CN110791543A - Method for identifying action target of natural product medicine - Google Patents

Method for identifying action target of natural product medicine Download PDF

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CN110791543A
CN110791543A CN201910942137.1A CN201910942137A CN110791543A CN 110791543 A CN110791543 A CN 110791543A CN 201910942137 A CN201910942137 A CN 201910942137A CN 110791543 A CN110791543 A CN 110791543A
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drug
protein
sample
detected
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李静
戚欣
买小圆
唐薇
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Ocean University of China
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Abstract

The invention provides a method for identifying a natural product drug action target. The invention comprises the following steps: 1) adding the drug to be detected and the control solvent into the effective cells respectively, and incubating together; 2) placing at 30-80 deg.C, heat treating for 3-10min, cracking, centrifuging to obtain supernatant; 3) electrophoresis and transfer printing; 4) respectively staining by using broad-spectrum protein antibodies or dyes, comparing, and searching for bands with protein stability differences; 5) and carrying out electrophoresis on the drug protein sample to be detected, and carrying out mass spectrometry on the bands with protein stability difference and verifying to obtain a target spot. The invention relates to a natural product drug action target recognition method based on protein thermal stability and broad spectrum molecular dyeing, which can efficiently recognize potential drug targets at low cost in a label-free manner, the dosage of natural products is small, and the discovery of the potential drug targets can not only promote the research of disease action mechanisms and pharmacology, but also provide guidance information for the potential side effects of drugs and the commercialization of drugs.

Description

Method for identifying action target of natural product medicine
Technical Field
The invention relates to the technical field of drug target identification, in particular to a method for identifying a natural product drug action target.
Background
The natural product refers to components or metabolites thereof in animals, plant extracts (plant extracts for short) or insects, marine organisms and microorganisms, and a plurality of endogenous chemical components in human bodies and animals, and mainly comprises proteins, polypeptides, amino acids, nucleic acids, various enzymes, monosaccharides, oligosaccharides, polysaccharides, glycoproteins, resins, colloidal substances, lignin, vitamins, fats, oils, waxes, alkaloids, volatile oils, flavones, glycosides, terpenes, phenylpropanoids, organic acids, phenols, quinones, lactones, steroids, tannins, antibiotics and other naturally-occurring chemical components. Natural products with unique chemical properties and a wide range of biological activities often have novel modes of action that are hard to imagine when designing synthetic libraries, and become an important source of novel drug leads. From 1981 to 2014, 33% of all approved natural product drugs are natural products or their direct semisynthetic derivatives. Due to the great difference of ecological environment, the chemical structure and biological activity of marine natural products are richer than those of terrestrial organisms, and the marine natural products are an important source of marine medicines. By the end of 2012, about 2.5 million marine natural products have been found throughout the world. Since 2007, new marine natural products are published in average quantities over 1000 years, and 50% of these marine natural products have been found to have various biological activities. More encouraging, marine natural products with an ever-new framework have been reported.
The early definition of the action target of the new drug or the active compound is beneficial to carrying out optimization and reconstruction aiming at the target, establishing a proper drug evaluation model and searching corresponding indication patients, improving the success rate of drug research and development and promoting the discovery and innovation of the new drug. The natural product, particularly the marine natural product, has the characteristics of difficult acquisition, low yield, complex structure, difficult synthesis and marking and diverse activity, and due to the diversity of the structure and the activity, the search of the action target of the natural product is one of the main bottlenecks of the research and development of new drugs, thereby greatly limiting the druggability of the natural product in China. At present, no very effective natural product target discovery method exists. The most reported methods are chemical biological methods, including affinity purification methods: the natural product can be directly linked with different mediums such as agarose beads through covalent linkage, or the natural product and biotin can be coupled through chemical reaction. The method is suitable for natural product compounds with higher activity, and the greatest disadvantage of the method is that the natural product needs to be modified, and the activity of the natural product can be damaged. The application of technologies such as light coupling and the like also increases the probability of target discovery, and many natural products are difficult to synthesize completely from the beginning, so that the application of the method is limited. The above methods also require a relatively large amount of natural products, and trace extraction of natural products does not meet the requirements of the above methods. In addition, the reported methods also include some basic biological methods, such as genetic screening method, transcriptome, phage display, yeast triple hybrid, proteomics, but these methods have difficulty in achieving wide screening of unknown target proteins. The new method reported at present is based on the activity-based protein mass spectrometry (ABPP) and the target stability of Drug Affinity Reaction (DARTS), which both greatly increase the opportunity of target identification. However, the broad-spectrum application of ABPP is greatly limited by the characteristics of being only suitable for a target with catalytic activity, covalent modification and the like; DARTS has the dependence of conformational change and the defect of being incapable of identifying low-abundance targets.
Disclosure of Invention
The invention aims to provide a method for identifying a natural product drug action target, which solves the problems that the prior art has the dependence of conformational change and can not identify low-abundance targets, and the like, so that the method can not be applied to the identification of the natural product drug action target.
In order to solve the technical problem, the technical scheme of the invention is realized as follows: the method comprises the following steps:
1) collecting a sample, and dissolving a drug to be detected in a solvent to obtain an experimental sample; taking another solvent as a control sample; respectively adding the experimental sample and the control sample into the effective cells, and incubating for 1-5h to respectively obtain a drug group sample to be detected and a solvent control group sample;
2) performing heat treatment, namely placing the drug group sample to be detected and the solvent control group sample at 30-80 ℃, performing heat treatment for 3-10min, cracking, centrifuging, and taking supernate to respectively obtain a drug protein sample to be detected and a solvent control protein sample;
3) performing electrophoresis and transfer printing, namely performing electrophoresis and transfer printing on the drug protein sample to be detected and the solvent control protein sample to obtain a drug transfer printing group to be detected and a solvent control transfer printing group;
4) dyeing, namely dyeing the drug transfer printing group to be detected and the solvent control transfer printing group by adopting a broad-spectrum protein antibody or dye, comparing, and searching for bands with protein stability differences;
5) and (3) performing mass spectrometry, namely performing electrophoresis on another drug protein sample to be detected, performing mass spectrometry on the bands with protein stability difference, and verifying to obtain a target point.
The method comprises the steps of incubating a solvent control drug and a drug to be detected with effective cells respectively to obtain suspension cells, wherein digestion is needed if adherent cells exist, and digestion is not needed if the adherent cells exist; then heat treating the cells, treating for different time at different temperature, cracking, finally performing electrophoresis and transfer printing, dyeing by using broad-spectrum molecular dye and antibody, searching for different protein band positions, cutting off adhesive tapes from corresponding positions, and performing mass spectrometry to search for action targets of the drugs. The invention relates to a natural product drug action target spot recognition method based on protein heat stability and broad spectrum molecular dyeing, which comprises an active natural product, wherein the natural product does not need to be marked in advance, and the natural product has small dosage and trace dosage, and the dosage is less than 2 mg; the protein target of the invention comprises intracellular low-abundance target molecules, has the characteristic of widely searching natural product targets, can efficiently identify potential drug targets at low cost in a label-free manner, and can promote the research of disease action mechanism and pharmacology and provide guidance information for the potential side effect of the drug and the commercialization of the drug when finding the potential drug targets. The effective cells of the invention refer to those cells which are found by activity screening in advance and have phenotype change after the action of the drug to be detected, the screening methods are the prior art, and different screening methods are available for different diseases; the method for identifying the action target of the natural product medicament is not only suitable for identifying the target of natural product active compounds and medicaments, but also comprises the target identification of active compounds and chemical medicaments from other sources.
As a preferred embodiment, in the step 4), the specific operation of dyeing is: a) preparing 5% solution of skimmed milk powder with 1 × TBST buffer solution; b) taking a drug transfer group to be detected and a solvent control transfer group, adding the solution obtained in the step a), and incubating for 1-2h at room temperature on a shaking table; c) washing with 1 × TBST buffer solution, adding protein antibody or dye, standing at 4 deg.C, and incubating overnight; d) coating an ECL luminescent reagent or a dye substrate under the condition of keeping out of the sun, and developing by adopting an imaging system; e) comparing the transfer printing group of the drug to be tested with the solvent control transfer printing group, searching for a strip with protein stability difference, and recording the molecular weight. The invention adopts the skim milk powder as the milk to distinguish specific cells from non-specific cells, and adopts the broad-spectrum protein antibody or dye to dye, so the applicability is wide, the operation is simple, the development is obvious, and the experimental effect is good; in the present invention, ECL luminescent reagent is coated for development when protein antibody is used, and dye substrate is coated for development when dye is used. TBST buffer solution is an isotonic buffer salt solution commonly used in biology, is mainly used for washing reagents such as antibodies and the like which are not specifically bound on a membrane in a Western Blot experiment, and is a ready-to-use 1 XPBST buffer solution which can be directly used. ECL chemiluminescent reagents are a new generation of Luminol-based chemiluminescent substrate reagents that are catalyzed by horseradish peroxidase (HRP) to undergo a chemical reaction that fluoresces and the result can be revealed by X-ray film sheeting and other imaging techniques or detected using a Luminometer.
In a preferred embodiment, the protein antibody is any one of a tyrosine protein kinase antibody, a serine/threonine protein kinase antibody silver stain, and biotin-labeled lectin. The protein antibody has wide application range, convenient use and good effect. The search for target proteins on cell membranes of the invention is preferably a biotin-labeled lectin antibody, of which ricin lectin RCA I is preferred.
As a preferred embodiment, in step c), when adding the protein antibody, the protein antibody is a primary antibody, and the primary antibody further comprises the following operations after incubating overnight: taking a secondary antibody, and diluting the secondary antibody by 2000-fold with 1 xTBST buffer solution; and (3) washing the transfer printing group of the drug to be detected and the solvent control transfer printing group by adopting a 1 xTBST buffer solution, adding the diluted secondary antibody into the washed transfer printing group of the drug to be detected and the solvent control transfer printing group in a shaking table at room temperature within 1-2h, slowly shaking at room temperature, and incubating for 1-2 h. When the protein antibody is selected, primary antibody is used for incubation, and then secondary antibody is used for incubation.
As a preferred embodiment, in the step 3), the specific operation of electrophoresis is: A) taking SDS-PAGE gel, wherein the SDS-PAGE gel comprises 3-5% concentrated gel and 8-12 separating gel, coating and forming pores; B) adding 8-10 mu L of a drug protein sample to be detected or a solvent control protein sample into each hole, and adding protein pre-staining Marker into the side holes; C) placing in ice water bath, performing electrophoresis at 60-80V for 20-30min, after the protein pre-staining Marker is completely separated, increasing the voltage to 120-130V, and continuing electrophoresis. The invention adopts polyacrylamide gel electrophoresis, wherein, SDS-PAGE gel is the existing widely used gel; the protein pre-staining Marker is a reagent and can be directly purchased and used. The electrophoresis operation method is simple and has good experimental effect. In the present invention, the transfer is preferably carried out by transferring the charged protein onto a Nitrocellulose (NC) film by a semi-dry transfer method, and the transfer time is about 1 to 2 hours depending on the desired molecular weight.
As a preferred embodiment, in the step 2), the specific operation of the heat treatment is: s1 enriched cells: washing the drug group sample to be detected and the solvent control group sample to respectively obtain a drug group cell to be detected and a solvent control group cell; s2 gradient heating: respectively heating the cells of the drug group to be detected and the cells of the solvent control group in a gradient manner to 30-80 ℃ to obtain cell suspension of the drug group to be detected and cell suspension of the solvent control group; s3 cell lysis: cracking the cell suspension of the drug group to be detected and the cell suspension of the solvent control group, centrifuging and collecting supernatant; s4 sample collection: adding equal volume of Loading Buffer into the supernatant respectively, resuspending, decocting in boiling water for 15-20min, standing at-20 deg.C, and storing. The invention adopts a gradient heating method for heat treatment, and the Loading Buffer is a sample Loading Buffer solution and mainly has two functions, namely, the indicator bromophenol blue and the xylene cyanide FF play a role in indicating and display the progress of electrophoresis so as to stop the electrophoresis at the right time; second, the component glycerol increases the density of the sample to a level greater than the TAE, thereby settling into the wells and preventing the sample from drifting out of the wells.
In a preferred embodiment, the lysis is any one of repeated freeze thawing with liquid nitrogen, lysis with cell lysate and ultrasonic disruption lysis. The cracking method of the invention has various cracking modes, wherein when liquid nitrogen is adopted for repeated freeze thawing cracking, repeated freeze thawing is usually required for three times; of course, lysis with cell lysates or sonication can also be used.
In a preferred embodiment, in step S2, the cells of the drug to be tested and the cells of the solvent control group are heated to 40-60 ℃ in a gradient manner. In the heat treatment process of the present invention, the temperature may preferably be 40-60 ℃, and most proteins are unstable in this temperature range, while Tubulin as an internal reference may still be detected.
As a preferred embodiment, in step 1), the specific operation of sample collection is: sa cell plating: digesting and counting effective cells in good growth state and logarithmic growth phase with pancreatin, inoculating, and inoculating at 37 deg.C and 5% CO2Culturing for 24h under the condition; adding Sb cells: setting the experimental sample and the control sample, respectively, and adding the effective cells cultured in step Sa at 37 deg.C and 5% CO2Continuously culturing for 1-3h under the condition; sc-collected cells: removing the culture medium, adding cold PBS, washing, adding pancreatin for digestion, and uniformly blowing to make all adherent cells suspended. The sample collection is obtained by adding medicine into the cell seed plate and the cells and collecting the cells, only the adherent cells are digested in the digestion process, and the non-adherent cells do not need to be digested; the cold PBS is a precooled PBS buffer solution, and the PBS buffer solution is phosphate buffer saline (phosphate buffer saline) which is generally used as a solvent and plays a role in dissolving a protective reagent; it is a buffer solution which is most widely used in biochemical research, and the main component of the buffer solution is Na2HPO4、KH2PO4NaCl and KCl.
As a preferred embodiment, in the step 5), the specific operation of mass spectrometry is: and (3) taking a drug protein sample to be detected, carrying out electrophoresis according to the step 3), wherein the sample loading amount during electrophoresis is 20-30 mu L, cutting off adhesive strips near the difference, rinsing with ultrapure water, and carrying out mass spectrometry. The invention obtains the protein with the molecular weight of the protein stability difference by mass spectrometry, combines with the research results of some cell signal channels, verifies and locks the target protein, thereby determining the target point.
Compared with the prior art, the invention has the beneficial effects that: the invention relates to a natural product drug action target recognition method based on protein heat stability and broad spectrum molecular dyeing, wherein the natural product drug comprises an active natural product, the natural product does not need to be marked in advance, and the natural product has small dosage, trace dosage and dosage less than 2 mg; the protein target of the invention comprises intracellular low-abundance target molecules, has the characteristic of widely searching natural product targets, can efficiently identify potential drug targets at low cost in a label-free manner, and can promote the research of disease action mechanism and pharmacology and provide guidance information for the potential side effect of the drug and the commercialization of the drug when finding the potential drug targets.
Drawings
FIG. 1 is a process flow diagram of a method for identifying a natural product drug action target according to the present invention;
FIG. 2 is a graph showing the results of Muriceidine A-4 inhibiting proliferation of various tumor cells;
FIG. 3 is a graph showing the effect of Muriceidine A-4 on the proliferation inhibition of MDA-MB-231 cells;
FIG. 4 is a graph showing the effect of Muriceidine A-4 on a portion of the signaling protein in MDA-MB-231 cells;
FIG. 5 is a graph of the binding of Muriceidine A-4 to a glycoprotein in MDA-MB-231 cells;
FIG. 6 is a graph of the binding of Muriceidine A-4 to transferrin receptor in MDA-MB-231 cells;
FIG. 7 shows FeSO4Effect on Muriceidine A-4 induced apoptosis of MDA-MB-231 cellsA drawing;
FIG. 8 shows FeSO4Results of the effect on Muriceidine A-4 induced MDA-MB-231 apoptosis are shown.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Referring to the attached figure 1, the method for identifying the action target of the natural product medicament comprises the following steps:
1) collecting a sample, and dissolving a drug to be detected in a solvent to obtain an experimental sample; taking another solvent as a control sample; respectively adding the experimental sample and the control sample into the effective active cells, and incubating for 1-5h to respectively obtain a drug group sample to be detected and a solvent control group sample;
2) performing heat treatment, namely placing the drug group sample to be detected and the solvent control group sample at 30-80 ℃, performing heat treatment for 3-10min, cracking, centrifuging, and taking supernate to respectively obtain a drug protein sample to be detected and a solvent control protein sample;
3) performing electrophoresis and transfer printing, namely performing electrophoresis and transfer printing on the drug protein sample to be detected and the solvent control protein sample to obtain a drug transfer printing group to be detected and a solvent control transfer printing group;
4) dyeing, namely dyeing the drug transfer printing group to be detected and the solvent control transfer printing group by adopting a broad-spectrum protein antibody or dye, comparing, and searching for bands with protein stability differences;
5) and (3) performing mass spectrometry, namely performing electrophoresis on another drug protein sample to be detected, performing mass spectrometry on the bands with protein stability difference, and verifying to obtain a target point.
Preferably, in the step 4), the dyeing operation is as follows: a) preparing 5% solution of skimmed milk powder with 1 × TBST buffer solution; b) taking a drug transfer group to be detected and a solvent control transfer group, adding the solution obtained in the step a), and incubating for 1-2h at room temperature on a shaking table; c) washing with 1 × TBST buffer solution, adding protein antibody or dye, standing at 4 deg.C, and incubating overnight; d) coating an ECL luminescent reagent or a dye substrate under the condition of keeping out of the sun, and developing by adopting an imaging system; e) comparing the transfer printing group of the drug to be tested with the solvent control transfer printing group, searching for a strip with protein stability difference, and recording the molecular weight.
Further, the protein antibody is any one of a tyrosine protein kinase antibody, a serine/threonine protein kinase antibody, a protein silver stain and biotin-labeled lectin.
Specifically, in the step c), when the protein antibody is added, the protein antibody is a primary antibody, and the primary antibody further comprises the following operations after the overnight incubation: taking a secondary antibody, and diluting the secondary antibody by 2000-fold with 1 xTBST buffer solution; and (3) washing the transfer printing group of the drug to be detected and the solvent control transfer printing group by adopting a 1 xTBST buffer solution, adding the diluted secondary antibody into the washed transfer printing group of the drug to be detected and the solvent control transfer printing group in a shaking table at room temperature within 1-2h, slowly shaking at room temperature, and incubating for 1-2 h.
More preferably, in the step 3), the electrophoresis specifically operates as follows: A) taking SDS-PAGE gel, wherein the SDS-PAGE gel comprises 3-5% concentrated gel and 8-12 separating gel, coating and forming pores; B) adding 8-10 μ L of drug protein sample to be detected or solvent control protein sample into each hole, and adding protein pre-staining Marker into the side holes; C) placing in ice water bath, performing electrophoresis at 60-80V for 20-30min, after the protein pre-staining Marker is completely separated, increasing the voltage to 120-130V, and continuing electrophoresis.
Further, in the step 2), the specific operation of the heat treatment is as follows: s1 enriched cells: washing the drug group sample to be detected and the solvent control group sample to respectively obtain a drug group cell to be detected and a solvent control group cell; s2 gradient heating: heating the cells of the drug group to be detected and the cells of the solvent control group to 30-80 ℃ in a gradient way respectively, and obtaining cell suspension of the drug group to be detected and cell suspension of the solvent control group; s3 cell lysis: cracking the cell suspension of the drug group to be detected and the cell suspension of the solvent control group, centrifuging and collecting supernatant; s4 sample collection: adding equal volume of Loading Buffer into the supernatant respectively, resuspending, decocting in boiling water for 15-20min, standing at-20 deg.C, and storing.
More specifically, the lysis is any one of repeated freeze thawing of liquid nitrogen, lysis of cell lysate and ultrasonic disruption lysis.
Preferably, in step S2, during the gradient heating, the cells of the drug group to be tested and the cells of the solvent control group are heated to 40-60 ℃ respectively by gradient heating.
Still further, in the step 1), the specific operation of collecting the sample is as follows: sa cell plating: digesting and counting effective cells in good growth state and logarithmic growth phase with pancreatin, inoculating, and inoculating at 37 deg.C and 5% CO2Culturing for 24h under the condition; adding Sb cells: setting the experimental sample and the control sample, respectively, and adding the effective cells cultured in step Sa at 37 deg.C and 5% CO2Continuously culturing for 1-3h under the condition; sc-collected cells: removing the culture medium, adding cold PBS, washing, adding pancreatin for digestion, and uniformly blowing to make all adherent cells suspended.
More specifically, in the step 5), the mass spectrometry includes: and (3) taking a drug protein sample to be detected and a solvent control protein sample, carrying out electrophoresis according to the step 3), wherein the sample loading amount during electrophoresis is 20-30 mu L, cutting off adhesive strips near the difference, rinsing with ultrapure water, and carrying out mass spectrometry.
Example one
Compound early activity screening and signal path research
(1) Muriceidine A-4 with novel structure inhibits the proliferative activity of MDA-MB-231 cells
First, the MTT method was used to examine the effect of Muriceidine A-4 on the proliferation of various tumor cells. As shown in FIGS. 2 and 3, it can be seen from FIG. 2 that Muriceidine A-4 has proliferation inhibitory effect on all 13 human cancer cells, and Muriceidine A-4 has the strongest inhibitory effect on MDA-MB-231 cells; as can be seen in FIG. 3, Muriceidine A-4 was time and dose dependent on the proliferation inhibition of MDA-MB-231 cells at different times and concentrations, with an IC50 of 1.15. mu. mol/L.
(2) Effect of Muriceidine A-4 on activation of proliferation-related signaling molecules in MDA-MB-231 cells
Further examining the effect of Muriceidine A-4 on proliferation-related signaling molecules in MDA-MB-231 cells, the experimental results are shown in FIG. 4. As can be seen from FIG. 4, Muriceidine A-4 can inhibit the phosphorylation of AKT, Src, ERK and STAT3 in MDA-MB-231 cells.
Example two
(1) Searching differential protein of Muriceidine A-4 and MDA-MB-231 after cell action
Muriceidine A-4 has a significant inhibitory effect on signal molecules associated with proliferation in the cytoplasm of MDA-MB-231 cells, and may be associated with the action of compounds on cell receptor proteins associated with proliferation. protein glycosylation is one of the most common post-translational modifications of proteins, and membrane receptor proteins tend to have higher glycosylation modifications.lectin is a carbohydrate-binding protein in which ricin agglutinin (RCA I) binds specifically to glycoproteins having α, β -D galactose in the carbohydrate chain, while α, β -D galactose is present widely in most glycoproteins.firstly, by the property of improved thermal stability when binding compounds and proteins, MDA-231 cells after Muriceidine A-4 action are treated at different temperatures, the cells are lysed and a Westerbloting experiment is performed, using RCA I bound avidin antibodies to bind glycoproteins bound to MDA-4 and MDA-MB-231 cells, and it is found that after Muriceidine A-4 action, the thermal stability of molecules bound to biotinylated glycoproteins in MDA-MB-231 cells is increased as shown by a graph 80, 82, and a graph is shown by the results of the binding avidin antibodies bound glycoproteins having a certain galactose residue.
In order to further determine the glycoprotein bound to the muricidine A-4 in the MDA-MB-231 cells, after SDS-PAGE electrophoresis, a gel strip near 80kd is cut off and subjected to protein mass spectrometry detection, 142 proteins are identified in total, and the results of the partially highly related proteins are shown in Table 1, and as can be seen from Table 1, a Transfern receptor (TfR1) is a receptor protein, which indicates that the TfR1 may be a target protein bound to the muricidine A-4 in the MDA-MB-231 cells.
TABLE 1 partial protein species identified by protein Mass Spectrometry
Figure BDA0002223214340000101
The experiment comprises the following specific steps:
(I) sample Collection
(1) Cell plating: the effective cell-MDA-MB-231 with good growth state and in logarithmic growth phase is digested by pancreatin and counted (suspension cells do not need to be digested), inoculated into a 6-well plate, placed in a cell culture box, and subjected to 37 ℃ and 5% CO2Culturing for 24h under the condition;
(2) cell dosing: dissolving a drug to be detected, namely Muriceidine A-4, in a solvent to obtain an experimental sample; taking another solvent as a control sample; adding the effective cells into experimental sample and control sample, respectively, placing in cell incubator at 37 deg.C and 5% CO2Co-incubating for 3h under the condition;
(3) collecting cells: removing the culture medium, adding cold PBS, washing for 3 times, adding pancreatin for digestion, uniformly blowing, and suspending all adherent cells to respectively obtain a drug group sample to be detected and a solvent control group sample.
(II) Heat treatment
(1) Enriching cells: washing the drug group sample to be detected and the solvent control group sample, transferring the washed samples to a PCR tube, and respectively obtaining the drug group cell to be detected and the solvent control group cell;
(2) gradient heating: respectively placing the cells of the drug group to be detected and the cells of the solvent control group in a gradient heating PCR instrument, heating at a gradient temperature, and setting the temperature between 30 and 80 ℃ to obtain cell suspension of the drug group to be detected and cell suspension of the solvent control group;
(3) cell lysis: transferring the cell suspension of the drug group to be detected and the cell suspension of the solvent control group in the PCR tube to an EP tube, adding 0.5 mu L PMSF, immersing in liquid nitrogen for repeated freeze thawing three times, completing the lysis, immediately transferring to a low-temperature freezing centrifuge, centrifuging, and collecting the supernatant;
(4) collecting samples: and respectively adding equal-volume Loading Buffer into the supernatant for resuspension, boiling in boiling water for 15min, placing in a refrigerator at the temperature of-20 ℃, and storing for later use to obtain a drug protein sample to be detected and a solvent control protein sample.
(III) electrophoresis and transfer
(1) Taking SDS-PAGE gel, generally 5% of concentrated gel and 8% of separation gel, coating and forming holes;
(2) adding 8-10 mu L of drug protein sample to be detected or solvent control protein sample into each hole, and adding protein pre-staining Marker on the side holes;
(3) placing the electrophoresis tank in an ice-water bath, firstly carrying out electrophoresis for 25min at 70V, after the protein pre-dyeing Marker is completely separated, increasing the voltage to 125V, and continuing electrophoresis;
(4) the charged protein was transferred onto a Nitrocellulose (NC) membrane using a semi-dry transfer membrane method for 2 h.
(IV) dyeing
1) Weighing skim milk powder by a balance, preparing a 5% solution by adopting a 1 xTBST buffer solution, placing an NC membrane in an antibody incubation box, adding the solution, placing on a shaker, and incubating for 2h at room temperature;
2) washing away skimmed milk powder with 1 × TBST, adding broad-spectrum dye-biotinylated lectin RCA I, placing in 4 deg.C environment, and incubating overnight; if the antibody is used, washing away the unbound primary antibody by using 1 xTBST buffer solution, adding 1.5mL of secondary antibody diluted 4000 times by using 1 xTBST after washing, shaking the table at room temperature, slowly shaking the table for 2h, adding the secondary antibody, placing the secondary antibody on the shaking table, and incubating the secondary antibody at room temperature and slowly shaking the table for 2 h;
3) under the condition of keeping out of the sun, an ECL luminescent reagent is coated on the NC membrane, and a multifunctional imaging system is adopted for developing;
4) compared to the control, bands of protein stability differences were looked for and molecular weight size was recorded.
(V) Mass Spectrometry
(1) Performing SDS-PAGE electrophoresis in the same way, increasing the sample loading amount to 25 mu L, and cutting off adhesive tapes near the difference;
(2) the strip was transferred to a 0.5mL EP tube, rinsed twice with ultrapure water and analyzed by mass spectrometry.
(VI) analysis for target protein
According to the mass spectrum result, proteins with different molecular weights are searched for in protein stability difference, and then combined with the results of some cell signaling pathway researches, the target protein is verified to be locked, namely, TfR1 is the target protein combined with Muriceidine A-4 in MDA-MB-231 cells.
EXAMPLE III
Verification that TfR1 is a target protein for binding to Muriceidine A-4 in MDA-MB-231 cells
To further confirm that TfR1 is the target protein for binding to muricidine a-4 in MDA-MB-231 cells, the binding of muricidine a-4 to intracellular TfR1 was observed using TfR1 antibodies using a thermostability assay, with the results shown in fig. 6; as can be seen in FIG. 6, the binding of Muriceidine A-4 to TfR1 increased the thermal stability of TfR1, indicating that Muriceidine A-4 binds to TfR 1.
To verify whether Muriceidine A-4-induced MDA-MB-231 growth inhibition and apoptosis were associated with TfR1, FeSO was used4MDA-MB-231 cells are pre-treated for 24h to reduce the level of TfR1, and Muriceidine A-4 is added for 24h to act, and the result is shown in figure 7; as can be seen from FIG. 7, after Muriceidine A-4 treatment, TfR1 expression was reduced and c-PARP expression was significantly increased, indicating that MDA-MB-231 cells were apoptotic and then FeSO, compared to the control group4After the pretreatment is used for reducing the expression of TfR1, the apoptosis level of MDA-MB-231 cells is obviously reduced, which indicates that FeSO4Reduces Muriceidine A-4 mediated apoptosis. This suggests that Muriceidine A-4 mediated apoptosis of MDA-MB-231 cells is associated with TfR1, and further demonstrates that TfR1 is a target protein for Muriceidine A-4 binding in MDA-MB-231 cells. As shown in FIG. 8, it can be seen from FIG. 8 that the results have significant difference (P < 0.05).
Therefore, compared with the prior art, the invention has the beneficial effects that: the invention relates to a natural product drug action target recognition method based on protein heat stability and broad spectrum molecular dyeing, wherein the natural product drug comprises an active natural product, the natural product does not need to be marked in advance, and the natural product has small dosage, trace dosage and dosage less than 2 mg; the protein target of the invention comprises intracellular low-abundance target molecules, has the characteristic of widely searching natural product targets, can efficiently identify potential drug targets at low cost in a label-free manner, and can promote the research of disease action mechanism and pharmacology and provide guidance information for the potential side effect of the drug and the commercialization of the drug when finding the potential drug targets.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Claims (10)

1. A method for identifying action targets of natural product drugs is characterized by comprising the following steps: the method comprises the following steps:
1) collecting a sample, and dissolving a drug to be detected in a solvent to obtain an experimental sample; taking another solvent as a control sample; respectively adding the experimental sample and the control sample into the effective cells, and incubating for 1-5h to respectively obtain a drug group sample to be detected and a solvent control group sample;
2) performing heat treatment, namely placing the drug group sample to be detected and the solvent control group sample at 30-80 ℃, performing heat treatment for 3-10min, cracking, centrifuging, and taking supernate to respectively obtain a drug protein sample to be detected and a solvent control protein sample;
3) performing electrophoresis and transfer printing, namely performing electrophoresis and transfer printing on the drug protein sample to be detected and the solvent control protein sample to obtain a drug transfer printing group to be detected and a solvent control transfer printing group;
4) dyeing, namely dyeing the drug transfer printing group to be detected and the solvent control transfer printing group by adopting a broad-spectrum protein antibody or dye, comparing, and searching for bands with protein stability differences;
5) and (3) performing mass spectrometry, namely performing electrophoresis on another drug protein sample to be detected, performing mass spectrometry on the bands with protein stability difference, and verifying to obtain a target point.
2. The method for identifying the natural product drug action target according to claim 1, wherein the step 4) comprises the following steps:
a) preparing 5% solution of skimmed milk powder with 1 × TBST buffer solution;
b) taking a drug transfer group to be tested and a solvent contrast transfer group, adding the solution obtained in the step a), and incubating for 1-2h at room temperature on a shaking table;
c) washing with 1 × TBST buffer solution, adding protein antibody or dye, standing at 4 deg.C, and incubating overnight;
d) coating an ECL luminescent reagent or a dye substrate under the condition of keeping out of the sun, and developing by adopting an imaging system;
e) comparing the transfer printing group of the drug to be tested with the solvent control transfer printing group, searching for a strip with protein stability difference, and recording the molecular weight.
3. The method for identifying natural product drug action targets according to claim 2, wherein the method comprises the following steps:
the protein antibody is any one of a broad-spectrum tyrosine protein kinase antibody, a serine/threonine protein kinase antibody, a protein silver stain and biotin-labeled lectin.
4. The method for identifying natural product drug action targets according to claim 2, wherein the method comprises the following steps:
in step c), when protein antibody is added, the protein antibody is a primary antibody, and the primary antibody further comprises the following operations after overnight incubation:
taking a secondary antibody, and diluting the secondary antibody by 2000-fold with 1 xTBST buffer solution; and (3) washing the transfer printing group of the drug to be detected and the solvent control transfer printing group by adopting a 1 xTBST buffer solution, adding the diluted secondary antibody into the washed transfer printing group of the drug to be detected and the solvent control transfer printing group in a shaking table at room temperature within 1-2h, slowly shaking at room temperature, and incubating for 1-2 h.
5. The method for identifying the natural product drug action target according to any one of claims 1 to 4, wherein the electrophoresis in the step 3) is specifically performed by:
A) taking SDS-PAGE gel, wherein the SDS-PAGE gel comprises 3-5% concentrated gel and 8-12 separating gel, coating and forming pores;
B) adding 8-10 mu L of a drug protein sample to be detected or a solvent control protein sample into each hole, and adding protein pre-staining Marker into the side holes;
C) placing in ice water bath, performing electrophoresis at 60-80V for 20-30min, after the protein pre-staining Marker is completely separated, increasing the voltage to 120-130V, and continuing electrophoresis.
6. The method for identifying the natural product drug action target according to claim 1, wherein the heat treatment in the step 2) comprises the following specific operations:
s1 enriched cells: washing the drug group sample to be detected and the solvent control group sample to respectively obtain a drug group cell to be detected and a solvent control group cell;
s2 gradient heating: respectively heating the cells of the drug group to be detected and the cells of the solvent control group in a gradient manner to 30-80 ℃ to obtain cell suspension of the drug group to be detected and cell suspension of the solvent control group;
s3 cell lysis: cracking the cell suspension of the drug group to be detected and the cell suspension of the solvent control group, centrifuging and collecting supernatant;
s4 sample collection: adding equal volume of Loading Buffer into the supernatant respectively, resuspending, decocting in boiling water for 15-20min, standing at-20 deg.C, and storing.
7. The method for identifying natural product drug action targets according to claim 6, wherein the method comprises the following steps:
the cracking is any one of repeated freeze thawing of liquid nitrogen, cracking of cell lysate and ultrasonic disruption cracking.
8. The method for identifying natural product drug action targets according to claim 6, wherein the method comprises the following steps:
in step S2, during gradient heating, the cells of the drug group to be tested and the cells of the solvent control group are heated to 40-60 ℃ in a gradient manner.
9. The method for identifying the natural product drug action target according to claim 1, wherein in the step 1), the specific operation of sample collection is as follows:
sa cell plating: digesting and counting effective cells in good growth state and logarithmic growth phase with pancreatin, inoculating, and inoculating at 37 deg.C and 5% CO2Culturing for 24h under the condition;
adding Sb cells: setting the experimental sample and the control sample, respectively, and adding the effective cells cultured in step Sa at 37 deg.C and 5% CO2Continuously culturing for 1-3h under the condition;
sc-collected cells: removing the culture medium, adding cold PBS, washing, adding pancreatin for digestion, and uniformly blowing to make all adherent cells suspended.
10. The method for identifying the natural product drug action target according to claim 1, wherein the mass spectrometry in the step 5) is specifically performed by:
and (3) taking a drug protein sample to be detected, carrying out electrophoresis according to the step 3), wherein the sample loading amount during electrophoresis is 20-30 mu L, cutting off adhesive strips near the difference, rinsing with ultrapure water, and carrying out mass spectrometry.
CN201910942137.1A 2019-09-30 2019-09-30 Method for identifying action target of natural product medicine Pending CN110791543A (en)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109872781A (en) * 2019-02-26 2019-06-11 哈尔滨工业大学 Drug target recognition methods based on Xgboost

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109872781A (en) * 2019-02-26 2019-06-11 哈尔滨工业大学 Drug target recognition methods based on Xgboost

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
MARTINEZ MOLINA D等: "The Cellular Thermal Shift Assay: A Novel Biophysical Assay for In Situ Drug Target Engagement and Mechanistic Biomarker Studies", 《ANNU REV PHARMACOL TOXICOL.》 *
THOMFORD NE等: "Natural Products for Drug Discovery in the 21st Century: Innovations for Novel Drug Discovery.", 《INT J MOL SCI》 *
买小圆等: "柳珊瑚来源的愈创木倍半萜生物碱化合物Muriceidine A体外抗肿瘤活性的研究", 《中国海洋药物》 *
王岩航 等: "基于磁性纳米颗粒载体的中药活性成分体内原位靶点鉴定方法学探索", 《中国中药杂志》 *

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