WO2011060598A1 - Procédé de détermination du coefficient d'absorption relatif de molécules de colorant, d'anticorps, de médicament et de promédicament sur les molécules polypeptidiques - Google Patents

Procédé de détermination du coefficient d'absorption relatif de molécules de colorant, d'anticorps, de médicament et de promédicament sur les molécules polypeptidiques Download PDF

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Publication number
WO2011060598A1
WO2011060598A1 PCT/CN2010/000019 CN2010000019W WO2011060598A1 WO 2011060598 A1 WO2011060598 A1 WO 2011060598A1 CN 2010000019 W CN2010000019 W CN 2010000019W WO 2011060598 A1 WO2011060598 A1 WO 2011060598A1
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molecule
polypeptide
molecules
drug
tested
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PCT/CN2010/000019
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English (en)
Chinese (zh)
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毛晓波
杨延莲
王琛
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国家纳米科学中心
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Publication of WO2011060598A1 publication Critical patent/WO2011060598A1/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
    • G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements

Definitions

  • the invention belongs to the field of biomedical technology, in particular, the invention relates to a method for measuring the relative adsorption constant of a molecule such as a dye, an antibody, a drug and a prodrug on a polypeptide by using a scanning tunneling microscopy (STM) technique. method.
  • STM scanning tunneling microscopy
  • X-ray crystal diffraction technology is now mature, but due to the difficult crystallinity of some important proteins (such as membrane proteins, amyloid), it is difficult to obtain high-quality crystallographic data to study the interaction between drug molecules and proteins. .
  • the binding site of the drug on the target protein is difficult to ensure periodicity, so it is still difficult to analyze the interaction between the drug molecule and the protein.
  • NMR technology can accurately resolve the molecular structure in solution, avoiding the difficulty of crystallization, but the molecular quality that can be resolved is limited.
  • step 4) Normalize the amount of adsorption of the molecule to be tested on the labeled molecule obtained in step 3) into unit 1, and determine the relative adsorption constant of the molecule to be tested on the polypeptide molecule.
  • the preparation method of the assembly comprises the following steps: i) thoroughly mixing the polypeptide molecule with the labeling molecule to form a mixed solution;
  • step i) adding the mixed droplet obtained in the step i) to a conductive substrate such as graphite, a metal substrate such as gold, silver, copper or platinum, and a surface of a semiconductor substrate such as silicon to form an assembly.
  • a conductive substrate such as graphite
  • a metal substrate such as gold, silver, copper or platinum
  • a surface of a semiconductor substrate such as silicon
  • the polypeptide molecule forms an assembly with the labeling molecule, and the scanning tunneling microscope image of the assembly obtained by removing the solvent at the solid/gas interface can be removed, and the scanning tunneling microscope image of the solvent obtained at the substrate/solution interface can be retained.
  • the preparation method of the assembly comprises the following steps: i) thoroughly mixing the polypeptide molecule, the labeling molecule and the molecule to be tested to form a mixed solution;
  • step i) adding the mixed droplet obtained in the step i) to a conductive substrate such as graphite, a metal substrate such as gold, silver, copper or platinum, and a surface of a semiconductor substrate such as silicon to form an assembly.
  • a conductive substrate such as graphite
  • a metal substrate such as gold, silver, copper or platinum
  • a surface of a semiconductor substrate such as silicon
  • the polypeptide molecule, the labeling molecule and the molecule to be tested form an assembly, and the scanning tunneling microscope image of the assembly obtained by removing the solvent at the solid/gas interface can be removed, and the scanning tunneling microscope image of the solvent obtained at the substrate/solution interface can be retained.
  • ultrasound is used for thorough mixing.
  • the conductive substrate is graphite
  • the graphite is preferably a new cleavage highly oriented graphite.
  • Highly oriented graphite has an atomically flat surface and is stable in many environments, making it suitable for scanning tunneling microscopy studies.
  • the step of removing the residual liquid on the surface of the substrate is further included, and preferably, the residual liquid on the surface of the substrate may be removed by blowing an inert gas such as nitrogen.
  • the scanning tunneling microscope image in which the solvent is obtained at the solid/gas interface can be removed, and the scanning tunneling microscope image in which the solvent obtains the assembly at the substrate/solution interface can be retained.
  • the molecule to be tested is a dye, an antibody, a drug or a prodrug molecule or the like.
  • the dye is a phthalocyanine, a porphyrin molecule and a derivative thereof, Congo red, sulphonin, curcumin and derivatives thereof;
  • the antibody is an amyloid polypeptide molecule antibody;
  • the drug is pyridine, a nitrogen heterocyclic molecule such as pyrimidine, pyrazine, imidazole, pyrrole or the like, and derivatives thereof, such as 4'4-bipyridine, vinylpyridine, tripyridine, Congo red, curcumin;
  • the prodrug is sulfonamide .
  • the polypeptide molecule is pentaalanine, octaphenylalanine and octameric histidine.
  • Ben The invention is exemplified by pentaalanine, octaphenylalanine and octameric histidine, and other polypeptides such as beta amyloid and fragments thereof, amylin polypeptide and fragments thereof, prion fragment polypeptide, oligomerization may also be used.
  • the labeling molecule is pyridine, pyrimidine, pyrazine, imidazole, pyrrole nitrogen heterocyclic molecule and derivatives thereof, such as 4,4-bipyridine, vinylpyridine, tripyridine, pyrimidine, etc., preferably,
  • the standard " ⁇ has a 4,4-bipyridine.
  • the object of the present invention is to develop a new method based on scanning tunneling microscopy to study the interaction of proteins or polypeptides with small molecules at the molecular level, and to determine dyes, antibodies, drugs and prodrugs waiting for molecules.
  • the relative adsorption constant on the polypeptide is to develop a new method based on scanning tunneling microscopy to study the interaction of proteins or polypeptides with small molecules at the molecular level, and to determine dyes, antibodies, drugs and prodrugs waiting for molecules.
  • a method for measuring the relative adsorption constants of dyes, antibodies, drugs and prodrugs on a polypeptide by scanning tunneling microscopy obtaining an STM image of a self-assembled structure of a polypeptide molecule, or obtaining a polypeptide by co-adsorption with a labeled molecule - After labeling the STM image of the co-assembled structure of the molecule, adding dyes, antibodies, drugs and prodrugs to the test molecule, obtaining the binding site and the amount of adsorption of the molecule to be tested on the polypeptide, and studying the polypeptide at the molecular level The interaction of the molecules is measured, and the relative adsorption constant of the molecule to be tested on the polypeptide is determined.
  • One embodiment of the present invention determines the relative adsorption constants of dyes, antibodies, drugs, and prodrugs on a polypeptide by identifying the molecular assembly characteristics of the polypeptide, including the following steps:
  • Scanning tunneling 4 mirrors are used to measure the binding sites and adsorption quantities of dyes, antibodies, drugs or prodrugs in the high-resolution scanning tunnel fluoroscopy image on the peptide molecules, and are adsorbed. The relative relationship of constants.
  • the invention utilizes a scanning tunneling microscope to measure the relative adsorption constant of a molecule such as a dye, an antibody, a drug or a prodrug on a polypeptide, and can clearly recognize the directionality of the two-dimensional assembly of the polypeptide molecule, and on the basis of this, by adding a dye
  • the antibody, the drug or the prodrug molecule obtains the binding site and the amount of adsorption of the molecule on the polypeptide, and thereby obtains the relative adsorption capacity of the different polypeptides to the molecule, that is, the relative adsorption constant of the molecule on the polypeptide.
  • the invention utilizes scanning tunneling microscopy technology to study the interaction between drug molecules and protein molecules at the molecular level, the action sites and adsorption capacities of drug molecules on protein molecules, and are not affected by protein crystallinity, and are not bound by The effect of signal strength.
  • the invention adopts a polypeptide molecule and a labeling molecule to form an assembly, or a polypeptide molecule, a labeling molecule and a molecule to be tested to form an assembly, which can remove the scanning tunneling microscope image of the solvent at the solid/gas interface, and can also retain the solvent on the substrate. / Solution interface to obtain a scanned tunneling microscope image of the assembly.
  • the highly oriented graphite used in the present invention has an atomically level surface and is stable in many environments, and is suitable for scanning tunneling microscope research.
  • the present invention is of great importance for analysing the pattern and binding nature of target protein interactions with drugs.
  • the present invention can be used for the determination of targets in the early research stage of new drugs, the adsorption capacity of different sequence proteins for drug molecules, and the like. Especially in the treatment of neurodegenerative diseases, study the aggregation mechanism of amyloid and its interaction mechanism with drug molecules, use molecular level evidence to find possible drug targets, and thus find and design effective regulator molecules, Drug molecules, etc. provide guidance.
  • DRAWINGS
  • Figure 1 is a co-assembly of three model peptides, pentaalanine (5Ala), octaphenylalanine (8Phe), and octameric histidine (8His) and 4,4,-bipyridine molecule (4Bpy). Scanning tunneling microscope image;
  • Figure 2 is a scanning tunneling microscope image of the adsorption of dye molecules sulfonic acid phthalocyanine on three polypeptide-labeled molecular assemblies, wherein A and B are represented in the 5Ala-4Bpy assembly, and C and D are represented in the 8Phe-4Bpy assembly. In the body, E is expressed in the 8His-4Bpy assembly;
  • Figure 3 is a graph showing the amount of dye molecular sulfonic acid phthalocyanine adsorbed in three assemblies from the scanning tunneling microscope images of Figures 1 and 2;
  • Figure 4 is a graph showing the different adsorption capacities of the three polypeptide assemblies for the dye molecule sulfo Stt phthalocyanine, i.e., the relative adsorption constant of the dye molecules on the polypeptide molecule.
  • the adsorption ability of the 8Phe polypeptide molecule to the dye molecule is normalized to 1, the adsorption capacity of the 8His and 5Ala polypeptide molecules for the dye molecule is 10.2 and 41.8, respectively;
  • Figure 5 is a scanning tunneling microscope image of the drug molecule Congo red adsorbed on the 5 Ala-4Bpy assembly
  • Figure 6 is a graph showing the amount of Congo red adsorbed on the polypeptide and 4Bpy from the scanning tunneling microscope image of Figure 5;
  • Figure 7 is a scanning tunneling microscope image of the adsorption of the prodrug molecule ThT on the 5Ala-4Bpy assembly. The best way to implement the invention
  • Example 1 Determination of Relative Adsorption Constants of Dye Molecules on Polypeptides Based on Scanning Tunneling Microscopy
  • Phthalocyanine-tetrasulfonic 2 the method of determination
  • the polypeptide molecules (5Ala, 8Phe and 8His) and the pyridine-based labeling molecule 4Bpy were first mixed in an aqueous solution, sonicated for 10 minutes, and after thorough mixing, 15 ⁇ l of the solution was taken out, dropped onto the surface of the newly cleaved graphite, and allowed to stand for 10 minutes. After the mixed molecular system is formed into an assembly on graphite and deposited on the surface, it is blown with high purity nitrogen gas.
  • the dye molecule sulfonic acid phthalocyanine was added to the above mixed solution (that is, a mixed aqueous solution of a polypeptide molecule (5 Ala, 8Phe and 8His) and a pyridine-based labeling molecule 4Bpy) for 10 minutes, and after thoroughly mixing, 15 ⁇ L of the solution was taken out. , drip onto the surface of the new cleavage graphite, let stand for 10 minutes, and blow dry with high purity nitrogen.
  • the amount of adsorption of the sulfonic acid phthalocyanine at different adsorption sites that is, the amount of adsorption on the three polypeptide molecules (5 Ala, 8Phe and 8His) and 4Bpy (as shown in Fig. 3).
  • the number of adsorbed molecules of the dye molecules on the polypeptide and 4Bpy can be separately counted.
  • the amount of dye molecules adsorbed on 4Bpy is returned.
  • the relative relationship of the adsorption capacity of the cross-acid phthalocyanine on different polypeptides is obtained.
  • the adsorption capacity of the dye molecule on 8Phe is the basic unit 1
  • the adsorption capacity of the sulfonic acid phthalocyanine in 8His is 10.2 times that of 8Phe
  • the adsorption capacity of the sulfonic acid phthalocyanine in 5Ala is 41.8 times that of 8Phe, as shown in the figure. 4 is shown.
  • Example 2 Determination of Relative Adsorption Constants of Drug Molecules on Polypeptides Based on Scanning Tunneling Microscopy
  • the measurement method is the same as that in the first embodiment.
  • the measurement method was the same as in Example 1.
  • a high-resolution STM image of the drug precursor molecule ThT adsorbed in the 5 Ala and 4Bpy two-component assembly system was obtained by scanning tunneling fluoroscopy (as shown in Fig. 7).
  • ThT does not adsorb on 4Bpy, the relative adsorption constant is extremely large.

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Abstract

La présente invention concerne un procédé de détermination du coefficient d'absorption relatif d'une molécule à déterminer sur les molécules polypeptidiques au moyen de la microscopie à effet tunnel, la molécule à déterminer étant une molécule de colorant, d'anticorps, de médicament, de promédicament et équivalents. Le procédé comprend les procédures suivantes : obtention par microscopie à effet tunnel (STM) d'une image d'une structure d'autoassemblage de molécule polypeptidique ou obtention par STM de l'image d'une structure d'assemblage synchrone de molécules marquées par une molécule peptidique par absorption synchrone avec une molécule marquée, puis ajout de la molécule à déterminer telle qu'une molécule de colorant, d'anticorps, de médicament, de promédicament et équivalents pour obtenir les emplacements de liaison et les quantités d'absorption de la molécule à déterminer sur la molécule peptidique, étude de l'interaction entre la molécule polypeptidique et la molécule à déterminer au niveau moléculaire et détermination du coefficient d'absorption relatif d'une molécule à déterminer sur la molécule polypeptidique. Le procédé est très important pour analyser le mode d'interaction et l'essence de liaison d'une protéine cible et d'un médicament.
PCT/CN2010/000019 2009-11-17 2010-01-05 Procédé de détermination du coefficient d'absorption relatif de molécules de colorant, d'anticorps, de médicament et de promédicament sur les molécules polypeptidiques WO2011060598A1 (fr)

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CN200910238164.7 2009-11-17
CN2009102381647A CN102062718B (zh) 2009-11-17 2009-11-17 测定染料、抗体、药物和药物前体分子在多肽分子上相对吸附常数的方法

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CN103536897B (zh) * 2012-07-16 2016-03-09 国家纳米科学中心 抑制淀粉样多肽聚集的复合物及其制备方法和应用
CN105826170B (zh) * 2016-04-20 2018-04-06 中国科学院新疆理化技术研究所 一种在石墨基底上构筑金属有机框架薄膜的方法
CN111292812B (zh) * 2020-01-21 2021-03-05 中国医学科学院基础医学研究所 一种研究蛋白质及药物-蛋白质复合体构象多态性的方法

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1468374A (zh) * 2000-10-09 2004-01-14 拜澳富斯毫微科学有限公司 通过力分层和力扫视评价结合力

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CA2491310C (fr) * 2002-07-15 2015-10-06 Board Of Regents, The University Of Texas System Compositions comprenant des derives de la duramycine impermeants vis-a-vis des parois cellulaires
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1468374A (zh) * 2000-10-09 2004-01-14 拜澳富斯毫微科学有限公司 通过力分层和力扫视评价结合力

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
LEANNA, GIANCARLO ET AL.: "Scanning Tunneling Microscopy of Molecular Adsorbates at the Liquid-Solid Interface: Functional Group Variations in Image Contrast", LANGMUIR, vol. 14, 22 January 1998 (1998-01-22), pages 1465 - 1471 *
LEI, SHENGBIN ET AL.: "Site Selective Adsorption and Templated Assembling: Effects of Organic-Organic Heterogeneous Interface Studied by Scanning Tunneling Microscopy", J.PHYS.CHEM.B, vol. 108, 30 December 2003 (2003-12-30), pages 1173 - 1175 *
MA, XIAOJING ET AL.: "Amyloid (1-42) Folding Multiplicity and Single-Molecular Binding Behavior Studied with STM", J.MOL.BIOL., vol. 388, 25 March 2009 (2009-03-25), pages 894 - 901, XP026085435, DOI: doi:10.1016/j.jmb.2009.03.054 *
MAO, XIAOBO ET AL.: "Molecular-Level Evidence of the Surface-Induced Transformation of Peptide Structures Revealed by Scanning Tunneling Microscopy", LANGMUIR, vol. 25, no. 16, 22 July 2009 (2009-07-22), pages 8849 - 8853 *

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