS À LA POSITION 2$g(a)

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
BARSONY JULIA ET AL.: "Development of a biologically active fluorescent-labeled calcitriol and its use to study hormone binding to the vitamin D receptor", ANALYTICAL BIOCHEMISTRY, vol. 229, no. 1, 1995, pages 68 - 79, XP002216119 *

Also Published As

Publication number Publication date
AU2003207115A1 (en) 2003-09-09
JPWO2003070697A1 (ja) 2005-06-09
JP4443933B2 (ja) 2010-03-31

Similar Documents

Publication Publication Date Title
Wang et al. A novel near-infrared styryl-BODIPY fluorescent probe for discrimination of GSH and its application in living cells
Leonard et al. Chemical ‘omics’ approaches for understanding protein cysteine oxidation in biology
Zhang et al. A novel near-infrared fluorescent probe for highly selective detection of cysteine and its application in living cells
CN113834925B (zh) 用于hdl和apoa1的纯化和检测的组合物和方法
Kang et al. A novel ratiometric fluorescent H2S probe based on tandem nucleophilic substitution/cyclization reaction and its bioimaging
Guo et al. Self-assembled nanosensor based on squaraine dye for specific recognition and detection of human serum albumin
Li et al. Biotin switch assays for quantitation of reversible cysteine oxidation
TWI286554B (en) Ferrocene compounds and uses thereof
Kukulage et al. Emerging chemistry and biology in protein glutathionylation
WO2022085762A1 (fr) Réactif pour la mesure d'une sensibilisation de la peau, composé et procédé pour la mesure d'une sensibilisation de la peau
Malachowska-Ugarte et al. Brassinosteroid-BODIPY conjugates: Design, synthesis, and properties
Zhi et al. Fluorescent probe based on coumarin derivative for the selective detection of cysteine in living cells
Yang et al. Estrogen receptor sensing in living cells by a high affinity turn-on fluorescent probe
Wu et al. The ratiometric fluorescent probes for monitoring the reactive inorganic sulfur species (RISS) signal in the living cell
Zhang et al. Labeling of polyprenylated macromolecules using mild Ene reactions
Mano et al. Development of a highly sensitive in vitro system to detect and discriminate between vitamin D receptor agonists and antagonists based on split-luciferase technique
Zacharias et al. Affinity and chemical enrichment strategies for mapping low‐abundance protein modifications and protein‐interaction networks
Singh et al. Binding and mechanistic studies of 5-HT7 specific benzothiazolone derivatives with bovine serum albumin: Spectroscopic and in silico studies
EP2939029B1 (fr) Dérivés de folate, particulièrement utiles dans le cadre du dosage de folate(s)
Johnson et al. Characterization of cyclic peptides containing disulfide bonds
Qian et al. Red emission ratio fluorescent probe for the activity of vanin-1 and imaging in vivo
Lu et al. Quantitation of irbesartan and major proteins in human plasma by mass spectrometry with time-of-flight analyzer
Miller et al. Methods in tubulin proteomics
JP4443933B2 (ja) 蛍光偏光法を用いたビタミンｄ３受容体結合活性測定方法
JP7328657B2 (ja) 化合物及び誘導体化試薬、並びに化合物の合成方法

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ OM PH PL PT RO RU SC SD SE SG SK SL TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LU MC NL PT SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
WWE Wipo information: entry into national phase

Ref document number: 2003569605

Country of ref document: JP

122 Ep: pct application non-entry in european phase