WO2011060598A1

WO2011060598A1 - Method for determining relative absorption coefficient of dye, antibody, drug and prod rug molecules on the polypeptide molecules

Info

Publication number: WO2011060598A1
Application number: PCT/CN2010/000019
Authority: WO
Inventors: 毛晓波; 杨延莲; 王琛
Original assignee: 国家纳米科学中心
Priority date: 2009-11-17
Filing date: 2010-01-05
Publication date: 2011-05-26
Also published as: CN102062718B; CN102062718A

Abstract

Disclosed is a method for determining the relative absorption coefficient of a molecule to be determined on the polypeptide molecules by means of scanning tunneling microscopic technology, the molecule to be determined is dye, antibody, drug, prodrug molecule and the like. The method comprises the following procedures: obtaining scanning tunneling microscopy (STM) image of a polypeptide molecule self-assembly structure or obtaining the STM image of a polypeptide molecule-tagged molecule synchronous-assembly structure by synchronous absorption with tagged molecule, and thereafter adding the molecule to be determined such as dye, antibody, drug, prodrug molecule and the like so as to obtain the binding locations and absorption quantities of the molecule to be determined on polypeptide molecule, studying the interaction between polypeptide molecule and the molecule to be determined at the molecule level and determining the relative absorption coefficient of a molecule to be determined on the polypeptide molecule. The method is very important to analyze interaction mode and binding essence of a target protein and a drug.

Description

Determination of dyes, antibodies, drugs and prodrug molecules on polypeptide molecules

Method for relative adsorption constant

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. Background technique

In drug design and development studies, it is extremely important to analyze the pattern and binding nature of target protein interactions with drugs. Generally, high-throughput protein crystallization and high-resolution X-ray crystal diffraction techniques, two-dimensional nuclear magnetic resonance technology can be used. Conduct research (see Daniel Picot, Patrick J. Loll et al., Nature 1994, 367, 243. and Aneta T. Petkova, Yoshitaka Ishii et al, Proc. Natl. Acad. Sci. USA 2002, 99, 16742.) .

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. . In addition, even if the co-crystallization of the protein and the drug molecule is obtained, 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.

There are also a large number of studies based on supercomputers that perform molecular simulations and visualizations to simulate the dynamic processes of interactions between proteins and proteins or proteins and small molecules, to analyze the structural basis of drug action, and to design new drug molecules. However, there are some inevitable gaps between computer simulation and experimental research. Therefore, it is urgent to develop experimental research methods to obtain the action sites and visual images of the interaction of drug molecules with proteins or polypeptides, and to study the adsorption capacity of drug molecules on proteins or polypeptides on a molecular scale. The present inventors have developed a new method for the determination of the relative adsorption constants of dyes, antibodies, drugs and prodrugs on a polypeptide using scanning tunneling microscopy. Summary of the invention

It is an object of the present invention to provide a method for determining the relative adsorption constant of a molecule to be tested on a polypeptide molecule, the method comprising the steps of:

1) assembling a polypeptide molecule and a labeling molecule to obtain a scanning tunneling microscope image of an assembly of the polypeptide molecule and the labeling molecule;

2) assembling the polypeptide molecule, the labeling molecule and the molecule to be tested to form an assembly to obtain a scanning tunneling microscope image of the molecule to be tested adsorbed on the polypeptide molecule and the labeled molecule;

3) comparing the scanning tunneling microscope images obtained in steps 1) and 2), and counting the adsorption amount of the molecules to be adsorbed on the polypeptide molecules and the labeled molecules;

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.

Preferably, in the step 1), 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;

Ii) 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.

Wherein, 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.

Preferably, in the step 2), 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;

Wherein, 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.

Preferably, in the preparation assembly, ultrasound is used for thorough mixing.

Preferably, in the preparation assembly, the conductive substrate is graphite, and 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.

Preferably, in the preparation assembly, 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.

Among them, 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.

Preferably, the molecule to be tested is a dye, an antibody, a drug or a prodrug molecule or the like.

Preferably, 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 .

Preferably, 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. Polypeptides, block oligomeric polypeptides, artificial sequence polypeptides, and the like.

Preferably, 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.

In summary, 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.

In 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:

1) preparing a solution of the polypeptide molecule (or blended with the labeled molecule), sonicating for 10 minutes;

2) in a solution of the polypeptide molecule, or a mixed solution of the polypeptide molecule and the labeled molecule, adding a dye, an antibody, a drug or a prodrug to wait for the molecular solution to be thoroughly mixed;

3) After mixing, 15 μl of the solution was dropped on the surface of the newly cleaved high-oriented graphite and allowed to stand for 10 minutes;

4) blowing off the solution remaining on the graphite surface with high purity nitrogen;

5) 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 has at least the following beneficial effects:

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. When 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

The present invention will be further described in detail below with reference to the drawings and specific embodiments. Example 1: Determination of Relative Adsorption Constants of Dye Molecules on Polypeptides Based on Scanning Tunneling Microscopy

1. The chemical structure of the substance used

The chemical structures of the peptide molecules (5Ala, 8Phe and 8His), the pyridine molecule (4Bpy) and the dye molecule sulfo Stt phthalocyanine are shown in the figure below:

Ώ H H Ώ H H

H ₂ N-CHC-N-CHC-N-CHC-N-CHC-N-CHC-OH

CH3 CH3 CH3 CH3 CH3

Pentameric alanine (5Ala)

H -

Octa-phenylalanine (8Phe)

OctaHis (8His)

- (: ■ ', N

4'4-bipyridyl ¹ ^ ( 4'4-bipyridyl, 4Bpy )

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 μl 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.

Commercially available multi-mode scanning probe microscopy (SPM, Nanoscope Ilia type, Veeco, USA), experimental conditions for atmospheric constant current mode, three polypeptides and 4Bpy two-component system were scanned separately to obtain high-resolution STM The image (shown in Figure 1) is used to compare the images after dye molecule addition and the amount of dye molecule adsorption.

Further, 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 μL 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.

High-resolution STM images of citrate-based phthalocyanine adsorbed in three polypeptide molecules (5Ala, 8Phe and 8His) and 4Bpy two-component assembly system were obtained by scanning tunneling microscopy (Fig. 2).

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).

In the scanning tunneling microscope image of Fig. 2, the number of adsorbed molecules of the dye molecules on the polypeptide and 4Bpy can be separately counted. Considering the adsorption capacity of the sulfonic acid phthalocyanine on 4Bpy, the amount of dye molecules adsorbed on 4Bpy is returned. As a result, 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, and 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

1. The chemical structure of the substance used

The chemical structure of the polypeptide molecule pentaalanine (5Ala), the pyridine-labeled molecule (4Bpy) and the drug molecule Congo red is shown in the following figure: H ί? Η Ώ Η Η ί?

H ₂ N-CHC-N-CHC-N-CHC-N-CHC-N-CHC-OH

CH ₃ CH ₃ CH ₃ CH ₃ CH ₃ pentaalanine (5Ala )

4,4-bipyridyl (4Bpy)

Congo red

2. The measurement method is the same as that in the first embodiment.

A high-resolution STM image of Congo red adsorbed in the 5Ala and 4Bpy two-component assembly system was obtained by scanning tunneling microscopy (shown in Figure 5). By comparing the amount of adsorption on Congo red peptides and 4Bpy, the relative adsorption capacity of Congo red on the peptide and labeling molecule 4Bpy is provided (as shown in Figure 6). Example 3: Determination of Relative Adsorption Constants of Prodrug Molecules on Polypeptides Based on Scanning Tunneling Microscopy

1. The chemical structure of the substance used

The chemical structure of the polypeptide molecule pentaalanine (5Ala), the pyridine-labeled molecule (4Bpy) and the prodrug molecule Thioflavin T (ThT), as shown below:

Pentameric alanine (5Ala) ―... i...― \

\::::::::/

4'4-bipyridyl (4Bpy)

Thioflavin T ( Thioflavin T, 2, the method of determination

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). By counting the amount of ThT adsorbed on the peptide and 4Bpy, it provides a basis for the relative adsorption capacity of ThT on the polypeptide and the labeled molecule 4Bpy. Since ThT does not adsorb on 4Bpy, the relative adsorption constant is extremely large.

Claims

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A method for determining the relative adsorption constant of a molecule to be tested on a polypeptide molecule, the method comprising the steps of:

1) assembling a polypeptide molecule and a labeling molecule to obtain a scanning tunneling mirror image of an assembly of the polypeptide molecule and the labeling molecule;

2) assembling a polypeptide molecule, a labeling molecule and a molecule to be tested to form an assembly, to obtain a scanning tunneling microscope image of the molecule to be tested adsorbed on the polypeptide molecule and the labeling molecule;

2. The method according to claim 1, wherein in the step 1), the method for preparing the assembly comprises the following steps:

i) thoroughly mixing the polypeptide molecule with the labeling molecule to form a mixed solution;

3. The method according to claim 1, wherein in the step 2), the method for preparing 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;

4. Method according to claim 2 or 3, characterized in that in said step i) ultrasound is used for thorough mixing.

The method according to claim 2 or 3, wherein in the step ii), the conductive substrate is graphite, and the graphite is preferably freshly cleaved high-oriented graphite.

The method according to claim 2 or 3, wherein in the step ii), the step of removing the residual liquid on the surface of the substrate, preferably using a blowing inert gas such as nitrogen The residual liquid on the surface of the substrate is removed in a gaseous manner.

7. The method according to claim 1, wherein the molecule to be tested is a dye, an antibody, a drug or a prodrug molecule.

8. The method according to claim 7, wherein the dye is a phthalocyanine, a porphyrin molecule and a derivative thereof, Congo red, sulphonin, curcumin and derivatives thereof; the antibody is a starch a polypeptide molecule; the drug is a nitrogen heterocyclic molecule such as pyridine, pyrimidine, pyrazine, imidazole or pyrrole and a derivative thereof, such as 4,4-bipyridine, vinylpyridine, tripyridine, Congo red, curcumin; The prodrug is flavin.

9. The method according to claim 1, wherein the polypeptide molecule comprises pentameric alanine, octapolyphenylalanine, octameric histidine, beta amyloid polypeptide and fragment thereof, and amylin polypeptide. And a fragment thereof, a prion fragment polypeptide, an oligomeric polypeptide, a block oligomeric polypeptide, and an artificial sequence polypeptide, preferably, the polypeptide molecule is pentaalanine, octaphenylalanine, and octameric histidine.

10. The method according to claim 1, wherein the labeling molecule is a pyridine, a pyrimidine, a pyrazine, an imidazole, a pyrrole nitrogen heterocyclic molecule and a derivative thereof, such as 4,4-bipyridine or vinylpyridine. , tripyridine, pyrimidine, etc., preferably, the labeling molecule is 4,4-bipyridine.