WO2015025856A1 - Procédé de mesure d'affinité de liaison entre cible et ligand et assistant réactif et trousse pour utilisation dans ledit procédé - Google Patents

Procédé de mesure d'affinité de liaison entre cible et ligand et assistant réactif et trousse pour utilisation dans ledit procédé Download PDF

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WO2015025856A1
WO2015025856A1 PCT/JP2014/071682 JP2014071682W WO2015025856A1 WO 2015025856 A1 WO2015025856 A1 WO 2015025856A1 JP 2014071682 W JP2014071682 W JP 2014071682W WO 2015025856 A1 WO2015025856 A1 WO 2015025856A1
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target substance
hydrophobic
ligand
binding affinity
lipid
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PCT/JP2014/071682
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English (en)
Japanese (ja)
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松岡　茂
道雄 村田
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国立大学法人大阪大学
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Priority to JP2015532865A priority Critical patent/JP6057265B2/ja
Publication of WO2015025856A1 publication Critical patent/WO2015025856A1/fr

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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
    • G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors
    • 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
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/55—Specular reflectivity
    • G01N21/552—Attenuated total reflection
    • G01N21/553—Attenuated total reflection and using surface plasmons
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2405/00—Assays, e.g. immunoassays or enzyme assays, involving lipids
    • G01N2405/04—Phospholipids, i.e. phosphoglycerides

Definitions

  • the present invention relates to a method for measuring the binding affinity between a target substance and a ligand, and reagent aids and kits for use in the method, and more particularly to chemistry such as isothermal titration calorimetry (ITC).
  • ITC isothermal titration calorimetry
  • the present invention relates to a method capable of more efficiently measuring the binding affinity between substances, and a reagent auxiliary agent and kit for use in the method.
  • a fatty acid binding protein FABP3 (fatty acid binding protein 3) having a function of transporting "foreign fatty acid” from “cell membrane” to "mitochondrion” is a soluble protein having a molecular weight of 15 kDa involved in intracellular transport of fatty acids. It binds to fatty acids of the molecule (Non-patent Document 1).
  • Non-patent Document 2 shows that such FABP3 exhibits a unique selective affinity for fatty acids.
  • the selective affinity (binding affinity) between a target substance and a ligand can be analyzed by using, for example, an isothermal titration calorimetry (ITC) method. It is difficult to make an exhaustive comparison under the same conditions using the method, especially because long-chain fatty acids are hydrophobic.
  • ITC isothermal titration calorimetry
  • a technology that enables comparison of binding affinity under the same conditions without affecting the type of target substance is desired.
  • An object of the present invention is to solve the above problems, and the object of the present invention is to measure the binding affinity between the target substance and the ligand without affecting the type of the target substance.
  • the present invention is a method for measuring the binding affinity between a target substance and a ligand, Mixing a target substance and a substance capable of constituting a lipid aggregate to form a lipid aggregate containing the target substance; Combining a lipid assembly containing the target substance with a ligand to prepare a measurement sample; and measuring a physical property change amount of the measurement sample;
  • a method comprising
  • the lipid aggregate is at least one molecular group selected from the group consisting of liposomes, bicelles, and dendrimers.
  • the substance that can constitute the lipid aggregate is a phospholipid.
  • the target substance is a hydrophobic compound.
  • the hydrophobic compound is selected from the group consisting of hydrophobic fatty acids and derivatives thereof and salts thereof, hydrophobic proteins, hydrophobic peptides, hydrophobic small molecules, hydrophobic DNA, and hydrophobic RNA. At least one compound.
  • the physical property change amount is measured by an isothermal titration calorimetry method or a surface plasmon resonance method.
  • the present invention is also a reagent auxiliary for use in measuring the binding affinity between a target substance and a ligand, which contains substances that can form lipid aggregates.
  • the substance that can constitute the lipid aggregate is a phospholipid.
  • the present invention also provides a kit for use in measuring the binding affinity between a target substance and a ligand, It is a kit containing the substance which can comprise a lipid assembly.
  • the binding affinity between the target substance and the ligand can be efficiently measured regardless of the physical properties (water solubility, etc.) of the target substance. Furthermore, according to the present invention, the obtained measurement results can be compared with each other regardless of the type of the target substance.
  • Method for measuring binding affinity of target substance and ligand In the method for measuring the binding affinity between a target substance and a ligand of the present invention, first, the target substance and a substance capable of constituting a lipid aggregate are mixed to form a lipid aggregate containing the target substance. Is done.
  • the target substance used in the present invention refers to the entire compound that requires measurement of the binding affinity with the ligand described later through the isothermal titration calorimetry (ITC) method or the surface plasmon resonance (SPR) method.
  • the target substance is not necessarily limited, and may be, for example, either a water-soluble compound or a hydrophobic compound.
  • the term “hydrophobic” used in the present specification means that it is insoluble in water, or has a solubility of, for example, 1 g or less, preferably 0.1 g or less with respect to 100 g of water at 20 ° C. Point and say.
  • the “hydrophobic compound” refers to a compound that exhibits such hydrophobicity.
  • the water-soluble compound refers to a compound that is soluble in water, that is, a compound other than the hydrophobic compound.
  • the hydrophobic compound is not particularly limited, and examples thereof include hydrophobic fatty acids and derivatives or salts thereof, hydrophobic proteins, hydrophobic peptides, hydrophobic low molecular compounds, hydrophobic DNA, hydrophobic RNA, and combinations thereof. It is done.
  • the hydrophobic fatty acid is a fatty acid having a hydrophobic fatty acid group or an acyl group which may be substituted with an aryl group such as a phenyl group or a halogen-substituted phenyl group.
  • the hydrophobic fatty acid has a long carbon number of 10 or more.
  • the upper limit of the carbon number which comprises the said fatty acid is not necessarily limited, For example, 50 or less, 45 or less, 40 or less, and 35 or less are mentioned.
  • the fatty acids can also be either saturated or unsaturated fatty acids, and unsaturated fatty acids include both monounsaturated fatty acids and polyunsaturated fatty acids (eg, ⁇ 3 and ⁇ 6 fatty acids).
  • the hydrophobic fatty acid derivatives include esters of the above-mentioned hydrophobic fatty acids (for example, glycerin fatty acid ester, fatty acid alkyl (C 1 -C 40 ) ester), fatty acid alkenyl (C 1 -C 40 ) ester, and fatty acid alkynyl (C 1 -C 40 ) ester and the like.
  • the salt of the hydrophobic fatty acid include sodium salts, potassium salts, calcium salts, magnesium salts, zinc salts, iron salts, and copper salts of the hydrophobic fatty acids.
  • any fatty acid including such a hydrophobic fatty acid can be used as a target substance.
  • fatty acids that can be used as target substances are not necessarily limited.
  • Derivatives of fatty acids and salts of fatty acids that can be used as target substances also include derivatives and salts
  • the hydrophobic protein is not particularly limited, and examples thereof include membrane proteins such as integrins; polypeptide poisons such as colicin A, ⁇ -hemolysin, prion, and amyloid ⁇ peptide.
  • the hydrophobic peptide is not particularly limited, and examples thereof include glycophorin A, gramicidin D, M2-TM, and p24-TM.
  • the hydrophobic low molecular weight compound is not particularly limited, and examples thereof include indole derivatives such as indomethacin, acemetacin, sulindac, progouritacin maleate, and pindolol; salicylic acid derivatives such as aspirin and diflunisal; ibuprofen, ketoprofen, naproxen, and pranoprofen.
  • Phenylpropionic acid derivatives such as: mefenamic acid, anthranilic acid derivatives such as aluminum flufenamic acid; benzothiazine derivatives such as piroxicam, ampiroxicam; thiophene acetic acid derivatives such as thiaprofenic acid; medroxyprogesterone acetate, chlormadinone acetate, danazol, fluorometholone, dexamethasone , Hydrocortisone, prednisolone, methylprednisolone, betamethasone and other steroid derivatives; folic acid Folic acid derivatives such as methotrexate; Taxane derivatives such as paclitaxel and docetaxel hydrate; Purine derivatives such as mercaptopurine; Pyrimidine derivatives such as fluorouracil and tegafur; Peptide drugs such as cyclosporin A; Enoxacin, norfloxacin, ofloxacin, levof
  • hydrophobic RNA examples include RNA and tRNA composed of 3 to 100 bases modified with a hydrophobic structure.
  • hydrophobic DNA examples include DNA composed of 3 to 100 bases modified with a hydrophobic structure.
  • the substance that can constitute the lipid aggregate used in the present invention is a substance that can form the lipid aggregate described later together with the target substance, and the binding affinity between the target substance of the present invention and the ligand. It functions as a component of a reagent auxiliary in the measurement of sex.
  • Examples of substances that can constitute lipid aggregates include phospholipids, glycolipids, sphingolipids, sterols, archiols, and cardiochiols.
  • DMPC dimyristoyl phosphatidylcholine
  • DOPC dioleoylphosphatidylcholine
  • 1-palmitoyl-2-oleoylphosphatidylcholine egg yolk phosphatidylcholine
  • soybean phosphatidylcholine phosphatidylcholine
  • phosphatidylethanolamine phosphatidylinositol
  • phosphatidylserine Phosphatidylglycerol diphosphatidylglycerol
  • phosphatidic acid cholesterol and sphingomyelin.
  • Dimyristoyl phosphatidylcholine is more preferred.
  • the mixing ratio of the target substance and the aggregate constituent substance is not necessarily limited, but the aggregate constituent substance is, for example, 0.5 mol to 500 mol, preferably 1 mol to 1 mol of the target substance.
  • the amount is 100 mol, more preferably 5 to 20 mol.
  • the amount of the aggregate constituent material is less than 0.5 mol, a lipid aggregate containing the target substance is not properly formed in water, and as a result, the actual measurement value of the binding affinity such as isothermal dropping heat measurement is not obtained. There is a risk that it will not be obtained.
  • the amount of the aggregate constituent substance exceeds 500 mol, the target substance contained in the lipid aggregate constituted in water becomes small, and the detection sensitivity of the binding affinity may be lowered.
  • the above target substance and aggregate constituent substance are put in a predetermined container such as a test tube. Then, for the purpose of uniformly mixing both substances, such as chloroform, methanol, ethanol, 1-propanol, 2-propanol, diethyl ether, ethyl acetate, acetonitrile, dimethyl sulfoxide, dimethylformamide and tetrahydrofuran, and combinations thereof
  • a predetermined container such as a test tube.
  • the organic solvent may be preliminarily contained in the reagent auxiliary as one of the components together with the aggregate constituent material.
  • the target substance and the aggregate constituent material are uniformly mixed in the organic solvent in the same manner as described above by mixing a reagent auxiliary containing the aggregate constituent substance and the organic solvent with the target substance. Can be dispersed.
  • An aqueous medium for example, water or a buffer solution
  • the amount of the aqueous medium to be added is not particularly limited, and an amount sufficient to form a lipid aggregate is added.
  • the aggregate constituent material forms a lipid aggregate in a form containing the target substance.
  • lipid aggregates thus formed include liposomes, bicelles (planar lipid bilayers), and dendrimers (spherical lipid vesicles) and combinations thereof.
  • the lipid aggregate formed using the target substance and aggregate constituent substances is considered to have the following shape, for example.
  • FIG. 1 is a schematic diagram for explaining a state where target substance molecules are localized in the liposome when a target substance and a substance that can form a lipid aggregate are mixed to form a liposome containing the target substance.
  • FIG. 1 is a schematic diagram for explaining a state where target substance molecules are localized in the liposome when a target substance and a substance that can form a lipid aggregate are mixed to form a liposome containing the target substance.
  • the target substance-containing liposome 100 is a substantially spherical lipid aggregate mainly composed of phospholipids, and includes an inner film 106 constituting an inner layer and an outer film 108 constituting an outer layer.
  • the hydrophobic group portion 104 of the target substance molecule 110 is oriented on the side where the liposome portion 120 having the above exists, and the hydrophilic group portion 102 is oriented to the outside of the liposome portion 120.
  • the liposome 100 becomes one kind of particles having hydrophilicity as a whole and is uniformly dispersed in the aqueous medium.
  • the lipid aggregate containing the target substance is combined with the ligand to prepare a measurement sample.
  • the term “ligand” used in the present specification includes the entire substance selected in expectation of binding to the target substance, and is not necessarily limited to a substance that specifically binds to the target substance. It is not limited to.
  • the method of the present invention can be used to evaluate the binding affinity between a specific target substance and a specific target substance. Therefore, the binding affinity does not necessarily have to be generated between the two, and even when the binding affinity is not generated, the method of the present invention can be used for the evaluation. Therefore, all target substances employed for performing the evaluation can be “ligands” in the present specification.
  • the ligand that can be used in the present invention is not particularly limited, and examples thereof include proteins, polysaccharides, low molecular compounds, peptides, RNA, and DNA.
  • the type of protein that can be used as a ligand is also not particularly limited.
  • fatty acid binding protein FABP1-12 fatty acid binding protein 1-12
  • outer membrane specific lipoprotein molecule chaperone LolA fatty acid binding protein 1-12
  • outer membrane specific lipoprotein Receptors LolB outer membrane specific lipoprotein Receptors LolB
  • lipoxygenase and cyclooxygenase.
  • the type of polysaccharide that can be used as a ligand is also not particularly limited, and examples thereof include hyaluronic acid, dextrin, and cellulose.
  • the type of low molecular weight compound that can be used as a ligand is also not particularly limited.
  • tirofiban and 2-pyrrolidin-1-yl-N- [4- [4- (2-pyrrolidin-1-yl-acetylamino] ) -Benzyl] -phenyl] -acetamide are also not particularly limited.
  • the type of peptide that can be used as a ligand is also not particularly limited, and examples thereof include insulin, glucagon-like peptide-1, vasopressin, and oxytocin.
  • RNA and DNA that can be used as ligands are also not particularly limited.
  • siRNA, miRNA, and nucleic acid drugs for example, cytomegalovirus retinitis drug “Vitra Mune”, age-related macular degeneration drug, "McGen”).
  • the mixing ratio of the lipid aggregate containing the target substance and the ligand is not necessarily limited.
  • the molar ratio of the target substance to be mixed and the ligand is preferably 1: 2 to 10: 1. More preferably, it is 1: 1 to 2: 1.
  • the lipid aggregate containing the target substance and the ligand are mixed in the presence of the aqueous medium.
  • Measured sample is prepared in this way.
  • the physical property change amount of the measurement sample is measured.
  • the physical property change amount of the measurement sample can be measured using a method known to those skilled in the art for measuring the binding affinity between predetermined substances (for example, between a normal target substance and a ligand). Examples of such methods include isothermal titration calorimetry (ITC) method and surface plasmon resonance (SPR) method.
  • ITC isothermal titration calorimetry
  • SPR surface plasmon resonance
  • the conditions, means, etc. used in such a measurement method may be those known to those skilled in the art.
  • the binding affinity between the ligand and the ligand can be easily measured using a known method as described above. Furthermore, in the present invention, the binding affinity between the ligand and the ligand can be measured under the same conditions regardless of whether the target substance is hydrophobic or hydrophilic. For this reason, it is also possible to evaluate the binding affinity for the same ligand for a plurality of target substances regardless of whether they are hydrophobic or hydrophilic. By making it possible to compare the binding affinity under the same conditions, for example, a large number of target substances can be efficiently evaluated in the field of drug development.
  • the kit of the present invention enables, for example, the measurement of the binding affinity between a target substance and a ligand not only to evaluate the binding affinity of the target substance but also to compare with other target substances. That is, the kit of the present invention includes a substance that can constitute a lipid aggregate.
  • the substance (aggregate constituting substance) that can constitute the lipid aggregate contained in the kit of the present invention is the same as described above.
  • the kit of the present invention is provided with an instruction manual that describes the addition amount of the target substance, the lipid aggregate formation method, the measurement conditions of the isothermal titration calorimetry (ITC) method and the surface plasmon resonance (SPR) method, and the like. May be.
  • the aggregate constituent material is contained in a predetermined container such as an ampoule.
  • the amount of the aggregate constituent material that can be accommodated in one ampule is not necessarily limited, but when the amount of the target substance to be used is 1 mol, for example, 0.5 mol to 500 mol, preferably 1 mol to The amount satisfies a ratio corresponding to 100 mol, more preferably 5 mol to 20 mol.
  • the detection sensitivity of binding affinity can be further improved.
  • the ampoule contains an organic solvent such as chloroform, methanol, ethanol, 1-propanol, 2-propanol, diethyl ether, ethyl acetate, acetonitrile, dimethyl sulfoxide, dimethylformamide and tetrahydrofuran, and combinations thereof. It may be.
  • the content of the organic solvent in the ampoule is not particularly limited, and any amount can be selected by those skilled in the art.
  • the kit of the present invention can be used, for example, as follows for measuring the binding affinity between a target substance and a ligand.
  • a predetermined amount of a target substance is added into an ampoule containing the above-mentioned assembly constituent substances constituting the kit.
  • the ampoule is then added with an amount of an aqueous medium (eg, water or buffer) sufficient to form a lipid assembly.
  • an aqueous medium eg, water or buffer
  • a plurality of ampoules in which such lipid aggregates are formed are prepared.
  • the ampule formed with the lipid aggregate may be stored in a predetermined place until actual use.
  • a ligand is added to each ampoule, and a measurement sample is prepared in the same manner as described above.
  • the binding affinity for the target substance is measured.
  • ITC isothermal titration calorimetry
  • SPR surface plasmon resonance
  • a substance that can form a lipid assembly is included in the kit in advance to measure the binding affinity between the target substance and the ligand. Eliminates by preparing the materials individually. For this reason, the complexity of measurement preparation can be further eliminated.
  • Example 1 Measurement of binding affinity of caprylic acid and FABP3 using liposome by ITC
  • DMPC dimyristoylphosphatidylcholine
  • buffer solution (20 mM Tris-HCl (pH 8.0), 100 mM NaCl) was added to the test tube at 36.9 ° C. to hydrate the contents, and the test tube was vortexed and then sonicated. To form a uniform suspension.
  • test tube contents were frozen at ⁇ 30 ° C. and then thawed at 40 ° C. This freezing and thawing operation was repeated three times. Then, the test tube is subjected to a vortex mixer to form a suspension, and the contents in the test tube are subjected to a polycarbonate filter (product name: Nukurepore) by a small volume extruder (product name: Lipo Sofast, manufactured by Central Scientific Trade Co., Ltd.). It was filtered 19 times with a filter (pore size 100 nm), manufactured by Nitto Kikai Finetech Co., Ltd. Most often, the test tube was vortexed to form fatty acid-containing liposomes with a more uniform suspension.
  • a polycarbonate filter product name: Nukurepore
  • a small volume extruder product name: Lipo Sofast, manufactured by Central Scientific Trade Co., Ltd.
  • the ITC device (manufactured by TA Instruments Japan Co., Ltd.) was started and the measurement temperature was set to 37 ° C. Next, 50 ⁇ L of fatty acid-containing liposomes and 210 ⁇ L of degassed 0.2 mM FABP3 (prepared by the method described in Non-Patent Document 2) were respectively added to the titration syringe (50 ⁇ L) and the sample cell (190 ⁇ L) of the ITC device. Filled. The measurement parameters (measurement frequency: 3 times; drop amount: 2 ⁇ L; titration interval: 210 seconds; measurement temperature: 37 ° C .; stirring speed: 250 rpm) of the ITC apparatus were set to obtain measurement values. The obtained results are shown in Table 1.
  • Examples 2 to 12 Measurement of binding affinity of various saturated fatty acids and FABP3 using liposomes by ITC
  • Fatty acid-containing liposomes were formed in the same manner as in Example 1 except that various saturated fatty acids (0.5 mmol) shown in Table 1 were used instead of caprylic acid, and isothermal titration calorimetry of the liposomes and FABP3 was performed. went. The obtained results are shown in Table 1.
  • Examples 13 to 26 Measurement of binding affinity of various unsaturated fatty acids and FABP3 using liposomes by ITC
  • examples except that various unsaturated fatty acids (0.5 mmol) shown in Table 1 were used instead of caprylic acid, and evaporation of chloroform / methanol and subsequent complete removal under reduced pressure were performed under a nitrogen atmosphere.
  • fatty acid-containing liposomes were formed, and isothermal titration measurement of the liposomes and FABP3 was performed. The obtained results are shown in Table 1.
  • thermodynamic parameters it was found that all fatty acids described in Examples 1 to 26 have an enthalpy-driven intermolecular interaction. That is, in the binding between these fatty acids and FABP3, the contribution of the hydrophobic intermolecular interaction due to dehydration of the binding site is reduced as a result, and the intermolecular interaction due to hydrogen bond formation and van der Waals force is dominant. It is considered to be a factor.
  • isothermal titration heat measurement can be performed on fatty acids that have been conventionally insoluble or hardly soluble in water under the same conditions as other water-soluble fatty acids.
  • profile differences among the fatty acids can be easily compared.
  • Examples 27 to 33 Measurement of binding affinity of various saturated fatty acids and FABP3 using liposomes by ITC
  • Examples 34 and 35 Measurement of binding affinity of various fatty acids and FABP3 using liposomes by ITC
  • buffer solution 20 mM Tris-HCl (pH 7.0), 100 mM NaCl
  • binding to FABP3 is possible even using liposomes prepared using fatty acids such as prostaglandin E2 which is one type of physiologically active substance and jasmonic acid which is one type of plant hormone-like substance. It can be seen that the affinity could be measured using an ITC apparatus.
  • Examples 36 to 38 Measurement of binding affinity of various unsaturated fatty acids and FABP3 using liposomes by SPR
  • Fatty acid-containing liposomes were prepared in the same manner as in Example 1 except that various unsaturated fatty acids (1.1 mmol) shown in Table 4 were used instead of caprylic acid.
  • SPR measurement was performed as follows using a surface plasmon resonance (SPR) device (GE Healthcare BIAcore T200).
  • CM5 carboxylated dextran matrix
  • CM5 sensor chip was washed three times for 2 minutes at a flow rate of 20 ⁇ L / min using a 50 mM NaOH aqueous solution.
  • a mixed solution (1: 1 v) of 0.1M N-hydroxysuccinimide (NHS) and 0.39M 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) was added to the CM5 sensor chip.
  • / V) 70 ⁇ L was added at a flow rate of 5 ⁇ L / min over 7 minutes to convert the carboxyl group on the chip to succinimidyl ester and to activate.
  • FABP3 (100 ⁇ g / mL) prepared by the method described in Non-Patent Document 2 was immobilized on the sensor chip at a flow rate of 2 ⁇ mL / min for 30 minutes. Unreacted succinimidyl ester on the sensor chip is treated with 1M ethanolamine hydrochloride (pH 8.5), and the sensor surface is then buffered (phosphate buffered saline (PBS), pH 7.4) with 3 Washed twice.
  • PBS phosphate buffered saline
  • the fatty acid-containing liposome prepared above was added at a flow rate of 10 L / min in PBS of pH 7.4 at a fatty acid concentration as shown in Table 2, and SPR measurement was performed. .
  • Example 39 Measurement of binding affinity of stearic acid and FABP3 using ITC by ITC
  • DMPC dimyristoyl phosphatidylcholine
  • DHPC dihexanoylphosphatidylcholine
  • the ITC device (manufactured by TA Instruments Japan Co., Ltd.) was started and the measurement temperature was set to 37 ° C. Next, 50 ⁇ L of the stearic acid-containing bicell degassed in the titration syringe (50 ⁇ L) and the sample cell (190 ⁇ L) of the ITC device, and 0.2 mM FABP3 degassed (prepared by the method described in Non-Patent Document 2). 210 ⁇ L was filled.
  • the measurement parameters (measurement frequency: 3 times; drop amount: 2 ⁇ L; titration interval: 210 seconds; measurement temperature: 37 ° C .; stirring speed: 250 rpm) of the ITC apparatus were set to obtain measurement values.
  • the obtained dissociation constant (Kd) was 7.64 ⁇ M, and the enthalpy change (dH) was ⁇ 19.58 kJ / mol.
  • the binding affinity for FABP3 to stearic acid which is insoluble in water could be measured using the ITC apparatus through the production of the above bicelle.
  • the present invention for example, studies on dynamic physiological function expression of cell membranes, such as structure formation / function expression of membrane proteins, domain formation by aggregate distribution of cell membrane constituent molecules, through elucidation of intermolecular interactions in hydrophobic parts of cell membranes Useful in development.
  • the results measured by the method of the present invention can be used as, for example, basic information on target compounds in the field of drug discovery.
  • Target substance-containing liposome 102 Hydrophilic group part 104 Hydrophobic group part 106 Inner coat 108 Outer coat 110 Target substance molecule 120 Liposome part

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Abstract

　La présente invention porte sur un procédé de mesure d'affinité de liaison entre cible et ligand, et un assistant réactif et une trousse pour utilisation dans ledit procédé. Le procédé de mesure selon la présente invention comprend une étape destinée à mélanger une cible et une substance apte à constituer un agrégat lipidique pour former un agrégat lipidique contenant ladite cible, une étape destinée à amener l'agrégat lipidique contenant la cible conjointement avec un ligand pour préparer un échantillon de mesure, et une étape destinée à mesurer la quantité de variation physique dans l'échantillon de mesure.
PCT/JP2014/071682 2013-08-21 2014-08-19 Procédé de mesure d'affinité de liaison entre cible et ligand et assistant réactif et trousse pour utilisation dans ledit procédé WO2015025856A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2022031835A (ja) * 2013-10-14 2022-02-22 ザ ユニバーシティー コート オブ ザ ユニバーシティー オブ エジンバラ 診断的および治療的使用を有するタンパク質

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008111713A (ja) * 2006-10-30 2008-05-15 Ulvac Japan Ltd 結合素子、バイオセンサ及び生体物質間の相互作用測定方法

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008111713A (ja) * 2006-10-30 2008-05-15 Ulvac Japan Ltd 結合素子、バイオセンサ及び生体物質間の相互作用測定方法

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
DAISUKE MATSUOKA ET AL.: "Difference of Fatty Acid Binding Modes between Wild-type FABP3 and Its Mutants", PROTEIN SCIENCE SOCIETY OF JAPAN, vol. 13 TH, 31 May 2013 (2013-05-31), pages 65 *
HIROO SASAKURA ET AL.: "Shibosan Donyu Liposome no Chosei to Bussei", JAPAN OIL CHEMISTS, vol. 41 ST, 19 September 2002 (2002-09-19), pages 172 *
KOHEI OTSUKA ET AL.: "Suiyosei Shibosan Mimic no Bunshi Sekkei to FABP Ketsugo Kassei", CSJ: THE CHEMICAL SOCIETY OF JAPAN KOEN YOKOSHU, vol. 93 RD, no. 3, 8 March 2013 (2013-03-08), pages 973 *
MARIA CARMEN TAN ET AL.: "Interaction kinetics of liposome-incorporated unsaturated fatty acids with fatty acid-binding protein 3 by surface plasmon resonance", BIOORG. MED. CHEM., vol. 22, no. 6, 15 March 2014 (2014-03-15), pages 1804 - 1808, XP028627442, DOI: doi:10.1016/j.bmc.2014.02.001 *
PETER BRECHER ET AL.: "Fatty acid transfer between multilamellar liposomes and fatty acid- binding proteins", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 259, no. 21, pages 13395 - 13401 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2022031835A (ja) * 2013-10-14 2022-02-22 ザ ユニバーシティー コート オブ ザ ユニバーシティー オブ エジンバラ 診断的および治療的使用を有するタンパク質
JP7320585B2 (ja) 2013-10-14 2023-08-03 ザ ユニバーシティー コート オブ ザ ユニバーシティー オブ エジンバラ 診断的および治療的使用を有するタンパク質

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