WO2015125851A1 - Développement de système de criblage de ligand pour des récepteurs de neurotransmetteur - Google Patents

Développement de système de criblage de ligand pour des récepteurs de neurotransmetteur Download PDF

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WO2015125851A1
WO2015125851A1 PCT/JP2015/054558 JP2015054558W WO2015125851A1 WO 2015125851 A1 WO2015125851 A1 WO 2015125851A1 JP 2015054558 W JP2015054558 W JP 2015054558W WO 2015125851 A1 WO2015125851 A1 WO 2015125851A1
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receptor
compound
group
mmol
labeling
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格 浜地
茂樹 清中
翔 若山
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国立大学法人京都大学
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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
    • G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
    • G01N33/9406—Neurotransmitters
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B11/00—Diaryl- or thriarylmethane dyes
    • C09B11/04—Diaryl- or thriarylmethane dyes derived from triarylmethanes, i.e. central C-atom is substituted by amino, cyano, alkyl
    • C09B11/06—Hydroxy derivatives of triarylmethanes in which at least one OH group is bound to an aryl nucleus and their ethers or esters
    • C09B11/08—Phthaleins; Phenolphthaleins; Fluorescein
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B11/00—Diaryl- or thriarylmethane dyes
    • C09B11/04—Diaryl- or thriarylmethane dyes derived from triarylmethanes, i.e. central C-atom is substituted by amino, cyano, alkyl
    • C09B11/10—Amino derivatives of triarylmethanes
    • C09B11/24—Phthaleins containing amino groups ; Phthalanes; Fluoranes; Phthalides; Rhodamine dyes; Phthaleins having heterocyclic aryl rings; Lactone or lactame forms of triarylmethane dyes
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B11/00—Diaryl- or thriarylmethane dyes
    • C09B11/28—Pyronines ; Xanthon, thioxanthon, selenoxanthan, telluroxanthon dyes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/02—Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)

Definitions

  • the present invention relates to a fluorescently labeled neurotransmitter receptor, a method for producing a labeled neurotransmitter receptor, a neurotransmitter receptor labeling agent, a compound useful as the labeling agent, and a neurotransmitter receptor ligand.
  • the present invention relates to a screening method.
  • Receptors that receive neurotransmitters are proteins that are widely known as important drug targets.
  • glutamate is widely accepted as one of important neurotransmitters, and its receptor glutamate receptor is a protein essential for neurotransmission.
  • Glutamate receptors are classified into AMPA, NMDA, and kainate types based on the characteristics of agonists.
  • AMPA receptors are proteins essential for neuronal activity, particularly memory and learning. Causes various diseases such as illness, stroke, and Alzheimer's disease. Therefore, AMPA type receptors have been positioned as important drug targets. So far, a plurality of selective agonists have been developed, but there are problems such as subtype selectivity.
  • Non-patent Document 1 Non-patent Document 1
  • Drug assays using full-length glutamate receptors typically involve evaluation of AMPA receptor activity (ion channel characteristics), but the evaluation method is complicated and has not been developed into a high-throughput evaluation method. Absent.
  • An object of the present invention is to provide a technique for clarifying a function as a ligand by assaying a neurotransmitter receptor ligand (agonist, partial agonist, antagonist) at high throughput.
  • the present invention relates to a fluorescently labeled neurotransmitter receptor, a method for producing a labeled neurotransmitter receptor, a neurotransmitter receptor labeling agent, a compound useful as the labeling agent, and a neurotransmitter receptor ligand.
  • a screening method is provided. Item 1. The following formula (II)
  • Rec-Nu represents a group in which a hydrogen atom has been eliminated from a neurotransmitter receptor (Rec-Nu-H).
  • Nu represents a nucleophilic group (Nu-H) of the neurotransmitter receptor.
  • a divalent group in which a hydrogen atom is eliminated from L 2 L 2 represents a divalent linking group, and Fl represents a labeling group. It is expected that the labeled neurotransmitter receptor interacts with the receptor, which has a basic structure represented by the formula and changes the fluorescence pattern between when the antagonist is bound and when the agonist is bound.
  • a substance that binds to a neurotransmitter receptor characterized in that a candidate substance is allowed to act, and the binding mode of the candidate substance and the receptor is detected based on a change in a signal emitted by a label, and is performed based on the result.
  • Screening method Item 2. Item 2. The screening method according to Item 1, wherein the candidate substance is allowed to act on cells having a labeled neurotransmitter receptor. Item 3. Item 3. The screening method according to Item 1 or 2, wherein the receptor is an AMPA receptor. Item 4. Formula (I) below
  • L 1 and L 2 each independently represent a divalent linking group
  • Lg represents a ligand for a neurotransmitter receptor (Rec-Nu-H) having a nucleophilic group (Nu-H).
  • Fl represents a labeling group
  • R 1 and R 2 are the same or different and represent a hydrogen atom or a substituent.
  • L 1 and L 2 each independently represent a divalent linking group
  • Lg represents a ligand for a neurotransmitter receptor (Rec-Nu-H) having a nucleophilic group (Nu-H).
  • Fl represents a labeling group
  • R 1 and R 2 are the same or different and represent a hydrogen atom or a substituent.
  • L 1 and L 2 each independently represent a divalent linking group
  • Lg represents a ligand for a neurotransmitter receptor (Rec-Nu-H) having a nucleophilic group (Nu-H).
  • Fl represents a labeling group
  • R 1 and R 2 are the same or different and represent a hydrogen atom or a substituent.
  • the neurotransmitter receptor (Rec-Nu-H) is reacted with the compound represented by the following formula (II):
  • Rec-Nu represents a group in which a hydrogen atom is eliminated from the neurotransmitter receptor (Rec-Nu-H).
  • Nu represents a hydrogen atom from a nucleophilic group possessed by the neurotransmitter receptor.
  • L 2 represents a divalent linking group, and Fl represents a labeling group.
  • a method for producing a receptor is
  • Receptor proteins that receive ligands such as neurotransmitters are important drug targets.
  • glutamate is widely known as an important neurotransmitter, and its receptor glutamate receptor is an essential protein for neurotransmission.
  • the present inventors have developed a technique for selectively labeling in the vicinity of a ligand binding site of a neurotransmitter receptor such as AMPA receptor which is a kind of glutamate receptor.
  • a ligand of a neurotransmitter receptor whose structure is greatly changed by the binding of a ligand, an agonist that activates the receptor and an inactivatable antagonist, for example, a signal emitted by a label such as a fluorescence response is different, Agonists and antagonists can be distinguished, for example, as changes in fluorescence.
  • this change can be used for a high-throughput detection system. From the above results, this research result means that a system capable of assaying agonists of any neurotransmitter receptor including, for example, AMPA receptor, at high throughput could be constructed. Even with ligands of neurotransmitter receptors other than AMPA receptors, the agonists that activate the receptors and the inactivatable antagonists differ in the signal emitted by a label such as a fluorescent response, and any neurotransmitter receptor agonist. Antagonists can be distinguished, for example, as fluorescence changes, and selective agonists can be screened at high throughput.
  • LDAI LigandgDirected Acyl imidazole
  • Ligand function analysis by labeled AMPAR-2 Agonist and Antagonist showed different response patterns, and it became clear that it functions as a biosensor that distinguishes Agonist and Antagonist.
  • Evaluate responses to various ligands Ligand-responsive dye dependence. Alexa488 and Oregon green show similar responses regardless of the function of the ligand, making them suitable for binding rate quantification.
  • Alexa568 and ATTO655 are suitable for categorizing ligands because they respond by distinguishing the function / action mechanism of the ligand.
  • LDAI ligand-directed acylimidazole compound
  • L 1 and L 2 each independently represent a divalent linking group
  • Lg represents a ligand for a neurotransmitter receptor
  • Fl represents a labeling group
  • R 1 and R 2 are the same or different.
  • the labeling reagent (labeling agent) of the present invention having an acylimidazole moiety binds to the neurotransmitter receptor (Rec-Nu-H) at the ligand (Lg) moiety, and in the receptor (Rec-Nu-H) Nucleophilic group (Nu-H) attacks the C atom of the carbonyl of acylimidazole, the N-CO bond is cleaved, the ligand having an imidazole group leaves the acceptor, and the labeling group (Fl) of the acceptor Nu and -CO-O- (L 2 ) linked via n2 group labeled receptor: Rec-Nu-CO-O- (L 2 ) n2 -Fl
  • nucleophilic group examples include NH 2 group at the terminal of Lys contained in the amino acid constituting the receptor, phenolic OH group of Tyr, SH group of Cys, imidazole group of His, etc.
  • NH 2 group at the terminal of Nu includes -NH-, -O-, -S-, etc., and -NH- is preferred.
  • Preferred examples include Alexa-488, Oregon green, Alexa-546, Alexa-568, ATTO655, and CypHer5E.
  • the interacting protein can be bound to the receptor by irradiating light when the interacting protein binds to the labeled neurotransmitter receptor. It is useful for detecting protein-protein interactions in receptors.
  • the labeled neurotransmitter receptor on the membrane surface is useful for such functional modification because the function of the protein such as intracellular transport is modified.
  • a water-soluble polymer such as PEG
  • the labeled neurotransmitter receptor increases the stability of membrane proteins by increasing the blood half-life or increasing the degree of solubilization. Such an effect can be expected.
  • photocrosslinking agent examples include aryl azides such as phenyl azide, highly reactive groups such as diazirine and benzophenone. Since these groups react rapidly with nearby compounds by light irradiation, they are useful for detecting protein-protein interactions between ligands and receptors.
  • water-soluble polymer examples include polyethylene glycol (PEG).
  • radioisotope examples include a radioisotope, preferably 3 H, 35 S and the like.
  • peptides include membrane-permeable peptides such as tat peptides, various homing peptides, various peptide ligands, and the like.
  • any neurotransmitter receptor is a target of labeling, and is not particularly limited. Examples include ligands: Glutamate receptor: binds glutamate as a neurotransmitter. Depending on the drug to be bound, it is divided into NMDA receptor, AMPA receptor and kainate receptor.
  • Muscarinic acetylcholine receptor acetylcholine, muscarinic / adenosine receptor: adenosine, caffeine / adrenergic receptor: adrenaline, noradrenaline / GABA receptor: GABA Cannabinoid receptor: Cannabis component and anandamide Cholecystokinin receptor: Cholecystokinin Dopamine receptor: Dopamine histamine receptor: Histamine opioid receptor: Opium component such as cocaine, morphine, heroin and endogenous peptide Ligand (enkephalin, endorphins, etc.) Serotonin receptor: Serotonin Somatostatin receptor: Somatostatin Nicotinic acetylcholine receptor: Acetylcholine, Nicotine glycine receptor: Glycine as a neurotransmitter, strykinin
  • receptor and ligand combinations are shown above, but the combination of the receptor to be labeled and the ligand that binds to the receptor is not limited to these, and the currently known neurotransmitter receptor and ligand combinations Either a combination or a combination of neurotransmitter receptors and ligands discovered in the future may be used.
  • L1 and L2 may be the same or different, and examples thereof include a divalent linking group.
  • the divalent linking group include —O—, —CO—, —COO—, —O—CO—, —NHCO—, —CONH—, — (CH 2 ) m1 — (m1 represents an integer of 1 to 6).
  • Arylene groups (particularly phenylene such as ortho, meta, para), heteroarylene groups, —NH—, — (CH 2 CH 2 O) m2 — (m2 represents an integer of 1 to 10), — (CH 2 CH (CH 3 ) O) m2 — (m2 represents an integer of 1 to 10) and the like may be used, and these may be used alone or in combination of two or more of the same or different.
  • the linking group may be constituted.
  • (terminal) CO, COO, O—CO—, CONH, NHCO, NH, etc. of the divalent linking group are bonded to COOH, NH 2, etc. such as Lg, Fl, amide, urethane, urea, etc. Can be formed.
  • Lg and Fl can be carried out by utilizing them when they have a functional group such as carboxylic acid (COOH) or an ester or active ester thereof, NH 2 , SH, OH or the like.
  • a linking acid functional group such as carboxylic acid (COOH) or its ester or active ester, NH2, SH, OH, etc. is introduced at the terminal and this functional group is used.
  • Lg and Fl can be introduced, for example, according to the following schemes 1 and 2.
  • Schemes 1 and 2 are methods for introducing Lg and Fl using amide bonds (CONH, NHCO), but Lg and Fl are introduced according to known methods using ether bonds, thioether bonds, amino bonds, and the like. can do.
  • R 1 , R 2 , L 1 , L 2 , Lg, Fl are as defined above.
  • CONH-L 1a and NHCO-L 1a represent specific embodiments of L 1 , L 2a represents a divalent linking group
  • Y 1 represents an activity such as OH, alkoxy, aryloxy, imidazolyl, 1-hydroxybenzotriazolyl (O-Bt), N-hydroxysuccinimidyl (OSu), etc. (Denotes a leaving group used as an ester. DSC stands for disuccinimidyl carbamate.)
  • the amide bond (CONH, NHCO) is formed by using a raw material amino compound (compound having —NH 2 group) and carboxylic acid, ester or active ester compound (compound having —CO—Y1 group).
  • the reaction proceeds advantageously by using about equimolar amounts and reacting at a temperature from room temperature to the boiling point of the solvent for 1 to 24 hours.
  • the solvent include methanol, ethanol, methylene chloride, chloroform, carbon tetrachloride, 1,2-dichloroethane, benzene, toluene, acetone, methyl ethyl ketone, ether, tetrahydrofuran and the like.
  • DSC is Fl-CONH-L 2a -OH or Fl-NHCO-L 2a -OH1 mol using excess from 1 mole excess of a base (triethylamine from a catalytic amount of pyridine, dimethylaminopyridine,
  • the target compound (2) can be obtained by reacting in the presence of diisopropylethylamine, DBU, etc.) from room temperature to a temperature at which the solvent boils for 1 to 24 hours.
  • AMPA receptor ligand AMPAR ligand
  • NMDA receptor ligand NNDAR ligand
  • GABA receptor ligand GABA receptor ligand
  • mGluR1 ligand metabotropic glutamate receptor ligand
  • the AMPA receptor ligand can be bound to L 1 by an amide bond, urethane bond or the like using a terminal amino group.
  • the NMDA receptor ligand can be bound to L 1 by an amide bond, an ester bond or the like using a terminal ⁇ or ⁇ unsaturated carboxyl group.
  • GABA receptor ligands can alkylate a 7-membered secondary amino group (NH) and bind to L 1 via an NC bond.
  • the mGluR1 ligand described above is a pyrimidine ring having a halogen atom introduced therein.
  • a divalent linking group L 1 such as NH—, O—, or S— can be introduced into this halogen atom position by nucleophilic substitution reaction.
  • L 1 can also be bound at a position where the function of the ligand is not lost. Examples of combinations of two or more divalent linking groups include the following:
  • terminal amino (NH) group exemplified above may form an amide bond with a COOH group such as Fl or Lg.
  • R 1 to R 2 examples include alkyl, cycloalkyl, alkoxy, alkenyl, halogen atom, OH, CN, NO 2 , COOH, NH 2 , phenyl, benzyl, acetylamino, acetyl, acetyloxy, methoxycarbonyl, And ethoxycarbonyl, butoxycarbonyl, trifluoromethyl and the like.
  • Alkyl includes linear or branched C 1-18 alkyl such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, hexyl, preferably Examples thereof include C 1-6 alkyl, more preferably C 1-4 alkyl.
  • Cycloalkyl includes C 3-10 cycloalkyl such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl, preferably C 3-8 cycloalkyl, more preferably C 5-6 cycloalkyl. A part of the ring may be substituted with a hetero element or may have a substituent.
  • Alkoxy includes linear or branched methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy, n-pentyloxy, isopentyloxy, hexyloxy, polyethylene glycol derivatives, etc.
  • C 1-18 alkoxy preferably C 1-6 alkoxy, more preferably C 1-4 alkoxy.
  • R 1 and R 2 are preferably hydrogen atoms.
  • the compound of the present invention can be synthesized according to the following scheme 3.
  • Solvents include chlorinated hydrocarbons such as methylene chloride, chloroform and dichloroethane, aromatic hydrocarbons such as benzene and toluene, esters such as ethyl acetate, ketones such as acetone, methyl ethyl ketone, and methyl isobutyl ketone, ethers, diisopropyl Examples include ethers such as pyrether and tetrahydrofuran, aliphatic or alicyclic hydrocarbons such as hexane and cyclohexane, DMF, DMSO, dioxane, and N-methylpyrrolidone. Examples of the base include triethylamine, diisopropylethylamine, pyridine, dimethylaminopyridine, DBU and the like.
  • the neurotransmitter receptor may be an isolated receptor, or compound (I) may be reacted directly with a membrane-bound receptor. Alternatively, cells expressing the receptor may be reacted with compound (I).
  • receptor may include all of a receptor protein or a fragment thereof having a ligand binding site and a nucleophilic group, a fragment of a cell membrane containing the receptor, and a cell expressing the receptor. As the cell, a cell in which a receptor gene is introduced to highly express the receptor can be preferably used.
  • the receptor When the receptor is composed of a plurality of proteins, not only a protein containing a ligand binding site but also a cell in which genes of all proteins constituting the receptor are introduced and highly expressed can be used.
  • a cell in which the receptor is highly expressed is preferable because a signal (fluorescence intensity or a change thereof) increases during ligand screening.
  • the reaction between the receptor and compound (I) can be carried out in a solvent, buffer solution or cell culture medium.
  • Compound (I) is preferably used in excess relative to the receptor.
  • Compound (I) labels the receptor when the receptor and the ligand (Lg) are bound, but non-specific labeling is suppressed.
  • the compound (I) can be removed by washing, whereby excess compound (I) can be removed, and nonspecific fluorescent labeling can be suppressed.
  • the reaction temperature is from room temperature to about 37 ° C., and the reaction time is about 30 minutes to 12 hours.
  • the solvent is water or a solvent in which compound (I) is dissolved, for example, lower alcohols such as methanol, ethanol, propanol, acetone, tetrahydrofuran (THF), dioxane, N-methylpyrrolidone, dimethylformamide (DMF), acetonitrile, dimethylacetamide, dimethyl Water-miscible solvents such as sulfoxide (DMSO) can be mentioned.
  • lower alcohols such as methanol, ethanol, propanol, acetone, tetrahydrofuran (THF), dioxane, N-methylpyrrolidone, dimethylformamide (DMF), acetonitrile, dimethylacetamide, dimethyl
  • DMF dimethylformamide
  • DMSO dimethyl Water-miscible solvents
  • the buffer include HEPES buffer, phosphate buffer, Tris buffer, and the like.
  • the cell expressing the receptor may be a cultured cell, and a solution of compound (I) is applied to the receptor-expressing cell (preferably human cell) in the tissue section or the receptor-expressing cell in the living body of a non-human mammal. It may be supplied and fluorescently labeled.
  • both the agonist and the competitive antagonist have increased fluorescence ( ⁇ , FIG. 10), and when the labeling group is Alexa568, In the case of competitive antagonists, the fluorescence decreases ( ⁇ ), and in the case of agonists and antagonists, no change in fluorescence occurs. Furthermore, in the case of ATTO655, the fluorescence increases when the ligand is an agonist ( ⁇ ), the fluorescence is transiently increased after a competitive antagonist ( ⁇ ⁇ ), and the fluorescence decreases when a non-competitive antagonist is present ( ⁇ ⁇ ). ⁇ ).
  • the labeled receptor of the present invention can be combined with several labeled receptors as necessary, for example, by changing the signal emitted by the label such as fluorescence, the ligand becomes an agonist, competitive antagonist, non-competitive.
  • the ligand becomes an antagonist (agonist + antagonist) at high throughput, for example, select Alexa488 having a large fluorescence change.
  • a fluorescently labeled receptor can be selected.
  • Example 1 Materials and Methods All chemicals and biochemical reagents were purchased from commercial sources and used without further purification. Thin layer chromatography (TLC) was performed using aluminum sheets (Merck Inc.) pre-coated with silica gel 60 F 254, visualized by fluorescence quenching or ninhydrin stain. Purification by chromatography was performed using flash column chromatography on silica gel 60 N (neutral, 40-50 ⁇ m, Kanto Chemical Co.).
  • Reverse phase HPLC was measured on a Hitachi Chromaster system equipped with a diode array and fluorescence detector and a YMC-Pack Triat C18 or ODS-A column. A linear gradient containing 0.1% TFA or 10 mM ammonium acetate (solvent A) and 0.1% aqueous TFA-containing acetonitrile or acetonitrile ((solvent B)) was used.
  • Biochemical experiments SDS-PAGE and Western blotting were performed using a Bio-Rad Mini-Protean III electrophoresis apparatus. Fluorescence and chemiluminescence signals were detected with a ChemiDoc XRS system equipped with a 520DF30 filter (ChemiDoc, Bio-Rad laboratory) and Imagequant LAS 4000 (GE Healthcare). As the confocal laser microscope, FV1000 (Olympus) or LSM710 (Carl Zeiss) was used.
  • HEK293T cells 10% FBS, penicillin (100 units / mL) and Dulbecco was added streptomycin (100 ⁇ g / mL)'s Modified Eagle Medium (DMEM, glucose 4.5 g / L) in a medium, in a humidified atmosphere of 5% CO 2
  • DMEM Modified Eagle Medium
  • GluR2 glutamate receptor
  • GluR2 in HEK293T cells Labeling agent (2 ⁇ M) was added to HEK293T cells transfected with the above gene in the presence or absence of NBQX (50 ⁇ M) in DMEM medium (glutamax, 25 mM HEPES, FBS (-)). ) was added and incubated at 17 ° C. for 4 hours. Cells were washed twice with HBS and lysed on ice using RIPA (radioimmunoprecipitation assay) buffer containing 1% protease inhibitor cocktail set III (Calbiochem®).
  • RIPA radioimmunoprecipitation assay
  • the resulting solution was mixed with 2 ⁇ SDS-PAGE buffer (pH 6.8, 125 mM Tris ⁇ HCl, 20% glycerol, 4% SDS and 0.01% bromophenol blue, 100 mM DTT) and vortexed at room temperature for 30 minutes.
  • the obtained sample was developed by SDS-PAGE and transferred to an Immun-Blot PVDF membrane (Bio-Rad).
  • the labeled product was detected with an anti-Fluorescein antibody (Invitrogen, ⁇ 2000) and an anti-rabbit IgG antibody-HRP complex (GE Healthcare, ⁇ 3000).
  • Immunodetection of GluR2 was performed using anti-HA antibody (Abcam) and anti-rabbit IgG antibody-HRP complex (GE Healthcare, ⁇ 5000).
  • the HRP signal was detected with LAS 4000 imaging system (FujiFilm) using ECL prime western blotting detection reagent (GE Healthcare).
  • the resulting solution was mixed with 2 ⁇ SDS-PAGE buffer (pH 6.8, 125 mM Tris ⁇ HCl, 20% glycerol, 4% SDS and 0.01% bromophenol blue, 100 mM DTT) and vortexed at room temperature for 30 minutes.
  • the obtained sample was developed by SDS-PAGE and transferred to an Immun-Blot PVDF membrane (Bio-Rad). Labeled products were detected with anti-Fluorescein antibody (Invitrogen, ⁇ 2000) or anti-Alexa488 antibody (Invitrogen, ⁇ 1000) and anti-rabbit IgG antibody-HRP complex (GE Healthcare, ⁇ 3000).
  • Immunodetection of GluR1 was performed using anti-GluR1 antibody (millipore) and anti-mouse IgG antibody-HRP complex (GE Healthcare, ⁇ 3000).
  • the HRP signal was detected with LAS 4000 imaging system (FujiFilm) using ECL prime western blotting detection reagent (GE Healthcare).
  • tert-Butyl (2- (2-(((perfluorophenoxy) carbonyl) oxy) ethoxy) ethyl) carbamate (2b) Bis (perfluorophenyl) carbonate (1.15 g, 2.93 mmol), TBAF (190 mg) in a stirred solution of tert-butyl (2- (2-hydroxyethoxy) ethyl) carbamate (500 mg, 2.44 mmol) in THF (24 ml) , 0.73 mmol). The reaction mixture was stirred for 24 hours at room temperature. The solution was diluted with CHCl 3 and washed with 1N NaOH. The organic layer was dried over Na 2 SO 4 , filtered and evaporated.
  • reaction mixture was stirred at room temperature for 1 hour.
  • Boc-Ape (5) -OH (215 mg, 0.99 mmol, 1.1 eq), HBTU (410 mg, 1.08 mmol, 1.0 eq), DIEA (744 ⁇ L, 4.50 mmol, 5.0 eq) in dry DMF (10 mL) And a mixture of histamine dihydrochloride (100 mg, 0.543 mmol, 0.60 eq) was stirred at room temperature for 3 hours and 25 minutes under a nitrogen atmosphere. The solvent was distilled off under reduced pressure. The residue was then extracted with CHCl 3 (30 mL), and the organic layer was washed with 0.1N NaOH (30 mL ⁇ 3) and 30 mL saturated brine, dried over Na 2 SO 4 and evaporated under reduced pressure. did. The residue was purified by silica gel column chromatography (CHCl 3 : methanol: NH 3 water 10: 1: 1%) to obtain Compound 14c.

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Abstract

La présente invention concerne un procédé de criblage de substances qui se lient à des récepteurs de neurotransmetteur, qui ont une structure de groupe représentée par la formule (II) (dans la formule, Rec-Nu représente un groupe obtenu par séparation d'un atome d'hydrogène d'un récepteur de neurotransmetteur (Rec-Nu-H). Nu représente un groupe bivalent obtenu par séparation d'un atome d'hydrogène d'un groupe nucléophile (Nu-H) possédé par le récepteur de neurotransmetteur. L2 représente un groupe de liaison bivalent et Fl représente un groupe marqueur). Une substance candidate, dont les motifs fluorescents varient entre la situation dans laquelle un antagoniste est lié et celle dans laquelle un agoniste est lié, et qui est supposée interagir avec un récepteur de neurotransmetteur marqué est amenée à agir sur le récepteur, le mode de liaison de la substance candidate et du récepteur est détecté par la variation des signaux émis par un marqueur, et le criblage est effectué sur la base de ces résultats.
PCT/JP2015/054558 2014-02-19 2015-02-19 Développement de système de criblage de ligand pour des récepteurs de neurotransmetteur WO2015125851A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019168125A1 (fr) * 2018-03-01 2019-09-06 国立大学法人京都大学 Développement d'une nouvelle technique fondée sur une réticulation chimique permettant la visualisation de récepteurs in vivo

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002064772A1 (fr) * 2001-01-30 2002-08-22 Shionogi & Co., Ltd. Variant d'epissure du recepteur nmda humain
WO2004027424A1 (fr) * 2002-09-19 2004-04-01 Hamamatsu Photonics K.K. Methode d'analyse par fluorescence avec un anticorps fluorescent
WO2006070941A1 (fr) * 2004-12-28 2006-07-06 Tokyo University Of Science Nouveau procede de criblage de substance au moyen d'une sonde moleculaire fluorescente
JP4485475B2 (ja) * 2004-02-12 2010-06-23 独立行政法人科学技術振興機構 核内レセプターのアゴニスト・アンタゴニスト検出用プローブとそれを用いた核内レセプターに対するアゴニストおよびアンタゴニストのスクリーニング方法

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002064772A1 (fr) * 2001-01-30 2002-08-22 Shionogi & Co., Ltd. Variant d'epissure du recepteur nmda humain
WO2004027424A1 (fr) * 2002-09-19 2004-04-01 Hamamatsu Photonics K.K. Methode d'analyse par fluorescence avec un anticorps fluorescent
JP4485475B2 (ja) * 2004-02-12 2010-06-23 独立行政法人科学技術振興機構 核内レセプターのアゴニスト・アンタゴニスト検出用プローブとそれを用いた核内レセプターに対するアゴニストおよびアンタゴニストのスクリーニング方法
WO2006070941A1 (fr) * 2004-12-28 2006-07-06 Tokyo University Of Science Nouveau procede de criblage de substance au moyen d'une sonde moleculaire fluorescente

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
FUJISHIMA SHO-HEI ET AL.: "Ligand-Directed Acyl Imidazole Chemistry for Labeling of Membrane- Bound Proteins on Live Cells", J AM CHEM SOC, vol. 134, no. 9, pages 3961 - 3964, XP055221548 *
SHIGEYUKI NAMIKI ET AL.: "Optical glutamate sensor for spatiotemporal analysis of synaptic transmission", EUROPEAN JOURNAL OF NEUROSCIENCE, vol. 25, 2007, pages 2249.2259, XP055221551 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019168125A1 (fr) * 2018-03-01 2019-09-06 国立大学法人京都大学 Développement d'une nouvelle technique fondée sur une réticulation chimique permettant la visualisation de récepteurs in vivo

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